Foundation Medicine Inc Research Articles

Low PD-L1 expression, MAP2K2 alterations, and enriched HPV gene signatures characterize brain metastases in head and neck squamous cell carcinoma

BackgroundBrain metastasis (BM) is a rare but severe complication of head and neck squamous cell carcinoma (HNSCC), with limited knowledge of molecular characteristics and immunogenicity.MethodsWe analyzed 61 cases of HNSCC-BM from three academic institutions (n = 24) and Foundation Medicine Inc (FMI, n = 37). A subset of cases underwent next-generation sequencing, multiple immunofluorescence, and proximity ligation sequencing. Gene enrichment analysis compared alterations in FMI BM samples (n = 37) with local samples (n = 4082).ResultsDemographics included: median age of 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 67% human papillomavirus (HPV) +. ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%) were frequently altered in BM samples from academic centers (62% HPV/p16+). Structural rearrangements ranged from 9 to 90 variants by proximity ligation sequencing. BMs had low densities of CD8+, PD-1+, PD-L1+, and FOXP3 + cells, and 92% had PD-L1 combined positive scores < 1%. CDKN2A (40.5%), TP53 (37.8%), and PIK3CA (27.0%) alterations were common in the FMI BMs (51% HPV+). MAP2K2 alterations and HPV + signature were enriched in FMI BMs compared to local tumors (11.8% vs. 6.4%, P = 0.005 and 51.25% vs. 26.11%, P = 0.001 respectively), and pathogenic TSC1 inactivating mutations were enriched in local tumors (67.3% vs. 37.8%, P = 0.008). Median overall survival from BM diagnosis was 9 months (range 0–27).ConclusionsHNSCC patients with BM frequently have oropharyngeal primary sites and are HPV+. Common molecular alterations in BM samples, including targetable PIK3CA and ATM, were identified. MAP2K2 alterations were enriched and densities of immune cells were low, highlighting potential targets for further research and immunotherapy considerations.

Clinical, molecular, and immunologic profiling of brain metastases (BM) in head and neck squamous cell carcinoma (HNSCC).

6036 Background: BM is a rare complication of HNSCC that carries a high rate of morbidity and poor prognosis. Clinical risk factors, molecular characteristics, and the immunogenicity of HNSCC BM are not well defined, leaving a critical knowledge gap in this field. We performed one of the largest multi-institutional analyses summarizing the clinical, molecular, and immunologic profile of 61 cases of BM-HNSCC. Methods: We conducted a pooled analysis of the clinical characteristics pertaining to BM-HNSCC from 3 academic institutions (n=24). Next-generation sequencing (NGS) and immune profiling (IP) of primary and BM specimens was conducted on a subset of cases (n=19 and n=16, respectively); there were 3 paired samples for NGS and 0 for IP. Four samples (2 BM and 2 non-BM) were submitted to Phase Genomics, Inc for evaluation of structural variants in BM genomes by proximity ligation sequencing (PLS). These results were complimented by a comparative analysis of genomic alterations in an additional cohort of BM (n=37) and local samples (n=4082) submitted for NGS at Foundation Medicine, Inc (FMI). Statistical comparisons were done using Fisher’s exact testing of 2x2 contingency tables with p-values controlled for FDR by the Benjamini-Hochberg procedure. Results: Clinical features were as follows: median age at diagnosis 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 84% HPV+ or p16+. The most frequently altered genes in BM specimens (62% HPV/p16+) were ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%). BM and non-BM samples demonstrated significant levels of structural rearrangement ranging from 9 to 90 variants by PLS. IP identified lower densities of CD8+, PD1+, PDL1+, and FOXP3+ cells in BMs compared to primary tumors. PDL1 combined positive scores were &lt;1% in 12/13 unpaired samples (92%; 10 BM and 2 primary). The FMI BM-HNSCC cohort (51% HPV+) identified CDKN2A (40.5%), TP53 (37.8%), and PIK3CA (27.0%) as the most frequently altered genes. Enrichment analysis of the FMI cohort showed MAP2K2 alterations significantly enriched in BM (11.8% vs 6.4%, P=0.005) and TSC1 alterations significantly enriched in the local site (67.3% vs 37.8%, P=0.008). HPV+ was also significantly enriched in the BM cohort (51.25% vs 26.11%, P=0.001). Overall survival from BM diagnosis was 6m (range 0-27m). Conclusions: HNSCC patients with BM have higher-than-expected proportions of oropharyngeal primary site and HPV/p16-positivity. The most frequent molecular alterations in BM samples are also commonly found in non-BM HNSCC, including targetable PIK3CA alterations. MAP2K2 alterations were significantly enriched in BM compared to non-BM samples, which warrants further investigation. BM samples also tended to have lower markers of immunogenicity. This latter finding could have important clinical implications when considering immunotherapy or immune-modulating drugs.

Genomic landscape of metastatic clear cell renal cell carcinoma (mccRCC): A retrospective real-world analysis from Flatiron Health (FH)-Foundation Medicine, Inc. (FMI) clinico-genomic database (CGDB).

Genomic landscape of metastatic clear cell renal cell carcinoma (mccRCC): A retrospective real-world analysis from Flatiron Health (FH)-Foundation Medicine, Inc. (FMI) clinico-genomic database (CGDB).

Identification of prognostic genomic biomarkers in metastatic clear cell renal cell carcinoma (mccRCC): An analysis of the large retrospective real world clinico-genomic data from Flatiron Health (FH)-Foundation Medicine, Inc. (FMI) clinico-genomic database (CGDB).

Identification of prognostic genomic biomarkers in metastatic clear cell renal cell carcinoma (mccRCC): An analysis of the large retrospective real world clinico-genomic data from Flatiron Health (FH)-Foundation Medicine, Inc. (FMI) clinico-genomic database (CGDB).

Genetic ancestry-associated differences in genomic profiling and treatment patterns in pancreatic ductal adenocarcinoma (PDAC).

4138 Background: Prior epidemiological studies have explored race both as a population-level risk factor and as a factor influencing outcomes in PDAC. However, the impact of genetic ancestry on PDAC remains unclear. We assessed the genomic landscape, clinical care patterns, and outcomes across ancestries in a large and diverse PDAC cohort to determine the potential impact of genomics on ancestral disparities. Methods: A total of 24,948 patients diagnosed with PDAC who received tissue-biopsy based comprehensive genomic profiling (CGP; Foundation Medicine, Inc. (FMI)) as part of routine clinical care from Aug 2014-Sep 2023 were examined for genomic differences based on genetic ancestry which was determined using a SNP-based approach. The US-based de-identified Flatiron Health (FH)-FMI clinico-genomic database (CGDB) was used to assess testing and treatment patterns based on ancestry in 3,863 patients of European (EUR) and 414 patients of African (AFR) ancestry diagnosed with PDAC. Results: The genetic ancestry distribution in the overall cohort was 80.9% EUR, 10.0% AFR, 4.9% Admixed American, 3.5% East Asian, and 0.7% South Asian. AFR patients showed a slightly younger median age at biopsy than EUR (64 vs 67, p &lt; .001). KRAS, TP53, CDKN2A/ 2B and SMAD4 were the most commonly altered genes in the cohort, as expected. Compared to EUR, AFR patients were enriched in alterations in TP53 (85% vs 78%, p &lt; .001) and KRAS (95% vs 93%, p &lt; .001). While rare, alterations in GNAS, CHEK2, and BRAF were observed less frequently in AFR (1.7% vs 3.3%, 0.3% vs 1.7%, 0.9% vs 2.2%, respectively, all p &lt; .001). In the CGDB cohort, AFR patients were likely to have lower socioeconomic status (SES) compared to EUR patients (33% of AFR in the lowest SES quintile compared to 9% of EUR; p &lt; .001 across all quintiles). AFR patients were less likely to receive care at academic centers (AFR 14.8% vs EUR 22.2%, p &lt; .001) and less likely to receive an investigational drug during their cancer treatment course (5.9% vs 14.1%, p = .001). Rates of surgery (AFR 30.2% vs EUR 34.6%, p = .08), immunotherapy (3.2% vs 3.3%, p &gt; .99), and targeted therapy (4.1% vs 6.2%, p = .28) were not significantly different. While AFR patients comprised a significantly larger proportion of AFR+EUR CGP volume following the decision to implement Medicare coverage for NGS testing (10.6% after Mar 16, 2018 vs 6.6% prior, p &lt; .001), this remained below the expected based on share of the overall US population. Conclusions: To our knowledge, this is the largest study of genetic ancestry in PDAC to date. There were largely similar patterns of gene alterations across AFR and EUR ancestry groups suggesting non-genomic factors may underlie clinical outcomes disparities which have been reported in other studies. Notably, there were ancestry-based differences associated with SES and both CGP testing and clinical trial enrollment patterns, which may impact patient outcomes.

Abstract PO2-27-12: Genomic determinants of benefit to nivolumab (NIVO) plus low dose ipilimumab (IPI) among patients (pts) with hypermutated HER2-negative metastatic breast cancer (MBC): results of NIMBUS trial

Abstract Background: Genomic determinants of benefit to immune checkpoint inhibitors (ICI) among pts with MBC and high tumor mutational burden (TMB-H) are largely unknown. NIMBUS was an open-label, single-arm, multicenter, phase 2 study assessing the efficacy of NIVO 3 mg/kg intravenously every 14 days plus IPI 1 mg/kg IV every 6 weeks in pts with TMB-H HER2-negative MBC. The objective of this current study was to evaluate genomic profiling of pts included in NIMBUS and its association with benefit to ICI. Methods: In NIMBUS, eligible pts were required to have measurable HER2-negative MBC, TMB ≥9 Mut/Mb assessed by a cancer-gene panel evaluating &amp;gt; 300 genes and performed in a CLIA-certified laboratory, and 0-3 prior lines of chemotherapy in the advanced setting. The primary objective was overall response rate (ORR) according to RECIST 1.1. Secondary objectives include progression-free survival (PFS), and overall survival (OS). We performed genomic analyses on the pre-treatment tissue, and on baseline and on treatment plasma from pts run on the Foundation Medicine Inc (Boston, MA) FoundationOne®CDx (F1CDx®) and FoundationOne®Liquid CDx (F1LCDx®) panel, respectively. Results: A total of 30 pts (21 with estrogen receptor (ER)-positive and 9 with triple-negative breast cancer) were included in this study. After a median follow-up of 30.6 months, 6 pts (20%) achieved partial response (PR) for a confirmed ORR of 20% (95% CI: 7.7-38.6%). Exploratory analyses did not reveal differences in ORR based on ER status, PD-L1 status, or stromal TIL. The median PFS was 1.4 months (95% CI: 1.3 – 4.6), and the median OS was 16.9 months (95% CI: 7.1-not reached). Pts who achieved PR had a 3-year OS of 75% versus 25% in non-responders. A total of 29 pts (6 responders and 23 non-responders) had at least one pre-treatment genomic panel performed: 16 had both, 5 had pretreatment F1CDx only and 8 had F1LCDx panel only. Blood TMB (bTMB) was highly correlated with tissue TMB by F1CDx (r = 0.95). Using data from the most recent genomic panel available, (24 for baseline blood, and 5 for pretreatment tissue), the most frequently mutated genes ( &amp;gt; 30%) in this population were: TP53 (66%); PIK3CA (45%), ESR1 (35%), CDH1 (35%), ROS1 (31%). ESR1 and PTEN mutations were associated with absence of PR while PALB2 was associated with objective response. We observed a significant correlation between ESR1 mutations and a decrease in OS among pts with ER+ disease. Among the entire 29 pts, a negative correlation between PTEN with PFS and OS, and positive correlation between PALB2 with PFS was observed (Table). Among the 24 pts with baseline F1LCDx panel evaluable, the median TMB was 7.6 Mut/Mb ([interquartile range 0.0–113.8). Median TMB was 8.9 Mut/Mb and 41.7 Mut/mB among pts without and with PR, respectively. Baseline TMB &amp;gt; 20 mut/Mb was found in all pts with PR and in one of those without response. Paired serial blood samples demonstrated that among responding pts, TMB was significantly reduced from baseline to end of treatment (EOT) compared to those with no clinical benefit [n=16, p=0.0015]. Lastly, the tumor fraction (TF) estimates from 7 paired baseline to EOT samples demonstrated that all responders had a decrease or no change in TF compared to an increase in non-responders [n=7, p=0.017]. Conclusion: In the NIMBUS trial, all pts with PR to NIVO plus low dose IPI had a TMB &amp;gt; 20 Mut/Mb and had a decrease in TMB during treatment. ESR1 and PTEN mutations were associated with lack of benefit, while PALB2 mutation was associated with increased benefit. If validated, these results could help tailor the use of ICI among pts with TMB-H MBC. Association between genomic alterations in ESR1, PTEN and PALB2 and benefit to nivolumab and low dose ipilimumab among patients with HER2-negative MBC and TMB-H HR: hazard ratio; MBC: metastatic breast cancer; mOS: median overall survival (months); mPFS: median progression-free survival (months); TMB-H: tumor mutational burden above &amp;gt; 9 mutations/megabase. Citation Format: Romualdo Barroso-Sousa, Tyler Chinsky, Tianyu Li, Sangeetha Reddy, Leisha Emens, Beth Overmoyer, Saud AlDubayan, Hoyin Chu, Paulina Lange, Julie Kasparian, Ameer Basta, Molly DiLullo, Victoria Attaya, Melissa Hughes, Nancy Lin, Nabihah Tayob, Rinath Jeselsohn, Elizabeth Mittendorf, Sara Tolaney. Genomic determinants of benefit to nivolumab (NIVO) plus low dose ipilimumab (IPI) among patients (pts) with hypermutated HER2-negative metastatic breast cancer (MBC): results of NIMBUS trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-12.

439 Extracellular-in-frame deletions and kinase domain duplications are novel, gain-of-function mutations in fibroblast growth factor receptor genes in cancer

OBJECTIVES/GOALS: There are gain-of-function genomic alterations in FGFR genes that guide personalized treatment in some patients with cholangiocarcinoma (10%) and bladder cancer (30%) who can benefit from targeted therapies. We sought to evaluate other genomic alterations in cancer involving FGFRs and assess whether they are gain-of-function. METHODS/STUDY POPULATION: We collaborated with Foundation Medicine Inc (FMI), for the assessment of 300,000 sequenced tumors and a retrospective analysis of recent publications, to identify novel candidate FGFR alterations. We propose to transiently transfect HEK293T cells with an empty vector (EV), FGFR1-4 wild-type (WT), and these variants and use a luminescent-proximity based high-throughput assay, AlphaLISA, and Western blot to assess FGFR and phosphorylated downstream signaling proteins, FRS2, AKT and ERK, and their sensitivity to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200. RESULTS/ANTICIPATED RESULTS: Through our collaboration we identified &gt;100 novel candidate FGFR1-4 variants of unknown significance (VUS) including extracellular-in-frame deletions (EIDs), kinase domain duplications (KDDs), insertions/deletions (INDELs), short number variants (SNVs), and truncations. Immunoblot analysis confirmed the presence of desired EV, FGFR WT, and VUS’ in HEK293T cells. We anticipate the FGFR EIDs and KDDs to display an increased presence of in the respective pFGFR, pFRS2, pERK, and pAKT as compared to the EV and FGFR WT by both immunoblot and AlpahLISA analysis. Additionally, we anticipate the VUS’ to be sensitive to FGFR inhibitors: pemigatinib, erdafitinib, futibatinib, RLY-4008, and TYRA-200 using the AlphaLISA assay. DISCUSSION/SIGNIFICANCE: These findings suggest that the novel FGFR VUS’ are capable of constitutive activation of FGFR kinase activity, and they preliminary demonstrate that these newly identified FGFR alterations are therapeutically targetable. Thus, providing rationale for further clinical evaluation to identify new cohorts of FGFR inhibitor responders.

RAS/RAF Comutation and ERBB2 Copy Number Modulates HER2 Heterogeneity and Responsiveness to HER2-directed Therapy in Colorectal Cancer.

ERBB2-amplified colorectal cancer is a distinct molecular subtype with expanding treatments. Implications of concurrent oncogenic RAS/RAF alterations are not known. Dana-Farber and Foundation Medicine Inc. Colorectal cancer cohorts with genomic profiling were used to identify ERBB2-amplified cases [Dana-Farber, n = 47/2,729 (1.7%); FMI, n = 1857/49,839 (3.7%)]. Outcomes of patients receiving HER2-directed therapies are reported (Dana-Farber, n = 9; Flatiron Health-Foundation Medicine clinicogenomic database, FH-FMI CGDB, n = 38). Multisite HER2 IHC and genomic profiling were performed to understand HER2 intratumoral and interlesional heterogeneity. The impact of concurrent RAS comutations on the effectiveness of HER2-directed therapies were studied in isogenic colorectal cancer cell lines and xenografts. ERBB2 amplifications are enriched in left-sided colorectal cancer. Twenty percent of ERBB2-amplified colorectal cancers have co-occurring oncogenic RAS/RAF alterations. While RAS/RAF WT colorectal cancers typically have clonal ERBB2 amplification, colorectal cancers with co-occurring RAS/RAF alterations have lower level ERRB2 amplification, higher intratumoral heterogeneity, and interlesional ERBB2 discordance. These distinct genomic patterns lead to differential responsiveness and patterns of resistance to HER2-directed therapy. ERBB2-amplified colorectal cancer with RAS/RAF alterations are resistant to trastuzumab-based combinations, such as trastuzumab/tucatinib, but retain sensitivity to trastuzumab deruxtecan in in vitro and murine models. Trastuzumab deruxtecan shows clinical efficacy in cases with high-level ERBB2-amplified RAS/RAF coaltered colorectal cancer. Co-occurring RAS/RAF alterations define a unique subtype of ERBB2-amplified colorectal cancer that has increased intratumoral heterogeneity, interlesional discordance, and resistance to trastuzumab-based combinations. Further examination of trastuzumab deruxtecan in this previously understudied cohort of ERBB2-amplified colorectal cancer is warranted.

Real-world treatment patterns in patients with BRCA 1/2-positive (BRCA+) metastatic castration-resistant prostate cancer (mCRPC) initiating first-line (1L) therapy.

52 Background: Patients with BRCA+ mCRPC have an aggressive disease course. Recently, they have been shown to be responsive to treatment with poly ADP-ribose polymerase inhibitors, such as niraparib, olaparib, and rucaparib, used in combination with abiraterone acetate and prednisone or prednisolone, or as monotherapy. This study aimed to describe real-world treatment patterns and outcomes among patients with BRCA+ mCRPC. Methods: De-identified electronic health records from the Flatiron Health – Foundation Medicine Inc. (FMI) Metastatic PC Clinico-Genomic Database (01/01/2011 – 06/30/2022) were used to select BRCA+ mCRPC patients initiating 1L therapy on or after 01/01/2019 or monotherapy with androgen deprivation therapy (ADT). Patients were classified as recipients of ADT monotherapy if they used ADT at mCRPC diagnosis and did not initiate any subsequent advanced PC therapy. Distribution, line of therapy (LOT) sequences, and reasons for censoring were described in 1L, and among patients who advanced to second-line (2L) therapy. Flatiron Health and FMI did not participate in data analyses. Results: A total of 98 treated patients with BRCA+ mCRPC were included (mean age 72 years, 58% White, 15% Black, 55% Medicare-insured), 19 of whom received ADT monotherapy (mean age 70 years, 53% White, 26% Black, 47% Medicare-insured). The top five treatment regimens for mCRPC were ADT monotherapy (19%), enzalutamide (ENZ; 14%), olaparib (OLA; 13%), abiraterone acetate (AA; 11%), and docetaxel (DOC; 10%), with 14% of patients receiving ≥1 medication. The main reasons for censoring in patients with ADT monotherapy were death (52.6%), loss to follow-up (26.3%), and end of data availability (21.1%). Among the 79 patients treated with other than ADT monotherapy in 1L, 43.0% (n=34) did not advance to 2L, of which 35.3% were lost to follow-up, 29.4% died (median time-to-death: 2.25 months), 29.4% were censored at the end of data availability, and 5.9% initiated a clinical trial drug. The most prescribed 2L therapies were DOC (22.2%), OLA (20.0%), AA (13.3%), and ENZ (11.1%). In those who advanced to 2L (n=45), the most common 1L → 2L treatment sequences were OLA → DOC (11.1%), ENZ → DOC (6.7%), AA → ENZ (6.7%), and AA → OLA (6.7%). In patients receiving 2L, 51.1% (n=23) did not advance to third-line therapy, of which 34.8% died, 34.8% were lost to follow-up, 26.1% were censored at the end of data availability, and 4.3% initiated a clinical trial drug. Conclusions: Among treated patients with BRCA+ mCRPC, ADT monotherapy, ENZ, OLA, AA, and DOC were most commonly used. The majority of ADT monotherapy patients died, while a substantial proportion of patients initiating 1L therapy died or initiated a clinical trial drug and did not advance to 2L, suggesting unmet need for more effective 1L treatment for patients with BRCA+ mCRPC.

Genomic Profiles and Clinical Outcomes of Penile Squamous Cell Carcinoma With Elevated Tumor Mutational Burden.

Tumor mutational burden (TMB) is a putative biomarker of efficacy for immune checkpoint inhibitor (ICI) therapies of solid tumors, but not specifically for penile squamous cell carcinoma (PSCC). To characterize biomarker features and ICI therapy outcomes associated with high TMB in PSCC in the routine clinical practice setting. In this cohort study, 397 PSCC cases were analyzed to identify genomic alterations in more than 300 cancer-associated genes and genomic signatures, including TMB, using a hybrid capture-based comprehensive genomic profiling assay. Tumor mutational burden was categorized as low (<10 mutations per megabase [mut/Mb]), high (10-19 mut/Mb), or very high (≥20 mut/Mb). Germline status of genetic alterations was predicted using a validated somatic-germline computational method. Clinical outcomes of patients with metastatic PSCC receiving first-line ICI were abstracted using the deidentified nationwide Clinico-Genomic Database (CGDB) from January 1, 2011, through December 31, 2022. Comprehensive genomic profiling was performed using FoundationOne and FoundationOne CDx assays from Foundation Medicine Inc. The spectrum of genetic alterations by TMB level in PSCC, the percentage of germline genetic alterations, and the outcome (overall survival with routine clinical treatment) by TMB of chemotherapy-naive patients with PSCC who received ICI treatment up front were assessed in this descriptive study. Among 397 patients (median [IQR] age, 65 [54-73] years; 266 [67.0%] of European, 83 [20.9%] of admixed American, and 34 [8.5%] of African or other genomic ancestry), the median (IQR) age (eg, 65 [53-73] years for low TMB vs 68 [61-78] years for TMB ≥10 mut/Mb) and genomic ancestry distribution (eg, European 228 of 339 [67.3%] for low TMB vs 38 of 58 [65.5%] for TMB ≥10 mut/Mb) were similar between TMB subgroups. There were 339 PSCC cases (85.4%) with low TMB, 40 cases (10.1%) with high TMB, and 18 cases (4.5%) with very high TMB. Comparisons of TMB of 10 mut/Mb or higher vs low TMB showed an enrichment of genetic alterations in PIK3CA (48.3% vs 18.3%; P < .001) and KMT2D (29.3% vs 7.7%; P < .001) and less frequent genetic alterations in CDKN2A (25.9% vs 45.7%; P = .05). Most genetic alterations did not co-occur. Human papillomavirus identification was more frequent as TMB increased: 28.3% for low TMB, 50.0% for high, and 72.2% for very high. In total, 95 of 1377 genetic alterations (6.9%) were germline. Of 10 patients identified from the CGDB receiving frontline ICIs, median (IQR) follow-up was 9.9 months. Four patients had overall survival with clinical treatment of more than 12 months, including 2 of 3 patients with TMB of 10 mut/Mb or higher. In this cohort study of advanced metastatic PSCC based on TMB levels, significant differences were observed for biomarkers in nearly 15% of patients with a TMB of 10 mut/Mb or higher. Germline testing and ICI-based therapy should be integrated into the management of selected PSCC cases.

Genomic characterization of thymic epithelial tumors in a real-world dataset

Thymic epithelial tumors (TETs) are rare neoplasms arising in the mediastinum, including thymic carcinomas and thymomas. Due to their rarity, little is known about the genomic profiles of TETs. Herein, we investigated the genomic characteristics of TETs evaluated in a large comprehensive genomic profiling database in a real-world setting. We included data from two different cohorts: Foundation Medicine Inc. (FMI) in the United States and the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) in Japan. Samples profiled were examined for all classes of alterations in 253 genes targeted across all assays. Tumor mutational burden (TMB) and microsatellite instability (MSI) were also evaluated. A total of 794 patients were collected in our study, including 722 cases from FMI and 72 cases from C-CAT. In the FMI data, CDKN2A (39.9%), TP53 (30.2%) and CDKN2B (24.6%) were frequently altered in thymic carcinoma, versus TP53 (7.8%), DNMT3A (6.8%), and CDKN2A (5.8%) in thymoma. TMB-high (≥10 mutations/Mb) and MSI were present in 7.0% and 2.3% of thymic carcinomas, and 1.6% and 0.3% of thymomas, respectively. Within C-CAT data, CDKN2A (38.5%), TP53 (36.5%) and CDKN2B (30.8%) were also frequently altered in thymic carcinoma, while alterations of TSC1, SETD2 and LTK (20.0% each) were found in thymoma. To the best of our knowledge, this is the largest cohort in which genomic alterations, TMB and MSI status of TETs were investigated. Potential targets for treatment previously unbeknownst in TETs are identified in this study, entailing newfound opportunities to advance therapeutic development.

Clinical utility of liquid-based comprehensive genomic profiling (CGP) in gastrointestinal stromal tumors (GIST).

11541 Background: GIST is the most common mesenchymal cancer of the digestive tract. Beyond surgery, treatment for GIST focuses largely on tyrosine kinase inhibitors (TKI), whose selection and potential resistance depend on select mutations. We present the molecular landscape of GIST utilizing tissue and liquid biopsies with emphasis on the clinical utility of liquid biopsy in advanced GIST. Methods: Liquid (FoundationOne Liquid CDx [F1LCDx]) and tissue (FoundationOne CDx) CGP was performed by hybrid capture, targeted NGS at Foundation Medicine Inc. Tissue and liquid samples from 2,198 and 147 patients, respectively, were analyzed. A cohort of 27 paired tissue and liquid samples were also evaluated. The levels of circulating tumor DNA (ctDNA) in liquid biopsies was quantified by tumor fraction (TF), with a TF algorithm incorporating aneuploidy, variant allele frequency, and canonical alterations detected on F1LCDx. Results: Tissue CGP (n = 2,198) revealed the following prevalence of primary driver alterations: KIT (77%), PDGFRA (8%), NF1 (6%), SDHA/B/C/D (SDHx, 3%) and BRAF (1%). Rates of molecular markers previously associated with worse prognosis included: CDKN2A (29%), RB1 (9%), TP53 (6%) and SETD2 (4%). 7% of cases had no reportable known pathogenic alterations in canonical GIST genes (wild-type GIST), while 2% of cases had a mutation in more than one driver. In a cohort of 147 liquid biopsies, TF was &lt; 1% in 68.0%, 1-10% in 18.4%, &gt; 10% in 13.6% of samples. In samples with elevated TF ( &gt; 10%), the prevalence of targetable driver alterations in KIT (89%), PDGFRA (4%) , NF1 (4%) and BRAF (4%) was comparable to the tissue prevalence. In liquid, 58% (39/67) of samples with a KIT-driver mutation had a co-occurring imatinib-resistant KIT alteration. In addition, 4/147 patients (3%) were predicted to harbor a germline KIT mutation, including one patient (0.6%) with a potential imatinib-resistant KIT D820G germline mutation and another with clinical suspicion of germline KIT L576P mutation due to the presence of multiple primary GISTs, hyperplasia of myenteric plexus and dysplastic skin nevi. In paired tissue/liquid samples, liquid detected 2/2 driver mutations found in tissue when liquid TF was &gt; 10%, and 5/6 in specimens with TF &gt; 1%. In the overall cohort, the relative prevalence of KIT exon 9 and 11 driver alterations was comparable in tissue vs liquid, while imatinib-resistance KIT exon 13 and 17 mutations were enriched in liquid samples. Conclusions: Known driver and TKI-resistant mutations of both somatic and potential germline origin are identified in peripheral blood ctDNA of GIST patients. Liquid biopsy shows high concordance to tissue in identifying driver mutations in the presence of elevated TF and may exhibit TKI-resistant specific alterations. This study indicates that liquid biopsy may be useful in the molecular classification of GIST during the medical management of advanced GIST patients.

Abstract 3395: Clinical impact of FGFR inhibitors on FGFR2 positive pancreatic cancer

Abstract Objectives: Here we describe the durable clinical outcomes of four FGFR2 fusion positive pancreatic cancer patients treated with FGFR inhibitors as well as a pancreatic specific analysis of FGFR alterations through our collaboration with Foundation Medicine, Inc. (FMI). Methods: We identified four patients with FGFR2 fusion positive metastatic pancreatic cancer through tumor RNAseq (OSU-SpARKFuse) which identifies gene fusions from cell free DNA (cfDNA). We assessed two tumor markers (Ca19-9 and FGFRDx, an FGFR-focused cell free DNA liquid biopsy assay) and various clinical outcomes (Best overall response, duration, and side effects to their treatments). Additionally, 10,146 tumor-tissue clinical cases of pancreatic cancers were assayed by hybrid-capture based comprehensive genomic profiling (Foundation Medicine, Inc.) to identify genomic alterations in &amp;gt;300 cancer-associated genes, MSI, and tumor mutation burden (TMB). Results: All four patients demonstrated durable clinical responses to various FGFR inhibitors. Patient 1 harbored an FGFR2-USP33 fusion and exhibited a partial response to pemigatinib for 25 months following progression on gemcitabine/abraxane and FOLFIRI chemotherapy. Patient 2 harbored an FGFR2-INA fusion with 12 months of stable disease to pazopanib after experiencing progression from chemotherapy. Patient 3 harbored an FGFR2-INA fusion and exhibited a 12+ month on-going, partial response to pemigatinib. Patient 4 harbored an FGFR2-CEP55 fusion and demonstrated stable disease on ponatinib for 15 months, a 24-month response to gemcitabine/platinum chemotherapy, and a 7+ month on-going partial response to infigratinib. Through the genomic profiling of 10,146 tumor-tissue clinical cases of pancreatic cancer, we identified 108 FGFR fusions resulting in a prevalence of 1%. We are currently investigating co-occurring and mutually-exclusive genomic alterations, including TMB and MSI-H status, for pancreatic cancer patients harboring FGFR alterations. Conclusions: This retrospective analysis provides clinical evidence that there is a subpopulation of KRAS wild-type FGFR2 fusion positive pancreatic patients who can have beneficial and lasting responses to FGFR inhibitors further suggesting a need to investigate and expand the availability of FGFR inhibitors to more cancer types. Citation Format: Leah Stein, Julie W. Reeser, Michele R. Wing, Karthikeyan Murugesan, Anoosha Paruchuri, Zachary Risch, Eric Samorodnitsky, Emily L. Hoskins, Amy Smith, Thuy Dao, Melissa Babcook, Muhammad Imam, Aharon Freud, Sameek Roychowdhury. Clinical impact of FGFR inhibitors on FGFR2 positive pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3395.

Time-to-next treatment (TTNT) and overall survival (OS) among homologous recombination repair (HRR) positive and HRR negative patients with metastatic castration-resistant prostate cancer (mCRPC) initiating first-line (1L) therapy.

80 Background: Several recurrent mutations that interfere with the HRR DNA damage signaling response pathway have been recently identified as novel biomarkers that may help optimize treatment for patients with mCRPC. There is limited real-world information on clinical outcomes, including TTNT and OS, among patients with mCRPC who initiate 1L, overall and by HRR mutation status in the United States. Methods: This study used the nationwide (de-identified) Flatiron Health – Foundation Medicine Inc. (FMI) Metastatic Prostate Cancer Clinico-Genomic Database (1/1/2011–12/31/2021). The de-identified data originated from approximately 280 US cancer clinics (~800 care sites). Patients who initiated 1L therapy (index date) on or after mCRPC diagnosis and had ≥1 HRR mutation test any time prior to or on the index date were included. Patients were excluded if they initiated a clinical trial drug in 1L or had &lt;12 months of clinical activity before the index date. Patients were classified as HRR+ (i.e., ATM, BRCA1, BRCA2, BRIP1, CDK12, CHEK2, FANCA, PALB2) or HRR-. HRR+ pathogenic mutations were any qualifying short variant, copy number loss, or rearrangement mutations. For TTNT, patients were followed from index date until the earliest of 2L initiation (outcome), end of clinical activity (including death) or end of data availability. For OS, patients were followed from index date until the earliest of death (outcome), end of clinical activity, or end of data availability. For both outcomes, HRR- patients were censored at first HRR+ mutation test. TTNT and OS were reported overall and by baseline HRR status using Kaplan-Meier methods. Flatiron Health and FMI did not participate in data analyses. Results: A total of 1,150 tested patients with mCRPC who initiated 1L therapy were included (mean age 70 years, 68% white, 59% initiated 1L on or after 2019). Overall, 257 patients (22%) were HRR+ by 1L initiation, of which 82 (7% overall) had a BRCA1/2 mutation. Overall, enzalutamide (31%), abiraterone acetate (30%), and docetaxel (20%) were the most used medications in 1L. The median TTNT was 8.0 months, overall, and was numerically shorter for HRR+ (6.8 months) than HRR- (8.2 months). Overall, 2L initiation rates were 15% at 3 months (HRR+: 17%; HRR-: 15%), 38% at 6 months (HRR+: 45%; HRR-: 36%), and 64% at 12 months (HRR+: 69%; HRR-: 62%). The median OS time was 24.9 months, overall, and was numerically shorter for HRR+ (23.0 months) compared to HRR- (25.8 months) patients. Overall, survival rates were 91% at 6 months (HRR+: 93%; HRR-: 91%), 78% at 12 months (HRR+: 75%; HRR-: 79%), and 51% at 24 months (HRR+: 47%; HRR-: 53%). Conclusions: Patients with HRR+ mCRPC were observed to have shorter survival and progress more rapidly from 1L to 2L therapy than HRR- patients, suggesting greater unmet need in these patients.

Pan-cancer Landscape of Programmed Death Ligand-1 and Programmed Death Ligand-2 Structural Variations.

Programmed cell death protein-1 (PD-1) receptor and ligand interactions are the target of immunotherapies for more than 20 cancer types. Biomarkers that predict response to immunotherapy are microsatellite instability, tumor mutational burden, and programmed death ligand-1 (PD-L1) immunohistochemistry. Structural variations (SVs) in PD-L1 (CD274) and PD-L2 (PDCD1LG2) have been observed in cancer, but the comprehensive landscape is unknown. Here, we describe the genomic landscape of PD-L1 and PD-L2 SVs, their potential impact on the tumor microenvironment, and evidence that patients with these alterations can benefit from immunotherapy. We analyzed sequencing data from cancer cases with PD-L1 and PD-L2 SVs across 22 publications and four data sets, including Foundation Medicine Inc, The Cancer Genome Atlas, International Cancer Genome Consortium, and the Oncology Research Information Exchange Network. We leveraged RNA sequencing to evaluate immune signatures. We curated literature reporting clinical outcomes of patients harboring PD-L1 or PD-L2 SVs. Using data sets encompassing 300,000 tumors, we curated 486 cases with SVs in PD-L1 and PD-L2 and observed consistent breakpoint patterns, or hotspots. Leveraging The Cancer Genome Atlas, we observed significant upregulation in PD-L1 expression and signatures for interferon signaling, macrophages, T cells, and immune cell proliferation in samples harboring PD-L1 or PD-L2 SVs. Retrospective review of 12 studies that identified patients with SVs in PD-L1 or PD-L2 revealed > 50% (52/71) response rate to PD-1 immunotherapy with durable responses. Our findings show that the 3'-UTR is frequently affected, and that SVs are associated with increased expression of ligands and immune signatures. Retrospective evidence from curated studies suggests this genomic alteration could help identify candidates for PD-1/PD-L1 immunotherapy. We expect these findings will better define PD-L1 and PD-L2 SVs in cancer and lend support for prospective clinical trials to target these alterations.

Detection of BRCA1, BRCA2, and ATM Alterations in Matched Tumor Tissue and Circulating Tumor DNA in Patients with Prostate Cancer Screened in PROfound.

Not all patients with metastatic castration-resistant prostate cancer (mCRPC) have sufficient tumor tissue available for multigene molecular testing. Furthermore, samples may fail because of difficulties within the testing procedure. Optimization of screening techniques may reduce failure rates; however, a need remains for additional testing methods to detect cancers with alterations in homologous recombination repair genes. We evaluated the utility of plasma-derived circulating tumor DNA (ctDNA) in identifying deleterious BRCA1, BRCA2 (BRCA), and ATM alterations in screened patients with mCRPC from the phase III PROfound study. Tumor tissue samples were sequenced prospectively at Foundation Medicine, Inc. (FMI) using an investigational next-generation sequencing (NGS) assay based on FoundationOne®CDx to inform trial eligibility. Matched ctDNA samples were retrospectively sequenced at FMI, using an investigational assay based on FoundationOne®Liquid CDx. 81% (503/619) of ctDNA samples yielded an NGS result, of which 491 had a tumor tissue result. BRCA and ATM status in tissue compared with ctDNA showed 81% positive percentage agreement and 92% negative percentage agreement, using tissue as reference. At variant-subtype level, using tissue as reference, concordance was high for nonsense (93%), splice (87%), and frameshift (86%) alterations but lower for large rearrangements (63%) and homozygous deletions (27%), with low ctDNA fraction being a limiting factor. We demonstrate that ctDNA can greatly complement tissue testing in identifying patients with mCRPC and BRCA or ATM alterations who are potentially suitable for receiving targeted PARP inhibitor treatments, particularly patients with no or insufficient tissue for genomic analyses.

Genomic characterization of thymic epithelial tumor from real-world data.

8587 Background: Thymic epithelial tumors (TETs), including thymic carcinomas and thymomas, are rare neoplasms arising in the mediastinum. Chemotherapy is still the mainstay of treatment and few therapeutic options are available for patients with advanced or metastatic TETs. Due to their rarity, the sample size in the previous reports about the genomic profiles of TETs was small and the results varied from study to study, which hinders the development of treatment. Herein, we investigated the comprehensive genomic characteristics of TETs evaluated in a large genomic database profiled in a real-world setting. Methods: Tissue biopsy-based comprehensive genomic profiling was performed in the course of routine clinical care. We included data from two different cohorts: Foundation Medicine Inc. (FMI) in the US (Frampton GM, et al. Nat Biotechnol 2013) and the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) in Japan, which is engaged in a national project for collecting genomic analysis and clinical results from patients. Samples profiled at Foundation Medicine were examined for all classes of alterations in 253 genes targeted across all assays. Tumor mutational burden (TMB) and microsatellite instability (MSI) were calculated as previously described (Chalmers ZR, et al. Genome Med 2017. Trabucco, et al. J Mol Diagn 2019). Patients’ background including histology, age, and sex were also investigated, and genetic alteration, TMB, and MSI were stratified by them. Results: A total of 794 patients were collected for our study, including 722 cases from FMI and 72 cases from C-CAT. In the FMI data, 414 cases of thymic carcinoma and 308 cases of thymoma were included. CDKN2A (39.9%), TP53 (30.2%) and CDKN2B (24.6%) were frequently altered in thymic carcinoma, versus TP53 (7.8%), DNMT3A (6.8%), CDKN2A (5.8%) and CDKN2B (4.6%) in thymoma. TMB-High (≥ 10muts/Mb) and MSI-High were present in 7.0% and 2.3% of thymic carcinomas, and 1.6% and 0.3% of thymomas, respectively. Comparison of the thymic carcinoma cohort based on age &lt; 60 vs 60+ years found a significant difference in prevalence of NFKBIA alterations (2.7% age &lt; 60 vs 11.7% age ≥ 60, p = 0.034), while a similar comparison of the thymoma cohort found no significant differences between age groups. An analysis based on sex did not find any significant differences between groups. 55 cases of thymic carcinoma and 17 cases of thymoma were included from C-CAT data. In thymic carcinoma, CDKN2A (27.3%), TP53 (23.6%) and CDKN2B (20.0%) were also frequently altered, while alterations of TSC1 (23.5%) and CD22, LTK, NOTCH1, KMT2A, SETD2, ATM (17.6% each) were found in thymoma. Conclusions: To the best of our knowledge, this is the largest cohort in which genomic alterations, TMB, and MSI status of TETs were investigated. We suggest that several gene mutations, TMB, and MSI status might be potential targets for treatment and lead to therapeutic development opportunities, especially in thymic carcinoma.

Pan-cancer landscape of CD274 (PD-L1) and PDCD1LG2 (PD-L2) structural variations.

3133 Background: PD-1 receptor and PD-L1 ligand interactions are the target of immunotherapies across multiple tumor types. Established biomarkers that predict response to immunotherapy are microsatellite instability, tumor mutational burden, and PD-L1 immunohistochemistry. Structural variations in CD274 (PD-L1) and PDCD1LG2 (PD-L2) have been observed in cancer, but the comprehensive landscape is unknown. Here we describe the genomic landscape of CD274 and PDCD1LG2 structural variations, their potential impact on the tumor microenvironment, and evidence that patients with these alterations can benefit from immunotherapy. Methods: We analyzed sequencing data from 514 cancer cases with CD274 and PDCD1LG2 structural variations across 25 publications and data sources, including large pan-cancer sources: Foundation Medicine, Inc (FMI), The Cancer Genome Atlas (TCGA), and Oncology Research Information Exchange Network. To evaluate immune signature enrichment, we ran the software ImSig on gene expression data. We curated literature reporting clinical outcomes of patients harboring structural variations in CD274 and PDCD1LG2. Results: From 25 studies and datasets, we curated 514 cancer cases with structural variations in PD-L1 and PD-L2, including 158 duplications, 126 deletions, 97 inversions, 178 translocations, and 96 unclassified structural variations, totaling to 655 events. We observed breakpoint ‘hotspots’ in the 3’-untranslated regions (UTRs) of PD-L1 and PD-L2. Leveraging TCGA data, we observed, in CD274-rearranged tumors, significant upregulation in PD-L1 and PD-L2 expression and signatures for interferon signaling, macrophages, monocytes, T cells, and immune cell proliferation (each p &lt; 0.001, compared to CD274 non-rearranged, copy neutral tumors). Furthermore, retrospective review of 12 studies that included patients with structural variations in CD274 or PDCD1LG2, including duplications, inversions and copy number amplifications, revealed a 73% (52/71) response rate to PD-1 immunotherapy with durable responses. Conclusions: Our evaluation of CD274 and PDCD1LG2 structural variations shows that the 3’-UTR is frequently affected and is associated with increased expression of ligands and immune signatures. Enriched interferon signaling in CD274-rearranged tumors is of particular interest, as interferon exposure is known to drive PD-L1 and PD-L2 expression. Retrospective evidence from curated studies suggests that these genomic alterations could identify candidates for PD-1 or PD-L1 immunotherapy. We expect that these findings will better define CD274 and PDCD1LG2 structural variations in cancer and support our pan-cancer prospective clinical trial to target these alterations.

Biomarker Testing Patterns and Treatment Outcomes in Patients With Advanced Non-Small Cell Lung Cancer and MET Exon 14 Skipping Mutations: A Descriptive Analysis From the US.

Background MET exon 14 skipping mutation (METex14) is observed in ~3% of non-small cell lung cancer (NSCLC) cases and has been shown to be an independent poor prognostic factor associated with shorter overall disease-specific survival. Broad molecular testing can identify this biomarker in patients with advanced NSCLC (aNSCLC) and allow patients to be matched with the appropriate targeted therapy. This study examines biomarker testing patterns and clinical outcomes of chemotherapy and immuno-oncology (IO) monotherapy in aNSCLC patients with METex14.MethodsA descriptive retrospective study was conducted using the Flatiron Health–Foundation Medicine Inc. (FMI) clinico-genomic database. Patients with METex14 aNSCLC treated with systemic therapies were included in the biomarker testing analysis. The duration from specimen collection to reported results was assessed for PD-L1– and METex14-tested patients. Clinical outcomes were assessed in patients treated with chemotherapy or IO monotherapy. First-line (1L) and second-line (2L) real-world progression-free survival (rw-PFS) were estimated using Kaplan-Meier analysis.ResultsOf 91 METex14 patients eligible for the biomarker testing analysis, 77% and 60% received PD-L1 and FMI next-generation sequencing (NGS) testing within 3 months post aNSCLC diagnosis. Of those assessed for both PD-L1 and METex14 (n=9), the median duration between specimen collection and reporting was 1 week shorter for PD-L1 than for FMI NGS. Median 1L rw-PFS was 5.7 months (95% CI, 4.6-7.1) and 2.4 months (95% CI, 1.4-3.2) in patients receiving 1L chemotherapy (n=59) and IO monotherapy (n=18), with 3-month 1L rw-PFS rates of 78% and 33%. Median 2L rw-PFS was 3.5 months (95% CI, 1.9-11.1) and 4.7 months (95% CI, 2.8-12.9) in patients receiving 2L chemotherapy (n=16) and IO monotherapy (n=23), with 3-month 2L rw-PFS rates of 54% and 67%.ConclusionsThe median time from biopsy to test results appears 1 week shorter for PD-L1 than for FMI NGS. Chemotherapy and IO monotherapy were the most common regimens utilized but with limited PFS.

Comprehensive genomic profiling (CGP) of fibrolamellar oncocytic hepatoma (FLO) and conventional hepatocellular carcinomas (HCC): An observational study.

474 Background: FLO is a rare variant of liver cancer that disproportionately affects young adults and is frequently progressive and fatal as it is often detected in a clinically advanced stage. It is seldom associated with cirrhosis of liver, viral hepatitis or other risk factors associated with conventional HCC. Treatment options are largely limited to surgical resection, and there is dearth of effective targeted therapies for FLO. Methods: Comprehensive genomic profiling (CGP) was performed using the Foundation Medicine Inc. (FMI) data base on 63 FLO and 1,793 HCC clinically advanced cases between 6/2013-12/2020 using a hybrid capture-based assay of up to 324 genes a to detect genomic alterations (GA), tumor mutational burden (TMB) and microsatellite instability (MSI). PD-L1 expression in tumor cells (Dako 22C3) was measured by IHC and scored using the tumor proportion score (TPS) method. Results: The FLO patients (pts) were significantly younger than HCC pts (median age 20 vs. 64, respectively) and the male preponderance was similar. The HCC group featured significantly more GA/tumor than FLO group (3.74 vs 1.31 p&lt;.0001). In the currently untargetable GA group, CTNNB1, TERT and TP53 GA were significantly more frequent in HCC than FLO. GA in potentially targetable genes were extremely uncommon in both FLO and HCC with HCC featuring slightly more MTOR pathway targets ( PTEN, TSC2, NF1). GA in DNA damage and repair (DDR) pathway including BRCA2 were infrequent in both groups. GA in targetable kinases including EGFR, ERBB2, ALK, RET and PIK3CA were extremely uncommon in both groups. GA associated with intrahepatic cholangiocarcinoma (IDH1, FGFR2) were extremely uncommon in these tumors. Although the mean TMB was significantly higher in HCC than FLO, overall TMB was low with very few cases having TMB &gt; 10 mutations/Mb. PD-L1 expression was relatively low in both groups. GA in genes associated with immune checkpoint inhibitors (ICPI) drug response like PBRM1, CD274, MDM2, STK11 were rarely identified in both groups. Additional details are illustrated in the table. Conclusions: Comparison of CGP of FLO with HCC illustrates the multifarious nature of these cancers. In HCC, there is a high prevalence of GA in TERT, CTNNB1 and TP53. CGP identified certain targetable GA in the MTOR and DDR pathways and TMB was higher in HCC. These findings warrant further evaluation of clinically advanced FLO and HCC pts by CGP to identify possible targetable genomic pathways. [Table: see text]