NTRK Gene Fusions Research Articles

Abstract PR07: A pediatric phase 1 study of larotrectinib, a highly selective inhibitor of the tropomyosin receptor kinase (TRK) family: An updated analysis

Abstract Background: NTRK gene fusions occur in a broad range of pediatric and adult malignancies at low frequencies, but are present in the vast majority of infantile fibrosarcomas. Larotrectinib is the first selective small-molecule inhibitor of TRKA, B, and C in clinical development. Larotrectinib has demonstrated a high response rate and a favorable safety profile in children and adults with TRK-fusion positive cancers. Here we report an updated analysis from the pediatric phase 1 study of larotrectinib. Methods: This multicenter, rolling 6 phase 1 study enrolled pediatric patients with refractory solid or CNS tumors. Patients with locally advanced infantile fibrosarcoma who would require disfiguring surgery or limb amputation to achieve a complete surgical resection were also eligible. Measurable or evaluable disease by RECIST v1.1/RANO was required. Patients were dosed orally BID on a continuous 28-day schedule either by capsule or oral solution. Pharmacokinetic (PK)-directed intra-subject dose escalation was permitted, with exposures targeting the adult recommended phase 2 dose (RP2D) of 100 mg BID. The primary objective was to define the maximum tolerated dose (MTD) / RP2D; secondary objectives included pharmacokinetics (PK) and efficacy using RECIST v1.1/RANO. Results: As of July 17, 2017, 24 patients (17 with TRK fusions, 7 without TRK fusions) with a median age of 4.5 years (range 1 month-18 years) were enrolled. Patients had diverse TRK fusions (9 NTRK1, 1 NTRK2, and 7 NTRK3) in heterogeneous tumor diagnoses (8 infantile fibrosarcoma (IFS), 7 other sarcoma, and 2 papillary thyroid cancer). Patients were treated on 3 dose levels: 100 mg BID (DL1, n=4) and 150 mg BID (DL2, n=11) adult equivalent doses by SimCyp modeling, and 100 mg/m2/dose BID with a cap of 100 mg/dose (DL3, n=9). The RP2D was determined to be 100 mg/m2 (capped at 100 mg) BID based on PK, safety and efficacy. No MTD was defined. The most common AEs attributed to larotrectinib were increased AST, vomiting, and neutropenia. No grade 4/5 AEs were attributed to larotrectinib. Both capsule and solution formulations delivered similar dose-dependent PK. AUC0-24 at the RP2D was comparable to the adult RP2D across the pediatric age spectrum (<2, 2-11, and 12-18 years). No responses were seen in patients (n=7) without TRK fusions (all had PD as best response). Among patients with TRK fusions and measureable disease by RECIST (n=15), the investigator-assessed confirmed objective response rate was 93% (67% PR, 27% CR). 1 patient (7%) had SD with tumor shrinkage. All patients with TRK fusions (n=17) had a reduction in tumor burden regardless of histology or specific TRK fusion. With median follow-up of 6.5 (range 1.5+ to 12.5+) months, 14 patients with TRK fusions remain on treatment. Three patients have discontinued therapy, including two patients with responses that led to surgical resection; both had negative margins (R0 surgery) and one had a pathologic complete response. These patients remain on study and off larotrectinib without recurrence. Only one case of acquired resistance has been observed, which involved a TRKC G623R mutation. Conclusions: Larotrectinib demonstrated broad histology-independent efficacy in pediatric patients harboring TRK fusions and a favorable tolerability profile. A pediatric RP2D of 100 mg/m2/dose BID with a cap of 100 mg/dose has been defined. Blinded central review of responses and a minimum of 6 months of follow-up for all patients will be presented. Citation Format: Brian Turpin, Catherine M. Albert, Leo Mascarenhas, Noah Federman, Ramamoorthy Nagasubramanian, Mark Reynolds, Steven Smith, Scott Cruickshank, Michael C. Cox, Alberto S. Pappo, Douglas S. Hawkins, Steven G. DuBois, Theodore W. Laetsch. A pediatric phase 1 study of larotrectinib, a highly selective inhibitor of the tropomyosin receptor kinase (TRK) family: An updated analysis [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR07.

Potential Novel Therapy Targets in Neuroendocrine Carcinomas of the Breast

IntroductionNeuroendocrine carcinoma (NEC) of the breast is a rare, special type of breast cancer, reportedly constituting 2% to 5% of all breast cancers. Although breast NEC does not have a specific targeted therapy, several new targeted therapies based on specific biomarkers were recently investigated in the NEC of lung and in other types of breast carcinoma, which may provide guidance to their feasibility in breast NEC. Materials and MethodsTwenty breast NECs were profiled for biomarkers of therapy including antibody-drug conjugates (DLL3, TROP-2, and FOLR1), histone deacetylase (H3K36Me3) inhibitors, tropomyosin receptor kinases (NTRK1/2/3 gene fusions) targeted inhibitors, alkylating agents (MGMT), and immune checkpoint inhibitors (PD-L1, TMB, and MSI) using immunohistochemistry and DNA/RNA next-generation sequencing assays. ResultsPredictive expression of TROP-2, FOLR1, and H3K36Me3 were detected in different subsets of tumors and may pave the way for development of novel targeted therapies in some patients with breast NECs. There was no evidence of DLL3 expression, NTRK gene fusions, or MGMT hypermethylation. No biomarkers predictive of immune checkpoint inhibitor efficacy (programmed death-ligand 1 expression, tumor mutational burden, microsatellite instability) were identified. FGFR and CCND1 gene amplifications were detected in isolated cases. ConclusionsThis study identified several potential targets for novel therapies in breast NEC, including farletuzumab and mirvetuximab soravtansine (FOLR1), sacituzumab govitecan (TROP-2), and HDAC inhibitors (H3K36Me3). In some cases, CCND1 gene amplification may indicate the usefulness of investigational therapies. The reported results should serve as an early indication of potential clinical relevance in selected patients with breast NEC.

Abstract A29: The CUTO panel of patient-derived NSCLC cell lines reveals unique molecular characteristics and responses to targeted therapies

Abstract Cell lines generated from human tumors have been an invaluable tool in dissecting the underlying molecular mechanisms of cancer biology and for cancer drug development. The large library of original human lung cancer cell lines established by Minna and Gazdar, though instrumental in investigating various aspect of lung cancer biology, lacks examples of some of the diverse mutations responsible for this cancer. The CUTO (Colorado University Thoracic Oncology) cell line series, initiated in 2011, had the goal of curating a new panel of cells derived from NSCLC patients with distinct oncogenic drivers. By generating multiple, unique cell lines representing each oncogene driver subset, we believe that these lines would allow study of interpatient variability that underlie differential patient responses and duration of response to targeted therapies. The current CUTO panel consist of 33 cell lines with either 1) gene rearrangements in ALK, RET, ROS1 or NTRK1; 2) activating mutations in the ERBB gene family including exon 20 insertions in EGFR and ERBB2 (HER2) as well as rare mutations in EGFR; and 3) inactivation of the NF1 gene either as a concurrent mutation with other oncogenic alterations or as the sole driver mutation. For cells harboring gene rearrangements we have amassed four EML4-ALK lines, two KIF5B-RET cell lines, five ROS1 fusion lines (3 CD74-ROS1, 1 SDC4-ROS1, and 1 TPM3-ROS1), and one MPRIP-NTRK1 cell line. Our ERBB mutant lines include one cell line with HER2 exon 20 insertion, three lines with different exon 20 insertions in EGFR, and two EGFR mutant cell lines with compound mutations. We present our characterization of these cell lines in terms of their proliferation profiles and molecular signaling pathways in the presence of targeted inhibition. The derivation and characterization of these cell lines have facilitated the study of cell signaling in oncogene-driven cancer, have helped identify new resistance mechanisms, and have facilitated drug development for rare oncogenes such as NTRK1 gene fusions and EGFR exon 20 insertions. We expect that this growing library of cell lines will continue to further our understanding of oncogenic-driven tumor biology and provide mechanistic insight towards the development of novel therapeutics and drug combinations. Citation Format: Anh Tuan Le, Adriana Estrada-Bernal, Laura Schubert, Andrea Doak, Nan Chen, Kurtis Davies, Aria Vaishnavi, Mary Jackson, Vignesh Narayana, Kimi Kondo, John Mitchell, Michael Weyant, Tom Purcell, Paul Bunn, Ross Camidge, Janet Freeman-Daly, Robert Doebele. The CUTO panel of patient-derived NSCLC cell lines reveals unique molecular characteristics and responses to targeted therapies [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A29.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study

BackgroundGene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material.MethodsWe describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples.ResultsReproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies.ConclusionThis methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

Clinicopathologic Features of Non-Small-Cell Lung Cancer Harboring an NTRK Gene Fusion.

Gene rearrangements involving NTRK1/2/3 can generate fusion oncoproteins containing the kinase domains of TRKA/B/C, respectively. These fusions are rare in non-small cell lung cancer (NSCLC), with frequency previously estimated to be <1%. Inhibition of TRK signaling has led to dramatic responses across tumor types with NTRK fusions. Despite the potential benefit of identifying these fusions, the clinicopathologic features of NTRK fusion-positive NSCLCs are not well characterized. We compiled a database of NSCLC cases harboring NTRK fusions. We characterized the clinical, molecular, and histologic features of these cases with central review of histology. We identified 11 NSCLC cases harboring NTRK gene fusions verified by next-generation sequencing (NGS) and with available clinical and pathologic data, forming the study cohort. Fusions involved NTRK1 (7 cases) and NTRK3 (4 cases), with 5 and 2 distinct fusion partners, respectively. Cohort patients were 55% male, with a median age at diagnosis of 47.6 years (range 25.3-86.0) and a median pack year history of 0 (range 0-58). 73% of patients had metastatic disease at diagnosis. No concurrent alterations in KRAS, EGFR, ALK, ROS1, or other known oncogenic drivers were identified. Nine cases were adenocarcinoma, including 2 invasive mucinous adenocarcinomas and 1 adenocarcinoma with neuroendocrine features; one was squamous cell carcinoma; and one was neuroendocrine carcinoma. By collating data on 4872 consecutively screened NSCLC cases from unique patients, we estimate a frequency of NTRK fusions in NSCLC of 0.23% (95% CI 0.11-0.40). NTRK fusions occur in NSCLCs across genders, ages, smoking histories, and histologies. Given the potent clinical activity of TRK inhibitors, we advocate that all NSCLCs be screened for NTRK fusions using a multiplexed NGS-based fusion assay.

Cytoplasmic TrkA Expression as a Screen for Detecting NTRK1 Fusions in Colorectal Cancer

NTRK1 gene fusions, the targets of multikinase inhibitors, are promising therapeutic targets for colorectal cancer (CRC). However, screening methods for detecting NTRK1 gene fusions in CRC tissues have not been reported. In this study, we investigated the potential use of immunohistochemistry (IHC) for detecting NTRK1 gene fusions. We performed and compared IHC with fluorescence in situ hybridization (FISH) in 80 CRC patients. TrkA immunostaining was observed to be both membranous and cytoplasmic and was scored semiquantitatively using staining intensity and proportions. The tumors were observed to be NTRK1 gene fusion-positive when ≥20 out of 100 nuclei in FISH. A significant correlation between the IHC and FISH results for determination of the NTRK1 gene fusions was observed. We measured the cytoplasmic TrkA expression, which showed an area under the receiver operating characteristic (ROC) curve of 0.926 (range: 0.864-0.987, 95% CI, P=.001). By choosing 4.5 (sum of the intensity and proportion scores of cytoplasmic TrkA expression) as the cut-off value for the positive and negative NTRK1 gene fusion groups, the sensitivity and specificity for predicting lymph node metastasis were 100 and 83.8%, respectively (P=.001). Specifically, high cytoplasmic TrkA expression (sum of intensity and proportion scores >4) was associated with the presence of NTRK1 gene fusions (P<.0001, r=0.528). Taken together, our data showed that IHC for TrkA can be used as an efficient screening method for detecting NTRK1 gene fusions in CRC.

Larotrectinib for paediatric solid tumours harbouring NTRK gene fusions: phase 1 results from a multicentre, open-label, phase 1/2 study

Gene fusions involving NTRK1, NTRK2, or NTRK3 (TRK fusions) are found in a broad range of paediatric and adult malignancies. Larotrectinib, a highly selective small-molecule inhibitor of the TRK kinases, had shown activity in preclinical models and in adults with tumours harbouring TRK fusions. This study aimed to assess the safety of larotrectinib in paediatric patients. This multicentre, open-label, phase 1/2 study was done at eight sites in the USA and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion status; had a Karnofsky (≥16 years of age) or Lansky (<16 years of age) performance status score of 50 or more, adequate organ function, and full recovery from the acute toxic effects of all previous anticancer therapy. Following a protocol amendment on Sept 12, 2016, patients with locally advanced infantile fibrosarcoma who would require disfiguring surgery to achieve a complete surgical resection were also eligible. Patients were enrolled to three dose cohorts according to a rolling six design. Larotrectinib was administered orally (capsule or liquid formulation), twice daily, on a continuous 28-day schedule, in increasing doses adjusted for age and bodyweight. The primary endpoint of the phase 1 dose escalation component was the safety of larotrectinib, including dose-limiting toxicity. All patients who received at least one dose of larotrectinib were included in the safety analyses. Reported here are results of the phase 1 dose escalation cohort. Phase 1 follow-up and phase 2 are ongoing. This trial is registered with ClinicalTrials.gov, number NCT02637687. Between Dec 21, 2015, and April 13, 2017, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) with a median age of 4·5 years (IQR 1·3-13·3) were enrolled to three dose cohorts: cohorts 1 and 2 were assigned doses on the basis of both age and bodyweight predicted by use of SimCyp modelling to achieve an area under the curve equivalent to the adult doses of 100 mg twice daily (cohort 1) and 150 mg twice daily (cohort 2); and cohort 3 was assigned to receive a dose of 100 mg/m2 twice daily (maximum 100 mg per dose), regardless of age, equating to a maximum of 173% of the recommended adult phase 2 dose. Among enrolled patients harbouring TRK fusion-positive cancers, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. Adverse events were predominantly grade 1 or 2 (occurring in 21 [88%] of 24 patients); the most common larotrectinib-related adverse events of all grades were increased alanine and aspartate aminotransferase (ten [42%] of 24 each), leucopenia (five [21%] of 24), decreased neutrophil count (five [21%] of 24), and vomiting (five [21%] of 24). Grade 3 alanine aminotransferase elevation was the only dose-limiting toxicity and occurred in one patient without a TRK fusion and with progressive disease. No grade 4 or 5 treatment-related adverse events were observed. Two larotrectinib-related serious adverse events were observed: grade 3 nausea and grade 3 ejection fraction decrease during the 28-day follow-up after discontinuing larotrectinib and while on anthracyclines. The maximum tolerated dose was not reached, and 100 mg/m2 (maximum of 100 mg per dose) was established as the recommended phase 2 dose. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response as per Response Evaluation Criteria In Solid Tumors version 1.1; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response. The TRK inhibitor larotrectinib was well tolerated in paediatric patients and showed encouraging antitumour activity in all patients with TRK fusion-positive tumours. The recommended phase 2 dose was defined as 100mg/m2 (maximum 100 mg per dose) for infants, children, and adolescents, regardless of age. Loxo Oncology Inc.

Abstract P2-09-15: NTRK fusions in breast cancer: Clinical, pathologic and genomic findings

Abstract Background: The tropomysin receptor kinase A family includes the 3 NTRK1, NTRK2 and NTRK3 genes and plays major roles in neuronal development. The recent evidence of remarkable efficacy for kinase inhibitors (TKI) targeting NTRK across a wide variety of malignancies that harbor NTRK gene fusions has stimulated great interest in determining the type of cancers driven by these therapy defining NTRK genomic alterations. Methods: A consecutive series of 12,214 locally aggressive, relapsed and metastatic breast malignancies (mBM) were subjected to comprehensive genomic profiling (CGP) using DNA extracted from 40 µm of FFPE sections and adaptor ligation-based libraries to a mean coverage depth 719X for up to 315 cancer-related genes. The results were analyzed for all classes of genomic alterations (GA) including base substitutions, insertions and deletions, select fusions and rearrangements, and copy number changes. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA. Microsatellite instability (MSI) status was calculated by a customized algorithm. Results: 16 (0.13%) mBM (all female) harbored NTRK gene fusions. The median age was 51 years (range 34 to 70 years). There were 9 ductal carcinomas, 2 lobular carcinomas, 3 secretory carcinomas 1 metaplastic carcinoma and 1 angiosarcoma. Tumor stages at the time of sequencing were 12 Stage IV, 1 Stage III and 3 stage I (all 3 secretory carcinomas). In 9 cases, clinical receptor status was known: 3 (33%) were ER+/HER2- and 6 (66%) ER-/HER2- (all TNBC) with all 9 (100%) of cases HER2-. All 3 SCA were TNBC. 10 NTRK fusions involved NTRK1 featuring a variety of fusion partners (CGN, GATAD2B, LMNA, MDM4, PEAR1, and TPM3) and 6 involved NTRK3 (all ETV6 fusions). There were no NTRK2 fusions. NTRK fusion+ mBM featured a mean of 4.25 GA per sample. The most frequent non-fusion partner co-altered genes in this series of NTRK fusion+ mBM were: TP53 at 25%, IKBKE, PIK3C2B CCND1 at 19%, and AKT, PIK3CA, MYC, CDH1, CDKN2A, PTEN, FGF3, FGF4 and FGF19 all at 13%. The median TMB for NTRK fusion+ mBM was 0.9 mutations/Mb and no cases (0%) had a TMB &amp;gt; 10 mutations/Mb and no cases (0%) feared high microsatellite instability (MSI high). Clinical response assessment to NTRK TKI therapies in this series is ongoing. Conclusions: NTRK gene fusions although extremely uncommon in breast malignancies occur across a variety of tumor types, is universally HER2 negative, more frequent in TNBC than in ER+ tumors, is associated with a moderate frequency of additional genomic alterations and a complete absence of either high TMB or high MSI. This study confirms that a CGP assay, when applied to a large cohort of near universal clinically advanced disease can identify extremely rare alterations that can lead a small number of patients to highly effective precision therapies. Citation Format: Ross JS, Chung J, Elvin JE, Vergilio J-A, Ramkissoon S, Suh J, Severson E, Daniel S, Frampton GM, Fabrizio D, Hartmaier RJ, Albacker LA, Ali SM, Schrock AB, Miller VA, Stephens PJ, Gay LM. NTRK fusions in breast cancer: Clinical, pathologic and genomic findings [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-15.

Clinical benefit of entrectinib for patients with metastatic pancreatic cancer who harbor NTRK and ROS1 fusions.

521 Background: Despite recent improvements in chemotherapy for pancreatic cancer that have demonstrated improvements in survival, response rates remain at less than 50%. Distinct molecular subgroups of pancreatic cancer that harbor specific actionable molecular alterations have recently been identified. These include gene fusions of the receptor tyrosine kinases NTRK and ROS1. Entrectinib is a CNS-active, potent, and selective TRK and ROS1 inhibitor that has shown substantial clinical activity in patients with locally advanced or metastatic solid tumors that have NTRK or ROS1 fusions (Drilon A, et al. Cancer Discov. 2017;7[4]:400-409). Methods: We examined 3 cases of pancreatic cancer where an actionable gene rearrangement was identified: 2 with a TPR-NTRK gene fusion, and 1 with an SCL4-ROS1 gene fusion. All 3 patients were treated with entrectinib 600 mg daily in a phase 2 trial (NCT02568267). Results: Entrectinib was well tolerated with mostly grade 1-2 adverse events consisting primarily of arthralgias, myalgias, and fatigue. All 3 patients showed evidence of clinical improvement, with normalization of the tumor biomarker CA19-9 and confirmed partial responses as per RECIST v1.1 in the 2 patients with a TPR-NTRK fusion, 1 of which was associated with a significant decrease in metabolic activity as assessed by PET/CT scan. The patient with the SCL4-ROS1 fusion had stable disease for at least 6 months. All 3 patients had an improvement to, or maintenance of, a high quality of life while on study. At the time of data cutoff, 1 of the patients with a TPR-NTRK fusion remained on treatment &gt; 1 year. Conclusions: Entrectinib treatment was associated with response and prolonged disease control in these 3 patients with NTRK or ROS1 fusion-positive pancreatic cancer. NTRK and ROS1 gene fusions are rare in pancreatic cancer. The true incidence is unknown, as routine testing for gene fusions in pancreatic cancer is not yet standard practice. Given the benefit seen here with entrectinib and the clinical need for improved therapeutics, there may be value in broader and earlier molecular testing of patients with pancreatic cancer for these actionable fusions. Clinical trial information: NCT02568267.

Abstract A047: Molecular characterization of the malignancies with targetable NTRK gene fusions

Abstract Introduction: Targeted inhibitors of neurotropic tyrosine kinases are highly effective in selected patients with gene fusions involving NTRK1, NTRK2, or NTRK3 and are being actively investigated across cancer types. These fusions are consistently detected in rare cancer types (e.g., secretory breast carcinoma/MASC and infantile fibrosarcoma/congenital mesoblastic nephroma), but the occurrence of NTRK gene fusions in common cancer types is largely unexplored. Methods: Formalin-fixed, paraffin-embedded tumor tissues from 11,116 patients (Caris Life Sciences, Phoenix, AZ) were analyzed for 53 gene fusions (Archer FusionPlex) and additional sequencing of the 592 genes (NGS, Illumina). Total mutational load and gene copy number variations were calculated from the NGS results. NTRK protein (monoclonal rabbit antibody EPR17341, Abcam) and PD-L1 expression (SP142 or 22c3 monoclonal antibodies) were detected using immunohistochemistry (IHC). Results: Fusion assay identified 164 cases with a validated gene fusion (1.5% for the cohort), of which 23 cases (0.21% of the entire cohort) had an NTRK gene fusion. All 3 NTRK genes were involved in fusions with 13 different gene partners; TPM3:NTRK1 and ETV6:NTRK3 were the most common (6 cases each). Central nervous system malignancies had the highest frequency of NTRK fusions involving all 3 NTRK genes (11/400 cases, 2.8%), most commonly NTRK2 (7 cases fused with 7 different genes). NTRK2 fusions were exclusively detected in CNS malignancies (mostly GBM). Twelve non-CNS cases with NTRK1 or NTRK3 fusions included carcinomas of the respiratory tract (n=3), colon (n=2), thyroid (n=2), skin and cervix (n=1, each), uterine and soft tissue sarcoma (n=1, each), and one carcinoma of unknown primary (CUP) site. Strong (3+) and uniform (100% of cells) cytoplasmic/membranous expression of NTRK protein characterized NTRK1 fused cases (4 TPM3 and 1 MEF2D); NTRK2 fused cases were variably positive, while ETV6-NTRK3 cases were negative (0/6 cases). Of 23 NTRK fusion positive cases, eight had no other genomic alteration (pathogenic mutations or gene amplifications) which could be considered driving the cancer growth. Total mutational burden varied (1-37 somatic mutations per Mb) and was the highest in two MSI-H cases (CRC: TPM3-NTRK1 and CUP: ETV6-NTRK3). Additionally, PD-L1 expression was detected in 4 cases (lung and colon TPM3-NTRK1, GBM KCTD8-NTRK2 and CUP NTRK3:ETV6). Conclusion: Pathogenic NTRK genes fusions occur in a variety of cancers, but are generally rare (0.1%) outside primary CNS malignancies. NTRK may present as the only actionable target or co-occurring with other potentially targetable alterations (e.g., immune checkpoint inhibitors for PD-L1 positive or MSI-H cases). Uniform strong immunohistochemical staining for pNTRK may be a useful screening tool for NTRK1 gene fusion cancers outside the CNS. Citation Format: Zoran Gatalica, Jeffrey Swensen, Jeffery Kimbrough, Joanne Xiu. Molecular characterization of the malignancies with targetable NTRK gene fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A047.

Larotrectinib (LOXO-101).

One of the most challenging issues in oncology research and treatment is identifying oncogenic drivers within an individual patient's tumor which can be directly targeted by a clinically available therapeutic drug. In this context, gene fusions as one important example of genetic aberrations leading to carcinogenesis follow the widely accepted concept that cell growth and proliferation are driven by the accomplished fusion (usually involving former proto-oncogenes) and may therefore be successfully inhibited by substances directed against the fusion. This concept has already been established with oncogenic gene fusions like BCR-ABL in chronic myelogenous leukemia (CML) or anaplastic lymphoma kinase (ALK) in lung cancer, including special tyrosine kinase inhibitors (TKIs) which are able to block the activation of the depending downstream proliferation pathways and, consequently, tumor growth. During the last decade, the NTRK1, 2, and 3 genes, encoding the TRKA, B, and C proteins, have attracted increasing attention as another significant and targetable gene fusion in a variety of cancers. Several TRK inhibitors have been developed, and one of them, Larotrectinib (formerly known as LOXO-101), represents an orally available, selective inhibitor of the TRK receptor family that has already shown substantial clinical benefit in both pediatric and adult patients harboring an NTRK gene fusion over the last few years.

415O - Prevalence of NTRK gene fusions in a large cohort of Japanese patients with lung cancer

415O - Prevalence of NTRK gene fusions in a large cohort of Japanese patients with lung cancer

TRTH-10. PEDIATRIC PHASE 1/1B STUDY OF ENTRECTINIB IN PATIENTS WITH PRIMARY BRAIN TUMORS, NEUROBLASTOMA, AND NTRK, ROS1, OR ALK FUSIONS

AbstractThe 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 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.

Clinicopathologic features of non-small cell lung cancer (NSCLC) harboring an NTRK gene fusion.

11580 Background: Gene fusions involving NTRK1/2/3 can generate oncoproteins containing the kinase domains of TRKA/B/C, respectively. Inhibition of TRK signaling has led to dramatic responses across tumor types with NTRK fusions. An estimated 0.1 – 1% of NSCLCs harbor NTRK fusions. To date, clinical and radiographic responses to TRK inhibitors have been reported for 2 NTRK fusion-positive NSCLCs (Farago et al., 2015; Hong et al., 2016). Despite the potential benefit of identifying these fusions, the clinicopathologic features of NTRK fusion NSCLCs are not well characterized. Methods: Physicians across multiple institutions contributed deidentified cases to an NTRK fusion NSCLC database. A central pathologist (M.M.) reviewed tumor histology in cases with available tissue. Results: 10 NSCLC cases with NTRK gene fusions were identified. Of these, TRK kinase domain-containing potentially activating fusions were verified by next-generation sequencing (NGS) in 7, forming the study cohort. Fusions involved NTRK1 (6) and NTRK3 (1) with 6 different partners. Four (57%) patients were male. Median age at diagnosis was 47.6 years (range 27.9 – 86.0). The average smoking pack year history was 8.9 (range 0 to 30). Five (71%) presented with metastatic disease. No concurrent alterations in KRAS, EGFR, ALK, ROS1, or other known drivers were identified in the study cohort cases. On pathologic review of 4 cases, all were adenocarcinoma, including 2 invasive mucinous adenocarcinomas and 1 adenocarcinoma with neuroendocrine features. Of the 3 remaining non-study cohort cases, 1 was a non-kinase domain-containing NTRK1 fusion with a concurrent KRAS G12C mutation, 1 was an NTRK2 intragenic deletion disrupting the exon 18 3’ splice site, and 1 was an NTRK1 alteration detected by FISH but not verified by NGS and with a concurrent HER2 L755P mutation. Conclusions: NTRK fusions occur in both men and women across wide ranges in age and smoking history. We therefore suggest that all NSCLC adenocarcinomas without other oncogenic driver alterations be screened for NTRK fusions. Notably, not all NTRK alterations are activating, requiring validation of the specific position of the fusion.

P14.19 Preclinical and clinical efficacy of entrectinib in primary and metastatic brain tumors harboring NTRK, ROS1, or ALK gene fusions

P14.19 Preclinical and clinical efficacy of entrectinib in primary and metastatic brain tumors harboring NTRK, ROS1, or ALK gene fusions

Targeting TRK family proteins in cancer.

The tropomyosin receptor kinase (TRK) family includes TRKA, TRKB, and TRKC proteins, which are encoded by NTRK1, NTRK2 and NTRK3 genes, respectively. Binding of neurotrophins to TRK proteins induces receptor dimerization, phosphorylation, and activation of the downstream signaling cascades via PI3K, RAS/MAPK/ERK, and PLC-gamma. TRK pathway aberrations, including gene fusions, protein overexpression, and single nucleotide alterations, have been implicated in the pathogenesis of many cancer types, with NTRK gene fusions being the most well validated oncogenic events to date. Although the NTRK gene fusions are infrequent in most cancer types, certain rare tumor types are predominately driven by these events. Conversely, in more common histologies, such as lung and colorectal cancers, prevalence of the NTRK fusions is well below 5%. Selective inhibition of TRK signaling may therefore be beneficial among patients whose tumors vary in histologies, but share underlying oncogenic NTRK gene alterations. Currently, several TRK-targeting compounds are in clinical development. The ongoing Phase 2 trials with entrectinib and LOXO-101, two of the leading TRK inhibitors, are designed as 'basket trials', inclusive of patients whose tumors harbor NTRK gene fusions, independent of histology. Additional Phase 1 studies of other TRK inhibitors, including MGCD516, PLX7486, DS-6051b, and TSR-011, are underway. Interim data examining NTRK-rearranged tumors treated with entrectinib or LOXO-101 demonstrate encouraging activity, with patients achieving rapid and durable responses. Consequently, both drugs have achieved orphan designation from regulatory agencies, and efforts are underway to further expedite their development.

1460 - Clinical safety and activity from a phase 1 study of LOXO-101, a selective TRKA/B/C inhibitor, in solid-tumor patients with NTRK gene fusions

1460 - Clinical safety and activity from a phase 1 study of LOXO-101, a selective TRKA/B/C inhibitor, in solid-tumor patients with NTRK gene fusions

Detecting NTRK, ROS1, and ALK gene fusions in gastrointestinal tumor patients.

619 Background: The STARTRK-2 trial is a potentially registration-enabling Phase 2 global basket trial of the tyrosine kinase inhibitor entrectinib in patients with solid tumors harboring NTRK1, NTRK2, NTRK3, ROS1, or ALK gene fusions. Phase 1 studies of entrectinib reported a 79% ORR across multiple histologies in patients with gene fusions of these targets who were naïve to inhibitors of these targets, received an effective dose, and had extracranial disease. Patients harboring these fusions are typically rare in the cancer population ( &lt; 3%); however, they have been seen across a wide variety of GI tumors. In this presentation we report on the occurrence of these fusions in GI tumors, based on Ignyta’s internal and partner testing. We discuss the development of an assay for GI samples and immunohistochemistry (IHC) screening efficiency rates in a clinical cohort of 157 GI samples. Methods: The occurrence of NTRK, ROS1, and ALK gene fusions in GI tumors was studied from Ignyta internal and partner collaborations. We developed a 2-step diagnostic test to identify fusions in FFPE clinical specimens. IHC screening using a pan-receptor tyrosine kinase cocktail of antibodies precedes an RNA-based anchored multiplex PCR next generation sequencing (NGS) assay. Results: IHC positivity rates ranged from 0 to 24 percent depending on histology and averaging 8% positivity rate across 15 tumor locations. The screen is particularly effective in cholangiocarcinoma, colon, esophagus, gastric, peritoneum, rectum and small bowel. It was highest in pancreas (24%). Out of 157 GI samples, no instances were detected where the IHC was screened negative and gene fusions were observed by NGS. The IHC cocktail enriches detection of the patient population for gene fusions over 11-fold and has a 100% negative predictive value. Conclusions: This 2-step assay is an efficient method for detection of gene rearrangements in both high-volume clinical testing and studies of archival formalin-fixed paraffin-embedded specimens. Depending on how it is implemented in clinical trials, the staged approach can substantially decrease the costs and tissue expended for NGS. Screening efficiency may be improved even further as additional diagnostic antibodies become available. Clinical trial information: NCT02568267.

150O Clinical safety and activity from a phase 1 study of LOXO-101, a selective TRKA/B/C inhibitor, in solid-tumor patients with NTRK gene fusions

150O Clinical safety and activity from a phase 1 study of LOXO-101, a selective TRKA/B/C inhibitor, in solid-tumor patients with NTRK gene fusions

150O Clinical safety and activity from a phase 1 study of LOXO-101, a selective TRKA/B/C inhibitor, in solid-tumor patients with NTRK gene fusions

Background NTRK1, 2 and 3 gene fusions occur across a wide array oftumors. LOXO- 101 is an orally bioavailable, potent, ATP-competitive, selective pan-TRK inhibitor. Here, we report response and durability data for patients with NTRK fusions enrolled in 16C European Society for Medical Oncology 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected]16C European Society for Medical Oncology 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected] an ongoingPhaseI dose escalation trial. Updated pharmacokinetic (PK) and safetydata for all enrolled patients (pts) are also reported. Methods This is an ongoing, open-label, multicenter, 3 + 3 dose escalation Phase I study.LOXO-101isadministeredorallyasaone-ortwice-dailydoseforcontinuous28- day cycles. Response is measured by RECIST. Plasma is obtained for PK analysis. Safety information is collected on all patients and reported regardless of their attribution to the study drug. Results As of March 25, 2016, 43 pts with solid tumors have been enrolled, including sevenptswithNTRKfusionsacrossfivedifferenttumortypes.Sixofthesevenpatients were evaluable for response and all six have demonstrated a clinical response to LOXO- 101.Fiveofsixpatients(83%)haveachievedconfirmedRECISTpartialresponses.All sevenpatientsremainonstudywithadurationoftherapyfromonetofourteencycles. Noobjectiveanti-tumoractivityhasbeenobservedintreatedpatientswithoutanNTRK fusion. In total, 43 pts have been treated across five dose levels. Maximum plasma concentrationsofLOXO-101werereached30-60minutesfollowingdosing.The unbounddruglevelsofLOXO-101 appearsufficientforapproximately98%inhibition ofTRKA/B/Catpeakconcentrationsatalldoselevels.LOXO-101hasbeenwell tolerated. The maximum tolerated dose has not been reached, and the most common adverse events are Grade 1 and 2 fatigue (33%), constipation (23%) and dizziness (23%). Conclusions:LOXO-101 hasbeenwelltoleratedandhasshownpromisingandbroad anti-tumoractivityinpatientswithNTRKfusions.AllpatientswithNTRKfusionshave experienced objective tumor reductions and remain on study without disease progression.ThesedatasuggestthataLOXO-101 doseof100mgBIDiswell-tolerated andcapableofinducingdurablediseasecontrolinpatientswithNTRKgenefusions. Clinical trial indentification NCT02122913 Legal entity responsible for the study Loxo Oncology, Inc. Funding Loxo Oncology, Inc. Disclosure D.S.Hong:TravelpaidforbyLoxoOncology,Inc.S.Smith,S.Cruickshank: Consultant fees paid by Loxo Oncology, Inc. N. Nanda, M.C. Cox: Employee and stockholderofLoxoOncology,Inc.R.Doebele:ResearchgrantprovidedbyLoxo Oncology, Inc. All other authors have declared no conflicts ofinterest.