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PURPOSE Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS ( P = .04). The most frequently altered genes in the primary cohort were TP 53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort ( P = .015) and the validation cohort ( P = .012). CONCLUSION The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Ewing sarcoma (ES) is a primitive sarcoma defined by EWSR1-ETS fusions as the primary driver alteration. To better define the landscape of cooperating secondary genetic alterations in ES, we analyzed clinical genomic profiling data of 113 patients with ES, a cohort including more adult patients (18 years) and more patients with advanced stage at presentation than previous genomic cohorts.The data set consisted of patients with ES prospectively tested with the US Food and Drug Administration-cleared Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets large panel, hybrid capture-based next-generation sequencing assay. To assess the functional significance ofNovel subsets were defined by recurrent secondary alterations inOur findings open avenues to new insights into ES pathobiology and to novel therapeutic approaches in a subset of patients with ES.

PURPOSE The identification of novel oncogenic driver alterations and novel mechanisms of acquired resistance (AR) is the key for further development of personalized therapy. The current study investigates the potential role of YES1 amplification as a primary driver of tumorigenesis and of YES1/YAP1 amplifications as mediators of AR to ALK and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). METHODS Models of ectopic expression were established and characterized for YES1 and YAP1 in human bronchial epithelial cells and ALK fusion–positive (ALK+) and EGFR-mutant lung adenocarcinoma cell lines. MSK-IMPACT data for all lung adenocarcinoma cases and for ALK and EGFR TKI AR cases were surveyed for YES1 and YAP1 amplification. RESULTS We report response to SRC family kinase (SFK) inhibition in a patient whose lung cancer exhibited YES1 amplification, without any well-established primary driver alteration, suggesting that YES1 amplification can also function as a primary oncogenic driver. To investigate the possibility of YES1 as a primary driver in tumorigenesis, we established preclinical models of YES1 overexpression using human bronchial epithelial cells and normal human breast epithelial cells. We showed that YES1 overexpression conferred sensitivity to SFK TKIs and promoted EGF-independent growth in a YAP1-dependent manner. Analysis of clinical genomic sequencing data from cases of AR to EGFR and ALK inhibitors revealed acquired amplification of YAP1 in four cases. EGFR-mutant and ALK fusion–positive cells overexpressing YES1 or YAP1 were resistant to EGFR and ALK TKIs, respectively, but were sensitive to dual inhibition of the primary driver and YES1. CONCLUSION Our results demonstrate the therapeutic potential of SFK inhibition in primary tumorigenesis and AR driven by YES1/YAP1 signaling.

PURPOSE The clinical utility of cell-free DNA (cfDNA) as a biomarker for advanced clear cell renal cell carcinoma (ccRCC) remains unclear. We evaluated the validity of cfDNA-based genomic profiling in a large cohort of patients with ccRCC with matched next-generation sequencing (NGS) from primary tumor tissues. MATERIALS AND METHODS We performed paired NGS of tumor DNA and plasma cfDNA using the MSK-IMPACT platform in 110 patients with metastatic ccRCC. Tissues were profiled for variants and copy number alterations with germline comparison. Manual cross-genotyping between cfDNA and tumor tissue was performed. Deep sequencing with a higher sensitivity platform, MSK-ACCESS, was performed on a subset of cfDNA samples. Clinical data and radiographic tumor volumes were assessed to correlate cfDNA yield with treatment response and disease burden. RESULTS Tumor tissue MSK-IMPACT testing identified 582 genomic alterations (GAs) across the cohort. Using standard thresholds for de novo variant calling in cfDNA, only 24 GAs were found by MSK-IMPACT in cfDNA in 7 of 110 patients (6%). With manual cross-genotyping, 210 GAs were detectable below thresholds in 74 patients (67%). Intrapatient concordance with tumor tissue was limited, including VHL (31.6%), PBRM1 (24.1%), and TP53 (52.9%). cfDNA profiling did not identify 3p loss because of low tumor fractions. Tumor volume was associated with cfDNA allele frequency, and VHL concordance was superior for patients with greater disease burden. CONCLUSION cfDNA-based NGS profiling yielded low detection rates in this metastatic ccRCC cohort. Concordance with tumor profiling was low, even for truncal mutations such as VHL, and some findings in peripheral blood may represent clonal hematopoiesis. Routine cfDNA panel testing is not supported, and its application in biomarker efforts must account for these limitations.

PURPOSE Although MET exon 14 ( METex14)–altered lung cancers were first identified more than a decade and a half ago, the frequency of CNS metastatic disease remains poorly defined. Furthermore, the seminal trial of crizotinib in these patients (PROFILE 1001) did not report patterns of CNS response or progression. PATIENTS AND METHODS Patients with pathologically confirmed, advanced non–small-cell lung cancers (NSCLC) harboring a METex14 alteration by targeted DNA/RNA sequencing were studied. The incidence of brain metastases and the outcomes of MET inhibition with crizotinib were analyzed. RESULTS Eighty-three patients with METex14-altered metastatic NSCLC were identified. The incidence of CNS metastases at diagnosis was 17% (95% CI, 10% to 27%). The lifetime incidence was 36% (95% CI, 26% to 47%); 83% of patients had parenchymal disease, and 17% had leptomeningeal disease. The probability of having brain metastasis at 1, 2, and 3 years was 24%, 35%, and 38%, respectively. Fifty-four patients received crizotinib. The median time to radiologic CNS progression was 5.8 months (range, 3.7-20.0 months). Patterns of crizotinib progression were as follows: intracranial only in 10% of patients, intracranial and extracranial in 12%, and extracranial only in 78%. In patients with brain metastases before treatment, the median time on crizotinib was 7.5 months (range, 7.2-11.7 months). CONCLUSION CNS metastases, including leptomeningeal disease, occurred in more than a third of patients with METex14-altered lung cancers. In crizotinib-treated patients with or without CNS metastases, CNS failure was seen in less than a quarter of patients on progression.

PURPOSE Broad-panel sequencing of tumors facilitates routine care of people with cancer as well as clinical trial matching for novel genome-directed therapies. We sought to extend the use of broad-panel sequencing results to survival stratification and clinical outcome prediction. METHODS By using sequencing results from a cohort of 1,054 patients with advanced lung adenocarcinomas, we developed OncoCast, a machine learning tool for survival risk stratification and biomarker identification. RESULTS With OncoCast, we stratified this patient cohort into four risk groups on the basis of tumor genomic profile. Patients whose tumors harbored a high-risk profile had a median survival of 7.3 months (95% CI, 5.5 to 10.9 months) compared with a low-risk group with a median survival of 32.8 months (95% CI, 26.3 to 38.5 months) with a hazard ratio of 4.6 ( P < .001), far superior to any individual gene predictor or standard clinical characteristics. We found that comutations of both STK11 and KEAP1 are strong determinants of unfavorable prognosis with currently available therapies. In patients with targetable oncogenes (eg, EGFR, ALK, ROS1) who received targeted therapies, the tumor genetic background additionally differentiated survival with mutations in TP53 and ARID1A, which contributed to a higher risk score for shorter survival. CONCLUSION A mutational profile derived from broad-panel sequencing presents an effective genomic stratification for patient survival in advanced lung adenocarcinoma. OncoCast is available as a public resource that facilitates the incorporation of mutational data to predict individual patient prognosis and compare risk characteristics of patient populations.

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.

Novel resistance mechanisms to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer continue to be defined.1 With the recent approval of the third-generation EGFR TKI osimertinib as first-line treatment, and expanded use of large-panel next-generation sequencing (NGS)–based testing, many novel resistance mechanisms are emerging. Resistance to osimertinib can result from on-target mechanisms, such as the acquisition of second-site EGFR mutations, or from the activation of off-target mechanisms or bypass pathways, including acquired oncogenic fusions of RET, ALK, BRAF, and FGFR1,2 and amplification or mutation of HER2, BRAF, MEK, KRAS, PIK3CA, and MET.3 MET amplification is reported in 5% to 22% of EGFR TKI resistance.1,4 Preclinical studies have shown that acquired resistance to EGFR TKIs resulting from MET amplification can be reversed by combined therapy with EGFR and MET inhibitors.4 MET exon 14 skipping alteration (METex14) is present in 3% to 4% of lung adenocarcinomas,5 and the MET receptor lacking exon 14 shows decreased protein turnover because of loss of the ubiquitination site encoded by exon 14, resulting in aberrant MET activation and oncogenesis.6 In preclinical and clinical studies, responses to MET inhibitors, such as crizotinib, have been reported in patients with lung cancer with METex14 as a primary driver.6-11 However, it has not been previously implicated in acquired resistance to EGFR-TKIs. In this study, we used targeted NGS with Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT),12 immunohistochemistry, cell-free DNA testing, and fluorescence in situ hybridization to evaluate acquired resistance mediated by METex14. Furthermore, we used in vitro functional studies to establish METex14 as a novel mechanism of acquired resistance to EGFR TKIs. We used MSK-IMPACT, a large-panel NGS assay, to detect mutations, copy-number alterations, and select gene fusions involving up to 468 cancer-associated genes.12 METex14 was introduced into PC9 and H1975 cells as follows. Briefly, full-length METex14 was polymerase chain reaction amplified and subcloned into pLenti-CMV-blast lentiviral vector (plasmid 17451; Addgene, Cambridge, MA). The lentiviral plasmids were cotransfected with packaging plasmids into HEK 293 T cells using FuGENE HD (Promega, Madison, WI), and lentiviruses were generated. Cells were infected with lentivirus-expressing METex14 cDNA, followed by selection with blasticidin (20 µg/mL) for 8 days. The Data Supplement provides more detailed methods.

PURPOSE Matching patients to investigational therapies requires new tools to support physician decision making. We designed and implemented Precision Insight Support Engine (PRECISE), an automated, just-in-time, clinical-grade informatics platform to identify and dynamically track patients on the basis of molecular and clinical criteria. Real-world use of this tool was analyzed to determine whether PRECISE facilitated enrollment to early-phase, genome-driven trials. MATERIALS AND METHODS We analyzed patients who were enrolled in genome-driven, early-phase trials using PRECISE at Memorial Sloan Kettering Cancer Center between April 2014 and January 2018. Primary end point was the proportion of enrolled patients who were successfully identified using PRECISE before enrollment. Secondary end points included time from sequencing and PRECISE identification to enrollment. Reasons for a failure to identify genomically matched patients were also explored. RESULTS Data were analyzed from 41 therapeutic trials led by 19 principal investigators. In total, 755 patients were accrued to these studies during the period that PRECISE was used. PRECISE successfully identified 327 patients (43%) before enrollment. Patients were diagnosed with 29 tumor types and harbored alterations in 43 oncogenes, most commonly ERBB2 (21.3%), PIK3CA (14.1%), and BRAF (8.7%). Median time from sequencing to enrollment was 163 days (interquartile range, 66 to 357 days), and from PRECISE identification to enrollment 87 days (interquartile range, 37 to 180 days). Common reasons for failing to identify patients before enrollment included accrual on the basis of molecular alterations that did not match pre-established PRECISE genomic eligibility (140 [33%] of 428) and external sequencing not available for parsing (127 [30%] of 428). CONCLUSION PRECISE identified 43% of all patients accrued to a diverse cohort of early-phase, genome-matched studies. Purpose-built informatics platforms represent a novel and potentially effective method for matching patients to molecularly selected studies.

PURPOSE Non–clear-cell renal cell carcinoma (nccRCC) encompasses approximately 20% of renal cell carcinomas and includes subtypes that vary in clinical and molecular biology. Compared with clear cell renal cell carcinoma, nccRCC demonstrates limited sensitivity to conventional vascular endothelial growth factor– and mammalian target of rapamycin–directed agents, indicating a need for better therapies. Characterizing the genomic landscape of metastatic nccRCC variants may help define novel therapeutic strategies. PATIENTS AND METHODS We retrospectively analyzed tumor tissue from patients with metastatic nccRCC who consented to genomic analysis of their tumor and germline DNA. A hybridization capture–based assay was used to identify single nucleotide variants and small insertions and deletions across more than 340 cancer-associated genes with germline comparison. Clinical actionability of somatic mutations was assessed using OncoKB levels of evidence. Microsatellite instability (MSI) in the tumor was investigated. RESULTS Of 116 patients included in the analysis, 57 (49%) presented with de novo metastatic disease, and 59 (51%) presented with localized disease that later metastasized. Subtype classifications included unclassified (n = 41; 35%), papillary (n = 26; 22%), chromophobe (n = 17; 15%), translocation associated (n = 13; 11%), and other (n = 19; 16%). Of all tumors, 15 (13%) had putative driver somatic alterations amenable to targeted therapies, including alterations in MET, TSC1/2, and an ALK translocation. Of 45 patients who had germline testing, 11 (24%) harbored mutations, seven of which could potentially guide therapy. Of 115 available tumors for analysis, two (1.7%) had high and six (5%) had intermediate MSI status. CONCLUSION The mutation profiles of metastatic nccRCC vary by subtype. Comprehensive analysis of somatic mutations, germline mutations, and MSI, interpreted via an annotated precision oncology knowledge base, identified potentially targetable alterations in 22% of patients, which merits additional investigation.

PURPOSE To detect alterations in DNA damage repair (DDR) genes, measure homologous recombination deficiency (HRD), and correlate these findings with clinical outcome in patients with leiomyosarcoma (LMS). PATIENTS AND METHODS Patients with LMS treated at Memorial Sloan Kettering (MSK) Cancer Center who consented to prospective targeted next-generation sequencing with MSK-IMPACT were screened for oncogenic somatic variants in one of 33 DDR genes; where feasible, an experimental HRD score was calculated from IMPACT data. Progression-free survival (PFS) and overall survival (OS) were estimated after stratifying patients by DDR gene alteration status and HRD score. RESULTS Of 211 patients with LMS, 20% had an oncogenic DDR gene alteration. Univariable analysis of PFS in 117 patients who received standard frontline chemotherapy in the metastatic setting found that an altered homologous recombination pathway gene was significantly associated with shorter PFS (hazard ratio [HR], 1.79; 95% CI, 1.04 to 3.07; P = .035). Non- BRCA homologous recombination gene alteration was associated with shorter PFS (HR, 2.61; 95% CI, 1.35 to 5.04; P = .004) compared with BRCA-altered and wild-type homologous recombination genes. Univariable analysis of OS from diagnosis in the entire cohort of 211 patients found that age, tumor size, number of metastatic sites, localized disease, and non- BRCA homologous recombination gene alteration were significantly associated with OS. On multivariable analysis, non- BRCA homologous recombination pathway gene alteration remained significant (HR, 4.91; 95% CI, 2.47 to 9.76; P < .001). High HRD score was not associated with a different PFS or OS. CONCLUSION Patients with LMS with homologous recombination pathway gene alterations have poor clinical outcomes, particularly those with non- BRCA gene alterations. HRD score calculated from a targeted exome panel did not discern disparate clinical outcomes.

PURPOSE: Although MET exon 14 (METex14)–altered lung cancers were first identified more than a decade and a half ago, the frequency of CNS metastatic disease remains poorly defined. Furthermore, the seminal trial of crizotinib in these patients (PROFILE 1001) did not report patterns of CNS response or progression. PATIENTS AND METHODS: Patients with pathologically confirmed, advanced non–small-cell lung cancers (NSCLC) harboring a METex14 alteration by targeted DNA/RNA sequencing were studied. The incidence of brain metastases and the outcomes of MET inhibition with crizotinib were analyzed. RESULTS: Eighty-three patients with METex14-altered metastatic NSCLC were identified. The incidence of CNS metastases at diagnosis was 17% (95% CI, 10% to 27%). The lifetime incidence was 36% (95% CI, 26% to 47%); 83% of patients had parenchymal disease, and 17% had leptomeningeal disease. The probability of having brain metastasis at 1, 2, and 3 years was 24%, 35%, and 38%, respectively. Fifty-four patients received crizotinib. The median time to radiologic CNS progression was 5.8 months (range, 3.7-20.0 months). Patterns of crizotinib progression were as follows: intracranial only in 10% of patients, intracranial and extracranial in 12%, and extracranial only in 78%. In patients with brain metastases before treatment, the median time on crizotinib was 7.5 months (range, 7.2-11.7 months). CONCLUSION: CNS metastases, including leptomeningeal disease, occurred in more than a third of patients with METex14-altered lung cancers. In crizotinib-treated patients with or without CNS metastases, CNS failure was seen in less than a quarter of patients on progression.

Purpose Pediatric sarcomas provide a unique diagnostic challenge. There is considerable morphologic overlap between entities, increasing the importance of molecular studies in the diagnosis, treatment, and identification of therapeutic targets. We developed and validated a genome-wide DNA methylation–based classifier to differentiate between osteosarcoma, Ewing sarcoma, and synovial sarcoma. Methods DNA methylation status of 482,421 CpG sites in 10 Ewing sarcoma, 11 synovial sarcoma, and 15 osteosarcoma samples were determined using the Illumina Infinium HumanMethylation450 array. We developed a random forest classifier trained from the 400 most differentially methylated CpG sites within the training set of 36 sarcoma samples. This classifier was validated on data drawn from The Cancer Genome Atlas synovial sarcoma, TARGET-Osteosarcoma, and a recently published series of Ewing sarcoma. Results Methylation profiling revealed three distinct patterns, each enriched with a single sarcoma subtype. Within the validation cohorts, all samples from The Cancer Genome Atlas were accurately classified as synovial sarcoma (10 of 10; sensitivity and specificity, 100%), all but one sample from TARGET-Osteosarcoma were classified as osteosarcoma (85 of 86; sensitivity, 98%; specificity, 100%), and 14 of 15 Ewing sarcoma samples were classified correctly (sensitivity, 93%; specificity, 100%). The single misclassified osteosarcoma sample demonstrated high EWSR1 and ETV1 expression on RNA sequencing, although no fusion was found on manual curation of the transcript sequence. Two additional clinical samples that were difficult to classify by morphology and molecular methods were classified as osteosarcoma; one had been suspected of being a synovial sarcoma and the other of being Ewing sarcoma on initial diagnosis. Conclusion Osteosarcoma, synovial sarcoma, and Ewing sarcoma have distinct epigenetic profiles. Our validated methylation-based classifier can be used to provide diagnostic assistance when histologic and standard techniques are inconclusive.

Purpose With prospective clinical sequencing of tumors emerging as a mainstay in cancer care, an urgent need exists for a clinical support tool that distills the clinical implications associated with specific mutation events into a standardized and easily interpretable format. To this end, we developed OncoKB, an expert-guided precision oncology knowledge base. Methods OncoKB annotates the biologic and oncogenic effects and prognostic and predictive significance of somatic molecular alterations. Potential treatment implications are stratified by the level of evidence that a specific molecular alteration is predictive of drug response on the basis of US Food and Drug Administration labeling, National Comprehensive Cancer Network guidelines, disease-focused expert group recommendations, and scientific literature. Results To date, > 3,000 unique mutations, fusions, and copy number alterations in 418 cancer-associated genes have been annotated. To test the utility of OncoKB, we annotated all genomic events in 5,983 primary tumor samples in 19 cancer types. Forty-one percent of samples harbored at least one potentially actionable alteration, of which 7.5% were predictive of clinical benefit from a standard treatment. OncoKB annotations are available through a public Web resource ( http://oncokb.org ) and are incorporated into the cBioPortal for Cancer Genomics to facilitate the interpretation of genomic alterations by physicians and researchers. Conclusion OncoKB, a comprehensive and curated precision oncology knowledge base, offers oncologists detailed, evidence-based information about individual somatic mutations and structural alterations present in patient tumors with the goal of supporting optimal treatment decisions.

Purpose Ewing sarcoma (ES) and desmoplastic small round cell tumors (DSRCTs) are aggressive sarcomas molecularly characterized by EWSR1 gene fusions. As pathognomonic genomic events in these respective tumor types, EWSR1 fusions represent robust potential biomarkers for disease monitoring. Methods To investigate the feasibility of identifying EWSR1 fusions in plasma-derived cell-free DNA (cfDNA) from patients with ES and DSRCT, we evaluated two complementary approaches in samples from 17 patients with radiographic evidence of disease. The first approach involved identification of patient-specific genomic EWSR1 fusion breakpoints in formalin-fixed, paraffin-embedded tumor DNA using a broad, hybridization capture-based next-generation sequencing (NGS) panel, followed by design of patient-specific droplet digital polymerase chain reaction (ddPCR) assays for plasma cfDNA interrogation. The second approach used a disease-tailored targeted hybridization capture-based NGS panel applied directly to cfDNA, which included EWSR1 as well as several other genes with potential prognostic use. Results EWSR1 fusions were identified in 11 of 11 (100%) ES and five of six (83%) DSRCT cfDNA samples by ddPCR, whereas 10 of 11 (91%) and four of six (67%) were identified by NGS. The ddPCR approach had higher sensitivity, ranging between 0.009% and 0.018%. However, the hybrid capture–based NGS assay identified the precise fusion breakpoints in the majority of cfDNA samples, as well as mutations in TP53 and STAG2, two other recurrent, clinically significant alterations in ES, all without prior knowledge of the tumor genotype. Conclusion These results provide a compelling rationale for an integrated approach using both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.

RET rearrangements are identified in 1% to 2% of non-small-cell lung cancers (NSCLCs).1,2 In patients with advanced, RET-rearranged lung cancers, systemic therapy can be highly active. We demonstrated previously that pemetrexed-based chemotherapy can achieve an objective response rate of 45% and a median progression-free survival (PFS) of 19 months.3 Furthermore, the activity of targeted therapy has improved dramatically with the introduction of selective RET inhibitors to the clinic. In early-phase testing, objective response rates with LOXO-2924 and BLU-6675 are 68% (26 of 38) and 50% (seven of 14), respectively. These outcomes exceed the modest activity observed previously with multikinase inhibitors such as cabozantinib6 and vandetanib.7 In contrast, the activity of immunotherapy in RET-rearranged lung cancers has not been well characterized. This represents a clear unmet need, given that all prior regulatory approvals of immune checkpoint inhibitors, either alone or in combination with chemotherapy, and in stage III or IV disease, have technically included patients with RET-rearranged lung cancers.8,9 Furthermore, although increasing levels of programmed death-ligand 1 (PD-L1) expression and high tumor mutational burden (TMB) have been associated with benefit from immune checkpoint blockade,10 the immunophenotype of RET-rearranged lung cancers and the role of PD-L1 and TMB status in relation to benefit with immunotherapy remain poorly described. We set out to characterize these factors.

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.

Purpose We assessed the usefulness of real-time molecular profiling through next-generation sequencing (NGS) in predicting the tumor biology of advanced pancreatic neuroendocrine tumors (panNETs) and in characterizing genomic evolution. Methods Patients with metastatic panNETs were recruited in the routine clinical practice setting (between May 2014 and March 2017) for prospective NGS of their tumors as well as for germline analysis using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing platform. When possible, NGS was performed at multiple time points. Results NGS was performed in 96 tumor samples from 80 patients. Somatic alterations were identified in 76 of 80 patients (95%). The most commonly altered genes were MEN1 (56%), DAXX (40%), ATRX (25%), and TSC2 (25%). Alterations could be defined in pathways that included chromatin remodeling factors, histone methyltransferases, and mammalian target of rapamycin pathway genes. Somatic loss of heterozygosity was particularly prevalent (55 of 95 tested samples [58%]), and the presence of loss of heterozygosity resulted in improved overall survival (P = .06). Sequencing of pre- and post-treatment samples revealed tumor-grade progression; clonal evolution patterns were also seen (molecular resistance mechanisms and chemotherapy-associated mutagenesis). Germline genetic analysis identified clinically actionable pathogenic or likely pathogenic variants in 14 of 88 patients (16%), including mutations in high-penetrance cancer susceptibility genes ( MEN1, TSC2, and VHL). Conclusion A clinical NGS platform reveals pertubations of biologic pathways in metastatic panNETs that may inform prognosis and direct therapies. Repeat sequencing at disease progression reveals increasing tumor grade and genetic evolution, demonstrating that panNETs adopt a more aggressive behavior through time and therapies. In addition to frequent somatic mutations in MEN1 and TSC2, germline mutations in these same genes underlie susceptibility to panNETs and highlight the need to re-evaluate whether germline genetic analysis should be performed for all patients with panNETs.

Ewing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized byTo investigate the feasibility of identifyingThese results provide a compelling rationale for an integrated approach utilizing both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.