Zachary Hornby

NTRK1 rearrangement in colorectal cancer patients: evidence for actionable target using patient-derived tumor cell line

Background We have investigated the incidence of NTRK1 rearrangements in metastatic gastrointestinal cancer patients and demonstrated the potential for clinical response of these patients to targeted therapy. Methods We prospectively collected tumor tissue specimens for one year and simultaneously generated patient-derived tumor cells (PDCs). Specimens were initially screened for TrkA protein expression using TrkA immunohistochemistry (IHC). In the case of TrkA IHC positive results, samples were further examined by fluorescence in situ hybridization (FISH) and next generation sequencing (NGS) to confirm the presence of NTRK1 rearrangements. Results From January 2014 to December 2014, a total of 74 metastatic colorectal cancer (CRC) patients and 66 gastric cancer (GC) patients were initially screened by TrkA IHC. Two of the 74 CRC patients (2.7%) and one of the 66 GC patients (1.5%) were positive for TrkA expression by IHC. All three IHC positive cases had evidence of NTRK1 rearrangements by FISH. NGS was performed on the 3 IHC positive cases and confirmed TPM3-NTRK1 rearrangements in the two CRC cases. One GC patient with TrkA expression by IHC did not harbor an NTRK1 rearrangement. PDCs established from the NTRK1 positive CRC patients were positive for the NTRK1 rearrangement. Entrectinib, a pan-TRK inhibitor, profoundly inhibited cell proliferation of NTRK1-rearranged PDCs with such inhibition associated with inactivation of TrkA, and down-regulation of downstream signaling pathways. Conclusion TrkA IHC is an effective, initial screening method for NTRK1 rearrangement detection in the clinic. Inhibition of the TrkA kinase is a promising targeted therapy for cancer patients whose tumors harbor a NTRK1 rearrangement.

Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer

Background:Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies.Methods:We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR.Results:A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1–35 of CAD with exons 20–29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response.Conclusions:We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.

Entrectinib is a potent inhibitor of Trk-driven neuroblastomas in a xenograft mouse model

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.

Activity of Entrectinib in a Patient With the First Reported NTRK Fusion in Neuroendocrine Cancer

Despite advances in genomic analysis, the molecular origin of neuroendocrine tumors (NETs) is complex and poorly explained by described oncogenes. The neurotrophic TRK family, including NTRK1, 2, and 3, encode the proteins TRKA, TRKB, TRKC, respectively, involved in normal nerve development. Because NETs develop from the diffuse neuroendocrine system, we sought to determine whether NTRK alterations occur in NETs and whether TRK-targeted therapy would be effective. A patient with metastatic well-differentiated NET, likely of the small intestine, was enrolled on the STARTRK2 trial (ClinicalTrials.gov identifier: NCT02568267) and tissue samples were analyzed using an RNA-Seq next-generation sequencing platform. An ETV6:NTRK3 fusion was identified and therapy was initiated with the investigational agent entrectinib, a potent oral tyrosine kinase inhibitor of TRKA, TRKB, and TRKC. Upon treatment with entrectinib, the patient experienced rapid clinical improvement; his tumor response was characterized by initial tumor growth and necrosis. This is the first report of an NTRK fusion in NETs. Our patients response to entrectinib suggests that NTRK fusions can be important in the pathogenesis of NETs. Recent DNA-based genomic analyses of NETs may have missed NTRK fusions due its large gene rearrangement size and multiple fusion partners. The tumors initial pseudoprogression may represent a unique response pattern for TRK-targeted therapies. An effort to characterize the prevalence of NTRK fusions in NETs using optimal sequencing technology is important.

Abstract 2136: Entrectinib is effective against the gatekeeper and other emerging resistance mutations in NTRK-, ROS1- and ALK- rearranged cancers

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, entrectinib9s 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.

Response to Entrectinib in Differentiated Thyroid Cancer With a ROS1 Fusion

The detection of gene fusions in cancer provides insight into tumorigenesis and in some cases reveals potential therapeutic targets. There are now amultitude of small-molecule inhibitors that effectively target tumors harboring specific gene fusions. With successful paradigms in place for chronic myelogenous leukemia and non–smallcell lung cancer (NSCLC), discovery of fusion events can have an immediate and significant clinical impact. Gene fusions in ROS1, for example, predict sensitivity to the oral tyrosine kinase inhibitor crizotinib and have led to the approval of crizotinib for the treatment of NSCLC with an ROS1 fusion. ROS1 fusions have also been detected in multiple other cancer types, including glioblastomamultiforme, gastric cancer, and acral lentiginous melanoma, but response to ROS1 tyrosine kinase inhibitors in tumors other than lung cancer is not well characterized. Recently, a CCDC30-ROS1 fusion was identified in a case of papillary thyroid cancer; this patient was successfully treatedwith standard therapy.There have been no other reported cases of an ROS1 fusion in thyroid cancer. Although ROS1 fusions have been detected in various tumor types, none were identified in 226 cases analyzed by theMSKIMPACT (Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets) next-generation sequencing panel or in 498 cases included in an analysis of The Cancer Genome Atlas RNA sequencing data.Here, we provide the first report to our knowledge of an EZR-ROS1 fusion in a patient withmetastatic papillary thyroid cancer and confirm the role of this gene fusion as a therapeutic target by describing its response to entrectinib, an orally bioavailable, CNS-active, small-molecule inhibitor ofROS1 aswell asTRKA, TRKB, TRKC, and ALK.

Abstract A173: Potent anti-tumor activity of entrectinib in patient-derived models harboring oncogenic gene rearrangements of NTRKs

The Trk family of kinases, which include TrkA, TrkB and TrkC, encoded by NTRK1, NTRK2 and NTRK3, respectively, are high affinity receptors for the neurotrophin family of nerve growth factors. Dysregulated kinase activity of Trk family members due to chromosome rearrangements has been shown to be an oncogenic driver in a number of cancer types, including lung, colorectal, salivary gland, papillary thyroid, glioblastoma, melanoma and other tumors. Although the prevalence of such events is relatively low in most tumor types ( Entrectinib (formerly RXDX-101) is an orally available, potent and selective ATP-competitive pan-Trk, ROS1 and ALK inhibitor, with comparable, low nanomolar potency against kinase activity of TrkA, TrkB and TrkC in biochemical and cell based assays. In engineered BaF3 cells expressing clinically identified Trk fusion proteins, with various partners, entrectinib demonstrated potent anti-proliferative activity in the range of 2-5 nM, accompanied by inhibition of Trk phosphorylation and concomitant inactivation of downstream effectors such as PLCγ1, AKT and ERK. The clinical relevance of targeting Trk fusions by entrectinib was further demonstrated by several in vitro and in vivo studies involving patient-derived tumor cells (PDCs) and patient-derived xenografts (PDXs) determined to harbor (by NGS and FISH) and express (by IHC) Trk rearrangements. In 2-dimensional and 3-dimensional proliferation assays, entrectinib effectively inhibited proliferation of PDCs from a CRC patient positive for TPM3-NTRK1 fusion. In another independent study, entrectinib, at exposures significantly lower than clinically achievable levels, caused tumor regression in a PDX derived from a CRC patient positive for LMNA-NTRK1 fusion. All the functional readouts were correlated with changes in target and pathway biomarkers. In conclusion, our preclinical data demonstrate the potential of entrectinib as an effective treatment for Trk-fusion driven tumors of multiple histologies, which is now being demonstrated in ongoing clinical trials. Citation Format: Gang Li, Seung Tae Kim, Kyoung-Mee Kim, Jeeyun Lee, Mariangela Russo, Sandra Misale, Alberto Bardelli, Roopal Patel, Nicholas Cam, Ge Wei, Aaron Boomer, Danielle Murphy, Jason Christiansen, Robert Shoemaker, Zachary Hornby, Robert Wild. Potent anti-tumor activity of entrectinib in patient-derived models harboring oncogenic gene rearrangements of NTRKs. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A173.

Abstract B28: Overcoming drug resistance to Trk inhibition by rational combination of entrectinib and trametinib: from bench to bedside

Abnormal expression and constitutive activation of TrkA, TrkB and TrkC due to gene fusions are oncogenic drivers in many cancer types. Entrectinib, a potent, brain-penetrant Trk inhibitor, has demonstrated rapid, deep, and sustained clinical responses in patients with advanced or metastatic Trk-fusion-positive solid tumors across multiple histologies. For tyrosine kinase inhibitors, point mutations that disrupt the binding between the inhibitor and kinase domain of the target are a common mechanism of resistance. By design, entrectinib retains its potency against gatekeeper mutations in Trk, ROS1 and ALK. On the other hand, solvent front mutations, G595R in TrkA or G623R in TrkC (analogous to ROS1 G2032R and ALK G1202R), have been identified as resistance mutations in a clinical setting. Preclinically, upregulation of the MAPK pathway was observed following the introduction of such mutation in Trk-fusion-positive cancer cell lines. In vitro combination screening and in vivo efficacy study further demonstrated the potential for entrectinib-trametinib (a MEK inhibitor) combination to overcome the drug resistance mediated by solvent front mutations. In the clinic, a 34-year-old female patient with ETV6-NTRK3 positive mammary analog secretory carcinoma (MASC) who progressed despite multiple courses of prior multi-modal therapy, including crizotinib, experienced a rapid confirmed partial response (PR: 89% reduction at nadir) with entrectinib treatment. Seven months later, asymptomatic progressive disease (PD) was detected at a solitary tumor site, a biopsy of which showed a G623R solvent front mutation. After three more months on entrectinib, the patient experienced generalized progression and was in need of additional therapy. She was then treated with another Trk inhibitor with no clinical benefit. She then received palliative radiation therapy to symptomatic pleural/chest wall metastases. Supported by the preclinical data on combination therapy, a single patient protocol was subsequently developed to allow co-administration of entrectinib and trametinib. Significant tumor regression was achieved within the first eight weeks, including sustained resolution of tumor-associated pain and hypertrophic osteoarthropathy. In conclusion, we have identified a mitigation strategy, utilizing an approved agent combined with a well-characterized clinical stage agent, to overcome acquired Trk-inhibitor resistance, presumably by overcoming solvent front mutation-driven MAPK activation. The successful translation of a preclinical observation made at the bench to clinical practice at the bedside has greatly extended the duration of tumor regression and provided continued care to a Trk-fusion positive patient even after the emergence of resistance. This clinical observation will be further explored in a dedicated Phase 1/1b combination study. Citation Format: Ge Wei, Edna Chow Maneval, Vanessa Esquibel, Michael F. Berger, Sofia Haque, Roopal Patel, Colin Walsh, Zachary Hornby, Pratik Multani, Gary Li. Overcoming drug resistance to Trk inhibition by rational combination of entrectinib and trametinib: from bench to bedside [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B28.

Abstract CT030: 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

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 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

Abstract CT060: 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

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 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