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Background Cell-free DNA (cfDNA) analysis using peripheral blood represents an exciting, minimally invasive technology for cancer diagnosis and monitoring. The reliability of testing is dependent on the accuracy and sensitivity of specific molecular analyses to detect tumor-associated genomic variants and on the quantity and quality of cfDNA available for testing. Specific guidelines for standardization and design of appropriate quality programs focused specifically on cfDNA isolation are lacking, as are standardized quality control reagents. Content This report describes and illustrates quality control and quality assurance processes, supported by generation of in-house quality control material, to ensure the reliability of the preanalytical phase of cfDNA analysis. Summary We have developed a robust quality program to support high-volume automated cfDNA extraction from peripheral blood by implementing processes and procedures designed to monitor the adequacy of specimen collection, specimen stability, efficiency of cfDNA extraction, and cfDNA quality.

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

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of a novel ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(−) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.SIGNIFICANCE STATEMENTEndocrine resistant and metastatic breast cancer (BC) is a clinically significant problem. Our study of fulvestrant resistant cancer cells led to the discovery of a novel ERα isoform which we call ERα-LBD. Encoded by a truncated transcript variant (ESR1-LBD) and lacking the N-terminal domains (activation of transcription and DNA binding), ERα-LBD displays a unique role in BC tumorigenesis and progression by mechanisms that may involve metabolic and cell growth advantages, stemness and therapy resistance. Importantly, ESR1-LBD is preferentially expressed in human breast tumor tissues and may be used as prognostic marker in BC.

Paired T cell receptor and RNA single cell sequencing (scTCR/RNA-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scTCR/RNA-seq dataset of 162,062 single T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients who underwent resections for progressing lung cancers after immune checkpoint blockade (ICB). We found marked regional heterogeneity in tumor persistence that was associated with heterogeneity in CD4 and CD8 T cell phenotypes; regions with persistent cancer cells were enriched for follicular helper CD4 T cells (TFH), regulatory T cells (Treg), and exhausted CD8 T cells. Clonal analysis demonstrated that highly-expanded T cell clones were predominantly of the CD8 subtype, were ubiquitously present across all sampled regions, found in the peripheral circulation, and expressed gene signatures of ‘large’ and ‘dual-expanded’ clones that have been predictive of response to ICB. Longitudinal tracking of CD8 T cell clones in the peripheral blood revealed that the persistence of ubiquitous CD8 T cell clones, as well as phenotypically distinct clones with tumor-reactive features, correlated with systemic tumor control. Finally, tracking CD8 T cell clones across tissues revealed the presence of TCF-1+precursor exhausted CD8 T cells in tumor draining LNs that were clonally linked to expanded exhausted CD8 T cells in tumors. Altogether, this comprehensive scTCR/RNA-seq dataset with regional, longitudinal, and clonal resolution provides fundamental insights into the tissue distribution, persistence, and differentiation trajectories of ICB-responsive T cells that underlie clinical responses to ICB.

TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition1–8. With the recent approval of the first selective TRK inhibitor, larotrectinib, for patients with any TRK-fusion-positive adult or pediatric solid tumor, to identify mechanisms of treatment failure after initial response has become of immediate therapeutic relevance. So far, the only known resistance mechanism is the acquisition of on-target TRK kinase domain mutations, which interfere with drug binding and can potentially be addressable through second-generation TRK inhibitors9–11. Here, we report off-target resistance in patients treated with TRK inhibitors and in patient-derived models, mediated by genomic alterations that converge to activate the mitogen-activated protein kinase (MAPK) pathway. MAPK pathway-directed targeted therapy, administered alone or in combination with TRK inhibition, re-established disease control. Experimental modeling further suggests that upfront dual inhibition of TRK and MEK may delay time to progression in cancer types prone to the genomic acquisition of MAPK pathway-activating alterations. Collectively, these data suggest that a subset of patients will develop off-target mechanisms of resistance to TRK inhibition with potential implications for clinical management and future clinical trial design. A subset of patients treated with selective TRK inhibitors (including the newly approved larotrectinib) develop off-target resistance mediated by genomic acquisition of MAPK pathway-activating alterations, and may benefit from combined targeted therapy.

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.

Lineage plasticity contributes to therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon drives neuroendocrine (NE) and squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is lower than that of either LUAD or de novo LUSC patients. To date, little is known about the molecular effectors enhancing lineage plasticity and driving histological transdifferentiation due to the paucity of well annotated pre- and post-transdifferentiation clinical samples amenable for molecular analyses. Currently no specific therapies for LUSC or NE transdifferentiation prevention are available for patients at high risk of transformation. We performed multi-omic profiling of transdifferentiating clinical samples, as well as control never-transformed LUAD and de novo LUSC and small cell carcinomas, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Our data suggest that histological transdifferentiation is driven by epigenetic -rather than mutational- events, and indicate that transdifferentiated tumors retain molecular features of their previous LUAD state. Integrative analysis revealed biological pathways dysregulated specifically for distinct histological outcomes, including downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses revealed commonly dysregulated pathways for transdifferentiated tumors, including marked downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in EGFR-mutant patient-derived xenograft transdifferentiation models. These results identify common and histology-specific drivers and dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to constrain lineage plasticity and prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Jacklynn Egger, Juan Qiu, Elisa de Stanchina, Natasha Rekhtman, Brian Houck-Loomis, Richard P. Koche, Helena A. Yu, Triparna Sen, Charles M. Rudin. AKT pathway as a therapeutic target to constrain lineage plasticity leading to histological transdifferentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 658.

e21166 Background: In lung adenocarcinomas (LUADs), lineage plasticity drives neuroendocrine (NE) and squamous cell (LUSC) transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is remarkably lower than those of LUAD or de novo LUSC patients. The paucity of transforming clinical specimens amenable for molecular analyses has hindered the identification of histological transformation drivers, and to date no specific therapies aimed to prevent or delay transdifferentiation-led therapy relapse are available for patients at high risk of transformation. Methods: We performed multi-omic profiling of LUAD-to-LUSC and LUAD-to-NE transdifferentiating clinical samples, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Clinical findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Results: Our data supports that histological transdifferentiation from LUAD to LUSC or NE tumors is driven by epigenetic remodeling rather than by mutational events, and indicate that transdifferentiated tumors retain epigenomic features of their previous LUAD state. Integrative epigenomic, transcriptomic and protein analysis revealed divergent biological pathways dysregulated for each histological outcome, such as downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses identified commonly dysregulated pathways in both NE- and LUSC-transdifferentiating tumors, including remarkable downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in different EGFR-mutant patient-derived xenograft transdifferentiation models. Conclusions: These results identify common and divergent dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation.

Lineage plasticity, a capacity to reprogram cell phenotypic identity under evolutionary pressure, is implicated in treatment resistance and metastasis in multiple cancers. In lung adenocarcinomas (LUADs) amenable to treatment with targeted inhibitors, transformation to an aggressive neuroendocrine (NE) carcinoma resembling small cell lung cancer (SCLC) is a recognized mechanism of acquired resistance. Defining molecular mechanisms of NE transformation in lung cancer has been limited by a paucity of well annotated pre- and post-transformation clinical samples. We hypothesized that mixed histology LUAD/SCLC tumors may capture cancer cells proximal to, and on either side of, histologic transformation. We performed detailed genomic, epigenomic, transcriptomic and proteomic characterization of combined LUAD/SCLC tumors as well as pre- and post-transformation clinical samples. Our data support that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm in pre-transformation LUADs. Consistent shifts in gene expression programs in NE transformation include induction of several stem/progenitor cell regulatory pathways, including upregulation of PRC2 and WNT signaling, and suppression of Notch pathway activity. We observe induction of PI3K/AKT and an immunosuppressive phenotype in NE transformation. Taken together our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer.

Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 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 and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.

In many areas of oncology, we lack sensitive tools to track low-burden disease. Although cell-free DNA (cfDNA) shows promise in detecting cancer mutations, we found that the combination of low tumor fraction (TF) and limited number of DNA fragments restricts low-disease-burden monitoring through the prevailing deep targeted sequencing paradigm. We reasoned that breadth may supplant depth of sequencing to overcome the barrier of cfDNA abundance. Whole-genome sequencing (WGS) of cfDNA allowed ultra-sensitive detection, capitalizing on the cumulative signal of thousands of somatic mutations observed in solid malignancies, with TF detection sensitivity as low as 10−5. The WGS approach enabled dynamic tumor burden tracking and postoperative residual disease detection, associated with adverse outcome. Thus, we present an orthogonal framework for cfDNA cancer monitoring via genome-wide mutational integration, enabling ultra-sensitive detection, overcoming the limitation of cfDNA abundance and empowering treatment optimization in low-disease-burden oncology care. A new approach for whole-genome sequencing of plasma circulating tumor DNA allows for dynamic monitoring of disease burden and ultra-sensitive detection of minimal residual disease.

PURPOSECell-free DNA (cfDNA) analysis offers a noninvasive means to access the tumor genome. Despite limited sensitivity of broad-panel sequencing for detecting low-frequency mutations in cfDNA, it may enable more comprehensive genomic characterization in patients with sufficiently high disease burden. We investigated the utility of large-panel cfDNA sequencing in patients enrolled to a Phase I AKT1-mutant solid tumor basket study.METHODSPatients had AKT1 E17K-mutant solid tumors and were treated on the multicenter basket study (ClinicalTrials.gov identifier: NCT01226316 ) of capivasertib, an AKT inhibitor. Serial plasma samples were prospectively collected and sequenced using exon-capture next-generation sequencing (NGS) analysis of 410 genes (Memorial Sloan Kettering [MSK]-Integrated Molecular Profiling of Actionable Cancer Target [IMPACT]) and allele-specific droplet digital polymerase chain reaction (ddPCR) for AKT1 E17K. Tumor DNA (tDNA) NGS (MSK-IMPACT) was also performed on available pretreatment tissue biopsy specimens.RESULTSAmong 25 patients, pretreatment plasma samples were sequenced to an average coverage of 504×. Somatic mutations were called in 20/25 (80%), with mutant allele fractions highly concordant with ddPCR of AKT1 E17K ( r2= 0.976). Among 17 of 20 cfDNA-positive patients with available tDNA for comparison, mutational concordance was acceptable, with 82% of recurrent mutations shared between tissue and plasma. cfDNA NGS captured additional tumor heterogeneity, identifying mutations not observed in tDNA in 38% of patients, and revealed oncogenic mutations in patients without available baseline tDNA. Longitudinal cfDNA NGS (n = 98 samples) revealed distinct patterns of clonal dynamics in response to therapy.CONCLUSIONLarge gene panel cfDNA NGS is feasible for patients with high disease burden and is concordant with single-analyte approaches, providing a robust alternative to ddPCR with greater breadth. cfDNA NGS can identify heterogeneity and potentially biologically informative and clinically relevant alterations.

PURPOSE Fibroblast growth factor receptor (FGFR) 2 alterations, present in 5%-15% of intrahepatic cholangiocarcinomas (IHC), are targets of FGFR-directed therapies. Acquired resistance is common among patients who respond. Biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. We studied circulating tumor DNA (ctDNA) as a less invasive means of potentially identifying genomic mechanisms of resistance to FGFR-targeted therapies. MATERIALS AND METHODS Serial blood samples were collected from eight patients with FGFR-altered cholangiocarcinoma for ctDNA isolation and next-generation sequencing (NGS) throughout treatment and at resistance to anti-FGFR–targeted therapy. ctDNA was sequenced using a custom ultra-deep coverage NGS panel, incorporating dual index primers and unique molecular barcodes to enable high-sensitivity mutation detection. RESULTS Thirty-one acquired mutations in FGFR2, 30/31 located in the kinase domain, were identified at resistance in six of eight patients with detectable ctDNA. Up to 13 independent FGFR2 mutations were detected per patient, indicative of striking genomic concordance among resistant subclones. CONCLUSION ctDNA could be an effective means to longitudinally monitor for acquired resistance in FGFR2-altered IHC. The numerous acquired genetic alterations in FGFR2 suggest frequent polyclonal mechanisms of resistance that cannot be detected from single-site tissue biopsies.

High-throughput single-cell RNA sequencing has transformed our understanding of complex cell populations, but it does not provide phenotypic information such as cell-surface protein levels. Here, we describe cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), a method in which oligonucleotide-labeled antibodies are used to integrate cellular protein and transcriptome measurements into an efficient, single-cell readout. CITE-seq is compatible with existing single-cell sequencing approaches and scales readily with throughput increases.

IMPORTANCE: The combination of erlotinib and bevacizumab as initial treatment of epidermal growth factor receptor (EGFR [OMIM 131550])–mutant lung cancers improves progression-free survival (PFS) compared with erlotinib alone. Because osimertinib prolongs PFS compared with erlotinib, this trial was designed to study the combination of osimertinib and bevacizumab as first-line treatment. OBJECTIVES: To determine the safety and tolerability of osimertinib and bevacizumab combination treatment and assess the 12-month PFS of the combination in patients with metastatic EGFR-mutant lung cancers. DESIGN, SETTING, AND PARTICIANTS: From August 15, 2016, to May 15, 2018, 49 patients with metastatic EGFR-mutant lung cancers were enrolled in this interventional clinical trial, conducted at a single academic cancer center. In the phase 1 portion of the study, a standard 3 + 3 dose de-escalation design was used to determine the maximum tolerated dose of osimertinib and bevacizumab. In the phase 2 portion of the study, patients were treated at the maximum tolerated dose defined in the phase 1 portion. Statistical analysis was performed from August 1 to October 1, 2019. INTERVENTIONS: All patients received osimertinib, 80 mg daily, and bevacizumab, 15 mg/kg once every 3 weeks. MAIN OUTCOMES AND MEASURES: The primary objective of the phase 2 portion of the study was to determine the number of patients receiving the combination of osimertinib and bevacizumab who were progression free at 12 months. Secondary end points included overall response rate, median PFS, overall survival, and definition of the toxic effects of the combination treatment. RESULTS: Among the 49 patients in the study (34 women; median age, 60 years [range, 36-83 years]), PFS at 12 months was 76% (95% CI, 65%-90%). The overall response rate was 80% (95% CI, 67%-91%), and median PFS was 19 months (95% CI, 15-24 months). Of the 6 patients with measurable central nervous system disease, all had a partial or complete central nervous system response. Persistent detection of EGFR-mutant circulating tumor (ct)DNA at 6 weeks was associated with shorter median PFS (clearance at 6 weeks, 16.2 months [95% CI, 13 months to not reached]; and no clearance at 6 weeks, 9.8 months [95% CI, 4 months to not reached]; P = .04) and median overall survival (clearance at 6 weeks, not reached; and no clearance at 6 weeks, 10.1 months [95% CI, 6 months to not reached]; P = .002). Identified mechanisms of resistance included squamous cell transformation (n = 2) pleomorphic transformation (n = 1), and acquired EGFR L718Q (n = 1) and C797S (n = 1) mutations. CONCLUSIONS AND RELEVANCE: The combination of osimertinib and bevacizumab met the study’s prespecified effectiveness end point. Persistent EGFR-mutant circulating tumor DNA at 6 weeks was associated with early progression and shorter survival. A randomized phase 3 study comparing osimertinib and bevacizumab with osimertinib alone is planned. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02803203

Introduction: Circulating cell-free DNA (cfDNA) is a source of tumor-derived DNA to interrogate somatic alterations when tissue is not available or of insufficient quantity for analysis. At MSKCC, we have developed and validated MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), a targeted next-generation sequencing assay that can detect ultra-low frequency somatic variants in select exons and introns of 129 genes. MSK-ACCESS can identify mutations, copy number alterations, gene fusions, and MSI status in plasma and was recently approved by the NYS-DOH for clinical testing. Here, we present the results of the validation study and our clinical experience with MSK-ACCESS since June 2019. Methods: Target regions from 129 genes were selected to maximize coverage of actionable, oncogenic, and hotspot mutations based on the first 25,000 tumors sequenced using MSK-IMPACT, our institutional clinical sequencing assay. Plasma cfDNA and buffy-coat DNA were extracted from whole blood collected in cell-stabilizing tubes (STRECK BCT cell-free DNA tube). Unique molecular indexes were introduced during DNA library construction, allowing for error suppression from consensus reads collapsed by Marianas, an in-house-developed algorithm. These consensus reads enable variant calling at low allelic frequency (AF) based on a 10−6 background error rate. Results: Analytical validation of MSK-ACCESS demonstrated 93% accuracy (n=100 variants), 99% precision (n=153 variants), and 100% sensitivity based on an assay limit of detection of 0.5% AF (n=19 variants). Variants were detected down to 0.1% AF. To date, 240 clinical cfDNA and matched normal DNA pairs have been sequenced, analyzed for somatic alterations, and clinically reported to guide patient management. Most clinical cases were from lung (55%) or prostate (13%) cancers and submitted for diagnostic purposes (71%). Median raw coverage was 18,367X, and median consensus coverage was 1411X. Mutations were detected in 180 (75%) samples with a median variant AF of 1.8% (0.02% - 95%). Comparison of concurrent commercial plasma testing results to MSK-ACCESS revealed multiple variants that were of clonal hematopoiesis or germline origin incorrectly reported as somatic variants. In the lung cohort, 48 patients had tissue testing with MSK-IMPACT; among 32 patients with a driver alteration detected by MSK-ACCESS, 91% had the identical driver alteration reported by MSK-IMPACT. Additionally, MSK-ACCESS identified a MET exon 14 alteration in one lung cancer patient that led to protocol enrollment and partial response. Conclusions: Liquid biopsy testing using MSK-ACCESS reliably detected clinically actionable mutations, reducing the need for multiple biopsies. These results also illustrate the importance of analyzing a matched normal sample when interpreting cfDNA results and highlight the potential of using cfDNA analysis to guide treatment selection, assess for treatment response, and identify mechanisms of treatment resistance. This abstract is also being presented as Poster A20. Citation Format: A. Rose Brannon, Gowtham Jayakumaran, Monica Diosdado, Yu Hu, Anna Razumova, Fanli Meng, Emily Lebow, Juber Patel, Ian Johnson, Preethi Srinivasan, Maysun Hasan, Jenna-marie Dix, Aijazuddin Syed, Brian Houck-Loomis, Bob T. Li, Charles Rudin, David Solit, Marc Ladanyi, Maria Arcila, Dana Tsui, Ahmet Zehir, Michael Berger, Ryma Benayed. Validation and clinical implementation of MSK-ACCESS, an ultra-deep sequencing assay for noninvasive somatic mutation profiling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR08.

Lynch syndrome (LS), an inherited predisposition syndrome associated with an increased risk of colorectal, endometrial, and other cancers, is characterized by germline mutations in mismatch repair pathway genes, which typically lead to microsatellite instability (MSI) in the resulting tumors. The FDA approval of pembrolizumab for all advanced MSI-H solid tumors has led to increasing MSI assessment. The presence of MSI in LS-associated tumors provides a unique and transformative opportunity for early detection and disease monitoring in these patients. Here we describe an approach to detect MSI from plasma cfDNA using MSK-ACCESS, a custom capture “liquid biopsy” approved for clinical use by the NY State Department of Health. In addition to frequently mutated exons of 129 genes, MSK-ACCESS also includes 165 highly informative microsatellite loci, selected from over 1,000 microsatellite regions based on >25,000 tumors sequenced using MSK-IMPACT, an FDA-authorized tumor sequencing panel. A key challenge in detecting MSI from cfDNA is the lack of ground truth in these samples, as cfDNA obtained from patients with MSI-high tumors may not always exhibit sufficient tumor-derived DNA fragments. To address this, we developed a machine learning approach for cfDNA analysis trained on orthogonally validated tumors sequenced via MSK-IMPACT. We present Allelic Distance-based Microsatellite Instability Estimator (ADMIE), an approach to translate deviation in tumor/cfDNA from normal/buffy coat DNA at individual microsatellite loci to a binary MSI call. ADMIE achieved a cross-validation precision of 1.00 +/- 0.02 and recall of 0.99 +/- 0.07. We ran this on 44 plasma samples collected from over 30 patients with MSI tumors including colorectal, prostate, and gastric cancers across multiple time points. We also evaluated plasma from 70 patients with known MSS tumors and 46 healthy controls. None of the cfDNA from healthy controls or patients with MSS tumors were found to be MSI positive, indicating high specificity. To establish our limit of detection, we performed in silico dilution experiments leveraging patient samples and MSI signal of biologic origin to simulate different tumor fractions, establishing our limit of detection at 1%. Among patients with MSI-high tumors, we found the presence and magnitude of MSI in the cfDNA to be correlated with measurable response to treatment with immunotherapy. In these patients, we detected MSI in the cfDNA of 6/8 samples where at least one mutation was detectable in plasma above 0.2% at baseline. Among the 4/6 patients for whom we had additional time points post treatment, we did not detect any mutations or evidence of MSI. In one patient, MSK-ACCESS indicated the presence of a second primary tumor based on the detection of MSI and mutations in cfDNA completely independent from those identified in the previously sequenced tumor. Our results suggest that MSI can be reliably detected in cfDNA using MSK-ACCESS and the MSI signature can represent a marker of occult metastatic disease in LS. This abstract is also being presented as Poster A54. Citation Format: Preethi Srinivasan, Alicia Latham, Zalak Patel, John Ziegler, Maysun Hasan, Juber A. Patel, Ian Johnson, Ronak Shah, Fanli Meng, Xiaohong Jing, Grittney Tam, Rose Brannon, Andrea Cercek, Ahmet Zehir, Brian Houck-Loomis, Dana Tsui, Zsofia Stadler, Michael F. Berger. MSI detection in plasma cfDNA: MSI as a marker of disease burden [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR07.

Despite rapid developments in single cell sequencing technology, sample-specific batch effects, detection of cell doublets, and the cost of generating massive datasets remain outstanding challenges. Here, we introduce cell “hashing”, where oligo-tagged antibodies against ubiquitously expressed surface proteins are used to uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples. We demonstrate our approach by pooling eight human PBMC samples on a single run of the 10x Chromium system, substantially reducing our per-cell costs for library generation. Cell “hashing” is inspired by, and complementary to, elegant multiplexing strategies based on genetic variation, which we also leverage to validate our results. We therefore envision that our approach will help to generalize the benefits of single cell multiplexing to diverse samples and experimental designs.

Recent high-throughput single-cell sequencing approaches have been transformative for understanding complex cell populations, but are unable to provide additional phenotypic information, such as protein levels of well-established cell-surface markers. Using oligonucleotide-labeled antibodies, we integrate measurements of cellular proteins and transcriptomes into an efficient, sequencing-based readout of single cells. This method is compatible with existing single-cell sequencing approaches and will readily scale as the throughput of these methods increase.

Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.

A noted clinical application for liquid biopsy is as a non-invasive method of detecting and monitoring of minimal residual disease (MRD) in patients with cancer. The low concentration of circulating tumor DNA in blood, especially in early stage cancers, however, complicates the detection of tumor-derived cell-free DNA. To address this challenge, we have developed MSK-ACCESS (Analysis of Circulating cfDNA to Examine Somatic Status), a custom NGS assay covering selected exons from 129 cancer related genes for high-sensitivity detection of somatic mutations from plasma. Using ultra-high depth sequencing, with duplex unique molecular indexing (UMI), unique dual sample barcodes, and background error suppression, MSK-ACCESS is able to detect low-frequency (0.1%) variants with high confidence. The design of MSK-ACCESS leverages our dataset of more than 30,000 tumors profiled by our institutional tumor sequencing assay, MSK-IMPACT, ensuring that the majority of patients harbor multiple mutations that can be tracked in plasma. We have validated MSK-ACCESS using plasma samples collected from 40 healthy individuals and 70 cancer patients harboring a range of somatic mutations in 11 genes. Greater than 95% of mutations at allele fractions >0.1% were empirically detected, and we established the performance characteristics of the assay through intra- and inter-assay reproducibility tests and dilution experiments. We have initiated clinical trials in multiple tumor types to evaluate the benefit of early therapeutic intervention in patients where MSK-ACCESS can detect circulating tumor DNA following surgery. Tumor mutations revealed by MSK-IMPACT in surgically resected specimens will be monitored at regular intervals as evidence of MRD. As a proof of concept, we have applied MSK-ACCESS to monitor variants known from tissue tumor sequencing in pre- and post-surgical cfDNA samples from 9 colon adenocarcinoma patients. All samples were sequenced to an average total depth of approximately 20,000X coverage and subsequently collapsed to consensus sequences exhibiting an average noise level less than 0.0006%. Circulating tumor DNA was detected in 66%(6/9) of the pre-surgical samples. Of these samples, ctDNA was also detected in 50% (3/6) of the post-surgical samples. Overall, this study shows that MSK-ACCESS can be used to successfully detect MRD. Citation Format: Maysun M. Hasan, Juber Patel, Ian Johnson, Fanli Meng, Grittney K. Tam, Xiaohong Jing, Julie L. Yang, A. Rose Brannon, Jayakumaran Gowtham, Dennis P. Stephens, Monica Diosdado, Ryma Benayed, Ahmet Zehir, Chin-Tung Chen, Martin R. Weiser, Dana Tsui, Brian Houck-Loomis, Michael Berger. Tracking minimal residual disease in post-operative cell-free DNA using MSK-ACCESS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1387.

4096 Background: FGFR2 alterations are present in 14% of cholangiocarcinomas (CCA) and are promising targets of investigational FGFR-directed therapies. Cell-free DNA profiling has emerged as a non-invasive approach to monitor disease and longitudinally characterize tumor evolution. We describe the use of circulating tumor DNA (ctDNA) among patients (pts) with FGFR2-altered CCA receiving FGFR-targeted therapy in the identification of acquired FGFR2 mutations (mut) at resistance. Methods: Serial blood samples were collected from 8 pts with FGFR-altered CCA for ctDNA isolation and next generation sequencing. Plasma ctDNA collected at baseline and resistance to FGFR-targeted therapy were sequenced using a custom ultra-deep coverage cfDNA panel, MSK-ACCESS, incorporating dual index primers and unique molecular barcodes to enable background error suppression and high-sensitivity mut detection. The assay was enhanced to include all protein-coding exons and relevant introns of FGFR2. In 5/8 pts, genomic profiling of an initial tumor biopsy was performed. Results: 8 pts with FGFR2-altered CCA (7 gene fusions, 1 amplification) were treated with FGFR-targeted therapies. 7/8 pts exhibited stable disease or partial response. 19 total acquired mut in FGFR2 were detected at resistance in 5/8 pts (between 1-9 unique mut identified in each sample). All mut were located in the kinase domain. Conclusions: Acquired mut in FGFR2 are seen in pts who have developed resistance to targeted therapy. CtDNA can be used to identify these mut at the time of acquired resistance. The multitude of FGFR2 mut observed within individual pts suggest heterogeneity and evolutionary convergence of resistance mechanisms. Our results illustrate the utility of ctDNA as a less invasive way to monitor for signs of resistance and to identify other potential targetable alterations. [Table: see text]

Trimethylated histone H3 lysine 4 (H3K4) and H3K27 generally mark transcriptionally active and repressive chromatins, respectively. In most cell types, these two modifications are mutually exclusive, and this segregation is crucial for the regulation of gene expression. However, how this anticorrelation is achieved has not been fully understood. Here, we show that removal of the H3K27 trimethyl mark facilitates recruitment of SET1-like H3K4 methyltransferase complexes to their target genes by eliciting a novel interaction between histone H3 and two common subunits, WDR5 and RBBP5, of SET1-like complexes. Consistent with this result, H3K27 trimethylation destabilizes interactions of H3 with SET1-like complexes and antagonizes their ability to carry out H3K4 trimethylation of peptide (H3 residues 1 to 36), histone octamer, and mononucleosome substrates. Altogether, our studies reveal that H3K27 trimethylation of histone H3 represses a previously unrecognized interaction between H3 and SET1-like complexes. This provides an important mechanism that directs the anticorrelation between H3K4 and H3K27 trimethylation.

Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag . This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release.

The human immunodeficiency virus type 1 (HIV-1) Gag protein recruits Tsg101 to facilitate HIV-1 particle budding and release. In uninfected cells, the Hrs protein recruits the ESCRT-I complex to the endosome, also through an interaction with Tsg101, to promote the sorting of host proteins into endosomal vesicles and multivesicular bodies. Here, we show that the overexpression of the C-terminal fragment of Hrs (residues 391 to 777) or Hrs mutants lacking either the N-terminal FYVE domain (mutant dFYVE) or the PSAP (residues 348 to 351) motif (mutant ASAA) all efficiently inhibit HIV-1 Gag particle production. Expression of the dFYVE or ASAA mutants of Hrs had no effect on the release of Moloney murine leukemia virus. Coimmunoprecipitation analysis showed that the expression of Hrs mutant dFYVE or ASAA significantly reduced or abolished the HIV-1 Gag-Tsg101 interaction. Yeast-two hybrid assays were used to identify two new and independent Tsg101 binding sites, one in the Hrs coiled-coil domain and one in the proline/glutamic acid-rich domain. Scanning electron microscopy of HeLa cells expressing HIV-1 Gag and the Hrs ASAA mutant showed viral particles arrested in “lump-like” structures that remained attached to the cell surface. Together, these data indicate that fragments of Hrs containing the C-terminal portion of the protein can potently inhibit HIV-1 particle release by efficiently sequestering Tsg101 away from the Gag polyprotein.

In the nucleus of a cell, chromatin is structured into nucleosomes in which the DNA is wrapped around a histone core, condensing the DNA. In this form, DNA is not readily accessible for gene expression. Post-translational modification to the histone proteins, such as acetylation, regulates gene expression. Histone acetyltranferase (HAT) catlyzes the transfer of an acetyl group to a target lysine by aligning an acetyl-CoA group with a lysine on the unstructured histone tail region. The acetylated lysine tail then becomes a binding site for other factors. Bromodomain-containing proteins (BRD) recognize and bind to the acetylated lysine and recruit transcription factors and RNA polymerase II complex to the DNA. The transcriptional coactivator p300 contains both a HAT and BRD region and is known to play an important role in cell cycle and tumor suppression. The activity of the p300 HAT is in part dependent upon the activity of its BRD, and disruption of the BRD can significantly affect transcription of p300-r...

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly1,2. Pol is expressed exclusively as a Gag–Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon3. Both of these mechanisms occur infrequently and only affect 5–10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag–Pol ratio4–8. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.