NTRK1 Rearrangements Research Articles

Clinical and Molecular Characterization of Adult Glioblastomas in Southern Brazil.

We investigated 113 adult Brazilian patients with glioblastoma (GBM) for comparison with patients from distinct geographical areas and evaluation of suitability for novel targeted therapies. Patients were assessed for clinical features and tumor genomic characteristics such as ROS1 and NTRK1 rearrangements, KIT, PDGFRA, and KDR amplification, and RB1 deletion using multicolor fluorescence in situ hybridization. The majority of patients were male (53%), over 40 years (94%), with tumor located in single site (64%), in the right cerebral hemisphere (60%), and underwent partial resection (71%); 14% presented complications after surgery. The main clinical sign at diagnosis was focal abnormality (57%); frontal (31%); and temporal (20%) regions were most commonly affected. Median hospitalization time was 20 days, median survival was 175 days. One tumor was positive for rearrangement in NTRK1 and another in ROS1 (0.9% each). PDGFRA was amplified in 20% of cases, often co-amplified with KDR (>90%) and KIT (>60%). RB1 was deleted in 16% of patients. There was no association between these molecular abnormalities and patient survival. However, older age, complications after surgery, and right-sided tumors were independent variables associated with patient survival. This study contributes information on the molecular profile of glioblastomas in Latin America possibly supporting new target therapies.

Non‐secretory breast carcinomas lack NTRK rearrangements and TRK protein expression

Anti-TRK targeted therapies offer opportunities to treat patients with advanced NTRK1/2/3-rearranged cancers. Beyond NTRK-rearranged secretory breast carcinomas, little is known about NTRK rearrangements and the expression of TRK proteins in non-secretory breast carcinomas. We search for TRK proteins expressions using pan-TRK immunohistochemistry and NTRK1, NTRK2 and NTRK3 rearrangements using fluorescent in situ hybridization (FISH) tests in a set of tissue microarray included breast carcinomas. Only 1/339 invasive breast carcinomas, the only example of secretory subtype, was positive using pan-TRK immunohistochemistry and harboured a NTRK-rearrangement (NTRK1 positive FISH test). According to our results, druggable NTRK rearrangements and related-TRK proteins expression are not encountered in non-secretory breast carcinomas.

35P - Detection of ALK, RET, ROS1, NTRK1 and MET rearrangements and actionable mutations using next generation sequencing in patients with non-small cell lung cancer

35P - Detection of ALK, RET, ROS1, NTRK1 and MET rearrangements and actionable mutations using next generation sequencing in patients with non-small cell lung cancer

NTRK Fusions Define a Novel Uterine Sarcoma Subtype With Features of Fibrosarcoma.

Tropomyosin receptor kinase (Trk) inhibitors have shown high response rates in patients with tumors harboring NTRK fusions. We identified 4 NTRK fusion-positive uterine sarcomas that should be distinguished from leiomyosarcoma and undifferentiated uterine sarcoma. NTRK rearrangements were detected by fluorescence in situ hybridization (FISH) and/or targeted RNA or DNA sequencing in 4 undifferentiated uterine sarcomas with spindle cell morphology. Because of histologic overlap with leiomyosarcoma, TrkA and pan-Trk immunohistochemistry was performed in 97 uterine leiomyosarcomas. NTRK1 and NTRK3 FISH was performed on tumors with TrkA or pan-Trk staining. We also performed whole transcriptome RNA sequencing of a leiomyosarcoma with TrkA expression and targeted RNA sequencing of 2 additional undifferentiated uterine sarcomas. FISH and/or targeted RNA or DNA sequencing in the study group showed TPM3-NTRK1, LMNA-NTRK1, RBPMS-NTRK3, and TPR-NTRK1 fusions. All tumors were composed of fascicles of spindle cells. Mitotic index was 7 to 30 mitotic figures per 10 high power fields; tumor necrosis was seen in 2 tumors. Desmin, estrogen receptor, and progesterone receptor were negative in all tumors, while pan-Trk was expressed in all tumors with concurrent TrkA staining in 3 of them. TrkA and/or pan-Trk staining was also seen in 6 leiomyosarcomas, but these tumors lacked NTRK fusions or alternative isoforms by FISH or whole transcriptome sequencing. No fusions were detected in 2 undifferentiated uterine sarcomas. NTRK fusion-positive uterine spindle cell sarcomas constitute a novel tumor type with features of fibrosarcoma; patients with these tumors may benefit from Trk inhibition. TrkA and pan-Trk expression in leiomyosarcomas is rare and does not correlate with NTRK rearrangement.

NTRK and ALK rearrangements in mesothelioma and lung carcinoid.

e20558Background: Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK result in oncogenic fusion proteins that have been identified in many tumor types, including lung, colorectal, sali...

Abstract B33: CRISPR/Cas9 generation of Ret and Ntrk1 fusion oncogenes and novel in vitro sgRNA screening method

Abstract Chromosomal rearrangements of ALK, RET, ROS1, and NTRK1 collectively represent approximately 10% of oncogenic drivers in lung adenocarcinoma. These rearrangements result in the aberrant expression and constitutive activation of a chimeric fusion kinase, which promotes growth, proliferation and survival of the cancer cell. CRISPR/Cas9 technology can now be employed to generate these chromosomal rearrangements both in vitro and in vivo. sgRNAs can be designed to target the Cas9 endonuclease to introns within the 5′ and 3′ gene partners of the rearrangement. A unique advantage of this approach is that it is compatible with immunocompetent mouse models. We have developed a novel in vitro screening strategy to identify sgRNAs that successfully generated the intended rearrangements. Ba/F3 cells are a murine B-cell cell line whose proliferation is normally dependent on the exogenous addition of the cytokine interleukin 3 (IL3), but can be rendered IL3 independent if an oncogenic alteration is introduced. We hypothesized that IL3-independence in Ba/F3 cells could be used as a method to select for cells that had successfully created chromosomal rearrangements leading to oncogenic fusions. This strategy eliminates the need for single cell cloning to confirm that successful CRISPR genome editing has occurred. We transfected Ba/F3 cells with pairs of sgRNAs within the PX330 Cas9 containing plasmid and then withdrew IL3. Cells that became IL3 independent were tested for rearrangements. Using this screening system, we successfully generated Kif5b-Ret, Trim24-Ret, and Tpm3-Ntrk1 rearrangements in Ba/F3 cells. These rearrangements were confirmed using PCR or RT-PCR and sequencing using fusion-specific primers. Additionally, the Ret rearrangements and Ntrk1 rearrangements were far more sensitive to RET and TRK inhibitors, respectively, than parental Ba/F3 cells, further confirming that the Ba/F3 cells' growth was now dependent on signaling from the fusion kinase. RET protein expression was also detected with Western blotting in the two Ret rearranged Ba/F3 cell lines. Our future plans are to introduce adenovirus containing validated sgRNAs into immune-competent mice, and confirm that they generate tumors with the intended rearrangements. Citation Format: Laura Schubert, Anh T. Le, Stephen P. Malkoski, Raphael Nemenoff, Robert C. Doebele. CRISPR/Cas9 generation of Ret and Ntrk1 fusion oncogenes and novel in vitro sgRNA screening method [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B33.

The Novel Selective Pan-TRK Inhibitor ONO-7579 Exhibits Antitumor Efficacy Against Human Gallbladder Cancer In Vitro.

We previously reported that brain-derived neurotrophic factor (BDNF)/neurotrophic receptor tyrosine kinase 2 (NTRK2/TRKB) signaling contributes to induction of malignant phenotype of gallbladder cancer (GBC). Recently, pan-TRK inhibitors have been evaluated and their dramatic clinical activity is being shown for a variety of cancer types harboring an NTRK rearrangement in phase I trials. ONO-7579 is an oral pan-TRK inhibitor currently under investigation in phase I/II clinical trial for TRK-rearranged solid tumors. In this study, we evaluated the anticancer effect of ONO-7579 using GBC cells with or without KRAS mutant, NOZ, TYGBK-1. Our study showed that ONO-7579 had a suppressive effect on GBC proliferation in TYGBK-1, and on invasive potential and vascular endothelial growth factor expression in TYGBK-1 and NOZ. Our data indicated that ONO-7579 could be a promising treatment option for patients with GBC.

Mechanisms of Resistance to NTRK Inhibitors and Therapeutic Strategies in NTRK1-Rearranged Cancers.

Neurotrophic receptor tyrosine kinase 1 (NTRK1) gene rearrangement leads to constitutive activation of NTRK1, which induces high-transforming ability. NTRK-rearranged cancers have been identified in several cancer types, such as glioblastoma, non-small cell lung cancer, and colorectal cancer. Although there are currently no clinically approved inhibitors that target NTRK1, several tyrosine kinase inhibitors (TKI), such as entrectinib and LOXO-101, are in clinical trials. The purpose of this study was to identify potential mechanisms of resistance to NTRK inhibitors and find potential therapeutic strategies to overcome the resistance. We examined the sensitivity of TPM3-NTRK1-transformed Ba/F3 cells and TPM3-NTRK1-harboring KM12 cells to multiple NTRK inhibitors. Acquired NTRK inhibitor-resistant mutations were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3-TPM3-NTRK1 cells or by the establishment of NTRK-TKI-resistant cells from KM12 cells continuously treated with NTRK-TKIs. We identified multiple novel NTRK-TKI resistance mutations in the NTRK1 kinase domain, including G595R, and insulin growth factor receptor type 1 (IGF1R) bypass pathway-mediated resistance. After identifying the resistance mechanisms, we performed drug screening with small-molecule inhibitors to overcome the resistance. As a result, we found that ponatinib and nintedanib effectively inhibited the survival of TPM3-NTRK1-G667C but not G595R mutants, both of which showed resistance to entrectinib or larotrectinib (LOXO-101). Furthermore, cabozantinib with an IGF1R inhibitor such as OSI-906 could overcome bypass pathway-mediated resistance. We developed a comprehensive model of acquired resistance to NTRK inhibitors in cancer with NTRK1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance. Mol Cancer Ther; 16(10); 2130-43. ©2017 AACR.

Beyond ALK and ROS1: RET, NTRK, EGFR and BRAF gene rearrangements in non-small cell lung cancer.

The discovery of gene rearrangements involving the receptor tyrosine kinase genes ALK and ROS1 has revolutionized management of the subset of non-small cell lung cancers characterized by these alterations. The oncogenic fusion proteins expressed in these tumors drive cancer cell growth and survival, and targeted inhibition of this signaling can lead to dramatic and durable responses in patients. While the best characterized gene fusions in non-small cell lung cancer (NSCLC) involve ALK and ROS1, fusions involving other kinases including RET, NTRK, EGFR and BRAF are now established as additional targetable drivers. Here we review data supporting the roles of these fusions as oncogenic drivers, and the potential for targeting these fusions for improved clinical outcomes. These discoveries should encourage multiplexed molecular profiling of lung cancers using next-generation platforms which identify these gene fusions in order to expand treatment options for patients.

433PD - Comprehensive genomic profiling of metastatic and relapsed thyroid gland carcinomas is associated with tumor type and reveals new routes to targeted therapies

Background: We queried whether CGP could differentiate the mTC subtypes of papillary (PTC), follicular (FTC), medullary (MTC) and anaplastic (ATC) carcinomas and their potential responses to targeted and immunotherapies. Methods: CGP was performed on FFPE samples using hybridization-captured, adaptor ligation based libraries to a mean coverage depth of > 500X for up to 315 cancer-related genes. Total mutational burden (TMB) was determined on 1.1 megabases of sequenced DNA as previously described (PMID: 28420421). Results: 778 clinically advanced mTC were studied. Median age across the subtypes was similar with male patients slightly more frequent than females except for PTC. ATC had the highest median GA/sample at 4.34. BRAF was a target option for 73% of PTC and 39% of ATC, and RET for MTC in 81% of cases. Oncogenic rearrangements of RET, BRAF, ALK, and NTRK1 were detected in 8%, 3%, 1%, and 1% of PTC cases, respectively, and in 1%, 1%, 0%, and 1% of ATC cases, respectively, but not detected in any cases of FTC or MTC. At 66%, TP53 GA were most frequent in ATC. TERT GA were 58-67% in PTC, FTC and ATC and 0% in MTC. TMB was extremely low for all 4 mTC tumor types with only 3 mTC (<1%) having ≥ 20 mut/Mb. Examples of mTC with responses to targeted therapies will be presented.Table433PDPTCFTCMTCATCNo. Patients40877113180Median age (years)59605464Gender (F/M)215/19337/4046/6788/92GA/tumor2.753.011.964.34Significant genes alteredBRAF TERT TP53 RETTERT NRAS TP53 PTEN HRASRET VHL MEN1 HRAS CCND1TP53 TERT BRAF NRAS PIK3CA NF1 NF2 PTENTP53 GA Frequency11%16%2%66%RET GA Frequency9%0%81%2%hTERT Frequency58%67%1%61%BRAF GA Frequency73%7%0%39%BRAF, RET, ALK, or NTRK rearrangemens13%0%0%3%Total Mutational Burden ≥10 mut/Mb2%1%2%3%Opportunity for Targeted TherapiesHigh (BRAF, oncogenic fusions)LowHigh (RET)Moderate (BRAF, oncogenic fusions) Open table in a new tab Conclusions: Refractory mTC patients are generally older than patients with classic localized primary PTC, and feature relatively more males. Advanced stage PTC, and to a lesser extent ATC, is frequently driven by BRAF GA or oncogenic rearrangements. Relapsed MTC is nearly universally driven by RET GA, whereas FTC has no dominant driver GA identified. TMB appears to be low for all subtypes of MTC, suggesting low potential for immunotherapies. Legal entity responsible for the study: Jeffrey S. Ross Funding: None Disclosure: J.S. Ross: Employee, leader and stock owner in Foundation Medicine. L.M. Gay, P. Vanden Borre, N. Almog, A.B. Schrock, J-A. Vergilio, J. Suh, S. Ramkissoon, E. Severson, S. Daniel, S.M. Ali, J.A. Elvin: Employee and stock owner in Foundation Medicine. V.A. Miller, P.J. Stephens: Employee, leader stock ower in Foundation Medicine. All other authors have declared no conflicts of interest.

Identification and characterization of a novel SCYL3-NTRK1 rearrangement in a colorectal cancer patient.

In colorectal cancer patients, chromosomal rearrangements involving NTRK1 gene (encoding the TRKA protein) are shown in a small subset of patients and are associated with the constitutive activation of the kinase domain of TRKA. In turn, activated TRKA-fusion proteins are associated with proliferation and survival in colorectal cancer tumors.Here we report the identification and functional characterization of a new SCYL3-NTRK1 fusion gene in a 61-year-old colorectal cancer patient. To our knowledge, this fusion protein has never been previously documented in oncological patients. We show that this novel fusion is oncogenic and sensitive to TRKA inhibitors.As suggested by other pieces of evidence, entrectinib - an orally available pan-TRK, ROS1 and ALK inhibitor - may have particular efficacy in patients with NTRK rearrangements. Therefore, screening for rearrangements involving NTRK genes may help identifying a subset of patients able to derive benefit from treatment with entrectinib or other targeted inhibitors.

Abstract LB-123: Identification of the molecular mechanisms of resistance to NTRK inhibitors and the potential therapeutic strategies in NTRK1-rearranged cancers

Abstract Neurotrophic receptor tyrosine kinase 1 (NTRK1) gene rearrangement leads to constitutive activation of NTRK1, which induces high transforming ability. NTRK-rearranged cancers have been identified in several cancer types, such as glioblastoma, non-small-cell lung cancer, and colorectal cancer. Although there are currently no clinically available inhibitors that target NTRK1, several tyrosine kinase inhibitors (TKIs), such as entrectinib and LOXO-101, are in clinical trials. To identify potential mechanisms of resistance to NTRK inhibitors and find potential therapeutic strategies to overcome the resistance, we examined the sensitivity of TPM3-NTRK1-transformed Ba/F3 cells and TPM3-NTRK1-harboring KM12 cells to multiple NTRK inhibitors. Then, acquired NTRK inhibitor-resistant mutations were screened by N-ethyl-N-nitrosourea (ENU) mutagenesis with Ba/F3-TPM3-NTRK1 cells or by the establishment of NTRK-TKI-resistant cells from KM12 cells continuously treated with NTRK-TKIs. After identifying the resistance mechanisms, we performed drug screening with small-molecular inhibitors to overcome the resistance. As the results, we identified multiple novel NTRK-TKI resistance mutations in the NTRK1 kinase domain, including G595R. As a result of drug screening, we found that cabozantinib and a few TKIs effectively inhibited the survival of TPM3-NTRK1-G667C but not G595R mutants, both of which showed resistance to entrectinib or LOXO101. Furthermore, cabozantinib with an insulin growth factor receptor type 1 (IGF1R) inhibitor such as OSI906 could overcome bypass pathway-mediated resistance. Thus, in this study, we developed a comprehensive model of acquired resistance to NTRK inhibitors in cancer with NTRK1 rearrangement. In addition, we found drugs and a combination therapy to overcome the identified resistance to NTRK1-TKI. Citation Format: Ryohei Katayama, Miho J. Fuse, Tomoko Oh-hara, Hayato Ogura, Naoya Fujita. Identification of the molecular mechanisms of resistance to NTRK inhibitors and the potential therapeutic strategies in NTRK1-rearranged cancers [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 LB-123. doi:10.1158/1538-7445.AM2017-LB-123

ALK, ROS1, and NTRK Rearrangements in Metastatic Colorectal Cancer.

ALK, ROS1, and NTRK fusions occur in 0.2% to 2.4% of colorectal cancers. Pioneer cases of metastatic colorectal cancer (mCRC) patients bearing rearrangements who benefited from anti-ALK, ROS, and TrkA-B-C therapies have been reported previously. Here we aimed at characterizing the clinical and molecular landscape of ALK, ROS1, and NTRK rearranged mCRC. Clinical features and molecular characteristics of 27 mCRC patients bearing ALK, ROS1, and NTRK rearranged tumors were compared with those of a cohort of 319 patients not bearing rearrangements by means of Fisher's exact, χ2 test, or Mann-Whitney test as appropriate. Overall survival curves were estimated with the Kaplan-Meier method and compared using the log-rank test. A Cox proportional hazard model was adopted in the multivariable analysis. Deep molecular and immunophenotypic characterizations of rearranged cases, including those described in The Cancer Genome Atlas database, were performed. All statistical tests were two-sided. Closely recalling the "BRAF history," ALK, ROS1, and NTRK rearrangements more frequently occurred in elderly patients (P = .02) with right-sided tumors (P < .001) and node-spreading (P = .03), RAS wild-type (P < .001), and MSI-high (P < .001) cancers. All patients bearing ALK, ROS1, and NTRK fusions had shorter overall survival (15.6 months, 95% confidence interval [CI] = 0.0 to 20.4 months) than negative patients (33.7 months, 95% CI = 28.3 to 42.1 months), both in the univariate (hazard ratio [HR] = 2.17, 95% CI = 1.03 to 4.57, P < .001) and multivariable models (HR = 2.33, 95% CI = 1.10 to 4.95, P = .02). All four evaluable patients with rearrangements showed primary resistance to anti-epidermal growth factor receptor agents. Frequent association with potentially targetable RNF43 mutations was observed in MSI-high rearranged tumors. ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.

Targeting NTRK fusion in non-small cell lung cancer: rationale and clinical evidence.

In the era of personalized medicine, the identification of targetable genetic alterations represented a major step forward in anticancer therapy. NTRK rearrangements represent the molecular driver of a subset of solid tumors, including 3% of non-small-cell lung cancers (NSCLCs). Preliminary data indicate that molecularly selected NSCLC patients harboring NTRK fusions derive an unprecedented clinical benefit from Trk-directed targeted therapies. The aim of this review is to describe the molecular biology of NTRK signaling pathway and to summarize the preclinical data on novel Trk inhibitors, touching upon the clinical development of these inhibitors for the treatment of advanced NSCLC, which have already shown encouraging anticancer activity and acceptable safety profile in early phase I clinical trials.

Identification of NTRK3 Fusions in Childhood Melanocytic Neoplasms

Spitzoid neoplasms are a distinct group of melanocytic tumors. Genetically, they lack mutations in common melanoma-associated oncogenes. Recent studies have shown that spitzoid tumors may contain a variety of kinase fusions, including ROS1, NTRK1, ALK, BRAF, and RET fusions. We report herein the discovery of recurrent NTRK3 gene rearrangements in childhood melanocytic neoplasms with spitzoid and/or atypical features, based on genome-wide copy number analysis by single-nucleotide polymorphism array, which showed intragenic copy number changes in NTRK3. Break-apart fluorescence in situ hybridization confirmed the presence of NTRK3 rearrangement, and a novel MYO5A-NTRK3 transcript, representing an in-frame fusion of MYO5A exon 32 to NTRK3 exon 12, was identified using a rapid amplification of cDNA ends-based anchored multiplex PCR assay followed by next-generation sequencing. The predicted MYO5A-NTRK3 fusion protein consists of several N-terminal coiled-coil protein dimerization motifs encoded by MYO5A and C-terminal tyrosine kinase domain encoded by NTRK3, which is consistent with the prototypical structure of TRK oncogenic fusions. Our study also demonstrates how array-based copy number analysis can be useful in discovering gene fusions associated with unbalanced genomic aberrations flanking the fusion points. Our findings add another potentially targetable kinase fusion to the list of oncogenic fusions in melanocytic tumors.

SC05.03 Novel Tyrosine Kinase Inhibitors in Lung Cancer

SC05.03 Novel Tyrosine Kinase Inhibitors in Lung Cancer

Identification of Existing Drugs That Effectively Target NTRK1 and ROS1 Rearrangements in Lung Cancer.

Efforts to discover drugs that overcome resistance to targeted therapies in patients with rare oncogenic alterations, such as NTRK1 and ROS1 rearrangements, are complicated by the cost and protracted timeline of drug discovery. In an effort to identify inhibitors of NTRK1 and ROS1, which are aberrantly activated in some patients with non-small cell lung cancer (NSCLC), we created and screened a library of existing targeted drugs against Ba/F3 cells transformed with these oncogenes. This screen identified the FDA-approved drug cabozantinib as a potent inhibitor of CD74-ROS1-transformed Ba/F3, including the crizotinib-resistant mutants G2032R and L2026M (IC50 = 9, 26, and 11 nmol/L, respectively). Cabozantinib inhibited CD74-ROS1-transformed Ba/F3 cells more potently than brigatinib (wild-type/G2032R/L2026M IC50 = 30/170/200 nmol/L, respectively), entrectinib (IC50 = 6/2,200/3,500 nmol/L), and PF-06463922 (IC50 = 1/270/2 nmol/L). Cabozantinib inhibited ROS1 autophosphorylation and downstream ERK activation in transformed Ba/F3 cells and in patient-derived tumor cell lines. The IGF-1R inhibitor BMS-536924 potently inhibited CD74-NTRK1-transformed compared with parental Ba/F3 cells (IC50 = 19 nmol/L vs. > 470 nmol/L). A patient with metastatic ROS1-rearranged NSCLC with progression on crizotinib was treated with cabozantinib and experienced a partial response. While acquired resistance to targeted therapies is challenging, this study highlights that existing agents may be repurposed to overcome drug resistance and identifies cabozantinib as a promising treatment of ROS1-rearranged NSCLC after progression on crizotinib. Clin Cancer Res; 23(1); 204-13. ©2016 AACR.

PUB007 Novel TKIs in Non-Small Cell Lung Cancer

The presence of EGFR activating mutations and ALK chromosomic rearrangements with corresponding tyrosine kinase inhibitors (TKIs) has revolutionized the treatment strategies of patients with non-small cell lung cancer (NSCLC). Although tremendously initial response and manageable toxicity profiles, acquired resistance inevitably develops after approximately 1 year treatment with EGFR-TKIs (erlotinib and gefitinib) and ALK inhibitor (crizotinib). Encouragingly, third-generation EGFR-TKIs including AZD9291, CO1686 and brigatinib have showed striking efficacy overcoming acquired resistance driven by T790M secondary mutations. Other novel third-generation EGFR-TKIs such as ASP8274, EGF816, PF-06747775 and avitinib are also being investigated in early-stage clinical trials and the survival and safety data will be released in the near future. Another promising novel EGFR-TKI, namely AZD3759 has showed promising response in patients with brain metastases and leptomeningeal disease. Recently, EAI045, an EGFR allosteric inhibitor, in combination with cetuxmab exhibit antitumor activity in mouse models of lung cancer driven by L858R/T790M/C797S, a resistant mutation of AZD9291. Meanwhile, second-generation ALK inhibitors (ceritinib and alectinib) have entered clinical applications for NSCLC patients with ALK rearrangements after failure of crizotinib and third-generation ALK inhibitors (lorlatinib and ASP3026) are also being evaluated in clinical trials overcoming known ALK resistant mutations. On the other hand, with the development of high-throughput sequencing, called next-generation sequencing (NGS) and genomic technologies, more novel molecular targets such as MET 14 exon skipping splicing mutations have been identified as potential therapeutic targets and simultaneously analyzing hundreds of molecular alterations have turned out reality with limited tumor tissues. In the recent years, the emergence of numbers of oncogenic drivers other than EGFR mutations and ALK rearrangements has divided NSCLC into multiple distinct subtypes amenable to corresponding targeted therapy. For instance, dabrafenib either as monotherapy or in combination with MEK inhibitor (trametinib) has displayed promising antitumor activity and manageable safety profile in patients with BRAF V600E mutations, accounting for around 1.4% of patients with NSCLC. Other novel molecular targets maybe serving as oncogenic drivers including mutations in HER2 (neratinib and pyrotinib) and PI3KCA (BKM120 and GDC0941), ROS1 (entrectinib, foretinib and lorlatinib), RET (XL184) and NTRK (entrectinib) rearrangements and FGFR1 gene amplification (AZD4547, Lenvatinib and FP-1039) are being evaluated either in preclinical settings or early-stage clinical trials. Section not applicable Section not applicable Section not applicable

Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing.

Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens.

Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer.

Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors. We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements.