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ABSTRACT Background There has been a paradoxical rise in young‐onset gastric cancer (YOGC), defined as gastric cancer (GC) diagnosed before age 50. Precursor lesions may contribute to pathogenesis, though their role in progression to different histologic subtypes is unclear. The impact of self‐reported race is also poorly characterized and may be unreliable as a proxy for genetic differences. We aimed to compare differences in histology and genetic ancestry between YOGC and average‐onset gastric cancer (AOGC). Methods This retrospective cohort included all patients with GC at Memorial Sloan Kettering (MSK) from January 2013 to March 2021. Data on demographics, tumor characteristics, and precursor lesions were collected. Genetic ancestry was inferred from MSK‐Integrated Mutation Profiling of Actionable Cancer Targets panel. Results Of 1685 individuals with GC, 290 had YOGC. Compared to AOGC, individuals with YOGC tended to be female, Hispanic, foreign‐born, and feature diffuse‐type histology. YOGC was less likely to have precursor lesions, including intestinal metaplasia (20% vs. 37%, p

Abstract Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture-based next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms.

Comprehensive genomic sequencing is becoming a critical component in the assessment of hematologic malignancies, with broad implications for patient management. In this context, unequivocally discriminating somatic from germline events is challenging but greatly facilitated by matched analysis of tumor:normal pairs. In contrast to solid tumors, conventional sources of normal control (peripheral blood, buccal swabs, saliva) could be highly involved by the neoplastic process, rendering them unsuitable. In this work we describe our real-world experience using cell free DNA (cfDNA) isolated from nail clippings as an alternate source of normal control, through the dedicated review of 2,610 tumor:nail pairs comprehensively sequenced by MSK-IMPACT-heme. Overall, we find nail cfDNA is a robust source of germline control for paired genomic studies. In a subset of patients, nail DNA may have tumor DNA contamination, reflecting unique attributes of the hematologic disease and transplant history. Contamination is generally low level, but significantly more common among patients with myeloid neoplasms (20.5%; 304/1482) compared to lymphoid diseases (5.4%; 61/1128) and particularly enriched in myeloproliferative neoplasms with marked myelofibrosis. When identified in patients with lymphoid and plasma-cell neoplasms, mutations commonly reflected a myeloid profile and correlated with a concurrent/evolving clonal myeloid neoplasm. For nails collected after allogeneic stem-cell transplantation, donor DNA was identified in 22% (11/50). In this cohort, an association with recent history of graft-vs-host disease was identified. These findings should be considered as a potential limitation for the use of nail as normal control but could also provide important diagnostic information regarding the disease process.

In biliary tract cancer HER2 alterations correlate with poor prognosis. Here, the authors present the results of a phase II clinical trial reporting the efficacy and safety of the tyrosine kinase inhibitor neratinib in patients with HER2-mutation positive advanced biliary tract cancers.

Mutations in BRCA1/2 are associated with a homologous recombination deficiency phenotype in BRCA-associated cancers. Reversion mutations can restore BRCA1/2 function and result in treatment resistance in these cancer-types. Here, the authors show that, in select cases, reversion mutations in BRCA1/2 can indicate prior BRCA-mediated tumorigenesis in non-canonical histologies.

Abstract Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(−) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.

Sarcomas are rare tumours with many different subtypes and clinical outcomes; a broader knowledge of their genetic features is required. Here, the authors analyse 2138 soft tissue and bone sarcomas across 45 subtypes using MSK-IMPACT targeted sequencing and find genomic groups that are distinct from histological subgroups.

The results of the phase 1/2 LIBRETTO-001 clinical trial has recently established the efficacy of the RET inhibitor selpercatinib in RET-driven cancers. Here, the authors characterize the molecular determinants of response and resistance in 72 LIBRETTO-001 lung and thyroid cancer patients treated at a single site.

Cell free DNA (cfDNA) and circulating tumor cell free DNA (ctDNA) from blood (plasma) are increasingly being used in oncology for diagnosis, monitoring response, identifying cancer causing mutations and detecting recurrences. Circulating tumor RB1 DNA (ctDNA) is found in the blood (plasma) of retinoblastoma patients at diagnosis before instituting treatment (naïve). We investigated ctDNA in naïve unilateral patients before enucleation and during enucleation (6 patients/ 8 mutations with specimens collected 5-40 minutes from severing the optic nerve) In our cohort, following transection the optic nerve, ctDNA RB1 VAF was measurably lower than pre-enucleation levels within five minutes, 50% less within 15 minutes and 90% less by 40 minutes.

Understanding the molecular and phenotypic profile of colorectal cancer (CRC) in West Africa is important for early detection and treatment. Here, the authors use a multigene next-generation sequencing panel to identify genomic differences in Nigerian CRCs compared to those from TCGA and MSKCC cohorts.

Clonal haematopoiesis (CH) has been associated with altered inflammatory profiles and increased risk of cardiovascular and malignant diseases. Here, the authors analyze patient data from two different cohorts and show that CH is associated with severe infections and severe Covid19.

Objectives Because retinoblastoma diagnosis is usually made with the indirect ophthalmoscope without biopsy clinical errors continue to occur worldwide. Because cf RB1 is detectible in plasma of children with retinoblastoma, we wondered if it was present in the blood of pseudoretinoblastomas with the hope of ultimately developing a blood based test to aid clinicians in the diagnosis of retinoblastoma. The goal of this project was to see if circulating plasma RB1 cfDNA could be detected in the blood of patients with pseudoretinoblastoma.Methods and analysis Plasma cfDNA for circulating RB1 cfDNA was assayed with MSKCC’s next generation sequencing, N.Y. State Approved assay called ACCESS to evaluate somaticmutations in 40 patients with pseudoretinoblastoma.Results No plasma cfDNA RB1 was detected in the blood (plasma) of 40 patients with pseudoretinoblastoma.Conclusion Plasma cfDNA RB1 is commonly detectible in retinoblastoma patients but not in patients with a diverse group of pseudoretinoblastomas.

Abstract To find metastatic recurrence biomarkers of triple-negative breast cancer (TNBC) treated by neoadjuvant chemotherapy and anti-EGFR antibodies (NAT), we evaluated tumor genomic, transcriptomic, and immune features, using MSK-IMPACT assay, gene arrays, Nanostring technology, and TIL assessment on H&E. Six patients experienced a rapid fatal recurrence (RR) and other 6 had later non-fatal recurrences (LR). Before NAT, RR had low expression of 6 MHC class I and 13 MHC class II genes but were enriched in upregulated genes involved in the cell cycle-related pathways. Their TIL number before NAT in RR was very low (

Liquid biopsies allow the non-invasive detection of somatic mutations from tumours. Here, the authors develop and test MSK-ACCESS, an NGS-based clinical assay for identifying low frequency mutations in 129 genes and describe how it benefits patients in the clinic.

Abstract Background Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth. Methods Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES). Results cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score

There is an unmet clinical need for simple, accessible biomarkers to select patients who are more likely to respond to immune checkpoint therapy. Here the authors show that a lower neutrophil-to-lymphocyte ratio is associated with better overall and progressive-free survival, as well as higher rate of response, in a multi-cancer cohort of patients treated with immune checkpoint inhibitors.

Patients with solid cancers have high rates of clonal haematopoiesis associated with increased risk of secondary leukemias. Here, by using peripheral blood sequencing data from patients with solid non-hematologic cancer, the authors profile the landscape of mosaic chromosomal alterations and gene mutations, defining patients at high risk of leukemia progression.

Abstract Purpose The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell‐free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. Methods Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra‐ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA‐cleared tumor sequencing assay, MSK‐IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. Results Tumor‐guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. Conclusion Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow‐up schemes, and risk of metastasis.

In lung cancer, relatively few germline mutations are known to impact risk. Here the authors looked at rare variants in 39,146 individuals and find novel germline mutations associated with risk, as well as implicating ATM and a new candidate gene for lung cancer risk.

Purpose: Circulating tumor DNA (ctDNA) is released by many tumors into the plasma. Its analysis has minimal procedural risk and, in many cancers, has the potential for clinical applications. In retinoblastoma, the clinical correlations of ctDNA in eyes treated without enucleation have not been studied. This purpose of this study was to determine how the ctDNA RB1 variant allele frequency (VAF) changes in patients with unilateral retinoblastoma after intra-arterial chemotherapy (IAC) treatment. Variant allele frequency is a proxy for tumor fraction. Design: Case series from a single tertiary cancer referral center. Participants: Five patients with retinoblastoma with at least 1 measurable ctDNA plasma specimen both at the time of active intraocular retinoblastoma before IAC and after at least 1 IAC cycle. Methods: Circulating tumor DNA RB1 was detected and VAF was measured before and after IAC treatment. Clinical correlations were made using clinical examination, fundus photography, ultrasound, and OCT. Main Outcome Measures: Comparison of ctDNA RB1 VAF before and after IAC treatment for retinoblastoma and concordance of ctDNA RB1 detectability with activity of intraocular disease. Results: Twenty-three ctDNA specimens were included from 5 patients. The 5 baseline RB1 VAFs ranged from 0.27% to 4.23%. In all patients, the subsequent post–intra-arterial RB1 VAF was lower than baseline (0.0%–0.17%). At 4 months (2 months after IAC completion), the ctDNA consistently was negative in the patients who demonstrated clinically inactive intraocular disease. Conclusions: In this small cohort, a decremental decrease in ctDNA RB1 VAF was found after IAC, suggesting that relative VAF changes could be a biomarker of treatment response.

Purpose: Analysis of circulating tumor DNA (ctDNA) in the plasma of patients with retinoblastoma and simulating lesions. Design: Retrospective cross-sectional study of the association of plasma ctDNA from retinoblastoma and simulating lesions with disease course. Participants: Fifty-eight Memorial Sloan Kettering Cancer Center patients with retinoblastoma comprising 68 plasma ctDNA samples and 5 with retinoblastoma-simulating lesions. Methods: The ctDNA analyzed with hybridization capture and next-generation sequencing in blood (plasma) of patients who had retinoblastoma or simulating lesions were evaluated for association with clinical course of the disease. Main Outcome Measures: Presence or absence of molecular aberrations in the RB1 gene and correlations with clinical features. Results: RB1 cell-free DNA (cfDNA) was detected in 16 of 19 patients with newly diagnosed, untreated intraocular retinoblastoma and in 3 of 3 patients with newly diagnosed, untreated metastatic disease. It was also present in 3 patients with recurrent intraocular disease before therapy, but was not present in patients with recurrent disease who received intra-arterial chemotherapy, nor in 21 patients who had undergone enucleation for unilateral disease. In 1 patient who had delayed treatment (insurance reasons) and showed rapid growth of the intraocular tumor, the variant allele frequency increased in 1 month from 0.34% to 2.48%. No RB1 mutations were detected in the cfDNA from plasma of patients with simulating lesions (3 with Coats disease and 1 with persistent fetal vasculature [PFV]). In 2 patients, we identified 2 independent RB1 mutations in plasma. Conclusions: Mutations in RB1 were found in the cfDNA from blood of patients with newly diagnosed, untreated retinoblastoma and in patients who showed disease recurrence in the eye after prior treatment, but not in unilateral retinoblastoma after enucleation Levels of ctDNA increase in patients with progressive disease who did not receive any treatment. High plasma cfDNA levels were detected in patients with newly diagnosed metastatic disease, and these levels decreased after systemic chemotherapy was administered. Further validation is needed for measuring the somatic alterations in cfDNA from blood in retinoblastoma that could provide a promising method of monitoring patients in the future.

Abstract Background The growing number of Next Generation Sequencing (NGS) tests is transforming the routine clinical diagnosis of hereditary cancers. Identifying whether a cancer is the result of an underlying disease-causing mutation in a cancer predisposition gene is not only diagnostic for a cancer predisposition syndrome, but also has significant clinical implications in the clinical management of patients and their families. Methods Here, we evaluated the performance of MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) in detecting genetic alterations in 76 genes implicated in cancer predisposition syndromes. Output from hybridization-based capture was sequenced on an Illumina HiSeq 2500. A custom analysis pipeline was used to detect single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variants (CNVs). Results MSK-IMPACT detected all germline variants in a set of 233 unique patient DNA samples, previously confirmed by previous single gene testing. Reproducibility of variant calls was demonstrated using inter- and intra- run replicates. Moreover, in 16 samples, we identified additional pathogenic mutations other than those previously identified through a traditional gene-by-gene approach, including founder mutations in BRCA1, BRCA2, CHEK2 and APC, and truncating mutations in TP53, TSC2, ATM and VHL. Conclusions This study highlights the importance of the NGS-based gene panel testing approach in comprehensively identifying germline variants contributing to cancer predisposition and simultaneous detection of somatic and germline alterations.

The clinical significance of BRAF alterations in well-differentiated (WD) metastatic pancreatic neuroendocrine tumor (panNET) is unknown, but BRAF-mutated panNET could represent a subset characterized by an identifiable and clinically actionable driver. Following the identification of two patients with WD metastatic panNET whose tumors harbored BRAF mutations, we queried the MSK-IMPACT series of 80 patients with WD metastatic panNET for additional mutations in BRAF, and in other genes involved in RAS/ RTK/ PI3K signaling pathways. BRAF mutations were identified in six samples (7.5%): two tumors harbored V600E mutations, one tumor each expressed K601E, T599K, and T310I mutations, and one tumor expressed both G596D and E451K BRAF. Few additional actionable driver alterations were identified. To determine the ERK activating capability of four BRAF mutations not previously characterized, mutant constructs were tested in model systems. Biochemical characterization of BRAF mutations revealed both high and low activity mutants. Engineered cells expressing BRAF K601E and V600E were used for in vitro drug testing of RAF and MEK inhibitors currently in clinical use. BRAF K601E demonstrated reduced sensitivity to dabrafenib compared to BRAF V600E, but the combination of RAF plus MEK inhibition was effective in cells expressing this mutation. Herein, we describe the clinical course of a patient with BRAF K601E and a patient with BRAF V600E WD metastatic panNET, and the identification of four mutations in BRAF not previously characterized. The combined clinical and biochemical data support a potential role for RAF and MEK inhibitors, or a combination of these, in a selected panNET population.