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BACKGROUND: Adding trastuzumab to first-line chemotherapy improves overall survival in patients with HER2-positive metastatic gastric cancer. We assessed the safety and efficacy of pembrolizumab in combination with trastuzumab and chemotherapy in first-line HER2-positive metastatic esophagogastric (EG) cancer. METHODS: This was an open-label, non-randomized, single-arm, investigator-initiated single center phase 2 trial in patients aged 18 years or older with HER2-positive mEG cancer. Eligible patients had measurable or evaluable non-measurable disease at baseline, Eastern Cooperative Oncology Group performance status of 0, 1, or 2, and baseline left ventricular ejection fraction of ≥ 53%. Patients were eligible to receive an initial induction cycle of intravenous (IV) pembrolizumab (200 mg flat dose) and IV trastuzumab 8 mg/kg loading dose. For subsequent cycles, patients received IV oxaliplatin 130 mg/m(2) or cisplatin 80 mg/m(2) on day 1, oral capecitabine 850 mg/m(2) twice a day for 2 weeks followed by 1 week off, or IV 5-FU 800 mg/m(2)/day on day 1 to 5, and IV pembrolizumab 200 mg flat dose and trastuzumab 6 mg/kg, both administered on day 1 of each 3-week cycle. The primary endpoint was 6-month progression-free survival (PFS). Pre-treatment tumor and plasma samples were collected for confirmation of HER2 status and exploratory biomarker analysis by targeted and whole exome sequencing. This trial is registered with Clinicaltrials.gov, number NCT02954536 (ongoing, closed to enrollment). FINDINGS: Between Nov 11, 2016, and Jan 23, 2019, 37 patients were enrolled. At the time of data cutoff on Aug 6, 2019, the median follow-up among survivors was 13 months (range 6–31). The primary endpoint was achieved; 26 of 37 (70%, 95% CI 54–83%) patients were progression-free at 6 months. The overall response rate among 35 patients with measurable disease was 91% (32 patients, 95% CI 78–97%), including 6 (17%) complete responses, 26 (74%) partial responses, and 3 (8%) stable disease. The median PFS was 13.0 months (95% CI 8.6–NA), median overall survival (OS) was 27.2 months (95% CI 18.8–NA), and the 12-month OS rate was 80% (95% CI 68–95%). The most common treatment-related adverse event of any grade was neuropathy, which affected 36 (97%) of the 37 patients. Treatment-related grade 3 or 4 adverse events occurred in 21 (67%) patients, and serious adverse events occurred in 2. Two patients discontinued treatment due to treatment-related adverse events, and 4 discontinued pembrolizumab due to immune-related adverse events. ERBB2 amplification or focal gain was detected in 21 of 32 patients (66%) patients who had tumor sequencing and in 18 of 33 patients (54%) whose circulating tumor DNA (ctDNA) was sequenced. Tissue and ctDNA sequencing were concordant for presence or absence of ERBB2 amplification in 27 of 29 patients (93%). A decline in tumor-matched ctDNA was observed after the first dose of pembrolizumab and trastuzumab in 13 of the 16 patients with tumor-matched pre-treatment ctDNA. Patients with ERBB2 amplification in both tumor and ctDNA had longer PFS; median 16.4 (n = 14, 95% CI 13.0–NA) versus 6.2 months (n = 12, 95% CI 5.9–NA) in those who did not (p = 0.013). INTERPRETATION: Pembrolizumab can be safely combined with trastuzumab and chemotherapy and has promising activity in HER2-positive mEG cancer irrespective of PD-L1 status. Plasma-based ctDNA should be explored in future randomized studies as a predictive biomarker. A randomized phase 3 clinical trial assessing the efficacy and safety of pembrolizumab versus placebo in combination with trastuzumab and chemotherapy in first-line HER2-positive mEG cancer is currently underway.

An increasing number of studies are describing potential uses of circulating tumour DNA (ctDNA) in the care of patients with colorectal cancer. Owing to this rapidly developing area of research, the Colon and Rectal–Anal Task Forces of the United States National Cancer Institute convened a panel of multidisciplinary experts to summarize current data on the utility of ctDNA in the management of colorectal cancer and to provide guidance in promoting the efficient development and integration of this technology into clinical care. The panel focused on four key areas in which ctDNA has the potential to change clinical practice, including the detection of minimal residual disease, the management of patients with rectal cancer, monitoring responses to therapy, and tracking clonal dynamics in response to targeted therapies and other systemic treatments. The panel also provides general guidelines with relevance for ctDNA-related research efforts, irrespective of indication., The analysis of ctDNA obtained from low-volume blood samples has the potential to transform the management of patients with colorectal cancer. Nevertheless, research priorities and minimum standards for sample collection and analysis in this area are currently missing. In this Position Paper, the NCI Colon and Rectal–Anal Task Forces provide a set of recommendations designed to address these challenges and accelerate the implementation of ctDNA in the management of patients with colorectal cancer.

BACKGROUND: Trastuzumab with cisplatin and fluoropyrimidine is the standard treatment in metastatic HER2-positive gastric or gastroesophageal (GE) junction adenocarcinoma, however there is limited data on the efficacy of trastuzumab in combination with a three-drug regimen in this setting. We examined the efficacy and safety of modified docetaxel, cisplatin and 5 fluorouracil (mDCF) plus trastuzumab in a single-arm multicenter phase II trial. METHODS: Previously untreated patients with HER2-positive metastatic gastric or GE junction adenocarcinoma were treated with mDCF and trastuzumab every 2 weeks. The primary endpoint was 6-month progression free survival (PFS); secondary endpoints included objective response rate, overall survival (OS), and toxicity. RESULTS: We enrolled 26 patients with metastatic HER2-positive gastric or GE junction adenocarcinoma between February 2011 and June 2015. The median age of patients was 62 years; 96% had a Karnofsky performance status equal to or greater than 80%. With a median follow-up of 25.4 months, the 6-month PFS was 73% (95% CI, 51% - 86%). The objective response rate was 65%, the median PFS was 13 months (95% CI, 6.4 – 20.7) and the median OS was 24.9 months (95% CI, 14.4 – 42.5). Grade 3/4 toxicities included neutropenia (42%), fatigue (23%), and hypophosphatemia (15%). There were no episodes of febrile neutropenia. CONCLUSION: The combination of mDCF and trastuzumab is effective and safe in patients with metastatic HER2-positive gastric or GE junction adenocarcinoma and can be considered as an option for selected patients. This trial is registered at ClinicalTrials.gov, number .

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. Although TRK fusions occur in fewer than 1% of all solid tumors, inhibition of TRK results in profound therapeutic responses, resulting in Breakthrough Therapy FDA approval of the TRK inhibitor larotrectinib for adult and pediatric patients with solid tumors, regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and the clinical effects of targeting TRK in hematologic malignancies are unknown. Here, through an evaluation for TRK fusions across more than 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma, and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively, TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition.

BackgroundFusions involving one of three tropomyosin receptor kinases (TRK) occur in diverse cancers in children and adults. We evaluated the efficacy and safety of larotrectinib, a highly selective TRK inhibitor, in adults and children who had tumors with these fusions.MethodsWe enrolled patients with consecutively and prospectively identified TRK fusion-positive cancers, detected by molecular profiling as routinely performed at each site, into one of three protocols: a phase 1 study involving adults, a phase 1-2 study involving children, or a phase 2 study involving adolescents and adults. The primary end point for the combined analysis was the overall response rate according to independent review. Secondary end points included duration of response, progression-free survival, and safety.ResultsA total of 55 patients, ranging in age from 4 months to 76 years, were enrolled and treated. Patients had 17 unique TRK fusion-positive tumor types. The overall response rate was 75% (95% confidence interval [CI], 61 to 85) according to independent review and 80% (95% CI, 67 to 90) according to investigator assessment. At 1 year, 71% of the responses were ongoing and 55% of the patients remained progression-free. The median duration of response and progression-free survival had not been reached. At a median follow-up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Adverse events were predominantly of grade 1, and no adverse event of grade 3 or 4 that was considered by the investigators to be related to larotrectinib occurred in more than 5% of patients. No patient discontinued larotrectinib owing to drug-related adverse events.ConclusionsLarotrectinib had marked and durable antitumor activity in patients with TRK fusion-positive cancer, regardless of the age of the patient or of the tumor type. (Funded by Loxo Oncology and others; ClinicalTrials.gov numbers, NCT02122913 , NCT02637687 , and NCT02576431 .).

The incidence of esophagogastric cancer is rapidly rising, but only a minority of patients derive durable benefit from current therapies. Chemotherapy as well as anti-HER2 and PD-1 antibodies are standard treatments. To identify predictive biomarkers of drug sensitivity and mechanisms of resistance, we implemented prospective tumor sequencing of patients with metastatic esophagogastric cancer. There was no association between homologous recombination deficiency defects and response to platinum-based chemotherapy. Patients with microsatellite instability–high tumors were intrinsically resistant to chemotherapy but more likely to achieve durable responses to immunotherapy. The single Epstein–Barr virus–positive patient achieved a durable, complete response to immunotherapy. The level of ERBB2 amplification as determined by sequencing was predictive of trastuzumab benefit. Selection for a tumor subclone lacking ERBB2 amplification, deletion of ERBB2 exon 16, and comutations in the receptor tyrosine kinase, RAS, and PI3K pathways were associated with intrinsic and/or acquired trastuzumab resistance. Prospective genomic profiling can identify patients most likely to derive durable benefit to immunotherapy and trastuzumab and guide strategies to overcome drug resistance. Significance: Clinical application of multiplex sequencing can identify biomarkers of treatment response to contemporary systemic therapies in metastatic esophagogastric cancer. This large prospective analysis sheds light on the biological complexity and the dynamic nature of therapeutic resistance in metastatic esophagogastric cancers. Cancer Discov; 8(1); 49–58. ©2017 AACR. See related commentary by Sundar and Tan, p. 14. See related article by Pectasides et al., p. 37. This article is highlighted in the In This Issue feature, p. 1

BRAF V600E colorectal cancers are insensitive to RAF inhibitor monotherapy due to feedback reactivation of receptor tyrosine kinase signaling. Combined RAF and EGFR inhibition exerts a therapeutic effect, but resistance invariably develops through undefined mechanisms. In this study, we determined that colorectal cancer progression specimens invariably harbored lesions in elements of the RAS–RAF–MEK–ERK pathway. Genetic amplification of wild-type RAS was a recurrent mechanism of resistance in colorectal cancer patients that was not seen in similarly resistant melanomas. We show that wild-type RAS amplification increases receptor tyrosine kinase-dependent activation of RAS more potently in colorectal cancer than in melanoma and causes resistance only in the former. Currently approved RAF inhibitors inhibit RAF monomers but not dimers. All the drug-resistant lesions we identified activate BRAF V600E dimerization directly or by elevating RAS-GTP. Overall, our results show that mechanisms of resistance converge on formation of RAF dimers and that inhibiting EGFR and RAF dimers can effectively suppress ERK-driven growth of resistant colorectal cancer. Cancer Res; 77(23); 6513–23. ©2017 AACR.

Activating neurotrophic tyrosine receptor kinase (NTRK) fusions, typically detected using nucleic-acid based assays, are highly targetable and define certain tumors. Here, we explore the utility of pan-TRK immunohistochemistry (IHC) to detect NTRK fusions. NTRK rearrangements were detected prospectively using MSK-IMPACT, a DNA-based next-generation sequencing assay. Transcription of novel NTRK rearrangements into potentially functional fusion transcripts was assessed via Archer Dx fusion assay. Pan-Trk IHC testing with mAb EPR17341 was performed on all NTRK rearranged cases and 20 cases negative for NTRK fusions on Archer. Of 23 cases with NTRK rearrangements, 15 had known activating fusions. Archer detected fusion transcripts in 6 of 8 novel NTRK rearrangements of uncertain functional significance. Pan-Trk IHC was positive in 20 of 21 cases with NTRK fusion transcripts confirmed by Archer. The discordant negative case was a mismatch repair- deficient colorectal carcinoma with an ETV6-NTRK3 fusion. All 20 additional Archer-negative cases had concordant pan-TRK IHC results. Pan-Trk IHC sensitivity and specificity for transcribed NTRK fusions was 95.2% and 100%, respectively. All positive IHC cases had cytoplasmic staining while the following fusion partner-specific patterns were discovered: all 5 LMNA-NTRK1 fusions displayed nuclear membrane accentuation, all 4 TPM3/4 fusions displayed cellular membrane accentuation, and half (3/6) of ETV6-NTRK3 fusions displayed nuclear staining. Pan-Trk IHC is a time-efficient and tissue-efficient screen for NTRK fusions, particularly in driver-negative advanced malignancies and potential cases of secretory carcinoma and congenital fibrosarcoma. Pan-Trk IHC can help determine whether translation occurs for novel NTRK rearrangements.

Microsatellite instability (MSI), a well-established driver pathway in colorectal carcinogenesis, can develop in both sporadic and hereditary conditions via different molecular alterations in the DNA mismatch repair (MMR) genes. MMR protein immunohistochemistry (IHC) is currently widely used for the detection of MMR deficiency in solid tumors. The IHC test, however, can show varied staining patterns, posing challenges in the interpretation of the staining results in some cases. Here we report a case of an 80-year-old female with a colonic adenocarcinoma that exhibited an unusual “null” IHC staining pattern with complete loss of all 4 MMR proteins (MLH1, MSH2, MSH6, and PMS2). This led to subsequent MLH1 methylation testing and next generation sequencing which demonstrated that the loss of all MMR proteins was associated with concurrent promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. These molecular findings, in conjunction with the patient's age being 80 years and the fact that the patient had no personal or family cancer history, indicated that the MMR deficiency was highly likely sporadic in nature. Thus, the stringent Lynch syndrome type surveillance programs were not recommended to the patient and her family members. This case illustrates a rare but important scenario where a null IHC phenotype signifies complex underlying molecular alternations that bear clinical management implications, highlighting the need for recognition and awareness of such unusual IHC staining patterns.

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

The-Cancer-Genome-Atlas data on colorectal carcinoma have provided a comprehensive view of the tumor’s genomic alterations and their tumorigenic roles. Tumor morphology, however, has not been fully integrated into the analysis. The aim of this study was to explore relevant associations between tumor morphology and the newly characterized genomic alterations in colorectal carcinoma. Two-hundred-and-seven colorectal carcinomas that had undergone whole exome sequencing as part of The-Cancer-Genome-Atlas project and had adequate virtual images in the cBioPortal for Cancer Genomics http://www.cbioportal.org/ constituted our study population. Upon analysis, a tight association between “microsatellite instability-high histology” and microsatellite instability-high (p

Establishing ERBB2 [human epidermal growth factor receptor 2 (HER2)] amplification status in breast and gastric carcinomas is essential to treatment selection. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) constitute the current standard for assessment. With further advancements in genomic medicine, new clinically relevant biomarkers are rapidly emerging and options for targeted therapy are increasing in patients with advanced disease, driving the need for comprehensive molecular profiling. Next-generation sequencing (NGS) is an attractive approach for up-front comprehensive assessment, including ERBB2 status, but the concordance with traditional methods of HER2 assessment is not well established. The Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay, a hybrid capture-based NGS assay interrogating the coding regions of 410 cancer-related genes, was performed on manually macrodissected unstained sections from formalin-fixed, paraffin-embedded breast (n = 213) and gastroesophageal (n = 39) tumors submitted for clinical mutation profiling. ERBB2 status was assessed using a custom bioinformatics pipeline, and NGS results were compared to IHC and FISH. NGS ERBB2 amplification calls had an overall concordance of 98.4% (248/252) with the combined IHC/FISH results in this validation set. Discrepancies occurred in the context of low tumor content and HER2 heterogeneity. ERBB2 amplification status can be reliably determined by hybridization capture-based NGS methods, allowing efficient concurrent testing for other potentially actionable genomic alterations, particularly in limited material.

The WNT pathway is a fundamental regulator of intestinal homeostasis, and hyperactivation of WNT signaling is the major oncogenic driver in colorectal cancer. To date, there are no described mechanisms that bypass WNT dependence in intestinal tumors. Here, we show that although WNT suppression blocks tumor growth in most organoid and in vivo colorectal cancer models, the accumulation of colorectal cancer–associated genetic alterations enables drug resistance and WNT-independent growth. In intestinal epithelial cells harboring mutations in KRAS or BRAF, together with disruption of TP53 and SMAD4, transient TGFβ exposure drives YAP/TAZ-dependent transcriptional reprogramming and lineage reversion. Acquisition of embryonic intestinal identity is accompanied by a permanent loss of adult intestinal lineages, and long-term WNT-independent growth. This work identifies genetic and microenvironmental factors that drive WNT inhibitor resistance, defines a new mechanism for WNT-independent colorectal cancer growth, and reveals how integration of associated genetic alterations and extracellular signals can overcome lineage-dependent oncogenic programs. Significance: Colorectal and intestinal cancers are driven by mutations in the WNT pathway, and drugs aimed at suppressing WNT signaling are in active clinical development. Our study identifies a mechanism of acquired resistance to WNT inhibition and highlights a potential strategy to target those drug-resistant cells. This article is highlighted in the In This Issue feature, p. 1426

Background Angiogenesis is critical to gastroesophageal adenocarcinoma growth and metastasis. Regorafenib is a multikinase inhibitor targeting angiogenic and stromal receptor tyrosine kinases. We evaluated whether regorafenib augments the antitumor effect of first-line chemotherapy in metastatic esophagogastric cancer. Materials and methods Patients with previously untreated metastatic gastroesophageal adenocarcinoma received 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) every 14 days and regorafenib 160 mg daily on days 4 to 10 of each 14-day cycle. The primary endpoint was 6-month progression-free survival (PFS). To identify predictive biomarkers of outcome, we examined correlations between genomic characteristics of sequenced pretreatment tumors and PFS. Results Between August 2013 and November 2014, 36 patients with metastatic esophagogastric cancer were accrued to this single-center phase II study (NCT01913639). The most common grade 3-4 treatment-related adverse events were neutropenia (36%), leucopenia (11%) and hypertension (8%). The 6-month PFS was 53% (95% confidence interval [CI], 38%-71%), the objective response rate was 54% (95% CI, 37%-70%), and the disease control rate was 77% (95% CI, 67%-94%). Next-generation sequencing did not identify any genomic alterations significantly correlated with response, and there was no association between homologous recombination deficiency and PFS with platinum-based chemotherapy. Conclusion Regorafenib (one week on-one week off schedule) is well tolerated in combination with first-line FOLFOX but does not improve 6-month PFS relative to historical control. Implications for practice Prognosis for metastatic esophagogastric cancer remains poor despite modern systemic therapy regimens. This phase II trial indicates that the combination of regorafenib and FOLFOX is well tolerated but does not add to the efficacy of first-line chemotherapy in metastatic esophagogastric cancer. Notably, recently reported data suggest potential synergy between regorafenib and the PD-1 inhibitor nivolumab. As this study demonstrates that regorafenib plus FOLFOX is safe, and combined chemotherapy and immunotherapy show favorable toxicity profiles, future studies combining immunotherapy with regorafenib and chemotherapy may be feasible.

How pancreatic ductal adenocarcinoma (PDA) cells stimulate CXCR4 to exclude T cells and resist T cell checkpoint inhibitors is not known. Here, we find that CXCL12, the ligand for CXCR4 that is produced by the cancer-associated fibroblast, “coats” human PDA and colorectal cancer cells as covalent heterodimers with keratin 19 (KRT19). Modeling the formation of the heterodimer with three proteins shows that KRT19 binds CXCL12 and transglutaminase-2 (TGM2), and that TGM2 converts the reversible KRT19-CXCL12 complex into a covalent heterodimer. We validate this model by showing that cancer cells in mouse PDA tumors must express KRT19 and TGM2 to become coated with CXCL12, exclude T cells, and resist immunotherapy with anti-PD-1 antibody. Thus, PDA cells have a cell-autonomous means by which they capture CXCL12 to mediate immune suppression, which is potentially amenable to therapy.One Sentence SummaryCancer cells in pancreatic ductal adenocarcinoma use transglutaminase-2 to assemble a coating comprised of covalent CXCL12-keratin 19 heterodimers that excludes T cells and mediates resistance to inhibition of the PD-1 T cell checkpoint.

Oncogenic fusions involving NTRK1, NTRK2, and NTRK3 with various partners are diagnostic of infantile fibrosarcoma and secretory carcinoma yet also occur in lower frequencies across many types of malignancies. Recently, targeted small molecular inhibitor therapy has been shown to induce a durable response in a high percentage of patients with NTRK fusion–positive cancers, which has made the detection of NTRK fusions critical. Several techniques for NTRK fusion diagnosis exist, including pan-Trk IHC, FISH, reverse transcription PCR, DNA-based next-generation sequencing (NGS), and RNA-based NGS. Each of these assays has unique features, advantages, and limitations, and familiarity with these assays is critical to appropriately screen for NTRK fusions. Here, we review the details of each existing methodology.

Purpose: To investigate genomic differences between urothelial carcinomas of the upper tract (UTUC) and bladder (UCB), with a focus on defining the clonal relatedness of temporally distinct tumors. Experimental Design: We prospectively sequenced tumors and matched germline DNA using targeted next-generation sequencing methods. The cohort included 195 UTUC patients and 454 UCB patients. For a subgroup of 29 patients with UTUC and a history of a subsequent UCB, both tumors were analyzed to assess their clonal relatedness. Results: With the progression to higher UTUC clinical state, there were fewer alterations in the RTK/RAS pathway but more alterations in TP53/MDM2. Compared with UCB, TP53, RB1, and ERBB2 were less frequently altered in UTUC (26% vs. 46%, 3% vs. 20%, 8% vs. 19%, respectively; Q < 0.001), whereas FGFR3 and HRAS were more frequently altered (40% vs. 26%, 12% vs. 4%, respectively; Q < 0.001). On the basis of an integrated analysis of tumor mutational burden, MSIsensor score and mutational signature, 7.2% of UTUC tumors were classified as MSI-high/MMR-deficient (MSI-H/dMMR). The risk of bladder recurrence after UTUC was significantly associated with mutations in FGFR3, KDM6A, CCND1, and TP53. Comparison of UCB with corresponding UTUC tumors from the same patient supports their clonal relatedness. Conclusions: UTUC and UCB exhibit significant differences in the prevalence of common genomic alterations. In individual patients with a history of both tumors, UCB and UTUC were always clonally related. Genomic characterization of UTUC provides information regarding the risk of bladder recurrence and can identify tumors associated with Lynch syndrome.

PURPOSE: Microsatellite instability-high (MSI-H) colorectal carcinomas (CRCs) show high rates of response to immune checkpoint inhibitors (IOs). B2M mutations and protein loss have been proposed as causes of resistance to IOs, yet they are enriched in MSI-H CRC. We aimed to characterize B2M-mutant, IO-naive CRC. PATIENTS AND METHODS: All CRCs with results for Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets, a next-generation sequencing assay that interrogates > 400 genes for mutations as well as MSI status, were surveyed for B2M mutations. All B2M-mutant CRCs were assessed for expression of B2M, major histocompatibility complex class I, and programmed death-1 ligand (PD-L1) via immunohistochemistry and average CD3(+) and CD8(+) tumor-infiltrating lymphocyte counts against a control group of MSI-H B2M wild-type CRCs. RESULTS: Fifty-nine (3.4%) of 1,751 patients with CRC harbored B2M mutations, with 84% (77 of 92) of the mutations predicted to be truncating. B2M mutations were significantly enriched in MSI-H CRCs, with 44 (24%) of 182 MSI-H CRCs harboring B2M mutations (P < .001). Thirty-two of 44 B2M-mutant CRCs with available material (73%) had complete loss of B2M expression, whereas all 26 CRCs with wild-type B2M retained expression (P < .001). B2M mutation status was not associated with major histocompatibility complex class I expression, KRAS or BRAF mutation, tumor-infiltrating lymphocyte level, or PD-L1 expression after adjustment for MSI status. Of 13 patients with B2M-mutant CRC who received programmed death-1 or PD-L1 IOs, 11 (85%) achieved clinical benefit, defined as stable disease or partial response using Response Evaluation Criteria in Solid Tumors criteria. CONCLUSION: B2M mutations occur in approximately 24% of MSI-H CRCs and are usually associated with loss of B2M expression. Most patients with B2M-mutant MSI-H CRC with loss of protein expression obtain clinical benefit from IOs.

We report the emergence of the novel MEK1V211D gatekeeper mutation in a patient with BRAFK601E colon cancer treated with the allosteric MEK inhibitor binimetinib and the anti-EGFR antibody panitumumab. The MEK1V211D mutation concurrently occurs in the same cell with BRAFK601E and leads to RAF-independent activity but remains regulated by RAF. The V211D mutation causes resistance to binimetinib by both increasing the catalytic activity of MEK1 and reducing its affinity for the drug. Moreover, the mutant exhibits reduced sensitivity to all the allosteric MEK inhibitors tested. Thus, this mutation serves as a general resistance mutation for current MEK inhibitors; however, it is sensitive to a newly reported ATP-competitive MEK inhibitor, which therefore could be used to overcome drug resistance. Significance: We report a resistance mechanism to allosteric MEK inhibitors in the clinic. A MEK1V211D mutation developed in a patient with BRAFK601E colon cancer on MEK and EGFR inhibitors. This mutant increases the catalytic activity of MEK1 and reduces its affinity for binimetinib, but remains sensitive to ATP-competitive MEK inhibitors. This article is highlighted in the In This Issue feature, p. 1143

Introduction: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are oncogenic drivers in various tumor types. Larotrectinib is a first-in-class, highly selective, central nervous system (CNS)-active TRK inhibitor approved in over 40 countries, including the US, for the treatment of adult and pediatric patients with TRK fusion cancer. Larotrectinib was approved by the FDA in 2018 based on efficacy and safety outcomes in a primary analysis cohort of 55 patients with TRK fusion cancer from 3 clinical trials (Drilon A et al. NEJM. 2018). Here, we report long-term efficacy follow-up for larotrectinib and molecular data on co-occurring mutations in this primary analysis cohort. Methods: Patients with non-primary CNS TRK fusion cancer treated with larotrectinib were identified from 3 clinical trials (NCT02122913, NCT02576431, NCT02637687). NTRK gene status was determined by local molecular testing. Adult and pediatric patients received larotrectinib 100 mg and 100 mg/m2 twice daily, respectively. The primary endpoint was investigator-assessed objective response rate (ORR) (per RECIST v1.1). Next-generation sequencing was performed on pre-treatment tumor samples from a subset of patients with evaluable tumor material (n=31) using the FoundationOne® CDx assay. The data cut-off was July 15, 2019. Results: A total of 55 patients with 17 different tumor types were included in the primary analysis cohort. Median age was 45 years (range 0.3-76 years). Patients had gene fusions involving NTRK1 (45%), NTRK2 (2%), or NTRK3 (53%). For 31 patients the detailed genomic profile, including co-occurring mutations, will be presented. Forty-four (80%) patients had received prior systemic therapy, with a median of 2 prior therapies (range 0-10). ORR with larotrectinib was 80% (95% confidence interval [CI] 67-90%); 13 (24%) patients had a complete response (CR), 31 (56%) patients had a partial response (PR), 5 (9%) patients had stable disease, and 6 (11%) patients had progressive disease. With longer follow-up, best response for 4 patients improved from PR to CR. Median duration of response and progression-free survival were 35.2 months (95% CI 19.8-not estimable [NE]) and 25.8 months (95% CI 9.9-NE), respectively. Median overall survival (OS) was not reached (95% CI 44.4-NE) over a median follow-up of 32.5 months. The 36-month OS rate was 76% (95% CI 63-89%). In an expanded safety population (n=279), larotrectinib-related adverse events were mainly Grade 1-2. Conclusions: Larotrectinib demonstrated a robust response rate with long durability and extended survival benefits in adult and pediatric patients with TRK fusion cancer. These data highlight the importance of identifying NTRK gene fusions in patients with cancer. Citation Format: Alexander Drilon, Shivaani Kummar, Catherine M. Albert, Ramamoorthy Nagasubramanian, Jaclyn F. Hechtman, John A. Reeves, Georg Beckmann, Marion Rudolph, Justyna A. Wierzbińska, Laura Dima, Nicoletta Brega, Theodore W. Laetsch, David S. Hong. Long-term outcomes of patients with TRK fusion cancer treated with larotrectinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT020.

4058 Background: Pembrolizumab and trastuzumab (P&T) and chemotherapy demonstrated 27 month mOS, 13 month mPFS, and 91% response rate in first-line HER2-positive mEG cancer irrespective of PD-L1 status (Janjigian Lancet Oncology 2020). Biomarkers including 89Zr-trastuzumab PET, blood, and tumor analysis were correlated with progression-free survival. Methods: Twenty-five patients received P&T once 3 weeks prior to addition of chemotherapy to P&T. Pre-treatment tumor biopsies, 89Zr-trastuzumab PET scans, serial plasma ctDNA (Guardant360, Redwood City, CA) and CT scans were performed. Tumor-matched DNA alterations were identified by correlating ctDNA and tissue-NGS variant calls. Pre-, on-, and post-treatment biopsies were analyzed using WES and IHC (HER2, PD-L1). Biomarkers were correlated with mPFS and 6-month PFS, the primary endpoint. Results: Of patients with tumor-matched mutations ctDNA at baseline, 12 of 16 had a decline in their maxVAF by week 3, corresponding to a mPFS of 14.7 (11.0-NR) vs 5.9 (95% CI 4.1-NR) months (p=0.009) and a mOS of 29.7 (95% CI 27.2-NR) vs 7.71 (95% CI 6.6-NR) months (p=0.006). 9 of 12 (75%) patients with decline in ctDNA at 3 weeks post-P&T achieved the 6-month PFS primary endpoint while the 4 patients with no decline in ctDNA all progressed in under 6-months. Similarly, 7 of 9 (78%) patients who had a decline in CT-measurements in all disease sites achieved the 6 month PFS primary endpoint, versus 10 of 16 (62.5%) of patients who did not respond in all sites (p=0.66), suggesting that ctDNA is superior to CT as an early predictive biomarker of response. Lack of ERBB2 amplification (amp) by NGS in ctDNA and/or tumor was associated with lack of response to P&T alone prior to addition of chemotherapy. Interestingly, no lesions from patients lacking ERBB2 ctDNA amp (n=3) responded to induction P&T by CT, while lesions from 3/9 patients lacking ERBB2 tissue amp responded to P&T by 3-week CT, suggesting intrapatient HER2 heterogeneity. Eight patients also underwent 89Zr-Trastuzumab PET scans prior to P&T and up to 5 lesions per disease site were measured on serial CT scans. All 15 lesions with intense uptake (SUVmax>10) responded to P&T, but only 9/24 lesions with SUVmax89Zr-trastuzumab PET avidity are non-invasive predictive biomarkers of response to HER2-directed therapy. Evaluation of tumor immune environment digital spatial profiling is underway. Clinical trial information: NCT02954536.

10516 Background: Recently updated NCCN guidelines for management of germline MSH6 and PMS2 pathogenic/likely pathogenic (P/LP) variant carriers suggest a more modest phenotype with later onset colorectal cancer (CRC) and limited extra-colonic cancers compared to other Lynch Syndrome (LS) genes. However, data are limited, and a comprehensive understanding of the risk spectrum and age of cancer onset is critical for cancer screening and risk-reduction. We sought to characterize MSH6 and PMS2-associated cancers and age of diagnosis in those with mismatch repair deficient (MMRD)/ microsatellite instability-high (MSI-H) tumors, a hallmark of LS pan-cancer. Methods: Pts consented to IRB-approved protocols of tumor/germline sequencing or to a prospective registry of LS pts at a single institution from 2/2005-01/2021 were reviewed to identify those with germline heterozygous MSH6/ PMS2 P/LP variants; pts with constitutional MMRD (CMMRD) were excluded. In cancer-affected pts, tumors were evaluated for MSI and/or MMR protein expression via immunohistochemistry. Tumor types were tabulated, and clinical variables were correlated with MMR/MSI status using non-parametric tests. Results: We identified 243 pts (159 female, 94 male) with P/LP germline MSH6/PMS2 variants, and 186 (77%) pts had a confirmed cancer [ MSH6 111/148 (75%); PMS2 75/95 (79%)]. Overall, 51 (21%) pts had multiple primary cancers, 35 (24%) in MSH6 and 16 (17%) in PMS2 (p = 0.20), resulting in 261 total tumors, 160 in MSH6 and 101 in PMS2. Of the 191 tumors with molecular assessment, 118 (62%) were MMRD/MSI-H, including CRC (n = 54), endometrial (EC, n = 34), small bowel (SBA, n = 6), ovarian (OC, n = 5), urothelial (n = 5), pancreato-biliary (n = 4), gastroesophageal (n = 3), non-melanoma skin (n = 3), prostate (n = 2), breast (n = 1), and brain (n = 1). While CRC and EC were more likely to be MMRD/MSI-H (79% each) compared to other cancers (37%) (p < 0.001 overall, p = 0.001 for MSH6, and p < 0.001 for PMS2), 25% of all MMRD/MSI-H tumors in both genes were comprised of non-CRC/EC cancers. Notably, there were 6 SBAs (5 in PMS2, 1 in MSH6), and all were MMRD/MSI-H. There were 17 OCs (12 in PMS2, 5 in MSH6), and of the 12 that underwent molecular assessment, 5 (41.7%) were MMRD/MSI-H (3 PMS2, 2 MSH6). Among MMRD/MSI-H CRC and EC, median age of diagnosis was 51.5 (range 27-80) and 55 (range 39-74) respectively, with 9/54 (17%) of CRC (4 in MSH6, 5 in PMS2) diagnosed < age 35, the suggested upper threshold for initiation of colonoscopy per NCCN. Conclusions: Despite being lower penetrance LS-associated genes, pts with MSH6/PMS2 P/LP variants remain at risk for a broad-spectrum of cancers and very early-onset CRC, with 17% of MMRD/MSI-H CRC presenting prior to upper threshold of initiation of colonoscopic screening per NCCN.

BACKGROUND Ovarian metastases from colorectal cancer (OM-CRC) often are unresponsive to chemotherapy and are associated with poor survival. To the authors' knowledge, the clinicopathologic and genomic predictors of OM-CRC are poorly characterized and optimal clinical management remains unclear. METHODS Women with a histopathological diagnosis of OM-CRC who were treated at Memorial Sloan Kettering Cancer Center from 1999 to 2015 were identified. Next-generation somatic mutation profiling (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets [MSK-IMPACT]) was performed on 38 OM-CRC cases, including 21 matched tumor pairs/trios. Regression models were used to analyze variables associated with progression-free survival and overall survival (OS). RESULTS Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), SMAD family member 4 (SMAD4), and neurotrophic receptor tyrosine kinase 1 (NTRK1) mutations were more frequent in cases of OM-CRC than in instances of CRC occurring without OM. SMAD4 and lysine methyltransferase 2D (KMT2D) mutations were associated with reduced OS. Matched multisite tumor sequencing did not identify OM-specific genomic alterations. Of the 195 patients who underwent oophorectomy for OM-CRC (median age, 49 years with a progression-free survival of 9.4 months and an OS of 23 months from oophorectomy), 76% had extraovarian metastasis (EOM). In multivariable analysis, residual disease after surgery (R2 resection) was associated with worse survival. Patients with EOM were less likely to achieve R0/R1 surgical resection status (complete macroscopic resection without clinical/radiological evidence of disease) (48% vs 94%). However, if R0/R1 resection status was achieved, both patients with (35.9 months vs 12 months) and without (43.2 months vs 14.5 months) EOM were found to have better OS. Among 114 patients with R0/R1 resection status, 23 (20%) had no disease recurrence, including 10 patients (9%) with > 3 years of follow-up. CONCLUSIONS Loss-of-function alterations in SMAD4 are frequent and predictive of worse survival in patients with OM-CRC. Similar to oligometastatic CRC to the lung or liver, surgical resection of OM-CRC is associated with a better outcome only if all macroscopic metastatic disease is resected. Cancer 2016. © 2016 American Cancer Society.

Immune checkpoint blockade targeting the programmed death-1 (PD-1) pathway has shown efficacy in several types of cancers including mismatch-repair-deficient colorectal carcinoma. In some tumor types, programmed death-ligand 1 (PD-L1) expression detected by immunohistochemistry has shown utility as a predictive marker for response to anti-PD-1 therapies. This utility, however, remains to be determined in colorectal carcinoma. In addition, although tumor-infiltrating lymphocytes have been associated with better prognosis in colorectal carcinoma, the prognostic value of PD-1 expression in these lymphocytes and its interaction with PD-L1 expression still await investigation. To address these questions, we performed a pilot study to evaluate the patterns of PD-L1 and PD-1 immunohistochemical expression on colorectal carcinoma cells and their tumor-infiltrating lymphocytes, respectively. Using tissue microarray, we found that 5percnt; (19sol;394) of colorectal carcinomas exhibited high tumor PD-L1 expression, and 19percnt; (76sol;392) had elevated numbers of PD-1-positive tumor-infiltrating lymphocytes. PD-L1 levels correlated with PD-1 levels (Plt;0.001), and mismatch-repair-deficient tumors had significantly higher rates of high PD-L1 and PD-1 expression when compared with mismatch-repair-proficient tumors (18percnt; vs 2percnt; and 50percnt; vs 13percnt;, respectively; Plt;0.001 for both). Staining intensity was also stronger for both markers in mismatch-repair-deficient tumors. Furthermore, we observed that among patients with mismatch-repair-deficient colorectal carcinoma, PD-1sol;PD-L1 expression stratified recurrence-free survival in an inter-dependent manner: an association between high PD-1-positive tumor-infiltrating lymphocytes and improved recurrence-free survival (Pequals;0.041) was maintained only when the tumors had low-level PD-L1 expression (Pequals;0.006); patients whose tumors had both high PD-1-positive tumor-infiltrating lymphocytes and high PD-L1 expression had a significantly worse recurrence-free survival (Plt;0.001). Thus, our results not only provide a foundation for further assessment of PD-L1 immunohistochemistry as a predictive marker for anti-PD-1 therapy in colorectal carcinoma, they also shed light on the prognostic impact of tumor-infiltrating lymphocytes in different subsets of mismatch-repair-deficient colorectal carcinomas.

ARID1A is a chromatin remodeling gene that is mutated in a number of cancers including colorectal carcinoma (CRC). Loss of ARID1A has been associated with an adverse outcome in some types of cancer. However, literature data have not been consistent. Major limitations of some outcome studies include small sample size and heterogeneous patient population. In this study, we evaluated the prognostic value of ARID1A in a homogeneous group of early stage CRC patients, a population where prognostic markers are particularly relevant. We collected a retrospective series of 578 stage I or II CRCs. All patients underwent surgery with curative intent and without neoadjuvant or adjuvant therapy. ARID1A expression was analyzed by immunohistochemistry using tissue microarray. We found ARID1A loss in 49 of 552 analyzable tumors (8.9%). Compared with the ARID1A-retained group, cases with ARID1A loss were associated with female sex (P < .001), mismatch-repair protein deficiency (P < .001), poor differentiation (P < .001), lymphovascular invasion (P = .001), and higher pT stage (P = .047). However, at a median follow-up of 49 months, ARID1A loss did not correlate with overall, disease-specific, or recurrence-free survival. This is the first systematic analysis to evaluate the prognostic significance of ARID1A in stage I/II CRCs, and our data indicate that ARID1A loss lacks prognostic significance in this population despite its association with other adverse features. Such data are clinically relevant, as efforts are ongoing in identifying markers that can detect the small but significant subset of early stage CRCs that will have a poor outcome.

Targeted therapy for metastatic colorectal carcinoma consists of anti-EGFR therapy for patients with RAS/RAF wild-type tumors. However, the response rate remains low, suggesting the presence of alternative drivers possibly also representing potential therapeutic targets. We investigated receptor tyrosine kinase (RTK) alterations and MAP2K1 (MEK1) mutations in a large cohort of colorectal carcinoma patients studied by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and The Cancer Genome Atlas, focusing on amplifications, fusions, and hotspot mutations in RTK genes and MAP2K1. RTK gene amplifications were confirmed with FISH and immunohistochemical (IHC) staining. Among 751 colorectal carcinoma cases with next-generation sequencing data, 7% and 1% of colorectal carcinoma harbored RTK alterations and MAP2K1 hotspot mutations (n = 7), respectively. RTK-altered cases had fewer concurrent RAS/RAF mutations (P = 0.003) than RTK/MAP2K1 wild-type colorectal carcinoma. MAP2K1-mutated colorectal carcinoma showed no RAS/RAF mutations. ERBB2 (n = 32) and EGFR (n = 13) were the most frequently altered RTKs, both activated by amplification and/or hotspot mutations. Three RTK fusions were identified: NCOA4-RET, ERBB2-GRB7, and ETV6-NTRK3. Only 1 of 6 patients with an RTK or MAP2K1 alteration who received anti-EGFR and/or anti-ERBB2 therapy demonstrated stable disease; the rest progressed immediately. Overall, RTK alterations and MAP2K1 mutations occur in approximately 8% of colorectal carcinoma. In spite of the usual absence of RAS/RAF mutations, response to anti-EGFR and/or anti-ERBB2 therapy was poor in this limited group. Larger studies are warranted to further define these kinase alterations as novel therapeutic targets in colorectal carcinoma and as negative predictors of response to anti-EGFR therapy. Implications: Targetable kinase alterations were identified in a subset of advanced colorectal carcinoma patients, preferentially associated with wild-type RAS/RAF, and may predict poor response to standard anti-EGFR therapy. Mol Cancer Res; 14(3); 296–301. ©2015 AACR.

233 Background: Large-scale sequencing has identified multiple oncogenic drivers and molecularly defined subsets in esophagogastric cancer. However, besides therapies for HER2-positive and microsatellite instability-high disease, few genomic biomarker-driven treatments are currently approved. Patient-derived xenografts (PDXs) have emerged as promising preclinical models that capture the heterogeneity and biology of human tumors. Therefore, we established a comprehensive collection of esophagogastric cancer PDXs and performed next-generation sequencing (NGS) to genomically characterize these models. Methods: Starting in 2010, we developed an ongoing program for generating esophagogastric cancer PDXs from fresh tumor specimens that are acquired from surgical resections or biopsies and implanted into NOD scid gamma (NSG) mice either subcutaneously into flanks or orthotopically into the gastric wall. We reviewed clinical and pathologic characteristics of patients from whom established PDXs were derived, including stage, histology, HER2 status and treatment history. To identify oncogenic DNA alterations, NGS was performed on PDX material using MSK-IMPACT, a capture-based NGS platform. Results: From April 2010 to August 2018, we implanted 270 tumor samples, of which 112 were successfully engrafted (41.4%) including 57 gastric adenocarcinomas, 25 gastroesophageal junction adenocarcinomas, 23 esophageal adenocarcinomas, 4 squamous cell carcinomas and 3 small cell/high-grade neuroendocrine tumors. PDXs were generated from both primary tumors (n = 67, 59.8%) and metastases (n = 45, 40.2%), with many PDXs established from patients with metastatic disease who had progressed on standard therapy (n = 50, 44.6%). In addition, a large number of PDXs were derived from patients initially diagnosed with HER2-positive esophagogastric adenocarcinoma (n = 68, 60.7%). NGS of these PDXs demonstrated frequent alterations in TP53, receptor tyrosine kinases ( ERBB2, EGFR), cell-cycle mediators ( CDK12, CCNE1, CCND3) and histone methyltransferases ( KMT2C, KMT2D), consistent with clinical sequencing. Conclusions: Comprehensive molecular profiling demonstrates that esophagogastric cancer PDXs recapitulate the genomic heterogeneity and tumor biology of patients. This panel represents one of the largest described collections of esophagogastric cancer PDXs and serves as a powerful platform to investigate mechanisms driving tumor growth and metastasis, identify predictive biomarkers for treatment responsiveness and develop novel genomically-driven therapeutic strategies.

250 Background: While the rate of esophagogastric (EG) cancer is declining, early onset (EO) gastric cancer prior to age 50 is rising. It is unknown whether EO-EG cancer represents a distinct entity. This study investigates the clinical and molecular characteristics of EO compared with average onset (AO)-EG cancers. Methods: We reviewed clinical and molecular features of gastric (G), esophageal (E) and gastroesophageal junction (GEJ) cancer in patients treated at MSKCC between 2005 and 2018. We defined early onset as age < 49, based on the age cutoff for urgent endoscopy referral. Clinical symptoms at diagnosis, primary tumor location, histology, HER2 and MSI status and molecular alterations were compared using Fisher’s exact test. Benjamini-Hochberg method was used to decrease the false discovery rate. Results: We analyzed 738 pts with EG cancer (age < 49 n=151; age >50 n=587). Race and sex were different with more Asian (19% vs. 9%), fewer Caucasian (62% vs. 81%) ( P

Oncogenic kinase activation by gene fusions can promote cancer development and tumor progression, however, kinase fusions have not been characterized extensively in breast cancer. In this study, we characterized kinase fusions in a large cohort of 4854 breast cancer patients using clinical DNA and/or RNA next generation sequencing platforms. Twenty-seven cases harboring kinase fusions were identified, including 11 FGFR (5 FGFR2, 3 FGFR3, 3 FGFR1), 5 BRAF, 4 NTRK1, 2 RET, 2 ROS1, 1 ALK, 1 ERBB2, and 1 MET. Eight patients with a history of endocrine therapy had available pre-treatment samples, of which six were negative for kinase fusion, and ESR1 hotspot mutations were not observed in any of these kinase fusion-positive samples. These findings suggest a potential role for kinase fusions in endocrine therapy resistance and prompted us to model the kinase fusions in human breast cancer cell lines. Ectopic expression of LMNA-NTRK1 fusion kinase activated growth factor signaling cascades, including PI3K-AKT and MAPK-ERK pathways, and promoted hormone-independent growth in MCF7 and T47D cells. Enforced expression of the LMNA-NTRK1 fusion conferred resistance to the ER antagonist fulvestrant and combined treatment of fulvestrant and the Trk inhibitor larotrectinib completely blocked the growth of LMNA-NTRK+ breast cancer cells. Similarly, expression of the EML4-ALK fusion also activated growth factor signaling pathways and caused resistance to estrogen depletion and induced sensitivity to the ALK inhibitor, Ceritinib. Treatment of xenografted LMNA-NTRK1 expressing tumors confirmed the efficacy of the combined treatment of antiestrogen and NTRK1 inhibition in vivo. Two patients with acquired LMNA-NTRK1 fusions and metastatic disease received larotrectinib and demonstrated clinical benefit. Overall, our findings demonstrate that kinase fusions promote endocrine resistance in ER-positive breast cancer, and suggest that fusion screening in advanced breast cancer, particularly those with ER-positive breast cancer at progression on hormone therapy can identify rare tumors harboring targetable kinase fusions. Citation Format: Bo Liu, Dara S. Ross, Alison M. Schram, Pedram Razavi, Stephen M. Lagana, Yanming Zhang, Maurizio Scaltriti, Jacqueline F. Bromberg, Marc Ladanyi, David M. Hyman, Alexander Drilon, Ahmet Zahir, Ryma Benayed, Jaclyn F. Hechtman, Sarat Chandarlapaty. Kinase fusions drive endocrine resistance in estrogen receptor-positive breast cancer [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 5280.

The study was undertaken to determine if microsatellite instability (MSI) status influences the composition of the tumor microbiome in primary colon carcinoma. 426 unique primary, stage I-III colon biopsy or resection specimens (234 right-sided, 192 left-sided) were sequenced by MSK-IMPACT, a large panel next generation sequencing (NGS) assay. MSI status [150 MSI-H; 276 microsatellite stable (MSS)] was determined using the MSISensor algorithm, which is clinically validated to detect MSI status from NGS data. We have developed a technique for identifying the presence of microbial species in tumor tissue being tested with NGS by analysis of non-human DNA read sequences. The method has been validated and determined to have comparable accuracy to reference microbial tests for both viruses and bacteria. We identified 13 bacterial species that are enriched in MSI-H colon carcinoma compared to MSS (odds ratio, 95% confidence interval greater than 1 with Bonferonni adjustment) (Table 1). We also observed that MSI-H is more common in right-sided tumors compared to left-sided tumors (53% vs. 13%, p=1.0E-4). To understand if sidedness of the tumor was a significant confounder, we performed stratified analysis of bacteria enrichment for right and left sided colon carcinoma. 8 of the 13 species remained significantly enriched in MSI-H tumors independent of sidedness; 9 of 13 in right-sided tumors; and 12 of 13 in left-sided tumors. Non-significant confidence intervals are underlined in Table 1. While Fusobacterium nucleatum has previously been reported to be enriched in MSI-H colon cancer, the broader spectrum of species enriched in MSI-H colon carcinoma we observed in our study has not been reported. The different microbiome suggests that either these bacteria play a role in development of MSI-H tumors or a unique immune environment exists in MSI-H tumors. The relationship between this unique microbiome and MSI status is the focus of further investigation. Table 1:Bacteria enriched in MSI-H colon cancerMSI-HMSI-HMSI-HMSSMSSMSSBacteria speciesLocationPositiveNegativeTotalPositiveNegativeTotalOdds ratioBonferroni adjusted 95% confidence intervalFusobacterium nucleatumTotal/right/left54/44/1096/81/15150/125/2538/20/18238/89/149276/109/1673.5/2.4/5.5(1.6-7.7)/(1.3-4.4)/(2.2-14.1)Bacteroides fragilisTotal/right/left47/35/12103/90/13150/125/2537/16/21239/93/146276/109/1672.9/2.3/6.4(1.3-6.5)/(1.2-4.4)/(2.6-15.9)Prevotella intermediaTotal/right/left27/22/5123/103/20150/125/2514/10/4262/99/163276/109/1674.1/2.1/10.2(1.4-12.5)/(1.01-4.7)/(2.5-41.1)Prevotella melaninogenicaTotal/right/left20/18/2130/107/23150/125/256/3/3270/106/164276/109/1676.9/5.9/4.8(1.5-31.9)/(1.7-20.8)/(0.8-30.0)Odoribacter splanchnicusTotal/right/left26/20/6124/105/19150/125/2513/5/8263/104/159276/109/1674.2/4.0/6.3(1.4-13.3)/(1.4-11.0)/(2.0-20.0)Selenomonas sputigenaTotal/right/left33/31/2117/94/23150/125/2522/15/7254/94/160276/109/1673.3/2.1/2.0(1.3-8.4)/(1.04-4.1)/(0.4-10.2)Bacteroides xylanisolvensTotal/right/left32/25/7118/100/18150/125/2521/8/13255/101/154276/109/1673.3/3.2/4.6(1.3-8.6)/(1.4-7.3)/(1.6-13.0)Prevotella denticolaTotal/right/left20/16/4130/109/21150/125/259/3/6267/106/161276/109/1674.6/5.2/5.1(1.2-17.2)/(1.5-18.3)/(1.3-19.6)Leptotrichia buccalisTotal/right/left17/13/4133/112/21150/125/256/5/1270/104/166276/109/1675.8/2.4/31.6(1.2-27.2)/(0.8-7.0)/(3.4-296.4)Prevotella sp. oral taxon 299Total/right/left14/12/2136/113/23150/125/252/1/1274/108/166276/109/16714.1/11.5/14.4(1.2-161.7)/(1.5-89.7)/(1.3-165.6)Porphyromonas gingivalisTotal/right/left13/10/3137/115/22150/125/252/0/2274/109/165276/109/16713.0/19.9/11.3(1.1-150.8)/(1.1-343.0)/(1.8-71.1)Streptococcus anginosusTotal/right/left19/16/3131/109/22150/125/2510/4/6266/105/161276/109/1673.9/3.9/3.7(1.1-14.1)/(1.2-11.9)/(0.9-15.7)Fretibacterium fastidiosumTotal/right/left15/13/2135/112/23150/125/256/3/3270/106/164276/109/1675.0/4.1/4.8(1.03-24.3)/(1.1-14.8)/(0.8-30.0) Citation Format: Chad Michael Vanderbilt, Ajaratu Keshinro, Chin-Tung Chen, Esther Babady, Michael F. Berger, Ahmet Zehir, Marc Ladanyi, Jaclyn F. Hechtman, Zsofia K. Stadler, Jinru Shia, Martin R. Weiser. Unique tumor microbiome in microsatellite instability high (MSI-H) colon carcinoma [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 6095.

TRK inhibitors achieve marked tumor-agnostic efficacy in TRK fusion-positive cancers and consequently are now an established standard of care. Little is known, however, about the demographics, clinical outcomes, response to alternative standard therapies, or genomic characteristics of TRK fusion-positive cancers. Utilizing a center-wide screening program involving more than 26,000 prospectively sequenced patients, genomic and clinical data from all cases with identified TRK fusions were extracted. An integrated analysis was performed of genomic, therapeutic, and phenomic outcomes. In total, we identified 76 cases with confirmed TRK fusions (0.27% overall prevalence) involving 48 unique rearrangements and 17 distinct cancer types. The presence of a TRK fusion was associated with depletion of concurrent oncogenic drivers (p=4.4E-7) and lower tumor mutation burden (p=4.2E-9), with the exception of colorectal cancer where TRK fusions co-occur with microsatellite instability (MSI-H). Longitudinal profiling in a subset of patients indicated that TRK fusions were present in all sampled timepoints in 82% (14/17) of cases. Progression-free survival on first-line therapy, excluding TRK inhibitors, administered for advanced disease was 9.6 months (95% CI: 4.8-13.2). The best ORR achieved with chemotherapy containing-regimens across all lines of therapy was 63% (95% CI: 41-81). Among 12 patients treated with checkpoint inhibitors, the only response observed was in an MSI-H colorectal patient. TRK fusion-positive cancers can respond to alternative standards of care, although efficacy of immunotherapy in the absence of other predictive biomarkers (MSI-H) appears limited. TRK fusions are present in tumors with simple genomes lacking in concurrent drivers that may partially explain the tumor-agnostic efficacy of TRK inhibitors. Citation Format: Ezra Y. Rosen, Debra A. Goldman, Jaclyn F. Hechtman, Ryma Benayed, Alison M. Schram, Emiliano Cocco, Sophie Shifman, Yixiao Gong, Ritika Kundra, James P. Solomon, Alberto Bardelli, Maurizio Scaltriti, Alexander Drilon, Alexia Iasonos, Barry S. Taylor, David M. Hyman. Landscape and outcome of TRK fusion-positive Cancers [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 16.

4559 Background: Pembrolizumab can be safely combined with trastuzumab and chemotherapy and has promising activity with median OS 27 months and 91% objective response rate in HER2-positive mEG cancer irrespective of PD-L1 status (NCT02954536; Janjigian ESMO 2019). Tumor biopsies and blood samples were collected in this phase II trial to identify molecular and immune predictors of response and resistance to PTC. Methods: Pre-treatment and post-progression biopsies were analyzed using WES and IHC (HER2, PD-L1). Peripheral blood was collected pre-treatment, every 9 weeks on-treatment and at progression for plasma ctDNA (Guardant 360, Guardant Health, Redwood, CA). Tumor-matched DNA alterations were identified by correlating ctDNA and solid tumor WES results. Landmark PFS analysis was used to compare ctDNA clearance status at 9 weeks post-treatment. Results: Baseline ctDNA was analysed from 31 of 37 patients of whom 84% (26/31) had tumor-matched ctDNA detected at baseline. Patients who cleared ctDNA at 9 weeks (n = 17/23) achieved a longer median PFS than those who did not - mPFS 12.3 months (95% CI 7.44-NA vs 3.9 months (95% CI 2.01-NA) (log-rank p = 0.02). On serial blood monitoring of 16 patients with eventual radiographic progression, ctDNA re-appearance preceded CT detection in 8 (50%) patients. WES was completed in 31 patients with pre-treatment, and 12 patients post-progression, including matched samples from 10 patients. Loss of HER2 over-expression/amplification was noted in 44% (7/16) of post-progression samples by IHC/FISH (2 IHC 0/1, 5 FISH-). In paired post-progression samples on WES, we observed loss of ERBB2 in 2 patients, and new amplifications of CCND1/3, FGF3/4/19, CDK6/12, KRAS, MYC, and MET, as well as mutations in KRAS, PIK3CD and PIK3RA. Plasma analysis at progression demonstrated copy number increases and/or new amplifications in MET, CKD6, PIK3CA, KRAS, FGFR2, EGFR and CCDN1 as well as KRAS, RB1, PTEN, NF1, NOTCH1, BRAF, and FGFR1 mutations. Conclusions: The majority of patients with previously untreated HER2 positive mEG have detectable plasma ctDNA at baseline. The re-appearance of ctDNA during therapy may serve as an early predictor of progression and help identify genetic drivers of acquired resistance. Loss of ERBB2 over-expression/amplification and activating MAPK alterations occur at PTC progression. Evaluation of tumor immune environment by multiplex IHC and additional ctDNA analysis is underway.

1527 Background: PMS2-associated Lynch syndrome (LS) may have a more modest phenotype than that associated with other mismatch repair (MMR) genes ( MLH1, MSH2, MSH6, EPCAM). Recent studies suggest limited extra-colonic cancers, and modified risk-reducing measures can be provided. Understanding the spectrum of risk is of critical importance as some LS-associated cancers do not have effective screening, requiring risk-reducing surgery (endometrial, ovarian). As MMR-deficiency (MMRD)/ microsatellite instability (MSI) is associated with LS pan-cancer, we sought to characterize PMS2-associated malignancies according to MMR/MSI status. Methods: Review of cancer patients (pts) consented to an IRB-approved protocol of tumor/germline next-generation sequencing (NGS) identified 43 germline heterozygous PMS2 mutation carriers. Tumors were evaluated for MSI via MSIsensor and/or corresponding MMR protein expression via immunohistochemical staining (IHC). Clinical variables were correlated with MMR/MSI status, comparing via Chi-square or standard T-test. Results: There were > 10 tumor types; 69.8% (30/43) were extra-colonic cancers (endometrial (n = 4), ovarian (n = 6), small bowel (n = 3), urothelial (n = 2), pancreas (n = 3), prostate (n = 3), breast (n = 3), brain (n = 3), biliary (n = 1), spindle cell sarcoma (n = 1), and hepatoblastoma (n = 1)). 46.5% (20/43) of tumors were MMRD/MSI. 61.5% (8/13) of colorectal cancers (CRC) were MMRD/MSI, compared to 40% (12/30) of extra-colonic tumors. All endometrial and small bowel cancers were MMRD/MSI. Of 6 ovarian cancers, 3 were clear-cell, 1 endometrioid, and 2 high-grade serous (HGS). The only MMRD/MSI ovary tumor was HGS. 73.9% (17/23) of pts with MMRP/MSS tumors had recurrent/metastatic disease vs 30% (6/20) of pts with MMRD/MSI tumors ( p= 0.004). Mean age at diagnosis did not differ significantly between MMRP/MSS and MMRD/MSI groups (49 vs. 57, respectively, p= 0.146). 11.6% (5/43) of pts had a prior cancer, with only one patient having prior CRC. Pts with extra-colonic tumors were less likely to meet clinical pt and family history LS testing criteria than those with CRC (63.3% (19/30) vs. 7.7% (1/13); p< 0.001). Conclusions: While PMS2-related LS may have a more modest clinical phenotype, in this single-institution study, 60% (12/20) of patients with MMRD/MSI tumors presented with extra-colonic cancers. We caution counseling pts with PMS2-associated LS about reduced extra-colonic risk until more complete information about penetrance, spectrum, and age distribution of cancer is available.

Background NTRK1, NTRK2 and NTRK3 fusions are present in a plethora of malignancies across different histologies. These fusions represent the most frequent mechanism of oncogenic activation of these receptor tyrosine kinases, and biomarkers for the use of TRK small molecule inhibitors. Given the varying frequency of NTRK1/2/3 fusions, crucial to the administration of NTRK inhibitors is the development of optimal approaches for the detection of human cancers harbouring activating NTRK1/2/3 fusion genes. Materials and methods Experts from several Institutions were recruited by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) to review the available methods for the detection of NTRK gene fusions, their potential applications, and strategies for the implementation of a rational approach for the detection of NTRK1/2/3 fusion genes in human malignancies. A consensus on the most reasonable strategy to adopt when screening for NTRK fusions in oncologic patients was sought, and further reviewed and approved by the ESMO TR and PM WG and the ESMO leadership. Results The main techniques employed for NTRK fusion gene detection include immunohistochemistry, fluorescence in situ hybridization (FISH), RT-PCR, and both RNA-based and DNA-based next generation sequencing (NGS). Each technique has advantages and limitations, and the choice of assays for screening and final diagnosis should also take into account the resources and clinical context. Conclusion In tumours where NTRK fusions are highly recurrent, FISH, RT-PCR or RNA-based sequencing panels can be used as confirmatory techniques, whereas in the scenario of testing an unselected population where NTRK1/2/3 fusions are uncommon, either front-line sequencing (preferentially RNA-sequencing) or screening by immunohistochemistry followed by sequencing of positive cases should be pursued.

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplification or overexpression, but intrinsic and acquired resistance are common. We conducted a phase II study of afatinib, an irreversible pan-HER kinase inhibitor, in trastuzumab-resistant EG cancer. We analyzed pretreatment tumor biopsies and, in select cases, performed comprehensive characterization of postmortem metastatic specimens following acquisition of drug resistance. Afatinib response was associated with coamplification of EGFR and ERBB2. Heterogeneous 89Zr-trastuzumab PET uptake was associated with genomic heterogeneity and mixed clinical response to afatinib. Resistance to afatinib was associated with selection for tumor cells lacking EGFR amplification or with acquisition of MET amplification, which could be detected in plasma cell-free DNA. The combination of afatinib and a MET inhibitor induced complete tumor regression in ERBB2 and MET coamplified patient-derived xenograft models established from a metastatic lesion progressing on afatinib. Collectively, differential intrapatient and interpatient expression of HER2, EGFR, and MET may determine clinical response to HER kinase inhibitors in ERBB2-amplified EG cancer. Significance: Analysis of patients with ERBB2-amplified, trastuzumab-resistant EG cancer who were treated with the HER kinase inhibitor afatinib revealed that sensitivity and resistance to therapy were associated with EGFR/ERBB2 coamplification and MET amplification, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones. See related commentary by Klempner and Catenacci, p. 166. This article is highlighted in the In This Issue feature, p. 151

Purpose: Prior molecular profiling of hepatocellular carcinoma (HCC) has identified actionable findings that may have a role in guiding therapeutic decision-making and clinical trial enrollment. We implemented prospective next-generation sequencing (NGS) in the clinic to determine whether such analyses provide predictive and/or prognostic information for HCC patients treated with contemporary systemic therapies. Experimental Design: Matched tumor/normal DNA from patients with HCC (N = 127) were analyzed using a hybridization capture–based NGS assay designed to target 341 or more cancer-associated genes. Demographic and treatment data were prospectively collected with the goal of correlating treatment outcomes and drug response with molecular profiles. Results: WNT/β-catenin pathway (45%) and TP53 (33%) alterations were frequent and represented mutually exclusive molecular subsets. In sorafenib-treated patients (n = 81), oncogenic PI3K–mTOR pathway alterations were associated with lower disease control rates (DCR, 8.3% vs. 40.2%), shorter median progression-free survival (PFS; 1.9 vs. 5.3 months), and shorter median overall survival (OS; 10.4 vs. 17.9 months). For patients treated with immune checkpoint inhibitors (n = 31), activating alteration WNT/β-catenin signaling were associated with lower DCR (0% vs. 53%), shorter median PFS (2.0 vs. 7.4 months), and shorter median OS (9.1 vs. 15.2 months). Twenty-four percent of patients harbored potentially actionable alterations including TSC1/2 (8.5%) inactivating/truncating mutations, FGF19 (6.3%) and MET (1.5%) amplifications, and IDH1 missense mutations ( Conclusions: Linking NGS to routine clinical care has the potential to identify those patients with HCC likely to benefit from standard systemic therapies and can be used in an investigational context to match patients to genome-directed targeted therapies. See related commentary by Pinyol et al., p. 2021

Mutations of Hepatocyte-Nuclear-Factor-1-Homeobox-A gene and loss of Liver-Fatty-Acid-Binding-Protein are well documented in hepatocellular adenoma. However, the role of Hepatocyte-NuclearFactor-1-Homeobox-A mutations in hepatocellular carcinoma remains to be determined. In this study, all hepatocellular neoplasms evaluated by our institutional Memorial Sloan Kettering-Integrated Mutational Profiling of Actionable Clinical Targets assay or the Cancer Genome Atlas sequencing, and cases reported in the literature, were queried for Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations. Together, 11 of 672 (1.6%) hepatocellular carcinomas harbored Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations. The single case from our institution (n=153) was extremely well differentiated, arising in a background of adenomatosis. Both the adenoma and carcinoma component contained the same 2 somatic Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations (p. E32* and L214Q), with loss of Liver-Fatty-Acid-Binding-Protein. From the literature, 2 of 146 (1.4%) hepatocellular carcinomas had Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations, and both arose in a background of adenomatosis. Information on pre-existing adenoma for the remaining cases (8/373, from The Cancer Genome Atlas) was not available. Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations in carcinomas were associated with negative viral hepatitis status (p=0.004), mutually exclusive with Catenin-Beta-1 hotspot mutations, and trended to occur more in females (p=0.06) and without cirrhosis (p=0.03). Grade was not associated with Hepatocyte-Nuclear-Factor-1-Homeobox-A status (p=0.28). Somatic Hepatocyte-Nuclear-Factor-1Homeobox-A mutations occur in approximately 1–2% of hepatocellular carcinoma, often in a background of adenomatosis. Our findings suggest that malignant transformation of Hepatocyte-Nuclear-Factor-1Homeobox-A mutated hepatocellular adenoma occurs, albeit infrequently. Hepatocellular adenomas with Hepatocyte-Nuclear-Factor-1-Homeobox-A mutation or adenomatosis with loss of Liver-Fatty-Acid-Binding-Protein warrant thorough sampling and examination.

Purpose: Various genetic driver aberrations have been identified among distinct anatomic and clinical subtypes of intrahepatic and extrahepatic cholangiocarcinoma, and these molecular alterations may be prognostic biomarkers and/or predictive of drug response. Experimental Design: Tumor samples from patients with cholangiocarcinoma who consented prospectively were analyzed using the MSK-IMPACT platform, a targeted next-generation sequencing assay that analyzes all exons and selected introns of 410 cancer-associated genes. Fisher exact tests were performed to identify associations between clinical characteristics and genetic alterations. Results: A total of 195 patients were studied: 78% intrahepatic and 22% extrahepatic cholangiocarcinoma. The most commonly altered genes in intrahepatic cholangiocarcinoma were IDH1 (30%), ARID1A (23%), BAP1 (20%), TP53 (20%), and FGFR2 gene fusions (14%). A tendency toward mutual exclusivity was seen between multiple genes in intrahepatic cholangiocarcinoma including TP53:IDH1, IDH1:KRAS, TP53:BAP1, and IDH1:FGFR2. Alterations in CDKN2A/B and ERBB2 were associated with reduced survival and time to progression on chemotherapy in patients with locally advanced or metastatic disease. Genetic alterations with potential therapeutic implications were identified in 47% of patients, leading to biomarker-directed therapy or clinical trial enrollment in 16% of patients. Conclusions: Cholangiocarcinoma is a genetically diverse cancer. Alterations in CDKN2A/B and ERBB2 are associated with negative prognostic implications in patients with advanced disease. Somatic alterations with therapeutic implications were identified in almost half of patients. These prospective data provide a contemporary benchmark for guiding the development of targeted therapies in molecularly profiled cholangiocarcinoma, and support to the use of molecular profiling to guide therapy selection in patients with advanced biliary cancers. Clin Cancer Res; 24(17); 4154–61. ©2018 AACR.

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.

The PI3K/AKT/mTOR pathway is activated through multiple mechanisms in colorectal carcinoma. Here, the clinicopathologic and molecular features of AKT1 E17K–mutated colorectal carcinoma in comparison with PIK3CA-mutated colorectal carcinoma are described in detail. Interestingly, in comparison with PIK3CA mutants, AKT1 E17K was significantly associated with mucinous morphology and concurrent BRAF V600E mutation. Among PIK3CA mutants, exon 21 mutations were significantly associated with BRAF V600E mutation, MSI-H status, and poor differentiation, while exon 10 mutations were associated with KRAS/NRAS mutations. Three of four AKT1 mutants with data from both primary and metastatic lesions had concordant AKT1 mutation status in both. Both AKT1- and PIK3CA-mutant colorectal carcinoma demonstrated frequent loss of PTEN expression (38% and 34%, respectively) and similar rates of p-PRAS 40 expression (63% and 50%, respectively). Both patients with AKT1 E17K alone had primary resistance to cetuximab, whereas 7 of 8 patients with PIK3CA mutation alone experienced tumor shrinkage or stability with anti-EGFR therapy. These results demonstrate that AKT1 E17K mutation in advanced colorectal carcinoma is associated with mucinous morphology, PIK3CA wild-type status, and concurrent RAS/RAF mutations with similar pattern to PIK3CA exon 21 mutants. Thus, AKT1 E17K mutations contribute to primary resistance to cetuximab and serve as an actionable alteration. Implications: This first systematic study of AKT1 and PIK3CA hotspot mutations and their association with cetuximab resistance and BRAF V600E mutation has important ramifications for the development of personalized medicine, particularly in identifying patient candidates for PI3K or AKT inhibitors. Mol Cancer Res; 13(6); 1003–8. ©2015 AACR.

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.

Glioblastoma is the most common malignant primary brain tumor in adults. Despite aggressive initial treatment, recurrence is inevitable and almost always fatal. Treatment options for recurrent disease are limited and do not substantially improve survival. In recent years, molecular characterization of gliomas has revealed recurrent somatic mutations, structural rearrangements, and epigenetic alterations. These genomic alterations have implications for prognosis and response to treatment. IDH1/2 mutations are associated with a more favorable prognosis among glioblastomas, whereas tumors with MGMT promoter hypermethylation are more likely to respond to treatment with alkylating agents.1,2 The pattern of genomic alterations can also provide insight into the origin and evolution of the tumor. For example, the presence of coexistent mutations in IDH1 and ATRX suggest a secondary glioma that has progressed from a lower-grade glioma.3-5 In addition to informing prognosis and diagnostic classification, some genomic alterations in glioma represent potential therapeutic targets. Specifically, recurrent alterations in one of several receptor tyrosine kinases (RTKs), including EGFR, PDGFRA, MET, and FGFR1/2/3, have been observed in the majority of glioblastomas.6 Unfortunately, efforts to develop genomically targeted therapy for the treatment of glioblastoma have been unsuccessful to date.7 It remains unknown the extent to which the lack of activity of targeted therapy may be attributed to so-called key alterations that do not represent true clonal drivers that are critical to tumor growth and progression compared with the simple inability of existing drugs to achieve therapeutic exposures within the CNS. In addition to these more common genomic alterations, less common fusions that involve the kinase domain of tropomyosin receptor kinases (TRKs; A/B/C) encoded by the genes, NTRK1, NTRK2, and NTRK3 have also been identified in gliomas.8-11 In other cancer types, selective inhibition of these TRK fusions has resulted in remarkable efficacy12; however, it remains unknown whether this therapeutic strategy is effective in primary brain tumors. Here, we report the case of a young woman with recurrent glioblastoma treated with larotrectinib, a first-in-class, potent, highly selective TRK inhibitor. To our knowledge, this is the first report of the use of a TRK inhibitor in a patient with a TRK fusion–positive glioblastoma.

Mutations in succinate dehydrogenase complex genes predispose to familial paraganglioma-pheochromocytoma syndrome (FPG) and gastrointestinal stromal tumors (GIST). Here we describe cancer patients undergoing agnostic germline testing at Memorial Sloan Kettering Cancer Center and found to harbor germline SDHA mutations. Using targeted sequencing covering the cancer census genes, we identified 10 patients with SDHA germline mutations. Cancer diagnoses for these patients carrying SDHA germline mutations included neuroblastoma (n = 1), breast (n = 1), colon (n = 1), renal (n = 1), melanoma and uterine (n = 1), prostate (n = 1), endometrial (n = 1), bladder (n = 1), and gastrointestinal stromal tumor (GIST) (n = 2). Immunohistochemical staining and assessment of patient tumors for second hits and loss of heterozygosity in SDHA confirmed GIST as an SDHA-associated tumor and suggests SDHA germline mutations may be a driver in neuroblastoma tumorigenesis.

The incidence of other primary neoplasms in gastrointestinal stromal tumor (GIST) patients is relatively high. Our aim was to better characterize the clinicopathologic and molecular relationships in a cohort of GIST patients.All GIST patients with tumor samples sent for molecular testing were identified via electronic medical records. Clinicopathologic characteristics of GIST and additional primary malignancies were analyzed.Of 260 patients, 50 (19 %) had at least one additional primary malignancy. In 33 patients, separate primary neoplasms predated their GIST diagnosis and most commonly included: prostate (n = 9), breast (n = 8), and hematologic (n = 5). Renal (n = 4) and hematologic (n = 3) malignancies were the most frequent cancers identified after GIST diagnosis. The majority (8 of 12, 66 %) of malignancies diagnosed after GIST were found incidentally. Patients who developed other malignancies after GIST more often had KIT exon 11 mutations (100 vs. 66 %, P = 0.01). In comparison to patients with only GIST, patients with a second primary neoplasm of any chronology had GISTs with increased mitotic rate (≥5 per 50 high-power fields) (P = 0.0006). Literature review revealed colorectal cancer, gastric, prostate, renal, leukemia, and desmoid-type fibromatosis as the most common secondary neoplasms.Nineteen percent of GIST patients develop other malignancies. This is the first report to describe a relationship between additional primary malignancy and both mutation and mitotic rate of GIST. Although the basis of these relationships remains to be investigated, caution in the clinical management of GIST patients with additional lesions is warranted.

BACKGROUND. Gastric adenocarcinoma is a heterogenous disease that results from complex interactions between environmental and genetic factors, which may contribute to the disparate outcomes observed between different patient populations. This study aimed to determine whether genomic differences exist in a diverse population of patients by evaluating tumor mutational profiles stratified by race. METHODS. All patients with gastric adenocarcinoma between 2012 and 2016 who underwent targeted next-generation sequencing of cancer genes by the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets platform were identified. Patient race was categorized as Asian, African American, Hispanic, or Caucasian. Fisher’s exact test was used to examine differences in mutation rates between racial designations for the most common mutations identified. The p values in this study were adjusted using the false discovery rate method. RESULTS. The study investigated 595 mutations in 119 patients. The DNA alterations identified included missense mutations (66%), frame-shift deletions (13%), and nonsense mutations (9%). Silent mutations were excluded. The most frequently mutated genes were ARID1A, CDH1, ERBB3, KRAS, PIK3CA, and TP53. Of these, TP53 was the most frequently mutated gene, affecting 50% of patients. The proportion of patients with TP53 mutations differed significantly between races (p = 0.012). The findings showed TP53 mutations for 89% (16/18) of the African American patients, 56% (10/18) of the Asian patients, 43% (9/21) of the Hispanic patients, and 40% (25/62) of the Caucasian patients. CONCLUSIONS. Significantly higher rates of TP53 mutations were identified among the African American patients with gastric adenocarcinoma. This is the first study to evaluate tumor genomic differences in a diverse population of patients with gastric adenocarcinoma.