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Purpose:Oncogenic kinase fusions are targetable with approved and investigational therapies and can also mediate acquired resistance (AR) to targeted therapy. We aimed to understand the clinical validity of liquid biopsy comprehensive genomic profiling (CGP) to detect kinase fusions pan tumor.Experimental Design:CGP was performed on plasma and tissue samples during clinical care. All exons plus selected introns of 16 kinases involved in oncogenic fusions (ALK, BRAF, EGFR, ERBB2, FGFR1/2/3, MET, NTRK1/2/3, PDGFRA/B, RAF1, RET, and ROS1) were sequenced to capture fusions, including well-characterized and novel breakpoints. Plasma circulating tumor DNA (ctDNA) fraction was estimated to inform sensitivity.Results:Of 36,916 plasma cases, 32,492 (88%) had detectable ctDNA. Kinase fusions were detected in 1.8% of ctDNA-positive cases (571/32,492) and were most prevalent in patients with cholangiocarcinoma (4.2%), bladder cancer (3.6%), and non–small cell lung cancer (NSCLC; 3.1%). Of the 63 paired patient samples that had tissue and ctDNA specimens collected within 1 year and with estimated plasma ctDNA fraction >1%, fusions were detected in 47 of 51 (92%) liquid specimens with a fusion in the tissue sample. In 32 patients with fusions detected in liquid but not in tissue, 21 (66%) had evidence of putative acquired resistance.Conclusions:Targetable kinase fusions are identified in ctDNA across cancer types. In pairs with tissue-identified fusions, fusion detection in ctDNA is reliable with elevated ctDNA fraction. These data support the validity of CGP to enable ctDNA-based fusion detection for informing clinical care in patients with advanced cancer.

Supplementary Data from The Pan-Tumor Landscape of Targetable Kinase Fusions in Circulating Tumor DNA

Background The amounts of circulating cell-free DNA (cfDNA) and circulating-tumor DNA (ctDNA) present in peripheral blood liquid biopsies can vary due to preanalytic/analytic variables. In this study, we examined the impact of patient age, sex, stage, and tumor type on cfDNA yield, ctDNA fraction, and estimated ctDNA quantity from a large cohort of clinical liquid biopsy samples. Methods We performed a retrospective analysis of 12 139 consecutive samples received for liquid biopsy (FoundationOne® Liquid) clinical testing. Results Significant differences in both cfDNA yield and estimated ctDNA quantity were observed based on the underlying tumor type that initiated the liquid biopsy analysis and the stage of the patient (P Conclusions In this study, we show that ctDNA quantity varied significantly based on patient age, sex, stage, and tumor type, which could offer an explanation as to why certain liquid biopsy specimens are more likely to fail sequencing or provide clinically meaningful results. In addition, this could affect future clinical decisions on the blood sample volumes required to allow successful liquid biopsy testing.

PURPOSEMET exon 14 ( METex14) skipping alterations are oncogenic drivers in non–small-cell lung cancer (NSCLC). We present a comprehensive overview of METex14 samples from 1,592 patients with NSCLC, associated clinicogenomic characteristics, potential mechanisms of acquired resistance, treatment patterns, and outcomes to MET inhibitors.METHODSHybrid capture–based comprehensive genomic profiling (CGP) was performed on samples from 69,219 patients with NSCLC. For treatment patterns and outcomes analysis, patients with advanced METex14-altered NSCLC were selected from the Flatiron Health-Foundation Medicine clinicogenomic database, a nationwide deidentified electronic health record–derived database linked to Foundation Medicine CGP for patients treated between January 2011 and March 2020.RESULTSA total of 1,592 patients with NSCLC (2.3%) were identified with 1,599 METex14 alterations spanning multiple functional sites (1,458 of 60,244 tissue samples and 134 of 8,975 liquid samples). Low tumor mutational burden and high programmed death ligand 1 expression were enriched in METex14-altered samples. MDM2, CDK4, and MET coamplifications and TP53 mutations were present in 34%, 19%, 11%, and 42% of tissue samples, respectively. Comparing tissue and liquid cohorts, coalteration frequency and acquired resistance mechanisms, including multiple MET mutations, EGFR, ERBB2, KRAS, and PI3K pathway alterations, were generally similar. Positive percent agreement with the tissue was 100% for METex14 pairs collected within 1 year (n = 7). Treatment patterns showed increasing adoption of MET inhibitors in METex14-altered NSCLC after receipt of CGP results; the real-world response rate to MET inhibitors was 45%, and time to treatment discontinuation was 4.4 months.CONCLUSIONDiverse METex14 alterations were present in 2%-3% of NSCLC cases. Tissue and liquid comparisons showed high concordance and similar coalteration profiles. Characterizing common co-occurring alterations and immunotherapy biomarkers, including those present before or acquired after treatment, may be critical for predicting responses to MET inhibitors and informing rational combination strategies.

PURPOSE Profiling of circulating tumor DNA (ctDNA) is increasingly adopted in the management of solid tumors, concurrent with increased availability of more comprehensive ctDNA panels. However, variable ctDNA shed can result in variable assay sensitivity. We studied the relationship between ctDNA tumor fraction (TF) and detection of actionable alterations across cancer types. METHODS A total of 23,482 liquid biopsies (LBx) submitted between September 2020 and October 2021 were sequenced using a hybrid capture panel that reports genomic alterations (GAs) and genomic biomarkers across 324 cancer-related genes. The primary end points were the prevalence of targetable GAs by cancer type and detection in relationship to ctDNA TF. Sensitivity of detection in LBx was assessed in 1,289 patients with available tissue results. RESULTS 94% (n = 22,130) of LBx had detectable ctDNA, with a median TF of 2.2%. LBx profiling detected GAs in National Comprehensive Cancer Network category 1 genes in 37% of lung, 30% of prostate, 36% of breast, and 51% of colon cancer cases. Potential germline GAs flagged on clinical reports were detected in genes including BRCA1/2, PALB2, CHEK2, and ATM. Polyclonal mutations in genes associated with resistance such as AR, ESR1, RB1, and NF1 were detected. The sensitivity of LBx to detect driver alterations identified in tissue biopsy from the same patient ranged from 58% to 86% but was consistently at or near 100% in cases with TF ≥ 10%. CONCLUSION Elevated ctDNA shed is associated with both high sensitivity and negative predictive value for detection of actionable GAs. The presence of elevated TF suggests adequate tumor profiling and may reduce the value of subsequent reflex to confirmatory tissue testing in patients with negative LBx results.

BackgroundAt diagnosis, the majority of patients with intrahepatic cholangiocarcinoma (IHCC) present with advanced disease and a poor prognosis. Comprehensive genomic profiling (CGP) early in the disease course may increase access to targeted therapies and clinical trials; however, unresolved issues remain surrounding the optimal biopsy type to submit for CGP.Patients and MethodsMutational frequencies between primary tumor biopsies (Pbx), metastatic biopsies (Mbx), and liquid biopsies (Lbx) in 1,632 patients with IHCC were compared.ResultsPotentially actionable alterations were found in 52%, 34%, and 35% of patients in the Pbx, Mbx, and Lbx cohorts, respectively. In Pbx, Mbx, and Lbx, FGFR2 rearrangements were found in 9%, 6%, and 4%, and IDH1 mutations were identified in 16%, 5%, and 9% patients, respectively. Moreover, alterations in FGFR2 and IDH1 were significantly associated with distinct ancestries, including 2.1-fold enrichment for FGFR2 rearrangements in patients with African ancestry and 1.5-fold enrichment for IDH1 mutations in patients with admixed American (Hispanic) ancestry. Finally, the publication of biomarker-driven clinical trials in IHCC correlated with changing CGP testing patterns. Significant correlations between patient characteristics and IHCC trial disclosures were observed, including a significant decrease from time between biopsy and CGP testing, and more frequent testing of primary versus metastatic samples.ConclusionOverall, because of the high likelihood of identifying actionable genomic alterations, CGP should be considered for the majority of patients with inoperable IHCC, and Lbx and Mbx can be considered as part of the diagnostic suite.Implications for PracticeComprehensive genomic profiling (CGP) should be considered for all patients with intrahepatic cholangiocarcinoma (IHCC) or suspected IHCC, as actionable alterations were commonly found in multiple genes and a wide variety of FGFR2 fusion partners were identified. The disclosure of IHCC trial data correlated with increased use of CGP, an encouraging trend that moves new therapeutic options forward for rare cancers with a rare biomarker. Although tissue from the primary lesion may identify actionable alterations at higher rates, CGP of a liquid biopsy or metastatic site can be considered, particularly if the primary tissue block is exhausted.

Purpose: Comprehensive genomic profiling (CGP) is of increasing value for patients with metastatic castration-resistant prostate cancer (mCRPC). mCRPC tends to metastasize to bone, making tissue biopsies challenging to obtain. We hypothesized CGP of cell-free circulating tumor DNA (ctDNA) could offer a minimally invasive alternative to detect targetable genomic alterations (GA) that inform clinical care. Experimental Design: Using plasma from 3,334 patients with mCRPC (including 1,674 screening samples from TRITON2/3), we evaluated the landscape of GAs detected in ctDNA and assessed concordance with tissue-based CGP. Results: A total of 3,129 patients (94%) had detectable ctDNA with a median ctDNA fraction of 7.5%; BRCA1/2 was mutated in 295 (8.8%). In concordance analysis, 72 of 837 patients had BRCA1/2 mutations detected in tissue, 67 (93%) of which were also identified using ctDNA, including 100% of predicted germline variants. ctDNA harbored some BRCA1/2 alterations not identified by tissue testing, and ctDNA was enriched in therapy resistance alterations, as well as possible clonal hematopoiesis mutations (e.g., in ATM and CHEK2). Potential androgen receptor resistance alterations were detected in 940 of 2,213 patients (42%), including amplifications, polyclonal and compound mutations, rearrangements, and novel deletions in exon 8. Conclusions: Genomic analysis of ctDNA from patients with mCRPC recapitulates the genomic landscape detected in tissue biopsies, with a high level of agreement in detection of BRCA1/2 mutations, but more acquired resistance alterations detected in ctDNA. CGP of ctDNA is a compelling clinical complement to tissue CGP, with reflex to tissue CGP if negative for actionable variants. See related commentary by Hawkey and Armstrong, p. 2961

PURPOSE Human epidermal growth factor receptor 2 (HER2) overexpression or amplification (ERBB2amp) are biomarkers for approved anti-HER2 therapies. ERBB2amp may better predict response compared with immunohistochemistry or in situ hybridization, and quantitative copy number (CN) may further stratify patients. We characterized ERBB2amp in advanced gastroesophageal adenocarcinomas (GEA) and hypothesized that increased CN was associated with better outcome to trastuzumab. METHODS Comprehensive genomic profiling, including assessment of ERBB2amp, was performed for 12,905 GEA tissue cases. Clinical outcomes were assessed using a clinicogenomic database linking deidentified electronic health record–derived clinical data to genomic data. Multivariable Cox proportional hazard models were used for real-world progression-free survival (rwPFS) comparisons. RESULTS ERBB2amp (CN ≥ 5) was detected in 15% (1,934 of 12,905) of GEA; median CN 22 (interquartile range 9-73). Median ERBB2 amplicon size was 0.27 megabase (interquartile range 0.13-0.95), and smaller amplicons were associated with higher CN (P < .001). In the clinicogenomic database, of 101 evaluable first-line trastuzumab-treated patients, ERBB2 CN was a significant predictor of rwPFS as a continuous variable (adjusted hazard ratio = 0.73; 95% CI, 0.60 to 0.89; P = .002), whereas ERBB2 CN was not predictive of rwPFS on chemotherapy (adjusted hazard ratio = 0.93; 95% CI, 0.73 to 1.20; P = .59). Among trastuzumab-treated patients, no significant associations with ERBB2 CN were observed for disease site, age, stage at advanced diagnosis, or most selected coalterations. CONCLUSION ERBB2amp was detected in 15% of GEA tissue samples, with significant diversity in ERBB2 CN and amplicon focality. ERBB2 CN was predictive of rwPFS as a continuous variable for patients treated with trastuzumab. Further studies exploring the clinical utility of quantitative ERBB2 CN, particularly in the setting of the evolving anti-HER2 landscape and combination therapies, are warranted., ERBB2 copy number is a quantitative biomarker for outcomes to anti-HER2 therapy in advanced gastroesophageal cancer.

The most useful biomarkers for clinical decision-making identify patients likely to have improved outcomes with one treatment vs another.To evaluate treatment class-specific outcomes of patients receiving immune checkpoint inhibitor (ICI) vs taxane chemotherapy by tumor mutational burden (TMB).This comparative effectiveness analysis of clinical variables and outcomes used prospectively defined biomarker-stratified genomic data from a deidentified clinicogenomic database. Data included men with previously treated metastatic castration-resistant prostate cancer (mCRPC) receiving ICI or single-agent taxane chemotherapy from January 2011 to April 2021 at approximately 280 US academic or community-based cancer clinics (approximately 800 sites of care). Data were analyzed from July to August 2021.Single-agent ICI or single-agent taxanes. Treatments were assigned at discretion of physician and patient without randomization. Imbalances of known factors between treatment groups were adjusted with propensity weighting.Prostate-specific antigen (PSA) response, time to next therapy (TTNT), and overall survival (OS).A total of 741 men (median [IQR], 70 [64-76] years) with mCRPC received comprehensive genomic profiling and were treated with ICI or single-agent taxane therapy. At baseline, the median (IQR) PSA level was 79.4 (19.0-254) ng/mL, 108 men (18.8%) had Eastern Cooperative Oncology Group Performance Status scores of 2 or greater, and 644 men (86.9%) had received prior systemic treatments for mCRPC. A total of 45 patients (6.1%) received ICI therapy and 696 patients (93.9%) received taxane therapy. Among patients with TMB of fewer than 10 mutations per megabase (mt/Mb) receiving ICI, compared with those receiving taxanes, had worse TTNT (median [IQR], 2.4 [1.1-3.2] months vs 4.1 [2.2-6.3] months; hazard ratio [HR], 2.65; 95% CI, 1.78-3.95; P .001). In contrast, for patients with TMB of 10 mt/Mb or greater, use of ICIs, compared with use taxanes, was associated with more favorable TTNT (median [IQR], 8.0 [3.4 to unknown] months vs 2.4 [2.4-7.3] months; HR, 0.37, 95% CI, 0.15-0.87; P = .02) and OS (median 19.9 [8.06 to unknown] months vs 4.2 [2.69 - 6.12] months; HR, 0.23; 95% CI, 0.10-0.57; P = .001). Among all 741 patients, 44 (5.9%) had TMB of 10 mt/Mb or greater, 22 (3.0%) had high microsatellite instability, and 20 (2.7%) had both. Treatment interactions with TMB of 10 mt/Mb or greater (TTNT: HR, 0.10; 95% CI, 0.32-0.31; P .001; OS: HR, 0.25; 95% CI, 0.076-0.81; P = .02) were stronger than high microsatellite instability alone (TTNT: HR, 0.12; 95% CI, 0.03-0.51; P = .004; OS: HR, 0.38; 95% CI, 0.13-1.12; P = .08).In this comparative effectiveness study, ICIs were more effective than taxanes in patients with mCRPC when TMB was 10 mt/Mb or greater but not when TMB was fewer than 10 mt/Mb. The results add validity to the existing TMB cutoff of 10 mt/Mb for ICI use in later lines of therapy, and suggest that ICIs may be a viable alternative to taxane chemotherapy for patients with mCRPC with high TMB.

A majority of patients with metastatic colorectal cancer (mCRC) experience recurrence post curative-intent surgery. The addition of adjuvant chemotherapy has shown to provide limited survival benefits when applied to all patients. Therefore, a biomarker to assess molecular residual disease (MRD) accurately and guide treatment selection is highly desirable for high-risk patients. This feasibility study evaluated the prognostic value of a tissue comprehensive genomic profiling (CGP)-informed, personalized circulating tumor DNA (ctDNA) assay (FoundationOne®Tracker) (Foundation Medicine, Inc., Cambridge, MA, USA) by correlating MRD status with clinical outcomes. ctDNA analysis was performed retrospectively on plasma samples from 69 patients with resected mCRC obtained at the MRD and the follow-up time point. Tissue CGP identified potentially actionable alterations in 54% (37/69) of patients. MRD-positivity was significantly associated with lower disease-free survival (DFS) (HR: 4.97, 95% CI: 2.67–9.24, p < 0.0001) and overall survival (OS) (HR: 27.05, 95% CI: 3.60–203.46, p < 0.0001). Similarly, ctDNA positive status at the follow-up time point correlated with a marked reduction in DFS (HR: 8.78, 95% CI: 3.59–21.49, p < 0.0001) and OS (HR: 20.06, 95% CI: 2.51–160.25, p < 0.0001). The overall sensitivity and specificity at the follow-up time point were 69% and 100%, respectively. Our results indicate that MRD detection using the tissue CGP-informed ctDNA assay is prognostic of survival outcomes in patients with resected mCRC. The concurrent MRD detection and identification of actionable alterations has the potential to guide perioperative clinical decision-making.

Background Biomarkers predicting second-generation novel hormonal therapy (NHT) benefit relative to taxanes are critical for optimized treatment decisions for metastatic castration-resistant prostate cancer (mCRPC) patients. These associations have not been reported simultaneously for common mCRPC genomic biomarkers. Objective To evaluate predictive associations of common genomic aberrations in mCRPC using an established comprehensive genomic profiling (CGP) system. Design, setting, and participants A retrospective cohort study used data from a deidentified US-based clinicogenomic database comprising patients treated in routine clinical practice between 2011 and 2020, evaluated with Foundation Medicine CGP in tissue biopsies obtained around the time of treatment decision. The main cohort included 180 NHT and 179 taxane lines of therapy (LOTs) from 308 unique patients. The sequential cohort comprised a subset of the main cohort NHT LOTs immediately followed by taxane from 55 unique patients. Outcome measurements and statistical analysis Prostate-specific antigen (PSA) response, time to next treatment (TTNT), and overall survival (OS) were assessed. Main cohort analyses were adjusted for known treatment assignment biases via inverse probability of treatment weighting (IPTW) in treatment interaction models. Results and limitations In the main cohort, patients with AR amplification (ARamp) or PTEN aberrations (PTENalt) had worse relative PSA response on NHT versus taxanes compared with patients without. Patients with ARamp, PTENalt, or RB1 aberrations (RB1alt) also had worse relative TTNT and OS on NHT but not on taxanes. In multivariable models for TTNT and OS adjusted via IPTW, ARamp, PTENalt, and RB1alt were shown as poor prognostic factors overall and demonstrated significant treatment interactions, indicating reduced hazards of therapy switch and death on taxanes versus NHT. Consistent associations favoring increased benefit from subsequent taxane despite prior NHT treatment line were observed only for ARamp in the sequential cohort, in which very few patients had RB1alt for assessment. Conclusions ARamp status is a candidate biomarker to predict poor effectiveness of NHT relative to taxanes in mCRPC in scenarios where both options are considered. Patient summary Specific alterations in the DNA of tumors may assist in choosing between novel oral hormonal therapies and standard chemotherapy in advanced prostate cancer patients.

Pathological and genomic profiling have transformed breast cancer care by matching patients to targeted treatments. However, tumors evolve and evade therapeutic interventions often through the acquisition of genomic mutations. Here we examine patients profiled with tissue (TBx) and liquid biopsy (LBx) as part of routine clinical care, to characterize the tumor evolutionary landscape and identify potential vulnerabilities in the relapsed setting. Real-world evidence demonstrates that LBx is utilized later in care and identifies associations with intervening therapy. While driver events are frequently shared, acquired LBx alterations are detected in a majority of patients, with the highest frequency in ER+ disease and in patients with longer biopsy intervals. Acquired mutations are often polyclonal and present at lower allelic fractions, suggesting multi-clonal convergent evolution. In addition to well-characterized resistance mutations (e.g., ESR1, NF1, RB1, ERBB2), we observe a diversity of rarer but potentially targetable mutations (e.g., PIK3CA, HRAS/NRAS/KRAS, FGFR1/2/3, BRAF) and fusions (e.g., FGFR1/2, ERBB2, RET), as well as BRCA1/2 reversions through a variety of mechanisms, including splice alterations and structural deletions. This study provides insights on treatment and selection-driven tumor evolution and identifies potential combinatorial treatment options in advanced breast cancer.

FoundationOne®CDx (F1CDx) is a United States (US) Food and Drug Administration (FDA)-approved companion diagnostic test to identify patients who may benefit from treatment in accordance with the approved therapeutic product labeling for 28 drug therapies. F1CDx utilizes next-generation sequencing (NGS)-based comprehensive genomic profiling (CGP) technology to examine 324 cancer genes in solid tumors. F1CDx reports known and likely pathogenic short variants (SVs), copy number alterations (CNAs), and select rearrangements, as well as complex biomarkers including tumor mutational burden (TMB) and microsatellite instability (MSI), in addition to genomic loss of heterozygosity (gLOH) in ovarian cancer. CGP services can reduce the complexity of biomarker testing, enabling precision medicine to improve treatment decision-making and outcomes for cancer patients, but only if test results are reliable, accurate, and validated clinically and analytically to the highest standard available. The analyses presented herein demonstrate the extensive analytical and clinical validation supporting the F1CDx initial and subsequent FDA approvals to ensure high sensitivity, specificity, and reliability of the data reported. The analytical validation included several in-depth evaluations of F1CDx assay performance including limit of detection (LoD), limit of blank (LoB), precision, and orthogonal concordance for SVs (including base substitutions [SUBs] and insertions/deletions [INDELs]), CNAs (including amplifications and homozygous deletions), genomic rearrangements, and select complex biomarkers. The assay validation of >30,000 test results comprises a considerable and increasing body of evidence that supports the clinical utility of F1CDx to match patients with solid tumors to targeted therapies or immunotherapies based on their tumor’s genomic alterations and biomarkers. F1CDx meets the clinical needs of providers and patients to receive guideline-based biomarker testing, helping them keep pace with a rapidly evolving field of medicine.

Background This study assessed the contrasting genomic profiles from the primary tumors (PTs), metastatic (MET) sites, and circulating tumor DNA (ctDNA) of patients with prostate cancer (PC). Methods A total of 1294 PC tissue specimens and 2462 ctDNA specimens underwent hybrid capture-based comprehensive genomic profiling (CGP). Specimens included tissue from PTs; MET biopsies from bone, liver (LIV), lung (LU), brain (BN), lymph node, and soft tissue sites; and ctDNA. Results Differences in alteration frequencies between PT, MET, and ctDNA specimens for selected genes were observed. TMPRSS2:ERG fusion frequencies were similar between PTs and MET sites (35% vs 33%) but varied among MET sites. Genomic alterations (GAs) in AR were lowest in PTs (2%) and highest in MET sites (from 24% in LU to 50% in LIV). BN had the highest genomic alterations/tumor (8) and enrichment for PTEN GAs. The BRCA2 GA frequency varied from 0% in BN to 15% in LIV. ERBB2 amplification was increased in MET sites in comparison with PTs. RB1 GAs were increased in LIV. Biomarkers potentially associated with an anti-PD(L)1 response included CDK12 GAs (16% in LU) and a microsatellite instability-high status (29% in BN). Analyses of ctDNA featured a broad spectrum of GAs similar to those detected across MET sites. Conclusions CGP of PTs, MET sites, and ctDNA in PC exhibited differences most likely associated with tumor progression, clonal evolution, and exposure to systemic therapies; ctDNA can also capture a broad range of potential therapeutic opportunities for patients with PC.

Purpose: Oncogenic kinase fusions are targetable with approved and investigational therapies and can also mediate acquired resistance (AR) to targeted therapy. We aimed to understand the clinical validity of liquid biopsy comprehensive genomic profiling (CGP) to detect kinase fusions pan tumor. Experimental Design: CGP was performed on plasma and tissue samples during clinical care. All exons plus selected introns of 16 kinases involved in oncogenic fusions (ALK, BRAF, EGFR, ERBB2, FGFR1/2/3, MET, NTRK1/2/3, PDGFRA/B, RAF1, RET, and ROS1) were sequenced to capture fusions, including well-characterized and novel breakpoints. Plasma circulating tumor DNA (ctDNA) fraction was estimated to inform sensitivity. Results: Of 36,916 plasma cases, 32,492 (88%) had detectable ctDNA. Kinase fusions were detected in 1.8% of ctDNA-positive cases (571/32,492) and were most prevalent in patients with cholangiocarcinoma (4.2%), bladder cancer (3.6%), and non–small cell lung cancer (NSCLC; 3.1%). Of the 63 paired patient samples that had tissue and ctDNA specimens collected within 1 year and with estimated plasma ctDNA fraction >1%, fusions were detected in 47 of 51 (92%) liquid specimens with a fusion in the tissue sample. In 32 patients with fusions detected in liquid but not in tissue, 21 (66%) had evidence of putative acquired resistance. Conclusions: Targetable kinase fusions are identified in ctDNA across cancer types. In pairs with tissue-identified fusions, fusion detection in ctDNA is reliable with elevated ctDNA fraction. These data support the validity of CGP to enable ctDNA-based fusion detection for informing clinical care in patients with advanced cancer.

Immune checkpoint inhibitors (ICPI) have revolutionized cancer therapy and provided clinical benefit to thousands of patients. Despite durable responses in many tumor types, the majority of patients either fail to respond at all or develop resistance to the ICPI. Furthermore, ICPI treatment can be accompanied by serious adverse effects. There is an urgent need for identification of patient populations that will benefit from ICPI as single agents and when used in combinations. As ICPI have achieved regulatory approvals, accompanying biomarkers including PD-L1 immunohistochemistry (IHC) and tumor mutational burden (TMB) have also received approvals for some indications. The ICPI pembrolizumab was the first example of a tissue-agnostic FDA approval based on tumor microsatellite instability (MSI)/deficient mismatch repair (dMMR) biomarker status, rather than on tumor histology assessment. Several other ICPI-associated biomarkers are in the exploratory stage, including quantification of tumor-infiltrating lymphocytes (TILs), gene expression profiling (GEP) of an inflamed microenvironment, and neoantigen prediction. TMB and PD-L1 expression can predict a subset of responses, but they fail to predict all responses to checkpoint blockade. While a single biomarker is currently limited in its ability to fully capture the complexity of the tumor-immune microenvironment, a combination of biomarkers is emerging as a method to improve predictive power. Here we review the steadily growing impact of comprehensive genomic profiling (CGP) for development and utilization of predictive biomarkers by simultaneously capturing TMB, MSI, and the status of genomic targets that confer sensitivity or resistance to immunotherapy, as well as detecting inflammation through RNA expression signatures.

448 Background: There is compelling rationale that detection of MRD following curative therapy may identify patients at high risk of relapse requiring intensified adjuvant therapy. Combining MRD detection with CGP creates an opportunity to offer MRD-guided treatment with precision cancer therapeutics. Here we analyze the observation arm of the IMvigor-010 study to understand the genomics of resected early stage bladder cancer and to validate CGP-informed personalized MRD detection in circulating tumor DNA (ctDNA). Methods: Using the resected tumor, tissue CGP was performed retrospectively with a 300+ gene assay, followed by MRD detection using FoundationOne Tracker (F1T). Briefly, coding, synonymous, and non-coding variants were selected from tumor tissue sequencing using an optimized algorithm that filters out non-tumor derived variants (germline, clonal hematopoiesis derived, sequencing artifacts). Tumor-informed personalized multiplex PCR-next generation sequencing (Natera) assay was designed and used to detect and quantify variant allelic frequency (VAF) in ctDNA from 182 patients. ctDNA levels were reported in mean tumor molecules per mL of plasma. F1T, a tissue-informed personalized monitoring assay, was performed on plasma samples collected at an MRD timepoint a median of 11 weeks post-surgery. Results: At the MRD timepoint, ctDNA was detected in 66/182 (36%). Focusing on the 66 ctDNA-positive patients, 58 had relapsed (88% PPV) at time of analysis. Median disease-free survival (DFS) from randomization was 3 months in ctDNA-positive vs not reached in ctDNA-negative population (HR = 5.7, 95% CI: 3.8-8.6, p (HR = 5.7, 95% CI: 3.4-9.7, p

547 Background: There is an unmet need to identify metastatic urothelial carcinoma (mUC) patients who might be spared chemotherapy in 1st line. Anti-PD-(L)1 immune checkpoint inhibitors (ICPI) alone without chemotherapy did not show superiority to platinum-based chemotherapy in ITT populations of DANUBE, KEYNOTE-361, and IMvigor130. However, DANUBE and IMvigor130 reported secondary subgroup analyses, both suggesting enhanced benefit for ICPI vs. chemotherapy in patients with tumor mutational burden (TMB) ≥ 10 (mutations/megabase), using same cutoff and assay as pan-tumor CDx for pembrolizumab approved in later lines of therapy. We sought to determine if TMB ≥ 10 identified a group of enhanced relative ICPI benefit (single-agent anti-PD[L]1 w/o chemo) in real-world settings where patients are less eligible for chemotherapy. Methods: Association of genomic data with clinical variables and outcomes in cohort of patients with mUC treated January 2011- April 2021. Longitudinal de-identified clinical data from approximately 280 U.S. academic or community-based cancer clinics were derived from electronic health records, curated via technology-enabled abstraction by Flatiron Health and linked to genomic testing by Foundation Medicine. 849 1st line mUC patients received either ICPI (n = 307) or chemotherapy (n = 542) at physician’s discretion in standard of care settings. All patients underwent genomic testing using Foundation Medicine comprehensive genomic profiling assays (FoundationOne© or FoundationOne©CDx). PFS and OS were assessed unadjusted and adjusted for imbalances using propensity scores. Results: 273 of 849 (32.2%) patients had TMB ≥ 10. Pre-therapy characteristics: patients assigned ICPI vs. chemotherapy had comparable TMB, primary disease site, histology, smoking status, and PD-L1 staining, but were generally older (median years: 72 vs. 67, p < 0.001), higher ECOG scores (p < 0.001), lower CrCl (median ml/min: 49.8 vs. 59.7, p < 0.001), and lower hemoglobin (median: 11.5 vs. 12.1, p < 0.001). Unadjusted, TMB ≥ 10 group showed more favorable PFS (HR: 0.72, 95%CI: 0.52 – 0.99, p = 0.041) and OS (HR: 0.70, 95%CI: 0.49 – 0.1, p = 0.048) for ICPI vs. chemotherapy despite imbalances favoring outcomes on chemotherapy. ICPI vs. chemotherapy outcomes adjusted for imbalances: TMB ≥ 10 group showed more favorable PFS (HR: 0.65, 95%CI: 0.45 – 0.95, p = 0.026) and OS (HR: 0.61, 95%CI: 0.39 – 0.93, p = 0.022), while TMB < 10 had comparable or worse PFS (HR: 1.30, 95%CI: 0.98 – 1.72, p = 0.06) and OS (HR: 1.03; 95%CI: 0.78– 1.34, p = 0.85). Conclusions: In real-world settings, 1st line mUC patients with TMB ≥ 10 have more favorable PFS and OS on single agent ICPI than chemotherapy, adding clinical validity to TMB as a predictive biomarker in patient populations less eligible for chemotherapy than reported trials.

162 Background: The most useful biomarkers for clinical decision-making identify patients likely to have improved outcomes on one treatment vs. another. To date, no study has compared the treatment class-specific outcomes of patients with metastatic castration-resistant prostate cancer (mCRPC) on Immune Checkpoint Inhibitor (ICPI) vs. taxane chemotherapy by tumor mutational burden (TMB, mutations/megabase). Methods: Association of genomic data with clinical variables and outcomes in cohort of patients with mCRPC treated January 2011- April 2021. Longitudinal de-identified clinical data from ̃280 U.S. academic or community-based cancer clinics were derived from electronic health records, curated via technology-enabled abstraction by Flatiron Health and linked to genomic testing by Foundation Medicine (FoundationOne or FoundationOne CDx assays). 45 patients (14 with TMB ≥ 10, 31 TMB < 10) received single-agent anti-PD1 axis ICPI, 696 (30 with TMB ≥ 10, 666 TMB < 10) received single-agent taxanes, at discretion of physician without randomization. For time to next therapy (TTNT) and overall survival (OS) assessments, imbalances between treatment groups were adjusted with propensity weighting. Results: Overall cohort: Median age: 70 (IQR: 64 – 76), median PSA: 79.4 (IQR: 19.0 – 254), 108 (18.8%) were ECOG 2+, 644 (86.9%) had received prior systemic treatments for mCRPC. Patients receiving ICPI vs. taxanes had comparable pre-therapy age, PSA, hemoglobin, alkaline phosphatase, prior second generation novel hormonal therapy use, and prior opioid use, but higher TMB (median 3.5, IQR: 1.7 – 15 vs. median 2.5, IQR 1.3 – 3.8, p < 0.001), higher ECOG scores (0, 1, 2+ respectively 13.9%, 55.6%, 30.6% vs. 29.4%, 52.6%, 18.8%, p = 0.057), and greater prior taxane use (73.3% vs. 53.7%, p = 0.01). Among patients with evaluable PSA response, no difference was observed on taxanes by TMB level. No patients had PSA decline ≥ 50% on ICPI if TMB < 10, 4 of 9 with TMB ≥ 10 had PSA decline ≥ 50%. TMB < 10 receiving ICPI vs. taxanes had worse TTNT (median 2.4 vs. 4.1 months; HR: 2.7, 95%CI: 1.7 – 4.0, p < 0.001) and numerically worse OS (median 4.2 vs. 6.0 months, HR: 1.08; 95%CI: 0.68– 1.7, p = 0.73). In contrast, for TMB ≥ 10 ICPI vs. taxane use was associated with more favorable TTNT (median 8.0 vs. 2.4 months; HR: 0.37, 95%CI: 0.15 – 0.87, p = 0.022) and OS (median 19.9 vs. 4.2 months; HR: 0.23, 95%CI: 0.10 – 0.57, p = 0.0085). Among all 741 patients, 44 had TMB ≥ 10, 22 had high microsatellite instability (MSI-H), 20 had both. Treatment interactions with TMB ≥ 10 (TTNT: p < 0.001, OS: p = 0.021) were stronger than MSI-H (TTNT: p = 0.0038, OS: p = 0.080). Conclusions: The results suggest ICPI may be a viable alternative to taxane chemotherapy for patients with mCRPC with TMB ≥ 10, adding validity to existing FDA approved platform and pan-tumor TMB score cutoff of 10.

187 Background: Detection of MRD following metastatic liver resection in advanced CRC patients is associated with poor prognosis with high rate of relapse. Nevertheless, there is currently no standard of care to guide further therapy after curative intent surgery. MRD detection has the promise to be implemented into standard of care and guide treatment decision making. Here we establish feasibility of MRD detection using Foundation Medicine’s novel tissue-informed personalized monitoring assay, FoundationOne Tracker (F1T), in mCRC patients undergoing surgical resection with curative intent. Methods: Tissue-based CGP was performed retrospectively on a cohort of 72 patients from the PREDATOR trial. Trackable patient-specific single nucleotide variants were selected using a novel computational approach negating the need for buffy coat sequencing to filter germline variants. Personalized multiplex PCR was used to detect and evaluate prognostic value of ctDNA from plasma collected at MRD timepoint post-surgery (median 27 days, range 8-99.5). Median follow-up of patients in the overall population was 10.7 months (range: 0.9-53.8 months). Survival analyses were performed using the Kaplan-Meier Estimator and Cox regression. Results: Post-surgical F1T analysis was successful on 96% of cases (69/72). CGP analysis revealed at least one driver mutation in 57% of samples (41/72) including KRAS/NRAS (46%) and BRAF mutations (3%). MRD was detected in 45% (31/69) of patients, of which 94% (29/31) had progressed at the time of the data cut. Median progression -free survival (PFS) was 3.2 months (2.1-7.1) in ctDNA-positive vs 28 months (20.9-NA) in ctDNA-negative population (HR 5, CI 2.7-9.3, p

The transcriptional co-activator YAP1 oncogene is the downstream effector of the Hippo pathway, which regulates tissue homeostasis, organ size, regeneration, and tumorigenesis. Multiple cancers are dependent on sustained expression of YAP1 for cell proliferation, survival, and tumorigenesis, but the molecular basis of this oncogene dependency is not well understood. To identify genes that can functionally substitute for YAP1, we performed a genome-scale genetic rescue screen in YAP1-dependent colon cancer cells expressing an inducible YAP1-specific shRNA. We found that the transcription factor PRDM14 rescued cell proliferation and tumorigenesis upon YAP1 suppression in YAP1-dependent cells, xenografts, and colon cancer organoids. YAP1 and PRDM14 individually activated the transcription of calmodulin 2 (CALM2) and a glucose transporter SLC2A1 upon YAP1 suppression, and CALM2 or SLC2A1 expression was required for the rescue of YAP1 suppression. Together, these findings implicate PRDM14-mediated transcriptional upregulation of CALM2 and SLC2A1 as key components of oncogenic YAP1 signaling and dependency.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B’’’, striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity., eLife digest Cells maintain a fine balance of signals that promote or counter cell growth and division. Two sets of enzymes – called kinases and phosphatases – contribute to this balance. In general, kinases “switch on” other proteins by tagging them with a phosphate molecule. This process is called phosphorylation. Phosphatases, on the other hand, dephosphorylate these proteins, switching them off. Cancer cells often have mutations that activate kinases to drive cancer growth. The same cells can have mutations that inactivate the phosphatases or reduce their abundance. The roles of phosphatases in cancer are still being studied. One major hurdle in this research is that it is not always clear how they recognize the proteins they dephosphorylate. Protein phosphatase 2A (or PP2A for short) is one of the phosphatases that is often mutated or deleted in human cancers. Even just reduced levels of PP2A can promote cancer. Kim, Berrios, Kim, Schade et al. used an experimental trick to decrease the phosphatase activity of PP2A in human cells growing in a dish. Biochemical analysis of these cells showed that, as expected, many proteins were now in their phosphorylated states. Unexpectedly, however, some proteins were dephosphorylated under these conditions. One of these proteins was called MAP4K4. In the case of MAP4K4, the dephosphorylated state contributes to the growth of the cancer cell. Kim et al. carried out further genetic and biochemical experiments to show that, in these cells, PP2A and MAP4K4 stay physically connected to one another. This connection was enabled by a group of proteins called the STRIPAK complex. The STRIPAK proteins directed the remaining PP2A towards MAP4K4. Low levels or activity of PP2A could, therefore, promote cancer in a different way. Taken together, PP2A is not a single phosphatase that always turns proteins off, but rather is a dual switch that turns off some proteins while turning on others. Future experiments will explore to what extent these findings also apply in tumors. Information about how mutations in PP2A affect human cancers could suggest new targets for cancer drugs.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B’’’, striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.

SUMMARY Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers., In Brief Advanced prostate cancers develop resistance to androgen receptor (AR)-targeting therapies. Hwang et al. show that resistant prostate cancers overexpress or amplify CREB5, mediating resistance to AR inhibition. CREB5 suppression reduces the viability of therapy-resistant patient-derived models, suggesting that CREB5 is a target in prostate cancer patients with limited treatment options., Graphical Abstract

Background: F1LCDx is an FDA-approved liquid biopsy test offering pan-tumor profiling of >300 genes with CDx indications for multiple cancer types. One key limitation is that sensitivity for detection of genomic alterations in ctDNA is impaired when the plasma tumor fraction (TF) is low. TF is known to vary in advanced cancer and has been correlated with overall survival (Stover, JCO, 2018). While some ctDNA assays use variant allele fraction (VAF) to quantify tumor content, such methods may be prone to interference from germline variants, copy number alterations and a limited number of mutations. We studied SNP aneuploidy to develop a variant-independent measure of TF which we hypothesized could inform assay sensitivity. Methods: For each liquid sample, coverages from heterozygous SNPs, across the genome, were used to measure allelic deviations from normal via a root-mean-square (RMS) metric. The relationship between RMS and TF was established based on a training data set of clinical samples with known TF that were diluted in silico. In this way, the RMS metric for any sample could be linked to a distribution of TF values. TF is reported when the lower bounds of the predicted TF distribution is >0%. Positive percentage agreement (PPA) of liquid NGS versus tissue genotype was studied in those with a TF reported vs TF not quantified. Results: For a pilot concordance analysis, we studied a real-world cohort of NSCLC patients where NGS was performed on both tissue and on a 70-gene ctDNA assay (FoundationOne®Liquid (F1L), median 314 days between results). Among 251 cases with tissue NGS showing an EGFR L858R or 19del mutation and a F1L result available, the EGFR mutation was detected 90% of the time (57/63) when TF was reported (range 13-74%) but was detected 51% of the time (96/188) when TF was not quantified. Next, we analyzed a cohort of 3 clinical trials in advanced cancers where tissue genotype was known and with F1LCDx results available, including studies of activating PIK3CA mutations in breast cancer, MET exon 14 mutations in NSCLC and BRCA1/2 mutations in prostate cancer. For the subset of patients with TF reported (range 9.6-72%), PPA was 94.7% (breast, n=38), 100% (NSCLC, n=19), and 90.1% (prostate, n=46), versus 66.7% (breast, n=33), 66.7% (NSCLC, n=45) and 89.6% (prostate, n=48) for TF not quantified. Conclusions: We describe a novel method to measure TF in plasma ctDNA based on aneuploidy, which may overcome some limitations of TF measurement based on VAF. When TF confirms tumor content, sensitivity of F1LCDx for short variant detection is excellent (90%-100%), suggesting reduced value from a reflex to tissue genotyping in the absence of a targetable genomic alteration. When TF is not quantified, sensitivity can be lower and the value of reflex to tissue genotyping may be heightened. Citation Format: Meijuan Li, Bernard Fendler, Lei Yang, Russell Madison, Brennan Decker, Ole Gjoerup, Kimberly McGregor, Aparna Aiyer, Jason Hughes, Priti Hegde, Christine Vietz, Geoffrey R. Oxnard. Utility of plasma tumor fraction (TF) to inform sensitivity of FoundationOne Liquid CDx (F1LCDx) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2231.

5062 Background: Loss-of-function genomic alterations (GAs) in MSH6 have been associated with a unique subtype of hypermutated mCRPC that is often microsatellite stable (MSS) and may occur in either a sporadic or familial Lynch Syndrome-like clinical setting. Methods: 5,617 mCRPC cases were sequenced to evaluate all classes of GA using a hybrid capture-based FDA-approved comprehensive genomic profiling (CGP) assay. Tumor mutational burden (TMB) was determined on 0.8 Mb of sequenced DNA and microsatellite instability high (MSI-High) was determined on 95 loci. MSI-low status was not assessed. Results: 78 (1.4%) mCRPC were MSH6mut (Table). MSH6mut mCRPC included 73.1% short variant mutations, 23.1% biallelic deletions, 2.6% genomic rearrangements, and 1.3% multiple GAs/sample. Co-mutation of MSH2 was found in 28% of MSH6mut cases vs. 2% in MSH6wt cases (P mut mCRPC, which was significantly greater than the 2% seen in MSH6wt cases (P mut cases, compared to 3% MSH6wt cases (P mut cases with neither MSI-High nor MMR mutational signature, 87% did not have biallelic loss of MSH6 or any other MMR gene, confirming that monoallelic pathogenic mutations are insufficient to cause the MMR-D phenotype. For subjects whose variants could be classified, 45% (19/42) of pathogenic MSH6 alleles were germline; of these, 58% (11/19) had neither MSI-High nor an MMR single nucleotide signature. MSH6mut cases had fewer TMPRSS2: ERG fusions (P =.01), but harbored significantly higher frequencies of GAs in AR (P =.0002), ATM (P =.04), PIK3CA (P =.0003), APC (P =.005), ERBB2 (P =.001), and CDK6 (p =.046), likely at least partially attributable to the higher TMB in MSH6mutcases (P mut mCRPC is a unique disease that often features a hypermutated genomic signature, although only 46% of cases exhibited MSI-high status. This complex phenotype highlights the potential utility of multiple rather than single biomarkers to understand tumor biology and determine patients who may benefit from immunotherapy.[Table: see text]

3009 Background: The increased use of ctDNA CGP has paralleled increased detection and interest in CH, which can confound CGP results from ctDNA or tissue, and can be associated with hematologic and cardiovascular morbidity. However, paired-depth sequencing of white blood cells (WBC) for confirmation of CH is not widely available. We here study the prevalence of inferred CH (iCH), which refers to incidental detection on routine clinical CGP of variants attributable to CH due to their known CH association and their negligible prevalence in solid tumors. Methods: A database of clinical CGP results was reviewed, including two 324-gene NGS panels for tumor tissue (FoundationOne CDx) and plasma ctDNA (FoundationOne Liquid CDx). Analysis was limited to NSCLC, breast, prostate, colorectal, and pancreatic cancers. iCH was defined as any pathogenic mutation in ASXL1, DNMT3A, and TET2, and prespecified mutations in JAK2, SF3B1, U2AF1, MYD88, IDH2, MPL, CBL. Variant allelic frequency (VAF) > 2% was considered clinically significant and VAF > 10% was considered high risk. Results: 100,905 total cases were studied; median age was 65 for tissue CGP and 68 for ctDNA. iCH was more commonly detected in ctDNA (1468/2891, 51%) than in tissue (9416/97993, 10%). Among cases with any iCH detected, multiple iCH mutations were seen more commonly in ctDNA (640/2891, 22%) than in tissue (987/98014, 1%). Focusing on clinically significant iCH ( > 2% VAF), prevalence remained higher in ctDNA (22%, 637) than in tissue (8%, 7878), while the higher sensitivity of ctDNA testing identified more low level iCH (< 2% VAF, 40% in ctDNA, 2% in tissue). Across cancer types, iCH > 2% was consistently more common in ctDNA (Table). As expected, prevalence of iCH > 2% increased with age (continuous variable, p < 0.001). High risk iCH ( > 10% VAF) was seen in 4% of total cases (most commonly ASXL1, TET2, DNMT3A); 1% of all cases had multiple clinically significant iCH variants ( > 2% VAF). Focusing on a subset of 439 cases with both tissue and ctDNA results (median 1.5 months between samples), 290 iCH mutations were detected in ctDNA (median VAF 1%) but only 38 in tissue (median VAF 9%). Conclusions: Inferred CH is common on somatic CGP of cancer patients, with a high prevalence in ctDNA likely due to the deeper sequencing depth and WBC contamination. For the minority of patients with high VAF iCH, further research is needed to understand whether this might be representative of an occult hematologic condition deserving of further evaluation.[Table: see text]

8555 Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy with limited therapeutic options. Immunotherapy has emerged as an effective therapeutic strategy, particularly in the non-epithelioid MPM subgroup. An improved understanding of the molecular profile of MPM may inform targeted therapeutic approaches and provide insights into factors underlying differential sensitivity to therapies. Methods: We performed comprehensive genomic profiling of MPMs from 980 patients, with histologic subtyping on a subset (n = 340; n = 235 epithelioid, n = 48 sarcomatoid, n = 57 biphasic). Analyses included quantifying tumor mutational burden (TMB), assessing microsatellite instability (MSI), and evaluating PD-L1 expression (Dako 22C3). Results: Median TMB of MPM was 1.74 (0.87-2.61) mutations per megabase (mut/mb). Median TMB was comparable for non-epithelioid (biphasic, sarcomatoid) vs epithelioid MPM (1.25 mut/mb vs 1.25 mut/mb, p = 0.43). In the overall cohort, inactivating alterations in the following genes were most prevalent: CDKN2A (49%), BAP1 (44%), CDKN2B (42%), MTAP (35%), and NF2 (33%). MTAP loss co-occurred with CDKN2A and CDKN2B loss in > 99% and 94% of cases, respectively. MTAP loss was observed in 28% of epithelioid vs 46% of non-epithelioid MPMs (p = 0.03). BAP1 alterations were detected in 51% of epithelioid vs 36% of non-epithelioid MPMs (p = 0.81). PD-L1 expression was assessed in 308 MPMs, among which 153 (49%) had PD-L1 ≥1%, including 35 (11%) with PD-L1 ≥50%. Among 164 specimens that underwent histologic subtyping and PD-L1 evaluation, PD-L1 was expressed in 46 (71%) non-epithelioid MPMs compared to 49 (51%) epithelioid MPMs (p = 0.03). Given the high prevalence of alterations involving the two genes, we evaluated impact of BAP1 and MTAP status on PD-L1 and TMB. TMB (1.74 mut/mb vs 1.74 mut/mb) and PD-L1 expression (50% vs 54%) were comparable in BAP1-altered vs wildtype specimens. Although TMB was comparable in MTAP-deficient and MTAP-intact tumors (1.25 mut/mb vs 1.74 mut/mb), more tumors with MTAP loss expressed PD-L1 (68% vs 44%, p = 0.0004). Conclusions: Compared to epithelioid MPM, non-epithelioid tumors demonstrate comparable TMB, higher PD-L1 expression, and enrichment for MTAP loss. As MTAP is frequently altered in MPM, further study is indicated to explore the relationship between MTAP status and MPM biology, including sensitivity to therapeutics such as immunotherapy and synthetic lethal approaches.

5054 Background: No established genomic biomarkers exist for guiding treatment decisions between novel hormonal therapy (NHT) vs taxane chemotherapy in mCRPC. However, specific alterations in AR have been associated to decreased responsiveness to NHT in this setting. Leveraging routine comprehensive genomic profiling (CGP) testing of mCRPC tissue samples, we hypothesized that patients (pts) with AR amplification ( ARamp) would have better outcomes on taxanes over NHT. Methods: Pts were selected from Flatiron Health (FH)-Foundation Medicine (FMI) clinico-genomic database (CGDB), a nationwide deidentified electronic health record database linked to CGP. Data originated from approximately 280 US cancer clinics (̃800 sites). CGP results (including analysis of AR and 15 other genomic biomarkers) were obtained from mCRPC tumor tissue collected up to 180 days before or 30 days after initiation of new systemic therapy between 1/1/11 - 6/30/20, and linked to PSA response, time to next therapy (TTNT) and overall survival (OS). Multivariable treatment interaction models were adjusted for drug assignment imbalances (line of therapy, prior NHT or taxane use, PSA, alkaline phosphatase, hemoglobin, albumin, years to CRPC, biopsy site) using inverse probability of treatment weighting via propensity scores. Results: Among 5754 evaluable mCRPC lines of therapy, 180 receiving NHT and 179 receiving taxanes met inclusion criteria, 359 total from 308 unique patients. Pts with ARamp vs no ARamp on NHT had worse PSA response (median +57.3% vs. -31.4%, p = 0.002), TTNT (HR: 2.03, p < 0.001), and OS (HR: 2.28, p < 0.001), but had no difference in outcomes on taxanes. Multivariable interaction Cox models found ARamp independently associated to better TTNT on taxanes vs. NHT (HR: 0.48, p = 0.010), similar to pts with RB1 alterations (HR: 0.46, p = 0.027). Consistent treatment interactions were seen with OS for ARamp (HR: 0.53, p = 0.025) and RB1 (HR: 0.32, p = 0.024). While CDK12 was not predictive, it independently associated with worse OS overall (HR: 2.25, p = 0.0011). In the 55 pts who received NHT followed by taxane immediately after, ARamp pre-NHT was associated with better TTNT on the subsequent taxane than on the initial NHT (HR: 0.40, p = 0.028). Of these, 33 had PSA responses evaluable, and ARamp pre-NHT was significantly associated with better PSA decline on the subsequent taxane, despite disadvantage of first progressing on NHT (OR: 10.9, p = 0.021). Conclusions: Genomic biomarkers routinely identified with CGP such as ARamp may aid in identifying mCRPC pts who are to obtain greater benefit from taxane chemotherapy instead of NHT. Prospective efforts are needed to further validate the utility of CGP for assisting treatment decisions for mCRPC patients.

142 Background: Robust biomarkers for personalization of NHT treatment decisions remains an unmet need. Most assessments of candidate biomarkers to predict NHT resistance have been conducted in clinical trials or academic centers, meriting additional validation in diverse community settings. We sought to correlate real-world outcomes on NHT with comprehensive genomic profiling (CGP)-reported alterations, hypothesizing that AR amplification ( ARamp) and deleterious genomic alterations (GAs) in BRCA2, PTEN, RB1, TP53 would correlate with worse outcomes on NHT. Methods: Following a prespecified analysis plan, pts were selected from Flatiron Health (FH)-Foundation Medicine (FMI) clinico-genomic database (CGDB), a nationwide deidentified electronic health record database linked to FMI CGP. Inclusion criteria: mCRPC diagnosis, treatment in FH network and CGP result between 1/1/11-3/30/20 where tissue collected prior to initiation of first line (1L) or second (2L) NHT (within 180 days for ARamp +/- comparison). A priori power analyses were conducted. Adjusted hazard ratios (aHR) from multivariable Cox proportional hazard models were utilized for TTD and OS comparisons from start of NHT including: GA groups, adjusted for age, line number, practice type, and left truncation. Results: Among 1626 evaluable pts, 397 received 1L (n = 287; 72%) or 2L (n = 110; 28%) NHT with majority treated in community setting (n = 297; 75%). Abiraterone (n = 242; 61%) and enzalutamide (n = 145; 39%) were most common NHTs. Incidence: ARamp (15%) and deleterious GA in TP53 (45%), PTEN (28%), RB1 (3%), and BRCA2 (8%). Cohort was strongly powered to assess TP53 & PTEN, moderately for ARamp & BRCA2, weakly for RB1. As hypothesized, ARamp correlated with worse TTD (aHR: 3.37 [1.26-9.0]) and OS (aHR: 4.92 [1.47-16.5]). BRCA2 GA correlated with improved OS (aHR: 0.41 [0.21-0.81]), but no differences in TTD (aHR: 1.25 [0.82-1.9]). RB1 GA had trends for worse OS (aHR: 2.0 [0.93-4.28]) and worse TTD (aHR – 1.41 [0.72-2.8]). TP53 GA had worse OS (aHR: 1.47 [1.1-2.0]), but no difference in TTD (aHR: 1.08 [0.85-1.4]), and PTEN GA did not correlate with TTD (aHR: 0.94 [0.72-1.2]) or OS (aHR: 1.01 [0.74-1.38]). Conclusions: ARamp is associated with worse TTD and OS in mCRPC pts treated with NHT in real-world, mostly community practice, consistent with prior academic center studies and trials data. Surprisingly, BRCA2 GA correlated with improved OS but not TTD. RB1 GA were directionally consistent with prior studies but underpowered. This study supports using CGP to inform decisions for escalation of non-NHT treatment use in conjunction with patient goals and predictive CGP biomarkers for other drugs. Additional biomarkers, multivariable models and, NHT vs taxane chemo predictive assessments will be reported at symposium.

163 Background: Herpesvirus HHV-8 has been detected in normal prostate tissue and has been linked to the acquisition of androgen-independent growth of prostate cancer cells in vitro, early development of anti-androgen therapy resistance, and more aggressive disease in a subset of patients with mCRPC. We used comprehensive genomic profiling (CGP) to test the hypothesis that HHV-8 associated mCRPC is a distinct molecular subset of mCRPC featuring altered AR signaling compared to HHV-8 negative mCRPC. Methods: Using a hybrid capture-based FDA-approved CGP assay, a series of 4,918 mCRPC were sequenced to evaluate all classes of genomic alterations (GAs). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. PD-L1 expression was determined by IHC (Dako 22C3). HHV-8 status was determined by CGP-based identification of unique viral reads. Results: Overall, 128 (2.6%) of the mCRPC cases harbored HHV-8 sequences (Table). The patient age distribution was similar in the HHV-8+ and HHV-8− groups. GAs in AR, predominantly amplifications, and RAD21 were slightly reduced in HHV-8+ mCRPC cases (P = .01 for both). The frequencies of GAs in genes associated with mCRPC including PTEN, BRCA2, ATM, CDK12, and the TMPRSS2: ERG fusion were similar in the two groups. HHV-8+ patients were less likely to be of European ancestry (P = 0.001). Putative biomarkers associated with immunotherapy response (MSI status, TMB, and PD-L1 expression) were also similar in the two groups. Conclusions: HHV-8+ mCRPC is a rare sub-type of mCRPC with lower frequency of AR GAs compared to HHV-8− mCRPC cases, suggesting a potential role of HHV-8 in inducing androgen-independent prostate cancer growth. Additional alterations associated with potentially enhanced PARP inhibitor response seen in HHV-8+ mCRPC (e.g., RAD21) suggest that early performance of CGP may inform precision treatment strategies to be tested in clinical trials. [Table: see text]

2 Background: Although SCC of the penile skin (pSCC) and the male urethral surface epithelium (uSCC) arise in nearby locations and can feature similar histology, their clinical manifestations, disease course, and surgical and medical treatment options are distinct. We performed CGP on pSCC and uSCC to examine genomic profiles differences. Methods: Tissues obtained from men with clinically advanced pSCC (n = 230) and uSCC (n = 17) underwent hybrid capture-based CGP to evaluate all classes of genomic alterations (GAs). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on up to 114 loci. PD-L1 expression was determined by IHC (Dako 22C3). Results: The median ages were similar in both groups. pSCC exhibited a slightly higher frequency of HPV-16/18 infection (29% vs. 12%, P = .16), although the TP53 mutation frequencies were nearly identical (55% vs. 59%, NS). CDKN2A inactivation (P = .08), CCND1 amplification trending higher and TERT promoter mutations (P = .01) were more frequent in pSCC, potentially indicating prior HPV infection. GAs in NOTCH1 were exclusively identified in pSCC. Potentially actionable GAs identified in both groups included PIK3CA activating mutations (TKIs) as well as pathogenic alterations in FBXW7 and PTEN (MTOR inhibitors). Rare BRCA1 and BRCA2 inactivation (PARP inhibitors) was seen in pSCC only. High-positive PD-L1 staining was elevated in pSCC (34 vs. 14%, P = .06). Although average TMB was similar in both groups, pSCC exhibited an elevated frequency of cases with CD274 ( PD-L1) amplification as well as TMB >10 mut/Mb which are on label for immune checkpoint inhibitor (ICPI) treatment. Conclusions: CGP of pSCC and uSCC identifies opportunities for both targeted and ICPI therapies. Compared to uSCC, pSCC had genomic features more similar to head and neck SCC including slightly increased cell-cycle perturbation, HPV infection, and NOTCH pathway signaling alterations. Further use of CGP in the treatment planning for pSCC and uSCC may be warranted. [Table: see text]

421 Background: PDAC has a propensity for early systemic dissemination and most pts with primary tumors radiographically confined to the pancreas harbor micro-metastases. cfDNA based CGP offers a non-invasive mechanism to identify actionable genomic targets in PDAC and account for serial clonal evolution in response to therapeutic selection pressure. We interrogated the Foundation Medicine database to characterize cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of pts with PDAC who underwent cfDNA based CGP between May 2016 and February 2020. cfDNA based CGP was done utilizing either the FoundationOne Liquid (F1-Liquid) or the Foundation-ACT (F-ACT) testing platform. Samples were interrogated for exonic and select intronic regions of cancer-related genes for both F1-Liquid (Exons: 70 genes; Select Introns: 7 genes) and F-ACT (Exons: 59 genes; Select Introns: 6 genes). Variant zygosity and somatic/germline status (SGZ) for short variant mutations was computationally predicted without matched normal tissue using an investigational method. A Gaussian Mixture Model (GMM) was used to identify latent molecular classes in samples harboring somatic, pathogenic variants (n=613); the Bayesian Information Criteria (BIC) was used for model selection. Results: We identified 1,009 pts with cfDNA based CGP - median age was 65, 55% were male, and median cfDNA concentration was 24 ng/µL. cfDNA yield had an influence on the detection of somatic alterations (p

422 Background: Early systemic dissemination is a salient feature of PDAC and the ability to reliably detect as well as monitor cfDNA based alterations may have predictive and/or prognostic value. However, a clear correlation between the clonality of the primary tumor and micro-metastatic disease has not been established in PDAC. We examined the correlation between T-CGP and cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of PDAC pts who had both T-CGP (October 2010 – March 2020) and cfDNA based CGP (May 2016 – March 2020), with an average of 645 days between tests (Range: 21 – 2101 days) For T-CGP, exonic regions and select intronic regions of cancer related genes were sequenced to a high uniform depth (>500X median coverage) using FoundationOne Heme (Exons: 315 genes; Select Introns: 28 genes) and FoundationOne CDx (Exons: 310 genes; Select Introns: 34 genes). cfDNA based CGP, was performed using either FoundationOne Liquid (Exons: 70 genes; Select Introns: 7 genes) or Foundation-ACT (Exons: 59 genes; Select Introns: 6 genes) platform. Results: Eighty-one pts were identified with both T-CGP and cfDNA based CGP - median age was 61 years, 59.3% (48/81) were male and 66.2% (49/74) had reported metastatic disease. The median tumor DNA content available for T-CGP analysis was 112.3 ng. The median cfDNA concentration was 1.7 ng/µL, and the number of reported ct-DNA based somatic alterations correlated significantly with the concentration of ct-DNA (Spearman’s R = 0.19, p < 10-8). 75/81 (92.6%) and 41/81 (50.6%) of pts had at least one pathogenic somatic alteration detected by T-CGP and cfDNA based CGP, respectively. KRAS, TP53 and CDKN2A were the most frequently altered genes in both T-CGP and cfDNA based CGP (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [ BRCA2, ATM, and CHEK2 alterations] were detected in 7.4% (6/81) of T-CGP and 7.4% (6/81) cfDNA based CGP, respectively. Two BRAF fusions were detected from samples tested with T-CGP, however these were not detected with the cfDNA assay. Conversely, an ALK-EML4 fusion was detected using both platforms. Conclusions: The concordance between T-CGP and cfDNA based CGP in HR-DDR gene alterations may be indicative of the germline status of some of these alterations; nevertheless, it suggests the predictive utility of cfDNA based CGP in PDAC. The differential prevalence of other gene alterations between the two modalities is likely reflective of the cfDNA yield available for CGP and to a lesser extent, clonal evolution. cfDNA based CGP holds promise as a complimentary predictive tool to T-CGP. [Table: see text]

337 Background: The association of BRCA tumor suppressor gene (TSG) inactivation and PARP inhibitor efficacy through a mechanism of synthetic lethality (SL) is now well-established. Recently, the PRMT5 arginine methyltransferase dependence in cells with MTAP (S-methyl-5'-thioadenosine phosphorylase) genomic alterations (GA) has been proposed as a new SL based anti-tumor strategy and under consideration for development for intrahepatic cholangiocarcinoma (IC). Methods: 2,170 cases of clinically advanced IC were submitted for comprehensive genomic profiling (CGP) to detect genomic alterations (GA), tumor mutational burden (TMB) and microsatellite instability (MSI) using a hybrid capture based assay (F1CDx). PD-L1 expression in tumor cells (Dako 22C3) was measured by IHC and scored using the tumor proportion score (TPS) method. Results: 328 (15%) ICs featured MTAP inactivation (MTAP-IC). 48% of MTAP-IC cases were female and 52% were male with a median age of 66 years (range 26-93 years). MTAP-IC featured 6.3 GA per sample. The median TMB was 2.5 mut/Mb with only 2% of cases with TMB ≥10 mut/Mb. No (0%) MTAP-IC cases were MSI-high. Of 112 MTAP-IC IHC stained for PD-L1, 4% were PD-L1 high (> 50%), 14% were PD-L1 low (1-49%) and 82% were PD-L1 negative. 99% of MTAP inactivation was by copy number (CN) loss and 1% was due to non-CN mutation. In MTAP-IC, 99% featured CDKN2A loss and 95% featured CDKN2B loss (9p21 co-deletion). TP53 was mutated in 31% and KRAS in 24% (3% G12C). Other currently non-targetable GA included ARID1A (19%), BAP1 (16%) and SMAD4 (13%). Among potentially targetable GA, the MTAP-IC featured 12% FGFR2 GA (8% rearrangements), 10% BRAF (8% V600E), 7% IDH1, 7% PIK3CA, 4% BRCA2, 4% ERBB2 (2% amplification) and 3% MET. Conclusions: Comprehensive genomic profiling elucidated that MTAP GA are frequent in IC and features relatively high GA/tumor, including 9p21 co-deletion, but relatively low TMB and PD-L1 expression. Isolated MTAP deletion without CDKN2A/B co-deletion was extremely rare. In addition to the potential targeting of PRMT5 in MTAP-IC tumors, co-altered genes known to be targets in IC such as FGFR2, IDH1 and BRAF are still prevalent in MTAP-IC, indicating potential for combining targeted therapies in some MTAP-IC patients.