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Entrectinib, a potent oral inhibitor of the tyrosine kinases TRKA/B/C, ROS1, and ALK, was evaluated in two phase I studies in patients with advanced or metastatic solid tumors, including patients with active central nervous system (CNS) disease. Here, we summarize the overall safety and report the antitumor activity of entrectinib in a cohort of patients with tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions, naïve to prior TKI treatment targeting the specific gene, and who were treated at doses that achieved therapeutic exposures consistent with the recommended phase II dose. Entrectinib was well tolerated, with predominantly Grades 1/2 adverse events that were reversible with dose modification. Responses were observed in non–small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1–NTRK1-rearranged lung cancer.Significance: Gene fusions of NTRK1/2/3, ROS1, and ALK (encoding TRKA/B/C, ROS1, and ALK, respectively) lead to constitutive activation of oncogenic pathways. Entrectinib was shown to be well tolerated and active against those gene fusions in solid tumors, including in patients with primary or secondary CNS disease. Cancer Discov; 7(4); 400–9. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 339

Neuroblastoma (NB) is one of the most common and deadly childhood solid tumors. These tumors are characterized by clinical heterogeneity, from spontaneous regression to relentless progression, and the Trk family of neurotrophin receptors plays an important role in this heterogeneous behavior. We wanted to determine if entrectinib (RXDX-101, Ignyta, Inc.), an oral Pan-Trk, Alk and Ros1 inhibitor, was effective in our NB model. In vitro effects of entrectinib, either as a single agent or in combination with the chemotherapeutic agents Irinotecan (Irino) and Temozolomide (TMZ), were studied on an SH-SY5Y cell line stably transfected with TrkB. In vivo growth inhibition activity was studied in NB xenografts, again as a single agent or in combination with Irino-TMZ. Entrectinib significantly inhibited the growth of TrkB-expressing NB cells in vitro, and it significantly enhanced the growth inhibition of Irino-TMZ when used in combination. Single agent therapy resulted in significant tumor growth inhibition in animals treated with entrectinib compared to control animals [p < 0.0001 for event-free survival (EFS)]. Addition of entrectinib to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals [p < 0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ]. We show that entrectinib inhibits growth of TrkB expressing NB cells in vitro and in vivo, and that it enhances the efficacy of conventional chemotherapy in in vivo models. Our data suggest that entrectinib is a potent Trk inhibitor and should be tested in clinical trials for NBs and other Trk-expressing tumors.

The STARTRK-NG (Studies of Tumor Alterations Responsive to Targeting Receptor Kinases - Next Generation) trial is a Phase 1/1b study of entrectinib in pediatric patients with cancer, including primary brain tumors, neuroblastoma, and other non-neuroblastoma, extracranial solid tumors harboring NTRK, ROS1, or ALK gene fusions (NCT02650401). Entrectinib is a potent oral, CNS-penetrant, inhibitor of the tyrosine kinases TRKA/B/C (encoded by the genes NTRK1/2/3, respectively), ROS1, and ALK with IC50s < 2 nM (biochemical kinase assay). Overall, gene fusions are rare in the cancer population (10% incidence) treatment-related adverse events have been fatigue/asthenia, dysgeusia, paresthesia, nausea, myalgia, diarrhea, dizziness, arthralgia, vomiting, and constipation; importantly, there has been no evidence of cumulative toxicity. In addition, we have treated a 20-month-old boy presenting with infantile fibrosarcoma metastatic to the brain, which harbored an ETV6-NTRK3 fusion. Tumor restaging following 1 month of therapy showed a significant tumor regression of CNS metastases accompanied by significant clinical improvement. Together, these data provide compelling evidence to identify pediatric patients harboring NTRK, ROS1, or ALK fusions for enrollment in the STARTRK-NG trial.

Here, we describe the dramatic response of a patient with an ETV6-NTRK3-driven mammary analogue secretory carcinoma to treatment with a pan-Trk inhibitor, and the development of acquired resistance linked to a novel NTRK3 mutation that interferes with drug binding. This case emphasizes how molecular profiling can identify therapies for rare diseases and dissect mechanisms of drug resistance., Background Mammary analogue secretory carcinoma (MASC) is a recently described pathologic entity. We report the case of a patient with an initial diagnosis of salivary acinic cell carcinoma later reclassified as MASC after next-generation sequencing revealed an ETV6-NTRK3 fusion. Patients and methods This alteration was targeted with the pan-Trk inhibitor entrectinib (Ignyta), which possesses potent in vitro activity against cell lines containing various NTRK1/2/3 fusions. Results A dramatic and durable response was achieved with entrectinib in this patient, followed by acquired resistance that correlated with the appearance of a novel NTRK3 G623R mutation. Structural modeling predicts that this alteration sterically interferes with drug binding, correlating to decreased sensitivity to drug inhibition observed in cell-based assays. Conclusions This first report of clinical activity with TrkC inhibition and the development of acquired resistance in an NTRK3-rearranged cancer emphasize the utility of comprehensive molecular profiling and targeted therapy for rare malignancies (NCT02097810).

Background: The STARTRK-NG (Studies of Tumor Alterations Responsive to Targeting Receptor Kinases - Next Generation) trial is a Phase 1/1b dose-escalation and expansion study of entrectinib in pediatric patients with cancer. Entrectinib is a potent oral, CNS-penetrant, inhibitor of the tyrosine kinases TRKA/B/C (encoded by the genes NTRK1/2/3, respectively), ROS1, and ALK with IC50s < 2 nM (biochemical kinase assay). Three adult studies (Phase 1 ALKA-372-001, Phase 1 STARTRK-1, and Phase 2 STARTRK-2) have enrolled hundreds of patients with advanced or metastatic solid tumors harboring TRKA/B/C, ROS1, or ALK molecular alterations, with or without CNS disease. Previously, we reported 400 mg/m2 daily as the adult body surface area (BSA)-based Recommended Phase 2 Dose (RP2D) (Patel et al, ASCO 2015). An objective response rate of 79% was seen in 24 tyrosine kinase inhibitor-naïve patients with TRK, ROS1, or ALK fusions who were treated at doses consistent with the RP2D (Drilon et al, AACR 2016). Gene fusions have been observed in a variety of adult solid tumor types, including non-small cell lung cancer, salivary gland cancers, soft tissue sarcomas and glioneuronal tumors. These fusions have also been found in cancers that affect the pediatric population, such as infantile fibrosarcoma, juvenile breast cancer, mesoblastic nephroma, intrinsic pontine gliomas, acute leukemias and anaplastic lymphoma. In addition, overexpression of TRKB and activating ALK point mutations have been observed in neuroblastoma. Thus, a pan-TRK, ROS1, and ALK inhibitor, like entrectinib, may potentially have broad therapeutic utility in pediatric patients. Methods: This is a multicenter, dose escalation study in pediatric patients (aged 2-21 years) with relapsed or refractory extracranial solid tumors (Phase 1), with additional expansion cohorts (Phase 1b) in patients with primary brain tumors harboring TRK, ROS1, or ALK molecular alterations inclusive of gene fusions, neuroblastoma, and other non-neuroblastoma, extracranial solid tumors harboring TRK, ROS1, or ALK gene fusions (NCT02650401). During dose escalation, a 3+3 schema will be used to determine the pediatric RP2D of entrectinib with a starting dose of 250 mg/m2 (approximately 60% of the adult BSA-based RP2D), administered orally once daily in repeated 4-week cycles, with concordant pharmacokinetics and pharmacodynamics studies. Up to four dose levels will be evaluated. Dose modifications, if necessary, will follow a protocol-specific dosing nomogram for each dose level. Once the pediatric RP2D is determined, the Phase 1b expansion cohorts will be opened simultaneously, and prospective molecular profiling will be performed to determine eligibility except for patients with neuroblastoma. Citation Format: Ami V. Desai, Garrett M. Brodeur, Jennifer Foster, Suzanne Shusterman, Amit J. Sabnis, Magaret Macy, Cynthia Wetmore, Ellen Basu, Zachary Hornby, Vanessa Esquibel, Edna Chow Maneval, Pratik S. Multani, Elizabeth Fox. STARTRK-NG: A phase 1/1b study of entrectinib in children and adolescents with advanced solid tumors and primary CNS tumors, with or without TRK, ROS1, or ALK fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT030. doi:10.1158/1538-7445.AM2017-CT030

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and comprises a heterogeneous group of diseases. A number of recurrent leukemogenic gene mutations or chromosomal rearrangements have been identified and clinically validated in AML. However, nearly 50% of AML patient samples lack any known AML driver mutations. Advances in molecular diagnostics have resulted in the identification of novel and actionable gene mutations or chromosomal rearrangements in these AML samples. The ETV6-NTRK3 fusion gene is one such rearrangement identified in samples from patients with AML. Fusion of ETV6 sequences to the tyrosine kinase domain of NTRK3 results in constitutive activation of the TRKC kinase and ETV6-NTRK3 expression has emerged as one of the key oncodrivers for leukemogenesis. Constitutive activation of TRK family tyrosine kinases has also been detected in a wide range solid tumor and hematologic malignancies, including lung, colorectal, salivary gland, sarcoma, thyroid, glioblastoma, melanoma, anaplastic large cell lymphoma (ALCL) and Philadelphia-like acute lymphoblastic leukemia. Entrectinib (RXDX-101) is an investigational, orally available, brain-penetrant, highly potent and selective kinase inhibitor with low nanomolar potency against TRKA/B/C, ROS1 and ALK kinase activities (encoded by NTRK1/2/3, ROS1 and ALK genes, respectively). In these studies, we have demonstrated sensitivity to entrectinib in AML cell lines with endogenous expression of the ETV6-NTRK3 fusion gene. Entrectinib treatment blocked cell proliferation and survival in vitro with sub-nanomolar EC50 values. Phosphorylation of the ETV6-TRKC fusion protein as well as phosphorylation of known TRKC downstream signaling effectors was inhibited by entrectinib treatment in a dose-dependent manner. Sensitivity to entrectinib was dependent on expression of the TRKC fusion protein. In xenograft models, entrectinib treatment at clinically relevant doses resulted in tumor regression, which was accompanied by elimination of residual cancer cells from the bone marrow. The clinical relevance of activated oncogenic tyrosine kinases resulting from chromosomal rearrangements has been validated by the efficacy of selective tyrosine kinase inhibitors. Our preclinical data demonstrate the potential of entrectinib as an effective treatment for patients with NTRK rearranged acute myeloid leukemias and provide rationale for the clinical development of entrectinib in molecularly defined hematologic malignancies. Entrectinib is currently the subject of an ongoing global Phase 2 basket study enrolling patients across multiple tumor histologies containing TRK, ROS1 or ALK fusions. Citation Format: Patrick Fagan, Maria Barrera, Colin Walsh, Danielle Murphy, Ian Silverman, Robert Shoemaker, Ge Wei, Zachary Hornby, Gary Li, Kristen M. Smith. Anti-tumor activity of entrectinib, a highly potent pan-TRK, ROS1 and ALK inhibitor, in molecularly defined acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5158. doi:10.1158/1538-7445.AM2017-5158

Background: Entrectinib is a potent, CNS-penetrant, oral inhibitor of the tyrosine kinases TRKA/B/C (encoded by the genes NTRK1/2/3, respectively), ROS1, and ALK with IC50s < 2 nM (biochemical kinase assay). Two Phase 1 studies ALKA-372-001 and STARTRK-1 have enrolled more than 130 patients with advanced or metastatic solid tumors harboring TRKA/B/C, ROS1, or ALK molecular alterations, with or without CNS disease. Previously, we reported an objective response rate of 79% in 24 tyrosine kinase inhibitor-naïve patients with TRK, ROS1, or ALK gene fusions who were treated at doses that achieved therapeutic exposures consistent with the Recommended Phase 2 Dose (RP2D) (Drilon et al, AACR 2016). Entrectinib was well tolerated, with predominantly Grades 1 or 2 adverse events that were reversible with dose modification. Responses were observed in NSCLC, colorectal cancer, mammary analog secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as > 2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1-NTRK1-rearranged lung cancer. Methods: STARTRK (Studies of Tumor Alterations Responsive to Targeting Receptor Kinases)-2 is a potentially registration-enabling Phase 2 basket study of entrectinib for the treatment of patients with advanced solid tumors that harbor a TRK, ROS1, or ALK gene fusion. In order to determine enrollment eligibility and assignment to a specific tumor type basket, patients are screened for gene fusions either locally, including by ctDNA, or centrally at Ignyta’s CLIA/CAP diagnostic laboratory using next generation sequencing. The study’s eligibility criteria were designed to maximize enrollment of these rare patients by allowing CNS disease, Eastern Cooperative Oncology Group (ECOG) performance status 2, and any prior line of therapy, with the exception of TRK, ROS1, or ALK inhibitors. Patients with ALK- or ROS1-rearranged NSCLC who had previously been treated with crizotinib and experienced CNS-only progression are also eligible. In addition, a “non-evaluable” basket allows enrollment of patients confirmed to have gene fusions who do not meet all the inclusion or exclusion criteria. Entrectinib is administered orally on a continuous daily dosing regimen, at a dose of 600 mg once-daily in repeated 4-week cycles. Safety is assessed by monitoring of adverse events, laboratory tests, and clinic visits. Tumor assessments (computed tomography (CT) or magnetic resonance imaging (MRI)) of the chest, abdomen, pelvis (depending on tumor type), plus bone and/or brain as applicable, are performed at the end of Cycle 1 and every 8 weeks thereafter. All CT and MRI scans are read by a central independent imaging laboratory using the Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 and the Response Assessment in Neuro-Oncology Criteria (RANO) or RANO - Brain Metastases (RANO-BM), as applicable, for patients with primary or secondary CNS disease, respectively. Blood and tissue are collected at the time of progression for biomarker analyses for potential mechanisms of resistance to entrectinib. Patients remain on study treatment until documented radiographic progression as assessed by blinded independent central review (BICR), development of unacceptable toxicity, or withdrawal of consent. Citation Format: Alexander Drilon, Kamalesh Kumar Sankhala, Stephen V. Liu, Byoung Chul Cho, Collin Blakely, Cheng E. Chee, Marwan Fakih, Jonathan Polikoff, Zachary Hornby, Lisa Schechet, David Luo, Edna Chow Maneval, Pratik S. Multani, Robert C. Doebele. STARTRK-2: A global phase 2, open-label, basket study of entrectinib in patients with locally advanced or metastatic solid tumors harboring TRK, ROS1, or ALK gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT060. doi:10.1158/1538-7445.AM2017-CT060

Targeted therapies that effectively cross the blood-brain barrier to treat primary and metastatic brain tumors represent a critical unmet medical need in neuro-oncology. NTRK, ROS1, and ALK gene fusions are seen in over 40 primary solid tumor histologies, many of which may be complicated by brain metastases. An inhibitor with demonstrated brain tumor efficacy could be beneficial for these patient populations. Entrectinib (RXDX-101), an orally available, selective and potent kinase inhibitor of TRK, ROS1, and ALK, is specifically designed to cross the blood-brain barrier and is being developed in part to address this need. STARTRK-2 (Studies of Tumor Alterations Responsive to Targeting Receptor Kinases - 2) (NCT02568267) is an open-label, multi-center, global phase 2 pivotal basket study of oral entrectinib in adult patients with locally advanced or metastatic solid tumors, including primary and secondary brain lesions, harboring a gene fusion in NTRK, ROS1, or ALK. While NTRK, ROS1, or ALK gene fusions are rare in non-small cell lung cancer (NSCLC), colorectal cancer, and primary brain tumors, NTRK fusions are more frequent in rare cancers such as infantile fibrosarcoma, secretory breast cancer and mammary analog secretory carcinoma (MASC) of the salivary gland. We report the activity of entrectinib against metastatic brain lesions in preclinical models and clinical subjects bearing these gene fusions. Entrectinib demonstrated a CNS penetration with a brain/blood ratio of 0.4 in mouse, 0.6 - 1.0 in rat, and 1.4 - 2.2 in dog. In a mouse model of intracranial ALK-fusion-driven lung cancer, entrectinib led to a survival benefit of 57 days vs. 34 days (p < 5x10e-4), after 10 days of oral treatment. In phase 1 clinical studies of entrectinib, a total of 119 patients have been treated with entrectinib (as of March 7, 2016). In the subpopulation of 24 patients positive for NTRK, ROS1, or ALK gene fusions who were naïve to prior TRK-inhibitor treatment specific for their respective fusion, effective therapeutic dosing of entrectinib resulted in a 79% response rate, with a well-tolerated safety profile. Regression of primary and metastatic brain tumors was demonstrated in 100% of NTRK fusion positive patients with CNS disease (3/3). In an NSCLC patient with SQSTM1-NTRK1 gene fusion, entrectinib treatment led to a rapid and sustained clinical response systemically and in 15-20 secondary brain lesions. In multiple ROS1-rearranged NSCLC patients, entrectinib treatment also resulted in robust regression of metastatic brain lesions. Finally, compassionate use of entrectinib in a young patient with infantile fibrosarcoma harboring an ETV6-NTRK3 fusion led to a confirmed RECIST response of multiple CNS metastases. Together, these data provide compelling evidence to identify patients with primary or metastatic brain tumors harboring NTRK, ROS1, or ALK fusions for enrollment in the STARTRK-2 trial.

Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK result in oncogenic fusion proteins that have been identified in many types of cancer, including lung, colorectal, salivary gland, sarcoma, papillary thyroid, glioblastoma, melanoma and other histologies. Entrectinib (RXDX-101) is an orally available, highly potent and selective ATP-competitive pan-Trk, ROS1 and ALK inhibitor. In preclinical studies, entrectinib effectively inhibits target kinase activity and cancer cell proliferation and in vivo tumor growth across various fusion partners and cancer types. More importantly, entrectinib's activity has been validated clinically in patients across multiple fusion partners and tissue histologies. Trk inhibitors, including entrectinib, have shown promising clinical activity in molecularly selected patients. Predictably, potential resistance mechanisms have also begun to emerge. For example, mutations in the Trk kinase domain were identified as one of the in vitro induced resistance mechanisms to the Trk inhibitor, Loxo-101. The three reported resistance mutations in the Ba/F3-MPRIP-NTRK1 cell line model treated with Loxo-101 were F589, G667 and V573. The F589 location on TrkA is equivalent to the gatekeeper mutations, L1196 location on ALK and L2026 location on ROS1. These gatekeeper mutations often arise as resistance mechanisms in patients treated with ALK and ROS1 inhibitors. To test the activity of entrectinib against these three reported NTRK1 mutations, we introduced mutated Trk proteins into Ba/F3 and cancer cell lines and performed dose-dependent proliferation studies. Entrectinib was able to inhibit proliferation of cells harboring each of these three mutations that confer resistance to other Trk inhibitors. Particularly, the IC50 values of entrectinib against kinase domain wildtype and gatekeeper mutated (F589) are essentially unchanged (low single-digit nM), which is consistent with the observation that entrectinib is also able to inhibit the gatekeeper mutation in ALK (L1196) in both cell based assays and in vivo tumor growth inhibition studies. In conclusion, our preclinical data suggest that entrectinib is an effective treatment for patients with NTRK-rearranged tumors, including cancers that harbor certain resistance mutations to other Trk inhibitors. Citation Format: Ge Wei, Elena Ardini, Roopal Patel, Nicholas Cam, Jason Harris, Jean-Michel Vernier, Nanqun Zhu, Litain Yeh, Robert Shoemaker, Pratik Multani, Zachary Hornby, Robert Wild, Gary G. Li. Entrectinib is effective against the gatekeeper and other emerging resistance mutations in NTRK-, ROS1- and ALK- rearranged cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2136.