NTRK1 Rearrangements Research Articles

Clinicopathologic and molecular characterization of NTRK-rearranged thyroid carcinoma (NRTC)

Primary thyroid neoplasms with actionable NTRK rearrangements are rare, and their clinical behavior, histologic characteristics, and molecular landscape are not well understood. We report an institutional series of eleven NTRK-rearranged thyroid carcinomas (NRTC) by performing clinicopathologic review and next-generation sequencing for targeted mutations and gene rearrangements. The NRTC encompass a histomorphologic spectrum of ten papillary thyroid carcinomas (PTC), including one with high-grade features, and one secretory carcinoma (SC), in ten adults and one adolescent. All NRTC were characterized by an unusual multinodular growth pattern, extensive lymphovascular invasion, and cervical lymph node metastases at initial presentation. Immunophenotypically, while most cases were positive for TTF1 and PAX8, the SC case was negative/weak for these markers and instead diffusely expressed GATA3, mammaglobin and S100. Observed gene rearrangements included ETV6-NTRK3 (n = 4, including the SC), TPR-NTRK1 (n = 2), RBPMS-NTRK3 (n = 2), SQSTM1-NTRK1 (n = 1), SQSTM1-NTRK3 (n = 1), and EML4-NTRK3 (n = 1). Mutation profiling revealed a concurrent TERT promotor mutation C228T in two (22%) patients and a novel frameshift MEN1 deletion in one. All patients received total thyroidectomy and radioactive iodine. Despite frequent development of persistent/recurrent disease (9 cases, 82%) and distant metastases (6 cases; 55%), no tumor-related death occurred over a median (range) follow-up of 44 (11 to 471) months. Three patients received NTRK inhibitor therapy, with the SC case showing complete resolution and two other patients experiencing 33% and 69.7% decrease of disease burden. Although the range of features is variable, NRTC appear to be clinically aggressive tumors with high metastatic rate but relatively low mortality with NTRK inhibitor therapy. The histologic findings of multinodular growth and extensive lymphovascular spread, seen in all NRTC, including PTC and SC, may serve as useful histomorphologic clues to prompt NTRK status testing. We also present the first report of concurrent TERT promotor activating mutation which did not appear to confer entrectinib resistance to NRTC.

Pan-TRK Immunohistochemistry: An Example-Based Practical Approach to Efficiently Identify Patients With NTRK Fusion Cancer.

Food and Drug Administration-approved TRK inhibitors with impressive overall response rates are now available for patients with multiple cancer types that harbor NTRK rearrangements, yet the identification of NTRK fusions remains a difficult challenge. These alterations are highly recurrent in extremely rare malignancies or can be detected in exceedingly small subsets of common tumor types. A 2-step approach has been proposed, involving a screening by immunohistochemistry (IHC) followed by a confirmatory method (fluorescence in situ hybridization, reverse transcriptase-polymerase chain reaction, or next-generation sequencing) in cases expressing the protein. However, there is no interpretation guide for any of the available IHC clones. To provide a pragmatic update on the use of pan-TRK IHC. Selected examples of the different IHC staining patterns across multiple histologies are shown. Primary literature review with PubMed, combined with personal diagnostic and research experience. In-depth knowledge of pan-TRK IHC will help pathologists implement a rational approach to the detection of NTRK fusions in human malignancies.

Clinicopathological features and immunohistochemical utility of NTRK-, ALK-, and ROS1-rearranged papillary thyroid carcinomas and anaplastic thyroid carcinomas

NTRK1/3, ALK, and ROS1 translocations have been reported in a minor subset of papillary thyroid carcinomas (PTCs). We aimed to elucidate the prevalence and clinicopathological characteristics of these gene rearrangements and the utility of immunohistochemistry (IHC) in PTC and anaplastic thyroid carcinoma (ATC). We screened nonradiation-exposed cases of 307PTCs and 16 ATCs by IHC for pan-Trk, ALK, and ROS1, followed by fluorescence in situ hybridization (FISH). In the PTC group, IHC for pan-Trk, ALK, and ROS1 was positive in 18 cases (5.9%), 1 case (0.3%), and 12 cases (3.9%), respectively. Among the pan-Trk IHC-positive cases (n=18), 2 cases (11.1%; 0.7% of all PTCs) had NTRK1 or NTRK3 gene rearrangement with conventional PTC histology. The ALK IHC-positive case (n=1) was the follicular variant of PTC with consistent ALK gene rearrangement. ROS1 gene rearrangement was not detectable in the ROS1 IHC-positive PTCs (0/12) by FISH. Most (approximately 70%) of the pan-Trk or ROS1 IHC-positive/FISH-negative cases had BRAF gene mutation with conventional PTC morphology. In the ATC group, neither ALK nor ROS1 IHC was positive, whereas pan-Trk IHC was positive in 1 case (6.3%) in which NTRK1 gene rearrangement was confirmed by FISH. These results suggest that NTRK, ALK, and ROS1 rearrangements are rare molecular events in nonradiation-exposed Japanese patients with PTC and ATC. Although IHC is not an entirely specific surrogate for these abnormalities and does not serve as a stand-alone companion diagnosis, the combined use of IHC and molecular testing may be helpful for determining promising therapeutic strategies with tyrosine kinase inhibitors.

Fine-needle aspiration cytomorphology of papillary thyroid carcinoma with NTRK gene rearrangement from a case series with predominantly indeterminate cytology.

Neurotrophic tyrosine kinase receptor (NTRK) rearrangement has been reported in a subset of papillary thyroid carcinoma (PTC) cases. Little is known about the cytomorphologic features of NTRK-rearranged PTC. We report an institutional series of 13 fine-needle aspiration (FNA) specimens of NTRK-rearranged PTC with a predominantly indeterminate cytology diagnosis. NTRK3 or NTRK1 rearrangements were detected on FNA or surgical specimens by next-generation sequencing. The 13 patients had a median age of 18 years; 10 patients were female and 3 patients were male. In 10 (77%) cases, cytology was indeterminate, and histopathologic follow-up was predominantly the follicular variant of PTC (n=8 [62%]), mostly infiltrative subtype. Of 12 FNA specimens available for review, a predominant loosely cohesive group pattern was the most commonly encountered architectural pattern (n=5 [41%]), followed by single cell (n=3 [25%]), thick cord (n=2 [17%]), and microfollicular pattern (n=2 [17%]). Background lymphocytic thyroiditis was observed in 9 cases. At the cellular level, the cytoplasm was moderate and granular, occasionally vacuolated. Classic PTC nuclear features (eg, nuclear enlargement, elongation, grooves, and nuclear membrane irregularity) were present but were often focal and subtle. Chromatin was often granular. Intranuclear pseudoinclusions were absent or rare. Our study demonstrates that most cases of NTRK rearrangement lack classic PTC cytomorphologic characteristics. Loosely cohesive groups and single cells with granular, sometimes vacuolated cytoplasm and subtle nuclear features are often seen on FNA specimens. Recognizing these characteristics may be helpful to preoperatively prompt molecular testing, including NTRK rearrangement analysis.

Congenital mesoblastic nephroma is characterised by kinase mutations including EGFR internal tandem duplications, the ETV6-NTRK3 fusion, and the rare KLHL7-BRAF fusion.

Congenital mesoblastic nephroma (CMN) is histologically classified into classic, cellular and mixed subtypes. The aims of this study were to characterise the clinical, pathological and molecular features of a series of CMNs, and to determine the utility of pan-Trk and epidermal growth factor receptor (EGFR) immunohistochemistry as surrogate markers for NTRK gene fusions and EGFR internal tandem duplications (ITDs). Twenty-two archival CMN cases (12 classic, five cellular, and five mixed) were tested for the ETV6-NTRK3 fusion and EGFR ITD transcripts by the use of reverse transcriptase polymerase chain reaction (PCR), and next-generation sequencing-based anchored multiplex PCR. All 12 classic CMNs had EGFR ITD. Of the five cellular CMNs, four had the ETV6-NTRK3 fusion and one had the KLHL7-BRAF fusion. Of the five mixed CMNs, four had EGFR ITD, and one had the ETV6-NTRK3 fusion. Pan-Trk immunoreactivity was 100% sensitive and 94.1% specific for the presence of NTRK rearrangement. However, EGFR staining was only 62.5% sensitive and 33.3% specific for EGFR ITD. EGFR ITD is a consistent genetic event in classic CMN. A majority of cellular CMNs have the ETV6-NTRK3 fusion. Rare cellular CMNs may harbour non-canonical mutations such as the KLHL7-BRAF fusion, which was found in one case. Mixed CMNs may have either EGFR ITD or the ETV6-NTRK3 fusion. Pan-Trk immunohistochemistry is a sensitive, albeit not perfectly specific, marker for NTRK rearrangement. EGFR immunohistochemistry is not helpful as a marker of EGFR ITD.

Detection of NTRK1/3 Rearrangements in Papillary Thyroid Carcinoma Using Immunohistochemistry, Fluorescent In Situ Hybridization, and Next-Generation Sequencing.

NTRK1/3 rearrangements have been reported in 2.3-3.4% of papillary thyroid carcinoma (PTC) and are regarded as potential therapeutic targets. Recently, the application of immunohistochemistry (IHC) to detect NTRK rearrangements has been widely discussed. The current study aimed to characterize the clinicopathological features of PTC with NTRK1/3 fusions, to examine the utility of pan-TRK IHC, and to compare IHC with fluorescent in situ hybridization (FISH) and next-generation sequencing (NGS). In a cohort of 525 consecutive PTC cases, 60 BRAFV600E-negative cases underwent complete analyses of FISH, and 12 (2.3%) cases with NTRK1/3 break-apart were found. A novel ERC1-NTRK3 fusion was identified by NGS in one case. Pathological features of non-infiltrative tumor border, clear cell change, and reduced nuclear elongation and irregularity were significantly more common in NTRK1/3-rearranged PTC when compared with 48 BRAFV600E-negative non-NTRK1/3 PTC cases. In whole tissue sections, pan-TRK IHC was positive in 3/7 (42.9%) cases with an ETV6-NTRK3 rearrangement including 2 cases with low percentage of stained tumor cells, 2/3 (66.7%) with non-ETV6 NTRK3 rearrangements, and 2/2 (100%) with NTRK1 rearrangements. All FISH-negative cases were negative for pan-TRK in tissue microarray sections. As a result, pan-TRK IHC showed a sensitivity of 58.3% and specificity of 100% for NTRK1/3 rearrangements in BRAFV600E-negative PTC. In conclusion, NTRK1/3-rearranged PTC shared some unique morphologic features. Pan-TRK IHC showed high specificity and moderate sensitivity for NTRK1/3-rearranged PTC and should be interpreted with caution due to staining heterogeneity. Based on the above findings, we propose an algorithm integrating morphology, IHC, and molecular testing to detect NTRK1/3 rearrangements in PTC.

Abstract 177: RNA sequencing based gene fusion detection with oncomine comprehensive assay plus

Abstract Introduction Detection of oncogenic fusions has been of great importance for understanding tumorigenesis and for precision oncology in enhancing diagnosis and selection of targeted therapies. Herein, we describe an extended Oncomine targeted RNA sequencing assay for detection of fusion transcripts and intragenic rearrangements (exon deletion/skipping). For multiple key driver genes we also supplemented the panel with a complementary transcript-based expression imbalance assay designed to identify gene fusions in a partner agnostic manner. Methods Based on evidence from Oncomine™ Knowledgebase and collaboration with leading oncology researchers, we designed an Ion AmpliSeq™ panel to target > 1,200 fusion breakpoints in > 50 driver genes, > 40 intragenic rearrangements (e.g., MET exon 14 skipping, ARv7, EGFRvIII) in 7 genes, and 5 RNA expression controls. In addition, the panel supports detection and reporting of non-targeted fusions (i.e., novel combinations of drivers and partners). We supplemented the panel with exon tiling expression imbalance assays, using amplicons tiling the exon junctions of ALK, RET, NTRK1, NTRK2 and NTRK3 to measure 3'/5' expression imbalance signatures. We developed a bioinformatic tool to call fusions from a normalized and corrected expression imbalance profile per gene (using a baseline from normal formaldehyde fixed paraffin embedded [FFPE samples]). We optimized the gene fusion algorithms and integrated them as workflows into the Ion ReporterTM Software to facilitate the summary of the results with relevant annotations, rich data visualizations and easily interpretable reports. Results We sequenced hundreds of positive and negative fusion samples including commercial reference standards, cell lines and FFPE clinical research samples on the Ion GeneStudioTM S5 sequencer. To assess the feasibility of the combined panel, we sequenced the Seraseq® FFPE tumor fusion RNA reference, 7 fusion positive cell lines with ALK, RET, ROS1, NTRK1, FGFR1, FGFR2 and FGFR3 rearrangements, and cohorts of FFPE samples using 20ng RNA as input and successfully detected the expected fusion isoforms or other RNA rearrangements in each sample. We applied the exon tiling fusion detection method for ALK, RET and NTRK1 and observed perfect concordance between the true isoform in the positive samples and the predicted breakpoint position and magnitude of 3'/5' expression imbalance indicated by the exon tiling method. Conclusions We developed an extended, multiplexed RNA panel for fusions and intragenic rearrangements that retains the simple workflow and fast turn-around time of previous Oncomine fusion panels and significantly expands the scope of fusion isoform detection including methods to detect gene fusions in a partner agnostic manner. For research use only. Not for use in diagnostic procedures. Citation Format: Amir Marcovitz, Rajesh K. Gottimukkala, Gary G. Bee, Jennifer M. Kilzer, Vinay K. Mital, Elain Wong-Ho, Chenchen Yang, Yu-Ting Tseng, Scott P. Myrand, Paul D. Williams, Seth Sadis, Fiona C. Hyland. RNA sequencing based gene fusion detection with oncomine comprehensive assay plus [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 177.

Evaluating entrectinib as a treatment option for non-small cell lung cancer

ABSTRACT Introduction Entrectinib, an oral pan-TRK, ALK, and ROS1 inhibitor is approved as a first-line treatment for NTRK-rearranged solid tumors and ROS1-rearranged non-small cell lung cancer (NSCLC). It has demonstrated clinical efficacy for patients harboring the relevant gene rearrangement in both systemic and intracranial disease, regardless of the tumor type. Areas covered In this review, the authors analyzed data from preclinical and clinical studies, the characteristics of entrectinib compared to those of other relevant inhibitors (currently available and/or under investigation), and the emerging resistance mechanisms. The authors then provide the readers with their future perspectives. Expert opinion Entrectinib has been well studied across many tumor types, including NSCLC with ALK, ROS1, and NTRK rearrangements. The drug has demonstrated favorable properties with oral administration, prolonged response duration, high intracranial efficacy, and a favorable toxicity profile. However, with acquisition of resistance and the development of newer generation TKIs, the optimal place for entrectinib in the landscape of targeted therapies for NSCLC warrants further validation.

Soft tissue tumors characterized by a wide spectrum of kinase fusions share a lipofibromatosis-like neural tumor pattern.

Gene fusions resulting in oncogenic activation of various receptor tyrosine kinases, including NTRK1-3, ALK, and RET, have been increasingly recognized in soft tissue tumors (STTs), displaying a wide morphologic spectrum and therefore diagnostically challenging. A subset of STT with NTRK1 rearrangements were recently defined as lipofibromatosis-like neural tumors (LPFNTs), being characterized by mildly atypical spindle cells with a highly infiltrative growth in the subcutis and expression of S100 and CD34 immunostains. Other emerging morphologic phenotypes associated with kinase fusions include infantile/adult fibrosarcoma and malignant peripheral nerve sheath tumor-like patterns. In this study, a large cohort of 73 STT positive for various kinase fusions, including 44 previously published cases, was investigated for the presence of an LPFNT phenotype, to better define the incidence of this distinctive morphologic pattern and its relationship with various gene fusions. Surprisingly, half (36/73) of STT with kinase fusions showed at least a focal LPFNT component defined as >10%. Most of the tumors occurred in the subcutaneous tissues of the extremities (n = 25) and trunk (n = 9) of children or young adults (<30 years old) of both genders. Two-thirds (24/36) of these cases showed hybrid morphologies with alternating LPFNT and solid areas of monomorphic spindle to ovoid tumor cells with fascicular or haphazard arrangement, while one-third (12/36) had pure LPFNT morphology. Other common histologic findings included lymphocytic infiltrates, staghorn-like vessels, and perivascular or stromal hyalinization, especially in hybrid cases. Mitotic activity was generally low (<4/10 high power fields in 81% cases), being increased only in a minority of cases. Immunoreactivity for CD34 (92% in hybrid cases, 89% in pure cases) and S100 (89% in hybrid cases, 64% in pure cases) were commonly present. The gene rearrangements most commonly involved NTRK1 (75%), followed by RET (8%) and less commonly NTRK2, NTRK3, ROS1, ALK, and MET.

NTRK and ALK rearrangements in malignant pleural mesothelioma, pulmonary neuroendocrine tumours and non-small cell lung cancer

ObjectivesGene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK have been identified in many types of cancer, including non-small cell lung cancer (NSCLC). Data in malignant pleural mesothelioma (MPM), lung neuroendocrine tumors (NETs) and small-cell lung cancer (SCLC) are lacking. Given the activity of NTRK, ROS-1 and ALK inhibitors in tumors harboring gene fusions, we sought to explore such rearrangements in these less common tumors in addition to NSCLC. MethodsArchival tumor tissue from patients with MPM, lung NETs, SCLC and NSCLC were used to create tissue microarrays. Immunohistochemistry (IHC) was performed using a cocktail of antibodies against TRK, ROS1 and ALK. IHC positive samples underwent RNA sequencing using the ArcherDX FusionPlex CTL diagnostic assay. Clinical data were obtained through retrospective chart review. ResultsWe performed IHC on 1116 samples: 335 MPMs, 522 NSCLCs, 105 SCLCs and 154 lung NETs. There were 23 IHC positive cases (2.1%) including eight MPMs (2.4%), eight NETs (5.2%), five SCLC (4.8%) and two NSCLC (0.4%).The following fusions were detected: one MPM with an NTRK ex10-TPM3 ex8, another MPM with an ALK ex20-EML4ex13, one lung intermediate-grade NET (atypical carcinoid) with an ALK ex20-EML4 ex6/intron6, and two NSCLCs with an ALK ex20-EML4 ex6/intron6 rearrangement. None of the patients received targeted treatment. ConclusionsTo our knowledge, we report for the first time NTRK and ALK rearrangements in a small subset of MPM. An ALK rearrangement was also detected in lung intermediate-grade NET (or atypical carcinoid). Our data suggest that IHC could be a useful screening test in such patients to ensure that all therapeutic strategies including targeted therapy are utilized.

The impact of next generation sequencing on sarcoma diagnosis.

e23528 Background: The rarity and heterogeneity of sarcoma has been complicating the diagnosis of sarcoma for years. Even expert pathologists of sarcoma could make mistakes in the diagnosis of this disease. The availability of Next Generation Sequencing (NGS) data enabled more accurate diagnosis of sarcoma. In this study, we systematically described the application of NGS on the diagnosis of sarcoma and the contribution of NGS to the diagnostic accuracy of sarcoma. Methods: A multi-center, retrospective study included 235 sarcoma patients’ tumor and paired normal samples that were sent from 56 hospitals to a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory, at Shanghai, China for Next Generation Sequencing (NGS) was performed. Using next generation sequencing based YS panel consisting 450 genes, these 235 sarcoma patients’ sample were sequenced and the NGS data was analyzed. The initial diagnosis without NGS information was reconsidered by expert pathologists. Results: Taking into consideration both the initial diagnosis and the NGS results, the final diagnosis of these 235 sarcoma cases included 8 low grade malignant fibromyxoid tumors, 11 dermatofibrosarcoma protuberans (DFSP), 38 myxoliposarcomas, 22 alveolar rhabdomyosarcomas, 11 alveolar soft tissue sarcoma, 2 desmoplastic small round cell tumors, 37 NTRK rearrangement spindle cell tumors, 40 Ewing’s sarcoma and 66 synoviosarcomas. In total, 29% initial diagnoses were changed according to NGS identified fusions, including 13% low grade malignant fibromyxoid tumors (1 FUS- CREB3L2 fusion), 27% DFSPs (3 COL1A1- PDGFB fusions), 11% myxoliposarcomas (3 FUS- DDIT3 fusions and 1 EWSR1- DDIT3 fusion), 14% alveolar rhabdomyosarcomas (2 PAX7- FOXO1 fusions and 1 FOXO1- LINC00598 fusion), 18% alveolar soft tissue sarcomas (2 ASPSCR1- TFE3 fusions), 50% desmoplastic small round cell tumor (1 EWSR1- WT1 fusion), 95% NTRK rearrangement spindle cell tumors, 13% Ewing’s sarcomas (3 EWSR1- FLI1 fusions and 2 EWSR1- ERG fusions) and 21% synoviosarcomas (9 SS18- SSX1 fusions and 5 SS18- SSX2 fusions). Conclusions: NGS would be highly recommended for accurate diagnosis of sarcoma, especially for NTRK rearrangement spindle cell tumor, the majority of which were confirmed according to NGS identified fusions.

Uterine mesenchymal tumours: recent advances.

Almost all uterine mesenchymal tumours have been historically classified as either smooth muscle or endometrial stromal neoplasms. Recent application of molecular techniques has identified numerous lesions with distinctive genetic abnormalities and clinicopathological characteristics. Newly discovered uterine sarcoma subtypes include high-grade endometrial stromal sarcomas with BCOR genetic abnormalities, fibrosarcoma-like uterine sarcomas with NTRK rearrangements and COL1A-PDGFRB fusions, as well as undifferentiated uterine sarcomas with SMARCA4 mutations. Novel PLAG1 and PGR fusions have been identified in subsets of myxoid and epithelioid leiomyosarcomas. Some uterine tumours resembling ovarian sex-cord tumour harbour GREB1 and ESR1 rearrangements. Histological and immunophenotypical features as well as underlying genetic abnormalities defining these lesions are discussed.

Mutational analysis of &gt;14,000 NGS tests from community oncology practices: The Sarah Cannon molecular landscape.

26 Background: Utilization of NGS-based testing for patients in community oncology settings continues to grow. A comprehensive analysis of the molecular landscape of &gt; 14,000 patients with commercial tissue-based and plasma-based NGS (tbNGS and pbNGS) testing from Sarah Cannon’s (SC) network of community oncologists between 2012 and 2018 was performed. These data aid clinical trial design/feasibility and identify rare and actionable mutations in the community oncology setting. Methods: Medical Oncologists in the SC network ordered NGS-based molecular profiling for their patients as standard of care. Data from multiple commercial vendors starting in 2012 were harmonized and analyzed. Results: On average, tbNGS (n = 8938) and pbNGS (n = 5419) tests revealed 14 and 4 mutations/sample, respectively. The top 10 mutated genes from both tbNGS and pbNGS - tests are in the table. The most predominant mutation-types detected were SNVs in both tbNGS and pbNGS tests, and gene amplifications were detected in ~40% of tbNGS and ~22% of pbNGS tests. MET amplifications (range: 2 - 40 copies (pbNGS) and 6 - 82 copies (tbNGS)) and ERBB2 amplifications (range: 2.1 - 90.2 copies (pbNGS) and 4 - 293 copies (tbNGS)) were detected in 1.6% (MET-tbNGS) and 4.6% (MET-pbNGS) and 4.2% (ERBB2-tbNGS) and 2.69% (ERBB2-pbNGS) of patients, respectively, and informed clinical trial enrollment. Of the fusions detected, 28 NTRK rearrangements were detected (NTRK1 – 16, NTRK2 – 5, NTRK3 – 7) by tissue-based testing and 3 (NTRK1) fusions were detected by plasma-based tests. Conclusions: These data describe the mutational landscape of NGS tests ordered within community oncology practices. SC’s data infrastructure links NGS-based testing data and clinical data to support real world evidence research, and highlights the need for healthcare IT systems to marry clinical and genomic data for clinical decision support and clinical research. [Table: see text]

1549P - NTRK1-3 genomic fusions in non-small cell lung cancer (NSCLC) determined by comprehensive genomic profiling

BackgroundThe Neurotrophic Tyrosine Kinase genes, NTRK1-3 are extremely rare drivers of a wide variety of malignancies. The recent pan-cancer approval of kinase inhibitor drugs targeting NTRK has sparked interest in the frequency of NTRK rearrangements found in NSCLC. MethodsComprehensive genomic profiling (CGP) was performed on FFPE samples from 42,791 NSCLC cases. Tumor mutational burden (TMB) was determined on 0.8-1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 loci. Results58 (0.1%) of the NSCLC featured genomic fusions or rearrangements in the NTRK1-3 genes (Table). The ages and genders of NTRK1-3 fusion+ cases were similar to NSCLC in general and did not enrich for younger patients. NTRK fusions were highest in adenocarcinomas (67%) and squamous cell carcinomas (SCC; 9%). The average genomic alterations (GA) per NTRK1-3 altered tumor ranged from 6.1 – 8.2 GA and was higher than that reported for EGFR, ALK and ROS1 driven NSCLC. The NTRK1-3 fusions involved a wide variety of fusion partners with NTRK1- IRF2BP2 (6) and NTRK1-TPM3 (9) most frequent. The levels of TMB and frequencies of PD-L1 expression were higher than EGFR, ALK and ROS1 altered cases. The STK11 GA frequencies were similar to NSCLC in general but lower than the frequency in lung adenocarcinoma only.Table1549PTableNTRK1NTRK2NTRK3Cases4756Median age/Range64 (22-88)68 (65-76)71 (63-74)Gender m/f23/243/25/1NSCLC Types Adenocarcinoma NSCLC NOS Large Cell Neuroendocrine SCC Sarcomatoid Large Cell31 10 2 2 1 14 14 2GA/tumor6.18.26.2TP5355%80%67%KRAS15%0%17%STK1111%20%0%MSI High0%0%0%Median TMB mut/Mb3.511.314.4TMB >10/20 mut/Mb22%/7%60%/20%67%/33%PD-L1 Expression low/high32%/16% (n=19)50%/50% (n=2)0%/33% (n=3) ConclusionsAlthough the addition of RNA sequencing may marginally increase their detection, based on this DNA-only CGP sequencing study, NTRK1-3 fusions are extremely rare in NSCLC. These NTRK1-3 fusion-driven NSCLC differ from other well-known driver associated NSCLC such as EGFR, ALK and ROS1 in their having higher GA/tumor, TMB and PD-L1 expression frequencies suggesting that immunotherapies may also be available for the care of these patients, possibly as a combination therapy. Legal entity responsible for the studyFoundation Medicine. FundingFoundation Medicine. DisclosureE.S. Sokol: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. S.E. Trabucco: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. D.X. Jin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. G.M. Frampton: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. J.A. Elvin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. J. Vergilio: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. J.K. Killian: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. N. Ngo: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. D. Lin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. S. Ramkissoon: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. E. Severson: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. S.M. Ali: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. A.B. Schrock: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. J. Chung: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. P. Reddy: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. K. McGregor: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. B.M. Alexander: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Foundation Medicine. J.S. Ross: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine. All other authors have declared no conflicts of interest.

PL01.01 Tumor-Agnostic Biologically Driven Treatments: An Endless Dream?

PL01.01 Tumor-Agnostic Biologically Driven Treatments: An Endless Dream?

Pan-TRK Immunohistochemistry: A Useful Diagnostic Adjunct For Secretory Carcinoma of the Breast.

Secretory carcinoma is a special-type breast carcinoma underpinned by a recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion. Immunohistochemistry (IHC) using a pan-TRK antibody has been recently shown to help identify NTRK rearrangements in other tumor types. The purpose of this study was to assess the diagnostic utility of pan-TRK IHC in secretory carcinoma of the breast. Pan-TRK IHC was performed using a rabbit monoclonal antibody on whole sections of 24 breast secretory carcinomas and tissue microarray sections of other breast carcinoma types (n=203) and histologic mimics (n=15). Cases were assessed for staining intensity and localization. The 24 patients with secretory carcinoma had a median age of 44 years and a median tumor size of 1.0 cm. ETV6 fluorescence in situ hybridization was positive in all cases tested (n=20). Twenty-three cases (95.8%) showed staining with pan-TRK, which was exclusively nuclear in 19, primarily nuclear with weak cytoplasmic staining in 3, and primarily cytoplasmic with focal nuclear staining in 1. The nuclear staining was diffuse in 17 and at least focally strong in 17. The only pan-TRK negative case was a core biopsy with limited tumor. Among the 203 nonsecretory carcinomas, 21 (10.3%) showed focal, weak nuclear staining in <5% of tumor cells and 1 (0.5%) showed focal membranous staining. All histologic mimics were negative. In conclusion, diffuse and/or at least focally strong nuclear pan-TRK staining is a sensitive and specific marker for secretory carcinoma of the breast.

Clinic-pathologic features and gene fusion pattern of ALK and ROS1 in non-small cell lung cancer show association with household coal combustion.

BackgroundLung cancer induced by burning coal can be etiologically and clinically different from lung cancer caused by smoking. Despite previous work, the gene fusion patterns in lung cancer patients affected by household coal combustion still deserve further study.MethodsNon-small cell lung cancer (NSCLC) patients exposed to household coal use (HCU) were recruited from rural areas in China’s Yunnan Province, certain areas in this region had notably high lung cancer rate nationwide. Reverse transcription-polymerase chain reaction (RT-PCR) was used for detection of ALK, ROS1, RET and NTRK1 rearrangements. Eighteen studies on ALK fusions were summarized and compared with present work.ResultsAmong the 205 patients, there were 112 (54.6%) coal users and 96 (46.8%) smokers, union set had 145 (70.7%) subjects, in which 63 (30.7%) were double-positive for HCU and smoking. HCU patients featured with younger age and advanced stage. Union set patients covered larger age span (range, 40–82 years old), showed clear early-onset, and made the majority of stage IIIA–IV cases. Double-positive individuals were mainly in later stage, but with wider age span (range, 38–75 years old). In addition, 18 patients (8.8%) had EML4-ALK rearrangement, with apparently higher-than-average variant 3 ratio (77.8% vs. 44%). Five ROS1 fusion cases (2.5%) were identified, all were CD74-ROS1 (E6/E34), and had HCU experience. ALK and ROS1 fusions were mutually exclusive. Both ALK fusions and total gene rearrangement events (ALK and ROS1) showed association with HCU and overall exposure (tobacco and coal). Suggesting there could be unique gene fusion patterns in lung cancer patients affected by coal use.ConclusionsPresent study found clinic-pathologic features and gene fusion patterns in NSCLC showed association with household coal combustion. Our findings may help evaluate the impact of coal use on the pathogenesis of lung cancer, and also highlight the significance of integrating different exposure histories into clinical and theoretical research.

Uterine and vaginal sarcomas resembling fibrosarcoma: a clinicopathological and molecular analysis of 13 cases showing common NTRK-rearrangements and the description of a COL1A1-PDGFB fusion novel to uterine neoplasms

Mesenchymal neoplasms of the uterus (corpus and cervix) encompass a heterogeneous group of tumors with differing morphologies, immunophenotypes and molecular alterations. With the advent of modern molecular techniques, such as next generation sequencing, newly defined genetic abnormalities are being reported in this group of neoplasms. Herein we report the clinicopathological and molecular features of a series of 13 spindle cell sarcomas of the uterus and vagina (10 cervix, 2 uterine corpus, 1 vagina) with morphology resembling fibrosarcoma. After targeted RNA-sequencing, dual FISH fusion and array-CGH analysis, 7 of 13 tumors exhibited NTRK rearrangements (6 TPM3-NTRK1 and 1 EML4-NTRK3) and 3 a COL1A1-PDGFB fusion; in the other 3 neoplasms, all of which were positive with S100 (2 diffuse, 1 focal), we identified no rearrangement. All the NTRK fusion-positive sarcomas were located in the cervix and exhibited diffuse staining with Trk while all the other neoplasms were negative. CD34 was diffusely positive in all 3 of the COL1A1-PDGFB fusion sarcomas. The latter molecular abnormality is identical to that commonly found in dermatofibrosarcoma protuberans and has not been reported previously in uterine mesenchymal neoplasms. We suggest that uterine sarcomas with a morphology resembling fibrosarcoma (and in which leiomyosarcoma and the known molecularly confirmed high-grade endometrial stromal sarcomas have been excluded) can be divided into 3 groups:- an NTRK fusion group, a COL1A1-PDGFB fusion group and a group containing neither of these molecular abnormalities which, on the basis of positive staining with S100, could be tentatively classified as malignant peripheral nerve sheath tumor, although additional molecular studies may identify specific genetic alterations necessitating a nomenclature change. We suggest a diagnostic algorithm when reporting such neoplasms. Identification of these newly described fusion-associated sarcomas is important given the potential for targeted treatments.

Mutational analysis of >14,000 solid tumor NGS tests from community oncology practices: The Sarah Cannon molecular landscape.

e18065 Background: Utilization of NGS-based testing for patients in community oncology settings continues to grow. A comprehensive analysis of the molecular landscape of &gt; 14,000 patients with commercial tissue-based and plasma-based NGS (tbNGS and pbNGS) testing from Sarah Cannon’s (SC) network of community oncologists between 2012 and 2018 was performed. These data aid clinical trial design/feasibility and identify rare and actionable mutations in the community oncology setting. Methods: Medical Oncologists in the SC network ordered NGS-based molecular profiling for their patients as standard of care. Data from multiple commercial vendors starting in 2012 were harmonized and analyzed. Results: On average, tbNGS (n = 8938) and pbNGS (n = 5419) tests revealed 14 and 4 mutations/sample, respectively. The top 10 mutated genes from both tbNGS and pbNGS - tests are in the table. The most predominant mutation-types detected were SNVs in both tbNGS and pbNGS tests, and gene amplifications were detected in ~40% of tbNGS and ~22% of pbNGS tests. MET amplifications (range: 2 - 40 copies (pbNGS) and 6 - 82 copies (tbNGS)) and ERBB2 amplifications (range: 2.1 - 90.2 copies (pbNGS) and 4 - 293 copies (tbNGS)) were detected in 1.6% (MET-tbNGS) and 4.6% (MET-pbNGS) and 4.2% (ERBB2-tbNGS) and 2.69% (ERBB2-pbNGS) of patients, respectively, and informed clinical trial enrollment. Of the fusions detected, 28 NTRK rearrangements were detected (NTRK1 – 16, NTRK2 – 5, NTRK3 – 7) by tissue-based testing and 3 (NTRK1) fusions were detected by plasma-based tests. Conclusions: These data describe the mutational landscape of NGS tests ordered within community oncology practices. SC’s data infrastructure links NGS-based testing data and clinical data to support real world evidence research, and highlights the need for healthcare IT systems to marry clinical and genomic data for clinical decision support and clinical research. [Table: see text]

Antitumor activity of larotrectinib in tumors harboring NTRKgene fusions: a short review on the current evidence.

The development of deep-sequencing methods is now unveiling a new landscape of previously undetected gene fusion across different tumor types. Chromosomal translocation involving the NTRK gene family occur across a wide range of cancers in both children and adults. Preclinical studies have demonstrated that chimeric proteins encoded by NTRK rearrangements have oncogenic properties and drive constitutive expression and ligand-independent activation. Larotrectinib (ARRY470, LOXO101, Vitrakvi) is a highly and potent inhibitor of TRKA, TRKB, and TRKC, and has demonstrated rema rkable antitumor activity against TRK-fusion-positive cancers with a favorable side-effect profile in phase I/II clinical trials. In November 2018, the US Food and Drug Administration granted accelerated approval to larotrectinib for adult and pediatric patients with solid tumors harboring NTRK gene fusions without known acquired resistance mutation. In this review, we discuss the clinical activity and safety profile of larotrectinib, focusing on the clinical trials that led to its first global approval.