Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

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1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Dombi E, Baldwin A, Marcus LJ, et al. Activity of selumetinib in neurofibromatosis type 1 related plexiform neurofibromas. N Engl J Med 2016;375: DOI: /NEJMoa

2 This supplement contains the following items: 1. Original protocol, most recent protocol for data included in this manuscript (amendment I), and a summary of changes for all amendments. 2. Original statistical analysis plan (section 5.4 of the protocol), final statistical analysis plan for data included in this protocol (amendment I), and a summary of changes

3 CTEP Protocol No: 8799 NIH CC Protocol No: Amendment: Version Date: IRB Approval Date: xx/xx/2010 A Phase 1 Study of the mitogen activated protein kinase kinase (MEK) 1 inhibitor AZD6244 hydrogen sulfate (Selumetinib sulfate) in children with neurofibromatosis type 1 (NF1) and inoperable plexiform neurofibromas (PN) Coordinating Center: Pediatric Oncology Branch, NCI Principal Investigator: Brigitte Widemann, M.D. (POB, NCI) * Lead Associate Investigator: Leigh Marcus, M.D. (POB, NCI) Trial Sponsor NCI, Cancer Therapy Evaluation Program NCI-supplied Agent AZD6244 (Astra-Zeneca), Selumetinib sulfate IND# 77, 782 NSC# NCI Protocol number: NIH Associate Investigators: Andrea Gillespie, R.N. (POB, NCI) Andrea Baldwin, P.N.P. (POB, NCI) Eva Dombi, MD (POB, NCI) ** Nalini Jayaprakash, M.S. (POB, NCI) ** Patricia Whitcomb, R.N. (POB, NCI) Staci Martin, Ph.D. (POB, NCI) *Pharmacology & Experimental Therapeutics Section Pediatric Oncology Branch, NCI 10 Center Drive Building 10, CRC, Room Bethesda, MD Phone: (301) /Fax: (301) widemanb@mail.nih.gov ** Individuals with no direct clinical care responsibilities PROPRIETARY and CONFIDENTIAL This protocol contains confidential information that should only be disclosed to those persons responsible for execution and organization of the trial and on condition that all such persons agree not to further disseminate it.

4 NCI protocol #; Version Date: Précis Background Patients with Neurofibromatosis 1 (NF1) have an increased risk of developing tumors of the central and peripheral nervous system, including plexiform neurofibromas (PN), which are benign nerve sheath tumors that are among the most debilitating complications of NF1. PN may be congenital and appear to have the fastest growth rate in young children. There are no standard treatment options for PN other than surgery, which is often difficult due to the encasement of vital structures, and extensive and invasive growth. PN are composed of neoplastic Schwann cells that lack NF1 gene expression resulting in upregulation of Ras, which initiates several signaling cascades regulating cell proliferation. AZD6244 hyd sulfate, a novel orally bioavailable mitogen activated protein kinase inhibitor, is a specific inhibitor of MEK 1, which is currently undergoing evaluation in adult cancers and children with brain tumors, and may mediate anti-tumor effects in PN by inhibition of downstream signaling of Ras. Objectives To determine the maximum tolerated dose (MTD) of oral AZD6244 hyd sulfate administered daily to pediatric patients with NF1 and inoperable PN. To define the acute and chronic toxicities, pharmacokinetics (PK), and pharmacodynamics (PD) of AZD6244 hyd sulfate. To determine the effect of AZD6244 hyd sulfate on the growth rate of PN. Eligibility Pediatric Patients (12-18 years) with NF1 and inoperable measurable PN that have the potential to cause significant morbidity. Design AZD6244 hyd sulfate will be administered orally BID on a continuous dosing schedule (28 days = 1 treatment cycle). Limited dose escalations will be performed to define the MTD based on tolerability of AZD6244 hyd sulfate during the first three treatment cycles. Disease status will be evaluated using volumetric MRI analysis at regular intervals. The plasma PK and PD of AZD6244 hyd sulfate will be evaluated. 2

5 NCI protocol #; Version Date: Table of Contents PRÉCIS INTRODUCTION OBJECTIVES BACKGROUND AND RATIONALE Neurofibromatosis Type I and Plexiform Neurofibromas Imaging and Measurement of Plexiform Neurofibromas Genetics and Biology of PN AZD Adult Studies Pediatric Phase I Trial Rationale for proposed pediatric phase I trial of AZD6244 in NF1 and PN ENROLLMENT PROCEDURES ELIGIBILITY CRITERIA Inclusion criteria Exclusion Criteria PRE-TREATMENT EVALUATION (SEE APPENDIX III) PATIENT REGISTRATION IMPLEMENTATION OF STUDY STUDY DESIGN Overall Trial Design Criteria for Dose Escalation Definition of Dose Limiting Toxicity (DLT) Criteria for Intra Patient Dose Escalation Definition of Maximum Tolerated Dose (MTD) Criteria for Starting Subsequent Treatment Cycles Duration of Treatment DRUG ADMINISTRATION TREATMENT MODIFICATIONS PHARMACOKINETICS CORRELATIVE/PHARMACODYNAMIC STUDIES BLOOD VOLUME FOR RESEARCH STUDIES ON STUDY EVALUATIONS (APPENDIX III) CONCURRENT THERAPIES CRITERIA FOR REMOVAL FROM TREATMENT OFF STUDY CRITERIA END OF TREATMENT EVALUATION (APPENDIX III) SUPPORTIVE CARE DATA COLLECTION AND EVALUATION DATA COLLECTION Case Report Forms RESPONSE CRITERIA TOXICITY CRITERIA STATISTICAL SECTION Subject Accrual Statistics and feasibility DATA AND CENTER AUDITS Data And Safety Monitoring Plan STORAGE, TRACKING AND HANDLING OF RESEARCH SPECIMENS HUMAN SUBJECTS PROTECTIONS

6 NCI protocol #; Version Date: RATIONALE FOR SUBJECT SELECTION PARTICIPATION OF CHILDREN RISKS AND BENEFITS CONSENT AND ASSENT PROCESS AND DOCUMENTATION DATA REPORTING PATIENT REGISTRATION CASE REPORT FORMS SAFETY REPORTING ADVERSE EVENTS ADVERSE EVENT REPORTING REQUIREMENTS NCI-IRB Reporting NCI-IRB Reporting of IND Safety Reports PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION AZD6244 HYDROGEN SULFATE (NSC ) AZD6244 hyd sulfate Administration AZD6244 hyd sulfate Toxicity Profile COLLABORATIVE AGREEMENT REFERENCES APPENDICES APPENDIX I: PERFORMANCE STATUS SCALES/SCORES APPENDIX II: PROTOCOL FOR REQUIRED MRI STUDIES OF PN APPENDIX III: REQUIRED STUDY EVALUATIONS APPENDIX IV: PHARMACOKINETIC WORKSHEET APPENDIX V: PERIPHERAL BLOOD MONONUCLEAR CELL SAMPLES (PBMCS) APPENDIX VI: DOSING NOMOGRAM FOR AZD6244 HYD SULFATE FOR PHASE I PEDIATRIC NF-1 TRIAL APPENDIX VIIA: ADHERENCE QUESTIONNAIRES APPENDIX VIIB: PATIENT DAILY MEDICATION DIARY FOR AZD6244 HYD SULFATE APPENDIX VIIC: ADHERENCE BARRIERS LIST FOR PATIENT DAILY MEDICATION DIARY APPENDIX VIID: RESPONSIBILITY FOR MEDICATION QUESTIONNAIRES APPENDIX VIII: SKIN TOXICITY

7 NCI protocol #; Version Date: Introduction 1.1 Objectives Primary Study Objectives: 1. To determine the maximum tolerated dose (MTD), extended tolerability, and recommended phase II dose of AZD6244 hyd sulfate administered orally every 12 hours on a continuous daily schedule for cycles of 28 days with no rest period between cycles in children and adolescents with neurofibromatosis type 1 (NF1) and inoperable plexiform neurofibromas (PN). The MTD will be defined based on toxicities observed during the first three treatment cycles. 2. To study the plasma pharmacokinetics (PK) of AZD6244 hyd sulfate at baseline and steady state. Secondary Study Objectives: 1. To determine the effect of AZD6244 hyd sulfate on the growth rate of PN using automated volumetric MRI analysis. 2. To study the pharmacodynamics (PD) of AZD6244 hyd sulfate by evaluating ERK phosphorylation in peripheral blood mononuclear cells (PBMCs) in peripheral blood samples, obtained pre-treatment and at steady state on treatment with AZD6244 hyd sulfate. 3. To measure adherence of AZD6244 hyd sulfate chronic dosing in this patient population. 4. To describe and define the toxicities in pediatric patients on chronic dosing of AZD6244 hyd sulfate. 1.2 Background and rationale Neurofibromatosis Type I and Plexiform Neurofibromas Neurofibromatosis type 1 (NF1) is a common autosomal dominant, progressive disorder with an incidence of 1:3500 (>80,000 persons affected in The United States) [1]. NF1 is characterized by diverse, progressive cutaneous, neurological, skeletal, and neoplastic manifestations with no standard drug treatment options available. Patients with NF1 have an increased risk of developing tumors of the central and peripheral nervous system including plexiform neurofibromas (PN) (27%), optic gliomas (15-20%), pheochromocytomas (1%), and malignant peripheral nerve sheath tumors, (5%) [2, 3]. PN are benign nerve sheath tumors that grow along the length of nerves and involve multiple branches of a nerve. Many PN are thought to be congenital, but may not be diagnosed until later in life. Early childhood, puberty and childbearing age in females are considered to be the periods of greatest risk for disease progression[4]. In the past, the rate of growth of this histologically benign neoplasm has been described as unpredictable and often episodic[2]. However, a recent analysis of PN using volumetric MRI analysis suggests consistent growth rate of PN within a 5

8 NCI protocol #; Version Date: patient, the median observation period of 34 months (see section 1.2.2)[5]. These tumors may cause significant morbidity by disfigurement, as well as compression of vital structures. As examples, PN may infiltrate the orbit, displace the globe and compromise vision; paraspinal tumors can compress the spinal cord and cause paralysis; tumors in the mediastinum may compress the trachea or great vessels; and tumors of the extremities can cause local nerve infiltration, progressive neurologic deficit and often unremitting pain[2, 4]. A review of 47 children and young adults with NF1 and PN entered on clinical trials at the NCI, Pediatric Oncology Branch showed that 25 patients (53%) underwent surgery of their PN prior to trial entry, 28 patients (60%) were disfigured by the PN, 16 patients (34%) required pain medications, 9 patients (19%) had neurologic symptoms, 5 patients (11%) had airway compromise, and 2 (4%) patients died as a result of PN progression[6]. Surgery is the only standard treatment of PN. However, up to 44% of tumors progress after the first surgery, most commonly in patients younger than ten years of age with head and neck tumors that could not be completely resected[4]. The unknown natural history of PN in NF1 and difficulties in measuring changes in size of these complex, large, and slow growing lesions has made it difficult to define the benefit of medical treatments for PN. However, a number of medical treatments including thalidomide[7], cis retinoic acid, interferon alfa 2b, methotrexate and vinblastine, PEG interferon alfa-2b, pirfenidone, and the farnesyltransferase inhibitor R115777[8] have been evaluated or are undergoing evaluation in early clinical trials for patients with NF1 and PN with the goal to reduce the growth rate or shrink these tumors. Published results are available for the phase I thalidomide trial and the phase I trial of R115777[7, 8]. In the thalidomide trial, 20 patients with NF1 and PN received thalidomide at doses up to 200 mg/day. Response was assessed by clinical measurements and 2-D radiographic measurements. Of 12 patients who completed 1 year of treatment, four showed a <25% reduction in tumor size, and seven showed symptomatic improvement with predominantly a decrease in pain. The phase I trial of the farnesyltransferase inhibitor R included seventeen patients with NF1 and PN. At the MTD of 200 mg/m 2 /dose, R was well tolerated, and patients with NF1 received a median of 10 treatment cycles (range 1-32). No objective tumor shrinkage was observed using conventional response criteria for solid tumors. The NCI POB coordinated a multi-center double-blinded, placebo-controlled, cross-over trial of tipifarnib (R115777) for children and young adults with progressive PN, which has recently been completed. Time to disease progression measured by volumetric MRI analysis was the primary trial endpoint. The median time to progression (PN volume increase by 20%) for 30 patients randomized to receive placebo as the first treatment was 10.6 months, and for patients randomized to receive tipifarnib as first treatment was 19.2 months (one sided alpha 0.129). Tipifarnib thus did not result in a greater or equal to doubling of the time to disease progression in children and young adults with NF1 and progressive PN. One of the key findings of this trial provided information regarding the time to progression of PN in patients who do not receive medical treatments directed at their PN, which is the first time this has been documented. This data will be helpful for the design of future trials, and holds rationale for time of radiographic evaluation in this trial[9]. The NCI, POB has coordinated a phase I trial of the antifibrotic agent pirfenidone. This trial used automated volumetric MRI analysis of PN to determine progression. Of 12 patients entered at the second dose level and phase II dose of 500mg/m 2 /dose TID on a continuous dosing 6

9 NCI protocol #; Version Date: schedule, 9 were removed from the protocol with disease progression after a median of 15 treatment cycles (range 7-21 cycles). Two patients completed 2 years of treatment with stable disease, and 1 patient was removed from the protocol for non-pirfenidone-related medical complications [10]. The NCI, POB has completed a phase II trial of pirfenidone for patients with progressive PN. The median TTP for patients treated with pirfenidone was 13.2 months and thus similar to the TTP of patients treated with placebo on the tipifarnib trial[9, 11]. In addition, a phase I trial of sorafenib, and phase II trials of PEG-intron and sirolimus for NF1 patients with inoperable PN are ongoing Imaging and Measurement of Plexiform Neurofibromas Tumor response criteria that are used for solid tumors are based on one-dimensional (1-D) and two-dimensional (2-D) tumor measurements[12, 13]. These methods have limited value in the assessment of treatment outcome for PN, which are frequently large, have a complex (nonspherical) shape, and have a slow, growth pattern. In order to reproducibly quantify the size of these complex lesions and detect small changes in the size over time, we used MR imaging characteristics of PN to develop an automated method of lesion detection and volume measurement [14]. Short T1-Inversion Recovery [15] MR images, on which PN are bright lesions compared with normal surrounding tissue, were used to develop a program for automated image analysis within MEDx (v3.41) software (Sensor Systems, Inc. Sterling, VA). Reproducibility and inter-observer variability of this automated method were determined by 2 observers who quantified volumes for PN of the orbit (n=2), face/neck (n=3), abdomen (n=1), and pelvis (n=3) on three different days. For each MR image (Fig. 1A), the tumor is roughly Figure 1: Axial MRI of pelvic plexiform neurofibroma. Steps of automated volumetric analysis A-D. A C B D outlined manually including a rim of low signal intensity normal tissue (Fig. 1B). The program then performs a histogram analysis of signal intensity pixel by pixel and a threshold that distinguishes high signal intensity tumor from normal tissue is defined (Fig. 1C). Tumor contours are then determined using a gradient image, connected component analysis and automatic edge following operation (Figure 1D). There is an option for reanalysis of MR images using an average or selected threshold. Tumor volume is calculated by summing the results from all images based on the resulting 2-D contours and slice thickness; and a report is generated. For comparison, PN volume was also determined by manually tracing the tumor borders on each MR image. The results of application of the automated method are shown in the table below. 7

10 NCI protocol #; Version Date: Observer 1 Observer 2 Mean tumor volume ml, median (range) 291 ( ) 290 ( ) Inter-day CV %, median (range) 3.6 ( ) 1.6 ( ) Median (range) % difference in volume between observers 6.4 ( ) Correlation automated vs. manual method, R This automated volumetric MRI analysis is applicable to most PN, has excellent intra- and interobserver reproducibility, and agrees with volumes determined by manual tumor tracing. This method is currently used at the NCI, POB to centrally evaluate PN volume on several multicenter clinical trials for children with NF1 and PN. In these clinical trials tumor progression is defined as an increase in tumor volume by 20%. This volume increase corresponds to much smaller changes in 1-D, or 2-D measurements as outlined in the table below. Response Criteria Disease progression (Increase) RECIST Diameter, 2r WHO Product, (2r) 2 NF1 PN trials Volume, 4/3 r Shaded areas show current criteria used to define disease progression by RECIST, WHO, and the ongoing clinical trials for NF1 and PN. On these clinical trials volumetric MRI analysis of PN has been feasible for most tumors. Analysis of patients entered on NCI POB trials showed that the growth rate of PN is variable among patients, but remains constant within patients, and that young children (less than 8 years of age) have a more rapid PN growth rate compared to older children [5]. This is demonstrated in the figure below. (A) The relationship between the slopes for percent change in PN volume and percent change in body weight per year. Values above the 45 degree line represent ratios of percent change in PN volume to percent change in body weight which are greater than 1. Closed symbols represent patients the median age (8.3 years) at study entry, and open symbols patients > 8.3 years. Percent change in PN volume (B) and body weight (C) per year as a function of patient age at baseline. The shaded area indicates the 20% change in PN volume required for documentation of disease progression in ongoing clinical trials. The line in B represents an exponential fit to the data. 8

11 NCI protocol #; Version Date: The analysis emphasizes the need to develop effective medical interventions for young children with NF1 and PN Genetics and Biology of PN The gene responsible for NF1 has been cloned and encodes a protein called neurofibromin[16]. Although the function of neurofibromin is not completely understood, it is known to include a GTPase activating protein (GAP) domain that regulates hydrolysis of Ras-GTP to Ras-GDP[17]. Histologically, PNs are composed of neoplastic Schwann cells accompanied by a varying number of cellular and noncellular components including fibroblasts, perineural cells, and mast cells. Recent studies have demonstrated that the neoplastic Schwann cells lack NF1 gene expression[18, 19]. Therefore, loss of neurofibromin is associated with elevated levels of activated ras. Activated ras results in the initiation of a cascade of signaling events such as activation of raf and MAPK that lead to increased cell proliferation. Several clinical trials with targeted agents aiming at reduction of PN growth have been conducted[20]. For example, farnesyltransferase inhibitors (FTI) were developed to prevent posttranslational Ras processing and transduction of proliferative signals, which is required for activity of mutant and wild type Ras. Inhibition of Ras activity was the rationale for evaluation of FTIs in cancer and NF1[21]. However, N-, and K-Ras can undergo an alternate lipid modification by geranyl-geranylation, and thus overcome the effect of FTI[22]. This is critical in light of recent data pointing towards K-ras as a key mediator of Ras activity in NF1 tumors[23, 24]. Thus additional strategies such as novel RAS inhibitors blocking all Ras isoforms [25] or agents blocking downstream pathways of RAS, such as ADZ6244, are being considered for development in NF1. Recently, Lauchle et al showed in an NF1 mouse model of myeloid leukemia that MEK inhibitors induced objective regression of many Nf1-deficient acute myeloid leukemias [26]. These data provide further support for evaluation of MEK inhibitors in NF1 related tumors AZD6244 AZD6244 is an orally bioavailable targeted agent against mitogen-activated protein kinase kinase (MEK) 1, with a concentration which resulted in 50% inhibition (IC 50 ) of 9

12 NCI protocol #; Version Date: approximately 10 to 14 nm in adult tumor moedels, including breast cancer and non small cell lung cancer (NSCLC) cell lines[27, 28]. MEK is a critical kinase in the mitogen-activated protein (MAP) kinase signal transduction pathway for many growth factor receptors that provide growth signals to cancer cells. AZD6244 inhibits the activity of isolated MEK to phosphorylate extracellular signal-regulated kinase (ERK) 2 in enzyme assays. AZD6244 hyd sulfate is a highly specific inhibitor of the MEK kinases[27]. Directly downstream in the MAPK pathway from MEK is ERK. Inhibition of ERK phosphorylation was found in tumors in which growth was inhibited by AZD6244, indicating that tumor phosphorylated ERK (perk) levels are a potential biomarker for AZD6244 activity in vivo. Thus, agents targeted at inhibition of the MAPK signaling pathway have a good rationale for evaluation in NF1 and inoperable PN. Detailed information regarding preclinical and clinical studies with AZD6244 is provided in the following pages, and data is directly from Investigator Brochure[29]. Preclinical Studies AZD6244 All preclinical information provided in this protocol is from the Investigator s Brochure. In vitro efficacy studies These studies were done using the free-base of AZD6244. The effect of AZD6244 on ERK phosphorylation and cell viability was determined in a panel of 10 cells lines, including various tumor types (breast, colorectal, lung, osteosarcoma, ovarian, pancreatic, and prostate), in which the mutational status of BRAF and KRAS is known. The potency of AZD6244 in inhibiting ERK1 and ERK2 phosphorylation was consistent among cell lines, ranging from to M. A panel of human tumor cell lines were exposed to varying concentrations of AZD6244. The IC 50 values ranged from <10nM to >10μM and most of the cell lines were sensitive to AZD6244 that contained either a BRAF or Ras gene mutation. N-desmethyl AZD6244, the primary active metabolite of AZD6244, has 3 to 5-fold greater activity of its parents drug, and is a more potent inhibitor if ERK phosphorylation[29]. In vivo efficacy studies The pharmacological effects of AZD6244 have been studied extensively by examining the anti-tumor activity of AZD6244 in different tumors in mice. In one such study, human lung Calu-6 tumor cells were implanted subcutaneously in the flanks of nude mice and the tumors were allowed to grow to approximately 0.2 cm 3. The mice were then treated for 25 days with AZD6244 (10, 25 or 100 mg/kg bid oral dosing) or control. Tumor growth was suppressed in all 3 dose groups during the treatment period, with regrowth following treatment withdrawal[30]. 10

13 NCI protocol #; Version Date: In a series of similar studies, AZD6244 showed significant anti-tumor activity in several tumor models, including human melanomas (LOX and A375v), human breast carcinomas (Zr-75-1 and MDA-MB-231), human pancreatic tumors (BxPC3, AsPC1, HPAC, MIA PaCa-2 and PANC 1), human lung cancer tumors (A549) and human colon carcinomas (HT29, Colon 26 tumors, Colo205, SW620, Lovo and HCT116)[28, 30-33]. However, AZD6244 was only minimally active in SK MEL-28 melanoma, PC3 prostate tumors or H460 lung tumors. Overall, these results demonstrate that AZD6244 has strong antitumor activity in multiple non-clinical tumor models. Combination studies AZD6244 has been combined with a number of therapeutic agents both in vitro and in vivo including standard cytotoxic drugs including irinotecan and docetaxel [31], which enhanced efficacy and synergy, cytarabine [34], which showed enhanced induction of apoptosis in AML cells, and novel targeted therapies [35, 36] using both KRAS mutant and wild type models. Gefitinib, vandetinib and taxotere combinations with AZD6244 have been tested in the KRAS mutant non-small cell lung cancer model A549a, all combinations are more effective than the respective monotherapies[31, 37]. Combinations between AZD6244 and the mtor kinase inhibitor AZD8055 achieves a greater anti-tumor effect than the respective monotherapies in all models tested. Calu-6 model combination of AZD6244 and cediranib was more effective than either agent alone in inhibiting tumor growth. Pharmacodynamic Markers in Tumor Samples The effects of single doses of AZD6244 upon the levels of perk in human Calu-6 xenograft tumors have been determined as a measure of AZD6244 activity against MEK, and related to plasma levels of AZD6244[31]. The levels of perk in the tumour cell cytoplasm and nucleus were determined by immunohistochemical (IHC) staining of formalin- fixed tissue sections, and western blot analysis of tumor protein lysates (Fig. 2). Following an acute dose of 25 mg/kg AZD6244, perk levels decreased by approximately 80% to 90% at 1, 2 and 4 hour post-dose, when plasma concentrations of AZD6244 were highest, and recovered to >50% of control levels by 24 h. The same trend is observed by western blot analysis though the perk signal was consistently, slightly higher than determined by IHC analysis. 11

14 NCI protocol #; Version Date: Figure 2. Inhibition of ERK phosphorylation by a single 25 mg/kg dose of AZD6244 in Calu-6 human lung cancer xenograft tumors Animal toxicities Toxicities in preclinical studies, more frequent with chronic dosing, are not consistent through all animals. They included mineralization, defined as extracellular deposits of calcium and phosphate crystals, in multiple tissues, including cornea, kidney, liver, myocardium, skeletal muscle, diarrhea, renal changes (tubular epithelial swelling and mild vacuolation) secondary to persistent diarrhea, hematopoietic atrophy, and dilation of the corpus spongiosum, which lead to urethral compression. Safety pharmacokinetic studies Individual high performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) assays for measurement of AZD6244, AZD6244 N-desmethyl and AZD6244 amide have been developed and validated, as well as combined assays to measure AZD6244 and N-desmethyl in a single assay, and a combined assay to measure AZD6244, N-desmethyl and the amide metabolites. All assays used have a coefficient of variation (CV)% <15% across the analytical range. The current preclinical and clinical assay range is μg/ml for AZD6244 and μg/ml for N-desmethyl AZD6244 and the amide metabolite. 14 C-labelled AZD6244 and N-desmethyl (AZ ) have been synthesized and used in metabolism studies. In the rat and monkey 1-month toxicity studies performed with AZD6244 free-base, no accumulation of AZD6244 was seen on multiple dosing at any dose level. In the mouse and monkey 1-month toxicity studies performed with AZD6244 there remained little or 12

15 NCI protocol #; Version Date: no accumulation of AZD6244 on multiple dosing in either species, however, in both with dosing of AZD6244, AZD6244 area under the plasma concentration-time curve from time 0 to 12 h (AUC 0-12 ) increased approximately in proportion to dose. Oral bioavailability following dosing with 5mg/kg of either AZD6244 free base or Hyd- Sulphate in captisol was 29% and 56% respectively. Toxicokinetic data from the mouse and monkey 26-week studies, performed with AZD6244 Hyd-Sulphate, largely supported the data previously seen in the 1-month studies with the salt form. In both species, AUC 0-12 increased approximately in proportion to dose, which ranged from 1 to 20mg/kg bid in mouse and 0.5 to 4mg/kg bid in monkey. In the monkey 26 week study, exposure to AZD6244 was similar for both males and females. Small increases in AZD6244 AUC 0-12 values of between 1.5- and 1.9-fold occurred between day 1 and 26, indicating that some accumulation may have occurred. The mean terminal elimination half-life of AZD6244 was approximately 6 hours with individual values ranging from 2.95 to 11 hours. Metabolism Metabolism of AZD6244 was investigated in vitro using hepatocytes from animals and humans. There was evidence of Phase 1 and 2 metabolism with the majority of metabolites detected as glucuronide conjugates. Phase 1 metabolism included N- demethylation, oxidative defluorination and loss of the side chain to form amide and acid metabolites. Inhibition studies indicated that CYP 1A2 was the enzyme primarily responsible for the formation of the N-desmethyl metabolite. Using expressed CYPs it was evident that CYPs 2C19 and 3A4 also metabolized AZD6244 in addition to 1A2. Preliminary investigation in vitro using human hepatocytes indicated that AZD6244 was a weak inducer of CYPs 3A, 1A and 2C9. At AZD6244 concentrations approximately 10-fold higher than those achieved in the clinic, the level of induction was <40% of positive control inducers. N-desmethyl AZD6244 (a pharmacologically active metabolite) was identified to be approximately 3 to 5-fold more active than the AZD6244 parent compound. The amide metabolite is up to 50-fold less active than AZD6244, and is therefore unlikely to significantly contribute to biological activity. The N-desmethyl metabolite was not detectable in rat and at only trace levels in the monkey, but was produced in mouse at circulating levels around 2-12% of parent compound. However, there was evidence that exposure to the metabolite was reduced on multiple dosing. Preliminary unvalidated data indicate that circulating levels of the amide metabolite are detected in both mouse and monkey, and there was a trend towards an increase in exposure on multiple dosing. The plasma concentrations of N-desmethyl metabolite is only about 10% of the parent compound AZD6244. Conversely the amide metabolite showed increased exposure on multiple dosing indicating some accumulation, which is possibly consistent with a longer 13

16 NCI protocol #; Version Date: terminal half-life[38]. However, it was not possible to confirm this. Table 1. Pharmacokinetic Summary Table Preclinical Model Dose (mg/kg) BID Duration C max (ng/ml) T max (hr) AUC 0-12 (ng hr/ml) F (%) Mouse 11 1 month month month mos mos / mos Monkey month month % month 96/99 259/ mos mos mos In all species, AZD6244 was predominantly associated with the plasma component of blood. AZD6244 was highly protein bound. Table 2. Plasma protein binding of AZD6244 and N-desmethyl AZD6244 AZD6244 was metabolized by CYPs 1A2, 2C19 and 3A4 and glucuronidation appeared to be a major clearance mechanism for AZD6244 and Phase 1 metabolites. AZD6244 was a weak direct inhibitor of CYP2C9; IC μm. N-desmethyl AZD6244 was a weak inhibitor of CYP1A2; IC μm. Toxicities in different species Studies were done in animals administered both forms of AZD6244: Hyd-Sulfate capsular form and free-base suspension mix and drink form or SBE-CD, Captisol form. Toxicities seen in the free-base suspension mix were in part attributed to vehicle. However, children on this trial will be administered Hyd-Sulfate capsule form only. 14

17 NCI protocol #; Version Date: Acute studies Administration of AZD6244 free-base at single oral doses of up to 300 mg/kg/day was well tolerated in rats with no treatment-related findings. Administration of AZD6244 free-base as 2 single oral doses, approximately 12 hours apart, at doses of up to 100 mg/kg/dose, was well tolerated in cynomolgus monkeys. Watery-liquid stool was observed in all dose groups, including vehicle control. Transient increases in serum ALT, AST and lactate dehydrogenase (LDH) were seen in all dose groups receiving AZD6244. In the 100 mg/kg bid dose group, the increases in serum enzymes (in particular ALT) were suggestive of mild hepatocellular injury. However, enzyme levels were normal by Day 14 and there were no histopathological changes in liver or skeletal muscle. Minimal lymphoid hyperplasia and follicular hypertrophy were seen in the spleen, primarily in the 100 mg/kg bid group, but were considered to be of little or no toxicological significance. Chronic studies Administration of AZD6244 free-base at daily doses 10, 30 or 100 mg/kg/day to rats for 1 month was well tolerated, but was associated with soft stools and increases in serum phosphorus, urea nitrogen and globulin, and decreased serum albumin. All of these changes showed reversibility on completion of a 1- month recovery period. Dosing with AZD6244 free-base also produced an increased incidence and severity of gastric mucosal mineralization (mineral deposition in the interstitum of the lamina propria of the mucosa of the glandular stomach without cell necrosis/apoptosis or significant inflammation) in males at all dose levels and in females dosed at >30 mg/kg/day. In addition, 1 male at 100 mg/kg/day showed tunica muscularis mineralization. Gastric mucosal mineralization was still apparent in several animals at the end of the recovery period. Twice-daily administration of AZD6244 free-base (12 hours apart) to cynomolgus monkeys at doses of 0, 3, 10 or 30 mg/kg/dose for 1 month, at a dose volume of 2 ml/kg/dose in SBE-CD vehicle, produced persistent watery-liquid stools in all groups, including vehicle controls. From Day 14, the volume of vehicle was reduced from 2 ml/kg/dose (600 mg/kg/dose) to 0.5 ml/kg/dose (150 mg/kg dose). This ameliorated the diarrhea in the control and low dose groups, although it persisted in mid- and high-dose groups for the remainder of the dosing period. The incidence of watery stools decreased in all animals during the recovery period. Treatment-related findings were largely secondary to the persistent watery-liquid stools and included dehydration (decreased skin turgor and reduced body weight), reduced serum albumin, increased serum globulin, increased serum blood urea nitrogen and creatinine, and decreased serum electrolytes, primarily in the 10 mg/kg bid and 30 mg/kg bid groups. Renal histological changes (mild/moderate renal tubular epithelial swelling and/or mild vacuolation), seen in 2 animals dosed at 30 mg/kg bid for which more severe indices of dehydration were apparent, were also considered secondary changes. Similar 15

18 NCI protocol #; Version Date: changes were not seen in animals at the end of a 1- month recovery period. A 7 day dose setting study and 1 and 6 month repeat dose studies, with AZD6244 Hyd- Sulfate have been conducted in mice. At 1- month the low dose of 11 mg/kg bid was well tolerated, although there may have been plasma alkaline phosphatase (ALP) and triglyceride changes seen for animals dosed at this levels, in addition to stomach glandular epithelial cell degeneration, mesenteric lymph node reactive lymphoid hyperplasia and mineralization in several tissues (cornea, with corneal epithelial degeneration, and liver in association with inflammatory or necrotic areas). Oral administration of AZD6244 to mice at 5 mg/kg bid for 6 months was well tolerated, with no treatment-related mortalities or adverse signs, slight reductions in bodyweight gain (females, only), minor hematology (elevated neutrophil and monocyte counts) and blood chemistry (increases in plasma phosphorus, males only) changes. Minimal to slight inflammation with hyperplasia was observed in the colon, cecum and rectum in a few animals, and minimal multifocal mineralization was recorded in the liver of one male. In animals dosed at 1 mg/kg bid for 26 weeks, the only findings considered possibly related to treatment were minimal inflammation with epithelial hyperplasia in the colon in one male and two females. In the 6 month study, AZD6244 Hyd-Sulfate was administered bid (approximately 12 hours apart) to cynomolgus monkeys at doses of 0, 0.5, 1.5 or 4 mg/kg bid. Dosing AZD6244 to cynomolgus monkeys for 26 weeks was associated with loose/liquid feces. This was predominantly seen in animals dosed at 4 mg/kg bid and in some cases was associated with signs of dehydration and/or body weight loss. Following dosing at 4 mg/kg bid, both sexes had reductions in plasma albumin and albumin/globulin ratio and higher mean beta and gamma globulins. Elevated plasma AST, and reduced cholesterol and calcium, were also apparent in one or both sexes. One high dose male also showed elevations in plasma phosphorus from weeks 4 to 26. Preclinical Reproductive Toxicity Genotoxic studies have not shown any overall effect level, but have shown micronuclei in mice in vivo. Chromosomal damage is used as a surrogate for genotoxicity, which is apparent as micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. Chronic genotoxic studies continue to be ongoing. Survival as estimated by mean and median age at death is approximately 15 years less than expected in individuals with NF1[29]. The reproductive capacity of individuals with NF-1 is expected to be normal. Less data are available for children with large PNs and high existing or potential for morbidity. Therefore, we believe that the potential benefits of therapy with AZD6244 justify the risks involved. Male fertility was also assessed by mating treated males from the 6 month mice study with additional groups of undosed females. Dosing of males for 10 weeks at doses of up to 20 mg/kg bid was not associated with any impairment of the mating performance 16

19 NCI protocol #; Version Date: or fertility. In females, dosing at up to 75 mg bid was well tolerated, with no evidence of maternal toxicity Adult Studies Below are the summaries of trials using AZD6244 hyd sulfate capsules, which have replaced the suspension formulation. Adult phase 1 A Phase I, open label, multi-center study to assess the safety, tolerability and pharmacokinetics of AZD6244 in patients with advanced solid refractory malignancies for which no standard therapy exists has been completed. Efficacy assessment in this study was an exploratory endpoint. The study was conducted in two parts. Part A (31 patients) of the study was a dose escalation study, was designed to provide adequate tolerability, safety, pharmacokinetic, and pharmacodynamic data. In Part A, the first cohort received a single 25 mg dose of the AZD6244 hyd sulfate capsule formulation of AZD6244 on Day 1, followed by twice daily dosing from Day 2 onwards. Other doses investigated were 50 mg, 75 mg and 100 mg. The aim of Part B (29 patients randomized) was to determine the relative oral bioavailability of the AZD6244 hyd sulfate capsule, and secondly to expand the safety, tolerability and preliminary efficacy profile of the MTD from Part A (75mg BID). In Part B, patients received either a single dose of capsule formulation or free-base suspension formulation on Day 1 and 8, followed by continuous twice daily dosing of capsule formulation from Day 9 onwards. AZD6244 was absorbed relatively quickly across all dose levels, with a median Tmax of 1.5 hours. Following the peak, AZD6244 concentrations declined multi-exponentially, with a mean T 1/2 ranging from 5 to 7 hours, which is consistent across dose levels. CL/F and Vss/F also remained largely consistent across the dose range, with mean values ranging from 12 to 23 L/h and 87 to 126 L respectively. Plasma N-desmethyl AZD6244 concentrations followed a similar pharmacokinetic profile to AZD6244, although exposure was much lower, with Cmax and AUC values generally <15% of parent, within each patient. The median Tmax was around 1.5 hours and T 1/2 was around 9 to 13 hours. There was minimal accumulation after single versus multiple twice daily dosing. Limited data are available for AZD6244 amide. Concentrations of this metabolite are very variable, both between subjects and within the same subject on different study days. The time to maximum concentration was much more variable than either parent or the N- desmethyl metabolite, ranging from 0.5 to 24 hours across patients. Although there was insufficient data to calculate a terminal half-life, the available data suggest this is longer than that of either AZD6244 and N-desmethyl. Dose-limiting toxicity (DLT) were grade 3 acneiform rash and pleural effusion. There was only 1 Grade 4 event in the study, an event of hypoglycaemia. Fatigue (65.7%) and acneiform dermatitis (60.0%) were the most frequent adverse events at the MTD, with 17

20 NCI protocol #; Version Date: resolution of fatigue upon discontinuation of treatment. A patient with metastatic melanoma bearing a V600E BRAF mutation achieved a complete response persisting after 15 months of therapy. Fifty-five patients had RECIST evaluable tumors. At the 75 mg dose 16/35 (45.7%) patients had stable disease for 6 weeks. One complete response was reported in a 30- year-old female patient with a BRAF mutation positive malignant melanoma receiving 75 mg twice daily of the AZD6244 hyd sulfate capsule in Part A, which is ongoing at two years of therapy with AZD6244. Nine patients in Part A and 13 patients in Part B had a best response of stable disease. Ten out of 55 (18.2%) patients (not including the patient who had a CR) had stable disease of 16 weeks. Seven patients in Part A and 12 patients in Part B had a best response of progressive disease, and 10 patients in Part A and 3 patients in Part B were not evaluable for response. The MTD was 75mg BID[38]. Table 3. Summary of Pharmacokinetic Parameters for AZD6244 A Phase I, open-label, multi-center study is underway to assess the safety, tolerability and pharmacokinetics of AZD6244 hyd sulfate in patients with advanced solid tumors when given in combination with the following chemotherapies: docetaxel, dacarbazine, erlotinib, temsirolimus. The primary objective is to define MTD of AZD6244 when administered twice daily with the afore mentioned chemotherapies. Ninety-eight patients 18

21 NCI protocol #; Version Date: have received therapy and recruitment is ongoing. MTD for the free-base suspension is 100mg po BID while MTD for AZD6244 hyd sulfate was 75mg po BID. A Phase I, open-label study to assess the effect of dosing AZD6244 hyd sulfate in the presence and absence of food in patients with advanced solid malignancies was conducted. Thirty-one patients were randomized and received a single dose of 75mg AZD6244 taken with food on Day 1, followed by a single dose of 75mg fasted on Day 8 or vice versa, followed by BID dosing of 75mg AZD6244 from Day 10. C max and AUC were reduced by 62% and 19% under fed conditions compared with exposure following AZD6244 taken fasted. The time to reach maximum concentration was delayed by approximately 3 hours following administration of 75 mg of AZD6244 in the presence of food. Mean increases in serum liver transaminases (ALT and AST), ALP and phosphate were generally observed during treatment with AZD6244. The majority of reported enzyme elevations (ALT, AST and ALP) occurring prior to disease progression either remained within normal limits or were a maximum increase of 1 CTCAE grade. The small increases in phosphate observed were generally towards the ULN. Best overall response was defined as the best response recorded during the study, based upon the calculated response of target lesions, the investigator s opinion of changes in non-target lesions and the appearance of new lesions. Of the 28 patients with RECIST evaluable tumors, 2 partial responses (7.1%) were observed. Both patients had skin/soft tissue tumors (melanoma) and were noted to still be alive and continuing study treatment when last restaged. In addition, 5 (17.9%) patients had stable disease for >100 days. A phase 1 single institution open label, dose-escalation trial evaluating the safety and tolerability of AZD6244 and IMC-A12 in subjects with advanced solid malignancies is ongoing with no current recruitments. AZD6244 is planned for 75mg BID with reductions to 50mg BID and 50mg once daily. The pediatric brain tumor consortium phase I clinical trial is underway for children aged years old with recurrent or refractory low grade gliomas (see Section 1.2.6). Adult phase 2 A Phase 2, double-blind, randomized study to assess the efficacy of AZD6244 hyd sulfate in combination with Dacarbazine compared with Dacarbazine alone in first line patients with BRAF mutation positive advanced cutaneous or unknown primary melanoma is underway. AZD mg or placebo is given twice daily in combination with dacarbezine. Recruitment has been completed and 77 patients have been randomized. Data are available for 7 patients dosed at 50 mg AZD6244 twice daily in combination with 1000 mg/m 2 dacarbazine on day 1 of a 21 day cycle, and for 4 patients dosed at 75 mg AZD6244 twice daily in combination with 1000 mg/m 2 dacarbazine on day 1 of a 21 day cycle; however currently there is only 1 patient from this regimen that has plasma concentration data for AZD6244 and N-desmethyl AZD6244 both alone and in combination. For the proposed regimen of twice daily dosing of 75 mg AZD6244 in 19

22 NCI protocol #; Version Date: combination with 1000 mg/m 2 dacarbazine on day 1 of a 21 day cycle, the exposure to dacarbazine appears to be unaltered compared to when given alone. Exposure to the dacarbazine metabolite (AIC) is similarly unaltered, and these findings are not altered when the AZD6244 dose was 75 mg twice daily. From the analysis of the AZD6244 data available from the 50 mg arm the exposure to AZD6244 appears to be unaltered compared to when given alone. The exposure to N-desmethyl AZD6244 is similarly unaffected. One out of 6 evaluable patients in the AZD mg BID plus dacarbazine cohort experienced a DLT of grade 4 thrombocytopenia. None of the 6 evaluable patients in the AZD mg BID plus dacarbazine cohort experienced an adverse event which would be classed as a DLT according to the protocol criteria. Therefore, the recommended Phase II dose for the combination of these agents is 75 mg BID AZD6244 hyd sulfate and 1000 mg/m 2 dacarbazine (IV infusion over 60 minutes on day 1 of each 21 day cycle). A Phase 2, double-blind, randomized, placebo-controlled study is ongoing to assess the efficacy of AZD6244 hyd sulfate in combination with Docetaxel compared with Docetaxel alone, in second line patients with KRAS mutation positive locally advanced or metastatic non small cell lung cancer (Stage IIIB-IV). AZD mg or placebo is administered twice daily in combination with docetaxel. Eighty patients have been enrolled and 28 randomized. Data are available for 5 patients dosed at 50 mg AZD6244 twice daily in combination with 75 mg/m 2 docetaxel on day 1 of a 21 day cycle, and for 13 patients dosed at 75 mg AZD6244 twice daily in combination with 75 mg/m 2 docetaxel on day 1 of a 21 day cycle. For the proposed regimen of twice daily dosing of 75 mg AZD6244 in combination with 75 mg/m 2 docetaxel on day 1 of a 21 day cycle, the exposure to either AZD6244 or docetaxel appears to be unaltered compared to when each is given alone. The exposure to N-desmethyl AZD6244 is similarly unaltered. These findings are not altered when the AZD6244 dose was 50 mg twice daily. The recommended Phase II dose for the combination of these agents is 75 mg BID AZD6244 hyd sulfate and 75 mg/m 2 docetaxel (IV infusion over 60 minutes on day 1 of each 21 day cycle, with reactive G-CSF if clinically indicated). To date, a total of 19 patients have received the combination of AZD6244 plus docetaxel. The most frequently reported adverse events (regardless of dose cohort, severity, drug causality or seriousness) were diarrhea (11/19 patients; 57.9%), rash (8/19; 42.1%), nausea and vomiting (7/19 patients each; 36.8%), fatigue, mucosal inflammation, and neutropenia (6/19 each; 31.6%) and constipation, epistaxis and edema peripheral (5/19 each; 26.3%). In addition to the rash AEs there were additional dermatological AEs of dermatitis acneiform and rash macular reported by a single patient each. Dermatological adverse events reported more frequently as starting during the first month on treatment were, in decreasing order of frequency: dermatitis acneiform, rash (maculopapular, erythematous, macular, papular, exfoliative), erythema, skin exfoliation, folliculitis and erysipelas. Dermatological adverse events reported as starting after more prolonged administration (>3 months on treatment) were dry skin, pruritis, skin fissures and paronychia. Other dermatological adverse events occurring at lower frequencies include palmar-plantar eythrodysaesthesia, alopecia and hair depigmentation. A single 20

23 NCI protocol #; Version Date: case of skin photosensitivity has been reported in a patient receiving AZD6244. The majority of dermatitis acneiform and rash adverse events started within 2 weeks of initiation of treatment. The majority of dermatological adverse events were CTCAE Grade 1 or 2. When a dermatological adverse event was CTCAE Grade 3 or greater, or was intolerable CTCAE Grade 2, a dose interruption and/or dose reduction was required to bring the CTCAE intensity to within tolerable limits, although this did not always result in total resolution of the symptoms while the patient continued to receive AZD6244. CTCAE Grade 1 or 2 rash events were sometimes treated by topical administration of antibiotics such as metronidazole, topical steroids, oral tetracycline and simple emollients by the study investigators. In some cases the intensity of the rash was observed to fluctuate in the absence of intervention. Adverse events of diarrhea, nausea and vomiting were reported across all studies with AZD6244. The majority of adverse events were CTCAE Grade 1 (for diarrhea and nausea) or CTCAE Grade 1 or 2 (for vomiting). In the above mentioned phase 1 dose finding study and in the fed versus fasted study, vomiting was reported by 7% and 10% of patients, respectively. In the phase 1 dose finding study, 4/56 (7.1%) patients required a serotonin 5-HT 3 receptor antagonist to control nausea. SAEs of nausea were reported by 3.3% of patients in the fed versus fasted study. On going phase 2 studies include a phase II trial of AZD6244 hyd sulfate in patients with V600E BRAF mutated melanomas, a phase 2 study of AZD6244 in cancers with BRAF mutations identified by prospective genotypic analysis, and a phase 2 evaluation of AZD6244 in the treatment of recurrent or persistent endometrial carcinoma Pediatric Phase I Trial The Pediatric Brain Tumor Consortium (PBTC) is conducting a phase 1 trial in children ages 13 to 21 years with histologically confirmed diagnosis of low grade gliomas (WHO Grades I & II), including patients with pilocytic astrocytoma; astrocytoma, low grade (Fibrillary astrocytoma); astrocytoma, low grade (Low-grade Astrocytoma, NOS); and optic pathway glioma. Five dose levels are planned (Table below) with dose level 1 as the starting dose: Dose Level Dose (mg/m 2 /dose q12h) 0 25 mg/m 2 /dose, BID (total daily dose 50 mg/m 2 /day) 1 33 mg/m 2 /dose, BID (total daily dose 66 mg/m 2 /day) 2 43 mg/m 2 /dose, BID (total daily dose 86 mg/m 2 /day) 3 56 mg/m 2 /dose, BID (total daily dose 112 mg/m 2 /day) 4 73 mg/m 2 /dose, BID (total daily dose 146 mg/m 2 /day) 5 95 mg/m 2 /dose, BID (total daily dose 190 mg/m 2 /day) 21

24 NCI protocol #; Version Date: As of December 2010, one patient experienced a DLT (grade 3 headache) at the starting dose level of 33mg/m 2 /dose BID. This dose level is now expanded to study three additional patients; currently 3 patients have been enrolled on this study to date Rationale for proposed pediatric phase I trial of AZD6244 in NF1 and PN AZD6244 is a rational agent for development in NF1 and inoperable PN (see section 1.2.3). We are proposing a separate phase I study of AZD6244 in children with NF1 and PN for the following reasons: 1. Need to identify a long-term tolerable dose because of the increased incidence of toxicities identified with longer treatment An important aspect of AZD6244 is that with prolonged administration, additional or cumulative toxicities have been seen. Long term safety profiles have been described based on adult phase I and II studies with a small population receiving therapy for greater than 6 months. Since medical treatment is not standard of care for PN in NF1, these patients are generally not pretreated with cytotoxic therapy. Therefore, they will likely be a healthier population than children with refractory cancers enrolled in phase I trials, and may be able to acutely better tolerate AZD6244; however, this must be balanced by the likelihood that they will be on AZD6244 for a longer duration than children with solid and hematologic malignancies. In the R phase I trial for children with refractory solid tumors or NF1 and inoperable PN, children with solid tumors received a median of 1 cycle (range 1-4 cycles) of R compared to 10 cycles (range 1-32) for children with NF1. The definition of a dose, which will be tolerated in children with NF1 for an extended time period, prior to the initiation of a phase II trial, is thus an important goal. It is very unlikely that this information can be obtained from pediatric patients with solid tumors because of limitations by progressive disease, prior therapy, and overall health status. For the farnesyl transferase inhibitor R115777, the MTD and DLT were identical for NF1 and refractory cancers[21], however, sorafenib was less well tolerated in children with NF1 compared to cancers[39], but all toxicities were reversible. 2. Differences in patient characteristics and tolerable dose of AZD6244 between children with refractory cancers and NF1 and PN The natural history of PN is less likely to be imminently life threatening, and PN are generally slow growing tumors[40]. Children with NF1 are less likely to accept acute or chronic mild to moderate toxicities in comparison to children with acutely life threatening illnesses. In addition, children with NF1 enrolled in phase I studies are generally younger than children with refractory malignancies enrolled in phase I studies with a median age of 8 years in comparison to 13 years for cancer patients, (cumulative data from NCI POB originated phase I trials)[6]. Therefore, toxicities specific to AZD6244 and pharmacokinetics may be different for children with NF-1 and children and adults and children with refractory solid tumors. 22

25 NCI protocol #; Version Date: Necessity to evaluate secondary endpoints in pilot fashion in an institution trial prior to development of a large multi-center phase II study. a. The pharmacodynamics of AZD6244 by evaluating ERK phosphorylation in PBMCs in peripheral blood samples, obtained pre-treatment and at steady state on treatment with AZD6244. b. Adherence to medication has been the focus of studies of individuals with HIV disease[41, 42], diabetes[43], asthma[44], and a variety of other chronic conditions. To date, this topic has not been explored in the literature among individuals with NF1. However, assurance of a patient s adherence is essential in determining the efficacy of a medication, especially in clinical trials. Research on adherence among children and adolescents should be considered in the context of the family. Environmental factors related to the child and his or her parents can impact a child s adherence to medication. Common barriers to adherence include forgetting to take the medication[44], wanting to avoid side effects, and issues surrounding the child s level of responsibility for treatment, including medication administration[41]. We will use tools such as pill counts and adherence diaries to evaluate the level of adherence to AZD6244 and to determine the specific barriers to adherence that will aid the development and implementation of AZD6244 to patients with NF1 and PN. In conclusion, AZD6244 hyd sulfate is currently undergoing evaluation in adults with a variety of malignancies including phase I and II studies. In the clinical trials with AZD6244 hyd sulfate mix and drink formulation, adverse events of diarrhea, nausea and vomiting have been reported frequently. Most cases of diarrhea started within the first 2 weeks of treatment, whereas onset of adverse events of nausea and vomiting was variable. However, the majority of episodes have been self-limiting or easily managed with anti-emetic and anti-diarrheal medication. Skin rash was reported as the most common adverse event, grade 3 being frequent, requiring dose holidays or reductions. The suggested treatment of skin toxicity is outlined in Appendix IX. Other toxicities reported included fatigue, edema, and dyspnea. Adverse events relating to visual function have been reported in most, but not all studies. There were no specific clinical findings reported from those patients that underwent an ophthalmological evaluation after reporting a visual disturbance adverse event. No adverse events of urinary retention or priapism have been reported in any Phase I or II study with patients receiving AZD6244 hyd sulfate. 2.0 Enrollment Procedures 2.1 Eligibility criteria Inclusion criteria 23

26 NCI protocol #; Version Date: Age: >12 years and 18 years of age at the time of study enrollment. We plan to amend the protocol to include children 3 to 12 years of age, once we receive approval by the Human Exposure Limits Committee (HELC) at Astra Zeneca. The age limits including young children were chosen because early childhood and puberty are considered to be the greatest risk for disease progression, and AZD6244 hyd sulfate may provide the most benefit to this young group of patients. In addition, an important objective of this study is to characterize the pharmacokinetics of AZD6244 hyd sulfate in the pediatric population since it has been well studied in adults. 2. Diagnosis: Patients with NF1 and inoperable PNs that have the potential to cause significant morbidity, such as (but not limited to) head and neck lesions that could compromise the airway or great vessels, brachial or lumbar plexus lesions that could cause nerve compression and loss of function, lesions that could result in major deformity (e.g., orbital lesions) or significant cosmetic problems, lesions of the extremity that cause limb hypertrophy or loss of function, and painful lesions. Histiologic confirmation of tumor is not necessary in the presence of consistent clinical and radiographic findings, but should be considered if malignant degeneration of a PN is clinically suspected. A PN is defined as a neurofibroma that has grown along the length of a nerve and may involve multiple fascicles and branches. A spinal PN involves two or more levels with connection between the levels or extending laterally along the nerve. In addition to PN, all study subjects must have either positive genetic testing for NF1 or have at least one other diagnostic criterion for NF1 listed below (NIH Consensus conference[45]: Six or more café-au-lait macules ( 0.5cm in prepubertal subjects or 1.5 cm in post pubertal subjects) Freckling in axilla or groin Optic glioma Two or more Lisch nodules A distinctive bony lesion (dysplasia of the sphenoid bone or dysplasia or thinning of long bone cortex) A first-degree relative with NF1 3. Measurable disease: Patients must have at least one measurable PN, defined as a lesion of at least 3 cm measured in one dimension. Patients who underwent surgery for resection of a PN are eligible provided the PN was incompletely resected and is measurable as per criteria above. 4. Prior Therapy: Patients with NF1 will only be eligible if complete tumor resection is not considered to be feasible without substantial risk or morbidity, or if a patient with a surgical option refuses surgery. 24

27 NCI protocol #; Version Date: Since there is no standard effective chemotherapy for patients with NF1 and PN, patients may be treated on this trial without having received prior medical therapy directed at their PN. Since AZD6244 hyd sulfate is not expected to cause substantial myelosuppression, there will be no limit to number of prior myelosuppressive regimen for PN or other tumor manifestations associated with NF1 such as optic glioma. Patients who have received previous investigational agents or biologic therapy, such as tipifarnib, pirfenidone, Peg-Intron, sorafenib, or other VEGFR inhibitors are eligible for enrollment. Growth factors that support platelet or white cell number or function must not have been administered within the past 7 days. Patients who received prior medical therapy for their PN must have recovered from the toxic effects of all prior therapy before entering this study. 5. Performance status: Patients 16 years of age must have a Karnofsky performance level of 70%, and children < 16 years old must have a Lansky performance of 70% (Appendix I). 6. Hematologic Function: Patients must have an absolute neutrophil count 1000/µl, hemoglobin 9g/dl, and platelet 100,000/µl.. 7. Hepatic Function: Patients must have bilirubin within 1.5 x the upper limit of normal for age, with the exception of Gilbert syndrome, and ALT within 1.5 x upper limit of normal. 8. Renal Function: Patients must have a creatinine clearance or radioisotope GFR 60ml/min/1.73 m 2 or a normal serum creatinine based on age described in the table below. Maximum Serum Age (years) Creatinine (mg/dl) <age <age > Informed Consent: Diagnostic or laboratory studies performed exclusively to determine eligibility for this trial must only be done after obtaining written informed consent from all patients or their legal guardians (if the patient is <18 years old). When appropriate, pediatric patients will be included in all discussions. This can be accomplished through one of the following mechanisms: a) the NCI, POB screening protocol, b) an IRB-approved institutional screening protocol or c) the study-specific protocol. 25

28 NCI protocol #; Version Date: Documentation of the informed consent for screening will be maintained in the patient s research chart. Studies or procedures that were performed for clinical indications (not exclusively to determine eligibility) may be used for baseline values even if the studies were done before informed consent was obtained. 10. Durable Power of Attorney (DPA): All patients >18 years of age will be offered the opportunity to assign DPA so that another person can make decisions about their medical care if they become incapacitated or cognitively impaired Exclusion Criteria 1. Pregnant or breast-feeding females are excluded due to potential risks of fetal and teratogenic adverse events of an investigational agent. Pregnancy tests must be obtained prior to enrollment on this study in girls, age 9 or older. Males or females of reproductive potential may not participate unless they have agreed to use an effective contraceptive method. Abstinence is an acceptable method of birth control. 2. Patients who anticipate the need for surgical intervention within the first three cycles (3 months), as surgical intervention during the period of DLT evaluation may affect analysis of adherence and/or make the subject inevaluable. 3. An investigational agent within the past 30 days. 4. Ongoing radiation therapy, chemotherapy, hormonal therapy directed at the tumor, immunotherapy, or biologic therapy. 5. Clinically significant uncontrolled unrelated systemic illness such as serious infections or significant cardiac, pulmonary, hepatic or other organ dysfunction. 6. Patients who in the opinion of the investigator may not be able to comply with the safety monitoring requirements of the study. 7. Inability to swallow capsules, since capsules cannot be crushed or broken. 8. Inability to undergo MRI and/or contraindication for MRI examinations following the MRI protocol (Appendix II). Prosthesis or orthopedic or dental braces that would interfere with volumetric analysis of target PN on MRI. 9. Prior treatment with AZD6244 hyd sulfate. 26

29 NCI protocol #; Version Date: Evidence of an optic glioma, malignant glioma, malignant peripheral nerve sheath tumor, or other cancer requiring treatment with chemotherapy or radiation therapy. 11. Presence of grade 1 cataract, as cataract was observed in preclinical studies with AZD6244 hyd sulfate. 12. Supplementation with vitamin E greater than 100% of the daily recommended dose. 2.2 Pre-Treatment Evaluation (see Appendix III) Pre-treatment blood test should be performed within 2 weeks and imaging studies with in 4 weeks prior to enrollment on the trial unless otherwise stated. The evaluation required to starting treatment is listed in table form in Appendix III. 1. History and physical examination: Complete history, including prior and concurrent therapy and all documented previous height and weight measurements and growth curve if available; physical examination including documentation of measurable disease, performance status (Appendix I), blood pressure, height, weight, body surface area (BSA), and signs and symptoms. a. Height: The patients should take off shoes and socks and heels should be placed against the wall with ankles together. Height should be measured in a standing position with a stadiometer. Two additional repeat measurements must be made with the patient stepping off the stadiometer in between each measurement. Height measurements should be taken at approximately the same time of day for each visit, when possible. The average of the 3 measurements must be plotted on a standardized growth chart. I n patients with known leg length discrepancy due to limb hypertrophy, height should be measured with the patient bearing weight on the limb without hypertrophy. b. BSA: BSA is calculated from the average of 3 repeated measurements of weight and height on the same day by the standard formula in use at the NCI: BSA= Weight (kg) *Height (cm) Hematology: Complete blood count with differential and platelets. 3. Chemistries: Electrolytes (including sodium, potassium, chloride, CO 2 ), calcium, phosphorus, magnesium, creatinine, BUN, glucose, ALT, bilirubin, urinalysis, total protein, albumin, and CPK. 27

30 NCI protocol #; Version Date: EKG/Echo: Electrocadiogram and echocardiogram to be performed within 2 weeks prior to enrollment on the trial. 5. Urine or serum pregnancy test for all females of childbearing potential (females greater than 9 years of age or those showing pubertal development). This test is to be performed within 72 hours prior to enrollment. 6. Radiographic evaluation (Appendix II): An MRI scan of the entire inoperable PN within 4 weeks of enrollment on study. In addition, if possible, an MRI scan of all known additional, measurable PN within 4 weeks of enrollment on study. The imaging protocol outlined in appendix II will be used each time MRI examinations are performed to assess the effects to AZD6244 hyd sulfate. 7. Ophthalmology evaluation: A complete ophthalmology evaluation will be performed pre treatment. Particular emphasis will be given to the evaluation of corneal opacification. 8. Pharmacokinetics: Plasma samples for the evaluation of AZD6244 hyd sulfate pharmacokinetics will be obtained prior to and after the first dose of AZD6244 hyd sulfate, and at steady state during the first treatment cycle only as outlined in Appendix IV. 9. Pharmacodynamics: a. Peripheral blood mononuclear cells (PBMCs) will be collected pre treatment and at steady state on treatment during cycle #1 to assess the effects of AZD6244 hyd sulfate on inhibition of downstream targets of MEK (ERK2 phosphorylation) using flow cytometry (Appendix V). 2.3 Patient Registration Patients must be registered by contacting Andy Gillespie, RN at the Pediatric Oncology Branch (POB) (Ms. Gillespie phone number: , gillesan@mail.nih.gov). When registering a patient, information about all entry criteria (e.g., laboratory results) must be available to allow for verification of eligibility. Dr. Brigitte Widemann (phone number: , bw42y@nih.gov), Dr. Leigh Marcus (phone number: , marculei@mail.nih.gov) must also be contacted to discuss the patient prior to entry on study. All patients will be registered with Harris (phone: , fax: ) by the POB research nurse, and an identification number will be assigned to each patient by the POB research nurse. The POB research nurse must be notified when a participating patient is removed from the protocol. 28

31 NCI protocol #; Version Date: Implementation of Study 3.1 Study Design Overall Trial Design This is a phase I trial of AZD6244 hyd sulfate administered orally BID (approximately every 12 hours) on a continuous dosing schedule for children with NF1 and inoperable PN. A cycle of therapy is considered to be 28 days with no rest periods in between cycles. This dose finding study is designed to determine the MTD and extended toxicity profile of AZD6244 hyd sulfate in pediatric patients with NF1 and inoperable PN. Toxicities observed for the first 3 cycles will be used to define the MTD given the likely long-term exposure to AZD6244 hyd sulfate in NF1 patients. Three patients will be entered at each dose level, and the MTD dose level will be expanded up to an additional six patients to a total of 9 patients if feasible. Once the Human Exposure Limits Committee (HELC) of the manufacturer approves inclusion of patients under 12 years of age, and the NCI IRB and sponsor approve an amendment to expand the enrollment to this age group, we plan to amend the protocol to include children 3-11 years old. The MTD dose level will be expanded, if feasible, to include 6 patients 12 years of age, and 6 patients > 12 years of age, if feasible, to more fully characterize the toxicities and pharmacokinetics of AZD6244 hyd sulfate at the MTD. The starting dose level will be 20 mg/m 2 /dose (approximately 50% of the adult recommended single agent phase II dose). This will be followed by up to three dose escalations, with the highest dose level exceeding the adult MTD by 1 dose level. Automated volumetric MRI analysis will be used to monitor PN growth rate, and progression will be defined as a 20% increase in PN volume. This trial will also evaluate the 1) plasma pharmacokinetics of AZD6244 hyd sulfate during cycle #1, 2) the pharmacodynamics of AZD6244 hyd sulfate by evaluating the effects of AZD6244 hyd sulfate on targets downstream of MEK in peripheral blood mononuclear cells (PBMCs), 3) and chronic daily dosing adherence of AZD6244 hyd sulfate in this patient population Criteria for Dose Escalation Cohorts of 3 to 6 patients will be treated with AZD6244 hyd sulfate at each dose level. When a minimum of three patients who are evaluable for toxicity have completed three cycles of therapy at a dose level without evidence of doselimiting toxicity (section 3.1.3), subsequent patients may be enrolled at the next higher dose level. If DLT is observed during cycles 1-3 in 1 patient from the initial cohort of 3 patients at a given dose level, an additional 3 patients will be entered at that dose level. If none of these additional patients experiences a DLT (1/6 with DLT), the dose will be escalated. If 1 of the additional patients experience a DLT ( 2/6 with DLT), the MTD has been exceeded, and the next lower dose level will be considered the MTD. 29

32 NCI protocol #; Version Date: If the MTD has been exceeded at the first dose level, then the subsequent cohort of patients will be treated at dose level -1, which will be a 30% dose reduction (see Section 3.2 and dosing nomogram Appendix VI) Definition of Dose Limiting Toxicity (DLT) Toxicity will be graded according to the NCI Common Terminology Criteria for Adverse Events v4.0 (CTCAE) ( Dose limiting Toxicity (DLT) will be defined as any grade 3 toxicity possibly, probably, or definitely related to AZD6244 hyd sulfate with the exception of the following: Any grade 3 transaminases that return to levels that meet initial eligibility criteria within 14 days of AZD6244 hyd sulfate interruption and that do not recur upon re-challenge of AZD6244 hyd sulfate. Any grade 3 electrolyte abnormality must be confirmed with a repeated measurement within hours. In addition, any grade 1 cataract will be considered dose limiting, any grade 2 urinary tract obstruction at least possibly related to AZD6244 hyd sulfate (and clearly not related to tumor), or any grade 2 AZD6244 hyd sulfate related toxicities of 7 days may be considered dose limiting if they are intolerable to the patient and cannot be controlled with standard supportive measures Criteria for Intra Patient Dose Escalation The rationale for intrapatient dose escalation is based on 2 primary assumptions: 1. NF1 and PN are progressive diseases which have significant impact on patients lives and lack alternative treatment options, 2. Starting at 50% of the adult MTD is less likely to provide benefit, while higher doses may be of some efficacy. Therefore patients who have completed three treatment cycles at the enrollment dose (during MTD assessment) may have the AZD6244 hyd sulfate dose escalated to the next highest dose level if: The patient does not have progressive disease as defined in section 5.2. The patient has not experienced AZD6244 hyd sulfate related toxicities > grade 1. Off study Criteria have not been met (Section 3.10). Patients starting treatment at dose level 1 may have up to two dose escalations (one every 3 cycles) as long as the criteria above have been met, and patients starting treatment at dose level 2 may escalate up to 1 dose level. Intrapatient dose escalation will not be performed if the patient is treated at the MTD. Intrapatient dose escalation may never exceed the adult MTD (dose level 3) Definition of Maximum Tolerated Dose (MTD) 30

33 NCI protocol #; Version Date: The MTD is defined as the dose level immediately below the dose at which 33% of patients in a cohort experience a DLT. In order to escalate a dose level, < 33% of patients in a cohort should have a DLT. At least 3 patients in a cohort must be evaluable for the definition of the MTD in order to escalate. If 1 out of 3 patients in a cohort experience a DLT, then up to 6 patients must be enrolled and evaluable for the definition of MTD prior to dose escalation. To determine extended tolerability, toxicities observed during the first 3 treatment cycles will be used to define the MTD. A patient will be considered evaluable for definition of the MTD if at least 85% of the prescribed dose has been administered to the patient during the first three treatment cycles based on adherence diary review (Appendix VIIb) and pill count of returned drug. If a discrepancy occurs, pill count will be used for adherence measurement. If a patient has less than 85% adherence and has not experienced DLT during cycles 1-3, the patient will be replaced in the cohort. In addition, any patient who receives one or more doses and experiences a DLT will be considered evaluable for definition of MTD. The cohort at the MTD should be expanded up to as many as 12 evaluable patients in order to gain experience with toxicities and pharmacokinetics of AZD6244 hyd sulfate over a broad range of patients. An attempt should be made to enroll at least 6 patients that are 12 years of age, and 6 patients that are years of age Criteria for Starting Subsequent Treatment Cycles Patients who complete a treatment cycle may receive another cycle at the same dose level if the patient has not experienced DLT, and has not experienced disease progression as defined in section Duration of Treatment In order to minimize potential risk due to long-term and cumulative toxicity, the total duration of treatment with AZD6244 hyd sulfate will be limited based on the following: 1) For patients who have documented disease progression within approximately 1.5 years prior to trial entry defined by 20% increase in size of PN by volumetric analysis (or 13% increase in the product of the longest 2 perpendicular diameters, or 6% increase in the longest diameter), there will be no limit to the duration of treatment as long as the patient meets the requirements for further treatment (section 3.1.6). 2) For patients with no previous documented history of disease progression within the 1.5 years prior to trial entry, the duration of the study will be limited to 2 years if no radiographic response (volume decreased by 20%) is observed. For patients who do show radiographic response (section 5.2), the treatment duration will not be limited unless the patient experiences subsequent disease progression or meets other off treatment criteria (section 3.9, 3.10). 31

34 NCI protocol #; Version Date: Drug Administration The AZD6244 hyd sulfate will be supplied in 10 mg and 25 mg capsules. AZD6244 hyd sulfate will be administered orally twice daily (approximately every 12 hours) continuously for 28 day cycles with no rest periods between cycles. Patients should be instructed to take the dose of AZD6244 hyd sulfate on an empty stomach (either 1 hour before or 2 hours after meals) with water only. Dosing will be performed based on body surface area (BSA), and doses will be rounded the nearest 10 mg using a dosing nomogram (See Appendix VI). AZD6244 hyd sulfate dosing will be capped at a BSA > 2 m 2. At follow up evaluations, AZD6244 hyd sulfate will be adjusted for changes in body surface area according to the dosing nomogram. The capsules cannot be crushed and have to be swallowed in whole. The starting dose will be 20 mg/m 2 /dose, which corresponds to approximately 50% of the adult recommended phase II dose. This will be followed by up to a maximum of 3 dose escalations, with the highest (dose level 4) exceeding the adult recommended phase II dose by 25%. Dose Escalation Schema Dose Level Dose (mg/m 2 /dose q12h) Equivalent fixed adult dose (mg) Percent change (%) ** * Based on BSA of 1.8 m 2, shaded area 50% of adult MTD ** Adult MTD Patients or their guardians will keep a diary (Appendix VIIb and c) to document the intake of each dose of AZD6244 hyd sulfate and potential side effects. The patient diary review and pill count are described in Section 3.7, item # Treatment Modifications If the patient experiences a dose limiting toxicity (as defined above), the protocol therapy will be withheld. If the toxicity resolves to meet study parameters or grade 1 within 21 days of drug discontinuation, the patient may resume treatment at a dose reduced by 30% or more. For example, if the patient is taking 60 mg po BID, then a 30% dose reduction should be made such that 60 mg x 0.30 = 18 mg, 60 mg -18 mg=42 mg, rounded down 32

35 NCI protocol #; Version Date: to 40 mg. The patient should resume dosing when able at 40 mg po BID. It is recommended to calculate the dose reduction this way rather than using the dosing nomogram due to overlap in dose levels at certain BSA. For example, if using the dosing nomogram, a patient with BSA 0.85m 2 and DLT at dose level 3 would be have been taking 30mg per dose. Without doing the 30% reduction calculation, the patient would be dose reduced to dose level 2, which corresponds to the same dose (BSA 0.85m 2 at dose level 2 is also 30mg per dose). Thus, calculating the dose reduction and not using the dosing nomogram will assure that patients who experience DLT will receive a meaningful dose reduction. Dose reduced for toxicity will not be re-escalated, even if there is minimal or no toxicity with the reduced dose. AZD6244 hyd sulfate doses held while the patient is recovering from toxicity should not be made up for and the cycle remains 28 days. If toxicity does not resolve to meet study parameters within 21 days of drug discontinuation, the patient must be removed from protocol therapy. If dose-limiting toxicity recurs in a patient who has resumed treatment at the reduced dose, the patient must be removed from protocol therapy. Patients removed from study therapy for toxicity will be followed until resolution of toxicity and off study criteria are met. 3.4 Pharmacokinetics Pharmacokinetic evaluation will be performed after the first dose of AZD6244 hyd sulfate, and at steady state in all consenting patients. On the first treatment cycle only, the second and third dose of AZD6244 hyd sulfate will not be administered during the pharmacokinetic sampling, which continues for 36 hours after the first dose. The fourth dose should be administered after 36-hour sample is drawn. A liquid chromatography/mass spectroscopy/mass spectroscopy (LC/MS/MS) will be used to analyze AZD6244 hyd sulfate. All assays used have a coefficient of variation (CV)% <15% across the analytical range. The current preclinical assay range is 0.01 to 5 g/ml for AZD6244 and to 0.5 g/ml for N-desmethyl AZD C-labelled AZD6244 and N-desmethyl AZD6244 have been synthesized and used in metabolism studies. The LC/MS/MS assay will be cross validated at the POB, and samples collected will be analyzed at the POB. For blood samples, 3 ml of whole blood will be collected in a heparinized tube (Becton Dickinson 6334 Green Top) according to the following schedule: 1) Relative to the first oral dose on day 1: before the first oral dose, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 10 to 12 h, 24 h, and 30 to 36 hours following the first oral dose. 2) In addition, one sample will be obtained on day (+/- 3 days) at the time of evaluation for cycle #2, prior to the first oral administration of the two daily doses of AZD6244 hyd sulfate. Along with the samples, a pharmacokinetic work sheet (Appendix IV) should be filled out indicating the time points of the samples. Details regarding sample collection, handling, and shipment instructions are in Appendix IV of the protocol. 33

36 NCI protocol #; Version Date: Correlative/Pharmacodynamic Studies Section 5.6 describes storage, tracking, and handling of research specimens. Peripheral Blood Mononuclear Cells: Peripheral blood mononuclear cells (PBMCs) will be collected pre treatment and at steady state on treatment to assess the effects of AZD6244 hyd sulfate on inhibition of downstream targets of MEK (ERK2 phosphorylation). ERK phosphorylation will be evaluated by flow cytometry analysis. Peripheral blood samples will be collected prior to treatment (pre-treatment sample), and once between day 27 and 28 (at the time of evaluation for cycle #2) (+/- 3 days) of treatment with AZD6244 hyd sulfate in cycle one. Details regarding sample collection, handling, and shipment instructions are in Appendix V. 3.6 Blood Volume for Research Studies Institutional guidelines for the maximum blood volume for research samples should be utilized. The table below details the blood volume obtained for pharmacokinetic and pharmacodynamic studies during the first course. Analysis #samples Sample volume (ml) Volume course #1 (ml) Pharmacokinetics PBMC Total volume 40 ml Given that only patients who can swallow capsules will be able to participate in this study, we do not anticipate that above studies will exceed the institutional limit of blood draw for research specimens. For small patients, the priority for research studies involving blood draws is outlined below: 1) Pharmacokinetics 2) PBMC studies 3.7 On Study Evaluations (Appendix III) 1. History and Physical Examination: History and physical examination with vital signs to be performed every other week for cycles 1-3, prior to each cycle for cycles 4-11, and then prior to every other treatment cycle (pre cycle 13, 15, 17, 19, 21, etc) in subsequent cycles. Height, weight, and BSA (see section 34

37 NCI protocol #; Version Date: ) should be performed prior to cycle 4, 6, 11 and then subsequently after every 6 treatment cycles (cycles 17, 23, 29 etc). 2. Performance status: Performance status (Appendix I) will be recorded at the time of disease evaluations with MRI, and must also be recorded if there is a change noted from baseline. 3. Hematology: Complete blood count, differential, and platelet count every other week for cycles 1-3, prior to each cycle for cycles 4-11, and then prior to every other treatment cycle (pre cycle 13, 15, 17, 19, 21, etc) in subsequent cycles. 4. Chemistries: Electrolytes (including sodium, potassium, chloride, CO 2 ), calcium, phosphorus, magnesium, creatinine, BUN, glucose, ALT, bilirubin, total protein and albumin, and CPK to be performed every other week for cycles 1-3, prior to each cycle for cycles 4-11, and then prior to every other treatment cycle (pre cycle 13, 15, 17, 19, 21, etc) in subsequent cycles. Urinalysis will be performed at the time of disease evaluations with MRI. 5. Urine or serum pregnancy test to female subjects 9 years and older prior to disease evaluations with MRI. 6. Ophthalmology evaluation for corneal opacification: Pre cycle 4, and subsequently at the time of disease evaluations with MRI. 7. EKG/ECHO: Prior to cycle 6, 11 and then after every 6 cycles (pre 17, 23, 29, etc). 8. Pharmacokinetics: As detailed in Appendix IV. 9. Pharmacodynamics: PBMCs to be performed pretreatment and at steady state in cycle 1 as detailed in Appendix IV. 10. Radiographic evaluation: Evaluate the index lesions by 3-D MRI prior to cycles 6, 11, and subsequently after every 6 treatment cycles (pre cycle 17, 23, 29 etc) as long as the patient remains on study (Appendix II). If patient does show a responsive disease (RD) according to section 5.2, response will be confirmed after 1-3 treatment cycles. 11. Adherence interviews will be conducted by a psychology staff member with each parent and patient (together) during the pre-study visit to identify potential barriers to adherence. Parents and patients (ages 8 and up) will be given an Adherence Questionnaire containing a list of barriers (see Appendix VIIa) and asked to identify the ones that have interfered with adherence to other medications in the past. Any barriers identified will be discussed to help 35

38 NCI protocol #; Version Date: the family identify and implement strategies for preventing problems with adherence. 12. Patient diary to monitor concurrent side effects and adherence should be reviewed prior to each treatment cycle for cycles 2 9, and subsequently prior to every other treatment cycle (pre cycle 11, 13, 15, 17, etc) (Appendix VIIb and c). Pill Counts: The patient s research nurse will conduct pill counts with each scheduled visit (prior to cycle 2, 4, 8, 12, and subsequently every 6 cycles), and subsequently at the time of disease evaluations with MRI. 13. The Responsibility for Medication Questionnaire (RMQ) (Appendix VIId) is a 9-item measure that assesses parents and patients perceptions of who holds responsibility for various medication-related tasks. If adherence is calculated as less than 90% according to pill count or the adherence diaries this measure will be administered to patients (ages 8 and older only) and parents. In addition, the parent and patient will meet with a psychology staff member to review the results of adherence and discuss any discrepancies between parent and child perceptions of responsibility. Barriers to adherence will be discussed to help the family identify strategies for overcoming these obstacles. For patients who undergo an intra-patient dose escalation, all evaluations listed above will be performed as if the patient were starting his/her first cycle and so forth as described in this section, with the exception of PK and PD. 3.8 Concurrent Therapies 1. Other cancer chemotherapy, radiation, immunotherapy, biologic therapy, hematopoetic growth factors, or investigational agents cannot be administered to patient while receiving AZD6244 hyd sulfate. 2. Doses of vitamin E greater than the 100% daily recommended doses are contraindicated. 3. The use of corticosteroids for control of symptoms related to the underlying NF1 or for other reasons will be allowed. 4. Throughout the study, patients should be instructed to avoid changes to, or the addition of concomitant medications, in particular any that may affect the metabolism of AZD6244, unless considered clinically indicated. 3.9 Criteria for Removal from Treatment 1. A patient may be removed from the protocol for the following non-medical or administrative reasons: a. Patient refusal of further treatments (reasons must be noted on the patient s CRF). 36

39 NCI protocol #; Version Date: b. It is deemed in the best interest of the patient. In this instance the PI should be notified and the reasons of withdrawal should be noted in the CRF. c. Serious protocol violation as determined by the PI. 2. Any patient who develops dose-limiting toxicity, which does not resolve to meet study parameters within 21 days of drug discontinuation or within the time frame defined in the DLT definition, whichever comes first. 3. If dose-limiting toxicity recurs in a patient who has resumed treatment at the reduced dose. 4. Any patient with clinical or radiographic evidence of progressive disease on treatment, as documented by a 20% increase in tumor volume on 3-D MRI compared to baseline, following any treatment cycle will be removed from the study. 5. A patient who develops a concurrent serious medical condition that might preclude or contraindicate the further administration of AZD6244 hyd sulfate will be removed from the study. A patient who becomes pregnant will be immediately taken off therapy. 6. A patient who will undergo complete surgical resection of their PN (thus rendering them with no evidence of disease). 7. Repeated eligibility laboratory studies (CBC with differential, bilirubin, ALT (SGPT) and serum creatinine) are outside the parameters required for eligibility prior to the start of AZD6244 hyd sulfate (See Section 2.1). Patients who are off protocol therapy are to be followed until they meet the criteria for Off Study (see below). Follow-up data will be required unless consent is withdrawn. Toxicities must be followed until stabilization or resolution prior to a subject being taken off study. Patients with no previous documented history of disease progression with in the 1.5 years prior to trial entry with no radiographic response on drug will be removed off protocol after 2 years of study entry (section 3.1.7) Off Study Criteria 1. Thirty days after the last dose of investigational agent. Patients taken off treatment for toxicity will be followed until resolution or stabilization of toxicity, or until 30 days after the last dose of AZD6244, whichever is later. 2. Death 3. Lost to follow-up 4. Withdrawal of consent for any further data submission 5. Entry onto another therapeutic study End Of Treatment Evaluation (Appendix III) The following tests and procedures should be performed, if possible, at the time a patient comes off treatment regardless of the reason for coming off treatment, unless the test or procedure has been performed in the past 2 weeks or within a time period described below. 37

40 NCI protocol #; Version Date: History and physical examination, vital sign including blood pressure, and performance status. 2. Laboratory: Complete blood count, differential, and platelet count, electrolytes (including sodium, potassium, chloride, CO2), calcium, phosphorus, magnesium, creatinine, BUN, glucose, ALT, bilirubin, urinalysis, total protein, and albumin. 3. MRI of the PN and any other progressing lesions using the MRI protocol identified in Appendix II within the past 4 weeks. 4.0 Supportive Care Appropriate antibiotics, blood product support, anti-emetics, and general supportive care will be used as indicated. An algorithm for interventions for the management of dermatological adverse events has been adapted from clinical experience of management of similar dermatological effects of the EGFR tyrosine kinase inhibitor agents[46, 47] (see Appendix IX). Major surgery unrelated to PN will require holding drug 1 week prior to surgery and until wound has healed completely. Diarrhea will be managed with the following regimen: loperamide (4 mg PO) at onset of symptoms, followed by 2 mg loperamide every 2 hours while awake (or 4 mg PO every 4 hours while sleeping) up to a maximum of 16 mg loperamide per day. Additional agents may be used concurrently if loperamide is not adequate to control diarrhea as a single agent. 5.0 Data Collection And Evaluation 5.1 Data Collection The POB, NCI will coordinate the clinical trial and data collection, and supply the data to the trial sponsor CTEP via the CDUS online system. Data will be entered into the electronic NCI, Center for Cancer Research (CCR) C3D database. Documentation and date of IRB approval must be provided to the POB, NCI prior to initial patient entry from each institution. The completed eligibility checklist prepared by central registration must be faxed to Ms. Andy Gillespie ( ) prior to patient entry onto the trial Case Report Forms A study number, not names or initials will identify all case report forms and patient diaries. This will be done to protect patient confidentiality. Unless otherwise stated, all forms should be submitted to: 38

41 NCI protocol #; Version Date: Pediatric Oncology Branch, NCI c/o Andy Gillespie, RN Fax: Phone: Ms. Gillespie Center Drive, Building 10, CRC Room Bethesda, MD Case Report Forms: Case report forms will be completed within 2 weeks of each required evaluation. Data will be entered into the NCI, CCR C3D database. Patient Diaries for side effects and adherence (Appendix VIII): Patients or their guardians must keep a diary to document the intake of each dose of AZD6244 hyd sulfate and potential side effects. These diaries will be forwarded to Ms. Andy Gillespie (fax: ) after each treatment cycle. A pill count will be performed with each restaging. Pharmacokinetic and pharmacodynamic studies: Blood samples and corresponding worksheets (Appendix IV, and V) will be sent to the NCI POB as described in the Appendices. Adherence Assessment: Pill counts at restaging, adherence diaries & questionnaire forms (Appendix VIIa-d) should be photocopied and sent to Ms. Andy Gillespie (fax: ). Protocol Violations: Protocol deviations and violations should be directly reported to Dr. Brigitte Widemann (phone: (301) , fax (301) , widemannb@mail.nih.gov) and to the NCI IRB. 5.2 Response Criteria Response evaluation is performed according to Appendix 2. Response evaluation will be based on the selected three most clinically relevant PN which will be followed by 3-D MRI analysis. Response is assessed at the time that follow-up 3D-MRI scans which are performed after cycles 6, 11, and then after every 6 treatment cycles. For the purpose of determining the level of response, measurements from the follow-up scans are compared to the tumor size in the pretreatment MRI scan using 3D data analysis. Responsive Disease (RD): A 20% reduction in the sum of the volume of all index lesions for 4 weeks. Stable Disease (SD): A <20% increase and <20% reduction in the sum of the volume of all index lesions for 4 weeks. Progressive Disease (PD): A 20% increase in the volume of at least one of the index PN compared to the pretreatment volume measured prior to the start of the current treatment phase. The appearance of new discrete subcutaneous neurofibromas does not qualify for disease progression. Worsening of existing symptoms or the appearance of new symptoms that persist for more than 7 days 39

42 NCI protocol #; Version Date: and that are felt to be definitely related to the PN should be evaluated by repeating the MRI. Patients should not be classified as having progressive disease solely on the basis of new or increased symptoms without discussing the case with the protocol PI. 5.3 Toxicity Criteria CTCAE term (AE description) and grade: The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site Statistical section Subject Accrual Subjects of both genders, from all racial and ethnic groups are eligible for this trial if they meet the criteria. To date, there is no information that suggests differences in absorption, metabolism, or disposition or disease response among racial or ethnic groups or between the genders, indicating that results of the trial will be applicable to all groups. Efforts will be made to extend the accrual to a representative population, but in a phase I trial which will accrue <30 patients, a balance must be struck between patient safety considerations and limitations on the number of individuals exposed to potentially toxic or ineffective treatments on the one hand and the need to explore gender, racial, and ethnic aspects of clinical research on the other. If differences in outcome that correlate to gender, racial, or ethnic identity are noted, accrual may be expanded or additional studies may be performed to investigate those differences more fully Statistics and feasibility This trial uses a conventional dose-escalation design to establish the MTD of AZD6244 hyd sulfate based on the severity of toxicity. The starting dose level of 20 mg/m 2 /dose was determined based toxicities observed in the adult phase II trials. This will be followed by up to three dose level escalations with the highest dose exceeding the adult MTD. The total number of dose levels will likely be four at a maximum, with the possibility of requiring one dose de-escalation. Cohorts of 3 to 6 patients will be treated on each dose level. The MTD is defined as the dose level immediately below the level at which 33% of patients in a cohort experience a DLT. The MTD will be defined based on toxicities observed during the first three treatment cycles in order to capture toxicities, which occur after more prolonged administration. A patient will be considered evaluable for definition of the MTD if at least 85% of the prescribed dose has been administered to the patient during the first three treatment cycles based on adherence diary review and 40

43 NCI protocol #; Version Date: pill count of returned drug. In addition, any patient who receives one or more doses and experiences a DLT will be considered evaluable for definition of the MTD. Patient accrual will also be expanded at the MTD to, if feasible, a total of 12 patients in order to attempt to include 6 patients 12 years of age, and 6 patients 13 years of age to more fully characterize the toxicities and pharmacokinetics of AZD6244 hyd sulfate at the MTD. If 3 patients are enrolled at each of three dose levels with 12 patients at dose level 4 (or MTD), the minimum number of patients to be accrued is 21. The maximum number of patients required to complete this trial if 6 patients are accrued at each of three dose levels with 12 at dose level 4 (or MTD) will therefore be 30. If enrollment is approximately 2 patients per month, the primary trial objectives will likely be determined within approximately 15 months. The primary objective of this study is to determine the tolerability, toxicity and MTD of AZD6244 hyd sulfate in pediatric patients with NF1 and inoperable PN. Pharmacokinetic and pharmacodynamic endpoints will be studied. As a phase 1 protocol, this study is largely exploratory, precluding formal statistical comparisons of treatment groups. When appropriate, summary statistics will be generated. Toxicity and tolerability will be measured using CTCAE-4.0 and summarized by dose level. Prolonged grade 2, 3 and 4 toxicities attributable to AZD6244 hyd sulfate will be presented as the frequency and percentage of patients for each dose level. Pharmacokinetic analysis will be conducted using non-compartmental methods and estimated pharmacokinetic parameters including AUC, clearance, half-life and volume of distribution presented for each dose level using summary statistics (mean, standard deviation, median and range). Accumulation of AZD6244 hyd sulfate will be evaluated by defining the ratio of the AUC 0-, which equals the AUC 0-12h at steady state, to the AUC 0-12h after the first dose. For pharmacodynamic endpoints, descriptive statistics for each dose level (mean, standard deviation, median and range) will be calculated for each surrogate endpoint measured. To assess the effects of inhibition of downstream targets for ERK2 phosphorylation will be evaluated in peripheral blood mononuclear cells before and at steady state of AZD6244 hyd sulfate administration. As the values for the correlative markers are all quantitatively measured, the average values obtained prior to treatment and at steady state will be compared using a Wilcoxon signed rank test by dose level. Correlations will be made to toxicity and response if feasible. With information obtained from patient diaries, adherence will be calculated as follows: {(scheduled doses missed doses) / scheduled doses} X 100. For pill counts, the number of pills returned to clinic will be subtracted from the number of pills dispensed at the previous visit. That number will be divided by the number of pills the patient should have taken according to their prescribed dosing schedule, and then multiplied by 100 to obtain the adherence percentage. The toxicities in pediatric patients on chronic dosing of AZD6244 hyd sulfate will be collected on all subjects during and after treatment and will be reported descriptively. 5.5 Data and Center Audits Missing or spurious information and protocol deviations will be communicated in a report. Protocol deviations, which may result in compromise to safely administer study 41

44 NCI protocol #; Version Date: drug, or to determine study endpoints will be included in the annual protocol review for NCI IRB. Volumetric MRI analysis used to determine disease progression will be performed centrally at the NCI, POB. Plasma pharmacokinetics and pharmacodynamics will also be evaluated centrally at the NCI, POB Data And Safety Monitoring Plan The PI will monitor the clinical outcome in each patient treated on the trial, and the overall safety and efficacy of the drug at each dose level. The NCI PI, protocol lead associate investigator, responsible research nurse, and data manager will meet weekly to review enrollment and observed toxicities. 5.6 Storage, Tracking and Handling of Research Specimens This study is a trial within the Pediatric Oncology Branch, NCI. Sample labeling, collection and initial processing will be conducted as outlined in the study Section 3.4, and 3.5. Storage of samples in designated monitored freezers (-70 C or -20 C or 4 C as indicated in the protocol) will occur in the Pharmacology & Experimental Therapeutics Section (PETS) Laboratory, POB, NCI on 1 West. Samples will be tracked using LabMatrix. Pharmacokinetic and PBMC ERK phosphorylation will be analyzed at the NCI. Samples will be identified using unique patient number (study number) and shipped as biological samples. Samples will be tracked and considered the responsibility of Brigitte Widemann, MD. Once analyzed for studies outlined in this protocol any remaining samples will be returned to the POB, NCI. The study will remain open and status reported to the NCI IRB until all samples have been analyzed, reported or destroyed. Unintentional loss or destruction of any samples will be reported to the NCI IRB as part of annual continuing reviews. Any use of samples not outlined in Section 3.4 or 3.5 will require prospective NCI IRB review and approval. 6.0 Human subjects protections 6.1 Rationale for subject selection Neurofibromatosis type 1 is a genetic disorder with a worldwide incidence of 1:3500 individuals. No groups, in regards to gender, and racial and ethnic groups, are being excluded from participation in the trial. Females who are pregnant or breastfeeding will not be eligible for the trial due to risks of fetal and teratogenic adverse events as seen animal studies. This trial is designed to define the qualitative toxicities and maximum tolerated dose of AZD6244 hyd sulfate in children with NF1 and PN and, therefore, children will be entered onto this research trial. 6.2 Participation of children This trial is designed to define the qualitative toxicities and maximum tolerated dose of AZD6244 hyd sulfate in children and adolescents with PN and, therefore, children will be 42

45 NCI protocol #; Version Date: entered onto this research trial. Pediatric oncologists who have extensive experience in performing investigational drug trials in children will conduct the trial. Patients enrolled at the POB, NCI will be cared for in the POB outpatient pediatric oncology clinic or outpatient setting and if patients require hospital admission, they will be cared for on the pediatric unit of the CRC by the POB staff. 6.3 Risks and benefits Patients under the age of 18 years will be entered on this trial. The primary risk to patients participating in this research study is from toxicity of AZD6244 hyd sulfate, an investigational agent. The protocol provides for detailed and careful monitoring of all patients to assess for toxicity and the dose escalation scheme is very conservative. Toxicity data from the current dose level will be collected and reviewed to ensure that there were no severe (dose-limiting) toxicities prior to escalating to the next higher dose level. A total of 3 cycles will be followed to determine dose-limiting toxicity due to the increase of possibility of chronic and/or cumulative toxicity such as rash. Also, in order to minimize potential risk due to long-term and cumulative toxicity, treatment will be limited according to Section Although the primary objective of this phase I trial is to define the qualitative toxicities and maximum tolerated dose of the drug, all patients entered on the trial will have NF1 and PN, which is a disease that this agent is hypothesized to benefit. AZD6244 hyd sulfate offers a potential for direct benefit although the likelihood of this may be small. The potential benefits from this therapy are disease stabilization or shrinkage and a reduction in symptoms caused by the tumor. Furthermore, by just entering the study, patients will be followed very carefully, in an organized coherent manner, which may also benefit their overall healthcare. 6.4 Consent and assent process and documentation The investigational nature and research objectives of this trial, the procedures and treatments involved and their attendant risks and discomforts and benefits, and potential alternative therapies will be carefully explained to the patient or the patient s parents or guardian if he/she is a child, and a signed informed consent document will be obtained prior to entry onto the study. The investigators are requesting a waiver from the IRB to allow only one parent to sign the informed consent to enter a child on the protocol. Because many patients must travel to the NIH from long distances at substantial expense, requiring both parents to be present for the consent process could be a financial hardship for many families. The PI or an associate investigator of the trial will obtain consent. Where deemed appropriate by the clinician and the child s parents or guardian, the child will also be included in all discussions about the trial and verbal assent will be obtained. The parent or guardian will sign the designated line on the informed consent attesting to the fact that the child has given assent. 7.0 Data Reporting All data collection and reporting will be coordinated through: Andy Gillespie, RN Pediatric Oncology Branch, National Cancer Institute 43

46 NCI protocol #; Version Date: Building 10-CRC, Rm MSC Center Drive Bethesda, MD Phone (301) ; FAX (301) PATIENT REGISTRATION See Section CASE REPORT FORMS The POB will provide case report forms for recording relevant clinical data for each patient enrolled on the trial (5.0). Patient diaries (Appendix VII) should be provided to all patients and their families and submitted after completion of each required evaluation. Data will be entered into the NCI, CCR C3D database by the POB. 7.3 SAFETY REPORTING ADVERSE EVENTS Adverse events are any unfavorable and unintended sign (including an abnormal laboratory finding) symptom or disease temporally associated with the use of a medical treatment or procedure regardless of whether it is considered related to the medical treatment or procedure (attribution of unrelated, unlikely, possible, probable or definite). All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days following the last dose of study drug. AEs that are considered treatment related, expected, continuing, but not resolvable by 30 days after treatment completion (e.g., alopecia) will not be followed after the 30-day period. An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following: Results in discontinuation from the study Is associated with clinical signs or symptoms Requires treatment or any other therapeutic intervention Is associated with death or another serious adverse event, including hospitalization. Is judged by the Investigator to be of significant clinical impact If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient s outcome. Life-threatening adverse events are any adverse event that places the patient or subject, in view of the investigator at an immediate risk of death from the reaction as it occurred, i.e., it does not include a reaction that had it occurred in a more severe form, might have caused death. 44

47 NCI protocol #; Version Date: Serious adverse events are any adverse event occurring at any dose that result in any of the following outcomes: Death, a life-threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant disability/incapacity, a congenital anomaly/birth defect, and other medically significant events. Suspected adverse reaction: Suspected adverse reaction means any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, reasonable possibility means there is evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug. Unexpected adverse events are any adverse events, which are not listed in the Investigator Brochure and informed consent for this trial. Known toxicities observed to date are listed in Section "Unexpected, also refers to adverse events or suspected adverse reactions that are mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned as occurring with the particular drug under investigation. Disability is a substantial disruption of a person s ability to conduct normal life functions. Protocol Deviation (NIH Definition): A protocol deviation is any change, divergence, or departure from the study design or procedures of a research protocol that is under the investigator s control and that has not been approved by the IRB. Protocol Violation (NIH Definition): Any change, divergence, or departure from the study procedures in an IRB-approved research protocol that has a major impact on the subject s rights, safety, or well-being and/or the completeness, accuracy or reliability of the study data. Unanticipated Problem: Any incident, experience, or outcome that: Is unexpected in terms of nature, severity, or frequency in relation to (a) the research risks that are described in the IRB-approved research protocol and informed consent document; Investigator s Brochure or other study documents, and (b) the characteristics of the subject population being studied; AND Is related or possibly related to participation in the research; AND Places subjects or others at a greater risk of harm (including physical, psychological, economic, or social harm) than was previously known or recognized. All observed or volunteered adverse events, regardless of causal relationship to study drug, will be recorded on the Adverse Event page(s) of the case report form. Events involving adverse drug reactions, illnesses with onset during the study, or exacerbation of pre-existing illnesses should be recorded. Objective test findings (e.g., electrocardiogram changes, abnormal laboratory test results) that result in a change in study drug dosage 45

48 NCI protocol #; Version Date: should also be recorded ADVERSE EVENT REPORTING REQUIREMENTS All observed or volunteered adverse events, regardless of treatment group or suspected causal relationship to study drug, will be recorded on the Adverse Event page(s) of the case report form. Adverse events will be identified and graded using the NCI Common Toxicity Criteria version 4 (CTCAE-4). Next it will be determined if the adverse event is related to the medical treatment (attribution). If so, it will be determined whether the adverse event is expected or unexpected. Using the guidelines outlined in this section, adverse events will then be reported to the NCI using a routine report (CDUS), and if required, the Adverse Event Expedited Reporting System (AdEERS), an electronic system for expedited submission of adverse event reports. The adverse event page will contain information if the reported event was expected or unexpected, and if the reported toxicity is included in the informed consent. A justification will be provided on the adverse event page if the observed toxicity in not included in the informed consent. For all adverse events, the investigator must pursue and obtain information adequate both to determine the outcome of the adverse event and to assess whether it meets the criteria for classification as a serious adverse event requiring immediate notification to the sponsor. Follow- up of the adverse event, even after the date of therapy discontinuation, is required if the adverse event or its sequelae persist. Followup is required until the event or its sequelae resolve or stabilize at a level acceptable to the investigator and sponsor. Reporting requirements may include the following considerations: 1) whether the patient has received an investigational or commercial agent; 2) the characteristics of the adverse event including the grade (severity), the relationship to the study therapy (attribution), and the prior experience (expectedness) of the adverse event; and 3) whether or not hospitalization or prolongation of hospitalization was associated with the event. An investigational agent is a protocol drug administered under an Investigational New Drug Application. In some instances, the investigational agent may be available commercially, but is actually being tested for indications not included in the approved package label. Commercial agents are those agents not provided under an IND but obtained instead from a commercial source. The NCI, rather than a commercial distributor, may on some occasions distribute commercial agents for a trial. Additionally, certain adverse events must be reported in an expedited manner to allow for optimal monitoring of patient safety and care. The following sections provide information about expedited reporting. STEPS TO DETERMINE IF AN ADVERSE EVENT IS TO BE REPORTED IN AN EXPEDITED MANNER 46

49 NCI protocol #; Version Date: Step 1: Identify the type of adverse event using the NCI Common Terminology Criteria for Adverse Events (CTCAE). The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site Step 2: Grade the adverse event using the NCI CTCAE. Step 3: Determine whether the adverse event is related to the protocol therapy Attribution categories are as follows: Unrelated, Unlikely, Possible, Probable, and Definite. Note: This includes all events that occur within 30 days of the last dose of protocol treatment. Any event that occurs more than 30 days after the last dose of treatment and is attributed (possibly, probably, or definitely) to the agent(s) must also be reported according to the instructions above. Step 4: Determine the prior experience of the adverse event. Expected events are those that have been previously identified as resulting from administration of the agent. An adverse event is considered unexpected, for expedited reporting purposes only, when either the type of event or the severity of the event is not listed in the current Agent Specific Adverse Event List (ASAEL), as listed in the drug information section of the protocol. Step 5: Review Table A in this section to determine if: There are any protocol-specific requirements for expedited reporting of specific adverse events that require special monitoring; and/or There are any protocol-specific exceptions to the reporting requirements. Table A. AdEERS Reporting Requirements for Adverse Events That Occur Within 30 Days 1 of the Last Dose of the Investigational Agent on Phase 1 Trials Unrelated Unlikely Possible Probable Definite 1 Phase 1 Trials Grade 1 Grade 2 Grade 2 Grade 3 Grade 3 Unexpected and Expected Not Required Not Required Unexpected Not Required 10 Calendar Days Expected Not Required Not Required Unexpected with Hospitalization 10 Calendar Days 24-Hour; 5 Calendar Days without Hospitalization Not Required 24-Hour; 5 Calendar Days with Hospitalization 10 Calendar Days 10 Calendar Days Expected without Hospitalization Not Required Not Required Grades 4 & 5 2 Unexpected and Expected 24-Hour; 5 Calendar Days 24-Hour; 5 Calendar Days Adverse events with attribution of possible, probable, or definite that occur greater than 30 days after 47

50 NCI protocol #; Version Date: the last dose of treatment with an agent under a CTEP IND require reporting as follows: AdEERS 24-hour notification followed by complete report within 5 calendar days for: Grade 3 unexpected events with hospitalization or prolongation of hospitalization Grade 4 unexpected events Grade 5 expected events and unexpected events Although an AdEERS 24-hour notification is not required for death clearly related to progressive disease, a full report is required as outlined in the table. December 15, 2004 Note: All deaths on study require both routine and expedited reporting regardless of causality. Attribution to treatment or other cause must be provided. Expedited AE reporting timelines defined: 24 hours; 5 calendar days The investigator must initially report the AE via AdEERS within 24 hours of learning of the event followed by a complete AdEERS report within 5 calendar days of the initial 24-hour report. 10 calendar days A complete AdEERS report on the AE must be submitted within 10 calendar days of the investigator learning of the event. Any medical event equivalent to CTCAE grade 3, 4, or 5 that precipitates hospitalization (or prolongation of existing hospitalization) must be reported regardless of attribution and designation as expected or unexpected with the exception of any events identified as protocol-specific expedited adverse event reporting exclusions. Any event that results in persistent or significant disabilities/incapacities, congenital anomalies, or birth defects must be reported via AdEERS if the event occurs following treatment with an agent under a CTEP IND. Use the NCI protocol number and the protocol-specific patient ID assigned during trial registration on all reports. Hospitalization for management of NF1 related complications unrelated to study drug will not require expedited reporting. WHEN TO REPORT AN EVENT IN AN EXPEDITED MANNER Some adverse events require notification within 24 hours (refer to Tables A/A1 and B) to NCI via the web at: (telephone the Investigational Drug Branch (IDB) at: within 24 hours of becoming aware of the event if the AdEERS 24-Hour Notification web-based application is unavailable) and by telephone call to the Study Chair. When the adverse event requires expedited reporting, submit the report within 5 calendar days of learning of the event. EXPEDITED REPORTING METHODS AdEERS Reporting: To report adverse events in an expedited fashion use the NCI s 48

51 NCI protocol #; Version Date: Adverse Event Expedited Reporting System (AdEERS) that can be found at An AdEERS report must be submitted by: o Electronically submit the report via the AdEERS Web-based application located at or DEFINITION OF ONSET AND RESOLUTION OF ADVERSE EVENTS o If an adverse event occurs more than once in a course (cycle) of therapy only the most severe grade of the event should be reported. O If an adverse event progresses through several grades during one course of therapy, only the most severe grade should be reported. O The duration of the AE is defined as the duration of the highest (most severe) grade of the Adverse Effects. O The resolution date of the AE is defined as the date at which the AE returns to eligibility criteria level or Grade 1 level; whichever level is greater (note that the resolution date may therefore be different from the date at which the grade of the AE decreased from its highest grade). If the AE does not return to baseline the resolution date should be recorded as ongoing. An adverse event that persists from one course to another should only be reported once unless the grade becomes more severe in a subsequent course. An adverse event which resolves and then recurs during a different course, must be reported each course it recurs NCI-IRB Reporting NCI-IRB Expedited Reporting of Adverse Events, Unanticipated Problems, and Deaths The Protocol PI will report to the NCI-IRB: All unexpected serious adverse events that are possibly, probably, or definitely related to the research All deaths, except deaths due to progressive disease All Protocol Violations or Deviations All Unanticipated Problems Reports must be received by the NCI-IRB within 7 working days of PI awareness via iris NCI-IRB Requirements for PI Reporting of Adverse Events at Continuing Review The protocol PI will report to the NCI-IRB: 49

52 NCI protocol #; Version Date: All Grade 2 unexpected events that are possibly, probably, or definitely related to the research; All Grade 3 and 4 events that are possibly, probably, or definitely related to the research; All Grade 5 events regardless of attribution; All Serious Events regardless of attribution. NOTE: Grade 1 events are not required to be reported NCI-IRB Reporting of IND Safety Reports Only IND Safety Reports that require a sponsor recommended change to the protocol or the consent form or in the opinion of the PI increases risks to study participants will need to be reported to the NCI IRB. 8.0 PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION 8.1 AZD6244 hydrogen sulfate (NSC ) Chemical name (IUPAC): 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3Hbenzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide Other Names: Selumetinib; ARRY ; AR ; AR Chemical Structure: Classification: Mitogen-activated protein kinase kinase (MEK) inhibitor CAS Registry Number: Molecular Formula: C 17 H 15 BrClFN 4 O 3 M.W.: Solubility: Solubility in Captisol approximately 1 mg/ml at 25 C. Very low aqueous solubility (3.4 μg/ml at ph 7.4). 50

53 NCI protocol #; Version Date: Mode of Action: The RAS/RAF/MEK/ERK pathway is an important mediator of many cellular processes including proliferation, survival, differentiation, apoptosis, motility, and metabolism. This pathway is often aberrantly activated in human tumors due to the overexpression of activated K-RAS, mutant b-raf, or other growth factor receptors. AZD6244 hyd sulfate is a selective mitogen-activated protein kinase kinase (MEK) inhibitor. By inhibiting MEK, AZD6244 hyd sulfate inhibits ERK phosphorylation. Thus, AZD6244 hyd sulfate may inhibit oncogenic growth signaling in tumor cells by targeting the RAS/RAF/MEK/ERK pathway. How Supplied: AZD6244 hydrogen sulfate is supplied as 10mg, and 25 mg, size 4, plain, white, hydroxypropylmethylcellulose (HPMC) capsule in white high density polyethylene (HDPE) containers with foil-lined, induction-sealed, child-resistant closures. Each bottle contains 60 capsules. Each capsule contains a dispersion of AZD6244 hydrogen sulfate in d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS; a water soluble form of vitamin E). Storage: Store the AZD6244 hydrogen sulfate capsules at room temperature (20 C-25 C). Brief excursions are permitted between 15 C and 30 C. Stability: Stability studies are ongoing. Route of Administration: Oral. Take AZD6244 hyd sulfate on an empty stomach (either 1 hour before or 2 hours after meals). AZD6244 hyd sulfate capsules should be taken with water only. Potential Drug Interactions: High vitamin E doses may potentiate warfarin s anticoagulant activity. Monitor PT/INR more frequently in patients receiving both warfarin and AZD6244 hydrogen sulfate capsules. Avoid concomitant intake of Vitamin E in excess of 100% of the recommended daily dose. Agent Ordering and Agent Accountability: NCI supplied agents may be requested by the Principal Investigator (or their authorized designee) at each participating institution. Pharmaceutical Management Branch (PMB) policy requires that agent be shipped directly to the institution where the patient is to be treated. PMB does not permit the transfer of agents between institutions (unless prior approval from PMB is obtained). The CTEP assigned protocol number must be used for ordering all CTEP supplied investigational agents. The responsible investigator at each participating institution must be registered with CTEP, DCTD through an annual submission of FDA form 1572 (Statement of Investigator), Curriculum Vitae, Supplemental Investigator Data Form (IDF), and Financial Disclosure Form (FDF). If there are several participating investigators at one institution, CTEP supplied investigational agents for the study should be ordered under the name of one lead investigator at that institution. Agent may be requested by completing a Clinical Drug Request (NIH-986) and faxing it 51

54 NCI protocol #; Version Date: to the Pharmaceutical Management Branch at (301) For questions about drug orders, transfers, returns, or accountability call (301) Monday through Friday between 8:30 am and 4:30 pm (ET) or anytime. Agent Inventory Records - The investigator, or a responsible party designated by the investigator, must maintain a careful record of the inventory and disposition of all agents received from DCTD using the NCI Drug Accountability Record Form (DARF). (See the NCI Investigator s Handbook for Procedures for Drug Accountability and Storage.) AZD6244 hyd sulfate Administration AZD6244 hyd sulfate is administered orally, twice daily on a continuous dosing schedule with no rest period between cycles. A cycle of AZD6244 hyd sulfate is defined as 28 consecutive days starting with the first day of treatment (day 1). The dose of AZD6244 hyd sulfate is determined from the patient s body surface area using the dosing nomogram (Appendix VI). Treatment will be administered in an outpatient setting. AZD6244 hyd sulfate should be taken with water (approximately 2 to 4 ounces for children < 12 and 4 to 8 ounces for 12 years). Capsules can be combined to accurately dose patients (e.g., a dose of 30 mg could be three 10 mg capsules) AZD6244 hyd sulfate Toxicity Profile Comprehensive Adverse Events and Potential Risks list (CAEPR) for AZD6244 Free base (NSC ), AZD6244 Hydrogen sulfate (NSC ) The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single list of reported and/or potential adverse events (AE) associated with an agent using a uniform presentation of events by body system. In addition to the comprehensive list, a subset, the Agent Specific Adverse Event List (ASAEL), appears in a separate column and is identified with bold and italicized text. This subset of Aes (ASAEL) contains events that are considered expected for expedited reporting purposes only. Refer to the CTEP, NCI Guidelines: Adverse Event Reporting Requirements for further clarification. Frequency is provided based on 391 patients. Below is the CAEPR for AZD6244 hyd sulfate. 1 Version 2.2, November 2, Adverse Events with Possible Relationship to AZD6244 (CTCAE 4.0 Term) [n= 391] EXPECTED Aes FOR ADEERS REPORTING Agent Specific Adverse Event List (ASAEL) Likely (>20%) Less Likely (<=20%) Rare but Serious (<3%) Expected BLOOD AND LYMPHATIC SYSTEM DISORDERS Anemia Anemia CARDIAC DISORDERS Left ventricular systolic dysfunction Left ventricular systolic dysfunction 52

55 NCI protocol #; Version Date: EYE DISORDERS Blurred vision GASTROINTESTINAL DISORDERS Abdominal distension Abdominal pain Constipation Diarrhea 2 Dry mouth Mucositis oral Nausea Vomiting GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS Edema face Edema limbs Fatigue Fever INVESTIGATIONS Alanine aminotransferase increased Aspartate aminotransferase increased METABOLISM AND NUTRITION DISORDERS Anorexia MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS Arthralgia Back pain NERVOUS SYSTEM DISORDERS Dizziness Headache PSYCHIATRIC DISORDERS Depression Insomnia RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS Cough Dyspnea SKIN AND SUBCUTANEOUS TISSUE DISORDERS Alopecia Dry skin Pruritus Rash acneiform Rash maculo-papular VASCULAR DISORDERS Hypertension Abdominal pain Constipation Diarrhea Nausea Vomiting Edema face Edema limbs Fatigue Fever Anorexia Headache Cough Dyspnea Dry skin Rash acneiform Rash maculo-papular 1 This table will be updated as the toxicity profile of the agent is revised. Updates will be distributed to all Principal Investigators at the time of revision. The current version can be obtained by contacting PIO@CTEP.NCI.NIH.GOV. Your name, the name of the investigator, the protocol and the agent should be included in the . 2 SBE-CD (Captisol, vehicle) in the mix and drink formulation is known to cause soft stools and/or diarrhea in rats and dogs; however, it is possible that some of these findings might be related to exacerbation of the vehicle effect by AZD

56 NCI protocol #; Version Date: Infection includes all 75 infection sites under the INFECTIONS AND INFESTATIONS SOC. Also reported on AZD6244 trials but with the relationship to AZD6244 still undetermined: CARDIAC DISORDERS Sinus bradycardia GASTROINTESTINAL DISORDERS Dyspepsia GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS Non-cardiac chest pain INFECTIONS AND INFESTATIONS Infection 3 INVESTIGATIONS Platelet count decreased METABOLISM AND NUTRITION DISORDERS Dehydration; Hypoalbuminemia; Hypokalemia MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS Musculoskeletal and connective tissue disorder - Other (joint swelling) NERVOUS SYSTEM DISORDERS Dysgeusia PSYCHIATRIC DISORDERS Confusion RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS Epistaxis; Hypoxia; Pneumonitis; Voice alteration SKIN AND SUBCUTANEOUS TISSUE DISORDERS Palmar-plantar erythrodysesthesia syndrome; Skin and subcutaneous tissue disorders Other (skin fissures) VASCULAR DISORDERS Thromboembolic event Note: AZD6244 hyd sulfate in combination with other agents could cause an exacerbation of any adverse event currently known to be caused by the other agent, or the combination may result in events never previously associated with either agent. 8.2 Collaborative Agreement Protocols that involve agent(s) covered by a collaborative agreement with a biotech/pharma company(ies) must incorporate the NCI/ DCTD Standard Language shown below. The agent(s) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI under a Collaborative Agreement (CRADA, CTA, CSA) between the Pharmaceutical Company(ies) (hereinafter referred to as Collaborator(s) ) and the NCI Division of Cancer Treatment and Diagnosis. Therefore, the following obligations/guidelines, in addition to the provisions in the Intellectual Property Option to Collaborator contained within the terms of award, apply to the use of the Agent(s) in this study: 1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor can Agent(s) be transferred or licensed to any party not participating in the clinical study. Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and shall be maintained as such by the investigators. The protocol documents for studies utilizing Agents contain confidential information and should not be shared or distributed without the permission of the NCI. If a copy of this protocol is requested by a patient or patient s family member participating on the study, the individual should sign a confidentiality agreement. A suitable model agreement can be downloaded from: 54

57 NCI protocol #; Version Date: For a clinical protocol where there is an investigational Agent used in combination with (an)other Agent(s), each the subject of different Collaborative Agreements, the access to and use of data by each Collaborator shall be as follows (data pertaining to such combination use shall hereinafter be referred to as Multi-Party Data ): a. NCI will provide all Collaborators with prior written notice regarding the existence and nature of any agreements governing their collaboration with NCI, the design of the proposed combination protocol, and the existence of any obligations that would tend to restrict NCI s participation in the proposed combination protocol. b. Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial by any other Collaborator solely to the extent necessary to allow said other Collaborator to develop, obtain regulatory approval or commercialize its own Agent. c. Any Collaborator having the right to use the Multi-Party Data from these trials must agree in writing prior to the commencement of the trials that it will use the Multi-Party Data solely for development, regulatory approval, and commercialization of its own Agent. 3. Clinical Trial Data and Results and Raw Data developed under a Collaborative Agreement will be made available to Collaborator(s), the NCI, and the FDA, as appropriate and unless additional disclosure is required by law or court order as described in the IP Option to Collaborator ( Additionally, all Clinical Data and Results and Raw Data will be collected, used and disclosed consistent with all applicable federal statutes and regulations for the protection of human subjects, including, if applicable, the Standards for Privacy of Individually Identifiable Health Information set forth in 45 C.F.R. Part When a Collaborator wishes to initiate a data request, the request should first be sent to the NCI, who will then notify the appropriate investigators (Group Chair for Cooperative Group studies, or PI for other studies) of Collaborator s wish to contact them. 5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is a DMC for this clinical trial. 6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the Group office for Cooperative Group studies or 55

58 NCI protocol #; Version Date: by the principal investigator for non-cooperative Group studies for immediate delivery to Collaborator(s) for advisory review and comment prior to submission for publication. Collaborator(s) will have 30 days from the date of receipt for review. Collaborator shall have the right to request that publication be delayed for up to an additional 30 days in order to ensure that Collaborator s confidential and proprietary data, in addition to Collaborator(s) s intellectual property rights, are protected. Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for courtesy review as soon as possible and preferably at least three (3) days prior to submission, but in any case, prior to presentation at the meeting or publication in the proceedings. Press releases and other media presentations must also be forwarded to CTEP prior to release. Copies of any manuscript, abstract and/or press release/ media presentation should be sent to: ncicteppubs@mail.nih.gov The Regulatory Affairs Branch will then distribute them to Collaborator(s). No publication, manuscript or other form of public disclosure shall contain any of Collaborator s confidential/ proprietary information. 56

59 NCI protocol #; Version Date: References 1. Friedman, J.M., Neurofibromatosis 1: clinical manifestations and diagnostic criteria. Journal of child neurology, (8): p ; discussion 571-2, Korf, B.R., Plexiform neurofibromas. American Journal of Medical Genetics, (1): p Korf, B.R., Malignancy in neurofibromatosis type 1. The oncologist, (6): p Needle, M.N., et al., Prognostic signs in the surgical management of plexiform neurofibroma: the Children's Hospital of Philadelphia experience, The Journal of pediatrics, (5): p Dombi, E., et al., NF1 plexiform neurofibroma growth rate by volumetric MRI: relationship to age and body weight. Neurology, (9): p Kim, A., et al., Characteristics of children enrolled in treatment trials for NF1- related plexiform neurofibromas. Neurology, (16): p Gupta, A., et al., Phase I study of thalidomide for the treatment of plexiform neurofibroma in neurofibromatosis 1. Neurology, (1): p Widemann, B.C., et al., Phase I trial and pharmacokinetic study of the farnesyltransferase inhibitor tipifarnib in children with refractory solid tumors or neurofibromatosis type I and plexiform neurofibromas. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, (3): p Widemann, B.C., Children's Tumor Foundation meeting in Portland, Oregon. Phase II randomized, flexible cross-over, double-blinded, placebo-controlled trial of the farnesyltransferase inhibitor (FTI) tipifarnib (R115777) in pediatric patients (pts) with neurofibromatosis type 1 (NF1) and progressive plexiform neurofibromas (PN) Babovic-Vuksanovic, D., et al., Phase I Trial of Pirfenidone in Children with Neurofibromatosis Type 1 (NF1) and Plexiform Neurofibromas (PN). In Press: Journal of Pediatric Neurology, Widemann, B.C., Children's Tumor Foundation meeting in Portland, Oregon. Phase II trial of pirfenidone in children and young adults with neurofibromatosis type 1 (NF1) and progressive plexiform neurofibromas (PN) Estey, E., et al., Therapeutic response in phase I trials of antineoplastic agents. Cancer treatment reports, (9): p Therasse, P., et al., New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. Journal of the National Cancer Institute, (3): p Solomon, J., et al., Automated detection and volume measurement of plexiform neurofibromas in neurofibromatosis 1 using magnetic resonance imaging. Computerized medical imaging and graphics : the official journal of the Computerized Medical Imaging Society, (5): p

60 NCI protocol #; Version Date: Monestiroli, S., et al., Kinetics and viability of circulating endothelial cells as surrogate angiogenesis marker in an animal model of human lymphoma. Cancer research, (11): p Viskochil, D., et al., Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell, (1): p Xu, G.F., et al., The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell, (3): p Kluwe, L., et al., NF1 mutations in neurofibromatosis 1 patients with plexiform neurofibromas. Human mutation, (3): p Serra, E., et al., Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations. Human molecular genetics, (20): p Packer, R.J., et al., Plexiform neurofibromas in NF1: toward biologic-based therapy. Neurology, (10): p Widemann, B.C., et al., Phase I trial and pharmacokinetic study of the farnesyltransferase inhibitor tipifarnib in children with refractory solid tumors or neurofibromatosis type I and plexiform neurofibromas. Journal of Cinical Oncology (3): p Rowinsky, E.K., J.J. Windle, and D.D. Von Hoff, Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development. J Clin Oncol, (11): p Khalaf, W.F., et al., K-ras is critical for modulating multiple c-kit-mediated cellular functions in wild-type and Nf1+/- mast cells. J Immunol, (4): p Dasgupta, B., et al., Glioma formation in neurofibromatosis 1 reflects preferential activation of K-RAS in astrocytes. Cancer Res, (1): p Barkan, B., et al., The Ras inhibitor farnesylthiosalicylic acid as a potential therapy for neurofibromatosis type 1. Clin Cancer Res, (18): p Lauchle, J.O., et al., Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. Nature, (7262): p Yeh, T.C., et al., Biological characterization of ARRY (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res, (5): p Garon, E.B., et al., Identification of common predictive markers of in vitro response to the Mek inhibitor selumetinib (AZD6244; ARRY ) in human breast cancer and non-small cell lung cancer cell lines. Mol Cancer Ther. 9(7): p Astra-Zeneca, Investigator Brochure. 26 March 2010(Edition 9). 30. Shannon, A.M., et al., The mitogen-activated protein/extracellular signalregulated kinase kinase 1/2 inhibitor AZD6244 (ARRY ) enhances the radiation responsiveness of lung and colorectal tumor xenografts. Clin Cancer Res, (21): p Davies, B.R., et al., AZD6244 (ARRY ), a potent inhibitor of mitogenactivated protein kinase/extracellular signal-regulated kinase kinase 1/2 kinases: mechanism of action in vivo, pharmacokinetic/pharmacodynamic relationship, 58

61 NCI protocol #; Version Date: and potential for combination in preclinical models. Mol Cancer Ther, (8): p Denton, C.L. and D.L. Gustafson, Pharmacokinetics and pharmacodynamics of AZD6244 (ARRY ) in tumor-bearing nude mice. Cancer Chemother Pharmacol. 33. Chang, Q., E. Chen, and D.W. Hedley, Effects of combined inhibition of MEK and mtor on downstream signaling and tumor growth in pancreatic cancer xenograft models. Cancer Biol Ther, (20): p Nishioka, C., et al., Inhibition of MEK signaling enhances the ability of cytarabine to induce growth arrest and apoptosis of acute myelogenous leukemia cells. Apoptosis, (9): p Jin, N., et al., Dual inhibition of mitogen-activated protein kinase kinase and mammalian target of rapamycin in differentiated and anaplastic thyroid cancer. J Clin Endocrinol Metab, (10): p Yang, S., et al., AZD6244 (ARRY ) enhances the therapeutic efficacy of sorafenib in mouse models of gastric cancer. Mol Cancer Ther, (9): p Yoon, Y.K., et al., KRAS mutant lung cancer cells are differentially responsive to MEK inhibitor due to AKT or STAT3 activation: implication for combinatorial approach. Mol Carcinog. 49(4): p Banerji, U., et al., The first-in-human study of the hydrogen sulfate (Hyd-sulfate) capsule of the MEK1/2 inhibitor AZD6244 (ARRY ): a phase I open-label multicenter trial in patients with advanced cancer. Clin Cancer Res. 16(5): p Kim, A., Phase I Trial of Sorafenib in children with NF1and inoperable plexiform neurofibromas Neuro-Oncology, (6): p. ii Dombi, E., et al., Progression Analysis of Plexiform Neurofibromas (PN) in an Ongoing phase II Clinical trial for Neurofibromatosis Type 1 (NF1). NNFF International Consortium for the Molecular Biology of NF1 and NF2, Mellins, C.A., et al., The role of psychosocial and family factors in adherence to antiretroviral treatment in human immunodeficiency virus-infected children. The Pediatric infectious disease journal, (11): p Van Dyke, R.B., et al., Reported adherence as a determinant of response to highly active antiretroviral therapy in children who have human immunodeficiency virus infection. Pediatrics, (4): p. e Patino, A.M., et al., Health beliefs and regimen adherence in minority adolescents with type 1 diabetes. Journal of pediatric psychology, (6): p Martin, S.C., et al., Comparison of Knowledge and Perceptions of Responsibility for Medication Adherence Between HIV-positive Children and their Caregivers. Society of Pediatric Psychology Meeting, National Institutes of Health Consensus Development Conference Statement: neurofibromatosis. Bethesda, Md., USA, July 13-15, Neurofibromatosis, (3): p Galimont-Collen, A.F., et al., Classification and management of skin, hair, nail and mucosal side-effects of epidermal growth factor receptor (EGFR) inhibitors. Eur J Cancer, (5): p

62 NCI protocol #; Version Date: Chou, L.S., et al., Managing dermatologic toxicities of epidermal growth factor receptor inhibitors. Clin Lung Cancer, Suppl 1: p. S

63 NCI protocol #; Version Date: Appendices Appendix I: Performance Status Scales/Scores Appendix II: Protocol for required MRI studies of PN Appendix III: Required study evaluations Appendix IV: Pharmacokinetic Worksheet Appendix V: Peripheral Blood Mononuclear Cell Samples (PBMCs) Appendix VI: Dosing Nomogram for AZD6244 hyd sulfate for Phase I Pediatric NF-1 trial Appendix VIIa: Appendix VIIb: Appendix VIIc: Appendix VIId: Adherence Questionnaires Patient Daily Medication Diary Barriers to Adherence List Responsibility for Medication Questionnaires Appendix VIII: Skin Toxicity Algorithm 61

64 NCI protocol #; Version Date: Appendix I: Performance Status Scales/Scores PERFORMANCE STATUS CRITERIA Karnofsky and Lansky performance scores are intended to be multiples of 10 Karnofsky (> 16 years of age) Lansky ( 16 years of age) Score Description Score Description 100 Normal, no complaints, no evidence of disease 90 Able to carry on normal activity, minor signs or symptoms of disease. 80 Normal activity with effort; some signs or symptoms of disease. 70 Cares for self, unable to carry on normal activity or do active work. 60 Required occasional assistance, but is able to care for most of his/her needs. 50 Requires considerable assistance and frequent medical care. 40 Disabled, requires special care and assistance. 30 Severely disabled, hospitalization indicated. Death not imminent. 20 Very sick, hospitalization indicated. Death not imminent. 10 Moribund, fatal processes progressing rapidly. 100 Fully active, normal. 90 Minor restrictions in physically strenuous activity. 80 Active, but tires more quickly 70 Both greater restriction of and less time spent in play activity. 60 Up and around, but minimal active play; keeps busy with quieter activities. 50 Gets dressed, but lies around much of the day; no active play, able to participate in all quiet play and activities. 40 Mostly in bed; participates in quiet activities. 30 In bed; needs assistance even for quiet play. 20 Often sleeping; play entirely limited to very passive activities. 10 No play; does not get out of bed. 62

65 NCI protocol #; Version Date: Appendix II: Protocol for required MRI studies of PN Prior to starting treatment on this study all known measurable tumors should be imaged with MRI if feasible, to obtain a baseline. Tumor spread in patients with NF1 can be very extensive, and may not allow for all lesions to be followed using 3D-MRI. The three most relevant lesions will be chosen as index lesions. The goal will therefore be to use 3D-MRI only to follow the inoperable PN (a maximum of three lesions), which will be defined as index lesion(s). Pre-study radiographic evaluation: Identify and select the inoperable PN (a maximum of three lesions) for 3-D MRI evaluation based on prior imaging studies. Should there be more than 3 inoperable PN, the three most clinically relevant PN will be followed by 3-D MRI analysis. Perform 3-D MRI sequences on the selected index lesions as outlined in the MRI acquisition protocols below. In addition, if possible, perform MRI of all additional measurable PN. On study radiographic evaluation: Unless clinically indicated otherwise obtain MRI of the index lesions only as outlined in the MRI acquisition protocol below prior to cycles 4, 8, 12, and then after every 6 cycles as long as the patient remains on study. MRI protocols: Depending on the location of the index lesions the Spine, Head/Neck or Trunk/Extremities protocols outlined on the following pages will be used. Modifications should be documented in the MRI protocols, and the same imaging protocol, and, if possible, the same MRI scanner, should be used for all subsequent MRI studies. Every attempt should be made to image the entire progressive PN. All MRI studies requested per protocol will be analyzed at the NCI POB under supervision of Dr. Eva Dombi, M.D. (phone , dombie@mail.nih.gov). The MRI data from each scan will be processed to assess the volume of the index plexiform neurofibroma(s). Each patient s volumetric measurement obtained from the initial MRI will serve as the baseline against which to assess incremental changes in volume that occur during the subsequent intervals. 63

66 NCI protocol #; Version Date: MRI Protocol- Spine STUDY ID NUMBER: Note: Only the series outlined below are required for volumetric analysis of plexiform neurofibromas and must be performed within protocol specifications as indicated below. Additional series may be obtained as indicated per institutional PI. 1. AXIAL FSEIR Axial FSEIR Protocol Specifications Echo Train Length 5 TR 6000 TE 34 TI 150 Slice Thickness 5 mm Skip 0 Matrix FOV 256x cm NEX 1 Frequency Direction Options R L Tailored RF, FC, PC, 0.75 FOV Actual Specifications Reason For Change 2. CORONAL FSEIR Coronal FSEIR Protocol Specifications Echo Train Length 5 TR 6000 TE 34 TI 150 Slice Thickness 5 mm Skip 0 Matrix FOV 256x cm NEX 1 Frequency Direction Options S I Tailored RF, FC, PC Actual Specifications Reason For Change 64

67 NCI protocol #; Version Date: MRI Protocol- Head/Neck STUDY ID NUMBER: Note: Only the series outlined below are required for volumetric analysis of plexiform neurofibromas and must be performed within protocol specifications as indicated below. Additional series may be obtained as indicated per institutional PI. 1. AXIAL FSEIR Axial FSEIR Protocol Specifications Echo Train Length 5 TR 6000 TE 34 TI 150 Slice Thickness 4 mm Skip 0 Matrix FOV 256x cm NEX 1 Frequency Direction Options 2. CORONAL FSEIR Coronal FSEIR A P Tailored RF, FC, 0.75 FOV Protocol Specifications Echo Train Length 5 TR 6000 TE 34 TI 150 Slice Thickness Skip Matrix FOV 5 mm 0 mm 256x cm NEX 1 Frequency Direction Options S I Tailored RF, PC Actual Specifications Actual Specifications Reason For Change Reason For Change 65

68 NCI protocol #; Version Date: MRI Protocol-Trunk/Extremities STUDY ID NUMBER: Note: Only the series outlined below are required for volumetric analysis of plexiform neurofibromas and must be performed within protocol specifications as indicated below. Additional series may be obtained as indicated per institutional PI. 1. AXIAL PLANE-STIR Axial Plane STIR Coil: Body Protocol Specifications Sequence: Fast Spin Echo (Turbo) FSEIR Echo Train Length 5 TR 6000 TE 15 TI 150 Slice Thickness 10 mm Skip 0 Matrix FOV No. of Excitations/ Sequence 512x160 40x No. of Acquisitions 1 Saturation None Actual Specifications Reason For Change 2. CORONAL PLANE STIR Coronal Plane STIR Coil: Body Protocol Specifications Sequence: Fast Spin Echo (Turbo) FSEIR Echo Train Length 5 TR 3400 TE 15 TI 150 Slice Thickness 5 mm Skip 0 Matrix FOV No. of Excitations/ Sequence 512x160 48x48 No. of Acquisitions 1 Saturation 1 None Actual Specifications Reason For Change 66

69 NCI protocol #; Version Date: Appendix III: Required study evaluations STUDIES TO BE OBTAINED Pre- Study During Cycle 1 Prior To Subsequent Cycles 1 End of Therapy Evaluation 2 History X Every other week Every other week cycles 2 3 X Prior to cycles 4 11, 13, 15, 17, etc Physical Exam, vital signs X Every other week Every other week cycles 2 3 X Prior to cycles 4 11, 13, 15, 17, etc Height, weight, BSA 3 X - Prior to cycle 4, 6, 11 and then - after every 6 cycles (pre 17, 23, 29, etc) Performance Status 4 X - Prior to cycle 6, 11 and then after X every 6 cycles (pre 17, 23, 29, etc) EKG/Echo X - Prior to cycle 6, 11 and then after - every 6 cycles (pre 17, 23, 29, etc) CBC, differential, platelets 5 X Every other week Every other week cycles 2 3 X Prior to cycles 4 11, 13, 15, 17, etc Pharmacokinetics 6 X X Prior to cycle Pharmacodynamics 7 X X Prior to cycle Urinalysis X - Prior to cycle 6, 11 and then after X every 6 cycles (pre 17, 23, 29, etc) Electrolytes including Ca++, PO 4, Mg++, X Every other week Every other week cycles 2 3 X BUN, creatinine, glucose, ALT, bilirubin, Total protein/albumin, CPK Prior to cycles 4 11, 13, 15, 17, etc PN Disease Evaluation 8 X - Prior to cycle 6, 11 and then after X every 6 cycles (pre 17, 23, 29, etc) 9 Ophthalmology evaluation X - Prior to cycle 4, 6, 11 and then after every 6 cycles (pre 17, 23, 29, etc) Pregnancy Test 10 X - Prior to radiographic tests: pre - cycle 4, 6, 11 and then after every 6 cycles (pre 17, 23, 29, etc) Adherence Questionnaire X Patient diary 11 - Daily Daily - Pill Count X 12 1 Day (+/- 3 days) of the preceding cycle. 67

70 NCI protocol #; Version Date: Should be performed, if possible, when the patient comes off treatment regardless of the reason, unless the test or procedure has been performed within the time period defined in Section BSA should be calculated from average of three measurements. 4 Appendix I, prior to cycles 1 and then only required to be recorded if there is a change in performance status during the study. 5 If patients develop Grade 4 anemia then CBC should be checked every 3 to 4 days until recovery to Grade 1. 6 See Appendix IV for timing of PK samples 7 See Appendix V for timing of PD and correlative studies 8 Volumetric MRI. See Appendix II. 9 If patient does show a responsive disease (RD) according to section 5.2, response will be confirmed after 1-3 treatment cycles 10 Patients of childbearing potential require a negative pregnancy test prior to starting treatment and prior to every radiographic scan which will presumably be prior to cycle 6, 11, and then after every 6 cycles (pre-cycle 17, 23, 29, etc). Patients of childbearing potential must use an acceptable method of birth control. Abstinence is an acceptable method of birth control. 11 Review patient diary weekly with adherence questionnaire prior to each treatment cycle for cycles 2-9, and subsequently prior to every other treatment cycle (pre cycle 11, 13, 15, 17, etc) by Ms. Andy Gillespie. 12 The patient s research nurse will conduct pill counts with each scheduled visit (prior to cycle 2, 4, 8, 12, and subsequently every 6 cycles). Caregivers and patients will be instructed to return all empty, partially full, and full bottles to their research nurse at each visit. 68

71 NCI protocol #; Version Date: Appendix IV: Pharmacokinetic Worksheet Study ID: BSA (m 2 ): Dose level (mg/m 2 ): Actual Dose: Time dose taken: Date: Sample # Day Date Hour Target Time Time Obtained 1 1 Pre dose hr post dose # * 1 10 to hr post dose Pre dose 1 day *Obtain sample within +/- 3days of day Obtain this sample at the time of evaluation for cycle #2 on the same day the peripheral blood is obtained for biologic studies (see Appendix V). Blood samples: 3 ml of whole blood will be collected in a heparinized tube (Green Top). If blood samples are taken via an indwelling central venous cannula, an appropriate amount of fluid should be removed from the cannula to clear the line before each blood sample is taken. Plasma: should be separated by centrifugation (10 min at 1000 g or 2500 rpm) within four hours after collection. Separated plasma will be capped by means of polyethylene stoppers and labeled with the registration number, subject ID number, date and time of sampling, and sample number. Plasma samples should be stored in the dark at -70 C. Shipment: Samples should be brought to: Ms. Nalini Jayaprakash, M.S. Pediatric Oncology Branch, NCI 10 Center Drive Building 10, CRC, Room Bethesda, MD

72 NCI protocol #; Version Date: Appendix V: Study ID: Peripheral Blood Mononuclear Cell Samples (PBMCs) BSA (m 2 ): Dose level (mg/m 2 ): Actual Dose: Time dose taken: Date: These samples will be obtained prior to starting course # 1 of treatment with AZD6244 hyd sulfate (pre-treatment sample), and once between day 27 to 28 (+/- 3 days) of treatment course #1 (steady state sample) at the time of evaluation for course #2, as outlined below: Pretreatment sample date: Steady state sample: date/day of cycle: Collection of Peripheral Blood: Collect 5 ml peripheral blood in EDTA. Shipment Of Peripheral Blood: Place peripheral blood in polypropylene screw top tube(s). Label tube with the study ID, and date and time it was drawn. Place tube(s) in container. Place the container with the conical tube in styrofoam box. Package sample as appropriate for biologic material. Bring the sample to: Ms. Nalini Jayaprakash, M.S. Pediatric Oncology Branch, NCI 10 Center Drive Building 10, CRC, Room Bethesda, MD

73 NCI protocol #; Version Date: Appendix VI: Dosing nomogram for AZD6244 hyd sulfate for phase I Pediatric NF-1 trial Dose Level Dose (mg/m 2 /dose) 1 20 BSA* Dose BSA* Dose BSA* Dose BSA* Dose BSA* Dose * BSA, body surface area in m 2. Actual dose in mg (capsule sizes 10, and 25 mg) administered BID approximately every 12 hours. The dosing nomogram will not be used to make dose reductions for toxicity due to overlap in dose levels for certain BSA. Section 3.3 describes dose reductions for toxicity. 71

74 NCI protocol #; Version Date: Appendix VIIa: Adherence Questionnaires Parent Adherence Questionnaire Patient Name: Date: Name of person filling out this form: Instructions: Please think about a time when your child has had to take medicine in the past. From the list below, please check all items that may have caused your child to miss a dose of medicine, or that interfered with your child being able to take all his or her medicine the way it was prescribed. In the past, your child missed a dose of medication because (check all that apply): 1 You were away from home. 2 You were busy with other things. 3 You forgot. 4 Your child had too many pills to take. 5 You wanted to avoid your child having side effects, such as diarrhea, nausea, or tiredness. 6 You did not want others to see your child taking medicine. 7 You had a change in your daily routine. 8 You were too tired or slept through your child s dose time. 9 Your child was too tired or slept through his/her dose time. 10 Your child felt sick or ill. 11 You felt depressed or overwhelmed. 12 You had a problem giving your child the medicine with a clear liquid (if required). 13 Your child had difficulty swallowing the pill(s). 14 You ran out of pills. 15 Your child didn t like the taste of the medicine. 16 You didn t think the medicine was working anyway. 17 You just didn t feel like it, needed a break. 18 You weren t around to help your child get the medicine. 19 Your child refused to take the medicine. 20 Your child was too busy. 21 Your child felt sad or stressed out. 22 Other (please specify): 72

75 NCI protocol #; Version Date: Child/Adolescent Adherence Questionnaire Patient Name: Date: Instructions: Please think about a time when you had to take medicine in the past. From the list below, please check all items that may have caused you to miss a dose of medicine. In the past, you missed a dose of medication because (check all that apply): 1 You were away from home. 2 You were busy with other things. 3 You forgot. 4 You had too many pills to take. 5 You wanted to avoid having side effects, such as diarrhea, nausea, or tiredness. 6 You did not want others to see you taking medicine. 7 You had a change in your daily routine. 8 You were too tired or slept through your dose time. 9 Your parent was too tired or slept through your dose time. 10 You felt sick or ill. 11 You felt sad or stressed out. 12 You had a problem taking the medicine with a clear liquid (if required). 13 You had difficulty swallowing the pill(s). 14 You ran out of pills. 15 You didn t like the taste of the medicine. 16 You didn t think the medicine was working anyway. 17 You just didn t feel like it, needed a break. 18 Your parent wasn t around to help you get the medicine. 19 You refused to take the medicine. 20 Other (please specify): 73

76 NCI protocol #; Version Date: Appendix VIIb: Patient Daily Medication Diary for AZD6244 hyd sulfate Patient Initials: Initials of person filling out this form: Dose Level: Patient Dose: mg Each dose of AZD6244 hyd sulfate should be taken 1 hour before meals or 2 hours after meals on an empty stomach. Take with water only. Please complete the following chart by indicating the date and time of each dose of medication your child takes. Use a new chart for EACH WEEK. For missed doses, enter a M in the space under AM or PM, then refer to the Adherence Barriers List and enter the item number(s) that best describe the reason(s) for the missed dose. Please document at the time the dose is taken; do NOT WAIT until the end of the week to complete this form. This form will be reviewed by the research team. Date AM PM AM PM AM PM AM PM AM PM AM PM AM PM Time Dose Taken: Reason for missed doses (see separate page): If side effect is present, please answer items 1 through 8 below by indicating the severity of side effects experienced by the patient each day over the past week. Circle one and only one number per item, per day. Leave blank if side effect is not present. 1 Mild 2 Moderate 3 Severe 1. Diarrhea Rash Location: Swelling 4. Shortness of breath Tiredness Blurred vision Other: Other: Mild = 1-3 times/day; Moderate = 4-6 times/day; Severe = 7 or more times/day Please fax completed forms to Ms. Andy Gillespie at after each treatment cycle. Study ID: Dose level/dose: Cycle #: Start Date: Reviewer s signature: Date reviewed: 74

77 NCI protocol #; Version Date: Appendix VIIc: Adherence Barriers List For Patient Daily Medication Diary Instructions: Please refer to this list when completing your Daily Medication Diary. Each time you (or your child) misses a dose of your (his or her)medicine, identify the items below that best describe the reason(s) for the missed dose. Then fill in the item numbers for those reasons in the space on the Daily Medication Diary that correspond to the date and time (AM or PM) of the missed dose (see example below). 1 You were away from home. 2 You were busy with other things. 3 You forgot. 4 Your child had too many pills to take. 5 You wanted to avoid your child having side effects, such as diarrhea, nausea, or tiredness. 6 You did not want others to see your child taking medicine. 7 You had a change in your daily routine. 8 You were too tired or slept through your child s dose time. 9 Your child was too tired or slept through his/her dose time. 10 Your child felt sick or ill. 11 You felt depressed or overwhelmed. 12 You had a problem giving your child the medicine with a clear liquid. 13 Your child had difficulty swallowing the pill(s). 14 You ran out of pills. 15 Your child didn t like the taste of the medicine. 16 You didn t think the medicine was working anyway. 17 You just didn t feel like it, needed a break. 18 You weren t around to help your child get the medicine. 19 Your child refused to take the medicine. 20 Your child was too busy. 21 Your child felt sad or stressed out. 22 Other (please specify): Example: For a dose missed on 10/3/06 in the evening, a M is entered in the Time Dose Taken row, and a 1 and 7 are entered in the space corresponding to that day and time to indicate the reasons for the missed dose. Date 10/2/06 10/3/06 10/4/06 AM PM AM PM AM PM Time Dose Taken:1 8:15 8:00 8:30 M 8:15 8:00 Reason for missed doses (see separate page):2 1,7 75

78 NCI protocol #; Version Date: Appendix VIId: Responsibility for Medication Questionnaires Patient s Hosp #: Parent Initials: Date: RMS - Parent I am going to read you different tasks or situations that relate to your child s medicine. Please tell me if: 1 = You are in charge of the task almost all of the time. 2 = You and your child share being in charge of the task. 3 = Your child is in charge of the task almost all of the time. Who is in charge of. 1. Remembering to take medicine when you and your child are at home. 2. Making sure your child s medicine does not run out. 3. Getting the medicine ready to take (for example, getting pills out of the container). 4. Remembering to bring medicine when you will be away from home. 5. Telling the doctor or calling the pharmacy to refill a prescription. 6. Reporting side effects of the medicine to the doctor. 7. Remembering to take medicine when you and your child are not at home. 8. Remembering what time your child is supposed to take the medicine. 9. Talking to the doctor about the medicine. 76

79 Patient s Hosp #: Date: NCI protocol #; Version Date: RMS Child/Adolescent I am going to read you different tasks or situations that relate to taking your medicine. Please tell me if: 1 = Your parent(s) are in charge of the task almost all of the time 2 = You and your parent(s) share being in charge of the task. 3 = You are in charge of the task almost all of the time. Who is in charge of. 1. Remembering to take medicine when you are at home. 2. Making sure your medicine does not run out. 3. Getting medicine ready to take (for example, getting pills out of the container). 4. Remembering to bring medicine when you will be away from home. 5. Telling the doctor or calling the pharmacy to refill a prescription. 6. Reporting side effects of medicine to the doctor. 7. Remembering to take medicine when you are not at home. 8. Remembering what time to take the medicine. 9. Talking to the doctor about the medicine. 77

80 NCI protocol #; Version Date: Appendix VIII: Skin Toxicity Stop AZD6244 and refer to a burn-wound unit. 78

81 A PHASE I/II STUDY OF THE MITOGEN ACTIVATED PROTEIN KINASE KINASE (MEK) 1 INHIBITOR SELUMETINIB (AZD6244; HYD SULFATE) IN CHILDREN WITH NEUROFIBROMATOSIS TYPE 1 (NF1) AND INOPERABLE PLEXIFORM NEUROFIBROMAS (PN) Coordinating Center: Pediatric Oncology Branch, NCI Principal Investigator: Brigitte Widemann, M.D. (POB, NCI) * Lead Associate Investigator: +Rachel Ershler, M.D. (POB, NCI) Trial Sponsor NCI, Cancer Therapy Evaluation Program NCI-supplied Agent Selumetinib (AZD6244 hyd sulfate), AstraZeneca IND# 77, 782 NSC# NCI Protocol # Local Protocol # C-0161 Amendment I NIH Associate Investigators: Trish Whitcomb, R.N. (OD, CCR, NCI) Andrea Gillespie, R.N. (OD, CCR, NCI) Andrea Baldwin, P.N.P. (POB, NCI) Eva Dombi, M.D. (POB, NCI) ** +Alessandra Brofferio, M.D. (NHLBI) Nalini Jayaprakash, M.S. (POB, NCI) ** Staci Martin, Ph.D. (POB, NCI) Joanne Derdak, N.P. (NCI, POB) John Glod, M.D., Ph.D. (NCI, POB) Paul Jarosinski, Pharm.D. (Pharmacy, CC, NIH)** Pam Wolters, Ph.D. (NCI, POB) +Ed Cowen, M.D. (DB, CCR, NIH) +Scott Paul, M.D. (RMD, CC, NIH)** Seth M. Steinberg, Ph.D. (Biostatistics, OD, CCR, NCI)** Beth Solomon (RMD, CC, NIH) Kathleen Farrell (RMD, CC, NIH) Michaele Smith (RMD, CC, NIH) +Carmen Brewer, M.D. (NIDCD, NIH)** Non-NIH Associate Investigators: Michael J. Fisher, M.D., CHOP, Philadelphia, PA. Brian Weiss, M.D., Cincinnati Children s Hospital Medical Center, Cincinnati, OH AeRang Kim, M.D., Ph.D., Children s National Medical Center, Washington, DC Jaishri, Blakeley, M.D., Johns Hopkins University, Baltimore, MD 1

82 Wade Clapp, M.D., Riley Hospital for Children, Indianapolis, IN Kent Robertson, M.D., Riley Hospital for Children, Indianapolis, IN David Ingram, M.D., Riley Hospital for Children, Indianapolis, IN** Michael Ferguson, M.D., M.S., Riley Hospital for Children, Indianapolis, IN Dr. Chris Plummer, BSc, Ph.D, BM, BCh, FRCP**, Consultant Cardiologist Department of Cardiology, Freeman Hospital Newcastle upon Tyne, NE7 7DN, UK Tel: (direct) (sec.) e mail: Chris.Plummer@nuth.nhs.uk, C.J.Plummer@ncl.ac.uk. *Pharmacology & Experimental Therapeutics Section Pediatric Oncology Branch, NCI ** Individuals with no direct clinical care responsibilities 10 Center Drive + Non-prescribing physician associate investigator Building 10, CRC, Room Bethesda, MD Phone: (301) /Fax: (301) widemanb@mail.nih.gov PROPRIETARY and CONFIDENTIAL This protocol contains confidential information that should only be disclosed to those persons responsible for execution and organization of the trial and on condition that all such persons agree not to further disseminate it. 2

83 PRÉCIS Background Patients with Neurofibromatosis 1 (NF1) have an increased risk of developing tumors of the central and peripheral nervous system, including plexiform neurofibromas (PN), which are benign nerve sheath tumors that are among the most debilitating complications of NF1. PN may be congenital and appear to have the fastest growth rate in young children. Surgery is the only standard treatment option available for PN. However, this is often difficult due to the encasement of vital structures, and extensive and invasive PN growth. PN are composed of neoplastic Schwann cells that lack NF1 gene expression. This results in upregulation of Ras, which initiates several signaling cascades regulating cell proliferation. Selumetinib (AZD6244 hyd sulfate), a novel orally bioavailable mitogen activated protein kinase inhibitor, is a specific inhibitor of MEK 1, which may mediate anti-tumor effects in PN by inhibition of downstream signaling of Ras. Selumetinib is currently undergoing evaluation in adult cancers and children with brain tumors. In this phase I study of selumetinib directed at NF1 PN, we have observed a degree of activity which has not been observed previously; therefore a phase II evaluation will be conducted. Objectives Phase I To determine the maximum tolerated dose (MTD) of oral selumetinib administered daily to pediatric patients with NF1 and inoperable PN. Based on the results of the dose escalation in this study, the current MTD has been determined as 20 mg/m 2 /dose. To be consistent in pediatric dosing, an additional dose level of 25 mg/m 2 /dose was added, which is the MTD recently determined in a study conducted by the Pediatric Brain Tumor Consortium (PBTC). Amendment H (November 2014): The MTD has been determined to be 25 mg/m 2 /dose. To define the acute and chronic toxicities and pharmacokinetics (PK) of selumetinib. Completed with amendment H. To determine the effect of selumetinib on the growth rate of PN. Phase II Primary objectives: To evaluate the confirmed partial and complete response rate (See section 5.2) of selumetinib using volumetric MRI analysis in children and young adults with NF1 and inoperable PN with PN related morbidity at the time of enrollment. Secondary objectives include the evaluation of: o the confirmed partial and complete response rate (See section 5.2) of selumetinib in the overall population of all patients treated (patients with and without PN related morbidity at the time of enrollment), and those with typical PN versus nodular PN versus solitary nodular PN (See section 3.1). o the long-term tolerability and safety of selumetinib o the duration of response o the effect of selumetinib on bone mineral density in patients at the NCI with decreased bone mineral density at the time of enrollment. o the effect of selumetinib on pain, quality of life, physical functioning, and disfigurement o the PN growth rate based on volumetric MRI analysis 3

84 o time to progression (TTP) and progression free survival (PFS) o the effects of selumetinib on the pharmacokinetics (PK) and pharmacodynamics (PD) in peripheral blood bone marrow derived precursor cells and cytokines. Eligibility Pediatric Patients ( 2 and 18 years) who are able to swallow intact capsules, with NF1 and inoperable measurable PN that cause or have the potential to cause significant morbidity. Design Selumetinib will be administered orally BID on a continuous dosing schedule (28 days = 1 treatment cycle). In the phase I portion, limited dose escalations will be performed to define the MTD based on tolerability of selumetinib during the first three treatment cycles. In the phase II portion, the recommended phase II dose level (RP2D) will be administered. Phase II: Patients will be enrolled on one of two strata: o Stratum 1: PN related morbidity present at enrollment PN related pain, disfigurement, or difficulty in physical functioning o Stratum 2: No PN related morbidity present at enrollment Patient reported and functional outcomes will be evaluated at regular intervals. Disease status will be evaluated using volumetric MRI analysis at regular intervals. The day 1 and steady state plasma PK and PD of selumetinib will be evaluated. 4

85 Participating Sites: The Children s Hospital of Philadelphia Principal Investigator: Michael J. Fisher, M.D. Associate Professor of Pediatrics Center for Childhood Cancer Research and Division of Oncology The Children's Hospital of Philadelphia Colket Translational Research Building - 10th Floor 3501 Civic Center Blvd Philadelphia, Pennsylvania Phone# (215) Fax# (215) fisherm@ .chop.edu IRB Contact: Name: Samantha Ferrante, M.S., CIP Title: IRB Analyst II Address: 3535 Market St., Suite 1200 Philadelphia, PA Phone: Fax: ferrante@ .chop.edu Pharmacy Contacts: Amy Kane, Pharm.D and Eileen Wu, Pharm.D, Investigational Drug Service Pharmacists Address: 34th and Civic Center Blvd. Pharmacy Room AW19, Philadelphia, PA Phone: or Fax: investigationalonco@ .chop.edu Cincinnati Children s Hospital: Principal Investigator: Brian Weiss, M.D. Associate Professor Division of Oncology Cincinnati Children's Hospital Medical Center 3333 Burnet Ave, MLC 7015 Cincinnati, OH Phone: (513) Pager: (513) Fax: (513) brian.weiss@cchmc.org Administrative Assistant: Sheila Mosier (513) sheila.mosier@cchmc.org Pharmacy Contact: Denise Lagory, RPh Investigational Drug Pharmacy Cincinnati Children's Hospital Medical Center 3333 Burnet Avenue, MLC B-1011 Cincinnati, OH Phone: (513) Fax: (513) Denise.Lagory@cchmc.orty IRB Contact: Robert Frenck, M.D.; Chair Cincinnati Children's Hospital Medical Center Institutional Review Board 3333 Burnet Avenue, MLC 5020 Cincinnati, OH Phone: (513) Fax: (513) Robert.Frenck@cchmc.org Children s National Medical Center Principal Investigator: AeRang Kim, M.D., Ph.D Center for Cancer and Blood Disorders Children's National Medical Center Address: 111 Michigan Ave, NW Washington, DC Phone: Fax: aekim@childrensnational.org Pharmacy: Investigational Drug Service (IDS) c/o Pharmacy Department, Room 4500 Children's National Medical Center 111 Michigan Avenue, NW Washington, DC ATTN: Sarah Donegan, Pharm.D. Telephone Number: (202) Fax Number: (202) sdonegan@childrensnational.org IRB Contact: Naomi Luban, M.D.; Chair Children s National Institutional Review Board 801 Roeder Road Suite 801 Silver Spring, MD Phone: Fax:

86 Johns Hopkins University Hospital: Principal Investigator: Jaishri Blakeley, M.D. Johns Hopkins University 1550 Orleans Street Suite IM16 Baltimore, MD Phone: Fax: Study Contact:Lindsay Blair, CRNP Address: 1550 Orleans Street Suite IM16 Baltimore, MD Phone: Fax: Pharmacy Contact: Anne DeLisa, PharmD, BCOP Address: Oncology IDS Pharmacy c/o Johns Hopkins Hospital 401 N Broadway, Rm 2430 Baltimore, MD Phone: ; Fax: adelisa@jhmi.edu IRB Contact: Address: Johns Hopkins IRB East Baltimore Campus (Central Office) 1620 McElderry St., Reed Hall - B130 Baltimore, MD Phone: Fax: jhmeirb@jhmi.edu Indiana University School of Medicine 705 Riley Hospital Dr., RI 2606 Indianapolis, IN micjferg@iu.edu Phone: Fax: Study Contact: Cindy Dwight, R.N., N.D. Pediatric Clinical Translational Research Indiana University School of Medicine 699 Riley Hospital Dr. RR 345 Indianapolis, IN cdwight@iu.edu Phone: Fax: Pharmacy Contact: Annette Head, Pharm.D Investigational Drug Services 550 University Blvd. IUSCC Room C2102 Indianapolis, IN ahead@iuhealth.org Phone: IRB Contact: John Baumann, PhD IU Institutional Review Board 980 Indiana Avenue Indianapolis, IN Phone: IRB@iu.edu Indiana University School of Medicine Riley Hospital for Children Principal Investigator: Dr. Kent A. Robertson, M.D., Ph.D Indiana University School of Medicine 705 Riley Hospital Dr., RM 2606 Indianapolis, IN krobert@iu.edu Phone: Fax: Co-Investigator: Michael Ferguson, M.D., M.S. 6

87 TABLE OF CONTENTS A PHASE I/II STUDY OF THE MITOGEN ACTIVATED PROTEIN KINASE KINASE (MEK) 1 INHIBITOR SELUMETINIB (AZD6244; HYD SULFATE) IN CHILDREN WITH NEUROFIBROMATOSIS TYPE 1 (NF1) AND INOPERABLE PLEXIFORM NEUROFIBROMAS (PN)... 1 PRÉCIS INTRODUCTION OBJECTIVES Primary Study Objectives: Secondary Study Objectives: BACKGROUND AND RATIONALE Neurofibromatosis Type I and Plexiform Neurofibromas Imaging and Measurement of Plexiform Neurofibromas Genetics and Biology of NF1 and PN Selumetinib Adult Studies Pediatric Phase I Trial Rationale for proposed pediatric phase I trial of selumetinib in NF1 and PN ENROLLMENT PROCEDURES ELIGIBILITY CRITERIA Inclusion criteria (As of amendment I: Enrollment on the phase I component is complete) Exclusion Criteria SCREENING EVALUATION (SEE APPENDIX III) SCREENING AND REGISTRATION PROCEDURES Screening Process Registration Process IMPLEMENTATION OF STUDY STUDY DESIGN Overall Trial Design Criteria for Dose Escalation Completed with amendment I Definition of Dose Limiting Toxicity (DLT) for phase I and toxicities requiring dose modification for phase II Criteria for Intra Patient Dose Escalation Completed with Amendment I Definition of Maximum Tolerated Dose (MTD) Completed with Amendment I Criteria for Starting Subsequent Treatment Cycles Duration of Treatment DRUG ADMINISTRATION TREATMENT MODIFICATIONS Cardiac Toxicity PHARMACOKINETICS AND PHARMACODYNAMICS Pharmacokinetics: A liquid chromatography/mass spectroscopy/mass spectroscopy (LC/MS/MS) will be used to analyze selumetinib Pharmacodynamics: BLOOD VOLUME FOR RESEARCH STUDIES ON STUDY EVALUATIONS (APPENDIX III)* Evaluations During Therapy

88 3.6.2 Off Therapy Evaluations Long Term Safety Follow Up CONCURRENT THERAPIES CRITERIA FOR REMOVAL FROM TREATMENT OFF STUDY CRITERIA END OF TREATMENT EVALUATION (APPENDIX IIIA/B) SUPPORTIVE CARE DATA COLLECTION AND EVALUATION DATA COLLECTION Case Report Forms RESPONSE CRITERIA TOXICITY CRITERIA STATISTICAL SECTION Subject Accrual Statistics and feasibility for Phase I Statistics and feasibility for Phase II Total Patient Accrual and Enrollment DATA AND CENTER AUDITS Data And Safety Monitoring Plan STORAGE, TRACKING AND HANDLING OF RESEARCH SPECIMENS Sample Data Collection Sample Storage and Destruction HUMAN SUBJECTS PROTECTIONS RATIONALE FOR SUBJECT SELECTION PARTICIPATION OF CHILDREN RISKS AND BENEFITS CONSENT AND ASSENT PROCESS AND DOCUMENTATION DATA REPORTING PATIENT REGISTRATION CASE REPORT FORMS SAFETY REPORTING DEFINITIONS CTEP ADVERSE EVENT REPORTING REQUIREMENTS of ALL SITES NCI Guidance for Reporting Expedited Adverse Events for Multi-Center Trials NCI-IRB Reporting at the Coordinating Center NCI-IRB Reporting of IND Safety Reports PHARMACEUTICAL INFORMATION AZD6244 HYDROGEN SULFATE (NSC ) Selumetinib Administration Selumetinib Toxicity Profile COLLABORATIVE AGREEMENT Agreement Type MULTI-INSTITUTIONAL GUIDELINES IRB APPROVALS AMENDMENTS AND CONSENTS IND ACTION LETTERS OR SAFETY REPORTS DRUG ORDERING REFERENCES APPENDICES

89 11.1 APPENDIX IA: PERFORMANCE STATUS SCALES/SCORES PHASE I/II PROVIDER SIGNATURE MOST RECENT COMPLETED CYCLE#/ DATE APPENDIX IB: BLOOD PRESSURE APPENDIX IIA: PROTOCOL FOR REQUIRED MRI STUDIES OF PN PHASE I/II APPENDIX IIB: PN LOCATION AND CLASSIFICATION FORM APPENDIX IIC: DEXA (ONLY AT NCI) APPENDIX III: REQUIRED STUDY EVALUATIONS Appendix IIIA: Phase I Required Evaluations / Enrollment on phase I complete as of amendment I Appendix IIIB: Phase II Required Evaluations APPENDIX IV: PHOTOGRAPHY OF VISIBLE PN PHASE II APPENDIX V: PLEXIFORM NEUROFIBROMA SYMPTOM CHECKLIST PHASE II APPENDIX VI: TARGET PN LOCATION AND ASSOCIATED MORBIDITIES PHASE II APPENDIX VII: FUNCTIONAL EVALUATION FOR ORBITAL PN OPHTHALMOLOGY EVALUATION PHASE II APPENDIX VIII: AIRWAY FUNCTION SLEEP STUDY PULMONARY FUNCTION TESTS (PFTs) APPENDIX IX: MOTOR/STRENGTH EVALUATIONS PHASE II APPENDIX IXA: ENDURANCE EVALUATION APPENDIX IXB: STRENGTH EVALUATION APPENDIX IXC: RANGE OF MOTION EVALUATION APPENDIX IXD: LEG LENGTH EVALUATION APPENDIX IXE: GROOVED PEGBOARD TEST APPENDIX X: BLADDER AND BOWEL PATIENT QUESTIONNAIRE PHASE II APPENDIX XA: OTHER PN APPENDIX XI: PATIENT-REPORTED OUTCOMES QUALITY OF LIFE, PAIN AND PHYSICAL FUNCTIONING ASSESSMENT MEASURES PHASE II Patient-Reported Outcome (PRO) Questionnaires Administered by Age of Patient and Respondent Phase II Study of Selumetinib in Children with NF1: PRO Checklists APPENDIX XI: PAIN MEDICATION SURVEY: APPENDIX XII: PHARMACOKINETIC WORKSHEET PHASE II APPENDIX XIII: PHARMACODYNAMIC STUDIES PHASE II Appendix XIIIA. Plasma biomarker (cytokine) assays Appendix XIIIB Endothelial Progenitor Cell APPENDIX XIV: PATIENT DAILY MEDICATION DIARY FOR SELUMETINIB PHASE I/II APPENDIX XV: LONG TERM EVALUATION PHASE II APPENDIX XVI: ADHERENCE PHASE I/II Selumetinib Capsule Count Form : Adherence Intervention 30 minutes APPENDIX XVII: DOSING NOMOGRAM FOR SELUMETINIB FOR PHASE I/II PEDIATRIC NF-1 TRIALPHASE I/II APPENDIX XVIII: MEDICATIONS TO AVOID APPENDIX XIX: CTEP MULTICENTER GUIDELINES

90 1 INTRODUCTION 1.1 OBJECTIVES Primary Study Objectives: Phase I To determine the maximum tolerated dose (MTD), extended tolerability, and recommended phase II dose of selumetinib administered orally every 12 hours on a continuous daily schedule for cycles of 28 days with no rest period between cycles in children and adolescents with neurofibromatosis type 1 (NF1) and inoperable plexiform neurofibromas (PN). The MTD will be defined based on toxicities observed during the first three treatment cycles. To study the plasma pharmacokinetics (PK) of selumetinib at baseline and steady state. Phase II To evaluate the confirmed partial and complete response rate (See section 5.2) of selumetinib using volumetric MRI analysis in children and young adults with NF1 and inoperable PN with PN related morbidity at the time of enrollment Secondary Study Objectives: Phase I To determine the effect of selumetinib on the growth rate of PN using automated volumetric MRI analysis. To measure adherence of selumetinib chronic dosing in this patient population. To describe and define the toxicities in pediatric patients on chronic dosing of selumetinib. Phase II To evaluate the confirmed partial and complete response rate (See section 5.2) of selumetinib in the overall population of all patients treated (patients with and without PN related morbidity at the time of enrolment), and those with with typical PN versus nodular PN versus solitary nodular PN (See section 3.1). To determine the long-term tolerability and safety of selumetinib. To determine the duration of response to selumetinib. To evaluate the effect of selumetinib on bone mineral density in patients at the NCI with impaired bone mineral density at the time of enrollment. To characterize the effect of selumetinib on pain, quality of life, and physical functioning. To determine baseline functional impairments secondary to PN, and the effect of selumetinib on functional outcomes depending on PN location. 10

91 To determine the effect of selumetinib on the PN growth rate based on volumetric analysis of MRI studies obtained prior to enrollment (if available and amenable to volumetric analysis). To determine time to progression (TTP) and progression free survival (PFS) in progressive PN ( 20% increase in PN volume within months prior to enrollment), and compare PFS to the placebo arm of the R randomized trial (01-C-0222). To determine day 1 and steady state pharmacokinetics of selumetinib. To evaluate bone marrow derived precursor cells and cytokines pre treatment and on treatment with selumetinib, and to assess if changes in cell and cytokine profiles correlate with imaging response to selumetinib. In patients who enroll on this trial with presence of an optic pathway tumor or other glioma not requiring treatment with chemotherapy or radiation therapy (see Section 2.1.2): To evaluate the effect of selumetinib on changes in the size of the optic pathway tumor or other glioma. 1.2 BACKGROUND AND RATIONALE Neurofibromatosis Type I and Plexiform Neurofibromas Neurofibromatosis type 1 (NF1) is a common autosomal dominant, progressive disorder with an incidence of 1:3500 (>80,000 persons affected in The United States) 1. NF1 is characterized by diverse, progressive cutaneous, neurological, skeletal, and neoplastic manifestations with no standard drug treatment options available. Patients with NF1 have an increased risk of developing tumors of the central and peripheral nervous system including plexiform neurofibromas (PN) (27%), optic gliomas (15-20%), pheochromocytomas (1%), and malignant peripheral nerve sheath tumors (5%) 2,3. PN are benign nerve sheath tumors that grow along the length of nerves and involve multiple branches of a nerve. Many PN are thought to be congenital, but may not be diagnosed until later in life. Early childhood, puberty. and childbearing age in females are considered to be the periods of greatest risk for disease progression 4. In the past, the rate of growth of this histologically benign neoplasm has been described as unpredictable and often episodic 2. However, a recent analysis of PN using volumetric MRI analysis suggests consistent growth rate of PN within a patient with a median observation period of 34 months (see Section 1.2.2) 5 These tumors may cause significant morbidity by disfigurement, as well as compression of vital structures. For example, PN may infiltrate the orbit, displace the globe, and compromise vision; paraspinal tumors may compress the spinal cord and cause paralysis; tumors in the mediastinum may compress the trachea or great vessels; and tumors of the extremities can cause local nerve infiltration, progressive neurologic deficit and often unremitting pain 2,4. A review of 47 children and young adults with NF1 and PN entered on clinical trials at the NCI, Pediatric Oncology Branch showed that 25 patients (53%) underwent surgery of their PN prior to trial entry, 28 patients (60%) were disfigured by the PN, 16 patients (34%) required pain medications, 9 patients (19%) had neurologic symptoms, 5 patients (11%) had airway compromise, and 2 (4%) patients died as a result of PN progression 6. Surgery is the only standard treatment of PN. However, 11

92 up to 44% of tumors progress after the first surgery, most commonly in patients younger than ten years of age with head and neck tumors that could not be completely resected 4. Prior to recent work, the unknown natural history of PN in NF1 and difficulties in measuring changes in size of these complex, large, and slow growing lesions made it difficult to define the benefit of medical treatments for PN. However, a number of medical treatments including thalidomide 7, cis retinoic acid, interferon alfa 2b, methotrexate and vinblastine, PEG interferon alfa-2b, pirfenidone, and the farnesyltransferase inhibitor R have been evaluated or are undergoing evaluation in early clinical trials for patients with NF1 and PN with the goal to reduce the growth rate or shrink these tumors. Published results are available for the phase I thalidomide trial, the phase I and II trials of R115777, pirfenidone, and the phase II trial of sirolimus in non progressive PN 7,8. In the thalidomide trial, 20 patients with NF1 and PN received thalidomide at doses up to 200 mg/day. Response was assessed by clinical measurements and 2-D radiographic measurements. Of 12 patients who completed 1 year of treatment, four showed a <25% reduction in tumor size, and seven showed symptomatic improvement with predominantly a decrease in pain. The phase I trial of the farnesyltransferase inhibitor R included seventeen patients with NF1 and PN. At the MTD of 200 mg/m 2 /dose, R was well tolerated, and patients with NF1 received a median of 10 treatment cycles (range 1-32). No objective tumor shrinkage was observed using conventional response criteria for solid tumors. The NCI POB coordinated a multi-center, double-blinded, placebo-controlled, cross-over trial (01-C-0222) of tipifarnib (R115777) for children and young adults with progressive PN, which has recently been completed. Time to disease progression, measured by volumetric MRI analysism was the primary trial endpoint. The median time to progression (PN volume increase by 20%) for 30 patients randomized to receive placebo as the first treatment was 10.6 months, and for patients randomized to receive tipifarnib as first treatment was 19.2 months (one sided alpha 0.129). Tipifarnib thus did not result in a greater or equal to doubling of the time to disease progression in children and young adults with NF1 and progressive PN. This trial provided information regarding the time to progression of PN in patients who did not receive medical treatments directed at their PN, which is the first time this has been documented. This data is critical and is the basis for time of radiographic evaluation in this trial 9. It also set the foundation for the use of volumetric MRI as a sensitive assessment of tumor in clinical trials. The NCI POB coordinated a phase I trial of the antifibrotic agent pirfenidone. This trial used automated volumetric MRI analysis of PN to determine progression. Of 12 patients entered at the second dose level and phase II dose of 500mg/m 2 /dose TID on a continuous dosing schedule, 9 were removed from the protocol with disease progression after a median of 15 treatment cycles (range 7-21 cycles). Two patients completed 2 years of treatment with stable disease, and 1 patient was removed from the protocol for non-pirfenidone-related medical complications 10. The NCI POB has completed a phase II trial of pirfenidone for patients with progressive PN. The median TTP for patients treated with pirfenidone was 13.2 months and thus similar to the TTP of patients treated with placebo on the tipifarnib trial 9,11. 12

93 Amendment I: NF1 trials update November 2014: A recent analysis of changes in PN volumes in patients with NF1 and PN enrolled on the NCI NF1 natural history study demonstrated that PNs appear to grow linearly over extended periods of time, with younger children having faster PN growth rates than older children 12. This analysis did not demonstrate acceleration of PN growth during puberty 12. Additionally, out of 112 PNs analyzed, 7 demonstrated a spontaneous slow decrease in tumor volume (mean decrease/year: 4.8%, range: %). These PNs were found in older patients (age at max volume: median 17.6 years, range: years) 12. In the phase I trial of sorafenib in children with inoperable PN, dose escalation was terminated due to dose-limiting tumor pain at the starting dose level, and a maximum tolerated dose could not be determined 13. In the Department of Defense NF Clinical Trials Consortium phase II trial of the mtor inhibitor sirolimus for non-progressive NF1 PN, no partial responses (PN volume decrease 20%) based on volumetric MRI analysis were observed 14. Evaluation of the effect of sirolimus in progressive PN also did not demonstrate partial responses, but a modest (3.5 months) increase in the TTP compared to the placebo control arm of the tipifarnib phase II trial was observed 15. In the phase II trial of PEG-intron for NF1 patients with inoperable PN, analysis of patients with progressive PN demonstrated a doubling in the median time TTP compared to tipifarnib (10.6 months) 16. In the phase II trial of the c-kit and PDGFR inhibitor imatinib conducted by Kent Robertson, PN volume decreases ranging from 20 to 40% were observed in 6 of 23 response evaluable patients 17. This trial used a different method of volumetric analysis and responses were only described in very small PN ( 20 ml). In contrast, most target PN enrolled on NCI clinical trials have much larger volumes: Tipifarnib: median PN volume 364 ml, range, ml; pirfenidone: median PN volume 349 ml, range, ml. A recent analysis of the growth rate of PN in patients enrolled on the NCI NF1 natural history study demonstrated different growth characteristics of typical PN and nodular appearing lesions, which can arise within and outside of PN, and lack the central dot sign typical for PN on standard MRI 18. In contrast to typical PN, the nodular neurofibromas do not demonstrate a decrease in growth rate with increasing age, and the nodular lesions are not typically detected in very young children. These findings suggest that the biology of typical PN and nodular neurofibromas may be different, which should be considered in interpreting the results of clinical trials directed at PN. Our analysis of patients with nodular lesions at the NCI demonstrates that most of these lesions are fluoro-deoxy glucose (FDG) avid on FDG-PET. When FDG-PET was used in conjunction with clinical symptoms to target candidates for biopsy, some of these lesions were consistent with either MPNST or atypical neurofibromas (ANF) 19. Atypical neurofibromas show increased cellularity and atypical nuclei, but rarely mitoses. Recently Dr. Eric Legius described that ANF harbor deletions in CDKN2A/B which are also present in MPNST but not PN 20. ANF thus may represent precursor lesions to MPNST. For these reasons we will report response separately for patients with nodular lesions and typical PNs. 13

94 1.2.2 Imaging and Measurement of Plexiform Neurofibromas Tumor response criteria that are used for solid tumors are based on one-dimensional (1-D) and two-dimensional (2-D) tumor measurements 21,22. These methods have limited value in the assessment of treatment outcome for PN, which are frequently large, have a complex (non-spherical) shape, and have a slow growth pattern. In order to reproducibly quantify the size of these complex lesions and detect small changes in the size over time, we used MR imaging characteristics of PN to develop an automated method of lesion detection and volume measurement 23. Short T1-Inversion Recovery 24 MR images, on which PN are bright lesions compared with normal surrounding tissue, were used to develop a program for automated image analysis within MEDx (v3.41) software (Sensor Systems, Inc. Sterling, VA). Reproducibility and inter-observer variability of this automated method were determined by 2 observers who quantified volumes for PN of the orbit (n=2), face/neck (n=3), abdomen (n=1), and pelvis (n=3) on three different days. For each MR Figure 1: Axial MRI of pelvic plexiform neurofibroma. Steps of automated volumetric analysis A-D. A C B D image (Fig. 1A), the tumor is roughly outlined manually including a rim of low signal intensity normal tissue (Fig. 1B). The program then performs a histogram analysis of signal intensity pixel by pixel and a threshold that distinguishes high signal intensity tumor from normal tissue is defined (Fig. 1C). Tumor contours are then determined using a gradient image, connected component analysis and automatic edge following operation (Figure 1D). There is an option for re-analysis of MR images using an average or selected threshold. Tumor volume is calculated by summing the results from all images based on the resulting 2-D contours and slice thickness and a report is generated. For comparison, PN volume was also determined by manually tracing the tumor borders on each MR image. The results of application of the automated method are shown in the table below. Observer 1 Observer 2 Mean tumor volume ml, median (range) 291 ( ) 290 ( ) Inter-day CV %, median (range) 3.6 ( ) 1.6 ( ) Median (range) % difference in volume between observers 6.4 ( ) Correlation automated vs. manual method, R

95 This automated volumetric MRI analysis is applicable to most PN, has excellent intra- and inter-observer reproducibility, and is consistent with volumes determined by manual tumor tracing. This method is currently used at the NCI POB to centrally evaluate PN volume on several multi-center clinical trials for children with NF1 and PN. In these clinical trials tumor progression is defined as an increase in tumor volume by 20%. This percent volume increase for spherical tumors corresponds to much smaller changes in 1-D or 2-D measurements as outlined in the table below. Response Criteria RECIST WHO NF1 PN trials Diameter, 2r Product, (2r) 2 Volume, 4/3 r 3 Progressive disease (% Increase) Shaded areas show current criteria used to define disease progression by RECIST, WHO, and the ongoing clinical trials for NF1 and PN. On these clinical trials, volumetric MRI analysis of PN has been feasible for most tumors. Analysis of patients entered on NCI POB trials showed that the growth rate of PN is variable among patients, but remains constant within patients, and that young children (less than 8 years of age) have a more rapid PN growth rate compared to older children 5. This is demonstrated in the figure below: (A) The relationship between the slopes for percent change in PN volume and percent change in body weight per year. Values above the 45 degree line represent ratios of percent change in PN volume to percent change in body weight which are greater than 1. Closed symbols represent patients the median age (8.3 years) at study entry, and open symbols patients > 8.3 years. Percent change in PN volume (B) and body weight (C) per year as a function of patient age at baseline. The shaded area indicates the 20% change in PN volume required for documentation of disease progression in ongoing clinical trials. The line in B represents an exponential fit to the data. Figure 2: Variable PN growth rate 15

96 The analysis emphasizes the need to develop effective medical interventions for young children with NF1 and PN. In 2013, consensus recommendations for the imaging of PN were issued by an international working group (REiNS) 25 and recommended volumetric tumor assessment as preferable to all other MRI analysis techniques. Efforts are ongoing within the REiNS imaging working group to validate a method of volumetric MRI analysis developed at Massachusetts General Hospital (MGH), which allows volumetric MRI analysis within a shorter duration 23. Using this method, the tumor surface is identified by a dynamic-threshold level set method starting with a seed initiation within the lesion and propagating shell expanding to the boundary. This method has not been used prospectively in clinical trials to date. As the first step we conducted a comparison between the NCI method and the MGH method to assess inter observer variation when applied to complex tumors, and perform a comparison of response evaluations on longitudinal data. In order to test the agreement in detecting change, the two methods were applied to 15 representative tumors of variable sizes (ranging from 88 to 3300 ml), locations (orbit, face, neck, chest, abdomen, pelvis, extremities), levels of complexity, and imaging characteristics (diffuse or well circumscribed). The tumors were measured at 3 time points and the percent change between time points was calculated. Preliminary results of this comparison were presented at a REiNS meeting in December 2014 and demonstrated good agreement with a median % difference in the baseline volume of 6.2%, and a median % difference in the volume for all 45 measurements (15 PN, each analyzed 3 times by an observer at NCI and MGH) of 9.1%. Statistical analysis demonstrated no significant numerical differences in the volumes or in the percent volume change between time points when comparing NCI and MGH results (Wilcoxon signed rank test). Additional analyses are ongoing and a prospective comparison of both methods is planned. We hope that upon completion of the validation, the MGH method will become more widely available for use in clinical trials Genetics and Biology of NF1 and PN The gene responsible for NF1 has been cloned and encodes a protein called neurofibromin 26. Although the function of neurofibromin is not completely understood, it is known to include a GTPase activating protein (GAP) domain that regulates hydrolysis of Ras-GTP to Ras-GDP 27. Histologically, PNs are composed of neoplastic Schwann cells accompanied by a varying number of cellular and non cellular components including fibroblasts, perineural cells, and mast cells. Recent studies have demonstrated that the neoplastic Schwann cells lack NF1 gene expression 28,29. Therefore, loss of neurofibromin is associated with elevated levels of activated Ras. Activated Ras results in the initiation of a cascade of signaling events such as activation of Raf and MAPK that lead to increased cell proliferation. Several clinical trials with targeted agents aiming at reduction of PN growth have been conducted 30. For example, farnesyltransferase inhibitors (FTI) were developed to prevent posttranslational Ras processing and transduction of proliferative signals, which is required for activity of mutant and wild type Ras. Inhibition of Ras activity was the rationale for evaluation of FTIs in cancer and NF1 31. However, N- and K-Ras can undergo an alternate lipid modification by geranyl-geranylation, and thus overcome the effect of FTI 32. This is critical in light of recent data pointing towards K-Ras as a key mediator of Ras activity in 16

97 NF1 tumors 33,34. Thus additional strategies such as novel RAS inhibitors blocking all Ras isoforms 35 or agents blocking downstream pathways of RAS, such as ADZ6244, are being considered for development in NF1. Recently, Lauchle et al showed that in an NF1 mouse model of myeloid leukemia, MEK inhibitors induced objective regression of many Nf1- deficient acute myeloid leukemias 36. This data provides further support for evaluation of MEK inhibitors in NF1 related tumors. NF1 and bony manifestations: NF1 is also associated with multiple skeletal and osseous anomalies including scoliosis, sphenoid wing dysplasia, tibial pseudarthrosis, spinal canal widening, and bone cysts. In addition, several studies have demonstrated impaired bone mineral density in patients with NF This is likely multifactorial and may be due to a combination of 25-hydroxyvitamin D deficiency, chronic disease, poor nutrition, and tumor burden. Neurofibromin has been shown to be an important regulator of bone mineralization, and inactivation impairs osteocyte development 40,41. Several preclinical studies in NF1 mouse models have demonstrated the importance of ERK-dependent signaling in normal bone formation, as well as improved osteoblast differentiation and bone healing with MEK inhibition 41, Selumetinib Selumetinib (AZD6244 hyd sulfate) is an orally bioavailable targeted agent against mitogen-activated protein kinase kinase (MEK) 1, with a concentration which resulted in 50% inhibition (IC50) of approximately 10 to 14 nm in adult tumor models, including breast cancer and non small cell lung cancer (NSCLC) cell lines 43,44. MEK is a critical kinase in the mitogen-activated protein (MAP) kinase signal transduction pathway for many growth factor receptors that provide growth signals to cancer cells. Selumetinib inhibits the activity of isolated MEK to phosphorylate extracellular signal-regulated kinase (ERK) 2 in enzyme assays. Selumetinib is a highly specific inhibitor of the MEK kinases 43. Directly downstream in the MAPK pathway from MEK is ERK. Inhibition of ERK phosphorylation was found in tumors in which growth was inhibited by selumetinib, indicating that tumor phosphorylated ERK (perk) levels are a potential biomarker for selumetinib activity in vivo. Thus, there is good rationale for evaluation of agents targeted at inhibition of the MAPK signaling pathway in NF1 and inoperable PN. Detailed information regarding preclinical and clinical studies with selumetinib is provided in the following pages, and data is directly from Investigator Brochure Preclinical Studies Selumetinib All preclinical information provided in this protocol is from the Investigator s Brochure. In vitro efficacy studies These studies were done using the free-base of AZD6244. The effect of selumetinib on ERK phosphorylation and cell viability was determined in a panel of 10 cells lines, including various tumor types (breast, colorectal, lung, osteosarcoma, ovarian, pancreatic, and prostate), in which the mutational status of BRAF 17

98 and KRAS is known. The potency of selumetinib in inhibiting ERK1 and ERK2 phosphorylation was consistent among cell lines, ranging from to M. A panel of human tumor cell lines were exposed to varying concentrations of selumetinib. The IC50 values ranged from <10nM to >10μM and most of the cell lines were sensitive to selumetinib that contained either a BRAF or Ras gene mutation. N-desmethyl AZD6244, the primary active metabolite of selumetinib, has 3 to 5-fold greater activity of its parents drug, and is a more potent inhibitor of ERK phosphorylation 45. In vivo efficacy studies The pharmacological effects of selumetinib have been studied extensively by examining the anti-tumor activity of selumetinib in different tumors in mice. In one such study, human lung Calu-6 tumor cells were implanted subcutaneously in the flanks of nude mice and the tumors were allowed to grow to approximately 0.2 cm 3. The mice were then treated for 25 days with selumetinib (10, 25 or 100 mg/kg bid oral dosing) or control. Tumor growth was suppressed in all 3 dose groups during the treatment period, with re-growth following treatment withdrawal 46. In a series of similar studies, selumetinib showed significant anti-tumor activity in several tumor models, including human melanomas (LOX and A375v), human breast carcinomas (Zr-75-1 and MDA-MB-231), human pancreatic tumors (BxPC3, AsPC1, HPAC, MIA PaCa-2 and PANC 1), human lung cancer tumors (A549) and human colon carcinomas (HT29, Colon 26 tumors, Colo205, SW620, Lovo and HCT116) 44, However, selumetinib was only minimally active in SK MEL-28 melanoma, PC3 prostate tumors, and H460 lung tumors. Overall, these results demonstrate that selumetinib has strong antitumor activity in multiple non-clinical tumor models. Preclinical studies update as of amendment I (November 2014): Recently, genetically engineered mouse models of NF1 and neurofibroma have become available and are being used to evaluate targeted agents in preclinical trials with the goal of selecting agents for clinical evaluations In collaboration with Dr. Nancy Ratner, the NCI POB is performing volumetric MRI analysis of the neurofibromas in mice in similar fashion to the analyses performed in clinical trials. The first agent in this model to demonstrate decrease in neurofibroma volumes was a MEK inhibitor (PD ) 54, and identical activity was observed over a range of doses ( mg/kg). Dr. Wade Clapp also evaluated selumetinib in his transgenic mouse PN model and observed PN shrinkage with comparable responses at two dose levels tested (2.5 and 10 mg/kg once daily on a continuous dosing schedule) (personal communication, Wade Clapp). Combination studies Selumetinib has been combined with a number of therapeutic agents both in vitro and in vivo including standard cytotoxic drugs such as irinotecan and docetaxel 47, which enhanced efficacy and synergy, and cytarabine 55, which showed enhanced induction of apoptosis in AML cells, as well as novel targeted therapies 56,57 using both KRAS mutant and wild type models. Gefitinib, vandetinib, and taxotere combinations with selumetinib have been tested in the KRAS mutant non-small cell lung cancer model A549a. All combinations are more effective than the respective monotherapies 47,58. The combination of selumetinib and the 18

99 mtor kinase inhibitor AZD8055 achieves a greater anti-tumor effect than the respective monotherapies in all models tested. Calu-6 model combination of selumetinib and cediranib was more effective than either agent alone in inhibiting tumor growth. Pharmacodynamic Markers in Tumor Samples The effects of single doses of selumetinib on the levels of perk in human Calu-6 xenograft tumors have been determined as a measure of selumetinib activity against MEK, and related to plasma levels of selumetinib 47. The levels of perk in the tumor cell cytoplasm and nucleus were determined by immunohistochemical (IHC) staining of formalin-fixed tissue sections, and western blot analysis of tumor protein lysates (Fig. 2). Following an acute dose of 25 mg/kg selumetinib, perk levels decreased by approximately 80% to 90% at 1, 2, and 4 hours post-dose, when plasma concentrations of selumetinib were highest, and recovered to >50% of control levels by 24 h. The same trend is observed by western blot analysis, though the perk signal was consistently slightly higher than determined by IHC analysis. 19

100 Figure 3. Inhibition of ERK phosphorylation by a single 25 mg/kg dose of AZD6244 in Calu-6 human lung cancer xenograft tumor Animal toxicities Preclinical toxicology studies In preclinical toxicology studies, there were differences in the toxicity profiles between the species used (rodents and primates). In rodents, soft tissue mineralization, defined as extracellular deposits of calcium and phosphate crystals in multiple tissues, including cornea, kidney, liver, myocardium, and skeletal muscle, was associated with changes in plasma inorganic phosphate, calcium, and/or albumin. Other rodent specific changes seen included gastrointestinal tract toxicity and hematopoietic atrophy, with associated anemia and reticulocytosis, and inflammatory changes in the liver. In primates, diarrhea was the dose limiting toxicity, which in some animals resulted in dehydration and electrolyte imbalance, which in some animals was associated with renal changes (tubular epithelial swelling and mild vacuolation). Reproductive toxicity was also seen in rodents, with adverse effects on embryofetal development and survival at dose levels that do not induce maternal toxicity. Preclinical pharmacokinetic studies Individual high performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) assays for measurement of selumetinib, AZD6244 N-desmethyl and AZD6244 amide have been developed and validated, as well as combined assays to 20

101 measure selumetinib and N-desmethyl in a single assay, and a combined assay to measure AZD6244, N-desmethyl, and the amide metabolites. All assays used have a coefficient of variation (CV)% <15% across the analytical range. The current preclinical and clinical assay range is μg/ml for selumetinib and μg/ml for N-desmethyl AZD6244 and the amide metabolite. 14 C-labelled AZD6244 and N-desmethyl (AZ ) have been synthesized and used in metabolism studies. In the rat and monkey 1-month toxicity studies performed with AZD6244 free-base, no accumulation of selumetinib was seen with multiple dosing at any dose level. In the mouse and monkey 1-month toxicity studies performed with selumetinib, there remained little or no accumulation of selumetinib with multiple dosing in either species. However, in both species with dosing of selumetinib, selumetinib area under the plasma concentration-time curve from time 0 to 12 h (AUC0-12) increased approximately in proportion to dose. Oral bioavailability following dosing with 5mg/kg of either AZD6244 free base or Hyd- Sulphate in captisol was 29% and 56%, respectively. Toxicokinetic data from the mouse and monkey 26-week studies performed with selumetinib largely supported the data previously seen in the 1-month studies with the salt form. In both species, AUC0-12 increased approximately in proportion to dose, which ranged from 1 to 20mg/kg bid in mouse and 0.5 to 4mg/kg bid in monkey. In the monkey 26 week study, exposure to selumetinib was similar for both males and females. Small increases in selumetinib AUC0-12 values of between 1.5- and 1.9-fold occurred between day 1 and 26, indicating that some accumulation may have occurred. The mean terminal elimination half-life of selumetinib was approximately 6 hours with individual values ranging from 2.95 to 11 hours. Metabolism Metabolism of selumetinib was investigated in vitro using hepatocytes from animals and humans. There was evidence of Phase 1 and 2 metabolism with the majority of metabolites detected as glucuronide conjugates. Phase 1 metabolism included N-demethylation, oxidative defluorination, and loss of the side chain to form amide and acid metabolites. Inhibition studies indicated that CYP 1A2 was the enzyme primarily responsible for the formation of the N-desmethyl metabolite. Using expressed CYPs it was evident that CYPs 2C19 and 3A4 also metabolized selumetinib. Preliminary investigation in vitro using human hepatocytes indicated that selumetinib was a weak inducer of CYPs 3A, 1A and 2C9. At selumetinib concentrations approximately 10-fold higher than those achieved in the clinic, the level of induction was <40% of positive control inducers. N-desmethyl AZD6244 (a pharmacologically active metabolite) was identified to be approximately 3 to 5-fold more active than the selumetinib parent compound. The amide metabolite is up to 50-fold less active than AZD6244, and is therefore unlikely to significantly contribute to biological activity. The N-desmethyl metabolite was not detectable in rat, and was detectable at only trace levels in the monkey, but was produced in mouse at circulating levels around 2-12% of parent compound. However, there was 21

102 evidence that exposure to the metabolite was reduced on multiple dosing. Preliminary unvalidated data indicate that circulating levels of the amide metabolite are detected in both mouse and monkey, and there was a trend towards an increase in exposure on multiple dosing. The plasma concentrations of N-desmethyl metabolite is only about 10% of the parent compound selumetinib. Conversely, the amide metabolite showed increased exposure on multiple dosing indicating some accumulation, which is possibly consistent with a longer terminal half-life 59. However, it was not possible to confirm this. In all species, selumetinib was predominantly associated with the plasma component of blood, and was highly protein bound. In humans, selumetinib was 96.1% protein bound, and N-desmethyl AZD6244 was 96.4% protein bound. Selumetinib was metabolized by CYPs 1A2, 2C19, and 3A4 and glucuronidation appeared to be a major clearance mechanism for selumetinib and Phase 1 metabolites. Selumetinib was a weak direct inhibitor of CYP2C9; IC μm. N-desmethyl AZD6244 was a weak inhibitor of CYP1A2; IC μm. Toxicities in different species Pre-clinical safety evaluation studies conducted using AZD6244 free-base included acute (single or bid dosing on a single day) and 1-month repeat-dose studies in rats and cynomolgus monkeys, in vitro and in vivo genetic toxicology studies (including mouse bone marrow micronucleus assays), and a battery of safety pharmacology studies (assessing behavioral, cardiovascular, respiratory and gastrointestinal function/tolerability). Pre-clinical safety evaluation studies were also conducted with selumetinib and included an in vivo genetic toxicology study (mouse micronucleus assay), an in vitro phototoxicity test, repeat-dose studies of up to 6 months duration in mice and cynomolgus monkeys, and reproductive toxicology studies in mice (assessing fertility, early embryonic development, and embryofetal development). Preclinical animal studies showed that dosing with selumetinib enhanced systemic exposure to selumetinib, in comparison to free base. Acute studies In the rat, single oral dosing was well tolerated with no adverse effects and the NOEL was 300 mg/kg. Two doses of selumetinib given 12 hours apart were well tolerated in the monkey, but produced transient mild elevations in serum enzymes (ALT, AST, LDH) at the highest dose tested. These changes resolved by Day 14 post-dose and were not associated with any microscopic histopathological findings. Chronic (repeat dose) studies Dosing of AZD6244 free-base or selumetinib for up to 1-month was associated with soft tissue mineralization (calcium) in rats and mice. In rats, dosing with AZD6244 free-base produced gastric mucosal mineralization. In mice, dosing with selumetinib for up to 1- month produced more widespread tissue mineralization, with multiple organs affected (cornea, kidney, liver, myocardium, skeletal muscle, glandular stomach), and with associated changes in plasma inorganic phosphate and albumin, as well as with changes in calcium. While changes in plasma inorganic phosphate, calcium, and albumin were seen in a 6-month mouse study with selumetinib, tissue mineralization was restricted to minimal multifocal mineralization in the liver, which occurred in only a small number of 22

103 animals. Dosing of selumetinib to cynomolgus monkeys for up to 6-months was not associated with soft tissue mineralization or changes in plasma inorganic phosphate, calcium, or albumin. In repeat-dose toxicity studies, administration of AZD6244 free-base was associated with soft faeces in rats and mineralization in monkeys, which was exacerbated by the dosing vehicle sulpha butyl ethyl β cyclodextrin (SBE-CD). In monkeys, persistent diarrhea resulted in several secondary changes, including dehydration, serum chemistry abnormalities, and renal tubular microscopic changes. SBE-CD is known to cause gastrointestinal disturbances in rats and dogs, and reducing the total volume of SBE-CD given to each monkey during the study reduced the occurrence and severity of mineralization, most notably in the control and low dose selumetinib groups. In studies with selumetinib in cynomolgus monkeys using lower volumes of SBE-CD, the dose limiting toxicity (DLT) was again due to gastrointestinal disturbance (fluid faeces) but this was not associated with any gastrointestinal tract pathology in the 1- and 6-month studies. In mice, dosing with selumetinib for up to 6 months produced gastrointestinal tract toxicity and haematopoietic atrophy, with associated anaemia and reticulocytosis. Dosing with selumetinib for up to 6 months was also associated with inflammatory changes in the liver, with hepatocellular necrosis and vacuolation seen at very high, non-tolerated doses on a 7-day dose setting study. In male mice, dosing of selumetinib for >8 to 15 weeks produced vascular engorgement of the corpus cavernosum of the bulbocavernosus muscle, which in some animals resulted in obstruction of the urinary tract and back flow pressure effects leading to premature death. The mechanism underlying these changes in mice is unknown; similar changes were not seen following dosing of selumetinib for 6 months in cynomolgus monkeys. With the exception of the tissue mineralization seen in rats and mice, the changes showed evidence of reversibility on cessation of dosing or following a 1- or 3-month recovery period. Preclinical Genetic Toxicity In preclinical studies, selumetinib showed no evidence of genotoxicity in vitro in bacteria or mammalian cells, but induced micronuclei in the bone-marrow of mice. Centromeric staining has shown that the induced micronuclei are primarily a consequence of aneugenicity, and this is entirely consistent with disruption of normal spindle function as a consequence of the known pharmacological action of selumetinib. Preclinical Reproductive Toxicity Reproductive toxicology data indicate that selumetinib can have adverse effects on embryofetal development and survival at dose levels that do not induce maternal toxicity and that do not adversely affect male or female fertility. The effects on embryofetal development and survival are considered to be a consequence of the pharmacological action of the selumetinib. 23

104 Carcinogenicity studies Rodent carcinogenicity studies have not been required to support the adult advanced cancer indications. However, an assessment of carcinogenic potential in rodents will be undertaken to support the paediatric development in NF Adult Studies Below are the summaries of trials using selumetinib capsules, which have replaced the suspension formulation. Adult phase 1 A Phase I, open label, multi-center study to assess the safety, tolerability and pharmacokinetics of selumetinib in patients with advanced solid refractory malignancies for which no standard therapy exists has been completed. Efficacy assessment in this study was an exploratory endpoint. The study was conducted in two parts. Part A (31 patients) of the study was a dose escalation study, and was designed to provide adequate tolerability, safety, pharmacokinetic, and pharmacodynamic data. In Part A, the first cohort received a single 25 mg dose of the capsule formulation of selumetinib on Day 1, followed by twice daily dosing from Day 2 onwards. Other doses investigated were 50 mg, 75 mg, and 100 mg. The aims of Part B (29 patients randomized) were to determine the relative oral bioavailability of the selumetinib capsule, and secondly to expand the safety, tolerability, and preliminary efficacy profile of the MTD from Part A (75mg BID). In Part B, patients received either a single dose of capsule formulation or free-base suspension formulation on Day 1 and 8, followed by continuous twice daily dosing of capsule formulation from Day 9 onwards. Selumetinib was absorbed relatively quickly across all dose levels, with a median Tmax of 1.5 hours. Following the peak, selumetinib concentrations declined multi-exponentially, with a mean T1/2 ranging from 5 to 7 hours, which is consistent across dose levels. CL/F and Vss/F also remained largely consistent across the dose range, with mean values ranging from 12 to 23 L/h and 87 to 126 L, respectively. Plasma N-desmethyl AZD6244 concentrations followed a similar pharmacokinetic profile to selumetinib, although exposure was much lower, with Cmax and AUC values generally <15% of parent, within each patient. The median Tmax was around 1.5 hours and T1/2 was around 9 to 13 hours. There was minimal accumulation after single versus multiple twice daily dosing. Limited data are available for AZD6244 amide. Concentrations of this metabolite are very variable, both between subjects and within the same subject on different study days. The time to maximum concentration was much more variable than either parent or the N-desmethyl metabolite, ranging from 0.5 to 24 hours across patients. Although there was insufficient data to calculate a terminal half-life, the available data suggest this is longer than that of either selumetinib and N-desmethyl. Dose-limiting toxicities (DLT) were grade 3 acneiform rash and pleural effusion. There was only 1 Grade 4 event in the study, an event of hypoglycemia. Fatigue (65.7%) and acneiform dermatitis (60.0%) were the most frequent adverse events at the MTD, with resolution of fatigue upon discontinuation of treatment. A patient with metastatic melanoma bearing a V600E BRAF mutation achieved a complete response persisting after 24

105 15 months of therapy. Fifty-five patients had RECIST evaluable tumors. At the 75 mg dose, 16/35 (45.7%) patients had stable disease for 6 weeks. One complete response was reported in a 30-yearold female patient with a BRAF mutation positive malignant melanoma receiving 75 mg twice daily of the selumetinib capsule in Part A, which is ongoing at two years of therapy with selumetinib. Nine patients in Part A and 13 patients in Part B had a best response of stable disease. Ten out of 55 (18.2%) patients (not including the patient who had a CR) had stable disease of 16 weeks. Seven patients in Part A and 12 patients in Part B had a best response of progressive disease, and 10 patients in Part A and 3 patients in Part B were not evaluable for response. The MTD was 75mg BID

106 Summary of Pharmacokinetic Parameters for AZD6244 (selumetinib) Banerji et al. Clin Cancer Res 2010;16: A Phase I, open-label, multi-center study was conducted to assess the safety, tolerability, and pharmacokinetics of selumetinib in patients with advanced solid tumors when given in combination with the following chemotherapies: docetaxel, dacarbazine, erlotinib, temsirolimus. The primary objective was to define the MTD of selumetinib when administered twice daily with the afore mentioned chemotherapies. One hundred-forty patients were enrolled, and selumetinib could be safely combined at a dose of 75 mg BID (docetaxel, dacarbacine, temsirolimus) or 100 mg BID (erlotinib) with the chemotherapy agents listed. A Phase I, open-label study to assess the effect of dosing selumetinib in the presence and absence of food in patients with advanced solid malignancies was conducted. Thirty-one patients were randomized and received a single dose of 75 mg selumetinib taken with food on Day 1, followed by a single dose of 75 mg fasted on Day 8 or vice versa, 26

107 followed by BID dosing of 75 mg selumetinib from Day 10. Cmax and AUC were reduced by 62% and 19% under fed conditions compared with fasting conditions. The time to reach maximum concentration was delayed by approximately 3 hours following administration of 75 mg of selumetinib in the presence of food. Mean increases in serum liver transaminases (ALT and AST), ALP and phosphate were generally observed during treatment with selumetinib. The majority of reported enzyme elevations (ALT, AST and ALP) occurring prior to disease progression either remained within normal limits or were a maximum increase of 1 CTCAE grade. The small increases in phosphate observed were generally towards the ULN. Best overall response was defined as the best response recorded during the study, based upon the calculated response of target lesions, the investigator s opinion of changes in nontarget lesions and the appearance of new lesions. Of the 28 patients with RECIST evaluable tumors, 2 partial responses (7.1%) were observed. Both patients had skin/soft tissue tumors (melanoma) and were noted to still be alive and continuing study treatment when last restaged. In addition, 5 (17.9%) patients had stable disease for >100 days. A phase I single institution open label, dose-escalation trial evaluating the safety and tolerability of selumetinib and IMC-A12 in subjects with advanced solid malignancies has completed. There were 30 evaluable subjects and the RP2D was selumetinib 50 mg bd + IMC-A12 12 mg/kg D1 and D15 q28d 60. Study D1532C00086 Preliminary data (study D1532C00086) suggests that subjects of Asian descent may experience a higher exposure of selumetinib than the majority of subjects of non Asian descent receiving an equivalent dose of selumetinib. It is recommended that investigators take this information into consideration when dosing patients of Asian descent. If, during the course of the selumetinib development program the dosing regimen being evaluated in this study is found not to be tolerated in a specific ethnic group, this ethnic group may later be excluded from this study. Investigators will be notified, and the protocol will be amended to reflect such findings. The data so far do not suggest a safety concern in any specific population. Adult phase II A Phase II, double-blind, randomized study to assess the efficacy of selumetinib in combination with Dacarbazine compared with Dacarbazine alone as first line in patients with BRAF mutation positive advanced cutaneous or unknown primary melanoma was conducted (D1532C0006). 77 patients were randomized, and selumetinib 75mg or placebo was given twice daily in combination with dacarbazine. This Phase II study showed an improvement in OS that did not reach statistical significance (HR 0.93, 80% CI [0.67, 1.28], 1-sided p-value , 66 events). The median OS was 424 days (13.9 months) in the selumetinib+dacarbazine group compared to 321 days (10.5 months) in the placebo+dacarbazine group. The addition of selumetinib to dacarbazine produced a statistically significant improvement in PFS as compared with dacarbazine alone (HR 0.63, 80% CI [0.47, 0.84], 1-sided p-value 0.021); equating to a 37% reduction 27

108 in the risk of progression. Proportional hazards were observed. The sensitivity analyses performed were consistent with the primary analysis for PFS. The safety analysis set included 89 patients who received at least 1 dose of selumetinib/placebo in combination with dacarbazine (44 in the selumetinib+dacarbazine group and 45 in the placebo+dacarbazine group). The median actual duration of selumetinib/placebo treatment was longer in the selumetinib+dacarbazine group as compared with the placebo+dacarbazine group (175 vs 105 days). The combination of selumetinib 75 mg bid with dacarbazine 1000 mg/m2 was less well tolerated than placebo+dacarbazine. However, the safety profile of the combination was generally consistent with the individual monotherapy profiles, and it is considered that the majority of AEs may be managed in accordance with routine clinical practice. A Phase II, double-blind, randomized, placebo-controlled study (D1532C00016) was conducted to assess the efficacy of selumetinib in combination with docetaxel compared with docetaxel alone, in second line patients with KRAS mutation positive locally advanced or metastatic non small cell lung cancer (Stage IIIB-IV). Selumetinib 75mg or placebo was administered twice daily in combination with docetaxel. A total of 86 patients received randomized treatment. Although statistical significance was not achieved for OS (HR 0.80; 80% CI 0.56, 1.14; one-sided p=0.21), a numerically greater median OS of 9.4 months in the selumetinib+docetaxel group compared with the placebo+docetaxel group (5.2 months) was reported. There was a statistically significant improvement in PFS (HR 0.58; 80% CI 0.42, 0.79; one-sided p=0.014). Median PFS was 5.3 months in the selumetinib+docetaxel group compared with 2.1 months in the placebo+docetaxel group. A total of 16 (37%) patients in the selumetinib+docetaxel group, and none in the placebo+docetaxel group (p<0.0001), had an objective response 61. Adult phase III AstraZeneca is currently conducting three phase III studies in adults in 1 st line uveal melanoma, 2 nd line KRAS mutant non-small cell lung cancer in combination with docetaxel, and in radio-iodine refractory differentiated thyroid cancer. In addition, there is a large externally sponsored program which includes over 30 different studies in multiple indications Pediatric Phase I Trial The Pediatric Brain Tumor Consortium (PBTC) conducted a phase I trial in children ages 3 to 21 years with histologically confirmed diagnosis of low grade gliomas (WHO Grades I & II), including patients with pilocytic astrocytoma; astrocytoma, low grade (Fibrillary astrocytoma); astrocytoma, low grade (Low-grade Astrocytoma, NOS); and optic pathway glioma. This trial has completed the primary objective, and the MTD is 25 mg/m 2 /dose BID 62. Preliminary activity has been observed with 8/38 sustained responses (1 complete and 7 partial). Of these, 5 had biology data: 3 had BRAF fusion, 1 had BRAFV600E mutation and 1 was negative for both. A phase II trial is ongoing. 28

109 1.2.7 Rationale for proposed pediatric phase I trial of selumetinib in NF1 and PN Selumetinib is a rational agent for development in NF1 and inoperable PN (see Section and Section 1.2.4). We are conducting a separate phase I study of selumetinib in children with NF1 and PN for the following reasons: 1. Need to identify a long-term tolerable dose because of the potential of increased incidence of toxicities identified with longer treatment There is limited information whether prolonged administration with selumetinib results in additional or cumulative toxicities. In adult phase I and II studies, a small population received therapy for greater than 6 months. Since medical treatment is not standard of care for PN in NF1, these patients are generally not pretreated with cytotoxic therapy. Therefore, they will likely be a healthier population than children with refractory cancers enrolled in phase I trials, and may be able to acutely better tolerate selumetinib. However, this must be balanced by the likelihood that they will be on selumetinib for a longer duration than children with solid and hematologic malignancies. In the R phase I trial for children with refractory solid tumors or NF1 and inoperable PN, children with solid tumors received a median of 1 cycle (range 1-4 cycles) of R compared to 10 cycles (range 1-32) for children with NF1. Thus, the definition of a dose that will be tolerated in children with NF1 for an extended time period is an important goal prior to the initiation of a phase II trial. It is very unlikely that this information can be obtained from pediatric patients with solid tumors because of limitations by progressive disease, prior therapy, and overall health status. For the farnesyl transferase inhibitor R115777, the MTD and DLT were identical for NF1 and refractory cancers 31. However, sorafenib was less well tolerated in children with NF1 compared to those with cancer 13, but all toxicities were reversible. 2. Differences in patient characteristics and tolerable dose of selumetinib between children with refractory cancers and NF1 and PN The natural history of PN is less likely to be imminently life threatening, and PN are generally slow growing tumors 5,63. Children with NF1 are less likely to accept acute or chronic, mild to moderate toxicities in comparison to children with acutely life threatening illnesses. In addition, children with NF1 enrolled in phase I studies are generally younger than children with refractory malignancies enrolled in phase I studies, with a median age of 8 years compared to 13 years for cancer patients (cumulative data from NCI POB originated phase I trials) 6. Therefore, toxicities specific to selumetinib and pharmacokinetics may be different for children with NF-1 than children and adults with refractory solid tumors. 3. Necessity to evaluate secondary endpoints in pilot fashion in an institution trial prior to development of a large multi-center phase II study. a. Adherence to medication has been the focus of studies of individuals with HIV 64,65, diabetes 66, asthma 67, and a variety of other chronic conditions. To date, this topic has not been explored in the literature among individuals with NF1. However, assurance of a patient s adherence is essential in determining the efficacy of a medication, especially in clinical trials. Research on adherence among children and adolescents should be considered in the context 29

110 of the family. Environmental factors related to the child and his or her parents can impact a child s adherence to medication. Common barriers to adherence include forgetting to take the medication 67, wanting to avoid side effects, and issues surrounding the child s level of responsibility for treatment, including medication administration 64. We will use tools such as pill counts and adherence diaries to evaluate the level of adherence to selumetinib and to determine the specific barriers to adherence that will aid in the development and implementation of selumetinib for patients with NF1 and PN. In conclusion, selumetinib is currently undergoing evaluation in adults with a variety of malignancies including phase I, II, and III studies. In the clinical trials with selumetinib mix and drink formulation, adverse events of diarrhea, and nausea and vomiting have been reported frequently. Most cases of diarrhea started within the first 2 weeks of treatment, whereas the onset of nausea and vomiting was variable. However, the majority of episodes have been self-limiting or easily managed with anti-emetic and anti-diarrheal medication. Skin rash was reported as the most common adverse event, grade 3 being frequent, requiring dose holidays or reductions. Other toxicities reported included fatigue, edema, and dyspnea. Adverse events relating to visual function have been reported in most, but not all studies. There were no specific clinical findings reported in patients that underwent an ophthalmological evaluation after reporting a visual disturbance adverse event. No adverse events of urinary retention or priapism have been reported in any Phase I or II study with patients receiving selumetinib Update on pediatric trial in NF1: As of amendment I (December 31, 2014): Twenty-four patients (13 M:11 F, median age 10.9 years, range 3-18) with a median target PN volume of 1634 ml (range 47-10,269 ml) have enrolled. Dose level 2 (30 mg/m 2 /dose) exceeded the MTD with dose-limiting toxicity (DLT) in 2/6 patients: grade (gr) 3 creatine kinase (CK) elevation (n=1), and gr 3 decrease in left ventricular ejection fraction (n=1). DL1 (20 mg/m 2 /dose) was tolerated with DLT in 2/12 patients: gr 3 cellulitis (n=1), and grade 3 urticaria (n=1). All DLTs were reversible. No other patients developed decrease in LVEF. The DL1 dose of 20 mg/m 2 /dose is similar to the MTD of 25 mg/m 2 /dose determined in the Pediatric Brain Tumor Consortium (PBTC) study of selumetinib for low grade gliomas. For consistency in treating pediatric patients, we therefore evaluated the PBTC MTD of 25 mg/m 2 /dose in the NF1 PN trial as dose level 1.5. Of six patients enrolled at this dose level, one patient experienced a grade 3 rash as DLT during cycle 2. No other DLTs were observed during cycles 1-3, and the 25 mg/m 2 dose level is thus the MTD and the recommended phase II dose for children with NF1 and PN. The most frequent selumetinib toxicities (all grades) are acneiform rash, asymptomatic CK elevation, nausea, vomiting, abdominal pain, diarrhea, and fatigue. Five patients developed paronychia that was deemed possibly related to selumetinib. Of these, 2 had grade 1 and 3 experienced grade 2. One patient enrolled on the phase I selumetinib trial developed asymptomatic grade 1 cataract in both eyes, not interfering with vision, which were not appreciated on prior exams. The patient had previously been treated with imatinib and retinoic acid, both of which are associated with cataracts. This patient also experienced clinical benefit with reduction in PN related pain; celebrex, which was administered at trial entry for PN related 30

111 pain, could be discontinued on treatment. As the patient derived clinical benefit, selumetinib was continued. A review by AstraZeneca of development of cataract in studies with selumetinib identified 4 cases, all of which had confounding factors (>60 years old, hypertensive, previous exposure to steroids). Based on the information in the most relevant clinical studies, cataract was reported for very few patients on selumetinib therapy, and due to the presence of alternative risk factors like hypertension, older age and steroid therapy, a causal link with selumetinib could not been established. Decreased LVEF was seen in only one patient, who did not experience recurrent decreased in LVEF after dose reduction. Reversible, asymptomatic reductions in LVEF have been reported in a small number of patients receiving selumetinib in studies with scheduled echocardiography assessments. Evidence of reversibility of LVEF changes, often without therapeutic treatment, while continuing selumetinib, has been demonstrated in patients with follow-up assessments available. Preliminary median (range) selumetinib C1 day 1 PK parameters were: AUC0-24h DL1 (n=8) 2118 ( ) ng h/ml, DL2 (n=5) 2702 ( ) ng h/ml; half-life DL1 6.8 h ( ), DL2 7.6 h ( ). All of the 24 patients enrolled have had at least one restaging MRI to date. Twelve of 24 patients had a confirmed partial response (8 of 12 at DL1, 2 of 6 at DL2, and 2 of 6 at DL 1.5). In addition, 1 patient at DL2 has an unconfirmed PR, and 3 patients at DL 1.5 have an unconfirmed PR with maximal decrease in PN volume at their most recent evaluation. The overall confirmed and unconfirmed PR rate is thus 66.6% (16/24 patients). The remaining patients had minor decreases in PN volume, but did not achieve a partial response. The maximum PN volume decrease observed was 46%. Responses have been maintained in patients who did not require dose reductions for toxicity. No patient has experienced progressive disease to date. One patient was removed after 2 years of treatment without a response to selumetinib. One patient was removed from treatment with stable disease after 2 dose reductions for DLT (grade 3 CPK). One additional patient withdrew from the study after 5 cycles. This patient had not experienced DLT or a response. Currently, the median cycle number is 18 (range, 6-43). Pain, quality of life (QOL), and function have not been evaluated prospectively on this trial, but several patients reported a decrease in pain, reduced need for pain medication, and several patients reported a functional improvement. Details regarding enrollment of patients and DLTs observed during cycles 1-3 and after cycle 3 are provided in the table below: 31

112 Dose Pt. Patients Grade 3 DLT (N) (mg/m 2 ) # evaluable CY CPK gr 3 (n=1), LVEF decrease gr 3 (N=1) Infection (N=1), Urticaria (N=1) 25 MTD Maculo-papular rash (N=1) DLT after Cy 3 Cellulitis gr 3 Cy 15 (N=1) CPK gr 3 Cy 40 (N=1) Cataract gr 1 (C 16 (N=1) CPK gr 3Cy 11 (N=1) CPK gr 3Cy 6 (N=1) Mucositis gr 2 Cy 8 and gr 3 Cy 16 (N=1) Mucositis: gr 2 Cy 8 and grade 3 Cy 16 (N=1, 2 dose reductions same patient) Mucositis gr 2 Cy 9 (N=1) Selumetinib thus has preliminary activity in children and adolescents with NF1 PN at doses as low as 50% of the adult recommended dose (75 mg BID) and a phase II trial is in development to assess objective response rate by 3D MRI, as well as pain, QOL, and functional changes prospectively. - 32

113 Figure 4: Median Progression Free Survival in Progressive Plexiform Neurofibromas in NF1 Enrollment on our trial was limited to patients 18 years old. However, 4 patients were years old at the time of enrollment, and 3 of them had a partial response, as described below suggesting that selumetinib may be able to shrink PN in older patients. Patient 1, an 18.5 year old has had a partial response (maximal shrinkage 45.9% at 31.8 months) at a dose of 20mg/m2. Patient 4, a 17 year old, had a partial response with maximal shrinkage 34.4% in 23.7 months at a dose of 30mg/m 2, with anecdotal improvement in pain and disfigurement. Patient 6, a 17.4 year old, had stable disease and clinical improvement in pain, and is now off-study (dose 30 mg/m 2 ). Patient 7, a 17.9 year old, had a partial response with maximal shrinkage 24% at 4.6 months with a dose of 30mg/m 2. Figure 4 demonstrates progression free survival of those nine patients with progressive PN at enrollment in comparison to previous treatment trials directed at progressive PN in NF Rationale for proposed phase II expansion cohort: The degree of activity observed in this phase I trial has not been observed in any prior PN trials utilizing other agents. The goal of the expansion is to more fully evaluate the efficacy and safety of selumetinib in patients with NF1 and PN. Patients will also be carefully monitored for long-term tolerability of selumetinib. Because of the absence of agents with documented activity in large PN, and in order to allow enrollment in the timeliest fashion, this trial will be expanded with a phase II amendment. Important secondary objectives include evaluating the effect of selumetinib on patient reported outcomes (pain and quality of life) and functional outcomes, as well as biologic markers. 33

114 2 ENROLLMENT PROCEDURES 2.1 ELIGIBILITY CRITERIA Inclusion criteria (As of amendment I: Enrollment on the phase I component is complete). 1. Age Phase I: 3 years and 18 years of age at the time of study enrollment, if able to swallow whole capsules. The age limits including young children were chosen because early childhood and puberty are considered to be the greatest risk for disease progression, and selumetinib may provide the most benefit to this young group of patients. In addition, an important objective of this study is to characterize the pharmacokinetics of selumetinib in the pediatric population since it has been well studied in adults. Age Phase II: 2 and 18 years. BSA 0.55 m 2, and able to swallow whole capsules. 2. Diagnosis: Patients with NF1 and inoperable PN, defined as PN that cannot be surgically completely removed without risk for substantial morbidity due to encasement of, or close proximity to, vital structures, invasiveness, or high vascularity of the PN. The PN has to cause significant morbidity, such as (but not limited to) head and neck lesions that could compromise the airway or great vessels, brachial or lumbar plexus lesions that could cause nerve compression and loss of function, lesions that could result in major deformity (e.g., orbital lesions) or are significantly disfiguring, lesions of the extremity that cause limb hypertrophy or loss of function, and painful lesions. Patients will be enrolled into stratum 1 or 2 based on PN related morbidity (as defined in Section 2.2). Histologic confirmation of tumor is not necessary in the presence of consistent clinical and radiographic findings, but should be considered if malignant degeneration of a PN is clinically suspected. A PN is defined as a neurofibroma that has grown along the length of a nerve and may involve multiple fascicles and branches. A spinal PN involves two or more levels with connection between the levels or extending laterally along the nerve. In addition to PN, all study subjects must have either positive genetic testing for NF1 or have at least one other diagnostic criterion for NF1 listed below (NIH Consensus conference 68 ): Six or more café-au-lait macules ( 0.5cm in prepubertal subjects or 1.5 cm in post pubertal subjects) Freckling in axilla or groin Optic glioma Two or more Lisch nodules A distinctive bony lesion (dysplasia of the sphenoid bone or dysplasia or thinning of long bone cortex) A first-degree relative with NF1 34

115 3. Measurable disease: Patients must have at least one measurable PN, defined as a lesion of at least 3 cm measured in one dimension. Patients who underwent surgery for resection of a PN are eligible provided the PN was incompletely resected and is measurable. Phase II: Measurability and suitability for volumetric MRI analysis of the target PN must be confirmed with the NCI POB prior to enrolling a patient. The target PN will be defined as the clinically most relevant PN, which has to be amenable to volumetric MRI analysis. PN will be classified as typical PN versus nodular PN versus solitary nodular PN prior to enrollment (See section 3.1) (Appendix IIB). 4. Prior Therapy: Patients with NF1 will only be eligible if complete tumor resection is not considered to be feasible without substantial risk or morbidity. Since there is no standard effective chemotherapy for patients with NF1 and PN, patients may be treated on this trial without having received prior medical therapy directed at their PN. Since selumetinib is not expected to cause substantial myelosuppression, there will be no limit to number of prior myelosuppressive regimen for PN or other tumor manifestations associated with NF1 such as optic glioma. Patients who have received previous investigational agents or biologic therapy, such as tipifarnib, pirfenidone, Peg-Intron, sorafenib, imatinib or other targeted therapies are eligible for enrollment. At least 4 weeks must have elapsed since receiving medical therapy directed at the PN. Patients who received prior medical therapy for their PN must have recovered from the acute toxic effects of all prior therapy to grade 1 CTCAEv4 before entering this study. Growth factors that support platelet or white cell number or function must not have been administered within the 7 days prior to enrollment. At least 6 weeks must have elapsed prior to enrollment since the patient received any prior radiation therapy. At least 4 weeks must have elapsed since any surgeries, with evidence of good wound healing. 5. Performance status: Patients 16 years of age must have a Karnofsky performance level of 70%, and children < 16 years old must have a Lansky performance of 70% (Appendix I). 6. Hematologic Function: Patients must have an absolute neutrophil count 1500/µl, hemoglobin 9g/dl, and platelets 100,000/µl. 7. Hepatic Function: Patients must have bilirubin within 1.5 x the upper limit of normal for age, with the exception of those with Gilbert syndrome, and AST/ALT within 3 x upper limit of normal. 35

116 8. Renal Function: Patients must have a creatinine clearance or radioisotope GFR 60ml/min/1.73 m 2 or a normal serum creatinine based on age, described in the table below. Age (years) Maximum Serum Creatinine (mg/dl) <age <age > Cardiac Function: Normal ejection fraction (ECHO or cardiac MRI) 53% (or the institutional normal; if a range is given then the upper value of the range will be used); QTcF 450 msec. 10. Adequate Blood Pressure defined as: A blood pressure (BP) the 95th percentile for age, height, and gender measured as described in (Appendix IB). Adequate blood pressure can be achieved using medication for treatment of hypertension. 11. Informed Consent: Diagnostic or laboratory studies performed exclusively to determine eligibility for this trial must only be done after obtaining written informed consent from all patients or their legal guardians (if the patient is <18 years old). When appropriate, pediatric patients will be included in all discussions. This can be accomplished through one of the following mechanisms: a) the NCI POB screening protocol, b) an IRB-approved institutional screening protocol, or c) the study-specific protocol. Documentation of the informed consent for screening will be maintained in the patient s research chart. Studies or procedures that were performed for clinical indications (not exclusively to determine eligibility) may be used for baseline values even if the studies were done before informed consent was obtained. 12. Willingness to avoid excessive sun exposure and use adequate sunscreen protection if sun exposure is anticipated. 13. Willingness to avoid the ingestion of grapefruit and Seville oranges (as well as other products containing these fruits, e.g. grapefruit juice or marmalade) during the study, as these may affect selumetinib metabolism Exclusion Criteria 1. Pregnant or breast-feeding females are excluded due to potential risks of fetal and teratogenic adverse events of an investigational agent. Pregnancy tests must be obtained prior to enrollment on this study in girls age 9 or older. Males or females of reproductive potential may not participate unless they have agreed to use an effective contraceptive method. Abstinence is an acceptable method of birth control. 36

117 2. Phase I: Patients who anticipate the need for surgical intervention within the first three cycles (3 months), as surgical intervention during the period of DLT evaluation may affect analysis of adherence and/or make the subject inevaluable. 3. Use of an investigational agent within the past 30 days. 4. Ongoing radiation therapy, chemotherapy, hormonal therapy directed at the tumor, immunotherapy, or biologic therapy. 5. Any evidence of severe or uncontrolled systemic disease, active infection, active bleeding diatheses, or renal transplant, including any patient known to have hepatitis B, hepatitis C, or human immunodeficiency virus (HIV) will be excluded. Patients with HIV who have adequate CD4 count, not requiring antiretroviral medication, may be enrolled. 6. Patients who, in the opinion of the investigator, may not be able to comply with the safety monitoring requirements of the study. 7. Inability to swallow capsules, since capsules cannot be crushed or broken. 8. Inability to undergo MRI and/or contraindication for MRI examinations following the MRI protocol (Appendix II). Prosthesis or orthopedic or dental braces that would interfere with volumetric analysis of target PN on MRI. 9. Refractory nausea and vomiting, chronic gastrointestinal diseases (e.g., inflammatory bowel disease), or significant bowel resection that would preclude adequate absorption. 10. Prior treatment with selumetinib or another specific MEK1/2 inhibitor (unless the subject meets criteria for re-treatment). 11. Evidence of an optic glioma, malignant glioma, malignant peripheral nerve sheath tumor, or other cancer requiring treatment with chemotherapy or radiation therapy. 12. Supplementation with vitamin E greater than 100% of the daily recommended dose. 13. Patients not achieving adequate blood pressure in spite of antihypertensive therapy for control of blood pressure. 14. Cardiac Function: a. Known inherited coronary disease b. Symptomatic heart failure (NYHA Class II-IV prior or current cardiomyopathy, or severe valvular heart disease) c. Prior or current cardiomyopathy d. Severe valvular heart disease e. History of atrial fibrillation 15. Ophthalmologic conditions: a. Current or past history of central serous retinopathy b. Current or past history of retinal vein occlusion 37

118 c. Known intraocular pressure (IOP) > 21 mmhg (or ULN adjusted by age) or uncontrolled glaucoma (irrespective of IOP); patients with controlled glaucoma and increased IOP who do not have meaningful vision (light perception only or no light perception) may be eligible after discussion with the study chair. d. Subjects with any other significant abnormality on ophthalmic examination (performed by an ophthalmologist) should be discussed with the Study Chair for potential eligibility e. Ophthalmological findings secondary to long-standing optic pathway glioma (such as visual loss, optic nerve pallor or strabismus) or longstanding orbito-temporal PN (such as visual loss, strabismus) will NOT be considered a significant abnormality for the purposes of the study 16. Known severe hypersensitivity to selumetinib or any excipient of selumetinib or history of allergic reactions attributed to compounds of similar chemical or biologic composition to selumetinib 17. Recent major surgery within a minimum of 4 weeks prior to starting study treatment, with the exception of surgical placement for vascular access. 18. Any unresolved chronic toxicity with CTC AE grade 2 from previous anti- NF1 therapy, except for alopecia. 19. Clinical judgement by the investigator that the patient should not participate in the study. 20. While not an exclusion criterion, unless considered clinically indicated, patients should avoid taking other additional non-study medications that may interfere with the study medications. In particular, patients should avoid medications that are known to either induce or inhibit the activity of hepatic microsomal isoenzymes CYP1A2, CYP2C19 and CYP3A4, as this may interfere with the metabolism of selumetinib (Appendix XVIII). 2.2 SCREENING EVALUATION (SEE APPENDIX III) Pre-treatment laboratory tests should be performed within 2 weeks, and imaging studies within 4 weeks, prior to enrollment on the trial unless otherwise stated. The evaluations required prior to starting treatment in the Phase I portion are listed in table form in Appendix IIIa, and the evaluations required prior to starting treatment in the Phase II portion are listed in Appendix IIIb. Phase II: Patients will be enrolled on one of two strata depending on the presence or absence of PN related morbidity at enrollment. Stratum 1: Patients with PN related morbidity, and stratum 2: Patients without PN related morbidity at the time of enrollment. In order to preliminarily assign patients to either stratum the Target PN Location and Associated Morbidity Form (Appendix VI) should be completed by the Site PI and sent to the NCI POB prior to enrollment. 38

119 For enrollment on stratum 1 of the trial, presence of PN related morbidity at enrollment will be required. This is defined as presence of: PN related pain PN related disfigurement PN related difficulty in function Patient without PN related morbidity at enrollment will be enrolled on stratum 2. Appendix VI will be completed by the participating site at the time of enrollment, and the completed Appendix will be sent to the NCI POB at the same time as the eligibility checklist is submitted. After enrollment, when patients have completed baseline evaluations including QOL, pain, and functional evaluations, the presence or absence of PN related morbidity will be confirmed, and the patient will be definitively assigned to stratum 1 or 2 prior to initiation of treatment with selumetinib. 1. History and physical examination: Complete history, including prior and concurrent therapy, all documented previous height and weight measurements and growth curves if available; physical examination including documentation of measurable disease, performance status (Appendix I), blood pressure (Appendix IB), heart rate, respiratory rate, O2 saturation with pulse oximetry, height, weight, body surface area (BSA), and baseline signs and symptoms. Blood pressure will be measured with an appropriate sized cuff at rest. If the blood pressure is outside of the normal limits, three serial blood pressures should be obtained and averaged to determine baseline blood pressure (Appendix IB). a. Height: The patients should take off shoes and socks, and heels should be placed against the wall with ankles together. Height should be measured in a standing position with a stadiometer. Two additional repeat measurements must be made with the patient stepping off the stadiometer in between each measurement. Height measurements should be taken at approximately the same time of day for each visit, when possible. The average of the 3 measurements should be plotted on a standardized growth chart. In patients with known leg length discrepancy due to limb hypertrophy, height should be measured with the patient bearing weight on the limb without hypertrophy. b. BSA: BSA is calculated from the average of 3 repeated measurements of weight and height on the same day by using a standard formula consistently at participating sites. 2. Hematology: Complete blood count with differential and platelets. 3. Chemistries: Electrolytes (including sodium, potassium, chloride, CO2), calcium, phosphorus, magnesium, creatinine, BUN, glucose, ALT, AST, bilirubin, urinalysis, total protein, albumin, and CPK. 4. EKG/Echo: Electrocardiogram and echocardiogram or cardiac MRI to be performed within 4 weeks prior to enrollment on the trial. 39

120 5. Urine or serum pregnancy test for all females of childbearing potential (females greater than 9 years of age or those showing pubertal development). This test is to be performed within 72 hours prior to enrollment. 6. Ophthalmology evaluation: A complete ophthalmology evaluation will be performed within 8 weeks pre treatment. Particular emphasis will be given to the evaluation of lens or corneal opacification and retinal changes. 7. Imaging evaluation (Appendix II): An MRI scan of the entire inoperable target PN within 4 weeks of enrollment on study. In addition, if possible, an MRI scan of all known additional measurable PN within 4 weeks of enrollment on study. The imaging protocol outlined in Appendix II will be used each time MRI examinations are performed to assess the effects to selumetinib. The MRI study of the target PN should be sent to the NCI POB (see address below). Imaging studies will be reviewed within 3 days of receipt and confirmation of eligibility based on the measurability of the PN and suitability for volumetric analysis, will be sent electronically to the site PI. Imaging studies should be sent to: Dr. Eva Dombi NCI Pediatric Oncology Branch 10 Center Drive Building 10, room Bethesda, MD Phone: dombie@mail.nih.gov 8. Completion of the Target PN Location and Associated Morbidity Form (Appendix VI). Fax form with attention to Trish Whitcomb to prior to registration. Patients will be assigned preliminarily to stratum 1 or stratum 2 based on review of the MRI study and of Appendix VI. 2.3 SCREENING AND REGISTRATION PROCEDURES Screening Process Eligibility will be determined during a pre-study evaluation period after subjects have signed a screening consent. As previously noted, this can be accomplished by a) the NCI POB screening protocol, b) an IRB-approved institutional screening protocol, or c) the study-specific protocol. A copy of the signed screening consent should be ed to the Coordinating Center s Research Nurse (Trish Whitcomb, RN at the Pediatric Oncology Branch (POB) (phone number: , e- mail: whitcomt@mail.nih.gov). Subject information must be entered onto a screening log. For subjects not enrolled, a brief reason will be entered onto the screening log. At time of screening, all patients >18 years of age will be offered the opportunity to assign DPA so that another person can make decisions about their medical care if they become incapacitated or cognitively impaired. 40

121 2.3.2 Registration Process Registration of Participants at the NCI All patients will be registered with NCI CCR Central Registration Office (CRO) Monday through Friday between 8:30 am and 5:00 pm EST (phone: , fax: ). The CRO will notify the NCI Research Nurse by or fax that the protocol registration form has been received. An identification number will be assigned to each patient by the CRO. After confirmation of eligibility at Central Registration Office, CRO staff will notify the CC pharmacy prior to the release of any investigational agents. Verification of Registration will be forwarded electronically via . A copy of this verification should be maintained with the patient s research records Registration of Participants at all Participating Sites All patients must be registered through the NCI Central Registration Office (CRO). The CRO is open from 8:30am to 5:30pm EST Monday through Friday, excluding federal holidays. A protocol registration form and cover memo will be supplied by the Coordinating Center, NCI CCR and updates will be provided by the NCI Research nurse as needed after amendment approvals are obtained by the site. Subject eligibility and demographic information is required for registration. To register a subject, send the completed registration checklist to Coordinating Center s Research Nurse, Trish Whitcomb, RN at the Pediatric Oncology Branch (POB) (phone number: , whitcomt@mail.nih.gov, fax: ). Please indicate on the protocol registration form whether the patient is screening or is eligible to start treatment. The Coordinating Center s Research Nurse will review the completed form with the Coordinating Center PI and submit the reviewed eligibility checklist via fax to the CRO at The CRO will notify the NCI Research Nurse by or fax that the protocol registration form has been received. The CRO will assign a unique patient/subject ID number for each subject that will be used to enter data into the C3D database. The Coordinating Center Principal Investigator will assign the dose level for patient enrollment. The Coordinating Center will notify the PI of the participating site of the patient registration and dose level by . Questions about eligibility should be directed to the Coordinating Center s Research Nurse, or the Principal Investigator. Technical questions about the eligibility checklist should be directed to the Central Registration Office ( ). After confirmation of eligibility at Central Registration Office, CRO staff will call the pharmacy of the enrollee s site to advise them of the acceptance of the patient on the protocol prior to the release of any investigational agents. Verification of Registration will be forwarded electronically via . A copy of this verification should be maintained with the patient s research records. The Coordinating Center research nurse must be notified prior to enrollment and also when a participating patient is removed from the protocol. Contact Trish Whitcomb, RN at the Pediatric Oncology Branch (POB) (phone number: , whitcomt@mail.nih.gov, fax: ) or Dr. Brigitte 41

122 Widemann (phone number: , regarding potential enrollees or removing participating patients from study. 3 IMPLEMENTATION OF STUDY 3.1 STUDY DESIGN Overall Trial Design This is a multi-center phase I and II trial of selumetinib administered orally BID (approximately every 12 hours) on a continuous dosing schedule to children with NF1 and inoperable PN. A cycle of therapy is considered to be 28 days with no rest periods in between cycles. The Phase I study is designed to determine the MTD and extended toxicity profile of selumetinib in pediatric patients with NF1 and inoperable PN. Toxicities observed for the first 3 cycles will be used to define the MTD given the likely long-term exposure to selumetinib in NF1 patients. Three patients will be entered at each dose level, and the MTD dose level will be expanded up to an additional six patients to a total of 9 patients if feasible. The MTD dose level will be expanded, if feasible, to include 6 patients 12 years of age, and 6 patients > 12 years of age, to more fully characterize the toxicities and pharmacokinetics of selumetinib at the MTD. The starting dose level will be 20 mg/m 2 /dose (approximately 50% of the adult recommended single agent phase II dose). This will be followed by up to three dose escalations, with the highest dose level exceeding the adult MTD by 1 dose level. As of August 2014, the MTD in this study was determined to be 20 mg/m 2 /dose (dose level 1). In order to be consistent with the MTD determined in the PBTC study of 25 mg/m 2 /dose. the phase I trial was expanded to evaluate six patients at the 25 mg/ m 2 /dose level (dose level 1.5). As of amendment I (November 2014) enrollment on this dose level is complete, and the 25 mg/m 2 /dose dose level was determined to be the MTD of the phase I study (see Section 1.2.7). Automated volumetric MRI analysis will be used to monitor PN growth rate, and progression will be defined as a 20% increase in PN volume. This trial will also evaluate the 1) the plasma pharmacokinetics of selumetinib during cycle #1, 2) the pharmacodynamics of selumetinib by evaluating the effects of selumetinib on targets downstream of MEK in peripheral blood mononuclear cells (PBMCs), 3) and chronic daily dosing adherence of selumetinib in this patient population. Complete as of amendment I. The Phase II component of this study will be conducted in children 2 and 18 years old (as long as they are able to swallow the intact capsules) with NF1 and inoperable PN to evaluate the safety and efficacy of selumetinib. The primary endpoint will be to evaluate the confirmed partial or complete response rate in patients with PN related morbidity (see section 5.2) using centrally read 3D MRI volumetric analysis. Secondary aims include evaluating the confirmed and partial response rate of selumetinib in the overall population of all patients treated (patients with and without morbidity at the time of enrollment), and those with typical PN versus nodular PN versus solitary nodular PN, determining the long-term tolerability and safety of selumetinib, duration of 42

123 response, and the effects of selumetinib on pain, quality of life, physical functioning, time to progression, and progression free survival. In addition, we will evaluate the effect of selumetinib on bone mineral density in patients at the NCI with impaired bone mineral density at the time of enrollment. At the time of enrollment, PN will be classified at the NCI by Dr. Dombi as typical PN versus nodular PN versus solitary nodular PN as follows (Appendix IIB): Typical PN: Nodular component of PN is <30% Nodular PN: Nodular component of PN is 30% Solitary nodular PN: The target lesion is a sinlge nodular lesion As improvements in PROs and function can only be assessed in patients with baseline PN related morbidity, patients will be enrolled on 1 of 2 strata based on the presence or absence of PN related morbidity at the time of enrollment. Stratum 1: PN related morbidity present at enrollment Stratum 2: No PN related morbidity present at enrollment Presence of morbidity at enrollment will be defined as presence of: PN related pain PN related disfigurement PN related difficulty in function (See Appendix VI) Fifty (50) evaluable patients (at least 30 of which will be between 6-18 years of age) will be enrolled on stratum 1 (section 5.4.3). For these patients, the effect of selumetinib on present PN related morbidity will be assessed. For patients enrolled on stratum 2, evaluations will be done to determine if new PN related morbidity develops. A pain medication survey will be completed for all patients prior to enrollment (Appendix XI). All patients will undergo regular pain and quality of life evaluations (Appendix XI), photographic documentation of the target PN, if it is visible (Appendix IV), and complete a symptom checklist (Appendix V). Evaluations to assess physical functioning will be performed based on the location of the target PN. Each patient will be included in one or more of the following groups at baseline, prior to treatment initiation, based on location of the target PN: A) Orbital PN: Functional evaluations Appendix VII B) Airway PN: Functional evaluations Appendix VIII and IXA C) PN causing or with potential to cause motor dysfunction: Functional and patient reported (PROMIS) evaluations: Strength and range of motion evaluations (Appendix IXB, IXC), a physical functioning PRO measure (Appendix XI, Section II). Patients with PN located in the lumbosacral plexus and below (i.e. lower extremities) will undergo leg length testing (Appendix IXD), and those with upper extremity PN will undergo the NF1 Grooved Pegboard test (Appendix IXE). Patients 5 years of age with lower extremity PN will undergo regular evaluation of endurance with the 6- Minute Walk-Run test (Appendix IXA). 43

124 D) PN causing or with potential to cause bowel and/or bladder dysfunction: PRO evaluations (Appendix X). E) PN causing or with potential to cause other dysfunction: PN not fitting into A-D category. Appropriate functional evaluations will be selected based on clinical assessment by the study team prior to starting treatment with selumetinib. For example, PN affecting (or with the potential to affect) speech and swallowing, including facial, tongue, and anterior neck/upper airway PN, or PN greater than 2.5 cm on radiographic imaging with possible compression of cranial nerves or organs affecting speech or swallowing functions, will trigger consultation with Beth Solomon in Speech Pathology at the NCI to select appropriate evaluations (Appendix XA). As another example, for PN affecting hearing, Dr. Carmen Brewer at the NCI will be consulted to select appropriate audiology evaluations. For PN causing other morbidities, the study team (either at NCI or at other sites) will jointly discuss with the NCI consultants (e.g. Beth Solomon or Dr. Brewer) and decide on appropriate functional evalautions prior to starting study therapy. Some PN may impact multiple functions: For example, a large mediastinal/neck/brachial plexus PN may result in interference with airway function, and result in motor weakness/decreased range of motion. In this case, the patient should undergo evaluations described for all relevant functions, including both airway and motor evaluations. Based on a review of patients with NF1 and PN evaluated at the NCI POB we expect that the patients with PN causing motor dysfunction will be the largest subgroup of PN for functional evaluation. Many patients with NF1 PN have prior MRI studies of their PN, which may be amenable to volumetric MRI analysis and allow determination of the PN growth rate prior to treatment with selumetinib. Therefore, for all patients, MRI studies obtained within 3 years (more if available) prior to enrollment on this trial should be submitted to the NCI and evaluated for volumetric analysis, if feasible (Appendix II). In patients for whom the PN growth rate prior to enrollment can be determined by volumetric MRI analysis, we will evaluate changes in PN growth rate on treatment with selumetinib. If patients have prior functional evaluations (i.e. those collected as part of the NF1 Natural History Study), these evaluations will be analyzed retrospectively to investigate the development of morbidity over time. In addition, the day 1 and steady state pharmacokinetics of selumetinib will be studied (Appendix XII), and the effect of selumetinib on bone marrow derived precursor cells and cytokines will be evaluated (Appendix XIII). These analyses will be performed to evaluate if changes in PN volume on treatment with selumetinib can be associated with changes in function, patient reported outcomes, and/or pharmacodynamic markers. Patients enrolled at the NCI will have a DEXA scan done at baseline to evaluate bone mineral density (BMD) (Appendix IIC). Patients with abnormal BMD will have a repeat DEXA scan at 1 year, and then as clinically indicated. 44

125 Some patients who enroll on this trial will have optic pathway tumors or other gliomas, which do not require treatment at the time of enrollment. In these patients, the effect of selumetinib on the size of the optic pathway tumor or other glioma will be assessed by comparing imaging studies (brain MRI) obtained prior to and during treatment with selumetinib. In patients with glioma at the time of enrollment on this trial, brain MRIs performed prior to and during treatment with selumetinib will be collected and sent to the NCI for central analysis of changes. Only imaging studies performed for clinical evaluation will be used for this analysis. MRI to document presence or absence of glioma prior to enrollment is not required. Selumetinib will be administered at the MTD determined in the ongoing phase I trial, which is 25 mg/m 2 /dose BID on a continuous doing schedule (one cycle=28 days) (see dosing nomogram Appendix XVII). PHASE II: SUMMARY OF OBJECTIVES AND OUTCOME MEASUREMENTS PRIMARY OBJECTIVE To evaluate the confirmed partial and complete response rate (See section 5.2) of selumetinib using volumetric MRI analysis in children and young adults with NF1 and inoperable PN with PN related morbidity at the time of enrollment. OUTCOME MEASUREMENTS Objective response rate (PR defined as PN decrease 20% compared to baseline) using centrally read 3D MRI volumetric analysis PATIENTS All REFERENCE Appendix II 45

126 SECONDARY OBJECTIVES Evaluation of the confirmed partial and complete response rate (See section 5.2) of selumetinib in the overall population of all patients treated (patients with and without PN related morbidity at the time of enrolment), and those with typical PN versus nodular PN versus solitary nodular PN. OUTCOME MEASUREMENTS Objective response rate (PR defined as PN decrease 20% compared to baseline) using centrally read 3D MRI volumetric analysis PATIENTS All REFERENCES Appendix II To determine the long-term tolerability and safety of selumetinib To determine the duration of response to selumetinib To evaluate the effect of selumetinib on bone mineral density in patients at the NCI with impaired bone mineral density at the time of enrollment. To determine the effect of selumetinib on pain To determine the effect of selumetinib quality of life - Detailed clinical All evaluation - Laboratory studies - EKG/ECHO or cardiac MRI - Ophthalmologic exams - Symptom Checklist - Patient Diary - AEs - Long term follow up evaluation - Duration of response using centrally read 3D MRI volumentric analysis - DEXA Pts enrolled at the NCI - NRS 11 - Pain Interference Index - Pain Medication Survey - PedsQL - QOL Background form Appendix I Appendix V Appendix XIV Appendix XV All Section 5.4 All All Appendix IIC Appendix XI Appendix XI 46

127 To determine the effect of selumetinib on physical functioning -6-Minute Walk-Run Test To determine the effect of selumetinib on functional outcomes depending on PN location: 1. Orbit PN - Functional Evaluations (Vision) 2. Airway PN - Functional evaluations (Sleep studies, PFTs) 3. Motor PN - Strength- Range of motion - Leg length evaluation - Grooved Pegboard test - PROMIS (Mobility and Upper extremity) 4. Bowel/bladder PN - Patient reported outcomes Pts with lower extremity or airway PN Pts with orbit PN Pts with airway PN Pts with motor PN Pts with bowel/ bladder PN Appendix IXA Appendix VII Appendix VIII Appendix IX Appendix XI Appendix X 5. PN causing other morbidity To determine the effect of selumetinib on disfigurement To determine the effect of selumetinib on the PN growth rate To determine time to progression (TTP) and progression free survival (PFS) in progressive PN PN specific functional evaluations determined in discussion with study team - Photography (+/- video) evaluation - Volumetric analysis of MRI studies obtained prior to enrollment (if available and amenable to volumetric analysis). - TTP and PFS in progressive PN ( 20% increase in PN volume within months prior to enrollment) using centrally read 3D MRI volumetric analysis Pts with PN Appendix XA for causing other PN affecting morbidity speech or swallowing, and as to be determined Patients with Appendix IV visible PN All Section 5.4 All Section

128 To compare PFS in progressive PN to the placebo arm of the R randomized trial (01- C-0222). To determine pharmacokinetics of selumetinib. To determine the pharmacodynamics of selumetinib - PFS in progressive PN ( 20% increase in PN volume within months prior to enrollment) using centrally read 3D MRI volumetric analysis according to the REiNS criteria compared to PFS in the placebo arm of the R randomized trial (01-C-0222). Day 1 and steady state PK - Bone marrow derived precursor cells and cytokines pre treatment and on treatment with selumetinib, and to assess if changes in cell and cytokine profiles correlate with imaging response to selumetinib - Peripheral blood cytokines and progenitor cells (see section 3.4.3) All Section 5.4 All All consenting pts (nonmandatory) Appendix XII Appendix XIII Criteria for Dose Escalation Completed with amendment I Cohorts of 3 to 6 patients will be treated with selumetinib at each dose level. When a minimum of three patients who are evaluable for toxicity have completed three cycles of therapy at a dose level without evidence of dose-limiting toxicity (Section 3.1.3), subsequent patients may be enrolled at the next higher dose level. If DLT is observed during cycles 1-3 in 1 patient from the initial cohort of 3 patients at a given dose level, an additional 3 patients will be entered at that dose level. If none of these additional patients experiences a DLT (1/6 with DLT), the dose will be escalated. If 1 of the additional patients experience a DLT ( 2/6 with DLT), the MTD has been exceeded, and the next lower dose level will be considered the MTD. If the MTD has been exceeded at the first dose level, then the subsequent cohort of patients will be treated at dose level -1, which will be a 30% dose reduction (see Section 3.2 and dosing nomogram Appendix V). 48

129 3.1.3 Definition of Dose Limiting Toxicity (DLT) for phase I and toxicities requiring dose modification for phase II Toxicity will be graded according to the NCI Common Terminology Criteria for Adverse Events v4.0 (CTCAE) ( Dose limiting Toxicity (DLT) for phase I, or toxicities requiring dose modification for phase II, will be defined as any grade 3 toxicity possibly, probably, or definitely related to selumetinib with the exception of the following: In addition: Any grade 3 elevation of transaminases (AST, ALT) that return to levels that meet initial eligibility criteria within 14 days of selumetinib interruption and that do not recur upon re-challenge of selumetinib. Grade 3 hypophosphatemia, hypokalemia, hypocalcemia or hypomagnesemia responsive to supplementation. Skin rash, which can be managed successfully (lessens to Grade 2) with supportive interventions (see Section 4). Asymptomatic grade 3 creatine kinase elevation, which remains <10 x ULN. Gastrointestinal toxicity, such as grade 3 diarrhea, nausea, or vomiting, which can be managed successfully (lessens to Grade 1) with supportive interventions within 72 hours of symptom onset. Grade 3 cataracts will be considered on a case by case basis with consultation with the study chair and ophthalmology. Cardiac toxicity, as defined in section 3.3.1, will be considered dose limiting. The following grade 2 toxicities will be considered dose-limiting: Any grade 2 urinary tract obstruction at least possibly related to selumetinib (and clearly not related to tumor), grade 2 retinal detachment, grade 2 pneumonitis. Any grade 2 selumetinib related toxicities of 7 days may be considered dose limiting if they are intolerable to the patient and cannot be controlled with standard supportive measures. Retinal vein thrombosis will be considered dose-limiting and result in permanent discontinuation of treatment with selumetinib Criteria for Intra Patient Dose Escalation Completed with Amendment I The rationale for intrapatient dose escalation is based on 2 primary assumptions: NF1 and PN are progressive diseases which have significant impact on patients lives and lack alternative treatment options, Starting at 50% of the adult MTD is less likely to provide benefit, while higher doses may be of some efficacy. Therefore patients who have completed three treatment cycles at the enrollment dose (during MTD assessment) may have the selumetinib dose escalated to the next highest dose level if: 49

130 The patient does not have progressive disease as defined in Section 5.2. The patient has not experienced selumetinib related toxicities > grade 1. Off study Criteria have not been met (Section 3.10). Patients starting treatment at dose level 1 may have up to two dose escalations (one every 3 cycles) as long as the criteria above have been met, and patients starting treatment at dose level 2 may escalate up to 1 dose level. Intrapatient dose escalation will not be performed if the patient is treated at the MTD. Intrapatient dose escalation may never exceed the adult MTD (dose level 3) Definition of Maximum Tolerated Dose (MTD) Completed with Amendment I The MTD is defined as the dose level immediately below the dose at which 33% of patients in a cohort experience a DLT. In order to escalate a dose level, < 33% of patients in a cohort should have a DLT. At least 3 patients in a cohort must be evaluable for the definition of the MTD in order to escalate. If 1 out of 3 patients in a cohort experience a DLT, then up to 6 patients must be enrolled and evaluable for the definition of MTD prior to dose escalation. To determine extended tolerability, toxicities observed during the first 3 treatment cycles will be used to define the MTD. A patient will be considered evaluable for definition of the MTD if at least 85% of the prescribed dose has been administered to the patient during the first three treatment cycles based on adherence diary review (Appendix VIb) and pill count of returned drug. If a discrepancy occurs, pill count will be used for adherence measurement. If a patient has less than 85% adherence and has not experienced DLT during cycles 1-3, the patient will be replaced in the cohort. In addition, any patient who receives one or more doses and experiences a DLT will be considered evaluable for definition of MTD. The cohort at the MTD should be expanded up to as many as 12 evaluable patients in order to gain experience with toxicities and pharmacokinetics of selumetinib over a broad range of patients. An attempt should be made to enroll at least 6 patients that are 12 years of age, and 6 patients that are years of age Criteria for Starting Subsequent Treatment Cycles Patients who complete a treatment cycle may receive another cycle at the same dose level if they have not experienced DLT (phase I) or toxicities requiring dose modification (Phase II, section 3.3), and have not experienced disease progression as defined in section 5.2. For patients who experienced DLT (phase I) or a toxicity requiring dose modification (phase II), toxicities must have recovered to grade 1 prior to initiating the next cycle. If toxicity does not resolve to meet study parameters within 21 days of drug discontinuation, the patient must be removed from protocol therapy, except in the event that the patient was receiving clear clinical benefit, as defined in Section 3.1.7, and recovery occurs within 3 months of stopping selumetinib. 50

131 3.1.7 Duration of Treatment In order to minimize potential risk due to long-term and cumulative toxicity, the total duration of treatment with selumetinib will be limited based on the following: 1) For patients who have documented disease progression within approximately 1.5 years prior to trial entry, defined as 20% increase in size of PN by volumetric analysis (or 13% increase in the product of the longest 2 perpendicular diameters, or 6% increase in the longest diameter), there will be no limit to the duration of treatment as long as the patient meets the requirements for further treatment (Section 3.1.6). However, treatment may be discontinued earlier at the discretion of the institutional PI if this is felt to be in the best interest of the patient. 2) For patients with no previous documented history of disease progression within the 1.5 years prior to trial entry, the duration of the study will be limited to 2 years if no imaging response (volume decreased by 20%) is observed. i. Patients on the phase I or II portion of the study who are removed from treatment after 2 years for reasons other than toxicity or progression with stable disease will continue to be monitored with MRI and volumetric analysis every 4-6 months. Should the PN demonstrate progression (volume increase 15%) within approximately 2 years of stopping selumetinib, treatment with selumetinib may be restarted with the goal to stop further PN growth. Patients will be re-consented on the study and must meet all eligibility criteria with the exception of prior treatment with selumetinib or another specific MEK 1/2 inhibitor prior to restarting therapy. In these patients, treatment may continue as long as the PN remains stable or responsive (<20% increase in the PN volume). ii. For patients who do show imaging response (Section 5.2), the treatment duration will not be limited unless the patient experiences subsequent disease progression or meets other off treatment criteria (Section 3.9). However, treatment may be discontinued earlier at the discretion of the institutional PI if this is felt to be in the best interest of the patient. 3) In determining additional treatment with selumetinib after resolution of DLT (phase I) or a treatment limiting toxicity (phase II) within a time period exceeding 21 days but 3 months, benefit will be defined as either a partial response ( 20% decrease in PN volume), or stable disease (<20% increase or < 20% decrease in PN volume) in a patient who enrolled on the trial with progressive disease (see section paragraph 1), or improvement of symptoms or function. 4) In patients who experience a PR and in whom the target PN becomes amenable to surgery, selumetinib may be continued following surgical resectionof the target PN, provided the target PN is still measurable. 51

132 5) Re-treatment: Patients on the phase I or II portion of the study who are removed from treatment after 2 years of therapy for reasons other than toxicity or progression, and who have stable disease, will continue to be monitored with MRI and volumetric analysis every 4-6 months. Should the PN demonstrate progression (volume increase 15%) within approximately 2 years of stopping selumetinib, treatment with selumetinib may be restarted with the goal to stop further PN growth. Patients will be re-consented on the study and must meet all eligibility criteria, with the exception of prior treatment with selumetinib or another specific MEK 1/2 inhibitor prior to restarting therapy. In these patients, treatment may continue as long as the PN remains stable or responsive (<20% increase in the PN volume). 3.2 DRUG ADMINISTRATION Selumetinib will be supplied in 10 mg (white) and 25 mg (blue) capsules. Selumetinib will be administered orally twice daily (approximately every 12 hours) continuously for 28 day cycles with no rest periods between cycles. Patients should be instructed to take the dose of selumetinib on an empty stomach (no food or drink other than water for 2 hours before and 1 hour after dosing) with water only. Dosing will be performed based on body surface area (BSA), and doses will be rounded the nearest 10 mg using a dosing nomogram (See Appendix XVII). Selumetinib dosing will be capped at a BSA 2 m 2. At follow up evaluations (Appendix IIIA/B), selumetinib will be adjusted for changes in body surface area according to the dosing nomogram. The capsules cannot be crushed and must be swallowed in whole. Phase I Only: The starting dose will be 20 mg/m 2 /dose, which corresponds to approximately 50% of the adult recommended phase II dose. After dose escalation to 30 mg/ m 2 /dose, the MTD in this study was determined to be 20 mg/m 2 /dose. At the same time the MTD in a PBTC study (ISS ) was determined to be slightly higher with 25 mg/m 2 /dose. To be consistent with pediatric dosing, the 25 mg/m 2 /dose was added as an additional dose level. Provided this dose is tolerated, the pediatric MTD of selumetinib will be 25 mg/m 2 /dose in NF1 and PN and refractory brain tumors. As of amendment I complete. Phase I: Dose Escalation Schema Dose Level Dose (mg/m 2 /dose q12h) Equivalent fixed adult dose (mg) Percent change (%)

133 MTD 1.5*** ** * Based on BSA of 1.8 m 2, shaded area 50% of adult MTD ** Adult MTD ***This dose level was added after determining 20 mg/m 2 /dose as the MTD in order to harmonize pediatric dosing with the MTD of 25 mg/m 2 /dose determined in the PBTC study. As of amendment I (November 2014): The pediatric MTD has been determined on this trial and is 25 mg/m 2 /dose (dose level 1.5). This dose will be used for the phase II study. All patients or their guardians will keep a diary (Appendix XIV (phase I) and XIV (phase II) to document the intake of each dose of selumetinib and potential side effects. The patient diary review and pill count are described in Appendix III. 3.3 TREATMENT MODIFICATIONS If the patient experiences a dose limiting toxicity (phase I) or a toxicity requiring dose modification (phase II) as defined above, the protocol therapy will be withheld. If the toxicity resolves to meet study parameters or grade 1 within 21 days of drug discontinuation or as described in Section 3.1.6, the patient may resume treatment at a dose reduced by 25-33% following the table below: First selumetinib dose reduction Selumetinib current dose (mg/dose BID) Selumetininb first dose reduction (mg/dose) Percent decrease (approximately) in selumetinib dose (%) BID AM-30 PM BID BID AM-20 PM AM-10 PM AM-10 PM QD even day 25 53

134 20 QD odd day Doses reduced for toxicity will not be re-escalated, even if there is minimal or no toxicity with the reduced dose, with the following exception: The dose may be increased to account for an increase in BSA at the time of on study evaluations (Appendix IIIA/B). Dosing should be rounded to the nearest 5-10 mg (see dosing nomogram Appendix XVII). In addition, should PN volume measurements subsequent to a dose reduction demonstrate increases in PN volume 10% but <20%, and selumetinib is well tolerated at this reduced dose, it will be permitted to resume dosing at the dose level prior to dose reduction. However, dosing will be performed BID for 5 days, followed by 2 days rest (25% dose decrease). The rationale for this is that a certain dose of selumetinib may be required for inhibition of ERK phosphorylation, and that potential activity will be lost with a dose reduction below this dose. The daily x 5 days followed by 2 days rest schedule may allow for continued inhibition of ERK phosphorylation for the majority of treatment days. Selumetinib doses held while the patient is recovering from toxicity should not be made up and the cycle remains 28 days or until recovered from toxicity. If toxicity does not resolve to meet study parameters within 21 days of drug discontinuation, the patient must be removed from protocol therapy, except in the event that the patient was receiving clear clinical benefit as defined in Section and recovery occurs within 3 months of drug discontinuation. For example, patients who experience dose limiting cardiotoxicity (decreased LVEF) and whose LVEF recovers to meet study parameters within >21 days but 3 months, may upon recovery, continue protocol therapy at the reduced dose provided they have previously experienced clinical benefit while receiving selumetinib (see Section 3.1.7). If dose-limiting toxicity recurs in any patient who has resumed treatment at the reduced dose, the dose may be reduced a second time using the same criteria with the exception of cardiotoxicity, for which only one dose reduction will be allowed. The criteria for DLTs other than cardiotoxicity are: the toxicity resolves to meet study parameters within 21 days of drug discontinuation (unless receiving clinical benefit as stated in Section 3.1.7), the dose is reduced 25-33%, may not be re-escalated, and doses held will not be made up. The second dose reduction should be performed using the table below: Second selumetinib dose reduction Current selumetinib dose after dose reduction 1 (mg/dose) Selumetininb second dose reduction ( mg/dose) Percent decrease (approximately) in selumetinib dose (%) 35 BID 25 BID AM-30 PM 25 AM- 20 PM BID 25 AM-20 PM 25 54

135 25 BID 25 AM-10 PM AM-20 PM 20 AM - 10 PM AM-10 PM 25 AM- None PM AM-10 PM 10 AM 10 PM QD even day 20 QD odd day 10 QD even day 10 QD odd day 33 If dose-limiting toxicity recurs after two dose reductions, the patient must be removed permanently from protocol therapy. Patients removed from study therapy for toxicity will be followed until resolution of toxicity and off study criteria are met. In addition, patients enrolled on the phase II portion of the study will undergo long-term safety evaluations (Appendix XV). 55

136 SELUMETINIB MODIFICATION DOSE STARTING DOSE: 25 mg/m 2 /dose PO BID Toxicity requiring dose modification (Section 3.3)? YES NO Resolves to grade 1 within 21 days YES Resolves within 3 months AND Clinical benefit attained* No resolution within 21 days AND No clinical benefit* Continue 25 mg/m 2 /dose BID Resume drug at 25-33% of previous dose Resume with dosing of 35mg BID STOP DRUG PERMANENTLY Volume increase? DLT? Resolves within 21 days Volume increase 20%, STOP DRUG PERMANENTLY YES Resume dosing at 25-33% of previous dose** YES Resolves within 3 months AND clinical benefit attained* STOP DRUG PERMANENTLY IF FURTHER DLT Volume increase 10%: but <20% from baseline: resume dosing at 25 mg/m 2 /dose PO BID X5 days DLT? No resolution within 21 days AND no clinical benefit* STOP DRUG PERMANENTLY NO Continue 25 mg/m 2 /dose PO BID X5 days Resolves to grade 1 within 21 days with Resume dosing of 25mg drug at 25-33% BID of previous dose** DLT? YES STOP DRUG PERMANENTLY ** Only one dose reduction allowed for cardiotoxicity YES Resolves within 3 months AND Clinical benefit attained* No resolution within 21 days AND no clinical benefit* STOP DRUG PERMANENTLY NO Continue dosing at 25-33% of initial dose * Clinical benefit: 20% decrease in PN volume stable disease (<20% increase or < 20% decrease in PN volume) with progressive disease at enrollment Improvement of symptoms or function 56

137 3.3.1 Cardiac Toxicity Cardiac toxicity will be managed as described below (where NCI LLN designates LVEF of 53%, participating sites may use their institutional normal): 1) Decline in LVEF from baseline of 10 percentage points but with a normal EF of 53% Patients who have an asymptomatic decline in LVEF from baseline of 10 percentage points but continue to have a normal EF of 53% (if a range is given then the upper value of the range will be used) will continue on treatment with selumetinib and have a repeat ECHO or cardiac MRI 3-6 weeks later, and then as described in Section Patients who have a symptomatic decline in LVEF from baseline of 10 percentage points but continue to have a normal EF of 53% (if a range is given then the upper limit of the range will be used) selumetinib will be held. A cardiology consult will be initiated to determine the etiology of the symptoms and to direct management. At the same time, the primary team will investigate other possible etiologies of the symptoms. The ECHO or cardiac MRI will be repeated in these patients after 3-6 weeks and as recommended by cardiology. If the patient is receiving benefit from selumetinib (see Section 3.1.7), and if symptoms were found to be related to heart failure, selumetinib will be restarted at a reduced dose. If symptoms were unrelated to heart failure, selumetinib may be resumed at full dose. 57

138 2) Decline in LVEF from baseline of 10 percentage points with an EF < 53% For patients who have a symptomatic or asymptomatic decline in LVEF from baseline of 10 percentage points to < 53% (if a range is given then the upper limit of the range will be used), selumetinib will be held. Treatment with an ACE inhibitor should be initiated as soon as possible, and these patients will be referred to cardiology for additional evaluation and treatment. The ECHO or cardiac MRI will be repeated on these patients after 3-6 weeks, and as recommended by cardiology until recovery from toxicity (LVEF 53%). ECHOs will then be performed as described in Section 3.6. If the LVEF returns to 53% within 3 months of discontinuation of selumetinib, and the patient is receiving benefit from selumetinib (see Section 3.1.7), and symptoms have resolved, selumetinib may be restarted at a reduced dose. 3) Decline in LVEF from baseline of < 10 percentage points with an EF < 53% For patients who have an asymptomatic decline in LVEF from baseline of < 10 percentage points to < 53% (if a range is given then the upper limit of the range will be used), selumetinib will be continued. Treatment with an ACE inhibitor should be initiated as soon as possible, and these patients will be referred to cardiology for additional evaluation and treatment. An ECHO or cardiac MRI will be performed after 3-6 weeks and as directed by cardiology. Treatment with selumetinib can be continued for as long as the decrease in LVEF remains less than 10 percentage points from baseline in these patients and patients remain asymptomatic. Patients who have a symptomatic decline in LVEF from baseline of < 10 percentage points to < 53% (if a range is given then the upper limit of the range will be used), selumetinib will be held. Treatment with an ACE inhibitor should be initiated as soon as possible, and these patients will be referred to cardiology for additional evaluation and treatment. The ECHO or cardiac MRI will be repeated in these patients after 3-6 weeks and as recommended by cardiology until recovery of LVEF to 53%. ECHOs will then be performed as described in Section 3.6. If the LVEF returns to 53% within 3 months of discontinuation of selumetinib, and the patient is receiving benefit from selumetinib (see Section 3.1.7), and symptoms resolve, selumetinib may be restarted based on cardiology recommendation. If symptoms were related to heart failure selumetinib will be restarted at reduced dose. If symptoms were unrelated to heart failure, selumetinib may be resumed at full dose. Recommendations for treatment with ACE inhibitors: For patients meeting criteria to start an ACE inhibitor the following suggestions for choice and dosing of ACE inhibitors before cardiology consultation are made. These can be modified by institutional guidelines or cardiology. Enalapril: start at 2.5 mg once daily or 0.1 mg/kg daily for children smaller than 25 kg. Lisinopril: (children >6yo; GFR >30) start at 0.07 mg/kg once daily. Captopril: start 0.3 mg/kg/day divided every 8-12 hours. The above doses are starting doses. ACE inhibitor doses should be titrated and reduced by 50% in patients with hypovolemia, hyponatremia, or decreased renal function, or if receiving diuretics. 58

139 After initiation, treatment with an ACE inhibitor should be continued for the duration of the study unless otherwise directed by cardiology. Monitoring for recurrent cardiac toxicity will be performed as described in Section PHARMACOKINETICS AND PHARMACODYNAMICS Pharmacokinetics: Phase I: Completed with amendment H Pharmacokinetic evaluation will be performed after the first dose of selumetinib, and at steady state in all consenting patients. On the first treatment cycle only, the second and third dose of selumetinib will not be administered during the pharmacokinetic sampling, which continues for 36 hours after the first dose. The fourth dose should be administered after 36-hour sample is drawn. For blood samples, 3 ml of whole blood will be collected in a lavender top tube according to the following schedule: 1) Relative to the first oral dose on day 1: before the first oral dose, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 10 to 12 h, 24 h, and 30 to 36 hours following the first oral dose. 2) In addition, one sample will be obtained on day (+/- 3 days) at the time of evaluation for cycle #2, prior to the first oral administration of the two daily doses of selumetinib. Differences in timing of the PK sample from the target time will be recorded on the PK sheet, but not reported as protocol deviation. Along with the samples, a pharmacokinetic work sheet (Appendix XII) should be filled out indicating the time points of the samples. Details regarding sample collection, handling, and shipment instructions are in Appendix XII of the protocol. Phase II: Pharmacokinetic evaluation will be performed in all patients during cycle 1, with dose 1 and at steady state prior to cycle #2 or #3 as described in Appendix XII. For each PK sample, 3 ml of whole blood will be collected in a lavender top tube according to the following schedule: 1) Relative to the first oral dose on day 1: before the first oral dose, 0.5 h, 1.5 h, 3 h, 6 h, and 10 to 12 h following the first oral dose. 2) In addition, on day (+/- 7 days) at the time of evaluation for cycle #2 or #3, samples will be obtained prior to the first oral administration of the two daily doses of selumetinib, and 0.5 h, 1.5 h, 3 h, and 6 h after administration of the first daily selumetinib dose. To ensure robust collection and interpretation of the steady state PK: 1) The patients should have taken both am and pm doses of selumetinib on the 2 days prior to the steady state PK day (e.g. if PK was on Day 28, then request the patient to have taken both doses on Days 25 and 26) if there are any missed dosing, then please request a delay in patient coming in for their PK until after 2 days of continuous BD dosing. 2) In addition, please record the time of the am and pm dose of selumetinib on the day before the steady state PK sampling day (e.g. if PK was on Day 28, then request the patient recall the time of am and pm dose on Day 27 and the site should record the dosing times on Day 28). These data should be databased to be traceable (Appendix XII). Differences in timing of the PK samples from the target time will be recorded on the PK sheet, but not reported as protocol deviation. Along with the samples, a pharmacokinetic work sheet (Appendix XII) should be filled out indicating the time points of the samples. Details regarding sample collection, handling, and shipment instructions are in Appendix XII of the protocol. Details 59

140 on the PK analysis will be documented in a PK Analysis Plan that will be issued prior to Database Lock. If IV access for PK analysis is unable to be obtained after reasonable attempt, patients may still enroll on the study without participating in the PK portion of the study A liquid chromatography/mass spectroscopy/mass spectroscopy (LC/MS/MS) will be used to analyze selumetinib. All assays used have a coefficient of variation (CV)% <15% across the analytical range. The current preclinical assay range is 0.01 to 5 g/ml for AZD6244 and to 0.5 g/ml for N- desmethyl AZD C-labelled AZD6244 and N-desmethyl AZD6244 have been synthesized and used in metabolism studies. Samples collected will be analyzed by Covance Bioanalytical Services LLC (with the support of AstraZeneca) Pharmacodynamics: To assess the effect of selumetinib on the tumor microenvironment, changes in peripheral blood bone marrow derived precursor cells and cytokines will be analyzed in all patients prior to initiation of treatment and at steady state on treatment as described in Appendix XIII Cytokine studies: Cytokine assays will be performed using multiplex simultaneous quantification of thirty soluble cytokines and growth factors including: VEGF, TGF-β, PDGF-AB/BB, MCP-1, MIP1α, MIP1β, TNFα, GM-CSF, EGF, IL-13, IL-1rα, IL-10, IL-4, IL6, FGF-2, and RANTES. Plasma samples for each subject at each treatment time point will be stored at -80 C and thawed once for simultaneous assay. Samples will be collected within two weeks prior to cycle 1, and as outlined in Appendix IIIB. Details are provided in Appendix XIII. Rationale: It is well recognized that cytokines mediate and regulate immunity and inflammation, which are important components of the biological milieu associated with cancer progression. Cytokines have been used as biomarkers in research for prognosis, and have been associated with symptoms and adverse outcomes in multiple conditions, including cancer. It has been previously shown that multiple cytokines/growth factors are altered in patients with plexiform neurofibromas as well as in a tumor murine model of NF1. Data from Wade Clapp s laboratory (Riley Hospital for Children, Indianapolis, IN) suggests that cytokine levels are associated with tumor progression and responsiveness to Gleevec therapy. The examination of cytokine patterns has been limited by traditional laboratory methods. Multiplex now permits the characterization of a broader array of cytokines in a single specimen. Because cytokines operate in integrated networks, analyzing multiple cytokines at once will provide a more complete understanding of patterns of response that may be associated with treatment response, and the prognosis of NF Circulating peripheral blood bone marrow progenitor cell assays: Polychromatic Flow Cytometry (PFC) & Identification of Circulating Progenitor Cells (CPCs): PFC for the identification of CPCs will be performed at three time points. Whole blood will be drawn in EDTA tubes and sent for processing to the Indiana University. Using PFC, the phenotypic detection and enumeration of CPCs will be performed, and the ratio of pro-angiogenic (i.e. CD133+) vs. nonangiogenic CPCs will be calculated on all samples. Data obtained will then be correlated with tumor 60

141 response. Samples will be collected within two weeks prior to cycle 1, and as outlined in Appendix XIII. 3.5 BLOOD VOLUME FOR RESEARCH STUDIES Institutional guidelines for the maximum blood volume for research samples should be utilized. The table below details the blood volume obtained for pharmacokinetic and pharmacodynamic studies during the first course in Phase I patients (With amendment I complete). Analysis #samples Sample volume (ml) Volume course #1 (ml) Pharmacokinetics Total volume 33 ml Phase II patients: Analysis #samples Sample volume (ml) Volume course #1 (ml) Pharmacokinetics Pharmacodynamics Cytokines Flow cytometry Total volume 57 ml Given that only patients who can swallow capsules will be able to participate in this study, we do not anticipate that above studies will exceed the institutional limit of blood draw for research specimens. For small patients, the priority for research studies will be samples for PK analysis over Pharmacodynamics. 3.6 ON STUDY EVALUATIONS (APPENDIX III)* Evaluations During Therapy The following assessments and evaluations will be performed in all patients participating in this study, unless otherwise noted. Evaluations for the Phase I portion of the study are in Appendix IIIA, and for the Phase II portion in Appendix IIIB. 1. History and Physical Examination: History and physical examination with vital signs (Appendix IB provides details for blood pressure measurements) to be performed according to Appendix IIIA for patients on Phase I portion and Appendix IIIB for patients on the Phase II portion. Height, weight, and BSA (see Section 2.2) should be performed also according to Appendix IIIA for patients on Phase I portion and 61

142 Appendix IIIB for patients on the Phase II portion. Patients on the phase II portion will have photographic documentation of visible PN according to Appendix IV. 2. Performance status: Performance status (Appendix IA) will be recorded at baseline and per Appendix IIIA/B. 3. Hematology: Complete blood count, differential, and platelet count will be evaluated according to Appendix IIIA for patients on Phase I portion and Appendix IIIB for patients on the Phase II portion. 4. Chemistries: Electrolytes (including sodium, potassium, chloride, CO2), calcium, phosphorus, magnesium, creatinine, BUN, glucose, ALT, AST, bilirubin, total protein and albumin, CPK, and urinalysis to be performed according to Appendix IIIA for patients on Phase I portion and Appendix IIIB for patients on the Phase II portion. CPK isoenzymes will be performed per Appendix IIIB 5. Urine or serum pregnancy test in female patients of childbearing potential as per institutional standards (at NIH subjects 9 years and older) prior to disease evaluations with MRI. 6. Ophthalmology evaluation for corneal and lens opacifications and retinal changes according to Appendix IIIA for patients on Phase I portion and Appendix IIIB for patients on the Phase II portion. 7. EKG and ECHO or cardiac MRI: According to Appendix IIIA for patients on Phase I portion and Appendix IIIB for patients on the Phase II portion Upon recovery from toxicity (to a LVEF of 53%), patients should be monitored with an ECHO or cardiac MRI every 2 3 cycles or as directed by the cardiologist. 8. Pharmacokinetics: Phase I: Plasma samples for the evaluation of selumetinib pharmacokinetics will be obtained in consenting patients prior to and after the first dose of selumetinib, and at steady state during the first treatment cycle in patients enrolled in Phase I (Appendix IV). As of amendment I (November 2014 complete). On the phase II portion, sampling will be obtained prior to and after the first dose of selumetinib, and at steady state prior to cycle 2 in consenting patients as detailed in Appendix XII. 9. Imaging evaluation: Evaluate the target lesions by 3-D MRI according to Appendix IIIA for patients on Phase I portion and Appendix IIIB for patients on the Phase II portion as long as the patient remains on study (Appendix II). If patient does show a partial response (PR) according to Section 5.2, response will be confirmed at the time of the next restaging study. 10. Adherence 62

143 Phase I: Interviews will be conducted by psychology or other staff member with each parent and patient (together) during the pre-study visit to identify potential barriers to adherence. Parents and patients (ages 8 and up) will be given an Adherence Questionnaire containing a list of barriers (see Appendix XVI) and asked to identify the ones that have interfered with adherence to other medications in the past. Any barriers identified will be discussed to help the family implement strategies for preventing problems with adherence. Complete as of amendment H (November 2014) Phase II: Interviews will be conducted by psychology or other staff member with each parent and patient (together) during the pre-study visit to identify potential barriers to adherence. Parents and patients (ages 8 and up) will be given an Adherence Questionnaire containing a list of barriers (see Appendix XVI) and asked to identify the ones that have interfered with adherence to other medications in the past. Any barriers identified will be discussed to help the family implement strategies for preventing problems with adherence. Phase I and II: If diary review and capsule count indicates that the administered dose deviates by 20 % from the prescribed dose, the study staff will review adherence with the patient and their family and initiate interventions to improve adherence (Appendix XVI). In addition, the patient (ages 8 and up) and parent will complete the Responsibility for Medication Questionnaire (RMQ; Appendix XVI), a 9-item measure that assesses perceptions of who holds responsibility for medication-related tasks. Discrepancies in perceptions will be addressed by the psychology staff member to try to resolve these prior to starting the study drug. 11. Patient diary to monitor concurrent side effects and adherence should be reviewed as described in Appendix IIIA for the phase I portion and in Appendix IIIB for the phase II portion of the trial. Capsule counts will be performed as described in Appendix IIIB. Caregivers are instructed to bring empty pill bottles to every visit. 12. Phase I only: The Responsibility for Medication Questionnaire (RMQ) (Appendix XVII) is a 9-item measure that assesses parents and patients perceptions of who holds responsibility for various medication-related tasks. If adherence is calculated as less than 90% according to pill count or the adherence diaries this measure will be administered to patients (ages 8 and older only) and parents. In addition, the parent and patient will meet with a psychology staff member to review the results of adherence and discuss any discrepancies between parent and child perceptions of responsibility. Barriers to adherence will be discussed to help the family identify strategies for overcoming these obstacles. Complete as of amendment I (November 2014) 13. Phase II only (Appendix IIIB) a. For all patients: i. Photographic documentation of visible target PN (Appendix IV) 63

144 ii. Symptom checklist completed by patients or their guardians (Appendix V). iii. Baseline PN location and morbidity form to be completed by site PI (Appendix VI) iv. Baseline PN location and classification form to be completed by Dr. Dombi at the NCI (Appendix IIB) b. Based on the baseline PN location and morbidity and the assessed potential for morbidity, functional evaluations will be selected to be performed at baseline and during the trial. Each patient will be included in one or more of the following groups for the purpose evaluations of physical functions: i. Orbital PN: Functional evaluations Appendix VII ii. Airway PN: Functional evaluations Appendix VIII iii. PN causing motor dysfunction: Functional and patient reported evaluations Appendix IXA-E and XI. For lower extremity PN evaluation of endurance using the 6-Minute Walk-Run Test (Appendix IXA) only in patients 5 years old at time of enrollment. iv. PN causing bowel and/or bladder dysfunction: Evaluations Appendix X v. PN causing other morbidity: 1. PN affecting speech/swallowing: Evaluations to be determined in consultation with Beth Solomon at the NCI (Appendix XA) 2. For PN affecting hearing, evaluations to be determined in consultation with Dr. Carmen Brewer at NCI ( brewerc@nidcd.nih.gov, phone: ) 3. PN causing or with potential for other dysfunction: PN not fitting into i-iv. Evaluations will be selected based on clinical assessment by the study team prior to intitiation of treatment. c. In addition, based on prior imaging studies (if available) the PN growth rate prior to enrollment will be determined. MRI studies of the target PN performed within 2-3 years prior to enrollment will be submitted to the trial coordinating site to determine the PN growth rate prior to treatment initiation with selumetinib. i. Imaging studies should be sent to: Dr. Eva Dombi NCI Pediatric Oncology Branch 10 Center Drive 64

145 Building 10, room Bethesda, MD Phone: d. Pharmacodynamic studies: Peripheral blood cytokines and progenitor cells will be obtained in consenting patients on the phase II portion only as detailed in Appendix XIII. Blood samples will be obtained prior to treatment with selumetinib, and prior to cycles after 2 and 5. Quality of Life and pain evaluations: This study will assess both general health-related qualify of life (QOL) and pain (pain intensity and pain interference) as patient-reported outcomes (PROs) as described in Appendix XI. If you have any questions about the administration of any of the PRO measures, please contact: Dr. Pam Wolters Phone: woltersp@mail.nih.gov e. Dexa scan at baseline and as described in Appendix IIIB only in patients enrolled at NCI. See Appendix III for allowable variations in scheduling. Appendix IIIA for Phase I and Appendix IIIB for Phase II For patients who undergo an intra-patient dose escalation on Phase I, all evaluations listed above will be performed as if the patient were starting his/her first cycle and so forth as described in this section, with the exception of PK and PD. Complete as of amendment I (November 2014) Off Therapy Evaluations The following tests and procedures should be performed, if possible, at the time a patient comes off treatment regardless of the reason for coming off treatment, unless the test or procedure has been performed in the past 2 weeks or within a time period described below. 1. History and physical examination, vital sign including blood pressure, and performance status. 2. Laboratory: Complete blood count, differential, and platelet count, electrolytes (including sodium, potassium, chloride, CO2), calcium, phosphorus, magnesium, creatinine, BUN, glucose, ALT, bilirubin, urinalysis, total protein, and albumin. 3. MRI of the PN and any other progressing lesions using the MRI protocol identified in Appendix IIB within the past 4 weeks. 4. Functional assessments, Pain, and QOL evaluations (See Appendix XI) should be done, if feasible Long Term Safety Follow Up There is no available information about the potential long-term effects of selumetinib. The long term outcome of expected adverse reactions in this pediatric population remains unknown. NF1 is 65

146 a genetic tumor predisposition syndrome, and children and young adults with NF1 may be at greater risk to develop secondary malignancies. Therefore, the proposed timing for long term safety follow up is 7 years following initiation of treatment or 5 years after study drug discontinuation, whichever is longer. Long term safety evaluations will only be performed in patients enrolled on the phase II component of this trial. This follow-up will include an annual health check and evaluations as described in Appendix IIIB. This time frame is considered sufficient to monitor the final outcomes of the adverse reactions that patients would experience during study therapy. If a potential safety concern was identified, this time frame may be extended. Annual assessment is described in Appendix XV. Additional assessments may be performed at the discretion of the Investigator if clinically indicated. 3.7 CONCURRENT THERAPIES 1. Other cancer chemotherapy, radiation, immunotherapy, biologic therapy, hematopoietic growth factors, or investigational agents cannot be administered to patient while receiving selumetinib. 2. Doses of vitamin E greater than the 100% daily recommended doses are contraindicated. Selumetinib capsules contain vitamin E in the form of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a water-soluble form of vitamin E which acts as a formulation excipient. Capsule Ingredient Maximum amount of Vitamin E per capsule 10 mg Active 32.4 mg 25 mg Active 35.9 mg a. The maximum daily dose of vitamin E that a study subject may receive from selumetinib is approximately mg/day (patients who receive 40 mg BID). Therefore: b. Patients should not take any supplemental vitamin E. High doses of vitamin E have been reported to cause bleeding and interfere with blood coagulation processes. 3. Selumetinib should be administered with caution in patients who are also receiving concomitant coumarin anticoagulant medications, e.g. warfarin. These patients should have their INR monitored / anticoagulant assessments conducted more frequently and the dose of the anticoagulant should be adjusted accordingly. 4. The use of corticosteroids for control of symptoms related to the underlying NF1 or for other reasons will be allowed. 5. Throughout the study, patients should be instructed to avoid changes to, or the addition of concomitant medications. Unless considered clinically indicated, patients should avoid taking other additional non-study medications that may interfere with the study medications. In particular, patients should avoid medications that are known to either induce or inhibit the activity of hepatic microsomal isoenzymes CYP1A2, CYP2C19 and CYP3A4, as this may interfere with the metabolism of selumetinib (Appendix XVIII). The study team should also check a frequently-updated medical reference for a 66

147 list of drugs to avoid or minimize use of what is listed in APPENDIX XVIII. Appendix XVIII (Patient Drug Information Handout and Wallet Card) should be provided to patients. 6. Guidance for management of patients with reductions in LVEF are contained in Sections and CRITERIA FOR REMOVAL FROM TREATMENT 1. A patient may be removed from treatment for the following non-medical or administrative reasons: a. Patient refusal of further treatments (reasons must be noted on the patient s CRF). b. It is deemed in the best interest of the patient. In this instance the PI should be notified and the reasons of withdrawal should be noted in the CRF. c. Serious protocol violation as determined by the PI. 2. Any patient who develops dose-limiting toxicity (phase I) or a toxicity requiring dose modification (phase II), which does not resolve to meet study parameters within 21 days of drug discontinuation or within the time frame defined in the DLT definition and section 3.1.7, whichever comes first. 3. If dose-limiting toxicity recurs in a patient who has resumed treatment after two dose reductions. 4. Any patient with clinical or imaging evidence of progressive disease on treatment, as defined in Section 5.2 following any treatment cycle will be removed from study treatment. 5. A patient who develops a concurrent serious medical condition that might preclude or contraindicate the further administration of selumetinib will be removed from study treatment. A patient who becomes pregnant will be immediately taken off therapy. 6. A patient who will undergo complete surgical resection of their PN (thus rendering them with no evidence of disease). Patients who are off protocol therapy are to be followed until they meet the criteria for Off Study (see below). Follow-up data will be required unless consent is withdrawn. Toxicities must be followed until stabilization or resolution prior to a subject being taken off study. Patients enrolled on the phase II portion of the study will continue with long-term safety evaluations after completing study treatment (see Appendix XV). Patients with no previous documented history of disease progression within the 1.5 years prior to trial entry with no radiographic response on drug will be removed from study treatment after 2 years of study entry (Section 3.1.7). However, if a patient removed from treatment for the reasons above develops PN volume increase 15% after discontinuation of selumetinib (See Section 3.1.7), this patient may restart selumetinib provided all other criteria for continuation of therapy apply. In these patients treatment may continue as long as the PN remains stable or responsive (<20% increase in PN volume). 67

148 3.9 OFF STUDY CRITERIA Patients who are removed from treatment permanently for toxicity should be monitored until resolution or stabilization of toxicity, or until 30 days after the last dose of selumetinib, whichever is later. Patients enrolled on the phase II component of the study will be considered to have completed the study after completion of long term follow up evaluations described in Appendix IIIB and XV and Section In addition, the following criteria will result in removal from study (phase I and II): 1. Death 2. Lost to follow-up 3. Withdrawal of consent for any further data submission 4. Noncompliance with study requirements 5. Completion of long term safety evaluations All subjects must be taken Off-Study through the NCI Central Registration Office (CRO). The CRO is open from 8:30am to 5:30pm EST Monday through Friday, excluding federal holidays. Contact numbers for the CRO Office are Phone: (301) or FAX: (301) An offstudy form will be supplied by the CRO Office or Coordinating Center, NCI CCR. Send the completed off-study form to the Coordinating Center s Research Nurse; Trish Whitcomb, RN at the Pediatric Oncology Branch (POB), phone number: , whitcomt@mail.nih.gov, fax: END OF TREATMENT EVALUATION (APPENDIX IIIA/B) The following tests and procedures should be performed, if possible, at the time a patient comes off treatment regardless of the reason for coming off treatment, unless the test or procedure has been performed in the past 2 weeks or within a time period described below. 5. History and physical examination, vital sign including blood pressure, and performance status. 6. Laboratory: Complete blood count, differential, and platelet count, electrolytes (including sodium, potassium, chloride, CO2), calcium, phosphorus, magnesium, creatinine, BUN, glucose, ALT, AST, bilirubin, urinalysis, total protein, and albumin. 7. MRI of the PN and any other progressing lesions using the MRI protocol identified in Appendix IIA within the past 4 weeks. 8. Functional assessments, Pain, and QOL evaluations should be done, if feasible. 4 SUPPORTIVE CARE Appropriate antibiotics, blood product support, anti-emetics, and general supportive care will be used as indicated. Guidelines for the management of specific toxicities, which may be modified based on institutional policies and guidelines, are provided below.dermatologic adverse events: 68

149 The use of medications for the supportive care of rash is permitted, provided that compliance with Section 3.7 regarding concomitant medications is observed. Early initiation of treatment for rashes is strongly recommended to minimize the duration and severity of the adverse event. For pediatric subjects, the following guidelines have been found to be useful: Acneiform rash: Experience with acneiform rash in the pediatric studies to date suggests that topical clindamycin gel or lotion applied BID, rather than steroids, is the most helpful for pustular rash (typically seen in the older child/adolescent). In severe cases, semisynthetic oral tetracyclines such as doxycycline or minocyline may also be useful for older children and adolescents, but should be avoided in children younger than 8 years old because of risk to tooth development. Eczematous rash/xerosis: Eczematous/dry skin rash and other macular (non-acneiform) rash should be treated with a moisturizer such as Cerave or Eucerin. A low potency topical steroid may also be used if symptomatic. Ketoconazole shampoo should be used for any rash involving the scalp. Paronychia: For patients who do not undergo drainage, silver nitrate may be used, as well as topical bactroban, steroids, and/or antifungals. Silver nitrate is only of value when there is open inflamed skin or granulation tissue (e.g. pyogenic-granuloma-like lesions). If the periungual skin is swollen but intact (whether infectious or non-infectious), silver nitrate is not recommended. Patients should be cautioned to avoid trauma to the area. Podiatry consult may be considered for partial nail removal. Patients who undergo incision and drainage and are found to have no infectious organisms on culture, should be treated as above. If infection is identified, patients may be treated with systemic antibiotics (oral tetracyclines). If paronychia recurs or develops in other fingers or toes, Flurandrenolide (e.g. Cordran) tape or topical steroid cream such as triamcinolone can be used in the morning and Bactroban and Nizoral topical ointments in the evening. For any questions regarding management of skin toxicity, please contact: Edward Cowen ( ec176r@nih.gov, phone ). Visual adverse events: All patients will have a detailed ophthalmologic evaluation at baseline. In patients who develop visual symptoms, a repeat ophthalmologic evaluation will be performed to include: best corrected visual acuity, intraocular pressure, slit lamp fundoscopy (photograph if abnormal); consider optical coherence tomography. If a diagnosis of retinal pigment epithelial detachment (PRED) or central serous retinopathy (CSR) is made, treatment with selumetinib will be held and repeat ophthalmologic evaluations will be performed until resolution. Treatment may be restarted after a dose reduction using the criteria described in Section If a diagnosis of retinal vein occlusion (RVO) is made, selumetinib will be discontinued permanently. For the development of cornea or lens opacification see section

150 Gastrointestinal adverse events: Diarrhea Patients should be made aware that they may experience diarrhea, and should be encouraged to record the number of stools and possible associated symptoms in the patient diary (Appendix XIV). Patients should be given loperamide to take home and should start taking loperamide immediately after the first episode of unformed, loose stool (in accordance with local regulations and practice). Additional agents may be used concurrently if loperamide is not adequate to control diarrhea as a single agent. In addition, the following dietary advice should be considered: BRAT diet (bananas, rice, apple sauce, toast, plain pasta), readily digestible food, avoidance of lactose-containing products and fried, fatty or spicy foods, increased fluid intake (8-10 glasses of clear fluids/day (including water, clear broth, fluids containing salt and sugar). Patients should be encouraged to seek advice early from their physician or study nurse if they have persistent diarrhea, diarrhea complicated by vomiting, or inability to take oral liquids. Gastrointestinal adverse events: Oral mucosistis Patients should be encouraged to follow a daily oral health care regimes, both before and during treatment with selumetinib. Patients with a healthy mouth may use non alcoholic mouthwash 4 to 6 times daily (e.g. after each meal), or according to the instructions, during the study. Saline mouthwashes (Sodium chloride 0.9%) are preferred in cases of stomatitis, and should be used at a different time than toothbrushing (e.g. after tea). Use of a mouthwash immediately after selumetinib intake is recommended. The tongue can be gently brushed (if not sore) with a soft toothbrush. Patients with, or at risk of, stomatitis should not use commercial/over-the-counter mouthwashes because of the alcohol content and astringency. Chlorhexidine mouthwashes are not recommended for the treatment of established stomatitis. The mouth should be regularly inspected by the patient and healthcare professionals. Teeth should be brushed twice daily with a fluoride toothpaste and soft toothbrush, in the morning before breakfast and last thing in the evening before bed, about 30 minutes after eating. The toothbrush should be replaced regularly (at least every 3 months). Patients with stomatitis should change their toothbrush every 4-6 weeks. Consider culture to rule out herpes simplex. Consider treating stomatitis at an early stage (CTCAE grade 1) or as soon as the patient complains of a sore mouth. Consider using an oral topical analgesic, with or without topical steroids, depending on the patient s clinical condition and the local standard medical practice. Pulmonary adverse events: Dyspnea Patients who develop dyspnea while receiving selumetinib should undergo standard clinical evaluations to rule out infectious etiology and pneumonitis. Selumetinib will be held for grade 2 pneumonitis, and may be restarted at a reduced dose following criteria for dose reduction in Section 3.3 and

151 Surgical procedures Major surgery unrelated to PN will require holding selumetinib 1 week prior to surgery and until wound has healed completely. 5 DATA COLLECTION AND EVALUATION 5.1 DATA COLLECTION The POB, NCI will coordinate the clinical trial and data collection, and supply the data to the trial sponsor CTEP via the CDUS online system. Data will be entered into the electronic NCI, Center for Cancer Research (CCR) C3D database. In addition, the Labmatrix database will be used to enter data collected on PRO and functional changes related to PN. Sites will enter data into paper CRFs, or electronic forms for final entry into the Labmatrix database. Documentation and date of IRB approval must be provided to the POB, NCI prior to initial patient entry from each institution. The completed eligibility checklist prepared by central registration must be completed as per Section 2.3 Screening and Registration Procedures Case Report Forms The C3D study number, not names or initials, will identify all case report forms and patient diaries. This will be done to protect patient confidentiality. Unless otherwise stated, all forms should be submitted to: Pediatric Oncology Branch, NCI c/o Trish Whitcomb, RN Fax: Phone: Center Drive, Building 10, CRC Room 1W-5750 Bethesda, MD After receipt at the NCI, data will be entered into the Labmatrix database. Case Report Forms: Data entry into C3D and the Labmatrix case report forms will be completed within 2 weeks of each required evaluation. The Labmatrix CRFs will be reviewed for completeness at the NCI in real time, and and then transmitted electronically into Lab Matrix.. Patient Diaries for side effects and adherence (Appendix XIV): Patients or their guardians must keep a diary to document the intake of each dose of selumetinib and potential side effects per Appendix IIIA/B. These diaries will be forwarded to Ms. Trish Whitcomb (fax: ) after the time points indicated in Appendix IIIA/B. The participating site should include a pill count as described in Appendix IIIA/B. Pharmacokinetic studies: Blood samples and the corresponding PK worksheet (Appendix XII) will be sent to Covance, and a copy of the PK worksheets will be sent to the NCI POB as described in Appendix XII. 71

152 Pharmacodynamic studies: Blood samples and the corresponding PD worksheets (Appendix XIIIA/B) will be sent to Indiana University, and a copy of the PD worksheets to the NCI POB as described in Appendix XIIIA/B Adherence Assessment: Pill count forms, adherence diaries & questionnaire forms (Appendix XVI) should be photocopied and sent to Ms. Trish Whitcomb (fax: ) as described in Appendix IIIA/B. Phase II only: Photographic documentation of PN (Appendix IV), PN symptom checklist (Appendix V), PN location and morbidity form (Appendix VI), functional evaluation forms (Appendix VII, and/or VIII, IX, X), and quality of life and pain forms (Appendix XI) should be submitted as described in the respective Appendices and Appendix IIIB. Protocol Violations: Protocol deviations and violations should be directly reported to Dr. Brigitte Widemann (phone: (301) , fax (301) , widemanb@mail.nih.gov) and to the NCI IRB. 5.2 RESPONSE CRITERIA Response evaluation is performed according to Appendix IIA. Response evaluation will be performed using 3-D MRI analysis. Phase I: Response is assessed at the time that follow-up 3D-MRI scans which are performed prior to cycles 6, 11, and then after every 6 treatment cycles. For the purpose of determining the level of response, measurements from the follow-up scans are compared to the tumor size in the pretreatment MRI scan using 3D data analysis. Responsive Disease (RD): A 20% reduction in the sum of the volume of all index lesions for 4 weeks. Stable Disease (SD): A <20% increase and <20% reduction in the sum of the volume of all index lesions for 4 weeks. Progressive Disease (PD): A 20% increase in the volume of at least one of the index PN compared to the pretreatment volume measured prior to the start of the current treatment phase. The appearance of new discrete subcutaneous neurofibromas does not qualify for disease progression. Worsening of existing symptoms or the appearance of new symptoms that persist for more than 7 days and that are felt to be definitely related to the PN should be evaluated by repeating the MRI. Patients should not be classified as having progressive disease solely on the basis of new or increased symptoms without discussing the case with the protocol PI. Phase II: Response is assessed using volumetric MRI analysis prior to cycles 5, 9, 13, 17, 21, 25, and then after every 6 cycles. Complete response (CR): Disappearance of the target lesion. Partial response (PR): Decrease in the volume of the target PN by 20% or more compared to the baseline. The PR is considered unconfirmed at the first detection, confirmed when observed again within 3 6 months, and sustained when the response is maintained for 6 months or longer. 72

153 Stable Disease (SD): Insufficient volume change to qualify for either PR or PD. Progressive Disease (PD): Increase in the volume of the target PN by 20% or more compared to baseline or the time of best response after documenting a PR. The appearance of new PN (with the exception of new discrete subcutaneous neurofibromas as noted below) or unequivocal progression of existing clinically relevant non target PN is also considered PD. The clinical appearance of new discrete subcutaneous neurofibromas does not qualify for disease progression. Worsening of existing symptoms or the appearance of new symptoms that persist for more than 7 days and that are felt to be definitely related to the PN should be evaluated by repeating the MRI. Patients should not be classified as having progressive disease solely on the basis of new or increased symptoms without discussing the case with the protocol PI. Response evaluable: Subjects who have completed any protocol derived therapy (as little as one dose) will be considered evaluable for response. As a secondary endpoint this study aims to evaluate changes in PRO and functional outcomes during treatment with selumetinib. Changes will be recorded and documented, but as there are, at present, no validated response criteria for the evaluations to be performed, no response criteria have been defined a priori and the analysis will be descriptive (see statistical section). 5.3 TOXICITY CRITERIA CTCAE term (AE description) and grade: The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site STATISTICAL SECTION Subject Accrual Subjects of both genders, from all racial and ethnic groups are eligible for this trial if they meet the criteria. Efforts will be made to extend the accrual to a representative population, but in a targeted patient population, a balance must be struck between patient safety considerations and limitations on the number of individuals exposed to potentially toxic or ineffective treatments on the one hand and the need to explore gender, racial, and ethnic aspects of clinical research on the other. If differences in outcome that correlate to gender, racial, or ethnic identity are noted, accrual may be expanded or additional studies may be performed to investigate those differences more fully Statistics and feasibility for Phase I This trial uses a conventional dose-escalation design to establish the MTD of selumetinib based on the severity of toxicity. The starting dose level of 20 mg/m 2 /dose was determined based toxicities observed in the adult phase II trials. This will be followed by up to three dose level escalations with the highest dose exceeding the adult MTD. The total number of dose levels will likely be four at a maximum, with the possibility of requiring one dose de-escalation. Cohorts of 3 to 6 patients will be treated on each dose level. The MTD is defined as the dose level immediately below the 73

154 level at which 33% of patients in a cohort experience a DLT. The MTD will be defined based on toxicities observed during the first three treatment cycles in order to capture toxicities, which occur after more prolonged administration. A patient will be considered evaluable for definition of the MTD if at least 85% of the prescribed dose has been administered to the patient during the first three treatment cycles based on adherence diary review and pill count of returned drug. In addition, any patient who receives one or more doses and experiences a DLT will be considered evaluable for definition of the MTD. After dose escalation to 30 mg/m 2 /dose, two of six patients experienced DLT and the MTD was determined to have been exceeded. The MTD in this trial was subsequently determined to be 20 mg/m 2 /dose. At the same time the MTD in a PBTC study was determined to be slightly higher with 25 mg/m 2 /dose. To be consistent with pediatric dosing, we are therefore adding the 25 mg/m 2 /dose as an additional dose level. Provided this dose is tolerated, the pediatric MTD of selumetinib will be 25 mg/m 2 /dose in NF1 and PN and refractory brain tumors. Patient accrual will be expanded at the MTD to, if feasible, a total of 12 patients in order to attempt to include 6 patients 12 years of age, and 6 patients 13 years of age to more fully characterize the toxicities and pharmacokinetics of selumetinib at the MTD. If 3 patients are enrolled at each of three dose levels with 12 patients at dose level 4 (or MTD), the minimum number of patients to be accrued is 21. The maximum number of patients required to complete this trial if 6 patients are accrued at each of three dose levels with 12 at dose level 4 (or MTD) will therefore be 30. If enrollment is approximately 2 patients per month, the primary trial objectives will likely be determined within approximately 15 months. The primary objective of the phase I portion of this study is to determine the tolerability, toxicity and MTD of selumetinib in pediatric patients with NF1 and inoperable PN. Pharmacokinetic and pharmacodynamic endpoints will be studied. As a phase 1 protocol, this study is largely exploratory, precluding formal statistical comparisons of treatment groups. When appropriate, summary statistics will be generated. Toxicity and tolerability will be measured using CTCAE- 4.0 and summarized by dose level. Prolonged grade 2, 3 and 4 toxicities attributable to selumetinib will be presented as the frequency and percentage of patients for each dose level. Pharmacokinetic analysis will be conducted using non-compartmental methods and estimated pharmacokinetic parameters including AUC, clearance, half-life and volume of distribution presented for each dose level using summary statistics (mean, standard deviation, median and range). Accumulation of selumetinib will be evaluated by defining the ratio of the AUC0-, which equals the AUC0-12h at steady state, to the AUC0-12h after the first dose. As the values for the correlative markers are all quantitatively measured, the average values obtained prior to treatment and at steady state will be compared using a Wilcoxon signed rank test by dose level. Correlations will be made to toxicity and response if feasible. With information obtained from patient diaries, adherence will be calculated as follows: [(scheduled doses missed doses) / scheduled doses] X 100. For pill counts, the number of pills returned to clinic will be subtracted from the number of pills dispensed at the previous visit. That number will be divided by the number of pills the patient should have taken according to their prescribed dosing schedule, and then multiplied by 100 to obtain the adherence percentage. The toxicities in pediatric patients on chronic dosing of selumetinib will be collected on all subjects during and after treatment and will be reported descriptively Statistics and feasibility for Phase II Primary objectives: The primary objective, to evaluate the confirmed partial and complete 74

155 response rate (see section 5.2) of selumetinib in children and young adults with NF1 PN using volumetric MRI analysis, will be assessed in the patients with PN related morbidity as the primary endpoint, and in the overall population of all patients treated (patients with and without PN related morbidity at the time of enrollment) as an important secondary endpoint. Subjects who have completed any protocol derived therapy (as little as one dose) will be considered evaluable for response for the primary phase II objective. Patients will be enrolled on 1 of 2 strata based on the presence (stratum 1) or absence (stratum 2) of PN related morbidity at baseline, prior to treatment. It is anticipated that a high fraction of all patients will have PN related morbidity, and it is of primary interest to report the response rates for those in stratum 1, and of nearly as great importance for patients in both strata combined. The primary endpoint will be the proportion of patients in stratum 1 in the phase II portion of the trial who achieve a confirmed partial response or complete response (see section 5.2), and will be presented with corresponding 2-sided exact 95% CIs. The primary analysis will be conducted after all patients have completed a minimum of 12 months follow-up, unless they have progressed prior to this. An analysis based on patients in both strata will also be presented in a similar fashion. The sample size for the primary objective, that is, in patients with PN related morbidity, will be based on a target response rate to exclude 15% with a lower two-sided 95% confidence bound. With 50 total evaluable symptomatic patients, an exact binomial test with a nominal one-sided 2.5% significance level will have 90% power to detect the difference between a null hypothesis response rate of 15% and an alternative hypothesis response rate of 36%. With 14 or more responses out of the 50 patients, the lower limit of the exact two-sided 95% confidence interval for the response rate will be 16.2% or greater. Thus, 14 or more responses in 50 evaluable patients is consistent with results which would statistically significantly exceed a 15% true response rate based on a two-sided confidence interval. The study will intend to enroll patients until a total of 50 evaluable patients with PN related morbidity have been enrolled (stratum 1), regardless of the number of asymptomatic patients enrolled during this same period. It is expected that no more than 75 total patients would enroll before 50 patients with PN related morbidity have enrolled. For this key secondary endpoint, with 75 patients in the overall population, 19 or more responses in these 75 patients (a response rate of 25%) would be associated with a lower bound of a two-sided 95% CI which exceeds 15.0%. If fewer than 75 are enrolled before 50 patients with PN related morbidity have enrolled, the lower 95% CI bound will also be constructed and discussed relative to ruling out a 15% response rate. Other Secondary objectives: Descriptive summaries of the secondary outcome variables will be presented at the time of the primary analysis as outlined below, and will be repeated in subsequent analyses to characterize the extent to which changes are maintained over a longer follow-up period. The overall response will be summarized by category (Unconfirmed PR, Confirmed PR, sustained PR, CR, SD or PD) for patients with PN related morbidity (stratum 1) as well as all patients together (stratum 1 and 2), as well as the duration of response, and the response rates for progressive (PN volume increase 20% within 15 months prior to enrollment) and non-progressive PN. See section 5.2 for response criteria. In addition, response rates will be reported separately for stratum 2 (no PN related morbidity at baseline), as well as for typical PN versus nodular PN versus solitary nodular PN (see section 3.1) as secondary endpoints. If feasible, the volumes of the nodular and typical components of the 75

156 target PN will be analyzed separately. This would allow analyzing the response to selumetinib in nodular and typical PN components within individual patients. Each of these will be reported with appropriate confidence intervals as well as the observed fractions; the response results for patients without PN related morbidity will be considered important as a secondary evaluation although recognizing that it may be based on fewer than 25 patients. In summary, because a minority of enrolled patients are expected to have no PN related morbidity, these patients will primarily be included as a part of the overall primary response rate, and any evaluations on these patients in this regard may be considered more secondary and hypothesis generating, while those for the overall group and the patients with PN related morbidity may be considered to be more definitive. This study will also intend to address a set of other secondary, exploratory objectives. Effect of selumetinib on pain, quality of life, and functional outcomes: Changes in pain, quality of life, and physical function will be assessed as described in Appendices V symptom checklist, VII- X functional evaluation forms, and XI pain, quality of life, and physical functioning PRO forms. Changes noted will be assessed, focusing on descriptive measures of the change and primarily using confidence intervals to describe the results. For functional evaluations, patients will be classified in 1 or more of the following categories at entry, based on the location of the target PN (Appendix VI): Orbital PN, Airway PN, PN causing or with the potential to cause motor dysfunction, PN causing or with the potential to cause bowel/bladder dysfunction, and other PN. Changes in the appropriate categories of impairment will be assessed for affected individuals and reported descriptively as a minimum. In particular, reductions in morbidity for stratum 1 patients will include analyses of any decreases in patient reported pain and functional improvements, whether by PRO or clinical evaluation, for the 4 major PN classifications (motor weakness, airway, orbital, bowel/bladder). If sufficient patients manifest a given condition, appropriate paired statistical tests or confidence intervals based on continuous or categorical changes will be used to further describe the potential effect of selumetinib on the particular condition. Specifically, once patient responses have been determined, the data on the changes over time in pain, QOL, and functional outcomes will be evaluated relative to the degree of decrease in PN volume. Depending on the magnitudes of responses identified, the patients may be grouped into categories based on the percent decrease in PN volume and summary statistics as well as formal statistical analyses may be performed to describe the changes in the PRO and functional outcomes as they relate to degree of response by volumetric MRI analysis. The results may also be compared formally or informally between categories formed on the basis of the degree of response. As these evaluations may be performed in a post hoc manner based on the response results obtained, the findings will need to be carefully presented in the context of the exploratory nature of the analyses undertaken. As a general strategy, in patients enrolled on stratum 1, improvement or worsening of PROs or function will be monitored, and in patients enrolled on stratum 2, the development of morbidities will be monitored. For patients with MRI studies amenable to volumetric MRI analysis prior to enrollment on this trial, we will compare the PN growth rates (Percent PN volume increase per year) prior to treatment and on treatment with selumetinib. For patients with functional evaluations prior to enrollment (for example in patients enrolled on the NCI NF1 natural history study 08-C-0079), these will be collected, and the development of morbidity retrospectively analyzed. 76

157 Time to progression will be evaluated using Kaplan-Meier curves; this will be informally compared to the PFS from the placebo arm of the R randomized trial conducted at the NCI, including potential use of a log-rank test and/or Cox model if the curves appear to be distinct from one another. Additionally, a subset of patients treated at the NCI will have DEXA scans done at baseline and at 1 year on treatment to evaluate the effect of selumetinib on bone mineral density. Each of these secondary endpoints will be assessed in a hypothesis generating manner, without formal adjustment for multiple comparisons but reported in the context of the number and type of evaluation being performed. Because children with NF1 may have an increased risk for long term toxicities of selumetinib or for the development of second neoplasms, a long-term safety follow-up for a duration of 7 years from the initiation of treatment or 5 years after completion of selumetinib, whichever takes longer, will take place. To the extent possible, all subjects treated on the phase II portion of this study will undergo the safety evaluations detailed in Appendix XV. These results will be summarized and any occurrences of second neoplasms or other serious conditions will be reported as described in Section 7.3. Futility Assessment: To ensure that the study does not continue in the event that responses are much more rare than anticipated, the trial will also include an early stopping rule. The phase II portion of the trial will no longer accrue patients as soon as it can be determined that there are no responses in the first 15 evaluable patients, regardless of how many patients have been enrolled. Thus, accrual is permitted until this determination has been made Total Patient Accrual and Enrollment The phase I portion of the study enrolled 24 patients and is complete. The phase II component of the study will require a total of 50 patients with PN related morbidity and no more than 75 total evaluable patients overall since a smaller subset of patients (up to 25) will be enrolled on stratum 2. It is expected that >2/3 will be symptomatic, and enrollment to phase II will end when 50 evaluable symptomatic patients have been enrolled. In order to allow for a small number of inevaluable patients, the accrual ceiling will be set at 80 patients for phase II. Although accrual may be brisk initially because of the likely presence of a group of patients waiting to receive treatment, this may slow down eventually. After approval of the amendment at participating sites we expect to enroll up to 50 patients per year. At least 30 patients will be between the ages of 6 and 18 years old. Since up to 80 total patients may be required for the Phase II portion in order to enroll 50 evaluable symptomatic patients, the maximum patient enrollment is 104 patients who will be enrolled on the phase I/II components, although the actual number enrolled will likely be less since the phase II portion will end enrollment when 50 evaluable symptomatic patients have been enrolled and a response determination made. Enrollment to the phase II portion will be completed within 1-2 year, but subjects will be followed for duration of sustained response, symptom or function improvement, and longer-term quality of life and progression free survival which may require follow up for up to 5 years after the last patient is enrolled. In addition, patients enrolled on the phase II portion of the study will be followed for long-term safety for 5 years after completing study drug treatment or a 77

158 duration of 7 years, whichever is longer (Appendix XV). This study is therefore expected to remain open for at least 9 years. 5.5 DATA AND CENTER AUDITS Missing or spurious information and protocol deviations will be communicated in a report. Protocol deviations, which may result in compromise to safely administer study drug, or to determine study endpoints will be included in the annual protocol review for NCI IRB. Volumetric MRI analysis used to determine disease progression will be performed centrally at the NCI, POB (Appendix IIA). Plasma pharmacokinetics and pharmacodynamics will also be evaluated as described in Appendices XII (PK), V-XI (pain, quality of life, function), XIII (blood progenitor and cytokines). Adherence will be monitored per Appendix XVI Data And Safety Monitoring Plan The PI will monitor the clinical outcome in each patient treated on the trial, and the overall safety and efficacy of the drug at each dose level including the phase II recommended dose. The NCI PI, protocol lead associate investigator, responsible research nurses, and data manager will meet weekly to review enrollment, observed toxicities, and responses. A monthly call will be held with the institutional PIs of the participating institutions. Long term safety will be assessed regularly and communicated with the NCI IRB, and with CTEP. Safety concerns, new information that might affect either the ethical and/or scientific conduct of the trial, or protocol violations will be immediately forwarded to the IRB of the participating site and the PI of the coordinating center in written form. This information will be conveyed to all participating investigators. This study is monitored by CTEP via quarterly data submissions in CDUS. On-site audits will be conducted at all participating sites by the Clinical Trials Monitoring Service (Theradex). 5.6 STORAGE, TRACKING AND HANDLING OF RESEARCH SPECIMENS This study will be coordinated by the Pediatric Oncology Branch, NCI. Sample labeling, collection, processing, and shipment will be conducted as in described in Section 3.4, Appendices XII (PK) and XIII (PD). S Samples will be processed and stored in Doug Figg s lab prior to shipping samples according to the applicable Appendix. Please page (Figg lab) for immediate pick-up upon drawing study samples. The Clinical Pharmacology Program (CPP) processing group in 10/5A09 at or can be contacted with any questions. The samples will be processed, barcoded, and stored in Dr. Figg s lab until requested by the investigator. Samples, which require shipment to outside institutions, will be sent as directed in the Appendices XII (PK) and XIII (PD) by Dr. Figg s laboratory with labels that specify Protocol ID, Patient study ID number, Patient Initials, Time Point, and collection date (DD-MMM-YYYY) and sample type, or the pre-printed labels as specified in Appendices XII (PK) for PK samples. 78

159 5.6.1 Sample Data Collection All samples sent to the Clinical Pharmacology Program (CPP) will be barcoded, with data entered and stored in the Patient Sample Data Management System (a.k.a LabSamples) utilized by the CPP. This is a secure program, with access to LabSamples limited to defined CPP personnel, who are issued individual user accounts. Installation of LabSamples is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. All CPP personnel with access to patient information annually complete the NIH online Protection of Human Subjects course. LabSamples creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without LabSamples access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location, and date samples are sent to the investigator or shipped to outside institutions. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.) Sample Storage and Destruction Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80 C according to stability requirements. These freezers are located onsite in the CPP and offsite at NCI Frederick Central Repository Services (Fisher Bioservices) in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in LabSamples. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned to the CPP. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval. Following completion of this study, samples will remain in storage as detailed above. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material. If, at any time, a patient withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed (or returned to the patient, if so requested), and reported as such to the IRB. Any samples lost (in transit or by a researcher) or destroyed due to unknown sample integrity (i.e. broken freezer allows for extensive sample thawing, etc.) will be reported as such to the IRB. Sample barcodes are linked to patient demographics and limited clinical information. This information will only be provided to investigators listed on this protocol, via registered use of the LabSamples. It is critical that the sample remains linked to patient information such as race, age, dates of diagnosis and death, and histological information about the tumor, in order to correlate genotype with these variables. The study will remain open and status reported to the NCI-IRB until all samples have been analyzed, reported or destroyed. Unintentional loss or destruction of any samples will be 79

160 reported to the NCI-IRB as part of annual continuing reviews. Any use of these samples for purposes not described in this protocol will require prospective NCI-IRB review and approval. Data from patient reported outcomes and functional endpoints will be sent to POB NCI for data analysis. All data will be identified using the study number; no personal identifying information should be sent to NCI. 6 HUMAN SUBJECTS PROTECTIONS 6.1 RATIONALE FOR SUBJECT SELECTION Neurofibromatosis type 1 is a genetic disorder with a worldwide incidence of 1:3500 individuals. No groups, in regards to gender, and racial and ethnic groups, are being excluded from participation in the trial. Females who are pregnant or breastfeeding will not be eligible for the trial due to risks of fetal and teratogenic adverse events as seen animal studies. This trial is designed to define the qualitative toxicities, maximum tolerated dose, and efficay of selumetinib in children with NF1 and PN and, therefore, children will be entered onto this research trial. 6.2 PARTICIPATION OF CHILDREN This trial is designed to define the qualitative toxicities, maximum tolerated dose, and efficacy of selumetinib in children and adolescents with PN and, therefore, children will be enrolled on this research trial. Pediatric oncologists who have extensive experience in performing investigational drug trials in children will conduct the trial. Patients enrolled at the POB, NCI will be cared for in the POB outpatient pediatric oncology clinic or outpatient setting, and if patients require hospital admission, they will be cared for on the pediatric unit of the CRC by the POB staff. 6.3 RISKS AND BENEFITS Patients under the age of 18 years will be entered on this trial. The primary risk to patients participating in this research study is from toxicity of selumetinib, an investigational agent. The protocol provides for detailed and careful monitoring of all patients to assess for toxicity and the dose escalation scheme is very conservative. Toxicity data from the current dose level will be collected and reviewed to ensure that there were no severe (dose-limiting) toxicities prior to escalating to the next higher dose level. A total of 3 cycles will be followed to determine doselimiting toxicity due to the increase of possibility of chronic and/or cumulative toxicity such as rash. Also, in order to minimize potential risk due to long-term and cumulative toxicity, treatment will be limited according to Section In the phase I portion, the primary objective of the phase I trial was to define the qualitative toxicities and maximum tolerated dose of selumetinib. In addition, 14 of 24 patients had a partial response. Responses have been maintained in patients who did not require dose reductions for toxicity, while no patient has experienced progressive disease to date. Therefore in the phase II portion we believe that selumetinib may offer a potential for direct benefit. The potential benefits from this therapy are disease stabilization or shrinkage and a reduction in symptoms caused by the tumor. Pain, quality of life (QOL), and function have not been evaluated prospectively in the patients on the Phase I portion of this trial, but several patients reported a decrease in pain, and one patient reported a functional improvement with improved ability to lift her arm. Furthermore, by just entering the study, patients will be followed very carefully, in an organized coherent manner, which may also benefit their overall healthcare. 80

161 Other risks: Patients of Asian descent may experience a higher exposure of selumetinib than the majority of subjects of non-asian descent receiving an equivalent dose of selumetinib. These patients will receive standard dosing but will be counselled regarding this higher drug exposure and urged to contact investigators if they are experiencing any side effects. If, during the course of the selumetinib development program the dosing regimen being evaluated in this study is found not to be tolerated in a specific ethnic group, this ethnic group may later be excluded from this study. Investigators will be notified, and the protocol will be amended to reflect such findings. The data so far do not suggest a safety concern in any specific population. The PRO measures will be reviewed by a member of the Health Psychology group, POB, NCI, who administered the forms to the patient. If any responses are noted that suggest a severe level of emotional distress (e.g., on the Emotional Functioning scale of the Adult PedsQL NF1 module), the responses will discussed with one of the AIs, Drs. Wolters or Peron, and if in agreement, the PI will be notified. Appropriate actions to follow up on the concerning responses could include discussing these concerns with the participant, referral for a psychiatry consult while at the NIH, and/or providing referrals for therapeutic services in the participant s local area. Therefore, this research study, at this time, involves greater than minimal risk but presents the prospect of direct benefit to the individual (child and adult) subjects involved in the research. 6.4 CONSENT AND ASSENT PROCESS AND DOCUMENTATION The investigational nature and research objectives of this trial, the procedures and treatments involved and their attendant risks and discomforts and benefits, and potential alternative therapies will be carefully explained to the patient or the patient s parents or guardian if he/she is a child, and a signed informed consent document will be obtained prior to entry onto the study. The investigators are requesting a waiver from the IRB to allow only one parent to sign the informed consent to enter a child on the protocol. Because many patients must travel to the NIH from long distances at substantial expense, requiring both parents to be present for the consent process could be a financial hardship for many families. The PI or an associate investigator of the trial will obtain consent. Where deemed appropriate by the clinician and the child s parents or guardian, the child will also be included in all discussions about the trial and verbal assent will be obtained. The parent or guardian will sign the designated line on the informed consent attesting to the fact that the child has given assent. 7 DATA REPORTING All data collection and reporting will be coordinated through: Pediatric Oncology Branch, NCI c/o Trish Whitcomb, RN Fax: Phone: Center Drive, Building 10, CRC Room 1W-5750 Bethesda, MD

162 7.1 PATIENT REGISTRATION See Section CASE REPORT FORMS The POB will provide case report forms for recording relevant clinical data for each patient enrolled on the trial (5.0). Patient diaries (Appendix XIV) should be provided to all patients and their families and reviewed as detailed in Appendix XVI. Data will be entered into the NCI, CCR C3D database by participating sites and into the Labmatrix database by the NCI personnel. 7.3 SAFETY REPORTING DEFINITIONS An adverse event is any reaction, side effect, or untoward event that occurs during the course of the clinical trial associated with the use of a drug in humans, whether or not the event is considered related to the treatment or clinically significant. For this study, AEs will include events reported by the patient, as well as clinically significant abnormal findings on physical examination or laboratory evaluation. A new illness, symptom, sign or clinically significant laboratory abnormality or worsening of a pre-existing condition or abnormality is considered an AE. All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days following the last dose of study drug. AEs that are considered treatment related, expected, continuing, but not resolvable by 30 days after treatment completion (e.g., alopecia) will not be followed after the 30-day period. An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following: Results in discontinuation from the study Is associated with clinical signs or symptoms Requires treatment or any other therapeutic intervention Is associated with death or another serious adverse event, including hospitalization Is judged by the Investigator to be of significant clinical impact If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient s outcome. Life-threatening adverse events are any adverse events that places the patient or subject, in view of the investigator, at an immediate risk of death from the reaction as it occurred, i.e., it does not include a reaction that had it occurred in a more severe form, might have caused death. Serious: An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious Adverse Event or if it compromises the safety, welfare or rights of subjects or others. Serious adverse events are any adverse events occurring at any dose that result in any of the following outcomes: Death, a life-threatening adverse event, inpatient hospitalization or 82

163 prolongation of existing hospitalization, a persistent or significant disability/incapacity, a congenital anomaly/birth defect. Important medical events that may not result in death, be lifethreatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Suspected adverse reaction: Suspected adverse reaction means any adverse event for which there is a reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, reasonable possibility means there is evidence to suggest a causal relationship between the drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than adverse reaction, which means any adverse event caused by a drug. Unexpected adverse events are any adverse events, which are not listed in the Investigator Brochure and informed consent for this trial, or are not listed at the specificity or severity that has been observed; or are not consistent with the risk information described in the general investigational plan or elsewhere in the current application. Known toxicities observed to date are listed in Section "Unexpected, also refers to adverse events or suspected adverse reactions that are mentioned in the Investigator Brochure as occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically mentioned as occurring with the particular drug under investigation. Disability is a substantial disruption of a person s ability to conduct normal life functions. Protocol Deviation (NIH Definition): A protocol deviation is any change, divergence, or departure from the IRB-approved research protocol. To accommodate changes in patients schedules, holidays, and provide flexibility for long term follow up, any change or divergence in the scheduling or conduct of examinations, tests and scans or sporadic missing vital signs or laboratory tests described in this protocol will NOT be considered a protocol deviation and will not require reporting to the NCI IRB or FDA unless it meets the definition of unanticipated problem or serious non-compliance. Deviations will require reporting to the NCI IRB if there is a pattern that elevates the event(s) to the level of an unanticipated problem, i.e. consistent disregard for meeting protocol requirements or scheduled assessments. Similarly, 1) isolated missed lab values or missed selumetinib doses will be documented, but not be reported as a protocol deviation, 2) isolated missing lab values will be noted, but not reported as protocol deviation, and 3) differences in the timing of the PK samples from the target time and missing PK samples will be recorded on the PK sheet, but not reported as protocol deviation. Routine deviations will be reported in aggregate at the time of the NCI IRB continuing review. Non-compliance (NIH Definition): The failure to comply with applicable NIH Human Research Protections Program (HRPP) policies, IRB requirements, or regulatory requirements for the protection of human research subjects. Unanticipated Problem: Any incident, experience, or outcome that: Is unexpected in terms of nature, severity, or frequency in relation to (a) the research risks that are described in the IRB-approved research protocol and informed consent document; Investigator s Brochure or other study documents, and (b) the characteristics of the subject population being studied; AND Is related or possibly related to participation in the research; AND 83

164 Suggests that the research places subjects or others at a greater risk of harm (including physical, psychological, economic, or social harm) than was previously known or recognized. All observed or volunteered adverse events, regardless of causal relationship to study drug, will be recorded on the Adverse Event page(s) of the case report form. Events involving adverse drug reactions, illnesses with onset during the study, or exacerbation of pre-existing illnesses should be recorded. Objective test findings (e.g., electrocardiogram changes, abnormal laboratory test results) that result in a change in study drug dosage should also be recorded CTEP ADVERSE EVENT REPORTING REQUIREMENTS of ALL SITES Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. The following list of AEs (Section 8.1.2) and the characteristics of an observed AE (Section ) will determine whether the event requires expedited reporting via the CTEP Adverse Event Reporting System (CTEP-AERS) in addition to routine reporting Adverse Event Characteristics CTCAE term (AE description) and grade: The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site For expedited reporting purposes only: - AEs for the agent that are bold and italicized in the CAEPR (i.e., those listed in the SPEER column, Section 8.1.2) should be reported through CTEP-AERS only if the grade is above the grade provided in the SPEER. - Other AEs for the protocol that do not require expedited reporting are outlined in section Hospitalization for management of complications unrelated to study drug will not require expedited reporting. Attribution of the AE: - Definite The AE is clearly related to the study treatment. - Probable The AE is likely related to the study treatment. - Possible The AE may be related to the study treatment. - Unlikely The AE is doubtfully related to the study treatment. - Unrelated The AE is clearly NOT related to the study treatment. 84

165 Expedited Adverse Event Reporting Expedited AE reporting for this study must use CTEP-AERS (CTEP Adverse Event Reporting System), accessed via the CTEP Web site ( The reporting procedures to be followed are presented in the NCI Guidelines for Investigators: Adverse Event Reporting Requirements for DCTD (CTEP and CIP) and DCP INDs and IDEs which can be downloaded from the CTEP Web site ( ts.htm). These requirements are briefly outlined in the tables below. In the rare occurrence when Internet connectivity is lost, a 24-hour notification is to be made to CTEP by telephone at Once Internet connectivity is restored, the 24-hour notification phoned in must be entered electronically into CTEP-AERS by the original submitter at the site. CTEP-AERS is programmed for automatic electronic distribution of reports to the following individuals: Principal Investigator and Adverse Event Coordinator(s) (if applicable) of the Corresponding Organization, the local treating physician, and the Reporter and Submitter. CTEP-AERS provides a copy feature for other recipients. The Coordinating Center of the Corresponding Organization is responsible for submitting to the CTSU documentation of AEs that they deem reportable for posting on the CTSU protocol web page and inclusion on the CTSU bi-monthly broadcast. Expedited Reporting Guidelines Use the NCI protocol number and the protocol-specific patient ID assigned during trial registration on all reports. Note: A death on study requires both routine and expedited reporting regardless of causality, unless as noted below. Attribution to treatment or other cause must be provided. Death due to progressive disease should be reported as Grade 5 Neoplasms benign, malignant and unspecified (incl cysts and polyps) - Other (Progressive Disease) under the system organ class (SOC) of the same name. Evidence that the death was a manifestation of underlying disease (e.g., radiological changes suggesting tumor growth or progression: clinical deterioration associated with a disease process) should be submitted. Phase 1 and Early Phase 2 Studies: Expedited Reporting Requirements for Adverse Events that Occur on Studies under an IND/IDE within 30 Days of the Last Administration of the Investigational Agent/Intervention 1, 2 FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) NOTE: Investigators MUST immediately report to the sponsor (NCI) ANY Serious Adverse Events, whether or not they are considered related to the investigational agent(s)/intervention (21 CFR ) An adverse event is considered serious if it results in ANY of the following outcomes: 1) Death 2) A life-threatening adverse event 3) An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for 24 hours 4) A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions 5) A congenital anomaly/birth defect. 6) Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 85

166 may be considered serious when, based upon medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. (FDA, 21 CFR ; ICH E2A and ICH E6). ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the NCI via electronic submission within the timeframes detailed in the table below. Hospitalization Resulting in Hospitalization 24 hrs Not resulting in Hospitalization 24 hrs Grade 1 and Grade 2 Timeframes 10 Calendar Days Not required Grade 3-5 Timeframes 24-Hour 5 Calendar Days NOTE: Protocol specific exceptions to expedited reporting of serious adverse events are found in the Specific Protocol Exceptions to Expedited Reporting (SPEER) portion of the CAEPR. Expedited AE reporting timelines are defined as: o 24-Hour; 5 Calendar Days - The AE must initially be submitted electronically within 24 hours of learning of the AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour report. o 10 Calendar Days - A complete expedited report on the AE must be submitted electronically within 10 calendar days of learning of the AE. 1 Serious adverse events that occur more than 30 days after the last administration of investigational agent/intervention and have an attribution of possible, probable, or definite require reporting as follows: Expedited 24-hour notification followed by complete report within 5 calendar days for: All Grade 3, 4, and Grade 5 AEs Expedited 10 calendar day reports for: Grade 2 AEs resulting in hospitalization or prolongation of hospitalization 2 For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-lives, rounded UP to the nearest whole day, after the agent/intervention was last administered. Footnote 1 above applies after this reporting period. Effective Date: May 5, Routine Adverse Event Reporting All Adverse Events must be reported in routine study data submissions. AEs reported expeditiously through CTEP-AERS must also be reported in routine study data submissions. Adverse event data collection and reporting, which are required as part of every clinical trial, are done to ensure the safety of patients enrolled in the studies as well as those who will enroll in future studies using similar agents. AEs are reported in a routine manner at scheduled times during the trial using C3D which sends data to CTEP. 86

167 Secondary Malignancy A secondary malignancy is a cancer caused by treatment for a previous malignancy (e.g., treatment with investigational agent/intervention, radiation or chemotherapy). A secondary malignancy is not considered a metastasis of the initial neoplasm. CTEP requires all secondary malignancies that occur following treatment with an agent under an NCI IND/IDE be reported expeditiously via CTEP-AERS. Three options are available to describe the event: Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic leukemia) Myelodysplastic syndrome (MDS) Treatment-related secondary malignancy Any malignancy possibly related to cancer treatment (including AML/MDS) should also be reported via the routine reporting mechanisms outlined in each protocol Second Malignancy A second malignancy is one unrelated to the treatment of a prior malignancy (and is NOT a metastasis from the initial malignancy). Second malignancies require ONLY routine AE reporting unless otherwise specified NCI Guidance for Reporting Expedited Adverse Events for Multi-Center Trials The participating site PI must immediately report to the Coordinating Center PI through the AERS system any serious adverse event, whether or not considered drug related, including those listed in the protocol or investigator brochure and must include an assessment of whether there is a reasonable possibility that the drug caused the event within 48 hours of PI awareness of the event. The participating site PI must also report any protocol deviations or violations (as defined by their IRB) to the Coordinating Center PI within 7 days of PI awareness. Participating centers must also submit the report to their respective IRB in accordance with their institutional policies NCI-IRB Reporting at the Coordinating Center NCI-IRB Expedited Reporting of Unanticipated Problems, and Deaths The Coordinating Center PI will be responsible for reporting all SAEs to the IRB of the Coordinating Center. The PI of the Coordinating Center will report to the NCI-IRB: All deaths, except deaths due to progressive disease All Protocol Deviations All Unanticipated Problems All serious non-compliance (non-compliance that increases risks or causes harm to participants, decreases potential benefits to participants, compromises the integrity of the NIH HRPP, or invalidates study data) Reports must be received by the NCI-IRB within 7 working days of PI awareness via iris NCI-IRB Requirements for PI Reporting at Continuing Review The Coordinating Center PI is responsible for reporting all AEs occurring on this study to the IRB 87

168 of the Coordinating Center at the time of the continuing review. The PI of the Coordinating Center will report to the NCI-IRB: 1. A summary of all protocol deviations in a tabular format to include the date the deviation occurred, a brief description of the deviation and any corrective action. 2. A summary of any instances of non-compliance 3. A tabular summary of the following adverse events: All Grade 2 unexpected events that are possibly, probably, or definitely related to the research; All Grade 3 and 4 events that are possibly, probably, or definitely related to the research; All Grade 5 events regardless of attribution; All Serious Events regardless of attribution. NOTE: Grade 1 events are not required to be reported NCI-IRB Reporting of IND Safety Reports Only IND Safety Reports that meet the definition of an unanticipated problem will be reported to the NCI IRB. (See Appendix XIX for responsibilities of the NCI PI as the PI of the Coordinating Center). 8 PHARMACEUTICAL INFORMATION 8.1 AZD6244 HYDROGEN SULFATE (NSC ) Chemical name (IUPAC): 6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3Hbenzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide Other Names: Selumetinib; ARRY ; AR ; AR Chemical Structure: Classification: Mitogen-activated protein kinase kinase (MEK) inhibitor CAS Registry Number: Molecular Formula: C17H15BrClFN4O3 M.W.:

169 Solubility: Solubility in Captisol approximately 1 mg/ml at 25 C. Very low aqueous solubility (3.4 μg/ml at ph 7.4). Mode of Action: The RAS/RAF/MEK/ERK pathway is an important mediator of many cellular processes including proliferation, survival, differentiation, apoptosis, motility, and metabolism. This pathway is often aberrantly activated in human tumors due to the overexpression of activated K-RAS, mutant b-raf, or other growth factor receptors. Selumetinib is a selective mitogen-activated protein kinase kinase (MEK) inhibitor. By inhibiting MEK, selumetinib inhibits ERK phosphorylation. Thus, selumetinib may inhibit oncogenic growth signaling in tumor cells by targeting the RAS/RAF/MEK/ERK pathway. How Supplied: Astra Zeneca supplies and CTEP, NCI, DCTD distributes AZD6244 hydrogen sulfate. The agent is supplied as size 4 hydroxypropylmethylcellulose (HPMC) capsules available in 10 mg (plain white) and 25 mg (blue) strengths. Capsules are packaged in white, high density polyethylene (HDPE) containers with induction-seals and child-resistant closures. Each bottle contains 60 capsules. AZD6244 hydrogen sulfate capsules contain a dispersion of drug in d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS; a water soluble form of vitamin E). Each 10 mg capsule contains 32.4 TPGS and each 25 mg capsule contains 35.9 mg TPGS. Storage: Storage of selumetinib 10mg capsules must be stored at room temperature (specifically below 30 C although the US pack says 20 to 25 C). The temperature of shipments must also be monitored to ensure the temperature is within the required limits (below 30 C). Data is available to support the 10mg capsule being stored at 30 C or less and that performance could be reduced if stored at temperatures higher than 30 C. Stability: Stability studies are ongoing. If a storage temperature excursion is identified, promptly return AZD6244 hydrogen sulfate to controlled room temperature and quarantine the supplies. Provide a detailed report of the excursion (including documentation of temperature monitoring and duration of the excursion) to PMBAfterHours@mail.nih.gov for determination of suitability. Route of Administration: Oral. Take selumetinib on an empty stomach (either 1 hour before or 2 hours after meals). Selumetinib capsules should be taken with water only. Potential Drug Interactions: High vitamin E doses may potentiate warfarin s anticoagulant activity. Monitor PT/INR more frequently in patients receiving both warfarin and AZD6244 hydrogen sulfate capsules. Avoid concomitant intake of Vitamin E in excess of 100% of the recommended daily dose. Agent Ordering and Agent Accountability: NCI supplied agents may be requested by the Principal Investigator (or their authorized designee) at each participating institution. Pharmaceutical Management Branch (PMB) policy requires that agent be shipped directly to the institution where the patient is to be treated. PMB does not permit the transfer of agents between institutions (unless prior approval from PMB is obtained). The CTEP assigned protocol number must be used for ordering all CTEP supplied investigational agents. The responsible investigator at each participating institution must be registered with CTEP, DCTD through an annual submission of FDA form 1572 (Statement of Investigator), Curriculum Vitae, Supplemental Investigator Data Form (IDF), and Financial Disclosure Form (FDF). If there are several participating investigators 89

170 at one institution, CTEP supplied investigational agents for the study should be ordered under the name of one lead investigator at that institution. Active CTEP-registered investigators and investigator-designated shipping designees and ordering designees can submit agent requests through the PMB Online Agent Order Processing (OAOP) application < >. Access to OAOP requires the establishment of a CTEP Identity and Access Management (IAM) account < > and the maintenance of an active account status and a current password. For questions about drug orders, transfers, returns, or accountability, call (240) Monday through Friday between 8:30 am and 4:30 pm (ET) or PMBAfterHours@mail.nih.gov anytime. Agent Inventory Records - The investigator, or a responsible party designated by the investigator, must maintain a careful record of the inventory and disposition of all agents received from DCTD using the NCI Drug Accountability Record Form (DARF). (See the NCI Investigator s Handbook for Procedures for Drug Accountability and Storage.) Selumetinib Administration Selumetinib is administered orally, twice daily on a continuous dosing schedule with no rest period between cycles (unless dose is modified as per section 3.3). A cycle of selumetinib is defined as 28 consecutive days starting with the first day of treatment (day 1). The dose of selumetinib is determined from the patient s body surface area using the dosing nomogram (Appendix XVII). Treatment will be administered in an outpatient setting. Selumetinib should be taken with water (approximately 2 to 4 ounces for children < 12 and 4 to 8 ounces for 12 years) in the fasted state (no food or drink other than water for 2 hours before dosing or 1 hour after dosing). Capsules can be combined to accurately dose patients (e.g., a dose of 30 mg could be three 10 mg capsules). Patient Precautions: Patients should avoid donating blood whilst participating in studies of selumetinib and for at least 12 weeks after receiving the last dose of the study medications or for longer if required in accordance with the prescribing information for the comparator / concomitant / compulsory administered medications. Reproductive toxicology data indicate that selumetinib has the potential for adverse effects on embryofoetal development and survival. Therefore: o Females of child-bearing potential must use acceptable, effective and reliable methods of contraception from the time of screening until at least 4 weeks after discontinuing study treatment or longer if required, depending on the prescribing information of the combination or concomitantly administered medications. Selumetinib must not be administered to pregnant or breast-feeding women. Conception while on treatment must be avoided. o Male patients with sexual partners who are pregnant or who could become pregnant (i.e. women of child-bearing potential) must use acceptable, effective and reliable methods of contraception during the study and for at least 12 weeks after 90

171 the last dose of selumetinib or for longer if required, depending on the prescribing information of the combination or concomitantly administered medications. o Male and females of childbearing potential: reliable methods of contraception should be used consistently and correctly Selumetinib Toxicity Profile Acceptable effective methods of contraception for women include implants, injectables, combined oral contraceptives, some intrauterine devices/systems and sterilisation including vasectomy of the partner, all being used in combination with barrier methods of contraception (e.g. condoms). Acceptable methods of contraception for men include the use of condoms with spermicidal foams/gels or prior vasectomy. True sexual abstinence is also an acceptable method of contraception (for both men and women). Selumetinib (AZD6244 Free base [NSC ], AZD6244 Hydrogen sulfate [NSC ]) The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single list of reported and/or potential adverse events (AE) associated with an agent using a uniform presentation of events by body system. In addition to the comprehensive list, a subset, the Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column and is identified with bold and italicized text. This subset of AEs (SPEER) is a list of events that are protocol specific exceptions to expedited reporting to NCI via AdEERS (except as noted below). Refer to the 'CTEP, NCI Guidelines: Adverse Event Reporting Requirements' for further clarification. Frequency is provided based on 665 patients. Below is the CAEPR for selumetinib (AZD6244). NOTE: Report AEs on the SPEER ONLY IF they exceed the grade noted in parentheses next to the AE in the SPEER. If this CAEPR is part of a combination protocol using multiple investigational agents and has an AE listed on different SPEERs, use the lower of the grades to determine if expedited reporting is required. Version 2.4, February 26, Adverse Events with Possible Relationship to Selumetinib (AZD6244) (CTCAE 4.0 Term) [n= 665] Specific Protocol Exceptions to Expedited Reporting (SPEER) (formerly known as ASAEL) Likely (>20%) Less Likely (<=20%) Rare but Serious (<3%) BLOOD AND LYMPHATIC SYSTEM DISORDERS Anemia Anemia (Gr 3) CARDIAC DISORDERS Febrile neutropenia 2 Left ventricular systolic dysfunction Left ventricular systolic dysfunction (Gr 2) GASTROINTESTINAL DISORDERS Abdominal pain Abdominal pain (Gr 2) Constipation Constipation (Gr 2) Diarrhea 3 Diarrhea 3 (Gr 3) Dry mouth 91

172 Mucositis oral Mucositis oral (Gr 2) Nausea Nausea (Gr 3) Vomiting Vomiting (Gr 2) GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS Edema face Edema face (Gr 2) Edema limbs Edema limbs (Gr 2) Fatigue Fatigue (Gr 2) INVESTIGATIONS Fever Fever (Gr 2) Alanine aminotransferase increased Alkaline phosphatase increased Aspartate aminotransferase increased Platelet count decreased Alanine aminotransferase increased (Gr 3) Aspartate increased (Gr 3) METABOLISM AND NUTRITION DISORDERS Anorexia Anorexia (Gr 2) Hypoalbuminemia Hypomagnesemia NERVOUS SYSTEM DISORDERS Dizziness PSYCHIATRIC DISORDERS Headache Headache (Gr 2) Insomnia RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS Cough Cough (Gr 2) Dyspnea Dyspnea (Gr 2) SKIN AND SUBCUTANEOUS TISSUE DISORDERS Dry skin Dry skin (Gr 2) Pruritus Rash acneiform Rash acneiform (Gr 3) aminotransferase Rash maculo-papular Rash maculo-papular (Gr 2) VASCULAR DISORDERS Hypertension 1 This table will be updated as the toxicity profile of the agent is revised. Updates will be distributed to all Principal Investigators at the time of revision. The current version can be obtained by contacting PIO@CTEP.NCI.NIH.GOV. Your name, the name of the investigator, the protocol and the agent should be included in the . 2 Febrile neutropenia/neutropenic infection has been observed primarily in trials combining selumetinib (AZD6244) and docetaxel. 3 SBE-CD (Captisol, vehicle) in the mix and drink formulation is known to cause soft stools and/or diarrhea in rats and dogs; however, it is possible that some of these findings might be related to exacerbation of the vehicle effect by selumetinib (AZD6244). 4 Infection includes all 75 sites of infection under the INFECTIONS AND INFESTATIONS SOC. 92

173 Also reported on selumetinib (AZD6244) trials but with the relationship to selumetinib (AZD6244) still undetermined: BLOOD AND LYMPHATIC SYSTEM DISORDERS - Blood and lymphatic system disorders - Other (hemorrhagic anemia) CARDIAC DISORDERS - Acute coronary syndrome; Cardiac disorders - Other (Takatsubo cardiomyopathy syndrome); Chest pain - cardiac; Heart failure; Palpitations; Right ventricular dysfunction; Sinus bradycardia EYE DISORDERS - Blurred vision; Extraocular muscle paresis; Eye disorders - Other (bilateral macular edema); Eye disorders - Other (black haze in line of vision); Eye disorders - Other (elevated intraocular pressure); Flashing lights; Glaucoma; Retinopathy GASTROINTESTINAL DISORDERS - Abdominal distension; Ascites; Colitis; Dyspepsia; Esophagitis; Flatulence; Gastric hemorrhage; Gastroesophageal reflux disease; Ileal stenosis GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Flu like symptoms; Non-cardiac chest pain HEPATOBILIARY DISORDERS - Hepatic failure; Hepatobiliary disorders - Other (cholangitis) INFECTIONS AND INFESTATIONS - Infection 4 INVESTIGATIONS - CPK increased; Creatinine increased; Electrocardiogram QT corrected interval prolonged; Neutrophil count decreased; Weight gain METABOLISM AND NUTRITION DISORDERS - Dehydration; Hypokalemia; Hyponatremia MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Arthralgia; Back pain; Generalized muscle weakness; Musculoskeletal and connective tissue disorder - Other (joint swelling); Musculoskeletal and connective tissue disorder - Other (rhabdomyolysis); Myalgia; Myositis; Pain in extremity NERVOUS SYSTEM DISORDERS - Cognitive disturbance; Depressed level of consciousness; Dysgeusia; Dysphasia; Ischemia cerebrovascular; Leukoencephalopathy; Memory impairment; Nervous system disorders - Other (numbness); Nervous system disorders - Other (spinal cord compression); Oculomotor nerve disorder; Peripheral sensory neuropathy; Reversible posterior leukoencephalopathy syndrome; Seizure PSYCHIATRIC DISORDERS - Confusion; Depression RENAL AND URINARY DISORDERS - Acute kidney injury; Proteinuria RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Adult respiratory distress syndrome; Epistaxis; Hypoxia; Pharyngolaryngeal pain; Pleural effusion; Pneumonitis; Sore throat; Voice alteration SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Pain of skin; Palmar-plantar erythrodysesthesia syndrome; Photosensitivity; Scalp pain; Skin and subcutaneous tissue disorders - Other (angular cheilitis, unilateral); Skin and subcutaneous tissue disorders - Other (skin fissures) VASCULAR DISORDERS - Hypotension; Thromboembolic event Note: Selumetinib (AZD6244) in combination with other agents could cause an exacerbation of any adverse event currently known to be caused by the other agent, or the combination may result in events never previously associated with either agent. 8.2 COLLABORATIVE AGREEMENT Agreement Type Protocols that involve agent(s) covered by a collaborative agreement with a biotech/pharma company(ies) must incorporate the NCI/ DCTD Standard Language shown below. 93

174 The agent(s) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI under a Collaborative Agreement (CRADA, CTA, CSA) between the Pharmaceutical Company(ies) (hereinafter referred to as Collaborator(s) ) and the NCI Division of Cancer Treatment and Diagnosis. Therefore, the following obligations/guidelines, in addition to the provisions in the Intellectual Property Option to Collaborator contained within the terms of award, apply to the use of the Agent(s) in this study: 1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor can Agent(s) be transferred or licensed to any party not participating in the clinical study. Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and shall be maintained as such by the investigators. The protocol documents for studies utilizing Agents contain confidential information and should not be shared or distributed without the permission of the NCI. If a copy of this protocol is requested by a patient or patient s family member participating on the study, the individual should sign a confidentiality agreement. A suitable model agreement can be downloaded from: 2. For a clinical protocol where there is an investigational Agent used in combination with (an)other Agent(s), each the subject of different Collaborative Agreements, the access to and use of data by each Collaborator shall be as follows (data pertaining to such combination use shall hereinafter be referred to as Multi-Party Data ): a. NCI will provide all Collaborators with prior written notice regarding the existence and nature of any agreements governing their collaboration with NCI, the design of the proposed combination protocol, and the existence of any obligations that would tend to restrict NCI s participation in the proposed combination protocol. b. Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial by any other Collaborator solely to the extent necessary to allow said other Collaborator to develop, obtain regulatory approval or commercialize its own Agent. c. Any Collaborator having the right to use the Multi-Party Data from these trials must agree in writing prior to the commencement of the trials that it will use the Multi-Party Data solely for development, regulatory approval, and commercialization of its own Agent. 3. Clinical Trial Data and Results and Raw Data developed under a Collaborative Agreement will be made available to Collaborator(s), the NCI, and the FDA, as appropriate and unless additional disclosure is required by law or court order as described in the IP Option to Collaborator ( Additionally, all Clinical Data and Results and Raw Data will be collected, used and disclosed consistent with all applicable federal statutes and regulations for the protection of human subjects, including, if applicable, the Standards for Privacy of Individually Identifiable Health Information set forth in 45 C.F.R. Part

175 4. When a Collaborator wishes to initiate a data request, the request should first be sent to the NCI, who will then notify the appropriate investigators (Group Chair for Cooperative Group studies, or PI for other studies) of Collaborator s wish to contact them. 5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is a DMC for this clinical trial. 6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the Group office for Cooperative Group studies or by the principal investigator for non-cooperative Group studies for immediate delivery to Collaborator(s) for advisory review and comment prior to submission for publication. Collaborator(s) will have 30 days from the date of receipt for review. Collaborator shall have the right to request that publication be delayed for up to an additional 30 days in order to ensure that Collaborator s confidential and proprietary data, in addition to Collaborator(s) s intellectual property rights, are protected. Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for courtesy review as soon as possible and preferably at least three (3) days prior to submission, but in any case, prior to presentation at the meeting or publication in the proceedings. Press releases and other media presentations must also be forwarded to CTEP prior to release. Copies of any manuscript, abstract and/or press release/ media presentation should be sent to: ncicteppubs@mail.nih.gov The Regulatory Affairs Branch will then distribute them to Collaborator(s). No publication, manuscript or other form of public disclosure shall contain any of Collaborator s confidential/ proprietary information. 9 MULTI-INSTITUTIONAL GUIDELINES The PI of the Coordinating Center for a trial is responsible to ascertain that no patients are entered on the trial at a participating institution without full IRB approval. Thus, the NCI IRB must approve the addition of each participating institution to the protocol and will require a copy of the local IRB approval from each participating institution before NCI IRB approval will be granted. In addition, this protocol will adhere to the policies and requirements of the CTEP Multicenter Guidelines. The specific responsibilities of the Principal Investigator and the Coordinating Center (Study Coordinator) and the procedures for auditing are presented in Appendix XIX. 9.1 IRB APPROVALS The PI will provide the NCI IRB and Central Registration Office with a copy of the participating institution s approved yearly continuing review. Registration will be halted at any participating institution in which a current continuing approval is not on file at the NCI IRB. 9.2 AMENDMENTS AND CONSENTS The CCR PI will provide the NCI IRB with copies of all amendments, consents and local IRB approvals from each participating institution. 95

176 9.3 IND ACTION LETTERS OR SAFETY REPORTS The CCR PI is responsible for distributing all IND Action Letters or Safety Reports received from CTEP to all participating institutions for submission to their individual IRBs for action as required. 9.4 DRUG ORDERING Each participating institution will order DCTD-supplied agents directly from CTEP. Agents may be ordered by a participating site only after the initial IRB approval for the site has been forwarded by the Coordinating Center to the CTEP PIO (PIO@ctep.nci.nih.gov). 96

177 10 REFERENCES 1. Friedman JM: Neurofibromatosis 1: clinical manifestations and diagnostic criteria. Journal of child neurology 17:548-54; discussion 571-2, , Korf BR: Plexiform neurofibromas. American Journal of Medical Genetics 89:31-37, Korf BR: Malignancy in neurofibromatosis type 1. The oncologist 5: , Needle MN, Cnaan A, Dattilo J, et al: Prognostic signs in the surgical management of plexiform neurofibroma: the Children's Hospital of Philadelphia experience, The Journal of pediatrics 131: , Dombi E, Solomon J, Gillespie AJ, et al: NF1 plexiform neurofibroma growth rate by volumetric MRI: relationship to age and body weight. Neurology 68:643-7, Kim A, Gillespie A, Dombi E, et al: Characteristics of children enrolled in treatment trials for NF1-related plexiform neurofibromas. Neurology 73:1273-9, Gupta A, Cohen BH, Ruggieri P, et al: Phase I study of thalidomide for the treatment of plexiform neurofibroma in neurofibromatosis 1. Neurology 60:130-2, Widemann BC, Salzer WL, Arceci RJ, et al: Phase I trial and pharmacokinetic study of the farnesyltransferase inhibitor tipifarnib in children with refractory solid tumors or neurofibromatosis type I and plexiform neurofibromas. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 24: , Widemann BC, Dombi E, Gillespie A, et al: Phase 2 randomized, flexible crossover, double-blinded, placebo-controlled trial of the farnesyltransferase inhibitor tipifarnib in children and young adults with neurofibromatosis type 1 and progressive plexiform neurofibromas. Neuro Oncol 16:707-18, Babovic-Vuksanovic D, Ballman K, Michels V, et al: Phase II trial of pirfenidone in adults with neurofibromatosis type 1. Neurology 67:1860-2, Widemann BC, Babovic-Vuksanovic D, Dombi E, et al: Phase II trial of pirfenidone in children and young adults with neurofibromatosis type 1 and progressive plexiform neurofibromas. Pediatr Blood Cancer 61: , Dagalakis U, Lodish M, Dombi E, et al: Puberty and plexiform neurofibroma tumor growth in patients with neurofibromatosis type I. J Pediatr 164:620-4, Kim A, Dombi E, Tepas K, et al: Phase I trial and pharmacokinetic study of sorafenib in children with neurofibromatosis type I and plexiform neurofibromas. Pediatr Blood Cancer 60: , Weiss B, Widemann BC, Wolters P, et al: Sirolimus for non-progressive NF1- associated plexiform neurofibromas: an NF clinical trials consortium phase II study. Pediatr Blood Cancer 61:982-6, Weiss B, Widemann B, Wolters P, et al: Sirolimus for Progressive NF1-Associated Plexiform Neurofibromas: An NF Clinical Trials Consortium Phase II Study. Neuo Oncology, In Press 97

178 16. Jakacki R, Dombi E, Steinberg S, et al: Preliminary results of a phase II trial of pegylated interferon-alfa-2b (PI) in pediatric patients with documented progression of neurofibromatosis type 1-related unresectable plexiform neurofibromas (PNF). Neuro-Oncology 14, Robertson KA, Nalepa G, Yang FC, et al: Imatinib mesylate for plexiform neurofibromas in patients with neurofibromatosis type 1: a phase 2 trial. Lancet Oncol 13: , Akshintala S, Dombi E, Baldwin A, et al: Growth patterns and predictors of plexiform neurofibroma (PN) growth in patients with neurofibromatosis 1 (NF1) based on volumetric Magnetic Resonance Imaging (MRI) analysis Children's Tumor Foundation. Washington, DC, Meany H, Dombi E, Reynolds J, et al: 18-fluorodeoxyglucose-positron emission tomography (FDG-PET) evaluation of nodular lesions in patients with neurofibromatosis type 1 and plexiform neurofibromas (PN) or malignant peripheral nerve sheath tumors (MPNST). Pediatr Blood Cancer 60:59-64, Beert E, Brems H, Daniels B, et al: Atypical neurofibromas in neurofibromatosis type 1 are premalignant tumors. Genes Chromosomes Cancer 50: , Estey E, Hoth D, Simon R, et al: Therapeutic response in phase I trials of antineoplastic agents. Cancer treatment reports 70: , Therasse P, Arbuck SG, Eisenhauer EA, et al: New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. Journal of the National Cancer Institute 92: , Solomon J, Warren K, Dombi E, et al: Automated detection and volume measurement of plexiform neurofibromas in neurofibromatosis 1 using magnetic resonance imaging. Computerized medical imaging and graphics : the official journal of the Computerized Medical Imaging Society 28: , Monestiroli S, Mancuso P, Burlini A, et al: Kinetics and viability of circulating endothelial cells as surrogate angiogenesis marker in an animal model of human lymphoma. Cancer research 61: , Dombi E, Ardern-Holmes SL, Babovic-Vuksanovic D, et al: Recommendations for imaging tumor response in neurofibromatosis clinical trials. Neurology 81:S33-40, Viskochil D, Buchberg AM, Xu G, et al: Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell 62:187-92, Xu GF, O'Connell P, Viskochil D, et al: The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell 62: , Kluwe L, Friedrich RE, Korf B, et al: NF1 mutations in neurofibromatosis 1 patients with plexiform neurofibromas. Human mutation 19:309, Serra E, Rosenbaum T, Winner U, et al: Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations. Human molecular genetics 9: ,

179 30. Packer RJ, Gutmann DH, Rubenstein A, et al: Plexiform neurofibromas in NF1: toward biologic-based therapy. Neurology 58: , Widemann BC, Salzer WL, Arceci RJ, et al: Phase I trial and pharmacokinetic study of the farnesyltransferase inhibitor tipifarnib in children with refractory solid tumors or neurofibromatosis type I and plexiform neurofibromas. Journal of Cinical Oncology 24: , Rowinsky EK, Windle JJ, Von Hoff DD: Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development. J Clin Oncol 17: , Khalaf WF, Yang FC, Chen S, et al: K-ras is critical for modulating multiple c-kitmediated cellular functions in wild-type and Nf1+/- mast cells. J Immunol 178: , Dasgupta B, Li W, Perry A, et al: Glioma formation in neurofibromatosis 1 reflects preferential activation of K-RAS in astrocytes. Cancer Res 65:236-45, Barkan B, Starinsky S, Friedman E, et al: The Ras inhibitor farnesylthiosalicylic acid as a potential therapy for neurofibromatosis type 1. Clin Cancer Res 12: , Lauchle JO, Kim D, Le DT, et al: Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. Nature 461:411-4, Dulai S, Briody J, Schindeler A, et al: Decreased bone mineral density in neurofibromatosis type 1: results from a pediatric cohort. J Pediatr Orthop 27:472-5, Lodish MB, Dagalakis U, Sinaii N, et al: Bone mineral density in children and young adults with neurofibromatosis type 1. Endocr Relat Cancer 19:817-25, Stevenson DA, Moyer-Mileur LJ, Murray M, et al: Bone mineral density in children and adolescents with neurofibromatosis type 1. J Pediatr 150:83-8, Kuhnisch J, Seto J, Lange C, et al: Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models. Bone 66:155-62, Ndong JD, Stevens DM, Vignaux G, et al: Combined MEK inhibition and BMP2 treatment promotes osteoblast differentiation and bone healing in Nf1 mice. J Bone Miner Res, Ono K, Karolak MR, Ndong Jde L, et al: The ras-gtpase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation. Hum Mol Genet 22: , Yeh TC, Marsh V, Bernat BA, et al: Biological characterization of ARRY (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res 13: , Garon EB, Finn RS, Hosmer W, et al: Identification of common predictive markers of in vitro response to the Mek inhibitor selumetinib (AZD6244; ARRY ) in human breast cancer and non-small cell lung cancer cell lines. Mol Cancer Ther 9: Astra-Zeneca: Investigator Brochure. 26 March Shannon AM, Telfer BA, Smith PD, et al: The mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 inhibitor AZD6244 (ARRY ) 99

180 enhances the radiation responsiveness of lung and colorectal tumor xenografts. Clin Cancer Res 15: , Davies BR, Logie A, McKay JS, et al: AZD6244 (ARRY ), a potent inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 kinases: mechanism of action in vivo, pharmacokinetic/pharmacodynamic relationship, and potential for combination in preclinical models. Mol Cancer Ther 6: , Denton CL, Gustafson DL: Pharmacokinetics and pharmacodynamics of AZD6244 (ARRY ) in tumor-bearing nude mice. Cancer Chemother Pharmacol 49. Chang Q, Chen E, Hedley DW: Effects of combined inhibition of MEK and mtor on downstream signaling and tumor growth in pancreatic cancer xenograft models. Cancer Biol Ther 8: , Gutmann DH, Hunter-Schaedle K, Shannon KM: Harnessing preclinical mouse models to inform human clinical cancer trials. J Clin Invest 116:847-52, Wu J, Dombi E, Jousma E, et al: Preclincial testing of sorafenib and RAD001 in the Nf(flox/flox) ;DhhCre mouse model of plexiform neurofibroma using magnetic resonance imaging. Pediatr Blood Cancer 58:173-80, Wu J, Williams JP, Rizvi TA, et al: Plexiform and dermal neurofibromas and pigmentation are caused by Nf1 loss in desert hedgehog-expressing cells. Cancer Cell 13:105-16, Yang FC, Ingram DA, Chen S, et al: Nf1-dependent tumors require a microenvironment containing Nf1+/-- and c-kit-dependent bone marrow. Cell 135:437-48, Jessen WJ, Miller SJ, Jousma E, et al: MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors. J Clin Invest 123:340-7, Nishioka C, Ikezoe T, Yang J, et al: Inhibition of MEK signaling enhances the ability of cytarabine to induce growth arrest and apoptosis of acute myelogenous leukemia cells. Apoptosis 14: , Jin N, Jiang T, Rosen DM, et al: Dual inhibition of mitogen-activated protein kinase kinase and mammalian target of rapamycin in differentiated and anaplastic thyroid cancer. J Clin Endocrinol Metab 94: , Yang S, Ngo VC, Lew GB, et al: AZD6244 (ARRY ) enhances the therapeutic efficacy of sorafenib in mouse models of gastric cancer. Mol Cancer Ther 8: , Yoon YK, Kim HP, Han SW, et al: KRAS mutant lung cancer cells are differentially responsive to MEK inhibitor due to AKT or STAT3 activation: implication for combinatorial approach. Mol Carcinog 49: Banerji U, Camidge DR, Verheul HM, et al: The first-in-human study of the hydrogen sulfate (Hyd-sulfate) capsule of the MEK1/2 inhibitor AZD6244 (ARRY ): a phase I open-label multicenter trial in patients with advanced cancer. Clin Cancer Res 16:

181 60. Wilky BA, Rudek MA, Ahmed S, et al: A phase I trial of vertical inhibition of IGF signalling using cixutumumab, an anti-igf-1r antibody, and selumetinib, an MEK 1/2 inhibitor, in advanced solid tumours. Br J Cancer, Janne PA, Shaw AT, Pereira JR, et al: Selumetinib plus docetaxel for KRAS-mutant advanced non-small-cell lung cancer: a randomised, multicentre, placebo-controlled, phase 2 study. Lancet Oncol 14:38-47, Banerjee A, Jakacki R, Onar-Thomas A, et al: A phase 1 study of AZD6244 in children with recurrent or refractory low-grade gliomas: A Pediatric Brain Tumor Consortium report. J Clin Oncol 32:5s (supp; abstr 10065, Korf BR: Plexiform neurofibromas. Am J Med Genet 89:31-7, Mellins CA, Brackis-Cott E, Dolezal C, et al: The role of psychosocial and family factors in adherence to antiretroviral treatment in human immunodeficiency virus-infected children. The Pediatric infectious disease journal 23: , Van Dyke RB, Lee S, Johnson GM, et al: Reported adherence as a determinant of response to highly active antiretroviral therapy in children who have human immunodeficiency virus infection. Pediatrics 109:e61, Patino AM, Sanchez J, Eidson M, et al: Health beliefs and regimen adherence in minority adolescents with type 1 diabetes. Journal of pediatric psychology 30: , Martin SC, Elliott DK, Toledo-Tamula MA, et al: Comparison of Knowledge and Perceptions of Responsibility for Medication Adherence Between HIV-positive Children and their Caregivers. Society of Pediatric Psychology Meeting, National Institutes of Health Consensus Development Conference Statement: neurofibromatosis. Bethesda, Md., USA, July 13-15, Neurofibromatosis 1: ,

182 11 APPENDICES IA: Performance score IB: Blood Pressure IIA: Imaging: MRI IIB: PN Location and Classification form IIC: Imaging: DEXA III: Screening and on study evaluations (3A phase I 3B phase II) IV: Photography V: Symptom checklist VI: PN location and morbidity form VII: Function: Orbit PN VIII: Function : Airway PN IX: Function: Motor PN - IXA: 6-Minute Walk-Run Test (motor PN lower extremity or Airway PN) - IXB: Strength evaluation (motor PN) - IXC: Range of Motion evaluation (motor PN) - IXD: Leg length evaluation (motor PN lower extremity) - IXE: NF1 Grooved Pegboard Test (motor PN upper extremity) X: Bladder and bowel evaluations -- XA: Function: Other PN XI: Patient Reported Outcomes: Pain and QOL (all patients) and physical functioning (motor PN) XII: Pharmacokinetics (PK) XIII: Pharmacodynamics: Blood progenitor cells and cytokines XIV: Diary A and B XV: Long term Follow up XVI: Adherence XVII: Dosing Nomogram XVIII: Medications to Avoid XIX: CTEP Multicenter Guidelines 102

183 11.1 APPENDIX IA: PERFORMANCE STATUS SCALES/SCORES PHASE I/II Patient C3D Study ID: Patient s Age: PERFORMANCE STATUS CRITERIA Karnofsky and Lansky performance scores are intended to be multiples of 10 Karnofsky (> 16 years of age) Lansky ( 16 years of age) Score Description Score Description 100 Normal, no complaints, no evidence of disease 90 Able to carry on normal activity, minor signs or symptoms of disease. 80 Normal activity with effort; some signs or symptoms of disease. 70 Cares for self, unable to carry on normal activity or do active work. 60 Required occasional assistance, but is able to care for most of his/her needs. 50 Requires considerable assistance and frequent medical care. 40 Disabled, requires special care and assistance. 30 Severely disabled, hospitalization indicated. Death not imminent. 20 Very sick, hospitalization indicated. Death not imminent. 10 Moribund, fatal processes progressing rapidly. 100 Fully active, normal. 90 Minor restrictions in physically strenuous activity. 80 Active, but tires more quickly 70 Both greater restriction of and less time spent in play activity. 60 Up and around, but minimal active play; keeps busy with quieter activities. 50 Gets dressed, but lies around much of the day; no active play, able to participate in all quiet play and activities. 40 Mostly in bed; participates in quiet activities. 30 In bed; needs assistance even for quiet play. 20 Often sleeping; play entirely limited to very passive activities. 10 No play; does not get out of bed. PERFORMANCE STATUS: Karnofsky Score: OR Lansky Score: 103

184 11.2 PROVIDER SIGNATURE MOST RECENT COMPLETED CYCLE#/ DATE APPENDIX IB: BLOOD PRESSURE Participant s C3D ID: Instructions: Blood pressure (BP) the 95th percentile for age, height, and gender is required for enrollment. Because measurement accuracy is dependent on the use of proper BP cuff size, BP cuff should be selected as follows: Cuff Type Infant Child Small Adult Adult Range: 8-11 cm cm cm cm Position the artery mark on the cuff on the patient's artery before inflation. Four blood pressure measurements will be taken simultaneously. Should the blood pressure be outside of the normal limits, 3 blood pressure measurements should be performed and the average used. BP measurement 1: BP measurement 2: BP measurement 3: AVERAGE OF 3 BP MEASUREMENTS: Provider Signature Date Please return via fax (without patient identifiers) to the NCI : Attention: Trish Whitcomb, RN:

185 BLOOD PRESSURE LEVELS FOR CHILDREN BY AGE AND HEIGHT PERCENTILE Blood pressure (BP) levels for BOYS Systolic Blood Pressure, mm Hg Diastolic Blood Pressure, mm Hg Age BP Percentile of Height Percentile of Height (years) Percentile 5th 10th 25th 50th 75th 90th 95th 5th 10th 25th 50th 75th 90th 95th 1 95th th th th th th th th th th th th th th th th th This table was taken from The Fourth Report on the Diagnosis, Evaluation, and Treatment of High Blood Pressure in Children and Adolescents PEDIATRICS Vol. 114 No. 2 August 2004, pp

186 Blood pressure (BP) levels for GIRLS Systolic Blood Pressure, mm Hg Diastolic Blood Pressure, mm Hg Age BP Percentile of Height Percentile of Height (years) Percentile 5th 10th 25th 50th 75th 90th 95th 5th 10th 25th 50th 75th 90th 95th 1 95th th th th th th th th th th th th th th th th th This table was taken from The Fourth Report on the Diagnosis, Evaluation, and Treatment of High Blood Pressure in Children and Adolescents PEDIATRICS Vol. 114 No. 2 August 2004, pp

187 11.3 APPENDIX IIA: PROTOCOL FOR REQUIRED MRI STUDIES OF PN PHASE I/II Prior to starting treatment on this study all known measurable PN should be imaged with MRI if feasible, to obtain a baseline. Extent of PN in patients with NF1 can be very substantial, and may not allow for all lesions to be followed using 3D-MRI. The clinically most relevant PN, which has to be amenable to volumetric MRI analysis, will be chosen as target lesion. Up to 2 additional PN can be followed as non target lesions with 3D analysis. PN will be classified at the NCI as typical PN versus nodular PN versus solitary nodular PN based on imaging characteristics. Pre-study imaging evaluation: Identify and select the inoperable target PN (plus a maximum of two additional non target PN) for 3-D MRI evaluation based on prior imaging studies. Should there be more than 3 inoperable PN, the three most clinically relevant PN will be followed by 3-D MRI analysis. Perform MRI sequences on the selected index lesions as outlined in the MRI acquisition protocols below. In addition, if possible, perform MRI of all additional measurable PN. On study imaging evaluation: Unless clinically indicated otherwise obtain MRI of the target and non-target PN only as outlined in the MRI acquisition protocol below at baseline and prior to cycles 5, 9, 13, 17, 21, 25 and then after every 6 cycles as long as the patient remains on study. MRI protocol: Images intended for volumetric analysis need to be performed without gaps between slices. The target tumor should be positioned close to the center of the imaging field and the outer edge of the tumor should be within the field of view. Peripheral nerve sheath tumors can be well visualized without the use of contrast agents on short TI inversion recovery (STIR) sequences because they have high signal intensity relative to normal tissues. The imaging protocol in the table below should be used for the imaging of the target and non target PN. Modifications should be documented in the MRI protocols, and the same imaging protocol, and, if possible, the same MRI scanner, should be used for all subsequent MRI studies. Every attempt should be made to image the entire PN. Axial STIR Volumetric sequence for PN Recommended Small PN/ Range Head-Neck Large PN/ Trunk-Extremity Echo Train Length TR TE

188 TI Slice Thickness 3-10 mm 3 mm 10 mm Skip Matrix 256x x x256 - FOV cm 22 cm 45 cm Phase FOV NEX Frequency Direction A P A P A P **Spinal/paraspinal PN should be imaged with the appropriate protocol based on tumor size. Tumors that include the neck and trunk, for which the majority of the tumor is in the trunk, should be imaged with the Large Trunk-Extremity PN protocol. ##Tumors < 5 cm in longest diameter would be considered small. Tumors > 10 cm longest diameter would be considered large. Tumors between 5-10 cm in longest diameter should be imaged with the appropriate protocol based on best FOV coverage. Please contact Dr. Dombi if there are questions regarding the appropriate coverage or imaging protocol. Dr. Dombi will confirm for each patient in screening that the MRI imaging is adequate and that the target PN is measurable and amenable to volumetric MRI analysis. All MRI studies will be analyzed at the NCI POB under supervision of Dr. Eva Dombi. Imaging studies including C3D ID and cycle # should be sent to: Dr. Eva Dombi, M.D. NCI POB 10 Center Drive Building 10, room Bethesda, MD Phone , dombie@mail.nih.gov The NCI POB will confirm receipt of the imaging study and report results of the volumetric MRI analysis to the site PI electronically within 7 days of receipt of the MRI. Evaluation of changes in known optic pathway tumors or other gliomas at enrollment: In patients enrolled with known optic pathway tumors or other gliomas, the brain MRI study performed closest prior to the time of enrollment, and all brain MRI studies performed after enrollment on the study during treatment with selumetinib should also be sent to Dr. Dombi for review. 108

189 11.4 APPENDIX IIB: PN LOCATION AND CLASSIFICATION FORM Participant s C3D ID: Date: To be completed by Dr. Eva Dombi or NCI team at baseline, prior to treatment initiation. PN Location Measurability (Yes/No) PN Classification ( typical PN, or nodular PN, or solitary nodular PN ) Target PN Non-Target PN #1 Non-Target PN #2 Typical PN: Nodular component of PN is <30% Nodular PN: Nodular component of PN is 30% Solitary nodular PN: The target lesion is a sinlge nodular lesion COMPLETED BY: DATE: 109

190 110 NCI protocol #11-C-0161; Version Date:8-3-15

191 Participant s C3D ID: Date: 11.5 APPENDIX IIC: DEXA (ONLY AT NCI) Patients enrolled at the NCI will have a DEXA scan done at baseline to evaluate bone mineral density (BMD). Those with abnormal BMD, will have a repeat DEXA done prior to cycle 13 (+/- 2 cycles), and then as clinically indicated. Select One: Baseline Prior to cycle 13 Other (Please specify): Height: Weight: DEXA Results: BMD Whole Body (Z) BMD Lumbar Spine (Z) BMD Femoral Neck (Z) COMPLETED BY: DATE: Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

192 11.6 APPENDIX III: REQUIRED STUDY EVALUATIONS Appendix IIIA: Phase I Required Evaluations / Enrollment on phase I complete as of amendment I STUDIES TO BE OBTAINED Pre- Study During Cycle 1 Prior To Subsequent Cycles 1 End of Therapy Evaluation 2 History X Every other week Every other week cycles 2 3 X Prior to cycles 4 11, 13, 15, 17, etc Physical Exam, vital signs X Every other week Every other week cycles 2 3 X Prior to cycles 4 11, 13, 15, 17, etc Height, weight, BSA 3 X - Prior to cycle 4, 6, 11 and then - after every 6 cycles (pre 17, 23, 29, etc) Performance Status 4 X - Only if change from baseline X EKG and X - Prior to cycle 4, 6, 11 and then - Echocardiogram 12 after every 6 cycles (pre 17, 23, 29, etc) CBC, differential, X Every other week Every other week cycles 2 3 X platelets 5 Prior to cycles 4 11, 13, 15, 17, etc Pharmacokinetics 6 X X Prior to cycle Urinalysis X - Prior to cycle 6, 11 and then after every 6 cycles (pre 17, 23, 29, etc) X Electrolytes including X Every other week Every other week cycles 2 3 X Ca++, PO 4, Mg++, BUN, Prior to cycles 4 11, 13, 15, 17, etc creatinine, glucose, ALT, bilirubin, Total protein/albumin, CPK PN Disease Evaluation 7 X - Prior to cycle 6, 11 and then after X every 6 cycles (pre 17, 23, 29, etc) 8 Ophthalmology evaluation 14 X - Prior to cycle 4, 6, 11 and then after every 6 cycles (pre 17, 23, 29, etc) Pregnancy Test 9 X - Prior to radiographic tests: pre - cycle 6, 11 and then after every 6 cycles (pre 17, 23, 29, etc) Adherence X Questionnaire 13 Patient diary 10 - Daily Daily - Pill Count X 11 1 Day (+/- 14 days) of the preceding cycle. 2 Should be performed, if possible, when the patient comes off treatment regardless of the reason, unless the test or procedure has been performed within the time period defined in Section BSA should be calculated from average of three measurements. 4 Appendix I, prior to cycle 1 and then only required to be recorded if there is a change in performance status during the study. 5 If patients develop Grade 4 anemia then CBC should be checked every 3 to 4 days until recovery to grade 1. 6 See Appendix IV for timing of PK samples. Differences in timing of the PK sample from the target time will be recorded on the PK sheet, but not reported as protocol deviation. 7 Volumetric MRI. See Appendix II. 8 If patient does show a responsive disease (RD) according to Section 5.2, response should be confirmed after 3-6 treatment cycles 9 Patients of childbearing potential require a negative pregnancy test prior to starting treatment and prior to every radiographic scan which will presumably be prior to cycle 6, 11, and then after every 6 cycles (pre-cycle 17, 23, 29, etc). Patients of childbearing potential must use an acceptable method of birth control. Abstinence is an acceptable method of birth control. 10 Review patient diary weekly with adherence questionnaire prior to each treatment cycle for cycles 2-9, and subsequently prior to every other treatment cycle (pre cycle 11, 13, 15, 17, etc) by the research team. 11 The patient s research team will conduct pill counts with scheduled visits prior to cycles 2 and 4. Caregivers and patients will be instructed to return all empty, partially full, and full bottles to their research team at each visit. 112

193 12 Patients who stop Selumetinib for cardiac toxicity should be monitored by ECHO or cardiac MRI every 3 to 6 weeks. Upon recovery from toxicity (to a LVEF of 53%), patients should be monitored with an ECHO or cardiac MRI every 2 3 cycles as directed by the cardiologist (Sections and 3.6). 13 Will only be performed if more than 20% discrepancy between diary and pill count. 14 Ophthalmology toxicity evaluation: Eye exam with emphasis on corneal and retinal changes 113

194 Appendix IIIB: Phase II Required Evaluations STUDIES TO BE OBTAINED Pre- Study During Cycle 1 Prior To Subsequent Cycles 1 History 4 X Preweek 3 Prior to cycles 2 5 (±3 days) Then every other cycle (Prior to cycles 7, 9, 11, 13, etc.) Physical Exam 5, vital signs 5 X Preweek 3 Prior to cycles 2 5 (±3 days) Then every other cycle (Prior to cycles 7, 9, 11, 13, etc.) Height, weight, BSA 6 X - Prior to cycles 2 5 Then every other cycle (Prior to cycles 7, 9, 11, 13, etc.) Performance Status 7 X - Prior to cycle 5, 9, 13, 17, 21, 25 Then after every 6 cycles (Prior to cycles 31, 37, 43, etc.) End of Therapy Evaluation 2 EKG X - As clinically indicated - Echocardiogram 8 X - Prior to cycle 5, 9, 13 Then after every 6 cycles (Prior to cycles 19, 25, 31 etc.) CBC, differential, platelets 9 X Prior to cycles 2 5 Then every other cycle (Prior to cycles 7, 9, 11, 13, etc.) Urinalysis X - Prior to cycle 5, 9, 13 Then after every 6 cycles (Prior to cycles 19, 25, 31 etc.) Electrolytes including Ca++, PO 4, Mg++, BUN, creatinine, glucose, ALT, AST, bilirubin, Total protein/albumin, amylase, lipase, CPK 10 X Prior to cycles 2 5 Then every other cycle (Prior to cycles 7, 9, 11, 13, etc.) PN location forms 11 X PN Disease Evaluation 12 X - Prior to cycle 5, 9, 13, 17, 21, 25 Then after every 6 cycles (Prior to cycles 31, 37, 43, etc.) Photography of visible PN (in consenting patients) 13 X - Prior to cycle 5, 9, 13, 17, 21, 25 Then after every 6 cycles (Prior to cycles 31, 37, 43, etc.) X X Long Term Follow Up 3 Annual with targeted questionnaire Annual - Annual X Annual -X Annually if LVEF is abnormal at end of therapy X X X X Annual At PI discretion Annual and if clinically indicated Q6 months x 2, then yearly x 2 then q2 years or as clinically indicated - Symptom Checklist 14 X - Prior to cycle 3, 5, 9, 13, 17, 21, 25 Then after every 6 cycles (Prior to cycles 31, 37, 43, etc.) Ophthalmology toxicity evaluation 15 X - Prior to cycle 7, 13, then yearly or more often as clinically indicated Pregnancy Test 16 X - Prior to imaging studies: Prior to cycle 5, 9, 13, 17, 21, 25 Then after every 6 cycles (Prior to cycles 31, 37, 43, etc.) X - X As clinically indicated - - Adherence Questionnaire 17 Patient diary and capusule count 18 RMQ only - Preweek 3 (±3 days) Only if pill count discrepancy >20% Prior to cycle 3, 5, 9, 13, 17, 21, 25 Then after every 6 cycles (Prior to cycles 31, 37, 43, etc.) Quality of Life and Pain evaluations 19 X - Prior to cycle 3, 5, 9, 13, then after every 12 cycles X - 114

195 (Prior to cycles 25, 37, etc.) Functional PRO X - Prior to cycle 3, 5, 9, 13, then after X - assessments 19 every 12 cycles (Prior to cycles 25, 37, etc.) Endurance evaluation 20 X Prior to cycle 5, 9, 13, then after every 12 cycles (Prior to cycles 25, 37, etc.) X - Functional Evaluations: X - Prior to cycle 5, 9, 13, then after X - Orbit PN, Airway PN, Motor PN, Bowel-Bladder every 12 cycles (Prior to cycles 25, 37, etc.) PN21, 22 DEXA 23 (NCI ONLY) X Prior to cycle 13 (+/- 2 cycles) - As clinically indicated if baseline is abnormal Pharmacokinetics 24 X X Prior to cycle 2 or PD: Cytokines Bone marrow derived precursor cells 25 X Prior to cycle 3, 5, 9, and 13, and if feasible, at time of progression - 1 Day (+/- 14 days) of the preceding cycle. 2 Should be performed, if possible, when the patient comes off treatment regardless of the reason, unless the test or procedure has been performed within the time period defined in Section Long term safety follow up is annually (+/- 2 months) for 7 years following initiation of treatment or 5 years after study drug discontinuation, whichever is the longer period. 4 Baseline history includes prior treatment for PN with dates. Interval history includes events and changes since last visit, review of all medications and allergies 5 A complete standard physical examination will be performed at baseline, subsequent physical exams will be targeted based on signs and symptoms of presenting patient. Vital signs: Heart rate, temperature, blood pressure (for eligibility see Appendix1B), respiratory rate, O2 saturation by pulse oximetry at baseline and then as indicated 6 BSA should be calculated from average of three measurements. Calculating BSA from < 3 measurements will be noted, but not reported as protocol deviation. 7 Appendix I and prior to the cycle indicated in the table. 8 Patients who stop selumetinib for cardiac toxicity should be monitored by ECHO or cardiac MRI every 3 to 6 weeks. Upon recovery from toxicity, patients should be monitored with an ECHO or cardiac MRI every 2-3 cycles as directed by the cardiologist (Sections and 3.6). 9 If patients develop Grade 4 neutropenia then CBC should be checked every 3 to 4 days until recovery to grade 1. Isolated missing lab values will be noted, but not reported as protocol deviation. 10 CPK isoenzymes should be drawn for CPK grade 2 (X1). This needs to be done only once. Isolated missing lab values will be noted, but not reported as protocol deviation. 11 PN location and morbidity form to be completed by site PI (Appendix VI). PN location and classification form to be completed by the NCI (Appendix IIB) 12 Volumetric MRI. See Appendix IIA. 13 Photography of visible PN in consenting patients. See Appendix IV 14 Symptom checklist. See Appendix V 15 Ophthalmology toxicity evaluation: Eye exam with emphasis on corneal, lens and retinal changes 16 Patients of childbearing potential as defined by institutional standards ( NIH 9 years) require a negative pregnancy test prior to starting treatment and prior to every radiographic scan which will presumably be prior to cycle 5, 9, 13, 17, 21, 25 and then after every 6 cycles.. Patients of childbearing potential must use an acceptable method of birth control. Abstinence is an acceptable method of birth control. 17 Adherence questionnaire (see Appendix XVI) should be completed if the diary review and pill count reveal an unexplained discrepancy >20%. 18 Patient diary and capsule count. See Appendix XIV 19 See Appendix XI for pain and quality of life evaluations for all patients and physical functioning PRO assessments for patients with PN affecting motor function 20 For patients with lower extremity or airway PN, see Appendix IXA for endurance evaluation (6-Minute Walk-Run test) 21 See Appendices VII, VIII, IX, X for functional evaluations 22 Patients with PN affecting motor function will have strength, range of motion (Appendix IX), and photography evaluations (Appendix IV), as well as a PRO measure (Appendix XI Section II). Patients with PN in the lumbosacral plexus or below (i.e. lower extremity) will undergo leg length testing (Appendix IXD). Those with upper extremity PN will undergo NF1 Grooved Pegboard testing (Appendix IXE) 23 Patients enrolled at the NCI only. See Appendix IIC 115

196 24 See Appendix XII for timing of PK samples. Differences in timing of the PK sample from the target time will be recorded on the PK sheet, but not reported as protocol deviation. 25 See Appendix XIII for PD studies: Cytokines and peripheral blood progenitor cells. a Yearly growth assessment requires integration of information including height measurements, puberty staging, bone age if clinically indicated, which should be plotted onto growth charts. Children with impaired growth velocity should be referred to a pediatric endocrinologist as clinically indicated for evaluation, and should be followed until they reach final adult height. 116

197 11.7 APPENDIX IV: PHOTOGRAPHY OF VISIBLE PN PHASE II Participant s C3D ID: Date: Current Cycle: Photography of visible PN will be performed at (select applicable time point) in consenting patients: Testing should be performed pre-treatment, and then prior to cycles 5, 9, 13, 17, 21, 25, and then after every 6 cycles. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Cycle 17 Cycle 21 Cycle 25 Other (Please specify): All photographs at each time point should be taken from the same distance, using similar lighting and camera settings, if possible. Baseline photographs should be present at the time of follow up photography to allow for the best possible comparison. Videotape may be used as well to document changes in function. Please Circle and Describe the location of each photograph: Description of Photo body location: #1: #2: #3: #4: #5: Completed By: - Please return with the pictures (without patient identifiers) to the NCI : Attention: Trish Whitcomb, RN: 10 Center Drive, Building 10, CRC, Room , Bethesda, MD Phone: ; fax: ; whitcomt@mail.nih.gov 117

198 11.8 APPENDIX V: PLEXIFORM NEUROFIBROMA SYMPTOM CHECKLIST PHASE II Participant s C3D ID: Date: Current Cycle: Testing should be performed pre-treatment, an then prior to cycles 3, 5, 9, 13,17, 21, 25 and then after every 6 cycles. To be completed by Patient if able, otherwise completed by the Parent/Guardian. To be completed by the same individual at each timepoint. Select one: Pre-treatment Prior to cycle 3 Prior to cycle 5 Prior to cycle 9 Prior to cycle 13 Prior to cycle 17 Prior to cycle 21 Prior to cycle 25 Other (Please specify): For each item below, please check one box (unless otherwise indicated) to describe how much each of these has been a problem for you in the past two weeks: 1) Fatigue/feeling tired Not at all A little Some Pretty much A lot 2) Sleep problems Not at all A little Some Pretty much A lot 3) Less appetite (eating less) 4) More appetite (eating more) Not at all A little Some Pretty much A lot Not at all A little Some Pretty much A lot 1) Headaches Not at all A little Some Pretty much A lot 2) Vision changes Not at all A little Some Pretty much A lot 3) Decreased hearing Not at all A little Some Pretty much A lot 4) Mouth Sores Not at all A little Some Pretty much A lot 5) Trouble swallowing Not at all A little Some Pretty much A lot 6) Choking Not at all A little Some Pretty much A lot 7) Snoring Not at all A little Some Pretty much A lot 8) Frequent awakenings at night Not at all A little Some Pretty much A lot 118

199 1) Cough Not at all A little Some Pretty much A lot 2) Wheezing Not at all A little Some Pretty much A lot 3) Difficulty breathing Not at all A little Some Pretty much A lot 1) Chest pain Not at all A little Some Pretty much A lot 2) Palpitations/fluttering Not at all A little Some Pretty much A lot 3) Shortness of breath with activity 4) Shortness of breath at rest Not at all A little Some Pretty much A lot Not at all A little Some Pretty much A lot 5) Swelling in hands/feet Not at all A little Some Pretty much A lot 1) Abdominal pain Not at all A little Some Pretty much A lot 2) Heartburn Not at all A little Some Pretty much A lot 3) Nausea Not at all A little Some Pretty much A lot 4) Vomiting Not at all A little Some Pretty much A lot 5) Diarrhea Not at all A little Some Pretty much A lot 6) Constipation Not at all A little Some Pretty much A lot 7) Stool incontinence Not at all A little Some Pretty much A lot 1) Pain with urination Not at all A little Some Pretty much A lot 2) Increased urinary frequency/urgency 3) Difficulty beginning urination Not at all A little Some Pretty much A lot Not at all A little Some Pretty much A lot 119

200 4) Urinary incontinence Not at all A little Some Pretty much A lot 1) Weakness Not at all A little Some Pretty much A lot If yes, where? 2) Muscle pain Not at all A little Some Pretty much A lot If yes, where? 1) Dizziness Not at all A little Some Pretty much A lot 2) Numbness Not at all A little Some Pretty much A lot 3) Tingling Not at all A little Some Pretty much A lot Completed By (Patient or Parent/Guardian): Please return with copy of the Symptom Checklist results (without patient identifiers) via fax to the NCI : Attention: Trish Whitcomb, RN:

201 Participant s (C3D) ID: Date: 11.9 APPENDIX VI: TARGET PN LOCATION AND ASSOCIATED MORBIDITIES PHASE II INSTRUCTIONS: To be completed by the Site PI at the time of enrollment on study and prior to starting treatment - Assess target PN location by clinical exam and imaging studies - List morbidities present using numbers below (see list) - If no morbidity present, list potential morbidities based on target PN site - Assign patient to Stratum 1 or 2 based on the presence/absence of PN-related morbidity PN Location Orbit Face Ear canal Tongue R L B/L Pain (Y/N) PN Associated Morbidities Present PN Morbidities (using numbers listed below)/ Comments Potential Morbidities Anterior neck/upper airway Posterior neck (cervical paraspinal) Mediastinum Intra-thoracic Thoracic/paraspinal/chest wall Liver Abdominal 121

202 Mesentery Posterior abdomen/pelvis (Lumbo-sacral plexus) Perineum/peri-rectal/bladder Upper arm (Brachial plexus) Forearm Hand Thigh/upper leg Lower leg Foot Other (please specify): Morbidities: 1- Vision loss 2- Facial dysfunction 3- Auditory loss 4- Difficulty swallowing 5- Abnormal speech 6- Airway obstruction 7- Respiratory compromise 8- Bladder dysfunction 9- Bowel dysfunction 10- Motor weakness 11- Decreased range of motion 12- Sensory deficit 13-PN related Disfigurement/appearance 13- Other (please describe): Site PI Morbidity Assignment: Motor Airway Vision Bowel/Bladder Other (please specify): 122

203 Site PI Assigned Stratum: Stratum 1 (PN-related morbidity present) Stratum 2 (No morbidity present) Please return with copy of the Target PN Location and Associated Morbidities results (without patient identifiers) via fax to the NCI : Attention: Trish Whitcomb, RN:

204 Participant s C3D ID: Date of Evaluation: APPENDIX VII: FUNCTIONAL EVALUATION FOR ORBITAL PN OPHTHALMOLOGY EVALUATION PHASE II Please perform, if feasible, eye exam as outlined below. Additional evaluations can be performed at discretion of the site PI. Testing should be performed pre-treatment, and then prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of the therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): Location of orbital PN? right left both Does patient have an optic pathway glioma? Yes No Examination Protocol 1. Distance Acuity: Testing should be performed using HOTV or Teller Acuity Cards as outlined below. Each eye should be tested separately with best correction in place. The same testing method must be used for each subject throughout their time on study. HOTV should be used for children old enough to perform reliably at the baseline pre-treatment visit. All other children should be tested using Teller Acuity Cards. For analysis, acuity will be corrected for normal at the age of testing by calculating an age-based acuity. Changes > 0.2 logmar will be considered significant. HOTV Testing HOTV testing should be performed using the HOTV PEDIG ATS protocol at 3 meters. Acuity should be reported in logmar. Teller Acuity Card Testing Teller Grating Acuity should be measures at 55 cm and reported in Cycles/cm (choose the best card achieved). Grating acuity will be converted to logmar by the study team. 2. Exophthalmometry Exophthalmometry measurements for proptosis should be made with the same equipment for each subject throughout their time on study. Measurements will be made three times during each visit and averaged. The type of exophthalmometer and the base between the orbital rims should be the same during each measurement and each visit unless the child has significant facial growth between visits. Measurement of the amount of proptosis will be reported in mm. For analysis, changes in proptosis > 2 mm will be considered significant. OD OS 124

205 Distance Acuity (best corrected): Teller Acuity (cycles/cm) HOTV (logmar) If acuity has declined, what is it due to: Progressive Orbital PN Progressive amblyopia (deprivation, anisometropic, stabismic, etc) Optic pathway glioma Other (describe): Progressive Orbital PN Progressive amblyopia (deprivation, anisometropic, stabismic, etc) Optic pathway glioma Other (describe): Exophthalmometry (mm) Measurement #1 Measurement #2 Measurement #3 Average Please return completed Appendix VII along with a copy of the report of the ophthalmology exam at the site (without patient identifiers) via fax to the NCI : Attention: Trish Whitcomb, RN:

206 11.11 APPENDIX VIII: AIRWAY FUNCTION Participant s C3D ID: Date: Current Cycle: PNs affecting the airway will be categorized as either upper airway/extrathoracic PNs (involving the hypopharynx, subglottis, and upper part of the trachea) or as lower airway/intrathoracic PNs (involving the airway below the thoracic inlet, which would include the lower part of the trachea, bronchi, and alveoli). All patients with airway PN (upper airway/extrathoracic and lower airway/intrathoracic) will undergo functional evaluations including sleep studies, and evaluation of endurance using the 6-Minute Walk-Run test (Appendix IXA). In addition, patients with upper airway/extrathoracic PN will undergo oscillometry, and patients with lower airway/intrathoracic PN will undergo spirometry. If the PN involves upper and lower airway, oscillometry, and spirometry should be performed, if feasible SLEEP STUDY Patients with airway tumors should be evaluated, if feasible, using sleep studies as a quantitative measure of respiratory progression. Testing should be performed pre-treatment, and then prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): Please record the following measures: Duration of sleep (hrs): Apnea hypopnea index (AHI) = Number of apnea/hypopnea events per hour: (ex: if 5 hrs of sleep with 20 events, AHI = 20/5 = 4) SpO2, oxygen saturation: Mean: Nadir: Percentage of total sleep with SPO2 saturation <90%: Percentage of total sleep with CO2 > 50 torr: Arousal index: Number of arousals per hour scored by change in EEG: Please return with full copy of sleep study results (without patient identifiers) via fax to the NCI Attention: Trish Whitcomb, RN:

207 Participant s C3D ID: Date: Current Cycle: PULMONARY FUNCTION TESTS (PFTs) Patients with pulmonary and/or airway tumors should be evaluated, if feasible, using Pulmonary Function Tests (PFTs) as a quantitative measure of respiratory progression. Oscillometry should be used for extrathoraci/upper airway lesions, and spirometry for intrathoracic/lower airway lesions. If the PN involves upper and lower airway, oscillometry, and spirometry should be performed, if feasible. Testing should be performed pre-treatment, and then prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): Spirometry: Please record the following measures: FEV1 (% Predicted ) FVC (% predicted ) FEV1/FVC In preschool children: FEV0.75 (% Predicted ) FEF25-75 Peak Expiratory Flow (PEF) SaO2 Oscillometry: Please record the following measures: Impulse Oscillometry (IOS): R10 (Hz) R20 (Hz) Please return with full copy of PFT/oscillometry results (without patient identifiers) via fax to the NCI: Attention: Trish Whitcomb, RN:

208 Participant s C3D ID: Date: Current Cycle: APPENDIX IX: MOTOR/STRENGTH EVALUATIONS PHASE II APPENDIX IXA: ENDURANCE EVALUATION All patients 5 years at enrollment, with lower extremity PN or airway PN, should undergo endurance testing using the 6-Minute Walk-Run Test. This evaluation should be performed by Rehabilitation Medicine, Physiatry, or Physical Therapy, if possible. Testing should be done pre-treatment and then prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): TOTAL DISTANCE (METERS) HEART RATE Before Run At Completion of Run After 5 Minute Cool-down BLOOD PRESSURE Before Run After Completion of Run After 5 Minute Cool-down VELOCITY (Total Distance (m)/6 Minutes) COMPLETED BY: DATE: Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

209 APPENDIX IXB: STRENGTH EVALUATION Participant s C3D ID: Date: Current Cycle: All patients with plexiform neurofibromas (PN) that cause motor dysfunction or weakness should undergo a focused evaluation of strength of the affected muscle groups. The contralateral side should be tested for comparison. This evaluation should be performed by Rehabilitation Medicine, Physiatry, or Physical Therapy, if possible, and should ideally be completed by the same examiner at each visit, if feasible. Please grade strength in the relevant muscle groups using Medical Research Council (MRC) grading of 0-5/5 (+/- may be used). Medical Research Council. Aids to the examination of the peripheral nervous system, Memorandum no. 45, Her Majesty's Stationery Office, London, 1981 Testing should be performed pre-treatment and prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): Manual Muscle/Strength Testing (include affected and at-risk muscle groups) POSITION RIGHT LEFT SUPINE Neck Flexors SIDELYING Gluteus Medius PRONE Gluteus Maximus Hamstrings Plantar flexors Neck extensors SITTING Trapezius Deltoids Biceps Triceps Wrist flexors Wrist extensors Finger extensors Finger flexors Interossei Iliopsoas Quadriceps Anterior tibial Gastrocnemius Extensor Hallucis Longus COMPLETED BY: DATE:Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

210 APPENDIX IXC: RANGE OF MOTION EVALUATION Participant s C3D ID: Date: Current Cycle: All patients with plexiform neurofibromas (PN) that cause motor dysfunction or weakness should undergo a focused evaluation of range of motion of the affected joints. The contralateral side should be tested for comparison. This evaluation should be performed by Rehabilitation Medicine, Physiatry, or Physical Therapy, and should ideally be completed by the same examiner at each visit, if feasible. Testing should be performed pre-treatment and prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 COMPLETED BY: Other (Please specify): RIGHT NECK LEFT Extension (Sidelying) Flexion (Sidelying) Lateral Flexion (Supine) Rotation (Supine) UPPER LIMB SHOULDER (Supine) Abduction (0-180) Internal Rotation (0-70) External Rotation (0-90) ELBOW (Supine) Flexion (0-50) Extension (0) WRIST (Supine) Flexion (0-80) Extension (0-70) LOWER LIMB HIP Flexion (0-120) (Supine) Extension (0) (Sidelying) External Rotation (0-60) (Supine) Internal Rotation (0-45) (Supine) Abduction (0-45) (Supine) KNEE (Supine) Flexion (0-145) Extension (0) ANKLE (Supine) Plantarflexion (0-50) Dorsiflexion (0-20) 1.1. DATE: Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

211 APPENDIX IXD: LEG LENGTH EVALUATION Participant s C3D ID: Date: Current Cycle: All patients with plexiform neurofibromas (PN) located in the lumbosacral plexus or below (i.e. lower limb) should undergo leg length measurements to evaluate for discrepancy. This evaluation should be performed by Rehabilitation Medicine, Physiatry, or Physical Therapy, and should ideally be completed by the same examiner at each visit, if feasible. Measurements should be taken from the Anterior Superior Iliac Spine to the Medial Malleolus of the ankle. The subject should be lying supine with hips and knees extended. Testing should be performed pre-treatment and prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): LEG LENGTH (cm) RIGHT LEFT Leg length discrepancy (difference between right and left): COMPLETED BY: DATE: Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

212 APPENDIX IXE: GROOVED PEGBOARD TEST All patients 5 years at enrollment with plexiform neurofibromas (PN) located in the upper extremities should undergo Grooved Pegboard Testing. Testing should be performed pre-treatment and prior to cycles 5, 9 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): Age: GROOVED PEGBOARD SCORE SHEET Dominant Hand (Circle one): LEFT RIGHT Dominant Hand Non-Dominant Hand Time (Sec) # of drops # of pegs placed X ± SD Z COMPLETED BY: DATE: Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

213 GROOVED PEGBOARD INSTRUCTIONS NCI protocol #11-C-0161; Version Date: This is a pegboard and these are the pegs. (The examiner points out each and then picks up one of the pegs and continues). All the pegs are the same, that is, they have a round side and a swuare side and so do the holes in the board. What you must do is match the groove of the peg with the groove in the board and put these pegs into the holes like this. (The examiner demonstrates by filling the top row. Then removes the pegs, putting them back into the tray. For the right hand trial, the examiner demonstrates that the pegs are placed from the patient s left to right, and from right to left for the left hand trial. The dominant hand trial is administered first, followed by the non-dominant hand trial). For adolescents (ages 9-0 to 14-12) and adults (age 15 years): When I say go, begin here and put the pegs into the board as fast as you can, using only your (dominant) hand. Fill the top row completely from this side to this side (from left to right for right-handed patients, from right to left for left-handed). Do not skip any. Fill each row the same way you filled the top row. Any questions? Ready, as fast as you can, go. (Repeat this task with the non-dominant hand) For children (ages 5-0 to 8-12) When I say go, begin here and put the pegs into the board as fast as you can, using only your (dominant) hand. Fill the top row completely from this side to this side (from left to right for right-handed patients, from right to left for left-handed). Do not skip any. Then go on to the second row and fill it the same way you filled the top row. Stop when you have completed the second row. Any questions? Ready, as fast as you can, go. (Repeat this tast with the non-dominant hand). 133

214 Participant s C3D ID: Date: Current Cycle: APPENDIX X: BLADDER AND BOWEL PATIENT QUESTIONNAIRE PHASE II Patients with bowel/bladder dysfunction or their guardians should complete the questionnaire below. Testing should be performed pre-treatment, and prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 Other (Please specify): To be completed by Patient if able, otherwise completed by the Parent/Guardian: 1. I pee in my underwear during the day: Never 1 day a week 2 3 days a week 4 5 days a week Every day 2. When I pee in my underwear, they are: I don t pee in my underwear Almost dry Damp Wet Soaked 3. In a normal day I go to the washroom to pee: 1 2 times 3 4 times 5 6 times 7 8 times More than 8 times 4. I feel that I have to rush to the washroom to pee: Never Less than half of the time Half of the time More than half of the time Every day 5. I hold my pee by crossing my legs or sitting down: Never Less than half of the time Half of the time More than half of the time Every day 6. It hurts when I pee: Never Less than half of the time Half of the time More than half of the time Every day 7. I wet my bed at night: Never 3 4 nights per week 1 2 nights per week 4 5 nights per week Every night 8. I wake up to pee at night: Never 3 4 nights per week 1 2 nights per week 4 5 nights per week Every night 9. When I pee, it stops and starts: Never Less than half of the time Half of the time More than half of the time Every day 10. I have to push or wait for my pee to start: Never Less than half of the time Half of the time More than half of the time Every day 134

215 11. I have bowel movements (poop): More than every day Every day Every other day Every 3 days 12. My stool (poop) is hard: Never Less than half of the time Half of the time More than half of the time Every day 13. I have bowel (poop) accidents in my underwear: Never 1 2 times per week 3 times per week 4 5 times per week Every day 14. How easy was it to answer these questions? Very easy Easy Neither easy nor difficult Difficult Very difficult Completed By (Patient or Parent/Guardian): Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

216 11.14 APPENDIX XA: OTHER PN 1. PN AFFECTING SPEECH/SWALLOW Speech Language Pathology Assessments: NCI protocol #11-C-0161; Version Date: Initial phone consultation with NCI Speech Pathology will be made based for PN: 1. affecting speech and swallowing (e.g. tongue, facial, anterior neck/upper airway) or with the potential to affect speech or swallowing; or 2. any one yes response to the following questions provided to the patient at the initial screening questionnaire: a. When eating or drinking, do you experience throat clearing? b. Do you feel food or liquid sticking in your neck when eating? c. Do you cough or choke when eating or within 20 minutes after eating? d. Have you noticed a change in your speech, voice or nasal quality? This mechanism will trigger the initial phone consultation with Beth Solomon, Speech Pathologist at the NCI. Review of the individual case with the primary team and Beth Solomon will determine recommended appropriate testing and may include: Review of swallowing functions for severity and frequency of symptoms Cranial nerve assessments for speech and swallowing function Diadochokinetic sampling of tongue function for speech and swallowing Digital speech and voice recordings for assessment of maximal phonation time, acoustic analysis and perceptual ratings. Additional instrumental assessments may be implemented following review of individual s functional status, plexifom neurofibroma location and size. Speech and swallowing functional assessments should be performed at pre-treatment, and prior to cycles 5, 9, 13, and then after every 12 cycles and at the end of therapy. Select One: Baseline Prior to: Cycle 5 Cycle 9 Cycle 13 For questions, please contact: Other (Please specify): Beth Solomon, MS, CCC-SLP Speech Language Pathologist, Rehabilitation Medicine Department 10 Center Drive 1_NE1455 Bethesda, MD Phone: BSolomon@cc.nih.gov 136

217 Participant s C3D ID: Date: Current Cycle: APPENDIX XI: PATIENT-REPORTED OUTCOMES QUALITY OF LIFE, PAIN AND PHYSICAL FUNCTIONING ASSESSMENT MEASURES PHASE II I. QOL and Pain Measures for Patients with all PN Types In participants with plexiform neurofibromas in all locations, this study will assess general healthrelated qualify of life (QOL) and pain (pain intensity and pain interference) using patient-reported outcomes (PROs) as described below. Description of QOL and Pain Measures General Health-Related Quality of Life Pediatric Quality of Life Inventory (PedsQL; Varni, 2001): The PedsQL 4.0 Generic Core Scales are multidimensional child self-report and parent proxy-report scales to assess health-related quality of life (QOL) in children, adolescents, and young adults ages 2 25 years. It is a brief standardized pediatric QOL scale with good reliability and validity, which includes both generic and disease specific modules. It consists of a 23-item core measure of global QOL that has four subscales: physical functioning, emotional functioning, social functioning, and school functioning. The domain of physical functioning will be used as a specific secondary outcome measure for this study, especially the parent proxy for younger children as it assesses children under 6 years of age. There are different forms for parents of patient s ages 2 18 years (toddler: 2 4; young child: 5 7; child: 8 12; adolescent: 13 18) and parallel self-report forms for patient s ages 5 25 years (young child: 5 7; child: 8 12; adolescent: 13 18; young adult: 18 25). It takes approximately 5-10 minutes to complete. For this study, children from 8 to 18 years of age will complete self-report measures of the PedsQL, and parents or legal guardians of children from 2 to 18 years of age will complete the parent proxy measures of the PedsQL. Pain Intensity The Numerical Rating Scale-11 (NRS-11) is a self-report segmented 11-point numeric scale that assesses pain severity (Hawker et al., 2011). It consists of a horizontal line with 0 representing no pain at the right end of the line and 10 representing worst pain you can imagine at the left end. Patients are asked to circle the one number from 0 to 10 that best describes their worst pain over the past week for the following: 1) overall pain, 2) overall tumor pain and 2) pain for a specific tumor they choose. It takes less than 1 minute to complete. The NRS-11 is recommended as a core outcome measure of pain intensity for clinical trials (Dworkin et al., 2005) in children with NF1 (Wolters, et al., 2013). Children, ages 8 to 18 years, will complete self-report measures of the NRS-11 for this study. Pain Interference The Pain Interference Index (PII) is a 6-item measure that assesses the degree to which pain has interfered with daily activities in the past week. This measure was developed in Sweden and 137

218 validated in Swedish with a group of children and adolescents with longstanding idiopathic pain (Wicksell, Melin, Lekander, & Olsson, 2009). The author (R. Wicksell) translated the measure into English, and members of the Neurobehavioral Group (S. Peron and P. Wolters) worked with the Swedish group to modify the wording to make it more readable and to shorten the time frame to one week, and we adapted it to create a parallel parent version. Dr. Wicksell and the Swedish research group are supportive of our efforts to validate the self-report and parent versions of the PII in NF1. Results of our validation studies in children with NF1 and their parents indicate that the internal consistency and construct validity of the PII are good. These data support the use of the PII to assess pain interference in youth with NF1 and PNs (Martin et al., submitted). For this study, children from 8 to 18 years of age will complete self-report PII, and parents or the legal guardian of children from 6 to 18 years of age will complete the parent proxy PII. Global Change in Pain The patient Global Impression of Change (GIC) Scale is a single item scale that evaluates the clinical significance of changes in pain intensity. We will administer an adapted version of the GIC Scale (Appendix ) at the follow-up PRO evaluations only (it should not be given pre-study because it assesses change in pain from baseline). A parallel parent proxy form also was developed. On the adapted GIC, patients (and their parents separately) will give their overall impression of change in level of the child s pain from before initiation of the MEK inhibitor to the current evaluation point. This global measure of change will be administered to children >8 years and for parents of children > 6 years of age at each PRO assessment through the first year (prior to cycle 13). The GIC Scale has been used in several research studies on chronic pain in adults and more recently children (Arnold et al., 2011; Mohammad et al., 2014). It has been recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) for clinical trials involving chronic pain (Dworkin et al., 2005; McGrath et al., 2008). The relationship between the numerical scale (NRS-11) and the qualitative scale (GIC) will be determined by breaking the participants into groups based on their qualitative response and determining the mean point difference associated with each qualitative label. This information will help evaluate the effects of the study drug on pain as well as provide data on clinically meaningful changes in pain. Pediatric Background Information The QOL Background Form should be completed by the parents of all patients ages 2-18 years at each evaluation to document information about demographics, use of pain medication, and NF1 disease severity. These data will be used to better interpret the PRO data. Administration of QOL and Pain Measures Participants will complete the following PRO measures at baseline (prior to starting treatment) and then prior to cycles 3, 5, 9, 13, and then after every 12 cycles. Self-report Children, 8 to 18 years of age, will complete the self-report measures of the PedsQL, NRS-11, and PII at baseline and all follow-up evaluations. The GIC scale will be done only at the followup evaluations after the other measures have been completed. Parent Proxy 138

219 Parents (or the legal guardian) of children, 2 to 18 years of age, will complete the parent proxy measures of the PedsQL and a background form. Parents (or the legal guardian) of children, 6 to 18 years of age, also will complete the parent proxy measures of the PII. At the follow-up evaluations only, they will be administered the GIC scale, which is to be completed after the other measures have been done. PATIENTS WITH MOTOR FUNCTION PN ONLY II. Physical Functioning PRO Measures only for Patients with a Motor PN In participants with plexiform neurofibromas affecting motor function, this study will administer a supplemental measure of physical functioning to further assess both mobility and upper extremity motor skills, as described below. Description of Supplemental Physical Functioning Measures Physical Functioning (Mobility and Upper Extremity) The Patient Reported Outcome Measurement Information System (PROMIS ), funded by the National Institutes of Health to provides clinicians and researchers access to efficient, precise, valid, and responsive parent and child reported measures of health and well being. The PROMIS Physical Functioning Scales assess the domains of Mobility and Upper Extremity Function and include mobility items such as I can could walk upstairs without holding on to anything and upper extremity items such as I can button my shirt or pants. Parent proxy items are parallel to child items. PROMIS pediatric eight-item short forms show good psychometric characteristics after large-scale testing (DeWitt et al., 2011). The pediatric parent proxy physical functioning scales also can be used for children ages 6-7 years (Varni et al., 2014). Pediatric self-report short forms will be administered to children from 8-18 years of age. Parallel parent proxy forms will be administered for children ages 6-18 years. The short forms consist of 8 items using a 5-point Likert scale format (i.e: 1 = unable to do, 5 = can do without any difficulty). Raw scores are converted to a T score, which is based on reference data from the US general population, where mean=50, SD=10. The 2 forms take approximately 2-3 minutes to complete. Administration of Supplemental Physical Functioning Measures Participants with motor PNs will complete the following PRO measures at baseline (prior to starting treatment) and then prior to cycles 3, 5, 9, 13, and then after every 12 cycles. Self-report Children, 8 to 18 years of age, will complete the self-report PROMIS Pediatric Physical Functioning Mobility and Upper Extremity Short Forms 8a Parent Proxy Parents (or the legal guardians) of children 6 to 18 years of age will complete the parent proxy PROMIS Physical Functioning Mobility and Upper Extremity Short Forms 8a. Additional guidelines for all evaluations will be sent to participating sites directly. 139

220 Please return the completed forms (without patient identifiers) via fax, using the attached fax cover sheet, to the NCI, Attention: Trish Whitcomb, RN: If you have any questions about the adminstration of these forms, please contact Dr. Pam Wolters at or References: DeWitt, E.M., Stucky, B.D., Thissen, D., Irwin, D.E., Langer, M., Varni, J.W., Jin-Shei Laig, Yeatts, K.B., DeWalt, D.A. (2011). Construction of the eight-item patient-reported outcomes measurement information system pediatric physical function scales: built using item response theory. Journal of Clinical Epidemiology, 64 (2011), Dworkin, RH, Turk, DC, Farrar, JT et al., (2005). Core outcome measures for chronic pain clinical trials: IMMPACT recommendations. Pain, 113, Hawker, G.A., Mian, S., Kendzerska, T., & French, M. (2011). Measures of Adult Pain. Arthritis Care & Research, 63 (S11), S240-S252. Varni JW, Seid M, Kurtin PS. (2001). PedsQL 4.0: Reliability and validity of the pediatric quality of life inventory version 4.0 generic core scales in healthy and patient populations. Med Care, 39, Varni JW, Burwinkle TM, Seid M. (2005). The PedsQL as a pediatric patient-reported outcome: Reliability and validity of the PedsQL measurement model in 25,000 children. Expert Rev Pharmacoecon Outcomes Res, 5: Varni JW, Thissen D, Stucky BD, Liu Y, Magnus B, Quinn H, Irwin DE, DeWitt EM, Lai JS, Amtman D, Gross HE, DeWalt AD. (2014). PROMIS Parent Proxy Report Scales for children ages 5-7 years: an item response theory analysis of differential item functioning across age groups. Quality of Life Research, 23: Wicksell RK, Melin L, Lekander M, Olsson GL. (2009). Evaluating the effectiveness of exposure and acceptance strategies to improve functioning and quality of life in longstanding pediatric pain - A randomized controlled trial. Pain, 141: Wolters, P.L., Martin, S., Merker, V.L., Gardner, K.L., Hingtgen, C.M., Tonsgard, J.H., Schorry, E.K., Baldwin, A. for the REiNS International Collaboration. (2013). Patient-reported outcomes in neurofibromatosis and schwannomatosis clinical trials. Neurology, 81, (Suppl 1), S6-S14. Martin, S., Nelson Schmitt, S., Wolters, P., Abel, B., Toledo-Tamula, M., Baldwin, A., Wicksell, R.K., Merchant, M., and Widemann, B. Development and Validation of the English Pain Interference Index and Pain Interference Index Parent Report. Manuscript submitted. 140

221 Patient-Reported Outcome (PRO) Questionnaires Administered by Age of Patient and Respondent Table 1. PROs administered to all patients by age and respondent Parent Questionnaires 8-12 (Child) Age of Patient (years) Background Information Parent report: Pediatric Background Form Pain Interference Parent proxy report: Pain Interference Index (6-7) Global Impression of Change (in pain) Parent report: Global Impression of Change Scale (6-7) General QOL Parent proxy report: PedsQL Acute form 2-4 (Toddler) 5-7 (Young Child) (follow-up evaluations only) 8-12 (Child) Table 2. PROs also administered to patients with motor tumors by age and respondent* Patient Questionnaire Age of Patient (years) Physical Functioning Patient self-report: PROMIS Mobility and Upper Extremity Short forms Parent Questionnaire Physical Functioning Patient Questionnaire Pain Intensity Patient self-report: Numeric Rating Scale-11 Pain Interference Patient self-report: Pain Interference Index Global Impression of Change (in pain) Patient self-report: Global Impression of Change Scale General QOL Patient self-report: PedsQL Acute form Age of Patient (years) Age of Patient (years) (follow-up evaluations only) (Teen) 141

222 Patient Questionnaire Parent proxy report: PROMIS Mobility and Upper Extremity Short forms NCI protocol #11-C-0161; Version Date: Age of Patient (years) (6-7) *In addition to the PROs administered to all patients, those with motor tumors also get the PROMIS measures. Administer items with a check mark under the correct age range of the patient 142

223 Phase II Study of Selumetinib in Children with NF1: PRO Checklists 143

224 Administration Instructions for Patient-Reported Outcome Measures (Phase II Selumetinib in children with NF1) Below are specific administration instructions for the pain, quality of life (QOL), and physical functioning PRO assessments required by the protocol. General administration procedures for the QOL/Pain questionnaires Fill out the patient/study ID#, date, and cycle number on each PRO form. Administer the QOL/pain questionnaires in person in the clinic. Please inform the parent/guardian that the same person needs to complete the PRO parent proxy forms each time. If the same parent cannot come to a particular clinic appointment, please give the forms to the other parent/guardian who came to the visit to give to the parent at home to complete and return in a postage-paid addressed envelope. The other option is to mail the forms to the parent on the day of the clinic visit with a postage-paid addressed envelope for the return and call the parent to let them know. The parent and patient should complete the forms separately. If a child patient needs help reading or marking an answer a staff person should help them, not their parent. When giving the forms to the participant, briefly explain the instructions for each of the QOL/pain questionnaires (see specific administration instructions below). Prior to administering the NRS-11 pain intensity questionnaire at any of the follow-up evaluations, refer back to the completed baseline form to identify the location of the tumor that the participant previously specified and then write this information on the blank line in the first question on the current form. Specifying the location will help ensure that they rate the severity of pain for the same tumor across all evaluations. Mention that you can read or explain any of the items if they need any help reading or if they have any questions. For participants who may have difficulty reading, it is permissible to read the items to them, or explain any items they have difficulty understanding, but the patients should answer the items themselves. One way to do this is to give the patient their own form on which to mark his/her answers while the staff person reads the items from another copy of the questionnaire. Immediately after the participants are finished filling out the questionnaires and before they leave the clinic, please check each form to make sure that the items are completed correctly (e.g., only one response circled) and that all the applicable items are answered. --If more than one answer is circled for an item, please have the individual choose one response. --If they forgot to answer an item, ask them to please try to answer it. Contact information For specific questions about administration of the forms or the QOL/pain data, please contact Pam Wolters at or woltersp@mail.nih.gov For any questions regarding the QOL/pain transmission forms please contact Trish Whitcomb at or whitcomt@mail.nih.gov 144

225 INTRODUCING THE STUDY and SPECIFIC ADMINISTRATION INSTRUCTIONS To begin, introduce yourself and say to parent and child: I have the pain and quality of life questionnaires for you and your child to complete for the study. The purpose of these questionnaires is to assess the effects of the treatment on (child s name) pain and everyday functioning. I will be asking each of you to complete these questionnaires before (child s name) starts treatment and then every 3 to 12 months during the study to evaluate the effects of the treatment on his/her pain and quality of life. Do you have any questions so far? OK, I m going to ask you to fill these forms out separately. First, I will take you (the parent) to a room and explain the instructions for each form. Then I will take you (the child) to a different room and help you do the questionnaires. Take them to separate rooms and explain the instructions below. Administration Instructions for each PRO Measure Children and parents should be administered the PRO forms separately. The child participant should complete the questions on their own, without help from a parent or significant other. The parent participant who will be completing the forms also should do them on their own without help from the child or another parent or relative. Please show the child and parent participant each questionnaire and briefly describe the instructions for each one. For younger children or any participants who have difficulty reading, the staff person administering the forms can read through each question, and wait for the child to circle his/her answer on the paper. If the child has motor problems and difficulty writing, the staff person can circle the answer the child says. Below are examples of how to explain the forms to the parent and patient. PARENTS/GUARDIANS: Background Form Provide the general instructions: This is a background form for you to give us general information about you and your child, such as about your child s educational history, pain medication, and severity of NF1 symptoms, which may help us better understand the results from the questionnaires. PII - Pain Interference Provide the general instructions: Here you will find a list of questions about how your child s pain gets in the way of his/her everyday activities. Please answer each question by circling a number between 0, which means pain does not interfere at all, and 6, which means pain interferes completely. Please note that we are asking about your child s activities during the past 7 days. PedsQL QOL Scale Provide the general instructions: This next questionnaire is a quality of life scale for children that assesses functioning four areas. Please read each item and answer how much of a problem each one has been for your child during the past 7 days, from 0 if it is NEVER a problem to 4 if it is ALMOST ALWAYS a problem. 145

226 PROMIS Physical Functioning Provide the general instructions: NCI protocol #11-C-0161; Version Date: These two questionnaires ask you about your child s physical functioning, which is how your child can move around and use his/her body, such as walking, playing, writing, and getting dressed. Read each question and then check one box per row that best describes how much trouble your child has doing each of these physical activities. Global Impression of Change Scale This has only one question to ask you about how much you think your child s pain has changed since starting on the MEK inhibitor for this study. The choices range from very much improved to very much worse. Please check only one box. For these questionnaires, there are no right or wrong answers, just try your best to answer them as honestly as possible. Do you have any questions about the forms? Please complete the forms in the order in which I have given them to you. I will give you some time to complete them and then I ll check back to see if you have any questions. Thank you! PATIENTS: NRS-11 - Pain Intensity Provide the general instructions: This is a questionnaire for you to show if you have pain and how much it hurts. There are 3 questions asking about different kinds of pain you might have. For each question, there is a line with numbers from 0 to 10, where 0 means no pain and 10 means the worst pain you can imagine. For each question, please circle the one number that best describes that pain at its worst during the past 7 days. For the last question, we want you to pick one tumor and then we will ask you to tell us how much that tumor hurts throughout the whole study. Which tumor do you want to pick? (The location of that tumor should be written on the form by the patient or staff person). PII - Pain Interference Provide the general instructions: Here you will find a list of questions about how your pain gets in the way of your everyday activities. Please answer each question by circling a number between 0, which means pain does not interfere at all, and 6, which means pain interferes completely in that activity. Please remember that we are asking about your activities during the past 7 days. PedsQL QOL Scale Provide the general instructions: 146

227 This questionnaire is a quality of life scale that assesses functioning in four areas. Please read each item and answer how much of a problem each one has been for you during the past 7 days, from 0 if it is NEVER a problem to 4 if it is ALMOST ALWAYS a problem. PROMIS Physical Functioning Provide the general instructions: These two questionnaires ask you about your physical functioning, which is how you can move around and use your body, such as walking, playing, writing, and getting dressed. Read each question and then check one box per row that best describes how much trouble you have doing these each of these physical activities. Global Impression of Change Scale Provide the general instructions: This is only one question to ask you about how much you think your pain has changed since starting on the MEK inhibitor for this study. The choices range from very much improved to very much worse. Please check only one box. For these questionnaires, there are no right or wrong answers, just do the best you can. Do you have any questions about the forms? I can read the questions to you if you would like. OK, please complete the forms in the order in which I have given them to you. I will give you some time to complete them and then I ll check back to see if you have any questions. (However, you may stay with the patient, especially younger children, to answer questions they may have or help with reading the form if needed). Thank you! **Please check back with the parent within minutes to see if he/she has any questions. When the parent and patient are done, immediately check each form to make sure that all the items are completed correctly (e.g., only one response circled) and that all the applicable items are answered. 147

228 Pediatric NF-1 PRO Background Information Sheet (For parents or primary caregivers to complete) Today s date: Child s date of birth: Child s sex: M F The parent/primary caregiver completing the parent questionnaires for this study is the child s: mother father other, please specify: Demographics: What is the total number of years of education completed by the parents/primary caregivers? Mother Father Write in the highest grade in school completed (from 1 12): Write in the number of years of college completed (from 1 4): Write in the number of years of graduate/professional school completed: Relationship of child to parent/primary caregiver: biological adopted foster extended family Special Education or Psychiatric Diagnosis: Has your child ever been diagnosed by a doctor or other health professional with any of the following special education or psychiatric diagnoses? (Please check each of the following yes or no). Attention deficit/hyperactivity disorder yes no Anxiety yes no Learning disability (specify: ) yes no Other (specify: ) yes no Depression yes no Current Alternative or Special Educational Services: Do you home school your child (by choice)? yes no Does your child receive any of the following services? (Please check each of the following yes or no). Self-contained special education class yes no Speech therapy yes no Pull-out services/resource room yes no Physical therapy yes no Mainstream class with aide yes no Occupational therapy yes no Homebound schooling (e.g., due to medical reasons) yes no Other: (specify: ) yes no School Performance: Your child s current overall school performance is: Above average Average Below Average If your child is too young for school, your child s overall development is: Above average Average Below Average Pain Medication: Is your child taking any medication for pain on a regular basis? yes no If yes, what kind of pain medication: over the counter (like Motrin or Tylenol) prescription both Visibility of tumor(s): When dressed, is your child s plexiform neurofibroma tumor(s) visible? (Please check one). mild: No visible tumor(s) outside of the normal clothing areas, and gait and posture appear normal when casually observed by others. moderate: Tumor(s) is visible on the neck, face, or hands, or other areas not typically covered by clothes, and/or gait or posture is somewhat affected. severe: Large tumor(s) is visible on the neck, face or hands, or other areas not typically covered by clothes and/or gait or posture is severely affected. Severity of NF-1 symptoms: How would you rate the symptoms of NF-1 that your child experiences? (Please check one). mild: Symptoms rarely affect physical well-being, daily functioning, or social life, such as neurofibroma(s) that are not visible and do not affect posture or gait noticeably, transient or mild pain that can be controlled, and/or mild learning disorders that generally do not affect activities of daily living. moderate: Symptoms moderately compromise daily functioning but are not severely disabling, such as external or internal neurofibroma(s), recurrent pain, problems with gait, posture, or vision, and/or learning disorders that may need intervention and somewhat affect activities of daily living. severe Symptoms significantly impact daily functioning, such as large internal or external neurofibroma(s) or other serious NF-1 tumors, significant pain that is not controlled, severe problems with gait, posture, or vision, and/or severe learning disorders that require intervention and greatly affect activities of daily living. 148

229 11.16 APPENDIX XI: PAIN MEDICATION SURVEY: Participant s ID: Date: To be completed by the patient of patient care giver for all patients (all ages). Please collect daily and as needed pain medications for 1 week prior to enrollment. Please include: Dates Pain medication name Frequency No Pain Medications used Sunday Monday Tuesday Wednesday Thursday Friday Saturday #1 #2 #3 #4 #5 COMPETED BY: DATE: Please return (without patient identifiers) via fax to the NCI, Attention: Trish Whitcomb, RN:

230 C3DID# Date: PedsQL Pediatric Quality of Life Inventory Acute Version Version 4.0 CHILD REPORT (ages 8-12) DIRECTIONS On the following page is a list of things that might be a problem for you. Please tell us how much of a problem each one has been for you during the past 7 days by circling: 0 if it is never a problem 1 if it is almost never a problem 2 if it is sometimes a problem 3 if it is often a problem 4 if it is almost always a problem There are no right or wrong answers. If you do not understand a question, please ask for help. 150

231 In the past 7 days, how much of a problem has this been for you About My Health and Activities (PROBLEMS WITH ) Never Almost Never NCI protocol #11-C-0161; Version Date: Sometimes Sometimes Often 1. It is hard for me to walk more than one block It is hard for me to run It is hard for me to do sports activity or exercise It is hard for me to lift something heavy It is hard for me to take a bath or shower by myself It is hard for me to do chores around the house I hurt or ache I have low energy Almost Always bout My Feelings (PROBLEMS WITH ) Never Almost Never Often. I feel afraid or scared I feel sad or blue I feel angry I have trouble sleeping I worry about what will happen to me ow I Get Along with Others (PROBLEMS WITH ) Never Almost Never. I have trouble getting along with other kids Other kids do not want to be my friend Other kids tease me I cannot do things that other kids my age can do It is hard to keep up when I play with other kids bout School (PROBLEMS WITH ) Never Almost Never Sometimes Sometimes Often Often. It is hard to pay attention in class I forget things I have trouble keeping up with my schoolwork I miss school because of not feeling well I miss school to go to the doctor or hospital Almost Always Almost Always Almost Always 151

232 C3DID# Date: PedsQL Pediatric Quality of Life Inventory Acute Version Version 4.0 TEEN REPORT (ages 13-18) DIRECTIONS On the following page is a list of things that might be a problem for you. Please tell us how much of a problem each one has been for you during the past 7 days by circling: 0 if it is never a problem 1 if it is almost never a problem 2 if it is sometimes a problem 3 if it is often a problem 4 if it is almost always a problem There are no right or wrong answers. If you do not understand a question, please ask for help. 152

233 In the past 7 days, how much of a problem has this been for you About My Health and Activities (PROBLEMS WITH ) Never Almost NCI protocol #11-C-0161; Version Date: It is hard for me to run It is hard for me to do sports activity or exercise It is hard for me to lift something heavy It is hard for me to take a bath or shower by myself It is hard for me to do chores around the house I hurt or ache I have low energy About My Feelings (PROBLEMS WITH ) Never Almost 2. I feel sad or blue I feel angry I have trouble sleeping I worry about what will happen to me How I Get Along with Others (PROBLEMS WITH ) Never Almost 2. Other teens do not want to be my friend Other teens tease me I cannot do things that other teens my age can do It is hard to keep up with my peers About School (PROBLEMS WITH ) Never Almost Sometimes Often Almost Never Always 1. It is hard for me to walk more than one block Sometimes Often Almost Never Always 1. I feel afraid or scared Sometimes Always Often Almost Never 1. I have trouble getting along with other teens Sometimes Always Often Almost Never 1. It is hard to pay attention in class I forget things I have trouble keeping up with my schoolwork I miss school because of not feeling well I miss school to go to the doctor or hospital

234 C3DID# Date: PedsQL Pediatric Quality of Life Inventory Acute Version Version 4.0 PARENT REPORT for CHILDREN (ages 8-12) DIRECTIONS On the following page is a list of things that might be a problem for your child. Please tell us how much of a problem each one has been for your child during the past 7 days by circling: 0 if it is never a problem 1 if it is almost never a problem 2 if it is sometimes a problem 3 if it is often a problem 4 if it is almost always a problem There are no right or wrong answers. If you do not understand a question, please ask for help. 154

235 In the past 7 days, how much of a problem has your child had with Physical Functioning (PROBLEMS WITH ) Never Almost Never NCI protocol #11-C-0161; Version Date: Walking more than one block Running Participating in sports activity or exercise Lifting something heavy Taking a bath or shower by him or herself Doing chores around the house Having hurts or aches Low energy level Feeling sad or blue Feeling angry Trouble sleeping Worrying about what will happen to him or her Other kids not wanting to be his or her friend Getting teased by other children Not able to do things that other children his or her age can do Often Keeping up when playing with other children Sometimes Emotional Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Feeling afraid or scared Social Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Getting along with other children School Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Paying attention in class Forgetting things Keeping up with schoolwork Missing school because of not feeling well Missing school to go to the doctor or hospital Almost Always Almost Always Almost Always Almost Always 155

236 C3DID# Date: PedsQL Pediatric Quality of Life Inventory Acute Version Version 4.0 PARENT REPORT for TEENS (ages 13-18) DIRECTIONS On the following page is a list of things that might be a problem for your teen. Please tell us how much of a problem each one has been for your teen during the past 7 days by circling: 0 if it is never a problem 1 if it is almost never a problem 2 if it is sometimes a problem 3 if it is often a problem 4 if it is almost always a problem There are no right or wrong answers. If you do not understand a question, please ask for help. 156

237 In the past 7 days, how much of a problem has your teen had with NCI protocol #11-C-0161; Version Date: Running Participating in sports activity or exercise Lifting something heavy Taking a bath or shower by him or herself Doing chores around the house Having hurts or aches Low energy level Almost Always 2. Feeling sad or blue Feeling angry Trouble sleeping Worrying about what will happen to him or her Almost Always 2. Other teens not wanting to be his or her friend Getting teased by other teens Not able to do things that other teens his or her age can do Keeping up with other teens Almost Always Physical Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Walking more than one block Emotional Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Feeling afraid or scared Social Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Getting along with other teens School Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Paying attention in class Forgetting things Keeping up with schoolwork Missing school because of not feeling well Missing school to go to the doctor or hospital Almost Always 157

238 C3DID# Date: PedsQL Pediatric Quality of Life Inventory Acute Version Version 4.0 PARENT REPORT for TODDLERS (ages 2-4) DIRECTIONS On the following page is a list of things that might be a problem for your child. Please tell us how much of a problem each one has been for your child during the past 7 days by circling: 0 if it is never a problem 1 if it is almost never a problem 2 if it is sometimes a problem 3 if it is often a problem 4 if it is almost always a problem There are no right or wrong answers. If you do not understand a question, please ask for help. 158

239 In the past 7 days, how much of a problem has your child had with NCI protocol #11-C-0161; Version Date: Running Participating in active play or exercise Lifting something heavy Bathing Helping to pick up his or her toys Having hurts or aches Low energy level Feeling sad or blue Feeling angry Trouble sleeping Worrying Other kids not wanting to play with him or her Almost Always Almost Always Almost Always 3. Getting teased by other children Not able to do things that other children his or her age can do Keeping up when playing with other children Physical Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Walking Emotional Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Feeling afraid or scared Social Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Playing with other children *Please complete this section if your child attends school or daycare School Functioning (PROBLEMS WITH ) Never Almost Never Sometimes Often 1. Doing the same school activities as peers Missing school/daycare because of not feeling well Missing school/daycare to go to the doctor or hospital Almost Always

240 C3DID# Date: PedsQL Pediatric Quality of Life Inventory Acute Version Version 4.0 PARENT REPORT for YOUNG CHILDREN (ages 5-7) DIRECTIONS On the following page is a list of things that might be a problem for your child. Please tell us how much of a problem each one has been for your child during the past 7 days by circling: 0 if it is never a problem 1 if it is almost never a problem 2 if it is sometimes a problem 3 if it is often a problem 4 if it is almost always a problem There are no right or wrong answers. If you do not understand a question, please ask for help. 160

241 In the past 7 days, how much of a problem has your child had with Physical Functioning (PROBLEMS WITH ) Never Almost Never NCI protocol #11-C-0161; Version Date: Running Participating in sports activity or exercise Lifting something heavy Taking a bath or shower by him or herself Doing chores, like picking up his or her toys Having hurts or aches Low energy level Emotional Functioning (PROBLEMS WITH ) Never Almost Never 2. Feeling sad or blue Feeling angry Trouble sleeping Worrying about what will happen to him or her Other kids not wanting to be his or her friend Getting teased by other children Not able to do things that other children his or her age can do Keeping up when playing with other children Sometimes Often 1. Walking more than one block Sometimes Often 1. Feeling afraid or scared Social Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Getting along with other children School Functioning (PROBLEMS WITH ) Never Almost Sometimes Often Never 1. Paying attention in class Forgetting things Keeping up with school activities Missing school because of not feeling well Missing school to go to the doctor or hospital Almost Always Almost Always Almost Always Almost Always 161

242 C3D ID # DATE: 162

243 C3D ID # DATE: 163

244 C3D ID # DATE: 164

245 C3D ID # DATE: 165

246 C3D ID # DATE: 166

247 C3D ID # DATE: 167

248 C3D ID # DATE: 168