Supplementary appendix

Transcription

1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Laetsch TW, DuBois SG, Mascarenhas L, et al. Larotrectinib for paediatric solid tumours harbouring NTRK gene fusions: phase 1 results from a multicentre, open-label, phase 1/2 study. Lancet Oncol 2018; published online March 29.

2 Methods Pharmacokinetic modelling of larotrectinib dose The doses assigned to patients enrolled to cohorts 1 and 2 were based upon age and body surface area using nomograms designed to achieve AUC equivalent to adults treated at doses of 100 mg twice-daily (BID) and 150 mg BID, respectively, as predicted by SimCyp modelling. Evaluating the pharmacokinetic data of patients enrolled to cohorts 1 and 2, it was apparent that the modelled doses resulted in lower AUC among young/small children than in older children and adults. Intrapatient dose escalation revealed generally dose-proportional increases in AUC. Following intrapatient dose escalation, the average larotrectinib dose in patients who achieved an AUC equal to that in adult patients was 100 mg/m 2 /dose BID. Based on the pharmacokinetics of larotrectinib in patients enrolled to cohorts 1 and 2, we calculated that a dose of 100 mg/m 2 BID, regardless of age, would achieve an AUC comparable to the adult RP2D. The protocol was amended to use this dose, with a cap of 100 mg/dose (the adult RP2D) for cohort 3. Following completion of enrolment to cohort 3, we analysed all patients treated on study with a dose of mg/m 2 which totalled 19 patients (2 from cohort 1 and 8 from cohort 2 who were dose-escalated; and all 9 enrolled to cohort 3). Median AUC0-24 of larotrectinib in cohort 3 was 3440, 4270, and 4790 ng*h/ml in patients aged <2, 2 11, and years, respectively, which was comparable to the median AUC in adults treated at the RP2D, which was 4460 ng*h/ml (supplementary figure 1). 1

3 Results Supplementary table 1: Participating centres Centre Principal investigator Number of patients enrolled University of Texas Southwestern Medical Center / Children s Health Theodore W. Laetsch, MD 5 Seattle Children s Hospital, University of Washington, Fred Hutchinson Cancer Research Center Catherine M. Albert, MD / Douglas S. Hawkins, MD 5 Children s Hospital Los Angeles, Keck School of Medicine, University of Southern California Leo Mascarenhas, MD 4 Cincinnati Children s Hospital Medical Center Brian Turpin, DO 2 Dana-Farber/Boston Children s Cancer and Blood Disorders Center and Harvard Medical School Steven G. DuBois, MD 2 Nemours Children's Hospital Ramamoorthy Nagasubramanian, MD 2 St Jude Children s Research Hospital Alberto S. Pappo, MD 2 University of California, Los Angeles Noah Federman, MD 2 2

4 Supplementary table 2: All larotrectinib-related treatment-emergent adverse events. No grade 4 or 5 treatment-related adverse events were observed. All patients (n=24) Cohort 3 (recommended phase 2 dose; n=9) All Grades Grades 1-2 Grade 3 All Grades Grades 1-2 Grade 3 At Least One Related TEAE 22 (92%) 21 (88%) 4 (17%) 8 (89%) 8 (89%) 2 (22%) Alanine aminotransferase increased 10 (42%) 9 (38%) 1 (4%) 3 (33%) 2 (22%) 1 (11%) Aspartate aminotransferase increased 10 (42%) 10 (42%) 0 4 (44%) 4 (44%) 0 Leukocyte count decreased 5 (21%) 5 (21%) 0 2 (22%) 2 (22%) 0 Neutrophil count decreased 5 (21%) 4 (17%) 1 (4%) 3 (33%) 2 (22%) 1 (11%) Vomiting 5(21%) 5 (21%) 0 2 (22%) 2 (22%) 0 Anaemia 4 (17%) 4 (17%) 0 2 (22%) 2 (22%) 0 Constipation 4 (17%) 4 (17%) 0 2 (22%) 2 (22%) 0 Hypoalbuminaemia 4 (17%) 4 (17%) 0 1 (11%) 1 (11%) 0 Nausea 4 (17%) 3 (13%) 1 (4%) 3 (33%) 2 (22%) 1 (11%) Blood creatinine increased 3 (13%) 3 (13%) 0 1 (11%) 1 (11%) 0 Fatigue 3 (13%) 3 (13%) 0 1 (11%) 1 (11%) 0 Blood alkaline phosphatase increased 2 (8%) 2 (8%) 0 1 (11%) 1 (11%) 0 Hyperkalaemia 2 (8%) 2 (8%) 0 1 (11%) 1 (11%) 0 Insomnia 2 (8%) 2 (8%) Protein total decreased 2 (8%) 2 (8%) 0 2 (22%) 2 (22%) 0 Abdominal pain 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Alopecia 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Anorexia and bulimia syndrome 1 (4%) 1 (4%) Anxiety 1 (4%) 1 (4%) Blood cholesterol increased 1 (4%) 1 (4%) Delirium 1 (4%) 1 (4%) Diarrhoea 1 (4%) 1 (4%) Dizziness 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Dry skin 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Ejection fraction decreased 1 (4%) 0 1 (4%) Flatulence 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Haematuria 1 (4%) 1 (4%) Hypernatraemia 1 (4%) 1 (4%) Hypertension 1 (4%) 1 (4%) Increased appetite 1 (4%) 1 (4%) Lymphocyte count decreased 1 (4%) 1 (4%) Pain in extremity 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Pharyngeal inflammation 1 (4%) 1 (4%) Platelet count decreased 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Sinus tachycardia 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Skin sensitisation 1 (4%) 1 (4%) 0 1 (11%) 1 (11%) 0 Weight increased 1 (4%) 0 1 (4%)

5 Supplementary table 3: HRQOL scores at baseline and before cycle 6 Mean +/- SD Still evaluable at Evaluable at baseline start of cycle 6 Infants (<2 years) / / Children and adolescents (2-18 years) / /

6 Supplementary figure 1: A. Capsule and liquid pharmacokinetics for larotrectinib in children. B. Larotrectinib area under the curve. Larotrectinib in plasma (ng/ml) A) B) 10 Adults (100 mg BID, n=29) Paediatric (liquid formulation) (100 mg/m 2, max 100 mg, BID, n=6) Paediatric (capsule formulation) IC 90 IC (100 mg/m 2, max 100 mg, BID, n=3) < Time (hours) (99 mg/m 2 ) (98 mg/m 2 ) (96 mg/m 2 ) Age (years) and mean BID dose Estimated plasma AUC 0 24 (ng*h/ml) 5000 n=6 n=9* n=5 n=29 Adult (100 mg) Population N C max (ng/ml) T max (h) AUC 0 24 (ng*h/ml) T 1/2 (h) Paeds liquid ± (0 5 1) 5570 ± ± 0 3 Paeds capsule ± (1 2) 6689 ± ± 0 2 Adult capsule ± ± ± 0 7 C max, AUC 0 24, and T 1/2 are mean ± standard deviation; T max is median (range) Age Range (years) N AUC 0 24 (ng*h/ml) Mean ± SD < ± * 5808 ± ± 2791 Adult ± 3520 *One patient included in both <2 and 2 11 year categories (due to aging while on study) 5

7 Supplementary figure 2: Waterfall plot of maximal change in tumour size in TRK fusion patients by independent radiology read. Bars are colour coded by NTRK fusion and partner (A) and histological diagnosis (B). 6

8 Supplementary figure 3: Kaplan-Meier plot of duration of response for patients with investigator assessed confirmed objective response 7

9 CLINICAL PROTOCOL LOXO-TRK A Phase 1/2 Study of the Oral TRK Inhibitor LOXO-101 in Pediatric Patients with Advanced Solid or Primary Central Nervous System Tumors Investigational Product: Protocol Number: Larotrectinib (LOXO-101) LOXO-TRK EudraCT #: Development Phase: 1 / 2 Sponsor: Loxo Oncology, Inc. 281 Tresser Boulevard, 9 th Floor Stamford, CT Current Version February 2017 Previous Version November 2016 Previous Version September 2016 Previous Version February 2016 Previous Version January 2016 Previous Version December 2015 Previous Version November 2015 Confidentiality Statement This document contains confidential information that is the property of Loxo Oncology, Inc. and may not be disclosed to anyone other than the recipient Clinical Investigator, research staff, and respective members of the Institutional Review Board/Independent Ethics Committee. This information cannot be used for any purpose other than the evaluation of conduct of the clinical investigation without the prior written consent of Loxo Oncology, Inc. Version 7.0 Confidential & Proprietary Page 1 of 123

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11 SYNOPSIS TITLE: A Phase 1/2 Study of the Oral TRK Inhibitor LOXO-101 in Pediatric Patients with Advanced Solid or Primary Central Nervous System Tumors PROTOCOL NUMBER: LOXO-TRK STUDY SITES: Approximately 20 institutions will be recruited for participation in this study. PHASE: ½ PHASE 1 OBJECTIVES: Primary: To determine the safety of oral larotrectinib (LOXO-101), including dose-limiting toxicity (DLT), in pediatric patients with advanced solid or primary central nervous system (CNS) tumors. Secondary: To characterize the pharmacokinetic (PK) properties of larotrectinib in pediatric patients with advanced solid or primary CNS tumors To identify the maximum tolerated dose (MTD) and/or the appropriate dose of larotrectinib for further clinical investigation in this patient population To describe the antitumor activity of larotrectinib in pediatric patients with advanced solid or primary CNS tumors To describe pain and health related quality of life (HRQOL) in pediatric patients with advanced solid or primary CNS tumors treated with larotrectinib Exploratory: To evaluate potential biomarkers of response and resistance to larotrectinib Version 7.0 Confidential & Proprietary Page 3 of 123

12 STUDY DESIGN: Phase 1 Dose Escalation This part of the study is a multicenter, open-label, Phase 1 study in pediatric patients with advanced solid or primary CNS tumors. Larotrectinib will be administered orally (PO) twice daily (BID), with the dose adjusted by body surface area (BSA). Body surface area will be determined by the Mosteller Formula ( ((height weight)/3600)) (Mosteller 1987). The initial starting dose level used in this study was based on a dose (100 mg BID) that has been previously tested in adults, which has not met dose limiting toxicity criteria in that population. For Cohorts 1 and 2, PK modeling (SimCyp ) was used to select a dose for pediatric patients that was predicted to equal the exposure achieved in adult patients taking a dose of 100 mg BID. The modeling takes into account body size differences and ontogeny of the enzymes that metabolize larotrectinib. Interim analysis of PK data from Cohorts 1 and 2 indicated that a body-surface area-based dose would provide more consistent PK. Therefore, doses for Cohort 3 and subsequent cohorts will be based on BSA without regard to the patient s age. Given this change in dosing strategy, the incremental increase in dose between Cohort 2 and Cohort 3 is more modest than typically used in early phase trials. The maximum dose will be no higher than the recommended Phase 2 dose of 100 mg BID in the adult Phase 2 trial (LOXO TRK 15002), regardless of the patient s BSA. Version 7.0 Confidential & Proprietary Page 4 of 123

13 Synopsis Table 1: Starting Doses of Larotrectinib Level Pediatric Dose Adult Equivalent Dose % of Adult Dose of 100 mg BID mg BID 50% -1 Refer to Table 13 through Table mg BID 75% mg BID 100% mg BID 150% mg/m 2 * NA NA mg/m 2 * NA NA mg/m 2 * NA NA Frequency Twice daily (BID) Cycle Length One cycle = 28 days continuous dosing Duration of Dosing Dosing continues until PD, unacceptable toxicity, or other reason for treatment discontinuation Abbreviations: BSA = body surface area; BID = twice daily; PD = progressive disease. * = The maximum dose will be no higher than the recommended Phase 2 dose of 100 mg BID in the adult Phase 2 trial (LOXO TRK 15002), regardless of the patient s BSA. When capsules are intended to be used for doses that are not divisible evenly by 25, the dose amounts will be rounded to the nearest 25 mg; (e.g., calculated doses of 12.5 mg to less than 37.5 mg will be rounded to 25 mg). Escalation will proceed through the planned 5 dose levels, or until the MTD is reached, or until the Sponsor determines that a suitable dose has been achieved based on PK exposure. At dose levels 3 and 4, at least 3 subjects with a BSA <1 m 2 for Cohort 3 and Cohort 4 will be enrolled before escalating the dose further. Dose level 5 will only be cleared by the Safety Review Committee (SRC) if at least 3 subjects with BSA <0.7 m 2 are enrolled and clear the DLT window. Over-enrollment to these cohorts may occur in order to meet this requirement. This trial will use a Rolling 6 dose escalation scheme (Skolnik, Barrett et al. 2008). A minimum of 3 patients and a maximum of 6 patients may be enrolled in an open cohort. All patients in a given cohort must have completed safety assessments through Cycle (C) 1, Day (D) 28, and received a minimum of 75% of the planned total dose in C1 (unless due to toxicity) to be eligible for the assessment of a DLT. A Safety Review Committee (SRC) will be convened for each cohort dose escalation decision, but will only be required to convene prior to a cohort escalation if there is a DLT reported in a cohort. A minimum of 3 patients in a given cohort must have completed 28 days of safety assessment in C1 without DLT, and a maximum of 1 DLT may be seen in 6 patients who have completed the DLT window before the next cohort initiates accrual. Escalation will proceed through all dose levels or until the Sponsor determines that a suitable dose has been achieved based on PK exposure or unacceptable DLT s as noted above. If 2 (>33%) of patients within a cohort experience a DLT, then further enrollment to that cohort will stop and the cohort data will be reviewed by the SRC. In order for the MTD to be determined, the SRC will evaluate whether the previous lower dose level will be considered the MTD, whether an intermediate dose level should be evaluated. Based on the interim evaluation of the safety and tolerability data of the previous cohort, additional patients may be enrolled in a previously studied cohort, provided the DLT rate was <33% in that cohort, or that accrual will take place at an intermediate dose level in order to more closely characterize DLT(s) and accurately identify the MTD. The MTD is defined as the highest dose level at which none of the first 3 treated patients, or no more than 1 of the first 6 treated patients, experiences a DLT. Dose Limiting Toxicity Definition A DLT is defined as any of the following treatment-emergent adverse events (TEAEs; a TEAE is defined as an adverse event [AE] that starts on or after the first administration of study medication) occurring in the first 28 days (i.e., in C1), and not reasonably attributed to patient s underlying disease or other medical condition and the Investigator determines the TEAE is related to larotrectinib: Version 7.0 Confidential & Proprietary Page 5 of 123

14 Grade 3 or higher nonhematologic toxicity, with the exception of Grade 3 fatigue or nausea Grade 3 or 4 vomiting or diarrhea will be considered DLT only if it persists for more than 48 hours despite standard of care treatment. Admission to the hospital of children <1 year of age with Grade 3/4 vomiting or diarrhea will be required until these events resolve to Grade 1. Any toxicity, regardless of the Common Terminology Criteria for Adverse Events (CTCAE) grade, resulting in discontinuation or dose reduction of treatment (with the exception of symptoms related to disease progression [PD]) Grade 4 thrombocytopenia or Grade 3 thrombocytopenia with Grade 1 or higher bleeding Grade 4 anemia lasting more than 7 days, despite supportive therapy, and not explained by underlying condition (transfusions are allowed during cycle 1) Grade 4 neutropenia, lasting more than 7 days, despite supportive therapy (C1 growth factor support not allowed for prophylaxis; Grade 3 uncomplicated febrile neutropenia will not be considered a DLT) Larotrectinib is administered in oral capsule or liquid formulation at the recommended Phase 2 dose. Each cycle consists of 28 days. MAJOR ELIGIBILITY CRITERIA: Inclusion Criteria 1. Phase 1: Between 1 and 21 years of age at C1D1 with a locally advanced or metastatic solid tumor or primary CNS tumor that has relapsed, progressed or was nonresponsive to available therapies and for which no standard or available systemic curative therapy, or: 1 month old with a diagnosis malignancy and with a documented NTRK fusion that has progressed or was nonresponsive to available therapies, and for which no standard or available curative therapy exists. or: Patients with locally advanced infantile fibrosarcoma who would require, in the opinion of the Investigator, disfiguring surgery or limb amputation to achieve a complete surgical resection Version 7.0 Confidential & Proprietary Page 6 of 123

15 2. Patients with primary CNS tumors or cerebral metastasis: a) Must be neurologically stable based on stable neurologic exam for 7 days prior to enrollment. b) Must have not required increasing doses of steroids within the 7 days prior to study entry to manage CNS symptoms c) Phase 2 only: Imaging study must be performed within 28 days of C1D1 while on stable dose steroid medication (if needed) for at least 7 days immediately before the imaging study. 3. Histologic verification of malignancy at original diagnosis or relapse, except in patients with intrinsic brain stem tumors, optic pathway gliomas, or patients with pineal tumors and elevations of cerebral spinal fluid (CSF) or serum tumor markers including alpha-fetoprotein or beta-human chorionic gonadotropin (HCG). 4. Evaluable and/or measurable disease a) Phase 1: Subjects must have evaluable and/or measurable disease as defined by RECIST, RANO or INRC. 5. Karnofsky (those 16 years and older) or Lansky (those younger than 16 years) performance score of at least Patients must have fully recovered from the acute toxic effects of all prior anti-cancer chemotherapy. a) Myelosuppressive chemotherapy: At least 21 days after the last dose of myelosuppressive chemotherapy (42 days if prior nitrosourea) b) Investigational agent or anticancer therapy other than chemotherapy within 2 weeks prior to planned start of larotrectinib or 5 half-lives, whichever is shorter, and with recovery of clinically significant toxicities from that therapy c) Hematopoietic growth factors: At least 14 days after the last dose of a long-acting growth factor (e.g., Neulasta) or 7 days for short-acting growth factor d) Immunotherapy: At least 42 days after the completion of any type of immunotherapy (except for steroids), e.g., immune checkpoint inhibitors and tumor vaccines e) X-ray therapy (XRT): At least 14 days after local palliative XRT (small port); At least 42 days must have elapsed if other substantial bone marrow (BM) radiation, including prior radio iodized metaiodobenzylguanidine ( 131 I-MIBG) therapy. f) Stem cell infusion without traumatic brain injury (TBI): No evidence of active graft versus host disease and at least 56 days must have elapsed after transplant or stem cell infusion. 7. For patients with tumors amenable to biopsy: A fresh biopsy or an archival tumor tissue sample must be available. If archived sample is not available, a fresh biopsy (at a primary or at one metastatic site) should be performed. 8. For patients without known bone marrow involvement: a) Absolute neutrophil count (ANC) /L and b) Platelet count /L (transfusion allowed) c) Hemoglobin 8.0 g/dl (transfusions allowed) 9. Patients with bone marrow involvement will not be evaluable for hematologic DLT and can enroll with: a) ANC /L and b) Platelet count /L (transfusions allowed) c) Hemoglobin 8.0 g/dl (transfusions allowed) 10. Adequate hepatic and renal function, defined as: a) Bilirubin (sum of conjugated + unconjugated) 1.5 upper limit of normal (ULN) for age (patients Version 7.0 Confidential & Proprietary Page 7 of 123

16 with documented Gilbert s Disease may be enrolled with Sponsor approval) b) SGPT (ALT) 1.5 upper limit of normal. c) Estimated glomerular filtration rate 30 ml/min/1.73 m 2 based on local institutional practice for determination, or a minimum serum creatinine as presented in Section Ability to comply with outpatient treatment, laboratory monitoring, and required clinic visits for the duration of study participation. 12. Willingness of male and female patients with reproductive potential to observe conventional and effective birth control for the duration of treatment and for 3 months following study completion. For male subjects with a pregnant partner, a condom should be used for contraception. For male subjects with a non-pregnant female partner of child-bearing potential and woman of child-bearing potential one of the following birth control methods with a failure rate of less than 1% per year when used consistently and correctly are recommended: a) Combined estrogen and progestogen containing hormonal contraception associated with inhibition of ovulation given orally, intravaginally, or transdermally b) Progestogen-only hormonal contraception associated with inhibition of ovulation given orally, by injection, or by implant c) Intrauterine device (IUD) d) Intrauterine hormone-releasing system (IUS) e) Bilateral tubal occlusion f) Vasectomized partner g) Sexual abstinence Alternative birth control methods with a failure rate of more than 1% per year could also be considered: a) Progestogen only oral hormonal contraception where inhibition of ovulation is not the primary mode of action b) Male or female condom with or without spermicide c) Cap, diaphragm, or sponge with spermicide Birth control methods unacceptable for this clinical trial are: a) Periodic abstinence (calendar, symptothermal, or post-ovulation methods) b) Withdrawal (coitus interruptus) c) Spermacide only d) Lactational amenorrhea method 13. Ability to swallow capsules, liquid or gastric access via a naso- or gastric tube Exclusion Criteria Patients meeting any of the following criteria are to be excluded from study participation: 1. Major surgery within 14 days (2 weeks) prior to C1D1. 2. Clinically significant active cardiovascular disease or history of myocardial infarction within 6 months prior to C1D1; ongoing cardiomyopathy; or current prolonged QT interval corrected for heart rate (QTc) interval >480 milliseconds. 3. Active uncontrolled systemic bacterial, viral, or fungal infection. 4. Malabsorption syndrome or other condition affecting oral absorption. 5. Current treatment with a strong cytochrome P450 3A4 (CYP3A4) inhibitor or inducer other than those allowed per Section Pregnancy or lactation. 7. Phase 2 only: Prior progression while receiving approved or investigational tyrosine kinase inhibitors targeting TRK, including entrectinib, crizotinib and lestaurtanib. Patients who received a tropomyosinrelated kinase (TRK) inhibitor for less than 28 days of treatment and discontinued because of intolerance remain eligible. Version 7.0 Confidential & Proprietary Page 8 of 123

17 PLANNED SAMPLE SIZE: Phase 1: For the dose escalation phase, it is anticipated that up to 36 patients will be required in order to define the MTD of larotrectinib. INVESTIGATIONAL DRUG: Larotrectinib for oral dosing is provided as a clear, yellow to reddish colored solution in a concentration of 20 mg/ml. In addition, for doses of 25 mg or greater, hard gelatin capsules of 25 or 100 mg larotrectinib may be utilized. Both capsules are opaque white in color and are distinguishable by size (capsule sizes 2 and 0, respectively). The solution will be provided to the patient in single-patient amber oval bottles and capsules will be provided to the site in bulk bottles of 72 capsules/bottle. TREATMENT PROCEDURES: Patients will begin BID dosing on C1D1 according to the assigned cohort. Each cycle will consist of 28 days of continuous dosing. Individual patients will continue larotrectinib BID dosing until PD, unacceptable toxicity, or other reason for treatment discontinuation. Subjects who have progressed may be allowed to continue larotrectinib if, in the opinion of the Investigator, the subject is deriving clinical benefit from continuing study treatment, and continuation of treatment is approved by the Sponsor. Patients treated with larotrectinib and who respond, either per RECIST or volumetric measurements, may enter a wait and see drug discontinuation period following the time of best response after a minimum of 6 cycles of treatment. Patients will continue to undergo diagnostic imaging per protocol-defined timelines during the drug holiday. Patients may be re-treated if they show evidence of PD after drug discontinuation. Patients who undergo surgical resection for local control may continue to receive larotrectinib after surgical recovery and discussion between the Investigator and the Sponsor. If the resection surgery results in negative margins and study treatment is stopped, clinical assessments and disease assessments, as described in Section 7.4, should be performed every 3 months following the last dose of study medication. These patients are permitted to restart study treatment if they experience PD after discussion with the Medical Monitor. All treated patients will undergo a safety follow-up visit at 28 days (± 7 days) after the last dose. Patients will be followed for long-term survival. STUDY ASSESSMENTS: The study will consist of a screening period, a treatment period, a safety follow-up visit, and long term follow up assessments. On-going safety, survival, and subsequent anticancer therapies will be assessed in the long term follow-up. Standard monitoring for safety are outlined in the protocol and will include physical examination and body weight, Karnofsky/Lansky score, clinical AEs, laboratory variables (e.g., hematology, serum chemistries, and urinalysis), electrocardiogram (ECG), and vital signs. Subjects will undergo radiologic evaluation of their disease on Day 1 of odd numbered cycles. Subjects are able to have an optional radiologic assessment at C2D1. Patients will have whole blood drawn for correlative genomic analysis during each scanning visit. PK blood sampling will occur in C1D1 and either C2D1 (Cohorts 1 and 2) or C4D1 (Cohorts 3 and higher). Blood for PK assessments will be collected as follows: Dose Escalation Cohorts 1 and 2: On C1D1 at 1 and 4 hours post-dose (allowable window of ± 10 minutes), pre-dose the second dose on C1D1, and on C2D1 at pre-dose, and at 1 and 4 hours postdose (allowable window of ± 10 minutes) in Cohorts 1 and 2. Dose Escalation Cohorts 3 and higher, Expansion Cohort, and Phase 2 efficacy cohorts: On C1D1 at 0.5, 1, 2, 4, 8 hours post dose, and pre-dose the second dose (allowable window of ± 10 minutes), C4D1 at pre-dose 0.5, 1, 2, 4, and 8 hours post-dose (allowable window of ± 10 minutes) and every subsequent 4th cycle (e.g., C8, C12, etc.) a pre-dose sample collected. Version 7.0 Confidential & Proprietary Page 9 of 123

18 Both of the twice daily doses of larotrectinib should be taken in the clinic on C1D1 and the first dose of the twice daily doses on PK sampling days to ensure a PK sample is collected pre-dose. If patients have an Ommaya Reservoir, CSF will also be collected for PK assessment on C1D1 in conjunction with the post-dose 1-hour PK sample. Patients undergoing a routine diagnostic or staging lumbar puncture will have CSF in addition to a blood sample collected for PK assessment anytime the lumbar puncture is performed. It is preferred but not required that the lumbar puncture not be performed within 1 hour following administration of larotrectinib. Pain and HRQoL Assessments will be collected every Cycle. In patients 3 years of age or older, pain will be assessed by the Wong-Baker Faces Scale. For all patients older than 1 month old, HRQoL will be assessed using the PedsQL-Core for the patient or their parent/caregiver. Study visits are required on D1 in all cycles. Central Radiology Review for Response: Patients with tumors who respond (CR, PR) to therapy or have long term stable disease (SD) ( 6 cycles) on protocol therapy will be centrally reviewed. All responses will be confirmed by a second scan 28 days after the initial response, per RECIST, RANO, or International Neuroblastoma Response Criteria (INRC), as appropriate. Copies of all subject scans, including the scan immediately prior to the eligibility scan, if available, will be submitted for central collection by Sponsor. Archived tumor tissue will be obtained for all subjects and banked by the Sponsor, in order to evaluate and confirm NTRK status in a centralized laboratory. Subjects without available archival tissue may use the fresh tumor biopsy tissue on a primary or metastatic site. At end of treatment (EOT), subjects may undergo an optional fresh tumor biopsy to evaluate tumor changes that may have resulted from treatment. Whole blood will be collected serially for genomic studies, and correlated with radiologic assessments and tumor tissue obtained through baseline fresh biopsy and EOT biopsy based on age and institutional guidelines, if available. Patients with sarcoma, including infantile fibrosarcoma, who receive larotrectinib and go on to an attempt at surgical resection will be re-staged at the time of surgery. Staging will be defined according to the tumor/node/metastasis system: T1 (localized to the organ or tissue of origin) or T2 (extending beyond the tissue or organ of origin) according to the invasion of contiguous organs; N0 or N1 and M0 or M1 according to the presence of lymph node or distant metastasis. Lymph node involvement should be evaluated clinically or by radiographic imaging and confirmed when necessary by cytological or histological biopsy. The status of resection margins will be classified according to the UICC-R classification and the Intergroup Rhadbomyosarcoma Staging (IRS) systems which is generally used for primary surgery I pediatric rhabdomyosarcomas. UICC-R R0 or IRS Group I will correspond to complete tumor resection with histologically free margins; UICC-R R1 or IRS II will correspond to macroscopic resection but invaded margins on histology; UICC-R R2 or IRS III correspond to macroscopic residual tumor after surgery (III B) or biopsy (III A). IRS IV corresponds to distant metastatic tumor. STATISTICAL METHODS: Sample Size Considerations: Phase 1 Dose Escalation Up to 6 patients evaluable for safety, with the exception of possible over enrollment in Cohorts 3, 4, and 5, will be enrolled in each dose cohort based on a Phase 1 rolling 6 design (Skolnik 2008). Each patient will be counted in only 1 dose cohort. Up to 36 patients may be required in order to define the MTD of larotrectinib. The actual number enrolled is dependent upon the observed safety profile, which will determine the number of patients per dose cohort, as well as the number of dose escalations required to achieve the MTD. Patients who miss 7 or more days of dosing in Cycle 1 for reasons unrelated to study drug toxicity will be considered to have inadequate study drug exposure to support dose escalation and will be replaced. Version 7.0 Confidential & Proprietary Page 10 of 123

19 Analysis Methods: Safety Analyses A SRC will be established to oversee the safety aspects of the study and to render dose escalation decisions in the dose escalation portion of the study. Specifically, the Committee will perform ongoing review of serious adverse events (SAEs) and other safety trends throughout the conduct of the study. The Committee membership will consist of the Sponsor s representatives and clinically qualified individuals from each active clinical site. The Committee will be convened as needed and decisions will be documented in written minutes. Safety analyses will be conducted using the Safety Analysis Set, which consists of all patients treated with one or more doses of study drug. The results of the safety analyses will be presented by study phase. For the Phase 1 portion, tabulations will be provided by dose cohort and overall summary. For Phase 2, tabulations will be provided by tumor type (infantile fibrosarcoma, other extra-cranial solid tumors, and primary CNS tumors) and overall summary. Some safety analyses may be performed based on all treated patients in Phase 1 and Phase 2 combined. TEAEs reported during the study will be tabulated and listed by Sponsor Dictionary for Regulatory Activities (MedDRA) System Organ Class (SOC) and Preferred Term (PT). Tables will display number and percentage of patients experiencing the event for the following categories: All AEs, AEs considered related to study drug, AEs by severity, DLTs, AEs occasioning treatment delay or discontinuation, and SAEs. For the dose escalation phase, the observed DLT rate in each dose cohort will be calculated by the proportion of patients who experienced DLT with a 2-sided 95% exact binomial CI. Hematology and serum chemistries will be summarized using conventional summary statistics (mean, standard deviation, median, and range) for the following: Baseline value, minimum and maximum post baseline values, average post baseline value, and last post baseline value. Standard shift tables will also be prepared presenting worst post baseline toxicity grade versus baseline. Vital signs will be summarized in a descriptive manner by calculating the mean, standard deviation, median, and range by time point in the same manner described for laboratory values. The Wilcoxon signed rank test may be used to assist in the identification of any systematic changes. Efficacy Analyses Version 7.0 Confidential & Proprietary Page 11 of 123

20 ORR will be estimated using disease-specific response criteria (e.g., RECIST 1.1 for patients with solid tumor). The estimate of the ORR will be calculated based on the maximum likelihood estimator (i.e., crude proportion of patients with best overall response of CR or PR. All responses will be confirmed by a second scan 28 days after the initial response. The estimate of the ORR will be accompanied by 1- and 2-sided CIs with various coverage probabilities (e.g., 80%, 95%). The primary analysis of ORR will be based on the responses determined by an independent radiology review committee. A secondary analysis will be based on the treating Investigator s assessment. Waterfall plots will be used to depict graphically the maximum decrease from baseline in the sum of longest diameters of target lesions. Spider plots and swimmer plots will be used to display the change in tumor burden over time for individual patients and the occurrence of clinical outcomes of interest (e.g., tumor response, PD, treatment discontinuation, death). DOR will be calculated for patients who achieve a CR or PR and is defined as the number of months from the start date of the response (and subsequently confirmed) to the first date that recurrent or PD is objectively documented. If a patient dies, irrespective of cause, without documentation of recurrent or PD beforehand, then the date of death will be used to denote the response end date. PFS is defined as the number of months from the date of the first dose of study drug to the earliest of documented PD or death without prior progression. For both DOR and PFS, the PD date and censoring date will be based on published conventions (FDA). OS is defined as the number of months from the date of the first dose of study drug to the date of death (due to any cause). Time-to-event endpoints will be summarized descriptively using the Kaplan-Meier method with 95% CIs calculated using Greenwood s formula. Median follow-up for each endpoint will be estimated according to the Kaplan-Meier estimate of potential follow-up. Progression-free survival rate at 6 and 12 months and corresponding 95% CIs will be estimated using the Kaplan-Meier method. Greenwood s formula will be used to calculate the standard errors of the Kaplan-Meier estimate and upper and lower limits of the 95% CI. Pharmacokinetic Analyses Plasma (and CSF where appropriate) concentrations of larotrectinib will be determined with a validated bioanalytical assay. The following PK parameters will be calculated where appropriate: Maximum drug concentration (C max), area under the concentration versus time curve from time 0 to t (AUC 0 t), apparent oral clearance of drug (CL/F), and CSF/plasma ratio. Summary statistics will be generated by dose cohort and across cohorts as appropriate. Patient Reported Outcomes Worsening or improvement of pain will be evaluated for patients 3 years of age or older using the Wong- Baker Faces Scale. Longitudinal changes in pain scale scores from baseline will be summarized in a descriptive manner. Similar analyses will be performed for the other measures of HRQoL evaluated. Version 7.0 Confidential & Proprietary Page 12 of 123

21 SCHEDULE OF ASSESSMENTS Evaluation Visit Window Screening (Day -28 to Day 0) Cycle 1 28-Day Cycles Cycles 2 through 12 Cycles 13 and higher End of Treatment (EOT) ± 2 Days ± 3 Days ± 3 Days ± 3 Days Safety Follow-Up Day 28 +/- 7 days Informed Consent X Sponsor, surgical, malignancy history X Urine or serum pregnancy test (if applicable) X Archived tumor tissue (or fresh biopsy) a X X a Physical examination and Karnofsky or Days 1, 8, 15, Day 1 Day 1 Lansky b X 22 X X Vital signs Days 1, 8, 15, Day 1 predose pre-dose Day 1 X 22 pre-dose X X Blood (and CSF, if appropriate) sample for PK (± 10 minutes) Blood (and CSF, if appropriate) sample for PK (± 10 minutes) for Cohort 3 and higher. Also to include Phase 2/Expansion patients. Cohorts 1 and 2 Day 1, 1h, 4h; pre-dose Day 1 second dose c Cohort 3 and subsequent 0.5, 1, 2, 4, 8 hours, and predose the second dose on Day 1 c Cohorts 1 and 2 Day 1, predose, 1h, 4h c Cohort 3 and subsequent Cycle 4 Day 1 pre-dose, 0.5, 1, 2, 4, 8 hours c Every forth cycle: Day 1 pre-dose Cohort 3 and subsequent cohorts Every fourth cycle: Day 1 pre-dose 12-lead ECG d X Hematology f Days 1, 8, 15, Day 1 Day 1 X X X 22 Long- Term Follow Up (LTFU) Every 3 months ± 1 month Version 7.0 Confidential & Proprietary Page 13 of 123

22 Evaluation Visit Window Screening (Day -28 to Day 0) Cycle 1 28-Day Cycles Cycles 2 through 12 Cycles 13 and higher End of Treatment (EOT) ± 2 Days ± 3 Days ± 3 Days ± 3 Days Safety Follow-Up Day 28 +/- 7 days Long- Term Follow Up (LTFU) Every 3 months ± 1 month Urinalysis h X Day 15 Day 1 Day 1 X [X] e Disease assessment i Day 1 (±7 days) Day 1 (±7 days) X Cycle 2 every third [X] j (optional) and cycle starting X n odd cycles at Cycle 14 Pain and HRQoL k Day 1 pre-dose Day 1 Day 1 X pre-dose pre-dose X Whole blood for cfdna analysis l Whole blood for genomic DNA analysis m X X Day 1 odd cycles Day 1 every third cycle starting at Cycle 14 Larotrectinib administration Patient dosing diary Adverse events Concomitant medications Note: This table is identical to the Schedule of Assessments located in Appendix A. cfdna = circulating free DNA; CNS = central nervous system; DNA = deoxyribonucleic acid; ECG = electrocardiogram; ECOG = Eastern Cooperative Oncology Group; EOT = end of treatment; h = hour; HRQoL = Health Related Quality of Life; PK = pharmacokinetics. Footnotes for Schedule of Assessments a Fresh tissue biopsy is required during Screening ONLY if archived tumor tissue is not available and tumor is amenable to biopsy. Tissue biopsy collected at the end of treatment (EOT) visits is optional. b Physical examination includes review of systems: Breast/chest, extremities, genitourinary, head/ears/eyes/nose/throat, lymph nodes, musculoskeletal, pulmonary, rectum, and skin, thorough neurologic assessment, body weight, and, height during Screening. Symptom-directed physical and neurological examinations, including measurement of weight and height, may be performed at other time points. Karnofsky should be assessed for those 16 years old or older, or Lansky should be assessed for those younger than 16 years. X Version 7.0 Confidential & Proprietary Page 14 of 123

23 c Blood for PK assessments will be collected on Cycle 1 Day 1 (C1D1) at 1 and 4 hours post-dose (allowable window of ± 10 minutes), predose the second dose on Day1 and on Cycle 2 Day 1 (C2D1) at predose, and at 1 and 4 hours post-dose (allowable window of ± 10 minutes) in Cohorts 1 and 2. In all subsequent cohorts, blood for PK assessments will be collected on C1D1 at 0.5, 1, 2, 4, 8 hours post-dose, and pre-dose the second dose (allowable window of ± 10 minutes) C4D1 will have a pre-dose as well as 0.5, 1, 2, 4, and 8 hours post-dose (allowable window of ± 10 minutes) Every subsequent 4 cycles (e.g., C8, C12, and so forth) will have a D1 pre-dose. Both of the twice daily doses of larotrectinib should be taken in the clinic on C1D1 and the first dose of the twice daily doses on PK sampling days to ensure a PK sample is collected pre-dose. If patients have an Ommaya Reservoir, cerebral spinal fluid (CSF) will also be collected for PK assessment on in conjunction with the post-dose 1-hour PK sample. If, after assessing C1D1 PK, the patient is not achieving a minimum target AUC 0-24 of 3500 ng/ml and is less than ½ of the target, and is not having dose limiting toxicities, intra-patient dose escalation may occur by 150% of prior dose level. Repeat PK sampling, similar to the C4D1 schedule, should occur on D1 of the next cycle, and further intra-patient dose escalations can occur in future cycles if the same conditions are met. d Electrocardiogram (ECG) should be done in triplicate. e Repeat the assessment only if the EOT result showed treatment-emergent abnormalities. f Hematology will include hemoglobin, hematocrit, red blood cell (RBC) count, white blood cell (WBC) count with differential (neutrophils [absolute count and percent] and lymphocytes, monocytes, eosinophils, basophils [percent]), and platelet count. g Serum chemistries (nonfasting) will include alkaline phosphatase, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), cholesterol, creatinine, glucose, lactate dehydrogenase (LDH), total and direct bilirubin, total protein, sodium, potassium, chloride, and bicarbonate. h Urinalysis will include complete urinalysis, including color, appearance, specific gravity, ph, glucose, bilirubin, ketones, occult blood, protein, leukocytes, nitrites and urobilinogen; if +2 protein is observed, a 24-hour urine collection will be taken in order to quantify proteinuria. i Tumor will be assessed by standard of care imaging modalities (CT and/or PET) or MRI (magnetic resonance imaging) of chest, abdomen, and pelvis, any other areas with suspected disease involvement. Patients enrolled with a history of CNS metastases should additionally have a head CT or MRI scan performed at each tumor assessment. Patients with locally advanced infantile fibrosarcoma should have an MRI or CT of the site of disease and a CT or X-Ray of the chest, plus any other areas of disease involvement. Patients with neuroblastoma will be followed with meta-iodobenzylguanidine (MIBG) scans, anatomic imaging, and bilateral bone marrow aspirates and biopsies. j Assessment should be performed if no disease assessment was performed in last 2 cycles. k Pain and Health Related Quality of Life (HRQoL) assessments will be collected every cycle. In patients 3 year of age or older, pain will be assessed by the Wong-Baker Faces Scale. For all patients HRQoL will be assessed using the PedsQL-Core for the patient or their parent/caregiver. l Whole blood for cfdna analysis should be obtained within 7 days following radiologic disease assessment, except for baseline/screening when it may be obtained any time prior to treatment on C1D1. Whole blood for cfdna analysis should be obtained at the EOT visit even if radiologic disease assessment is not performed. Patients less than 1-year-old will not have any blood drawn. Patients between 1 and 5 years of age will have 1 10 ml Streck tube drawn. Patients older than 5 years of age will have 2 10 ml Streck tubes drawn. Sites may draw fewer tubes based on local institutional policy for maximal blood draws in research subjects. m Whole blood for genomic DNA should be obtained any time during screening prior to beginning treatment. Patients greater than 5 years of age will have one PAXGene tube (8.5mL) drawn. Sites may draw fewer tubes based on local institutional policy for maximal blood draws in research subjects. n Date of progression, as assessed by treating Investigator and survival status (may be conducted by phone). Version 7.0 Confidential & Proprietary Page 15 of 123

24 TABLE OF CONTENTS SYNOPSIS... 3 Inclusion Criteria... 6 Exclusion Criteria... 8 SCHEDULE OF ASSESSMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES LIST OF ABBREVIATIONS AND DEFINITION OF TERMS INTRODUCTION NTRK Gene Family and TRK Tropomyosin-Related Kinase: Cancer Pathophysiology Tropomyosin-Related Kinase: Pediatric Cancers Congenital/Infantile Fibrosarcoma Congenital Mesoblastic Nephroma Neuroblastoma Study Population and Starting Dose Rationale Larotrectinib Chemistry and Description Pharmacodynamics Pharmacokinetics Safety Pharmacology Toxicology Rationale for Dose Selection in First in Human Adult Trial Anticipated Risks in Human Patients STUDY OBJECTIVES Phase 1 Objectives Primary Objective Secondary Objectives Exploratory Objectives Version 7.0 Confidential & Proprietary Page 16 of 123

25 2.2 Phase 2 Objectives Primary Objective Secondary Objectives Exploratory Objectives: INVESTIGATIONAL PLAN Phase Study Design Phase Study Design Investigational Sites SELECTION OF STUDY POPULATION Inclusion Criteria Exclusion Criteria ENROLLMENT PROCEDURES TREATMENT Investigational Product Larotrectinib Administration General Dosing Instructions Cycle Cycles 2 and Higher Dose Modifications Prior and Concomitant Medications General Allowed Concomitant Medications Prohibited Concomitant Medications Concurrent Palliative Radiotherapy and Elective Surgery Removal of Patients from Therapy or Assessment TESTS AND EVALUATIONS Screening Period Cycle Cycles 2 through Cycles 13 and Higher Version 7.0 Confidential & Proprietary Page 17 of 123

26 7.5 End of Treatment Visit Safety Follow-up Visit Long-Term Follow Up Procedures for Special Tests Tumor Measurements Pharmacokinetics Correlative Studies Pain and Health Related Quality of Life Collection of Radiographic Studies for Independent Review PLANNED ANALYSES Analysis Populations Determination of Sample Size Statistical Methods Demographics and Baseline Characteristics Safety Analyses Efficacy Analyses Pharmacokinetic Analyses Pain and Health Related Quality of Life ADVERSE EVENTS Safety Review Committee Grading and Intensity of Adverse Events Relationship to Study Drug Serious Adverse Event Reporting Serious Adverse Event Follow-up STUDY ADMINISTRATION Regulatory and Ethical Considerations Regulatory Authority Approval Ethics Approval Patient Informed Consent Investigator Reporting Requirements Data Management Study Monitoring Version 7.0 Confidential & Proprietary Page 18 of 123

27 10.4 Termination Records Retention Confidentiality of Information REFERENCES LIST OF TABLES Table 1 Estimated Frequencies of NTRK Fusions in Cancer Table 2 Pharmacokinetic Predictions with SimCyp Table 3 Preliminary Pharmacokinetic Parameters of Larotrectinib in Cancer Patients43 Table 4 PK Predictions with SimCyp Table 5 Treatment-Emergent Adverse Events in Study LOXO-TRK Table 6 Larotrectinib Dose Levels Table 7 Toxicities Requiring Dose Modification Table 8 Dose Level Reductions for Larotrectinib Table 9 NCI-CTCAE Definitions of Severity for Adverse Reactions Table 10 Schedule of Assessments Table 11 Karnofsky Performance Score ( 16 years old) Table 12 Lansky Performance Score (<16 years old) Table 13 DOSE LEVEL -2: 50 mg BID Adult Equivalent Dose Larotrectinib Pediatric Dose for Patients Who Can Swallow Capsules Table 14 DOSE LEVEL -2: 50 mg BID Adult Equivalent Dose Larotrectinib Pediatric Starting Dose for Patients Who Cannot Swallow Capsules Table 15 DOSE LEVEL -1: 75 mg BID Adult Equivalent Dose Larotrectinib Pediatric Dose for Patients Who Can Swallow Capsules Table 16 DOSE LEVEL -1: 75 mg BID Adult Equivalent Dose Larotrectinib Pediatric Starting Dose for Patients Who Cannot Swallow Capsules Table 17 DOSE LEVEL 1: 100 mg BID Adult Equivalent Dose Larotrectinib Pediatric Dose for Patients Who Can Swallow Capsules Table 18 DOSE LEVEL 1: 100 mg BID Adult Equivalent Dose Larotrectinib Pediatric Starting Dose for Patients Who Cannot Swallow Capsules Table 19 DOSE LEVEL 2: 150 mg BID Adult Equivalent Dose Larotrectinib Pediatric Dose for Patients Who Can Swallow Capsules Table 20 DOSE LEVEL 2: 150 mg BID Adult Equivalent Dose Larotrectinib Pediatric Starting Dose for Patients Who Cannot Swallow Capsules Table 21 DOSE LEVEL 3: 200 mg BID Adult Equivalent Dose Larotrectinib Pediatric Dose for Patients Who Can Swallow Capsules Table 22 DOSE LEVEL 3: 200 mg BID Adult Equivalent Dose Larotrectinib Pediatric Starting Dose for Patients Who Cannot Swallow Capsules Table 23 DOSE LEVEL 4: 300 mg BID Adult Equivalent Dose Larotrectinib Pediatric Dose for Patients Who Can Swallow Capsules Table 24 DOSE LEVEL 4: 300 mg BID Adult Equivalent Dose Larotrectinib Pediatric Starting Dose for Patients Who Cannot Swallow Capsules Version 7.0 Confidential & Proprietary Page 19 of 123

28 Table 25 Inhibitors of CYP3A Table 26 Inducers of CYP3A Table 27 Response Evaluation Criteria in Solid Tumors (RECIST) Guidelines for Tumor Response Table 28 Overall Response Criteria Table 29 RANO Response Criteria Incorporating MRI and Clinical Factor LIST OF FIGURES Figure 1 Mechanism of Fusion Oncogene Action (adapted from Vaishnavi 2015) Figure 2 Chemical Structure of Larotrectinib Sulfate Figure 3 Pharmacokinetics of HP-β-CD Solution Versus Clinical Capsule in Dogs: Identical Between the Two Formulations Figure 4 Preliminary Pharmacokinetics of Larotrectinib on Day 8 (Steady-State) in Patients Given 50 mg QD, 100 mg QD, 200 mg QD, 100 mg BID, or 150 mg BID of Larotrectinib LIST OF APPENDICES Appendix A Study Assessments Appendix B Karnofsky and Lansky Performance Status Scales Appendix C Dosing Calculation Tables Appendix D Inhibitors and Inducers of CYP3A Appendix E Tumor Measurements and Assessment of Disease Response: RECIST, Version Appendix F Response Assessment in Neuro-Oncology (RANO) Criteria for Primary CNS Malignancies Appendix G Pharmacokinetic Blood Sampling Schedule Version 7.0 Confidential & Proprietary Page 20 of 123

29 LIST OF ABBREVIATIONS AND DEFINITION OF TERMS Abbreviation or term AE AIEOP-STSC AKT ALP ALT ANC ASCO AST ATP AUC AUC 0-12 AUC 0-24 AUC 0 AUC 0-t BDNF BCRP BID BM BUN BSA C Definition adverse event Associazione Italiana Ematologia Oncologia Pediatrica Soft Tissue Sarcoma Committee protein kinase B alkaline phosphatase alanine aminotransferase absolute neutrophil count American Society for Clinical Oncology aspartate aminotransferase adenosine triphosphate area under the concentration versus time curve area under the curve time 0 to 12 hours area under the curve time 0 to 24 hours area under the concentration versus time curve from time 0 extrapolated to infinity area under the concentration versus time curve from time 0 to t brain-derived neurotrophic factor breast cancer resistance protein twice daily bone marrow blood urea nitrogen body surface area Cycle C1D1 Cycle 1 Day 1 C1D8 Cycle 1 Day 8 C1D28 Cycle 1 Day 28 C2D1 Cycle 2 Day 1 C4D1 Cycle 4 Day 1 CBR cfdna CI CIPA CL/F CLIA C max C min clinical benefit rate circulating free DNA confidence interval congenital insensitivity to pain with anhidrosis apparent oral clearance of drug Clinical Laboratory Improvement Amendments maximum drug concentration trough drug concentration Version 7.0 Confidential & Proprietary Page 21 of 123

30 Abbreviation or term CMN CNS COPD CR CRF CSF CT CTCAE CYP CYP3A4 D DD DLT DNA DOR ECG ECOG ecrf EDC EIAEDs EOT ERK Definition congenital mesoblastic nephroma tumors central nervous system chronic obstructive pulmonary disease complete response Case Report Form cerebral spinal fluid computerized tomography Common Terminology Criteria for Adverse Events cytochrome P450 cytochrome P450 3A4 Day dimerisation domain dose-limiting toxicity deoxyribonucleic acid duration of response electrocardiogram Eastern Cooperative Oncology Group electronic Case Report Form electronic data capture enzyme-inducing anti-epileptic drugs end of treatment extracellular signal-related kinases ETV6 ETS variant gene 6 FACES FAS Wong-Baker Faces Scale Full Analysis Set GAB1 GRB2-associated-binding protein 1 GCP GMP Good Clinical Practices Good Manufacturing Practices GRB2 growth factor receptor-bound protein 2 h HCG herg HP-β-CD HRQoL HSA hour human chorionic gonadotropin human ether-à-go-go related gene hydroxypropy-beta-cyclodextrin Health Related Quality of Life human serum albumin 131 I-MIBG radio iodized metaiodobenzylguanidine Version 7.0 Confidential & Proprietary Page 22 of 123

31 Abbreviation or term IB IC 50 ICH IEC IF IHC INRC IRB IRS IUD IUS KD LDH Larotrectinib (LOXO-101) MAP MAPK MASC MedDRA MEK MIBG MRI MTD MYCN NCI NGF NOAEL Definition Investigator s Brochure concentration at which 50% inhibition is achieved International Conference on Harmonisation Independent Ethics Committee infantile fibrosarcoma immunohistochemistry International Neuroblastoma Response Criteria Institutional Review Board insulin receptor substrate intrauterine device intrauterine hormone-releasing system kinase domain lactate dehydrogenase Investigational product mitogen-activated protein mitogen-activated protein kinase mammary analogue secretory carcinoma Sponsor Dictionary for Regulatory Activities mitogen-activated protein kinase meta-iodobenzylguanidine scans magnetic resonance imaging maximum tolerated dose myelocytomatosis viral -related oncogene National Cancer Institute nerve growth factor no-observable-adverse-effect-level NT-3, -4 neurotrophin-3 or -4 NTRK genes neurotrophic tyrosine kinase receptor genes NTRK1, -2, -3 human neurotrophic tyrosine kinase receptor gene or mrna, types 1, 2, and 3, coding for TRKA, TRKB, and TRKC, respectively ORR OS PD PedsQL-Core PET PFS overall response rate overall survival progressive disease Pediatrics Quality of Life - Core Module positron emission tomography progression-free survival Version 7.0 Confidential & Proprietary Page 23 of 123

32 Abbreviation or term P-gp PK PO PP PR PT PTT QD QTc RAF RANO RAS RBC REB RECIST RNA RT-PCR SAE SAS SD SEER SHC SIOP-MMT SOC SOS SRC STD 10 STSs T 1/2 TBI TEAE T max TRK ULN US Definition P-glycoprotein pharmacokinetic per os, orally Per-Protocol Set partial response prothrombin time or preferred term partial thromboplastin time once daily QT interval corrected for heart rate raf kinase Response Assessment in Neuro-Oncology (RANO) Criteria ras protein red blood cell(s) Research Ethics Board Response Evaluation Criteria in Solid Tumors ribonucleic acid reverse transcription polymerase chain reaction serious adverse event Safety Analysis Set stable disease or standard deviation Survey of Epidemiology and End Results Src Homology 2 Domain Containing Transforming Protein International Society of Pediatric Oncology Malignant Mesenchymal Tumor Committee system organ class son of Sevenless protein Safety Review Committee severely toxic dose in 10% of animals soft tissue sarcomas half-life traumatic brain injury treatment-emergent adverse event (a TEAE is defined as an AE that starts on or after the first administration of study medication) time to maximum plasma concentration tropomyosin-related kinase upper limit of normal United States Version 7.0 Confidential & Proprietary Page 24 of 123

33 Abbreviation or term VA V/F V z/f WBC WHO XRT Definition vincristine plus actinomycin-d volume of distribution apparent oral volume of distribution white blood cell(s) World Health Organization X-ray therapy Version 7.0 Confidential & Proprietary Page 25 of 123

34 1 INTRODUCTION 1.1 NTRK Gene Family and TRK In normal cells, the tropomyosin-related kinase (TRK) family of tyrosine kinase receptors are involved in the regulation of growth, differentiation, and apoptosis of neurons signal through associated tyrosine kinases (Nakagawara 2001). The TRK family of receptors, TRKA, TRKB, and TRKC, are encoded by the neurotrophic tyrosine kinase receptor (NTRK) genes NTRK1, NTRK2, and NTRK3, respectively, and are the preferred receptors of growth factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) (Nakagawara 2001). Following ligand binding, adjacent TRK receptors dimerize and become catalytically active by phosphorylating various tyrosine moieties within the cytoplasmic-facing region of the dimer counterpart. The propagation of these signals may stimulate growth, survival, and differentiation. Activated TRK receptors signal through many pathways, including the PI3 kinase pathway, phospholipase C-ɣ, the Erk 1 and 2 mitogen-activated protein (MAP) kinase pathways, and the Erk5 MAP kinase pathway (Rubin and Segal 2001). The human TRKA receptor is encoded by the NTRK1 gene. This kinase is a membranebound receptor that, upon activation by one of its ligands such as NGF or NT-3, phosphorylates itself and members of the mitogen-activated protein kinase (MAPK) pathway. The TRKA receptor is involved in cell differentiation and may play a role in determining sensory neuron subtype. Mice with global knockout for Ntrk1 or Ngf genes (the murine orthologs of human NTRK1 and NGF, respectively) can develop to birth, but are smaller and die early, possibly because of their defective neuronal development (Crowley, Spencer et al. 1994); (Smeyne, Klein et al. 1994). Germ line mutations in the NTRK1 gene in humans can lead to a defective TRKA receptor that has been associated with congenital insensitivity to pain, anhidrosis, self-mutilating behavior, mental retardation and cancer (Indo 2012). This condition, termed congenital insensitivity to pain with anhidrosis (CIPA) is an exceedingly rare clinical disorder and many patients do not survive childhood due to unchecked self-mutilation and trauma because of their insensitivity to pain. The human TRKB receptor is a protein that is encoded by the NTRK2 gene. This kinase is a transmembrane receptor which binds preferentially to BDNF and neurotrophin-4/5 (NT-4/5) (Skaper 2008). Mice lacking TRKB have drastically reduced numbers of sensory neurons, but the number of sensory neurons is not affected in mice lacking Bdnf or NT-4/5 (Skaper 2008). TRKB (-/-) knockout mice do not feed, and typically die within hours of birth (Snider 1994). Knockouts for Bdnf (-/-) have a milder phenotype expression, exhibiting head bobbing, spinning and hind limb extension during locomotion. These animals typically survive a few weeks. Vestibular ganglia in Bdnf (-/-) knockouts have a lack of neurons, typically >80% reduction (Snider 1994). Heterozygote Bdnf knockouts develop Version 7.0 Confidential & Proprietary Page 26 of 123

35 hyperphagia and obesity (Yeo, Connie Hung et al. 2004). Yeo and colleagues published a case-report of a male child with a de novo mutation of TRKB. The child was found to have a loss-of-function mutation in NTRK2 and demonstrated global developmental delay, impairment of short term memory, and impaired nociception, as well as severe early onset obesity (Yeo, Connie Hung et al. 2004). The TRKC receptor is encoded by the NTRK3 gene in humans. This kinase is a membranebound receptor which binds preferentially to NT-3 (Skaper 2008). Mice bred with either a TRKC (-/-) knockout exhibit abnormal movements and postures (Snider 1994). Due to the similarity of these movements to pseudoathetosis exhibited by humans with large fiber sensory neuropathies, these animals most likely are deficient in proprioceptive neurons (Snider 1994). Heterozygote knockouts for NT-3 exhibit a 50% reduction in muscle spindles, while homozygous knockouts lack development of proprioception-related sensory end organs and muscle spindles (Snider 1994). Mice with knockouts for TRKA, TRKB, or TRKC, the encoding proteins for NTRK1, NTRK2, or NTRK3 or the neurotrophins Ngf, Bdnf, NT-3, NT-4/5 fail to thrive or survive long-term if they are born alive (Crowley, Spencer et al. 1994, Smeyne, Klein et al. 1994, Snider 1994, Chen, Ye et al. 2005, Skaper 2008). In contrast, mice in which the Ntrk1 gene was ablated only in non-neuronal tissue were viable and appeared grossly normal (Coppola, Barrick et al. 2004). These data suggest that the TRKA receptor is essential for neuronal development, but somewhat dispensable in non-neuronal and post-development tissue in mice. Luikart and colleagues developed a conditional knockout using the cre/loxp system to study the effects of TRKB on synapse development (Luikart, Nef et al. 2005). Their work indicated that TRKB is required for presynaptic and postsynaptic sites during prenatal development. With homozygous TRKB knockouts, synapse numbers were significantly reduced (Luikart, Nef et al. 2005). Removal of TRKB after development did not affect the number of synapses (Luikart, Nef et al. 2005). Additional work by Chen and co-workers has confirmed that inhibition of TRK in postnatal mice does not affect development (Chen, Ye et al. 2005). Clinically, lestaurtinib (previously known as CEP-701), a multi-kinase inhibitor with potent pan-trk family inhibition, was tested in a Phase 1 trial that included pediatric patients with refractory, high-risk neuroblastoma (Minturn, Evans et al. 2011). Forty-seven patients were treated with a median of 2 cycles per patient and a range of 1 28 cycles. The median age was 10.7 years (range years). There were no reports of neurotoxicity or developmental delays (Minturn, Evans et al. 2011). 1.2 Tropomyosin-Related Kinase: Cancer Pathophysiology The first TRK oncogene, identified as a fusion with the tropomyosin gene in a colorectal cancer, follows the well-established paradigm of other oncogenic fusions by driving the growth of tumors via constitutive activation in its tyrosine kinase activity, thereby inducing cancer cell proliferation and initiating cancer-related downstream signaling pathways (Martin-Zanca, Hughes et al. 1986); (Vaishnavi, Le et al. 2015). A recent published analysis Version 7.0 Confidential & Proprietary Page 27 of 123

36 of the transcriptomes of nearly 7,000 tumors identified NTRK fusions in cancers including thyroid cancer, glioblastoma multiforme, lung adenocarcinoma, colon adenocarcinoma, head and neck squamous cell carcinoma, as well as soft tissue sarcoma (Stransky, Cerami et al. 2014). Table 1, below, summarizes the literature reports of NTRK fusions in cancer. Table 1 Estimated Frequencies of NTRK Fusions in Cancer Gene Fusion Cancer Estimated Frequency <4% 5 25% >25% NTRK1 Papillary thyroid cancer 1 NTRK1 Spitz neoplasms nevi 2 NTRK1 Glioblastoma 3 NTRK1 Intrahepatic cholangiocarcinoma 4 NTRK1 Lung large cell neuroendocrine cancer 5 NTRK1 Sarcoma 6 NTRK2 Astrocytoma 7 NTRK3 Mammary analogue secretory carcinoma (MASC) of the salivary glands Ref. 8 NTRK3 Secretory breast carcinoma 9 NTRK3 Congenital mesoblastic nephroma 10 NTRK3 Papillary thyroid cancer (post-radiation exposure) 11 NTRK3 Acute myeloid leukemia 12 NTRK3 Breast invasive carcinoma 6 NTRK3 Papillary thyroid cancer 13 NTRK3 Skin cutaneous melanoma 6 NTRK1/2 Lung adenocarcinoma 6, 14 NTRK1/3 Colorectal cancer 6 NTRK2/3 Brain low-grade glioma 6 NTRK2/3 Head and neck squamous cell carcinoma 6 NTRK1/2/3 Pontine glioma 15 Sources: 1. (Bongarzone, Vigneri et al. 1998) 2. (Brzezianska, Karbownik et al. 2006) (Wiesner, He et al. 2014) 3. (Kim, Lee et al. 2014) 4. (Ross, Wang et al. 2014) 5. (Fernandez-Cuesta, Peifer et al. 2014) 6. (Stransky, Cerami et al. 2014) 7. (Jones, Hutter et al. 2013) 8. (Bishop, Yonescu et al. 2013) 9. (Tognon, Knezevich et al. 2002) 10. (Argani, Fritsch et al. 2000) 11. (Atkinson and Brown 1985) (Rubin, Chen et al. 1998) (Leeman-Neill, Kelly et al. 2014) 12. (Kralik, Kranewitter et al. 2011) 13. (Leeman-Neill, Kelly et al. 2014) 14. (Vaishnavi, Capelletti et al. 2013) 15. (Wu, Diaz et al. 2014). The typical gene structure for an oncogenic fusion, like NTRK, is that the 3 region of a proto-oncogene (encoding the kinase domain) is juxtaposed to 5 sequences from an unrelated gene via an intra- or inter-chromosomal rearrangement. The resultant novel Version 7.0 Confidential & Proprietary Page 28 of 123

37 oncogene is both aberrantly expressed and has constitutive activation of the kinase domain. This results in activation of oncogenic downstream pathways (refer to Figure 1). Figure 1 Mechanism of Fusion Oncogene Action (adapted from Vaishnavi 2015) Source: (Vaishnavi, Le et al. 2015). Abbreviations: DD = dimerisation domain; KD = kinase domain; IRS = insulin receptor substrate; SHC = Src Homology 2 Domain Containing Transforming Protein; GRB2 = growth factor receptor-bound protein 2; SOS = son of Sevenless protein; RAS = ras protein; RAF = raf kinase; MEK = mitogen-activated protein kinase; ERK = extracellular signal-related kinases; GAB1 = GRB2-associated-binding protein 1; AKT = protein kinase B. Cancers with chromosomal rearrangements that result in fusion kinases have historically demonstrated oncogene addiction behavior and dramatic responses to relevant drug inhibitors (Weinstein 2002) (Shaw, Hsu et al. 2013). Fusion kinases involving ABL, ALK and ROS1 Version 7.0 Confidential & Proprietary Page 29 of 123