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Purpose: Even though BRAF fusions are increasingly detected in standard multigene next-generation sequencing panels, few reports have explored their structure and impact on clinical course., Experimental Design: We collected data from patients with BRAF fusion-positive cancers identified through a genotyping protocol of 97,024 samples. Fusions were characterized and reviewed for oncogenic potential (in-frame status, non-BRAF partner gene, and intact BRAF kinase domain)., Results: We found 241 BRAF fusion-positive tumors from 212 patients with 82 unique 5' fusion partners spanning 52 histologies. Thirty-nine fusion partners were not previously reported, and 61 were identified once. BRAF fusion incidence was enriched in pilocytic astrocytomas, gangliogliomas, low-grade neuroepithelial tumors, and acinar cell carcinoma of the pancreas. Twenty-four patients spanning multiple histologies were treated with MAPK-directed therapies, of which 20 were evaluable for RECIST. Best response was partial response (N = 2), stable disease (N = 11), and progressive disease (N = 7). The median time on therapy was 1 month with MEK plus BRAF inhibitors [(N = 11), range 0-18 months] and 8 months for MEK inhibitors [(N = 14), range 1-26 months]. Nine patients remained on treatment for longer than 6 months [pilocytic astrocytomas (N = 6), Erdheim-Chester disease (N = 1), extraventricular neurocytoma (N = 1), and melanoma (N = 1)]. Fifteen patients had acquired BRAF fusions., Conclusions: BRAF fusions are found across histologies and represent an emerging actionable target. BRAF fusions have a diverse set of fusion partners. Durable responses to MAPK therapies were seen, particularly in pilocytic astrocytomas. Acquired BRAF fusions were identified after targeted therapy, underscoring the importance of postprogression biopsies to optimize treatment at relapse in these patients., (©2024 American Association for Cancer Research.)

Cancer-associated venous thromboembolism (VTE) is a major source of oncologic cost, morbidity and mortality. Identifying high-risk patients for prophylactic anticoagulation is challenging and adds to clinician burden. Circulating tumor DNA (ctDNA) sequencing assays ('liquid biopsies') are widely implemented, but their utility for VTE prognostication is unknown. Here we analyzed three plasma sequencing cohorts: a pan-cancer discovery cohort of 4,141 patients with non-small cell lung cancer (NSCLC) or breast, pancreatic and other cancers; a prospective validation cohort consisting of 1,426 patients with the same cancer types; and an international generalizability cohort of 463 patients with advanced NSCLC. ctDNA detection was associated with VTE independent of clinical and radiographic features. A machine learning model trained on liquid biopsy data outperformed previous risk scores (discovery, validation and generalizability c-indices 0.74, 0.73 and 0.67, respectively, versus 0.57, 0.61 and 0.54 for the Khorana score). In real-world data, anticoagulation was associated with lower VTE rates if ctDNA was detected (n = 2,522, adjusted hazard ratio (HR) = 0.50, 95% confidence interval (CI): 0.30-0.81); ctDNA - patients (n = 1,619) did not benefit from anticoagulation (adjusted HR = 0.89, 95% CI: 0.40-2.0). These results provide preliminary evidence that liquid biopsies may improve VTE risk stratification in addition to clinical parameters. Interventional, randomized prospective studies are needed to confirm the clinical utility of liquid biopsies for guiding anticoagulation in patients with cancer., (© 2024. The Author(s).)

Introduction: No definitive answers currently exist regarding optimal first-line therapy for HER2-mutant NSCLC. Access to rapid tissue sequencing is a major barrier to precision drug development in the first-line setting. ctDNA analysis has the potential to overcome these obstacles and guide treatment., Methods: We retrospectively analyzed patients with metastatic HER2-mutant NSCLC who underwent prospective clinical ctDNA sequencing and received systemic therapy at Memorial Sloan Kettering Cancer Center (MSK) from January 2016 to September 2022. HER2 mutations were identified by next-generation sequencing through MSK-IMPACT, MSK-ACCESS or Resolution ctDx LungTM assay. Primary endpoints were time to the next treatment (TTNT) and overall survival (OS)., Results: Sixty-three patients were included in the primary analysis. Chemoimmunotherapy (33/63, 52.4 %) was the predominant first-line treatment with a median TTNT of 5.1 months (95 %CI 4.1 - 6.1) whereas 55.0 % (22/40) of patients who received second-line T-DXd obtained a median TTNT of 9.2 m (95 % CI, 0-22.2). Plasma ctDNA was tested before first-line therapy in 40 patients with a median OS of 28.0 months (95 % CI 21-34), in whom 31 patients (78.0 %) had detectable ctDNA. HER2 mutations were detected on ctDNA with a median turnaround time of 13 days, occasionally co-occurred with EGFR and MET alterations and were tracked longitudinally correlating with treatment response. Patients with detectable baseline ctDNA had significantly shorter OS (hazard ratio (HR), 5.25; 95 % CI, 1.2-23.9; p = 0.019)., Conclusion: Chemoimmunotherapy remains a major treatment option for metastatic HER2-mutant NSCLC. ctDNA can rapidly detect HER2 and co-mutations, and it has the potential to guide and monitor optimal first-line therapy. As a negative prognostic biomarker, detectable ctDNA at baseline would need to be taken into account for patient selection in future studies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bob T. Li has served as an uncompensated advisor and consultant to Amgen, AstraZeneca, Boehringer Ingelheim, Bolt Biotherapeutics, Daiichi Sankyo, Genentech, and Lilly. He has received research grants to his institution from Amgen, AstraZeneca, Bolt Biotherapeutics, Daiichi Sankyo, Genentech, Jiangsu Hengrui Pharmaceuticals, Lilly, Nuvalent, and Revolution Medicines. He has received academic travel support from Amgen. He is an inventor on three institutional patents at MSK (US62/685,057, US62/514,661, US63/424,813) and has intellectual property rights as a book author at Karger Publishers and Shanghai Jiao Tong University Press. Alexander Drilon declared as follows: Honoraria: 14ner/Elevation Oncology, Amgen, Abbvie, ArcherDX, AstraZeneca, Beigene, BergenBio, Blueprint Medicines, Bristol Myers Squibb, Chugai Pharmaceutical, EcoR1, EMD Serono, Entos, Exelixis, Helsinn, Hengrui Therapeutics, Ignyta/Genentech/Roche, Janssen, Loxo/Bayer/Lilly, Merus, Monopteros, MonteRosa, Novartis, Nuvalent, Pfizer, Prelude, Regeneron, Repare RX, Springer Healthcare, Takeda/Ariad/Millenium, Treeline Bio, TP Therapeutics, Tyra Biosciences, Verastem. Advisory Boards: Bayer, MonteRosa, Abbvie, EcoR1 Capital, LLC, Amgen, Helsinn, Novartis, Lilly, AnHeart Therapeutics. Consulting: MonteRosa, Innocare, Boundless Bio, Treeline Bio, Nuvalent, 14ner/Elevation Oncology, Entos, Prelude. Associated Research Paid to Institution: Foundatin Medicine, GlaxoSmithKlein, Teva, Taiho, PharmaMar. Equity: mBrace. Copyright: Selpercatinib-Osimertinib (filed/pending). Royalties: Wolters Kluwer; Other (Food/Beverage): Merck, Puma, Merus, Boehringer Ingelheim; CME Honoraria: Answers in CME, Applied Pharmaceutical Science, Inc, AXIS, Clinical Care Options, Doc Congress, EPG Health, Harborside Nexus, I3 Health, Imedex, Liberum, Medendi, Medscape, Med Learning, MedTalks, MJH Life Sciences, MORE Health, Ology, OncLive, Paradigm, Peerview Institute, PeerVoice, Physicians Education, Projects in Knowledge, Resources, Remedica Ltd, Research to Practice, RV More, Targeted Oncology, TouchIME, WebMD. Sarat Chandarlapaty has received institutional grant/funding from Daiichi-Sankyo, AstraZeneca, and Lilly, Shares/Ownership interests in Odyssey Biosciences, Effector Therapeutics, and Totus Medicines, and consultation/Ad board/Honoraria from AstraZeneca, Daiichi-Sankyo, Novartis, Neogenomics, Nuvalent, Blueprint, SAGA Diagnostics, and Effector Therapeutics. Jiannan Guo is a former employee of Resolution Bioscience. Eric Gagne, Kavita Garg and Frederick Baehner are employees of Exact Sciences. All remaining authors have declared no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Background and Objective: The source of tissue for genomic profiling of metastatic castration-resistant prostate cancer (mCRPC) is often limited to osseous metastases. To guide patient management, metastatic site selection and the technique for targeted bone biopsies are critical for identifying deleterious gene mutations. Our objective was to identify key parameters associated with successful large-panel DNA sequencing., Methods: We analyzed parameters for 243 men with progressing mCRPC who underwent 269 bone biopsies for genomic profiling between 2014 and 2018. Univariate and multivariate analyses were performed for clinical, imaging (bone scan; fluorodeoxyglucose [FDG] positron emission tomography [PET]; computed tomography [CT]; magnetic resonance imaging), and technical (biopsy site, number of samples, needle gauge) features associated with successful genomic profiling., Key Findings and Limitations: Overall, 159 of 269 biopsies (59%) generated sufficient tumor material for a genomic profile. Seventy (26%) of the failures were histopathologically negative for mCRPC and 40 (15%) had insufficient tumor for genomic profiling. Of 199 mCRPC samples submitted for molecular testing, 159 (80%) yielded a genomic profile. On univariate analysis, PSA, serum acid phosphatase, number of biopsy samples, FDG PET positivity, CT attenuation, and CT morphology were significantly associated with genomic profiling success. On multivariate analysis, higher FDG maximum standardized uptake value (odds ratio [OR] 7.51, 95% confidence interval [CI] 3.01-18.78; p < 0.001), higher number of biopsy samples (OR 4.73, 95% CI 1.49-15.02; p = 0.008), and lower mean CT attenuation (OR 0.4, 95% CI 0.18-0.89; p = 0.025) were significantly associated with sequencing success., Conclusions and Clinical Implications: In patients with mCRPC, bone biopsies from sites with metabolic activity and lower CT attenuation are associated with higher success rates for genomic profiling via a large-panel DNA sequencing platform., Patient Summary: We identified factors associated with successful genetic testing of bone tissue for patients with metastatic prostate cancer. Our findings may help in guiding the right scan technique and biopsy site for personalized treatment planning., (Published by Elsevier B.V.)

Purpose: Circulating tumor DNA (ctDNA) is released into the plasma by many cancers and offers clinical applications including noninvasive diagnostics. Histiocytosis results from myelogenous clonal expansion of histiocytes, predominantly driven by mutations in the mitogen-activated protein kinase pathway that are potentially detectable by ctDNA-based sequencing assays. However, ocular-involving histiocytosis is often a diagnostic challenge leading to delayed diagnosis and the need for invasive biopsy of sensitive ocular structures. The purpose of this study is to determine whether sequencing of plasma-derived ctDNA can noninvasively diagnose ocular-involving histiocytosis., Design: Single tertiary cancer referral center., Participants: Twenty-four adult patients with ocular-involving histiocytosis and ctDNA sequencing., Methods: Circulating tumor DNA was analyzed (via digital droplet polymerase chain reaction for BRAF V600E, and/or next-generation sequencing) and variant allele frequency was measured at initial presentation to our center. Patient demographics, clinical characteristics, and oncogenic mutations identified from tumor-based sequencing were recorded., Main Outcome Measures: Plasma-derived ctDNA detectability of pertinent driver mutations of histiocytosis., Results: At the initial presentation of 14 patients with ocular-involving histiocytosis, sequencing of plasma-derived ctDNA detected driver mutations for histiocytosis (BRAF V600E [10], KRAS [2], ARAF [1], and concurrent MAP2K1/KRAS [1]). Mutations found in circulating cell-free DNA were 100% concordant in 11 of 11 patients with mutations identified by solid tumor sequencing. Of 10 patients without driver mutation detected in ctDNA, 3 patients had alterations (CBL mutation or kinase fusion) not captured in the ctDNA sequencing assay, 3 were wildtype even by tumor sequencing; in 4 patients, tumor-based sequencing identified mutations (BRAF [2], MAP2K1 [2]) not detected in ctDNA. Detectable mutations in ctDNA were significantly more likely in patients with uveal infiltration ( P  = 0.036)., Conclusions: In this cohort, plasma-derived ctDNA was detectable and diagnostic in the majority of patients with ocular-involving histiocytosis. This suggests that if ocular histiocytosis is suspected (particularly if involving the uvea), noninvasive plasma-derived ctDNA analysis is a helpful diagnostic tool that may obviate the need to invasively biopsy sensitive ocular structures., Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (© 2024 by the American Academy of Ophthalmology.)

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.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in post-treatment settings can be crucial for relapse risk stratification in patients with B-cell and plasma cell neoplasms. Prior studies have focused on validation of various technical aspects of the MRD assays, but more studies are warranted to establish the performance characteristics and enable standardization and broad utilization in routine clinical practice. Here, the authors describe an NGS-based IGH MRD quantification assay, incorporating a spike-in calibrator for monitoring B-cell and plasma cell neoplasms based on their unique IGH rearrangement status. Comparison of MRD status (positive or undetectable) by NGS and flow cytometry (FC) assays showed high concordance (91%, 471/519 cases) and overall good linear correlation in MRD quantitation, particularly for chronic lymphocytic leukemia and B-lymphoblastic leukemia/lymphoma (R = 0.85). Quantitative correlation was lower for plasma cell neoplasms, where underestimation by FC is a known limitation. No significant effects on sequencing efficiency by the spike-in calibrator were observed, with excellent inter- and intra-assay reproducibility within the authors' laboratory, and in comparison to an external laboratory, using the same assay and protocols. Assays performed both at internal and external laboratories showed highly concordant MRD detection (100%) and quantitation (R = 0.97). Overall, this NGS-based MRD assay showed highly reproducible results with quantitation that correlated well with FC MRD assessment, particularly for B-cell neoplasms., Competing Interests: Disclosure Statement M.E.A. has served as a consultant and received honoraria from Biocartis US, Inc., Invivoscribe, Inc. Janssen Global Services, Bristol Myers Squibb, AstraZeneca, Roche, and Merck. C.H. has received honoraria from Blueprint Medicines, Hematopathology Advisory Board, and is an employee of Loxo Oncology, Inc. Y.H. and J.M. were employees of Invivoscribe, Inc., which developed and sells the commercial assay used in this paper. K.P.-D. received an honorarium from Invivoscribe not related to this study. M.R. has served as a consultant for BD Biosciences, Agios, and Celgene, as well as received contract research funding for Agios, Roche, BMS, and Bayer. A.D. has received consulting fees from Physicians' Education Resource, Seattle Genetics, Takeda, Roche, EUSA Pharma, Peerview Institute, Corvus Pharmaceuticals, and AbbVie, as well as research support from Roche and Takeda. C.V. has received consulting fees from DocDoc Pte. Ltd. and Paige.AI, Inc., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: Microsatellite instability (MSI) and mismatch repair (MMR) deficiency represent a distinct oncogenic process and predict response to immune checkpoint inhibitors (ICIs). The clinicopathologic features of MSI-high (MSI-H) and MMR deficiency (MMR-D) in lung cancers remain poorly characterized., Methods: MSI status from 5171 patients with NSCLC and 315 patients with SCLC was analyzed from targeted next-generation sequencing data using two validated bioinformatic pipelines., Results: MSI-H and MMR-D were identified in 21 patients with NSCLC (0.41%) and six patients with SCLC (1.9%). Notably, all patients with NSCLC had a positive smoking history, including 11 adenocarcinomas. Compared with microsatellite stable cases, MSI-H was associated with exceptionally high tumor mutational burden (37.4 versus 8.5 muts/Mb, p < 0.0001), MMR mutational signatures (43% versus 0%, p < 0.0001), and somatic biallelic alterations in MLH1 (52% versus 0%, p < 0.0001). Loss of MLH1 and PMS2 expression by immunohistochemistry was found in MLH1 altered and wild-type cases. Similarly, the majority of patients with MSI-H SCLC had evidence of MLH1 inactivation, including two with MLH1 promoter hypermethylation. A single patient with NSCLC with a somatic MSH2 mutation had Lynch syndrome as confirmed by the presence of a germline MSH2 mutation. Among patients with advanced MSI-H lung cancers treated with ICIs, durable clinical benefit was observed in three of eight patients with NSCLC and two of two patients with SCLC. In NSCLC, STK11, KEAP1, and JAK1 were mutated in nonresponders but wild type in responders., Conclusions: We present a comprehensive clinicogenomic landscape of MSI-H lung cancers and reveal that MSI-H defines a rare subset of lung cancers associated with smoking, high tumor mutational burden, and MLH1 inactivation. Although durable clinical benefit to ICI was observed in some patients, the broad range of responses suggests that clinical activity may be modulated by co-mutational landscapes., (Copyright © 2023 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

There is a continuing debate about the proportion of cancer patients that benefit from precision oncology, attributable in part to conflicting views as to which molecular alterations are clinically actionable. To quantify the expansion of clinical actionability since 2017, we annotated 47,271 solid tumors sequenced with the MSK-IMPACT clinical assay using two temporally distinct versions of the OncoKB knowledge base deployed 5 years apart. Between 2017 and 2022, we observed an increase from 8.9% to 31.6% in the fraction of tumors harboring a standard care (level 1 or 2) predictive biomarker of therapy response and an almost halving of tumors carrying nonactionable drivers (44.2% to 22.8%). In tumors with limited or no clinical actionability, TP53 (43.2%), KRAS (19.2%), and CDKN2A (12.2%) were the most frequently altered genes., Significance: Although clear progress has been made in expanding the availability of precision oncology-based treatment paradigms, our results suggest a continued unmet need for innovative therapeutic strategies, particularly for cancers with currently undruggable oncogenic drivers. See related commentary by Horak and Fröhling, p. 18. This article is featured in Selected Articles from This Issue, p. 5., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: Direct KRASG12C inhibitors are approved for patients with non-small cell lung cancers (NSCLC) in the second-line setting. The standard-of-care for initial treatment remains immune checkpoint inhibitors, commonly in combination with platinum-doublet chemotherapy (chemo-immunotherapy). Outcomes to chemo-immunotherapy in this subgroup have not been well described. Our goal was to define the clinical outcomes to chemo-immunotherapy in patients with NSCLC with KRASG12C mutations., Patients and Methods: Through next-generation sequencing, we identified patients with advanced NSCLC with KRAS mutations treated with chemo-immunotherapy at 2 institutions. The primary objective was to determine outcomes and determinants of response to first-line chemo-immunotherapy among patients with KRASG12C by evaluating objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). We assessed the impact of coalterations in STK11/KEAP1 on outcomes. As an exploratory objective, we compared the outcomes to chemo-immunotherapy in KRASG12C versus non-G12C groups., Results: One hundred and thirty eight patients with KRASG12C treated with first-line chemo-immunotherapy were included. ORR was 41% (95% confidence interval (CI), 32-41), median PFS was 6.8 months (95%CI, 5.5-10), and median OS was 15 months (95%CI, 11-28). In a multivariable model for PFS, older age (P = .042), squamous cell histology (P = .008), poor ECOG performance status (PS) (P < .001), and comutations in KEAP1 and STK11 (KEAP1MUT/STK11MUT) (P = .015) were associated with worse PFS. In a multivariable model for OS, poor ECOG PS (P = .004) and KEAP1MUT/STK11MUT (P = .009) were associated with worse OS. Patients with KRASG12C (N = 138) experienced similar outcomes to chemo-immunotherapy compared to patients with non-KRASG12C (N = 185) for both PFS (P = .2) and OS (P = .053)., Conclusions: We define the outcomes to first-line chemo-immunotherapy in patients with KRASG12C, which provides a real-world benchmark for clinical trial design involving patients with KRASG12C mutations. Outcomes are poor in patients with specific molecular coalterations, highlighting the need to develop more effective frontline therapies., (© The Author(s) 2023. Published by Oxford University Press.)

Abstract: Primary cutaneous gamma-delta T-cell lymphoma (PCGDTL) is a rare and diagnostically challenging primary skin lymphoma. We present a case of a 78-year-old otherwise healthy man who developed nonhealing nodules on his right posterior calf. Initial biopsy showed a dense, atypical, lymphoid infiltrate with gamma-delta and cytotoxic T-cell immunophenotypes. The diagnosis of PCGDTL was rendered; however, concurrent flow cytometry revealed expression of aberrant B-cell markers, including CD19 and cytoplasmic CD79a. Subsequent immunohistochemical studies corroborated this result. We report the extremely rare phenomenon of aberrant B-cell marker expression in PCGDTL, the first formally reported case to our knowledge., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

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., (© 2023. The Author(s).)

Nodal peripheral T-cell lymphomas (PTCL), the most common PTCLs, are generally treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based curative-intent chemotherapy. Recent molecular data have assisted in prognosticating these PTCLs, but most reports lack detailed baseline clinical characteristics and treatment courses. We retrospectively evaluated cases of PTCL treated with CHOP-based chemotherapy that had tumors sequenced by the Memorial Sloan Kettering Integrated Mutational Profiling of Actionable Cancer Targets next-generation sequencing panel to identify variables correlating with inferior survival. We identified 132 patients who met these criteria. Clinical factors correlating with an increased risk of progression (by multivariate analysis) included advanced-stage disease and bone marrow involvement. The only somatic genetic aberrancies correlating with inferior progression-free survival (PFS) were TP53 mutations and TP53/17p deletions. PFS remained inferior when stratifying by TP53 mutation status, with a median PFS of 4.5 months for PTCL with a TP53 mutation (n = 21) vs 10.5 months for PTCL without a TP53 mutation (n = 111). No TP53 aberrancy correlated with inferior overall survival (OS). Although rare (n = 9), CDKN2A-deleted PTCL correlated with inferior OS, with a median of 17.6 months vs 56.7 months for patients without CDKN2A deletions. This retrospective study suggests that patients with PTCL with TP53 mutations experience inferior PFS when treated with curative-intent chemotherapy, warranting prospective confirmation., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Accurate classification and risk stratification are critical for clinical decision making in patients with acute myeloid leukemia (AML). In the newly proposed World Health Organization and International Consensus classifications of hematolymphoid neoplasms, the presence of myelodysplasia-related (MR) gene mutations is included as 1 of the diagnostic criteria for AML, AML-MR, based largely on the assumption that these mutations are specific for AML with an antecedent myelodysplastic syndrome. ICC also prioritizes MR gene mutations over ontogeny (as defined in the clinical history). Furthermore, European LeukemiaNet (ELN) 2022 stratifies these MR gene mutations into the adverse-risk group. By thoroughly annotating a cohort of 344 newly diagnosed patients with AML treated at the Memorial Sloan Kettering Cancer Center, we show that ontogeny assignments based on the database registry lack accuracy. MR gene mutations are frequently observed in de novo AML. Among the MR gene mutations, only EZH2 and SF3B1 were associated with an inferior outcome in the univariate analysis. In a multivariate analysis, AML ontogeny had independent prognostic values even after adjusting for age, treatment, allo-transplant and genomic classes or ELN risks. Ontogeny also helped stratify the outcome of AML with MR gene mutations. Finally, de novo AML with MR gene mutations did not show an adverse outcome. In summary, our study emphasizes the importance of accurate ontogeny designation in clinical studies, demonstrates the independent prognostic value of AML ontogeny, and questions the current classification and risk stratification of AML with MR gene mutations., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Although in vivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10 -5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P < 0.005). Only three patients with undetectable constructs had disease progression at the time of sampling., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Malignancies involving the central nervous system present unique challenges for diagnosis and monitoring due to the difficulties and risks of direct biopsies and the low specificity and/or sensitivity of other techniques for assessment. In recent years, liquid biopsy of the cerebrospinal fluid (CSF) has emerged as a convenient alternative that combines minimal invasiveness with the ability to detect disease-defining or therapeutically actionable genetic alterations from circulating tumor DNA (ctDNA). Since CSF can be obtained by lumbar puncture, or an established ventricular access device at multiple time points, ctDNA analysis enables initial molecular characterization and longitudinal monitoring throughout a patient's disease course, promoting optimization of treatment regimens. This review outlines some of the key aspects of ctDNA from CSF as a highly suitable approach for clinical assessment, the benefits and drawbacks, testing methods, as well as potential future advancements in this field. We anticipate wider adoption of this practice as technologies and pipelines improve and envisage significant improvements for cancer care., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Maria E. Arcila has received honoraria or worked on advisory boards for Janssen Global Services, Bristol-Myers Squibb and AstraZeneca, speaker fees from Biocartis and Invivoscribe; has received CME honoraria from OncLive, PeerVoice, Physicians Education Resources, Peerview Institute and Clinical Care Options. All other authors have no other competing interests to declare., (Copyright © 2023. Published by Elsevier Inc.)

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Lineage infidelity is characteristic of mixed phenotype acute leukemia and is also seen in blast phase of chronic myeloid leukemia (CML), myeloid/lymphoid neoplasia with eosinophilia and gene rearrangements, and subtypes of acute myeloid leukemia. Driver genetic events often occur in multipotent progenitor cells in myeloid neoplasms, suggesting that multilineage output may be more common than appreciated. This phenomenon is not well studied in myelodysplastic syndrome (MDS) and non-CML myeloproliferative neoplasms (MPN)., Methods: We systematically evaluated phenotypic lineage infidelity by reviewing bone marrow pathology and flow cytometry (FC) studies of 1262 consecutive patients with a diagnosis of MDS and/or non-CML MPN. We assessed B- and T-cells in these patients by FC. When abnormal B-lymphoblast (ABLB) populations were detected, we additionally evaluated immature B-cells using a high sensitivity FC assay for B-lymphoblastic leukemia/lymphoma (B-ALL)., Results: We identified 9 patients (7 MDS, 7/713, 1%; 2 non-CML MPN, 2/312, 0.6%; 0 in MDS/MPN) with low-level ABLB populations (0.012%-3.6% of WBCs in marrow) with abnormal immunophenotypes. Genetic studies on flow sorted cell populations confirmed that some ABLB populations were clonally related to myeloid blasts (4/6, 67%). On follow-up, ABLB populations in 8/9 patients remained stable or disappeared. Only 1 case progressed to B-ALL., Conclusions: These findings demonstrate that phenotypically detectable abnormal immature B lineage output occurs in MDS and non-CML MPN, albeit rarely. While presence of ABLB does not necessarily reflect blast crisis, the underlying disease biology of our findings may ultimately be relevant to patient management and warrants further investigation., (© 2021 International Clinical Cytometry Society.)

Background: Patients with cancer have an increased risk for arterial thromboembolism (ATE). Scant data exist about the impact of cancer-specific genomic alterations on the risk for ATE., Objectives: The aim of this study was to determine whether individual solid tumor somatic genomic alterations influence the incidence of ATE., Methods: A retrospective cohort study was conducted using tumor genetic alteration data from adults with solid cancers who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets testing between 2014 and 2016. The primary outcome, ATE, was defined as myocardial infarction, coronary revascularization, ischemic stroke, peripheral arterial occlusion, or limb revascularization and identified through systematic electronic medical record assessments. Patients were followed from date of tissue-matched blood control accession to first ATE event or death, for up to 1 year. Cause-specific Cox proportional hazards regression was used to determine HRs of ATE for individual genes adjusted for pertinent clinical covariates., Results: Among 11,871 eligible patients, 74% had metastatic disease, and there were 160 ATE events. A significantly increased risk for ATE independent of tumor type was noted for the KRAS oncogene (HR: 1.98; 95% CI: 1.34-2.94; multiplicity-adjusted P  = 0.015) and the STK11 tumor suppressor gene (HR: 2.51; 95% CI: 1.44-4.38; multiplicity-adjusted P  = 0.015)., Conclusions: In a large genomic tumor-profiling registry of patients with solid cancers, alterations in KRAS and STK11 were associated with an increased risk for ATE independent of cancer type. Further investigation is needed to elucidate the mechanism by which these mutations contribute to ATE in this high-risk population., Competing Interests: This research was supported by a National Cancer Institute Cancer Center Support Grant (P30 CA008748). Dr Willeit was supported by a Dr. Johannes and Hertha Tuba Grant. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: ATE is an important cause of mortality in patients with solid cancer. Tumor somatic alterations in KRAS and STK11 were associated with an increased risk for arterial events. This effect was independent of cancer type and other cardiovascular disease risk factors. TRANSLATIONAL OUTLOOK: A better understanding of the genomic determinants of cancer-associated ATE could contribute to improved risk stratification and primary prophylaxis., (© 2023 The Authors.)

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., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following completing interests: I.J.D. is a consultant or advisory board member for Apexigen, Astra-Zeneca, Bristol-Myers, Squibb/Celgene, Day One, Fennec, QED, and Roche. M.E.A declares the following competing interests: AstraZeneca, Bristol-Myers Squibb, Invivoscribe, Janssen Global Services, Merck, Roche (Consultant); Biocartis, Invivoscribe (Speaker/Speaker’s Bureau). D.W.Y.T. received honoraria from Nanodigmbio, Cowen and BoA Merrill Lynch; Research funding from ThermoFisher Scientific, EPIC Sciences, Prostate Cancer Foundation; Travel, Accommodations, Expenses from Nanodigmbio. D.W.Y.T is a co-inventor on a provisional patent application for systems and methods for detecting cancer via cfDNA screening (PCT/US2019/027487), and an inventor on a provisional patent application for systems and methods for distinguishing pathological mutations from clonal hematopoietic mutations in plasma cell-free DNA by fragment size analysis. D.W.Y.T is currently an employee of PetDx. Inc. M.F.B. declares research funding from Grail; personal fees from Roche and PetDX; and a provisional patent for systems and methods for detecting cancer via cfDNA screening (PCT/US2019/027487). D.H.A., D.L.M., A.R.B., R.B., J.H.F., D.N.F., M.F.W., G.J., N.S., P.K., M.L., M.S.D., M.A.R., D.H.L. declare no conflict of interest. This does not alter out adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2023 Abramson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered., (© 2022 Wiley Periodicals LLC.)

Purpose: Gallbladder carcinoma (GBC) is an uncommon and aggressive disease, which remains poorly defined at a molecular level. Here, we aimed to characterize the molecular landscape of GBC and identify markers with potential prognostic and therapeutic implications., Experimental Design: GBC samples were analyzed using the MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) platform (targeted NGS assay that analyzes 505 cancer-associated genes). Variants with therapeutic implications were identified using OncoKB database. The associations between recurrent genetic alterations and clinicopathologic characteristics (Fisher exact tests) or overall survival (univariate Cox regression) were evaluated. P values were adjusted for multiple testing., Results: Overall, 244 samples (57% primary tumors and 43% metastases) from 233 patients were studied (85% adenocarcinomas, 10% carcinomas with squamous differentiation, and 5% neuroendocrine carcinomas). The most common oncogenic molecular alterations appeared in the cell cycle (TP53 63% and CDKN2A 21%) and RTK&#95;RAS pathways (ERBB2 15% and KRAS 11%). No recurrent structural variants were identified. There were no differences in the molecular landscape of primary and metastasis samples. Variants in SMAD4 and STK11 independently associated with reduced survival in patients with metastatic disease. Alterations considered clinically actionable in GBC or other solid tumor types (e.g., NTRK1 fusions or oncogenic variants in ERBB2, PIK3CA, or BRCA1/2) were identified in 35% of patients; 18% of patients with metastatic disease were treated off-label or enrolled in a clinical trial based on molecular findings., Conclusions: GBC is a genetically diverse malignancy. This large-scale genomic analysis revealed alterations with potential prognostic and therapeutic implications and provides guidance for the development of targeted therapies., (©2022 American Association for Cancer Research.)

Myeloid neoplasms and acute leukemias derive from the clonal expansion of hematopoietic cells driven by somatic gene mutations. Although assessment of morphology plays a crucial role in the diagnostic evaluation of patients with these malignancies, genomic characterization has become increasingly important for accurate diagnosis, risk assessment, and therapeutic decision making. Conventional cytogenetics, a comprehensive and unbiased method for assessing chromosomal abnormalities, has been the mainstay of genomic testing over the past several decades and remains relevant today. However, more recent advances in sequencing technology have increased our ability to detect somatic mutations through the use of targeted gene panels, whole-exome sequencing, whole-genome sequencing, and whole-transcriptome sequencing or RNA sequencing. In patients with myeloid neoplasms, whole-genome sequencing represents a potential replacement for both conventional cytogenetic and sequencing approaches, providing rapid and accurate comprehensive genomic profiling. DNA sequencing methods are used not only for detecting somatically acquired gene mutations but also for identifying germline gene mutations associated with inherited predisposition to hematologic neoplasms. The 2022 International Consensus Classification of myeloid neoplasms and acute leukemias makes extensive use of genomic data. The aim of this report is to help physicians and laboratorians implement genomic testing for diagnosis, risk stratification, and clinical decision making and illustrates the potential of genomic profiling for enabling personalized medicine in patients with hematologic neoplasms., (© 2022 by The American Society of Hematology.)

Purpose: Clinical relevance thresholds and laboratory methods are poorly defined for MET amplification, a targetable biomarker across malignancies., Experimental Design: The utility of next-generation sequencing (NGS) in assessing MET copy number alterations was determined in >50,000 solid tumors. Using fluorescence in situ hybridization as reference, we validated and optimized NGS analysis., Results: Incorporating read-depth and focality analyses achieved 91% concordance, 97% sensitivity, and 89% specificity. Tumor heterogeneity, neoplastic cell proportions, and genomic focality affected MET amplification assessment. NGS methodology showed superiority in capturing overall amplification status in heterogeneous tumors and defining amplification focality among other genomic alterations. MET copy gains and amplifications were found in 408 samples across 23 malignancies. Total MET copy number inversely correlated with amplified segment size. High-level/focal amplification was enriched in certain genomic subgroups and associated with targeted therapy response., Conclusions: Leveraging our integrated bioinformatic approach, targeted therapy benefit was observed across diverse MET amplification contexts., (©2022 American Association for Cancer Research.)

Circulating tumor DNA (ctDNA) sequencing guides therapy decisions but has been studied mostly in small cohorts without sufficient follow-up to determine its influence on overall survival. We prospectively followed an international cohort of 1,127 patients with non-small-cell lung cancer and ctDNA-guided therapy. ctDNA detection was associated with shorter survival (hazard ratio (HR), 2.05; 95% confidence interval (CI), 1.74-2.42; P < 0.001) independently of clinicopathologic features and metabolic tumor volume. Among the 722 (64%) patients with detectable ctDNA, 255 (23%) matched to targeted therapy by ctDNA sequencing had longer survival than those not treated with targeted therapy (HR, 0.63; 95% CI, 0.52-0.76; P < 0.001). Genomic alterations in ctDNA not detected by time-matched tissue sequencing were found in 25% of the patients. These ctDNA-only alterations disproportionately featured subclonal drivers of resistance, including RICTOR and PIK3CA alterations, and were associated with short survival. Minimally invasive ctDNA profiling can identify heterogeneous drivers not captured in tissue sequencing and expand community access to life-prolonging therapy., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Safe sampling of central nervous system tumor tissue for diagnostic purposes may be difficult if not impossible, especially in pediatric patients, and an unmet need exists to develop less invasive diagnostic tests., Methods: We report our clinical experience with minimally invasive molecular diagnostics using a clinically validated assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA). All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families., Results: We analyzed 64 CSF samples from 45 pediatric, adolescent and young adult (AYA) patients (pediatric = 25; AYA = 20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n = 10), medulloblastoma (n = 10), pineoblastoma (n = 5), low-grade glioma (n = 4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n = 4), retinoblastoma (n = 4), ependymoma (n = 3), and other (n = 5). Somatic alterations were detected in 30/64 samples (46.9%) and in at least one sample per unique patient in 21/45 patients (46.6%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (81.5% of samples from patients with disseminated disease were positive). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient's disease course., Conclusions: We identified three general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: (1) diagnosis and/or identification of actionable alterations; (2) monitor response to therapy; and (3) tracking tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population to improve care., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Salivary duct carcinoma (SDC) is an aggressive salivary gland malignancy with poor survival. Approximately 30% SDC harbor HER2 amplification and response to trastuzumab has been reported. However, a systematic approach for HER2 status assessment in this tumor type has not been established. A total of 67 tumor samples were evaluated for HER2 protein overexpression or ERBB2 gene amplification using at least 2 methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and/or targeted exome next-generation sequencing (NGS). NGS assessed ERBB2 copy number fold change (FC) and total copy number (TCN). HER2 status was first determined by IHC/FISH according to the 2018 ASCO/CAP breast cancer guidelines. FISH results, the "gold standard", were compared with the NGS results. All (15/15) IHC positive, 35% (6/17) equivocal, and no (0/19) IHC negative SDC were HER2 amplified by FISH. HER2 FISH signal/cell showed a good correlation with FC (Spearman correlation: 0.708, R 2 : 0.501, p < 0.0001) and TCN (Spearman correlation: 0.763, R 2 : 0.582, p < 0.0001). Receiver operating characteristics curve estimation showed an area under curve (AUC) of 0.975 for ERBB2 FC. FC cutoff of ≥1.8 corresponded to an accuracy of 95.2% for ERBB2 amplification (Youden's index: 0.84, sensitivity: 89.47%, specificity: 100%). FC < 1.3 could be reliably classified as ERBB2 not amplified and FC ≥ 1.3 and <1.8 as equivocal. TCN estimation showed AUC of 0.981. TCN cutoff of >6.0 corresponded to an accuracy of 92% for HER2 amplification (Youden's index: 0.81, sensitivity: 81.2%, specificity: 100%). TCN < 4 could be reliably classified as ERBB2 not amplified and TCN ≥ 4.0 and ≤6.0 as equivocal. FC and TCN were binarized with respective cutoffs of ≥1.8 and ≥6.0 and the proportion of agreement with FISH were 95% and 92%, respectively. The assessment of ERBB2 copy number by NGS is accurate and reliable with FC or TCN nearly equivalent to FISH in identifying HER2 amplified SDC., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Several kinase fusions are established targetable drivers in lung cancers. However, rapid and comprehensive detection remains challenging because of diverse partner genes and breakpoints. We assess the clinical utility and performance of a rapid microfluidic multiplex real-time PCR-based assay for simultaneous query of fusions involving ALK, ROS1, RET, and NTRK1/2/3, as well as MET exon 14 skipping, using a 3-hour automated process. Dual analytic strategies were utilized: fusion-specific amplification and 3' to 5' expression imbalance. One-hundred and forty-three independent, formalin-fixed, paraffin-embedded tumor samples (112 surgical specimens, 31 cytologic cell blocks) were analyzed: 133 with known kinase gene alterations and 10 negative samples based on clinically validated next-generation sequencing. Testing was successful in 142 (99%) cases. The assay demonstrated a sensitivity of 97% (28/29), 100% (31/31), 92% (22/24), 81% (22/27), and 100% (20/20) for ALK, RET, ROS1, and NTRK1/2/3 rearrangements and MET exon 14 skipping alterations, respectively, with 100% specificity for all. Concordant results were achieved in specimens aged up to 5 years, with >10% tumor, and inputs of at least 9 mm 2 (surgical specimens) and 9000 cells (cytologic cell blocks). The assay enables rapid screening for clinically actionable kinase alterations with quicker turnaround and lower tissue requirements compared with immunohistochemistry and molecular methods, while also circumventing the infrastructure dependencies associated with next-generation sequencing and fluorescence in situ hybridization., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: To evaluate factors associated with successful comprehensive genomic sequencing of image-guided percutaneous needle biopsies in patients with lung cancer using a broad hybrid capture-based next-generation sequencing assay (CHCA)., Methods: We conducted a single-institution retrospective review of image-guided percutaneous transthoracic needle biopsies from January 2018 to December 2019. Samples with confirmed diagnosis of primary lung cancer and for which CHCA had been attempted were identified. Pathologic, clinical data and results of the CHCA were reviewed. Covariates associated with CHCA success were tested for using Fisher's exact test or Wilcoxon ranked sum test. Logistic regression was used to identify factors independently associated with likelihood of CHCA success., Results: CHCA was requested for 479 samples and was successful for 433 (91%), with a median coverage depth of 659X. Factors independently associated with lower likelihood of CHCA success included small tumor size (OR = 0.26 [95% confidence interval (CI): 0.11-0.62, p  = 0.002]), intraoperative inadequacy on cytologic assessment (OR = 0.18 [95% CI: 0.06-0.63, p  = 0.005]), small caliber needles (≥20-gauge) (OR = 0.22 [95% CI: 0.10-0.45, p < 0.001]), and presence of lung parenchymal abnormalities (OR = 0.12 [95% CI: 0.05-0.25, p < 0.001]). Pneumothorax requiring chest tube insertion occurred in 6% of the procedures. No grade IV complications or procedure-related deaths were reported., Conclusions: Percutaneous image-guided transthoracic needle biopsy is safe and has 91% success rate for CHCA in primary lung cancer. Intraoperative inadequacy, small caliber needle, presence of parenchymal abnormalities, and small tumor size (≤1 cm) are independently associated with likelihood of failure., (© 2022 by the International Association for the Study of Lung Cancer.)

This study is the largest analysis of DNA viruses in solid tumors with associated genomics. To achieve this, a novel method for discovery of DNA viruses from matched tumor/normal next-generation sequencing samples was developed and validated. This method performed comparably to reference methods for the detection of high-risk (HR) human papilloma virus (HPV) (area under the receiver operating characteristic curve = 0.953). After virus identification in 48,148 consecutives samples from 42,846 unique patients, novel virus tumor associations were established by segregating tumor types to determine whether each DNA virus was enriched in each of the tumor types compared with the remaining cohort. All firmly established solid tumor-virus associations (eg, HR HPV in cervical cancer) were confirmed, and the novel associations discovered included: human herpes virus 6 in neuroblastoma, human herpes virus 7 in esophagogastric cancer, and HPV42 in digital papillary adenocarcinoma. These associations were confirmed in an independent validation cohort. HR HPV- and Epstein-Barr virus-associated tumors showed newly discovered genomic associations, including a lower tumor mutation burden. The study demonstrated the ability to study the role of DNA viruses in human cancer from clinical genomics data and established the largest cohort that can be utilized as a validation set for future discovery efforts., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The European Society for Medical Oncology (ESMO) held a virtual consensus-building process on epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer in 2021. The consensus included a multidisciplinary panel of 34 leading experts in the management of lung cancer. The aim of the consensus was to develop recommendations on topics that are not covered in detail in the current ESMO Clinical Practice Guideline and where the available evidence is either limited or conflicting. The main topics identified for discussion were: (i) tissue and biomarkers analyses; (ii) early and locally advanced disease; (iii) metastatic disease and (iv) clinical trial design, patient's perspective and miscellaneous. The expert panel was divided into four working groups to address questions relating to one of the four topics outlined above. Relevant scientific literature was reviewed in advance. Recommendations were developed by the working groups and then presented to the entire panel for further discussion and amendment before voting. This manuscript presents the recommendations developed, including findings from the expert panel discussions, consensus recommendations and a summary of evidence supporting each recommendation., Competing Interests: Disclosure AP received education grants, provided consultation, attended advisory board meetings and/or provided lectures for AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb (BMS), Daiichi Sankyo, eCancer, Eli Lilly, Janssen, Medscape, Merck Sharp & Dohme (MSD), Merck KGaA, Novartis, Pfizer, Roche/Genentech, Takeda, outside the submitted work. NL reports unrelated institutional research funding received from Amgen, Array, AstraZeneca, Bayer, BMS, Lilly, EMD Serono, Guardant Health, MSD, Pfizer, Roche, Takeda and unrelated personal fees (CME lectures, consulting) received from Bayer, Boehringer Ingelheim, BMS, Canadian Agency for Drugs and Technologies in Health, EMD Serono, MSD, Novartis, Sanofi Genzyme. SPo reports personal fees from Amgen, AstraZeneca, Bayer, BeiGene, Blueprint Medicines, BMS, Boehringer Ingelheim, Daiichi Sankyo, Guardant Health, Janssen, Lilly, Merck KGaA, Novartis, Roche, Takeda, Seattle Genetics, Turning Point Therapeutics, GlaxoSmithKline, outside the submitted work. KK reports personal fees from AstraZeneca, Roche, during the conduct of the study. MJA reports personal fees from AstraZeneca, Takeda, Roche, Alpha Pharmaceutical, Amgen, Lilly, ONO, MSD, Merck, outside the submitted work. MEA reports personal fees from Biocartis, Invivoscribe, Janssen Global Services, BMS, AstraZeneca, Roche, Merck, RMEI Medical Education, Clinical Care Options, PeerView Institute for medical education, outside the submitted work. OA reports personal fees from Pfizer, grants and personal fees from AstraZeneca, Boehringer Ingelheim, personal fees from Lilly, Merck, BMS, grants and personal fees from Roche, outside the submitted work. DP reports personal fees from AstraZeneca, BMS, Celgene, Daiichi Sankyo, Eli Lilly, Merck, Novartis, Pfizer, prIME Oncology, Peer CME, Roche, Samsung, AbbVie, Janssen, outside the submitted work; and clinical trials research (as principal investigator or co-investigator): AstraZeneca, AbbVie, BMS, Boehringer Ingelheim, Eli Lilly, Merck, Novartis, Pfizer, Roche, MedImmune, Sanofi-Aventis, Taiho Pharma, Novocure, Daiichi Sankyo, Janssen. FdM reports grants and personal fees from AstraZeneca, grants from Boehringer, personal fees from Roche, BMS, Novartis, MSD, Pfizer, Takeda, outside the submitted work. AMD reports personal fees from Roche, Eli Lilly, Pfizer, Pharmamar, Takeda, Boehringer Ingelheim, grants and personal fees from AstraZeneca, personal fees from Jansen, Chiesi, grants and personal fees from Amgen, personal fees from Bayer, Sanofi, outside the submitted work. RD reports personal fees from Pfizer, personal fees and non-financial support from Roche, non-financial support from AstraZeneca, personal fees from Novartis, Boehringer Ingelheim, Foundation Medicine, Karyopharm, outside the submitted work. CFF reports grants and other from AstraZeneca, other from MSD, grants and other from Elekta, during the conduct of the study. JF reports personal fees from Blueprint Medicines, Novartis, Janssen, AstraZeneca, outside the submitted work. EF reports personal fees from AbbVie, Amgen, AstraZeneca, Bayer, Blueprint Medicines, Boehringer Ingelheim, BMS, Eli Lilly, GlaxoSmithKline, Janssen, Medscape, Merck KGaA, MSD, Novartis, PeerVoice, Pfizer, prIME Oncology, Puma Biotechnology, Roche, Sanofi Genzyme, Springer, Takeda, Touch Medical, grants from Grant for Oncology Innovation (GOI), grants from Fundación Merck Salud, personal fees from CME Outfitters, BeiGene, Medical Trends, Peptomyc, Regeneron, from Syneos Health, outside the submitted work; and Grifols: independent member of the board. GC reports consultation fees from Pfizer, Roche, AstraZeneca, BMS, Daichii Sankyo, Seagen, Gilead, Novartis, Lilly, Ellipsis, Merck. RH reports consultation fees from AbbVie Pharmaceuticals, ARMO Biosciences, AstraZeneca, Bayer HealthCare Pharmaceuticals Inc., Bolt Biotherapeutics, BMS, Candel Therapeutics, Inc., Checkpoint Therapeutics, Cybrexa Therapeutics, DynamiCure Biotechnology, LLC, eFFECTOR Therapeutics, Inc., Eli Lilly and Company, EMD Serono, Foundation Medicine, Inc., Genentech/Roche, Genmab, Gilead, Halozyme Therapeutics, Heat Biologics, HiberCell, Inc., I-Mab Biopharma, Immune-Onc Therapeutics, Immunocore, Infinity Pharmaceuticals, Johnson & Johnson, Loxo Oncology, Merck and Company, Mirati Therapeutics, Nektar, Neon Therapeutics, NextCure, Novartis, Ocean Biomedical, Inc., Oncocyte Corp., Oncternal Therapeutics, Pfizer, Refactor Health, Inc., Ribbon Therapeutics, Sanofi, Seattle Genetics, Shire PLC, Spectrum Pharmaceuticals, STCube Pharmaceuticals, Inc., Symphogen, Takeda, Tesaro, Tocagen, Ventana Medical Systems, Inc., WindMIL Therapeutics, Xencor, Inc., research support from AstraZeneca, Eli Lilly and Company, Genentech/Roche, Merck and Company, and participation on board of Immunocore Holdings Limited, Junshi Pharmaceuticals. PAJ reports grants and personal fees from AstraZeneca, Boehringer Ingelheim, personal fees from Pfizer, Roche/Genentech, Chugai Pharmaceuticals, Loxo Oncology, grants and personal fees from Eli Lilly, personal fees from SFJ Pharmaceuticals, Voronoi, grants and personal fees from Daiichi Sankyo, personal fees from Biocartis, Novartis, Sanofi, grants and personal fees from Takeda Oncology, personal fees from Mirati Therapeutics, Transcenta, Silicon Therapeutics, Syndax, Nuvalent, Bayer, Eisai, Allorion Therapeutics, Accutar Biotech, AbbVie, grants from Revolution Medicines, grants from PUMA, grants from Astellas, outside the submitted work. In addition, PAJ is a co-inventor on a DFCI patent on EGFR mutations and receives postmarketing royalties from Lab Corp. TJ reports personal fees from Pfizer, AstraZeneca, BMS, Merck, MSD, Takeda, Boehringer Ingelheim, Roche, Ignyta, Novartis, Bayer, Amgen, Gilead, Puma Pharmaceuticals, outside the submitted work. TM reports grants and personal fees from AstraZeneca, Boehringer Ingelheim, Chugai, personal fees from Pfizer, during the conduct of the study; personal fees from BMS, grants and personal fees from Ono, MSD, personal fees from Novartis, grants and personal fees from Taiho, personal fees from Takeda, Amgen, Invitae, Merck Biopharma, Thermo Fisher, grants from Bridge Biotherapeutics, grants and personal fees from Ethicon, outside the submitted work. TM reports personal fees and other from AbbVie, Inc., ACEA Pharma, Alpha Biopharma Co. Ltd, Amgen, Amoy Diagnostics Co. Ltd, grants, personal fees and other from AstraZeneca, personal fees and other from BeiGene, Boehringer Ingelheim, grants, personal fees and other from BMS, personal fees and other from Blueprint Medicines Corporation, CStone Pharmaceuticals, Daiichi Sankyo, Eisai, Fishawack Facilitate Ltd, other from geneDecode, personal fees and other from Gritstone Oncology Inc., Guardant Health, Hengrui Therapeutics, Ignyta Inc., Incyte Corporation, personal fees from InMed Medical Communication, personal fees and other from Janssen, Lilly, Loxo Oncology, Lunit USA, Inc., personal fees from MD Health (Brazil), Medscape/WebMD, grants, personal fees and other from Merck Serono, MSD, personal fees and other from Mirati Therapeutics Inc., personal fees from MoreHealth, grants, personal fees and other from Novartis, personal fees and other from OrigiMed, personal fees from PeerVoice, Physicians' Education Resource, P. Permanyer SL, grants, personal fees and other from Pfizer, Inc., personal fees from PrIME Oncology, personal fees and other from Puma Biotechnology Inc., personal fees from Research to Practice, grants, personal fees and other from Roche, personal fees and other from Sanofi-Aventis R&D, grants, personal fees and other from Takeda, personal fees from Touch Medical Media, other from Virtus Medical Group, personal fees and other from Yuhan Corporation, other from AstraZeneca PLC, Hutchison Chi-Med, Sanomics Ltd, grants from Clovis Oncology, grants, personal fees and other from SFJ Pharmaceuticals, grants from Xcovery, personal fees and other from Curio Science, personal fees from Inivata, Berry Oncology, grants and personal fees from G1 Therapeutics Inc., other from Aurora, personal fees from Qiming Development (HK) Ltd, Daz Group, Lucence Health Inc., Merck Pharmaceuticals HK Ltd, Shanghai BeBirds Translation & Consulting Co., Ltd, Liangyihui Network Technology Co., Ltd, Taiho, personal fees and other from Gilead Sciences, Inc, Vertex Pharmaceuticals, personal fees from Covidien LP, outside the submitted work. NN reports personal fees from MSD, grants and personal fees from Qiagen, Biocartis, Incyte, Roche, BMS, Merck, Thermo Fisher, AstraZeneca, personal fees from Sanofi, Eli Lilly, Bayer, ArcherDx, grants and personal fees from Illumina, personal fees from Amgen, outside the submitted work. LPA reports grants from MSD, AstraZeneca, Pfizer, BMS, personal fees from Lilly, MSD, Roche, Pharmamar, Merck, AstraZeneca, Novartis, Servier, Amgen, Pfizer, Ipsen, Sanofi, Bayer, Blueprint, BMS, Mirati, Janssen, other from Genomica, Altum sequencing, outside the submitted work. SR reports personal fees from AstraZeneca, BMS, Amgen, GlaxoSmithKline, Merck, Takeda, Eisai, Lilly, outside the submitted work. LS reports grants and personal fees from AstraZeneca, grants from Novartis, personal fees from Janssen, grants and personal fees from Genentech, personal fees from Takeda, Pfizer, grants from Boehringer Ingelheim, outside the submitted work. JV reports honoraria for invited speaker from AstraZeneca, BMS, MSD, and Novartis; participation on advisory board of AstraZeneca, BMS, Boehringer Ingelheim, Daichi Sankyo, MSD, Pfizer, and Roche; grant from MSD. IIW reports grants and personal fees from Genentech/Roche, Bayer, BMS, AstraZeneca, Pfizer, HTG Molecular, personal fees from Asuragen, grants and personal fees from Merck, GlaxoSmithKline, Guardant Health, personal fees from Flame, grants and personal fees from Novartis, Sanofi, personal fees from Daiichi Sankyo, grants and personal fees from Amgen, personal fees from Oncocyte, MSD, Platform Health, grants from Adaptive, Adaptimmune, EMD Serono, Takeda, Karus, Johnson & Johnson, 4D, Iovance, Akoya, outside the submitted work. JW reports personal fees from Amgen, AstraZeneca, Bayer, Blueprint, grants and personal fees from BMS, personal fees from Boehringer Ingelheim, Chugai, Daiichi Sankyo, Ignyta, grants and personal fees from Janssen, personal fees from Lilly, Loxo, MSD, grants and personal fees from Novartis, Pfizer, personal fees from Roche, Seattle Genetics, Takeda, outside the submitted work. YLW reports personal fees from AstraZeneca, BeiGene, Boehringer Ingelheim, BMS, Eli Lilly, MSD, Pfizer, Roche, Sanofi, Novartis, Takeda; grants from AstraZeneca, Boehringer Ingelheim, BMS, Hengrui and Roche, outside the submitted work. JCHY reports personal fees and other from Amgen, grants, personal fees and other from AstraZeneca, personal fees and other from Bayer, Boehringer Ingelheim, BMS, Daiichi Sankyo, other from Eli Lilly, personal fees and other from Merck KGaA, Darmstadt, Germany, MSD, Novartis, personal fees from Ono Pharmaceuticals, Pfizer, personal fees and other from Roche/Genentech, Takeda Oncology, Yuhan Pharmaceuticals, other from Johnson & Johnson, Glaxo, Puma Technology, outside the submitted work. YY reports and contracted studies with ArcherDx, Chugai Pharma and Thermo Fisher Science; honoraria for lectures from MSD, Chugai Pharma, AstraZeneca, Pfizer, Roche/Ventana, Agilent/Dako, Thermo Fisher Science, ArcherDx, Novartis, Eli Lilly, Amgen and Sysmex; participation on advisory board of MSD, Chugai Pharma, AstraZeneca, Novartis, Amgen, Takeda and Daiichi-Sankyo. GP reports grants from Amgen, grants, personal fees and non-financial support from Merck, grants and non-financial support from AstraZeneca, grants and personal fees from Roche, BMS, grants from Lilly, grants and personal fees from MSD, Novartis, outside the submitted work. SPe received education grants, provided consultation, attended advisory board meetings and/or provided lectures for AbbVie, Amgen, AstraZeneca, Bayer, Biocartis, BioInvent, Blueprint Medicines Corporation, Boehringer Ingelheim, BMS, Clovis, Daiichi Sankyo, Debiopharm, Eli Lilly, F. Hoffmann-La Roche, Foundation Medicine, Illumina, Janssen, MSD, Merck Serono, Merrimack, Novartis, PharmaMar, Pfizer, Regeneron, Sanofi, Seattle Genetics, Takeda and Vaccibody, from whom she received honoraria (all fees to institution). All other authors have declared no conflicts of interest., (Copyright © 2022 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

Developments in genomics are profoundly influencing medical practice. With increasing use of genetic and genomic testing across every aspect of the health care continuum, patients and their families are increasingly turning to primary care physicians (PCPs) for discussion and advice regarding tests, implications, and results. Yet, with the rapid growth of information, technology, and applications, PCPs are finding it challenging to fill the gaps in knowledge and support the growing needs of their patients. A critical component in expanding PCP genomic literacy lies in the education of physicians in training and in practice. Although a framework for developing physician competencies in genomics has already been developed, the Association for Molecular Pathology is uniquely situated to actively utilize the skills of its members to engage and support PCPs in this effort. This report provides an overview and a suggested basic teaching framework, which can be used by molecular professionals in their individual institutions as a starting point for educational outreach., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Androgen receptor (AR) inhibitor therapy is a developing treatment for AR-positive breast cancer (BC) with ongoing clinical trials. AR splice variant-7 (AR-V7) is a truncated variant of AR that leads to AR inhibitor therapy resistance in prostate cancer; recent studies have identified AR-V7 in BC and theorized that AR-V7 can have a similar impact. This study assessed the prevalence and clinicopathologic features associated with AR-V7 in a large BC cohort. BC samples were evaluated by MSK-Fusion targeted RNAseq for AR-V7 detection and MSK-IMPACT targeted DNAseq, including triple-negative tumors with no driver alteration and estrogen receptor-positive/ESR1 wildtype tumors progressing on therapy. Among 196 primary and metastatic/recurrent cases (196 RNAseq, 194DNAseq), 9.7% (19/196) were AR-V7 positive and 90.3% (177/196) AR-V7 negative. All AR-V7 positive BC were AR-positive by immunohistochemistry (19/19). The prevalence of AR-V7 by receptor subtype (N = 189) was: 18% (12/67) in ER-/PgR-/HER2-negative BC, 3.7% (4/109) in ER-positive/HER2-negative BC, and 15.4% (2/13) in HER2-positive BC; AR-V7 was detected in one ER-positive/HER2-unknown BC. Apocrine morphology was observed in 42.1% (8/19) of AR-V7 positive BC and 3.4% (6/177) AR-V7 negative BC (P < 0.00001). Notably, AR-V7 was detected in 2 primary BC and 7 metastatic/recurrent BC patients with no prior endocrine therapy. We conclude that positive AR IHC and apocrine morphology are pathologic features that may indicate testing for AR-V7 is warranted in both primary and metastatic BC in the appropriate clinical context. The study findings further encourage the assessment of AR-V7 as a predictive biomarker for AR antagonist benefit in ongoing clinical BC trials., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Epstein-Barr virus (EBV) is associated with hematologic and solid tumors. We utilized a hybridization capture-based next-generation sequencing (NGS) platform targeting 400 genes associated with hematological malignancies to detect and quantify nontargeted viral-derived EBV reads that aligned to the EBV reference contig (NC&#95;007605). We evaluated 5234 samples from 3636 unique patients with hematological neoplasms and found that 100 samples (1.9%) in 93 unique patients had ≥6 EBV reads (range, 6 to 32,325; mean, 827.5; median, 54). Most (n = 73, 73%) represented known EBV-associated conditions, and the most common was post-transplant lymphoproliferative disorders (n = 21, 29%). Documented EBV viremia was found in 4 of 27 samples with a moderate quantity of EBV reads and conditions not known to be EBV associated, whereas suspected viremia or low-level activation was likely in the remaining 23 samples. A good correlation (Spearman r = 0.8; 95% CI, 0.74-0.85) was found between EBV reads by NGS and systematic semiquantitative EBV-encoded RNA in situ hybridization in 162 available samples, particularly at greater EBV involvement. An optimal threshold for significant morphologic EBV involvement was found to be ≥10 reads by the receiver operating characteristic analysis (area under the curve, 0.990; 95% CI, 0.9974%-1.000%). Thus, in addition to mutational analysis, hybrid-capture-based NGS panels can detect and quantitate off-target EBV-derived viral DNA, which correlates well with morphology., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Mutations of TP53 are observed in 5-10% of patients in myelodysplastic syndrome (MDS) and are associated with adverse outcomes. Previous studies indicate that the TP53 allelic state and variant allele frequency of TP53 mutation impact patient outcomes, but there is significant heterogeneity within this MDS subgroup. We performed retrospective review of clinicopathologic and genomic information of 107 patients with TP53 -mutated MDS. We assessed each mutation according to the phenotypic annotation of TP53 mutations (PHANTM) and analyzed the associations between predicted TP53 mutant function, represented by the PHANTM combined phenotype score, and overall survival (OS) using the log rank test and Cox regression. Our results indicated that patients with PHANTM combined phenotype score above the median (>1) had significantly shorter OS compared to those with scores below the median (median OS: 10.59 and 16.51 months, respectively, p = 0.025). This relationship remained significant in multivariable analysis (HR (95%CI): 1.62 (1.01-2.58), p = 0.044) and identified to have an independent prognostic influence, accounting for known risk such as IPSS-R and other standard risk variables. Our results suggest that the functional information of TP53 mutations, represented by PHANTM combined phenotype score, are associated with the clinical outcome of patients with TP53 -mutated MDS.

Although precision medicine has had a mixed impact on the clinical management of patients with advanced-stage cancer overall, for NSCLC, and more specifically for lung adenocarcinoma, the advances have been dramatic, largely owing to the genomic complexity and growing number of druggable oncogene drivers. Furthermore, although tumor tissue is historically the "accepted standard" biospecimen for these molecular analyses, there are considerable innate limitations. Thus, liquid biopsy represents a practical alternative source for investigating tumor-derived somatic alterations. Although data are most robust in NSCLC, patients with other cancer types may also benefit from this minimally invasive approach to facilitate selection of targeted therapies. The liquid biopsy approach includes a variety of methodologies for circulating analytes. From a clinical point of view, plasma circulating tumor DNA is the most extensively studied and widely adopted alternative to tissue tumor genotyping in solid tumors, including NSCLC, first entering clinical practice for detection of EGFR mutations in NSCLC. Since the publication of the first International Association for the Study of Lung Cancer (IASLC) liquid biopsy statement in 2018, several additional advances have been made in this field, leading to changes in the therapeutic decision-making algorithm for advanced NSCLC and prompting this 2021 update. In view of the novel and impressive technological advances made in the past few years, the growing clinical application of plasma-based, next-generation sequencing, and the recent Food and Drug and Administration approval in the United States of two different assays for circulating tumor DNA analysis, IASLC revisited the role of liquid biopsy in therapeutic decision-making in a recent workshop in October 2020 and the question of "plasma first" versus "tissue first" approach toward molecular testing for advanced NSCLC. Moreover, evidence-based recommendations from IASLC provide an international perspective on when to order which test and how to interpret the results. Here, we present updates and additional considerations to the previous statement article as a consensus from a multidisciplinary and international team of experts selected by IASLC., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: Liquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) can detect ALK fusions, though data on clinical utility of this technology in the real world is limited., Materials and Methods: Patients with lung cancer without known oncogenic drivers or who had acquired resistance to therapy (n = 736) underwent prospective plasma ctDNA NGS. A subset of this cohort (n = 497) also had tissue NGS. We evaluated ALK fusion detection, turnaround time (TAT), plasma and tissue concordance, matching to therapy, and treatment response., Results: ctDNA identified an ALK fusion in 21 patients (3%) with a variety of breakpoints and fusion partners, including EML4, CLTC, and PON1, a novel ALK fusion partner. TAT for ctDNA NGS was shorter than tissue NGS (10 vs. 20 days; p < 0.001). Among ALK fusions identified by ctDNA, 93% (13/14, 95% CI 66%-99%) were concordant with tissue evaluation. Among ALK fusions detected by tissue NGS, 54% (13/24, 95% CI 33%-74%) were concordant with plasma ctDNA. ctDNA matched patients to ALK-directed therapy with subsequent clinical response, including four patients matched on the basis of ctDNA results alone due to inadequate or delayed tissue testing. Serial ctDNA analysis detected MET amplification (n = 2) and ALK G1202R mutation (n = 2) as mechanisms of acquired resistance to ALK-directed therapy., Conclusion: Our findings support a complementary role for ctDNA in detection of ALK fusions and other alterations at diagnosis and therapeutic resistance settings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1 . Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically., Experimental Design: A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping ( n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS)., Results: Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion ( F11R - NRG2 ). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS -mutant IMAs, NRG1 -rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival ( P < 0.0001)., Conclusions: This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1 -rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset., (©2021 American Association for Cancer Research.)

Introduction: Pulmonary invasive mucinous adenocarcinomas (IMAs) often present with spatially separate lung lesions. Clonal relationship between such lesions, particularly those involving contralateral lobes, is not well established. Here, we used comparative genomic profiling to address this question., Methods: Patients with genomic analysis performed on two IMAs located in different lung regions were identified. Molecular assays included DNA-based next-generation sequencing (NGS) for 410 to 468 genes (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets), RNA-based NGS for 62 genes (Memorial Sloan Kettering-Fusion), or non-NGS assays., Results: Comparative genomic profiling was performed on two separate IMAs in 24 patients, of whom 19 had contralateral lesions. Tumors from all but one patient shared matching driver alterations, including KRAS (n = 19), NRG1 (n = 2), ERBB2 (n = 1) or BRAF (n = 1). In addition, in patients with paired tumors profiled by NGS (n = 12), shared driver alterations were accompanied by up to 4 (average 2.6) other identical mutations, further supporting the clonal relationship between the tumors. Only in a single patient separate IMAs harbored entirely nonoverlapping mutation profiles, supporting clonally unrelated, distinct primary tumors. Notably, in a subset of patients (n = 3), molecular testing confirmed a clonal relationship between the original resected IMAs and subsequent contralateral IMA presenting after an extremely long latency (8.1-11.7 y)., Conclusions: Comparative molecular profiling supports that nearly all separate pulmonary IMA lesions represent intrapulmonary spread arising from a single tumor and documents a subset with a remarkably protracted course of intrapulmonary progression. This study reinforces the unique biology and clinical behavior of IMAs while further highlighting the value of genomic testing for clarifying the clonal relationship between multiple lung carcinomas., (Copyright © 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Context.—: The increasing use of large panel next-generation sequencing technologies in clinical settings has facilitated the identification of pan-cancer biomarkers, which can be diagnostic, prognostic, predictive, or most importantly, actionable., Objective.—: To discuss recently approved and emerging pan-cancer and multihistology biomarkers as well as testing methodologies., Data Sources.—: The US Food and Drug Administration approval documents, National Comprehensive Cancer Network guidelines, literature, and authors' own publications., Conclusions.—: Since 2017, the US Food and Drug Administration has approved genotype-directed therapies for pan-cancer biomarkers, including microsatellite instability, neurotrophic receptor kinases fusions, and high-tumor mutation burden. Both the importance and rarity of these biomarkers have increased the prevalence of genomic profiling across solid malignancies. As an integral part of the management team of patients with advanced cancer, pathologists need to be aware of these emerging biomarkers, the therapies for which they determine eligibility, and the strengths and pitfalls of the available clinical assays.

Cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) offers unique opportunities for genomic profiling of tumors involving the central nervous system but remains uncommonly used in clinical practice. We describe our clinical experience using cfDNA from CSF for routine molecular testing using Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (targeting 468 cancer-related genes). In all, 148 cfDNA samples were assessed, comparing results of cfDNA versus genomic DNA (gDNA; gDNA from cell pellets) derived from the same CSF sample and the primary tumor. Of these, 71.6% (106/148) were successfully sequenced. Somatic alterations (mutations and fusions) were observed in 70.8% (75/106) of the samples; 97.3% (73/75) comprised variants confirming central nervous system involvement by a previously diagnosed tumor, 14.7% (11/75) had additional variants consistent with a therapy-related resistance mechanism, and 2.7% (2/75) had variants that independently diagnosed a new primary. Among samples with paired cfDNA and gDNA sequencing results, cfDNA was more frequently positive for at least one mutation [43.6% (55/126) versus 19.8% (25/126)] and harbored 1.6× more mutations (6.94 versus 4.65; P = 0.005), with higher mean variant allele fractions (41.1% versus 13.0%; P < 0.0001). Among mutation-positive cfDNAs, the corresponding gDNA was frequently negative (44.6%; 25/55) or failed sequencing (17.8%; 9/55). Routine molecular profiling of cfDNA is superior to gDNA from CSF, facilitating the capture of mutations at high variant allele frequency, even in the context of a negative cytology., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Importance: Recently, the benefit of adding daratumumab to the proteasome inhibitor-based, 3-drug combination of bortezomib, lenalidomide, and dexamethasone for patients with newly diagnosed multiple myeloma who underwent high-dose melphalan chemotherapy and autologous hemopoietic cell transplant was assessed. Here, we examine the addition of daratumumab to the second-generation proteasome inhibitor-based, 3-drug combination of carfilzomib, lenalidomide, and dexamethasone., Objective: To assess the safety and effectiveness of carfilzomib-lenalidomide-dexamethasone-daratumumab combination therapy for patients with newly diagnosed multiple myeloma, in the absence of high-dose melphalan chemotherapy and autologous hemopoietic cell transplant., Design, Setting, and Participants: Clinical and correlative pilot study at the Memorial Sloan Kettering Cancer Center in New York, New York. Patients with newly diagnosed multiple myeloma were enrolled between October 1, 2018, and November 15, 2019. The median follow-up from start of treatment was 20.3 months (95% CI, 19.2-21.9 months)., Interventions: Eight 28-day cycles with intravenous carfilzomib, 20/56 mg/m2 (days 1, 8, and 15); oral lenalidomide, 25 mg, (days 1-21); dexamethasone, 40 mg weekly, orally or intravenously (cycles 1-4), and 20 mg after cycle 4; and intravenous daratumumab, 16 mg/kg (days 1, 8, 15, and 22 [cycles 1-2]; days 1 and 15 [cycles 3-6]; and day 1 [cycles 7 and 8])., Main Outcomes and Measures: The primary end point was the minimal residual disease (MRD) rate, in the absence of high-dose melphalan chemotherapy and autologous hemopoietic cell transplant. Secondary end points included determining safety and tolerability, evaluating rates of clinical response per the International Myeloma Working Group, and estimating progression-free survival (PFS) and overall survival (OS) rates., Results: Forty-one evaluable patients were enrolled (median age, 59 years; range, 30-70 years); 25 (61%) were female, and 20 (49%) had high-risk multiple myeloma. The primary end point (MRD negativity in the bone marrow; 10-5 sensitivity) was achieved in 29 of 41 patients (71%; 95% CI, 54%-83%), and therefore the trial was deemed successful. Median time to MRD negativity was 6 cycles (range, 1-8 cycles). Secondary end points of the overall response rate and the very good partial response or complete response rate were 100% (41 of 41 patients) and 95% (39 of 41 patients), respectively. At 11 months of the median follow-up, the 1-year PFS rate and the OS rate were 98% (95% CI, 93%-100%) and 100%, respectively. Most common (≥2 patients) grade 3 or 4 adverse events were neutropenia (12 patients [27%]), rash (4 patients [9%]), lung infection (3 patients [7%]), and increased alanine aminotransferase level (2 patients [4%]). There were no deaths., Conclusions and Relevance: In this nonrandomized clinical trial, carfilzomib-lenalidomide-dexamethasone-daratumumab combination therapy was associated with high rates of MRD negativity in patients with newly diagnosed multiple myeloma and high rates of PFS.

Background Lenalidomide maintenance improves progression-free survival for patients with multiple myeloma, although its optimal duration is unknown. Clearance of minimal residual disease (MRD) in the bone marrow results in superior outcomes, although its attainment or sustainment does not alter clinical decision-making. Studies that have evaluated MRD serially are limited in length. We therefore aimed to evaluate longitudinal changes in MRD-status (dynamics) and their association with progression-free survival in patients with multiple myeloma., Methods: In this single-centre, single-arm, phase 2 study, we enrolled patients aged 18 years and older from the Memorial Sloan Kettering Cancer Center (New York, NY, USA) who had newly diagnosed multiple myeloma following unrestricted frontline therapy and an Eastern Cooperative Oncology Group Performance Status of 2 or lower, including patients who started maintenance before study enrolment. All participants received lenalidomide maintenance at 10 mg for 21 days of 28-day cycles until progression or unacceptable toxic effects for up to 5 years on protocol. The primary endpoint was progression-free survival at 60 months per protocol and key secondary endpoints were MRD rates after completion of the 12th, 24th, and 36th cycle of maintenance and the association between progression-free survival and annual measurement of MRD status. MRD was assessed from first-pull bone marrow aspirates at baseline and annually by flow cytometry per International Myeloma Working Group criteria, (limit of detection of at least 1 × 10 -5 ) up to a maximum of 5 years. Patients who completed at least four cycles of treatment were included in the analysis of the primary endpoint, and patients who had completed at least one dose of treatment on protocol were assessable for secondary endpoints. The study was registered at ClinicalTrials.gov, NCT02538198, and is now closed to accrual., Findings: Between Sept 8, 2015, and Jan 25, 2019, 108 patients (100 evaluable for the primary endpoint) were enrolled. Median follow-up was 40·7 months (95% CI 38·7-45·0). At 60 months, progression-free survival was 64% (95% CI 52-79). Median progression-free survival was unreached (95% CI unreached-unreached). MRD dynamics were assessed using 340 MRD assessments done over 5 years for 103 evaluable patients. Patients who sustained MRD negativity for 2 years (n=34) had no recorded disease progression at median 19·8 months (95% CI 15·8-22·3) past the 2-year maintenance landmark. By contrast, patients who lost their MRD-negative responses (n=10) were more likely to progress than those with sustained MRD negativity (HR infinite; p<0·0001) and those with persistent MRD positivity (HR 5·88, 95% CI 1·18-33·33; p=0·015) at the 2-year landmark. Haematological and non-haematological serious adverse events occurred in 19 patients (18%). The most common adverse events of grade 3 or worse were decreased lymphocyte count in 48 (44%) patients and decreased neutrophil count in 47 (44%) patients. One death occurred on study due to sepsis and heart failure and was considered unrelated to the study drug., Interpretation: Serial measurements of MRD allow for dynamic assessment of risk for disease progression. Early intervention should be investigated for patients with loss of MRD negativity. Sustained MRD positivity is not categorically an unfavourable outcome and might portend prolonged stability of low-level disease., Funding: Memorial Sloan Kettering and Celgene., Competing Interests: Declaration of interests MA reports personal fees from Invivoscribe, Biocartis, and AstraZeneca, outside the submitted work. BD reports personal fees from Medscape, outside the submitted work. ADo reports grants from Roche; grants and personal fees from Takeda; and personal fees from PeerView, Physician's Education Resource (PER), Seattle Genetics, and EUSA Pharma, outside the submitted work. SG reports personal fees and advisory role (scientific advisory board) from Actinnum, Celgene, Bristol Myers Squibb, Sanofi, Amgen, Pfizer, GlaxoSmithKline, JAZZ, Janssen, Omeros, Takeda, and Kite, outside the submitted work. HH reports grants from Celgene, during the conduct of the study; and grants from Celgene, Takeda, and Janssen, outside the submitted work. CH reports Honorarium from Invivoscribe, outside the submitted work. MH reports research funding from the Swedish Blood Cancer Foundation, the Karolinska Institute Foundations, and clinical trial collaborations with GlaxoSmithKline, Amgen, and Daiichi Sankyo. NK reports research funding through Amgen and participates in advisory board with Medimmune. OLah reports serving on Advisory Board for MorphoSys. OLan reports grants from Amgen, Janssen, and Takeda; Data Monitoring Committee from Janssen, Merck, and Takeda; and personal fees from Amgen, Janssen, GlaxoSmithKline, AstraZeneca, and The Binding Site, outside the submitted work. AML reports grants from Novartis, during the conduct of the study; grants from Bristol Myers Squibb; personal fees from Trillium Therapuetics; grants, personal fees and non-financial support from Pfizer; and grants and personal fees from Janssen, outside the submitted work. AML also has a patent US20150037346A1 with royalties paid. SM reports research funding from Allogene Therapeutics, Juno/Bristol Myers Squibb, Takeda Oncology, and Janssen Oncology; personal fees from Plexus communication, and Physician Education Resource, outside the submitted work. MS reports personal fees and other from Angiocrine Bioscience and Omeros Corporation; personal fees from McKinsey & Company, Kite, and i3Health, outside the submitted work. GLS reports research funding from Janssen and Amgen outside the submitted work. US reports personal fees from Physicians Educations Resources; grants and other from Celgene/Bristol Myers Squibb; other from Janssen; and grants from Parker Institute for Cancer Immunotherapy and Myeloma Crowd, outside the submitted work. ELS reports personal fees from Bristol Myers Squibb, Fate Therapuetics, Precision Biosciences, and Chimera Therapuetics, outside the submitted work. ELS has several patents for varying CAR T cells and antibody products for multiple myeloma with royalties paid to Bristol Myers Squibb. CT reports other from Janssen Research and Development outside the submitted work. KT reports non-financial support from The Binding Site; and grants and personal fees from Sebia, outside the submitted work. DJC, ADe, SD, HJL, SXL, KM, FM, KM, TP, LR, MR, and EHR declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: ROS1 fusions are oncogenic drivers in 1% to 3% of NSCLCs. The activity of immune checkpoint inhibitor (ICI) monotherapy or in combination with chemotherapy (chemotherapy with ICI [chemo-ICI]) in these tumors and their immunophenotype have not been systematically described., Methods: In this multi-institutional retrospective study, tumor programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB) were evaluated in patients with ROS1- rearranged NSCLC. Time-to-treatment discontinuation (TTD) and objective response rate (ORR) (Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1) were calculated for patients treated with ICI or chemo-ICI in the metastatic setting., Results: A total of 184 patients were identified. Among 146 assessable cases, PD-L1 expression was less than 1% in 60 (41%), 1% to 49% in 35 (24%), and greater than or equal to 50% in 51 tumors (35%). Of 100 (92%) TMB-assessable tumors, 92 had less than 10 mutations per megabase. TMB was significantly lower for ROS1- rearranged tumors (n = 97) compared with tumors with EGFR (n = 1250) or KRAS alterations (n = 1653) and all other NSCLC tumors (n = 2753) evaluated with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (median TMB = 2.6 versus 3.5, 7.0, and 6.1 mutations per megabase, p < 0.001). Among patients treated with ICI, median TTD was 2.1 months (95% confidence interval [CI]: 1.0-4.2 mo; n = 28) and ORR 13% (2 of 16 RECIST-assessable; 95% CI: 2%-38%). Among patients treated with chemo-ICI, median TTD was 10 months (95% CI: 4.7-14.1 mo; n = 11) and ORR 83% (5 of 6 RECIST-assessable; 95% CI: 36%-100%). There was no difference in PD-L1 expression ( p  = 0.91) or TMB ( p  = 0.83) between responders and nonresponders., Conclusions: Most ROS1- rearranged NSCLCs have low PD-L1 expression and TMB. The activity of ICI in these tumors is modest. In contrast, chemo-ICI can achieve meaningful activity., (© 2021 The Authors.)