Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: 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 CONTENTS SUPPLEMENTARY APPENDIX: Appendix Title Page List of investigators 3 Supplementary Methods 3 Overall Trial Design 3 Criteria for Dose Escalation (Standard 3+3 phase 1 dose escalation) 4 Definition of Dose Limiting Toxicity (DLT) 4 Criteria for Intra Patient Dose Escalation 5 Definition of Maximum Tolerated Dose (MTD) 6 Criteria for Starting Subsequent Treatment Cycles 7 Duration of Treatment 7 Treatment Modifications 8 Cardiac Toxicity 9 Recommendations for treatment with ACE inhibitors 11 Preclinical trial of selumetinib in the DhhCre;Nf1fl/fl genetically engineered mouse models (GEM) of NF1 neurofibromas 11 Supplementary Results 15 Effect of selumetinib on height and weight 15 Selumetinib Exposure 15 Supplementary Figures 16 Figure S1: Relationship of selumetinib dose (mg/m 2 ) and (A) maximal selumetinib concentration (C max ng/ml); (B) selumetinib exposure (AUC 0- ng h/ml); and (C) Relationship of age in years and apparent selumetinib clearance (CL/F L/m 2 /h) 16 1

3 Figure S2: Pharmacokinetics and pharmacodynamics of selumetinib in the mouse neurofibroma model Figure S3: Waterfall plot demonstrating response (black) and tumor growth in controls (white) in the preclinical trial of selumetinib Supplementary Tables 20 Table S1: Trial eligibility requirements 20 Table S2: On study evaluations 22 Table S3: Baseline and on study patient characteristics 25 Table S4: Selumetinib related adverse events 30 Table S5: Selumetinib cycle 1, dose 1 pharmacokinetic parameters Table S6: N-desmethyl selumetinib cycle 1, dose 1 pharmacokinetic parameters Table S7: Anecdotal evidence of improvement in pain, function, and appearance on the selumetinib trial Table S8: Comparison of selumetinib phase I trial results to other PN directed clinical trials Supplementary Appendix References 41 2

4 List of investigators: Eva Dombi, M.D., Andrea Baldwin, P.NP., Leigh J. Marcus, M.D., Michael J. Fisher, M.D., Brian Weiss, M.D., AeRang Kim, M.D., Ph.D., Patricia Whitcomb, R.N., Staci Martin, Ph.D., Lindsey E. Aschbacher-Smith, M.S., Tilat A. Rizvi, Ph.D., Jianqiang Wu, M.D., Rachel Ershler, M.D., Pamela Wolters, Ph.D., Janet Therrien, B.S., John Glod, M.D., Ph.D., Jean B. Belasco, M.D., Elizabeth Schorry, M.D., Alessandra Brofferio, M.D., Amy J. Starosta, Ph.D., Andrea Gillespie, R.N., Austin L. Doyle, M.D., Nancy Ratner, Ph.D., Brigitte C. Widemann, M.D. Supplementary Methods: Study design, definition of dose-limiting toxicities, and management of toxicities (copied from study protocol) Overall Trial Design This is a multi-center phase I trial of selumetinib 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 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. Automated volumetric MRI analysis will be used to 3

5 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 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. Criteria for Dose Escalation (Standard 3+3 phase 1 dose escalation) 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 (study protocol, 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 study protocol Section 3.2 and dosing nomogram Appendix V). 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 selumetinib with the exception of the following: 4

6 Any grade 3 transaminases 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. Any grade 3 electrolyte abnormality must be confirmed with a repeated measurement within hours. In addition, cardiac toxicity as defined in study protocol section 3.3.1, or any grade 1 cataract will be considered dose limiting. Any grade 2 urinary tract obstruction at least possibly related to selumetinib (and clearly not related to tumor), or 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. 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 selumetinib dose escalated to the next highest dose level if: The patient does not have progressive disease as defined in study protocol Section 5.2. The patient has not experienced selumetinib related toxicities > grade 1. Off study Criteria have not been met (study protocol 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 5

7 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) 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 (study protocol 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. 6

8 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 study protocol section 5.2. 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 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 study protocol Section However, treatment may be discontinued earlier at 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 radiographic response (volume decreased by 20%) is observed. For patients who do show radiographic response (study protocol Section 5.2), the treatment duration will not be limited unless the patient experiences subsequent disease progression or meets other off treatment criteria (study protocol Section 3.9, 3.10). However, treatment may be discontinued earlier at 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 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 7

9 or < 20% decrease in PN volume) in a patient who enrolled on the trial with progressive disease (see study protocol section paragraph 1), or improvement of symptoms or function. 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 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. Selumetinib 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, except in the event that the patient was receiving clear clinical benefit as defined in study protocol 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 8

10 >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 study protocol 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), dose is reduced 30%, dose may not be re-escalated, and doses held will not be made up. If dose-limiting toxicity recurs after two dose reductions, 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. Cardiac Toxicity Cardiac toxicity will be managed as described below: 1) Patients who have an asymptomatic decline in LVEF from baseline but continue to have a normal EF of 55% (if a range is given then the upper limit of the range will be used) will continue on treatment with selumetinib and have a repeat ECHO 3-6 weeks later, and as recommended by cardiology. Patients who have a symptomatic decline in LVEF from baseline but continue to have a normal EF of 55% (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 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 Study Protocol Section 3.1.7), and if symptoms were found to 9

11 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. 2) For patients who have a symptomatic or asymptomatic decline in LVEF from baseline of 10 percentage points to < 55% (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. ECHOs will be repeated on these patients every 3-6 weeks, and as recommended by cardiology until recovery from toxicity (LVEF 55%). If the LVEF returns to 55% within 3 months of discontinuation of selumetinib, and the patient is receiving benefit from selumetinib (see study protocol Section 3.1.7), and symptoms have resolved, selumetinib hyd sulfate may be restarted at a reduced dose. 3) For patients who have an asymptomatic decline in LVEF from baseline of < 10 percentage points to < 55% (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. ECHOs will be performed every 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 and patients remain asymptomatic. Patients who have a symptomatic decline in LVEF from baseline of < 10 percentage points to < 55% (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. ECHOs will be repeated in these patients every 3-6 weeks and as recommended by cardiology until recovery of LVEF to 55%. If the LVEF returns to 55% within 3 months of discontinuation 10

12 of selumetinib, and the patient is receiving benefit from selumetinib (see study protocol Section 3.1.7), and symptoms resolve, selumetinib may be restarted at reduced 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. 1) Enalapril: start at 2.5 mg once daily or 0.1 mg/kg daily for children smaller than 25 kg. 2) Lisinopril: (children >6yo; GFR >30) start at 0.07 mg/kg once daily. 3) Captopril: start 0.3 mg/kg/day divided every 8-12 hours. 4) 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. 5) 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 study protocol Section 3.6. Preclinical trial of selumetinib in the DhhCre;Nf1fl/fl genetically engineered mouse models (GEM) of NF1 neurofibromas Mouse Husbandry Mice were housed in temperature- and humidity-controlled facilities on a 12-hr dark light cycle with free access to food and water. The animal care and use committee of Cincinnati Children s Hospital Medical Center approved all animal use. The mice were in a mixed C57/129/FVBN 11

13 strain background and were interbred to obtain the required genotype. Mouse genotyping was carried out as described 1. Selumetinib Dosing Nf1fl/fl;DhhCre mice were treated with 10mg/kg selumetinib (from AstraZeneca, UK) or vehicle (control; 0.5% [w/v] methylcellulose solution with 0.2% [v/v] polysorbate 80 [Tween-80]), given twice daily via oral gavage, five days a week (5 days on, 2 days off) for 8 weeks. MRI and Neurofibroma Volumetric Measurement MRI in anesthetized Nf1 flox/flox ; DhhCre mice using a 7T Bruker Biospec (Ettlingen,Germany) and neurofibroma volumetric measurement were conducted as described 2,3. MRI, Statistical Analysis The effect of selumetinib 10 mg/kg BID (equivalent to 30 mg/m 2 in humans), 5 days on, 2 days off, in the DhhCre;Nf1fl/fl GEM neurofibroma model 1 was assessed with volumetric MRI analysis as previously described 2. Blood samples for pharmacokinetic analysis and tumor samples for pharmacodynamics studies were obtained as described previously 2. Neurofibroma shrinkage in each mouse from the last pre-treatment value to the post treatment value was compared using Fisher s exact test. Neurofibroma growth was compared by analyzing log transformed tumor volume data with a random effects model using the SAS mixed procedure 2. Plasma Collection We anesthetized tumor-bearing mice with iso-fluorane and collected blood by cardiac puncture. Blood samples were transferred to EDTA tubes on ice, and centrifuged within 30 min of collection at 4 C for 15 min at 14,000g. Plasma was stored at -80 C until analysis. 12

14 Pharmakokinetic Analysis Selumetinib was quantified in mouse plasma using midazolam (MDZ) as the internal standard and HPLC-MS/MS (Thermo TSQ Quantum Ultra). Selumetinib and MDZ were removed from plasma by protein precipitation with acetone. Selumetinib and MDZ were separated by a gradient mobile phase (acetonitrile: 5 mm ammonium acetate) with a C-18 column (Inertsil ODS-3;4.6 X 150 mm, 5 µm). The Q1/Q3 s (in positive mode) for selumetinib and MDZ were 483/249 and 326/291, respectively. The lower limit of quantification was 1 ng/ml using 20 ml of mouse plasma. Pharmacokinetic parameters for selumetinib including area under the curve (AUC) and the elimination rate constant, k el, were estimated (from average data) using noncompartmental methods with add-ins on Excel. Pharmacadynamic Analysis Tumors were harvested from mice after a final drug dose, snap frozen in liquid nitrogen and stored at -80 C. Tumors were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail (Halt TM Protease & Phosphatase Single-Use Inhibitor Cocktail 100X, Thermo Scientific, Florence, KY) using a Qiagen Tissue Ruptor. Protein concentration was estimated using Coo-massie1Plus Protein Assay Reagent (Bio-Rad, Berkeley, Ca). Proteins (20 mcg) were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis on 4 20% tris-glycine gel (Bio-Rad) and transferred to polyvinylidene diflouride membrane by electrophoresis. Membranes were blocked with 5% nonfat milk in 0.1% TBST to minimize nonspecific binding. Antibodies recognizing perk (1:5,000; #4370, Cell Signaling, Danvers, MA), ERK (1:5,000; #4695, Cell Signaling) were probed overnight at 4 C, followed by appropriate secondary HRP conjugated antibodies for 1 hr at room temperature. Antibody binding to the membrane was visualized using the Immobilon TM Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA). 13

15 Tissue Sections, Histology, and Immunohistochemistry Formalin-fixed neurofibromas were paraffin embedded. Paraffin blocks sectioned at 5 mm thickness, baked at 60 C for 1 h and air dried. Paraffin sections were deparaffinized and boiled in 0.01 M citrate buffer (ph 6.0) for antigen retrieval. Sections were quenched with 3% hydrogen peroxide to block endogenous peroxidases for 10 min, rinsed in PBS, and blocked in 10% normal goat serum with 0.3% Triton X-100. Sections were incubated overnight in primary antibody diluted in blocking medium; rabbit anti-iba1 (Wako Chemicals, Richmond, VA, 1:2,000), rabbit cleaved caspase 3 (CC3) (#9664S, 1:2000; Cell Signaling, Boston, MA), Ki67 (1:2000; NovaCastra). Sections were then incubated in appropriate biotinylated secondary antibodies (1:200; Vector). Microscopic images were acquired with Openlabsoftware suites on a Zeiss Axiovert 200.Cells were counted at 400X magnification in 4 high power fields per section, three sections per animal in at least three animals per condition. Statistical comparisons between control and neurofibroma samples were conducted using Student s t-test or One Way ANOVA. 14

16 Supplementary Results Effect of selumetinib on height and weight Potential effects of selumetinib on growth were monitored by comparing height and weight percentiles for age at baseline and most recent time point included in data analysis, and by calculating the annualized growth velocities and weight gain (increase in height or weight during treatment with selumetinib divided by duration of therapy in months, multiplied by 12) for each patient. There was no measurable effect of selumetinib on growth and weight during treatment. The mean height and weight percentiles for age at enrollment and after a median of 16.8 (±8.2) months on treatment with selumetinib were similar: Height 32.6% (±27.7) and 36.0% (±29.9), weight: 45.0% (±31.4), and 48.0% (±28.8), respectively. Patients grew on average 4.9 (±3.7) cm/year, and gained 4.1 (±4.0) kg/year. Selumetinib Exposure Selumetinib exposure was similar in patients with and without dose-limiting toxicities in cycles 1-3. Pharmacokinetic parameters of selumetinib in children on our study were similar to adults 4. At the 25 mg/m 2 dose level (corresponding to a fixed dose of 45 mg/dose based on an adult body surface area of 1.8 m 2 ) and the 30 mg/m 2 dose level (corresponding to a fixed adult dose of 54 mg), the mean selumetinib drug exposure was 2665 ng h/ml and 3434 ng h/ml, respectively in comparison to 3075 ng h/ml in adults receiving 50 mg selumetinib per dose 4. The mean maximal selumetinib concentration was 940 ng/ml at 25 mg/m 2 dose and 1057 ng/ml at 30 mg/m 2 dose compared to 1036 ng/ml at the adult 50 mg/dose dose level. 15

17 Figure S1: Relationship of selumetinib dose (mg/m 2 ) and (A) maximal selumetinib concentration (C max ng/ml); (B) selumetinib exposure (AUC 0- ng h/ml); and (C) Relationship of age in years and apparent selumetinib clearance (CL/F L/m 2 /h). The median increase in selumetinib exposure from dose level 1 to 1.5 was 1.15 (expected 1.25), from dose level 1.5 to 2 was 1.13 (expected 1.2), and from dose level 1 to 2 was 1.29 (expected 1.5). 16

18 Figure S2. Pharmacokinetics and pharmacodynamics of selumetinib in the mouse neurofibroma model. (A) Pharmacokinetics of selumetinib in mouse. Mice were administered a single dose of Selumetinib (10mg/kg via oral gavage). Blood was collected at 0.5, 2, 6, 10, and 14 hours after selumetinib dosing (n=3 per group per time point). (B) Pharmacodynamics of selumetinibtreated mice. Tumor-bearing Nf1 flox/flox ;DhhCre mice were dosed once with control vehicle; 0.5% [w/v] methylcellulose solution with 0.2% [v/v] polysorbate 80 [Tween-80], or Selumetinib10mg/kg in vehicle, p.o. Neurofibromas were rapidly removed, on ice, 0.5, 2 or 6h after dosing (n=3 mice/time point). Numbers indicate relative band intensity (measured with ImageJ) on film. Band intensity was calculated by dividing band intensities of antiphosphorylated-erk (P-ERK) by those of anti-total ERK (ERK1/2) for each sample; each ratio was then normalized to a control sample, which was set to 1.0. β-actin was used as an additional loading control. (C, D) Neurofibroma cell proliferation and cell death after selumetinib treatment. Nf1 flox/flox ;DhhCre mice were treated with 10mg/kg Selumetinib (AZD6244) given twice daily or vehicle (control), p.o. five days a week (5 days on, 2 days off) for 8 weeks. Neurofibromas were removed rapidly 0.5-2h after the final dose of Selumetinib, fixed, paraffin embedded, and sectioned. Sections were stained with antibodies detecting the proliferation marker Ki67 (C), or the apoptosis marker anti- cleaved caspase 3+ (D), using DAB as the chromogen. Graphs represent cell counts from 3-4 animals/group, 3 sections/sample and 3 areas/section. Error bars represent SEM. Statistical analysis was performed using Student s t-test. 17

19 18

20 Figure S3: Waterfall plot demonstrating response (black) and tumor growth in controls (white) in the preclinical trial of selumetinib. DhhCre;Nf1fl/fl were MRI scanned at 5 and 7 months of age to determine neurofibroma growth rate for statistical analysis. Mice were then treated for 2 months with vehicle or selumetinib, followed by MRI scan at 9 months of age. Each bar displays the percent change in neurofibroma volume between 7 and 9 months in a single mouse based on serial MRI imaging. A positive value indicates neurofibroma size increase; a negative value indicates neurofibroma shrinkage. 19

21 Table S1: Trial eligibility requirements Age: 3 years (until 9/ years old) and 18 years of age at the time of study enrollment Diagnosis: Patients with NF1 and inoperable plexiform neurofibromas (PN) 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. 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 NF1: In addition to PN, all study subjects must have either positive genetic testing for NF1 or meet diagnostic criteria for NF1 ( NIH Consensus conference) PN: Measurable: 3 cm in one dimension Inoperable: Complete PN resection is not considered feasible without substantial risk for morbidity Histologic confirmation not required in presence of consistent radiographic and clinical findings, but to be performed if malignant transformation was considered Performance status: Patients 16 years of age Karnofsky performance level of 70% Children < 16 years old Lansky performance of 70% Organ function: Recovered from prior therapy to grade 1 toxicity Hematologic function: Absolute neutrophil count 1,000/µL Hemoglobin 9.0 g/dl Platelet count 100,000/µL Hepatic function: Bilirubin within 1.5 x the normal limits except for patients with Gilbert syndrome Alanine aminotransferase 1.5-times the upper limit of normal Renal function: Age adjusted normal serum creatinine Cardiac function: Normal ejection fraction (echocardiogram) 20

22 Prior treatment, last dose of: Colony stimulating factors 1 week Radiation 6 weeks Investigational agent >30 days MRI imaging: Ability to undergo MRI evaluation of PN Exclusion criteria: Inability to swallow whole selumetinib capsules Ongoing hormonal-, immuno-, or chemotherapy directed at PN >1 prior myelosuppressive chemotherapy regimen Pregnancy or breastfeeding Significant systemic unrelated illness Presence of optic glioma, malignant glioma, malignant peripheral nerve sheath tumor, or other cancer requiring treatment with chemotherapy or radiation Inability to return for follow up visits Supplementation with vitamin E greater than 100% of the daily recommended dose 21

23 Table S2: On study evaluations End of Pre- During Therapy STUDIES TO BE OBTAINED Study Cycle 1 Prior To Subsequent Cycles 1 Evaluation 2 History X Every other week Physical Exam, vital signs X Every other week Every other week cycles 2 3 Prior to cycles 4 11, 13, 15, 17, etc Every other week cycles 2 3 Prior to cycles 4 11, 13, 15, 17, etc X X 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 and Echocardiogram 12 X - Prior to cycle 4, 6, 11 and then - CBC, differential, platelets 5 X Every other week after every 6 cycles (pre 17, 23, 29, etc) Every other week cycles 2 3 Prior to cycles 4 11, 13, 15, 17, etc X Pharmacokinetics 6 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, X Every other Every other week cycles 2 3 X Mg++, BUN, creatinine, glucose, week Prior to cycles 4 11, 13, 15, 17, etc ALT, bilirubin, Total protein/albumin, CPK 22

24 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 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 - Adherence Questionnaire X If indicated If indicated 10 cycle 6, 11 and then after every 6 cycles (pre 17, 23, 29, etc) Patient diary 11 - Daily Daily - Pill Count X 12 1 Day (+/- 3 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 study protocol, Section BSA should be calculated from average of three measurements. 4 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 study protocol Appendix IV for timing of PK samples 7 Volumetric MRI. See study protocol Appendix II. 8 If patient does show a responsive disease (RD) according to study protocol (Section 5.2) response should be confirmed after 1-3 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 23

25 (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 Indication for follow-up adherence questionnaire is less than 90% adherence upon diary review and pill count. 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 the research team. 12 The patient s research team 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 team at each visit. 13 Patients who stop selumetinib for cardiac toxicity should be monitored by ECHO every 3 to 6 weeks. Upon recovery from toxicity (to a LVEF of 55%), patients should be monitored with an ECHO every 2 3 cycles as directed by the cardiologist (see study protocol, Sections and 3.6) 24

26 Table S3: Baseline and on study patient characteristics Baseline characteristics On study characteristics Pt. Dose Age Sex PS Prior Baseline Target PN Dose-Limiting Maximal PN PN Morbidity at Complete No (mg/m 2 ) (Yr) (F, M) (%) PN Location Volume Growth Rate Toxicities (CTCAEv4) Shrinkage Baseline and on Cycles Therapy (ml) (%/ months ) Event Grade Cy. (% / months ) Selumetinib (N) M/S (N) M 90 0/0 Abdomen 2449 No PD -4% / 8m CK 3 40 PR -47% / 42m M 100 1/0 Neck, 211 No PD PR Appearance + 50 chest 0% / 16m -44% / 42m M 90 2/0 Pelvis, 4216 PD Cellulitis 3 15 SD Pain + 46 thigh +20% / 15m -17% / 9m Appearance Function motor F 100 1/0 Brachial 321 PD PR Function motor + 35 plexus + 177%/16m -30% / 10m Appearance F 100 2/0 Neck 507 PD Cellulitis 3 1 SD Pain % / 12m -15% / 11m Appearance Off treatment 25

27 M 80 0/5 Lumbar spine 104 N/A PR -36% / 27m Pain F 100 0/1 Chest 309 N/A PR % / 26m M 80 3/2 Pelvis, 8744 No PD PR Pain + 30 thighs +18% / 7m -37% / 21m Appearance + Function motor M 100 0/0 Chest, 260 PD Urticaria 3 1 PR - 30 abdomen +38% / 14m -25% / 20m F 90 1/0 Pelvis, 1621 No PD PR Pain + 30 leg +18% / 15m -38% / 26m Appearance M 80 2/0 Neck, chest 2280 N/A PR -24% / 22m Pain F 70 2/1 Neck, SD Pain 6 chest N/A -6% / 5m Appearance Off study F 80 1/1 Face, 788 N/A CK 3 10 PR Pain 40 neck -34% / 24m Appearance Function facial 26

28 F 90 2/2 Neck, 1646 No PD SD Appearance 29 chest +18% / 15m -14% / 5m Off treatment M 90 3/0 Whole 3408 No PD CK 3 2 SD Pain + 18 body +8% / 18m CK % / 5m Off study M 90 6/1 Pelvis, 7210 No PD LVEF 3 5 PR Appearance 30 thigh +18% / 16m -24% / 5m Function motor F 100 2/0 Orbit 47 PD Mucositis 2 8 SD Appearance % / 15m Mucositis % / 10m Function vision Off Mucositis 2 35 treatment M 80 3/1 Pelvis, 2091 No PD PR Appearance 37 thigh +19% / 15m -33% / 15m Pain Function motor M 90 4/4 Face, 1861 PD PR Appearance + 26 neck, +22% / 8m -35% / 20m Pain + chest Function motor F 80 1/0 Pelvis, 2158 PD CK 4 19 PR Appearance + 26 leg +105% / 18m -44% / 15m Pain + 27

29 M 90 1/6 Face, neck 207 N/A PR -26% / 9m Function motor + Appearance F 100 0/1 Orbit 29 N/A PR Appearance % / 21m M 70 2/0 Chest, 2238 PD Oral 2 9 SD Appearance 25 arm +23% / 16m mucositis -16% / 15m Pain + Function motor F 90 2/0 Head, 290 PD Rash 3 2 PR Appearance 23 face +49% / 16m -33% / 22m Abbreviations: PS= performance score: Karnofsky if 16 years old, Lansky if < 16 years old; PN= plexiform neurofibroma; M= number of prior PN directed medical treatments, which were: tipifarnib (N=3), prifenidone (N=3), peginterferon-alfa-2b (N=10), sirolimus (N=8), thalidomide (N=5), imatinib (N=6). S=number of prior PN related surgeries excluding biopsies/excisional biopsies; CK= creatine kinase; LVEF= left ventricular ejection fraction; PD= progressive disease prior to enrollment (=PN volume increase 20% within 18 months prior to enrollment); No PD= Not meeting criteria for PD prior to enrollment; N/A=insufficient data to determine progression status prior to enrollment. PN volume change within 18 months prior to study entry: Percent volume change / measured over months indicated. Months when maximal PN volume decrease was observed. PR=partial response; SD=stable disease. + Improvement in morbidity during treatment with selumetinib (see Supplemental Table S5). Patients removed from treatment with selumetinib: 28

30 Patients 5 and 11 lack of partial response, patient 6 lack of response and non-dose limiting toxicities, patient 8 after 2 dose reductions for intolerable mucositis, and patient 18 per patient request. 29

31 Table S4: Selumetinib related adverse events Cycles 1-3 Cycles 4 Dose Level (mg/m 2 /dose) 20 (n=12) 25 (n=6) 30 (n=6) 20 (n=12) 25 (n=6) 30 (n=6) Toxicity Grade CTCAEv Gastrointestinal Abdominal Pain Diarrhea Constipation Nausea Vomiting Anorexia Mucositis (Oral) Sore Throat Dry Mouth 1 1 Gastroesophageal Reflux 1 Disease Dermatologic Rash (Acneiform) Rash (Maculopapular) Pruritis Dry Skin Alopecia 1 2 Hair Color Lighter Hair Thinning

32 Urticaria 1 Erythema 1 1 Eczema 1 1 Hepatic Alkaline Phosphatase AST ALT Blood Bilirubin 1 1 Metabolic/Laboratory CPK Albumin Calcium Magnesium Magnesium 2 2 Sodium Sodium 1 1 Phosphorus Potassium 2 1 Glucose Constitutional Fatigue Fever 1 1 Hematologic White Blood Cell

33 Neutrophil Count Lymphocyte Count Lymphocyte Count Anemia Hemoglobin Platelets 1 1 Epistaxis 1 2 Neurologic Headache Dizziness 1 2 Sensory Neuropathy 2 1 Blurred Vision 1 1 Pain Back Pain Extremity Pain Non-cardiac chest pain 1 Pain (Other) Musculoskeletal Toxicity Myalgia Renal/Genitourinary Toxicity Creatinine Proteinuria Hematuria

34 Urinary Tract Pain 1 1 Urinary Tract Infection 1 Infection Paronychia/Nail Infection Skin Infection Infection: Cellulitis 1 1 Cardiac Hypertension Ejection Fraction Sinus Bradycardia 2 All possibly, probably, or definitively selumetinib related adverse events (dose-limiting and non-dose-limiting, worst grade per patient) during, and after the first three treatment cycles with exception of grade 1 adverse events that occurred only in 1 patient (<5 percent). Total number of cycles 1-3 completed = 72, total number of cycles 4 completed =

35 Table S5: Selumetinib cycle 1, dose 1 pharmacokinetic parameters Dose level Pt Age BSA Dose C max T max AUC 0-24h AUC 0- CL/F T 1/2 C trough (mg/m 2 ) (#) (years) (m 2 ) mg/dose mg/m 2 % dev. (ng/ml) (h) (ng h/ml) (ng h/ml) (L/h/m 2 ) (h) (ng/ml) Mean SD Median

36 Mean SD Median NO Abbreviations: Pt=patient; BSA= body surface area; dev=deviation; C max = maximal selumetinib concentration; T max = time to maximal selumetinib concentration; AUC= area under the concentration x time curve; CL/F= apparent clearance; T ½ = half-life; C trough = selumetinib trough concentrations on day of cycle 1. + Patients with dose-limiting toxicity during cycle

37 Table S6: N-desmethyl selumetinib cycle 1, dose 1 pharmacokinetic parameters Dose level Pt Age BSA Selumetinib Dose C max T max AUC 0-24h AUC 0- T 1/2 C trough (mg/m 2 ) (#) (yrs) (m 2 ) mg/dose mg/m 2 % dev. (ng/ml) (h) (ng h/ml) (ng h/ml) (h) (ng/ml) Mean SD Median

38 Mean SD Median N-desmethyl selumetinib trough concentrations on day of cycle 37

39 Table S7: Anecdotal evidence of improvement in pain, function, and appearance on the selumetinib trial Pt. No Baseline morbidity Improvement (months first noted) Improvement Category 2 Left neck pigmentation overlying PN 4 months: decrease in visible Appearance pigmentation overlying neck PN based on physical exam 3 Abdominal migraine 4 months: decrease frequency of Pain abdominal migraine based on patient report 6 Back pain 2 months: decrease back pain based on Pain 10 Decreased range of motion to right arm 14 Regular pain medication: oxycodone and gabapentin Visible buttock plexiform neurofibroma Unable to sit in wheelchair due to tumor bulk patient report 4 months: Increase in range of motion; able to raise arm above head and touch palms based on physical exam 12 months: Discontinued oxycodone Decreased visibility of buttock plexiform neurofibroma when unclothed Ability to sit comfortably in chair based on patient report Function Motor Pain Appearance Function Motor 16 Abdominal and leg pain 5 months: Decrease frequency in Pain abdominal pain and leg pain based on patient report 38

40 17 Scheduled celebrex for pain 8 months: decrease in pain, Celebrex Pain 19 Daily pain throughout the body Disfigurement secondary to plexiform neurofibroma Decreased range of motion to right arm 20 Chronic daily pain Decreased function, unable to walk for more than 5 minutes without pain, wheel chair use. Disfigurement 22 Disfigurement and proptosis secondary to PN 23 Pain to plexiform neurofibroma, guarding of plexiform neurofibroma. Unable to raise affected arm to shoulder level discontinued 7 months: pain reduced, Cymbalta discontinued, lyrica reduced Disfigurement improvement based on photography Increased range of motion to right arm based on physical exam 4 months: Decrease in pain, wean and discontinue neurontin, walking, less use of wheelchair 7 months: Disfigurement improved based on photography 7 months: Less proptosis, PN less disfiguring based on photography 3 months: Denies pain, does not guard plexiform neurofibroma based on parent report Able to raise affected arm at and above shoulder level based on photography and physical exam Pain Appearance Function Motor Pain Appearance Function Motor Appearance Pain Function Motor Note: All the data in this table were obtained from retrospective review of the patient s medical chart, including notes based on patient or parent report or investigator observations of the patients physical movement from the history and physical exam completed at each protocol evaluation and photography obtained on selected patient 39

41 Table S8: Comparison of selumetinib phase I trial results to other PN directed clinical trials Median (range) age, plexiform neurofibroma (PN) volume, median time to progression (TTP) and percent decrease in PN volumes in published phase II trials for progressive PN with similar eligibility criteria, identical response criteria, and central response evaluation using volumetric MRI analysis at the NCI. Comparison to patients with progressive PN enrolled on the selumetinib trial. The median cycle number on the selumetinib trial is 26 (range, 23-46) and no patient has experienced disease progression to date. Agent N Age PN volume TTP PN volume decrease (Years) (ml) (months) N 20% Max % Placebo (3-17.7) ( ) Tipifarnib (3-21.5) ( ) Pirfenidone (3-18.8) Sirolimus (3-45.4) 349 ( ) 186 ( ) Pegylated (6.9%) Not interferon alfa- ( ) ( ) provided 2b 8 Selumetinib 9 7 (5-14) 507 ( ) - 5 (56%) 47 40

42 Supplementary Appendix References 1. 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 2008;13: 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 2012;58: Jessen WJ, Miller SJ, Jousma E, et al. MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors. J Clin Invest 2013;123: 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. Clinical cancer research : an official journal of the American Association for Cancer Research 2010;16: 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 2014;16: 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 2014;61: Weiss B, Widemann BC, Wolters P, et al. Sirolimus for progressive neurofibromatosis type 1-associated plexiform neurofibromas: a 41

43 Neurofibromatosis Clinical Trials Consortium phase II study. Neuro Oncol 2015;17: Jakacki R, Dombi E, Steinberg SM, et al. Phase II trial of pegylated interferon alfa-2b in young patients with neurofibromatosis type 1 and unresectable plexiform neurofibormas. Neuro-Oncology Advance Access published August 10,