Short Variant Mutations Research Articles

Nature and distribution of methyl thioadenosine phosphorylase (MTAP) genomic loss in human tumors.

3067 Background: MTAP genomic loss, often but not exclusively a bi-allelic homozygous deletion, has recently emerged as important biomarker guiding a novel synthetic lethality mechanism for drugs in the class of PRMT5 and MAT2A inhibitors. In the current study, we assessed the nature and distribution of MTAP complete and partial loss in a variety of tumor samples. Methods: A total of 291,391 tissue samples underwent comprehensive genomic profiling (CGP) using a hybrid-capture method to evaluate all classes of genomic alterations (GA), and determine the microsatellite instability (MSI) status, tumor mutational burden (TMB), homologous recombination deficiency (HRD) score and genomic ancestry. The distribution of both complete and partial loss of MTAP was also determined. Results: MTAP GA were found in 8.6% of total with 9.3% genomic loss and 0.1% each short variant mutations and rearrangements. Important (not rare) tumor types with the most frequent MTAP loss included: 42.6% of glioblastoma (GBM), 13.4% of non-small cell lung cancer (NSCLC), 22.3% of pancreatic ductal and 24.9% of bladder urothelial carcinomas. The most frequently co-altered GA included CDKN2A (99.7%), CDKN2B (95.1%), TP53 (55.4%), KRAS (30.1%), TERT(20.5%) and PIK3CA (12%). In tumor types with frequent known targetable driver GA including short variant mutations in EGFR, ERBB2, BRAF, FGFR2-3and MET and fusions involving ALK, RET, ROS1 and NTRK1-3, MTAP loss was not associated with decreased frequencies of GA in these genes. The frequencies of MSI-high status was 0.3% and of TMB≥10 mut/Mb was 17%. In total, 63.7% of the MTAP loss cases involved deletion of all 8 MTAP exons (complete loss) with partial loss accounting for 36.3% of cases. All partial loss cases involved loss of multiple exons (35.8%) except for loss of only exon 8 (0.5%). There was no impact of MTAP loss status on genomic ancestry, cosmic trinucleotide signature and HRD score. The multiple exon loss frequencies included exons 2-8 loss in 24.7%, exons 5-8 loss in 4.1%, exons 6-8 loss in 3.3%, exons 3-8 loss in 1.6%, exons 7-8 in 1.1%, exon 4-8, 1-5, 2-7,2-5, 1-4, 2-6, 3-7 and 5-7 all at less than 1% frequencies. Conclusions: MTAPloss is a frequent GA of emerging clinical importance as the trials using PRMT5 and MTA-2 inhibitors progress. MTAPloss is frequent in common cancers of the brain, lung, pancreas and bladder and not associated with diminishment of other targetable driver mutations. Although one-third of MTAP loss is a partial loss, the partial loss cases involve near total (exons 2-8) loss and may well also be indicative of PRMT5/MAT2A inhibitor benefit. This study strengthens the opportunity to consider a tumor-agnostic approach to targeted therapies. [Table: see text]

A comprehensive analysis of POLE/POLD1 genomic alterations in colorectal cancer.

Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H), defined as TMB ≥ 10 mut/Mb, independent of deficient mismatch repair (dMMR) and microsatellite instability high (MSI-H) status. De-identified records of patients with colorectal cancer (CRC) profiled with the Tempus xT assay (DNA-seq of 595-648 genes at 500×) were identified from the Tempus Database. Among 9136 CRC samples profiled, the frequency of POLE/POLD1 genomic alterations was 2.4% (n = 217). Copy number loss was the most common genomic alteration (64%, n = 138) of POLE/POLD1, followed by copy number amplifications (18%, n = 40) and short variant mutations (18%, n = 39). The POLE/POLD1 mutated group presented with a higher frequency of TMB-H phenotype relative to wild type (WT; 22% vs. 9%, P < .001), with a median TMB of 127 mut/Mb in the TMB-H POLE/POLD1 subset. The TMB showed a dramatic contrast between POLE/POLD1 short variant mutations as compared to the group with copy number alterations, with a TMB of 159 mut/Mb vs 15 mut/Mb, respectively. Thus, the short variant mutations represented the so-called ultra-hypermutated phenotype. The POLE/POLD1 mutated group, as compared to WT, exhibited a higher rate of coexisting mutations, including APC, ALK, ATM, BRCA2, and RET mutations. Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers (ie, TMB, MMR, and MSI-H) and molecular co-alterations. Those with short variant mutations represented 18% of the POLE/POLD1 cohort and 0.4% of the total cohort examined. This group of patients had a median TMB of 159 mut/Mb (range 34-488), representing the ultra-hypermutated phenotype. This group of patients is important to identify given the potential for exceptional response to immune checkpoint inhibitors.

Abstract PO5-06-14: Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma

Abstract Background: Triple-negative (TN) invasive lobular carcinoma (ILC) of the breast is a rare entity, comprising roughly 2% of primary ILC. Clinicopathologic studies have shown that TN ILC is associated with more pleomorphism, higher nuclear grade, older patient age, and worse disease-related outcomes compared to estrogen receptor-positive (ER+) ILC. Intrinsic subtyping indicates that TN ILC less frequently expresses basal markers than TN invasive ductal carcinoma (IDC). This prompted investigation of the genomic landscape and clinical management of TN ILC to provide insight into potential therapeutic approaches. Methods: Twenty patients with TN ILC were included in this study. Three patients were treated at a single academic center and underwent comprehensive genomic profiling (CGP) of breast or metastatic tissue; clinicopathologic details were obtained from their electronic health records. Seventeen patients with TN ILC who underwent CGP of breast or metastatic biopsies were included from the Foundation Medicine, Inc. database. PD-L1 testing for all patients was determined by immunohistochemistry (IHC) using the VENTANA SP142 or Dako 22C3 commercial assays. HER2 expression was determined by IHC. Negative HER2 expression was defined as an IHC score of 0, or IHC 1+ or 2+ with non-amplified fluorescence in situ hybridization (FISH). Results: In the Foundation Medicine database of patients with TN ILC, the most frequent genomic alterations were in CDH1 (15/17 patients [88.2%]), TP53 (10/17 patients [58.8%]), and PIK3CA (10/17 patients [58.8%]). Short variant mutations in ERBB2 were found in 3/17 patients (17.6%). Fourteen patients were HER2-low (HER2 IHC 1+ or 2+ with non-amplified FISH). No cases were MSI high or expressed PD-L1 (8 of 17 cases underwent PD-L1 assessment by SP142). Median tumor mutational burden (TMB) was 5 muts/Mb. Among the three patients with TN ILC treated at a single center, the most common somatic mutations were in CDH1 and TP53, and no cases were MSI high or expressed PD-L1 (by SP142 or 22C3). All three patients had pleomorphic ILC, histologic grade 2 or 3, and HER2 IHC 0. One patient with mpT1cN1a(sn) ER+/HER2- ILC of the right breast in 2011 received adjuvant TAC (docetaxel, doxorubicin, cyclophosphamide), breast/axillary radiation, and ten years of adjuvant endocrine therapy. She was diagnosed with TN ILC of the left supraclavicular nodes in 2022; she did not exhibit a response to docetaxel and carboplatin with pembrolizumab, and she subsequently received palliative radiation followed by capecitabine. Another patient with locally advanced TN ILC progressed on the KEYNOTE-522 regimen, immediately developed metastases, and is currently receiving palliative sacituzumab govitecan. The third patient with cT3N1 TN ILC progressed on neoadjuvant therapy (through the I-SPY 2 trial) with oral paclitaxel, encequidar, and dostarlimab, followed by dostarlimab with dose-dense doxorubicin and cyclophosphamide. Her surgical pathology demonstrated ypT3N3a disease with 10 × 7 cm of tumor, and she developed skin metastases on the chest wall within weeks post-operatively. She received comprehensive chest wall and nodal radiation and is currently receiving palliative capecitabine with neratinib given a high variant allele frequency driver somatic ERBB2 mutation. Conclusions: The most common somatic mutations among TN ILC patients included in this study were in CDH1, TP53, and PIK3CA. Currently the management of TN ILC is extrapolated from that of TN IDC, however our patients did not respond to chemoimmunotherapy. The presence of HER2-low status or somatic ERBB2 mutations may provide opportunities for treatment with an anti-HER2 antibody-drug conjugate or tyrosine kinase inhibitor. Studies with larger sample sizes of TN ILC are needed for molecular profiling and assessment of outcomes with specific therapeutic approaches. Citation Format: Hemali Batra-Sharma, Smruthy Sivakumar, Prashanth Ashok Kumar, Ethan Sokol, Rebecca Shatsky, Jeffrey Ross. Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma [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 PO5-06-14.

Abstract 5536: Landscape and analysis of ERBB2 amplification and short variant mutations in large-scale Chinese patients with colorectal cancer

Abstract Background: Human epidermal growth factor receptor 2 (ERBB2) amplification is a rapidly emerging therapeutic target in CRC, in spite of a relatively low incidence. While ERBB2 amplification has been considered as a critical biomarker to predict the efficacy and safety of anti-ERBB2 therapy of CRC, its point mutation status is largely ambiguous.Methods: Blood or tissues samples from 2454 Chinese patients with CRC were collected, and the genomic landscape, clinical characteristics and genetic features of ERBB2 in these samples were retrospectively investigated using next-generation sequencing (NGS) analysis. Results: Of the 2454 colorectal cancer patients, 3.46% (85) carried ERBB2 amplification, 2.24% (55) carried ERBB2 mutation, and some patients harbored both ERBB2 amplification and point mutation at the same time. Microsatellite instability high (MSI-H) was found in approximately 32.7% (18/55) of patients with ERBB2-mutated CRC, but almost negative in ERBB2-amplified CRC. The median fold change of ERBB2 amplification was 30 (Range 4 to 245). Among the 16 mutation sites detected from 55 patients with ERBB2 point mutations, p.R678Q was the most common mutation site (28%), followed by p.V842I (24%) and p.S310F/Y (12%), which appeared to be slightly different from previous reports. ERBB2 mutation was more frequently found in CRC patients aged &amp;lt; 50 years. 50.9% of CRC patients with wild-type ERBB2 carried KRAS mutation, which was significantly higher than those with mutated ERBB2 (25.6%). A higher incidence of BRAF mutation was observed in patients with mutated-ERBB2 (8.5% vs. 2.3%). In addition, CRC patients with ERBB2 CNV showed a higher prevalence of TP53 mutation comparing with those with ERBB2 SNV (92.3% vs. 58.3%), while KRAS mutation exhibited a contrary trend (14.1% vs. 45.8%)Conclusions: The overall variation rate of ERBB2 is 5.4% in Chinese patients with CRC, which is accompanied by significantly different molecular pathological characteristics compared to patients with wild-type ERBB2, and different mutation types (CNV or SNV) display different molecular pathological characteristics. Citation Format: Zhiqin Chen, Shijun Yu, Yin Wu, Bei Zhang, Ding Zhang, Yong Gao, Ming Quan. Landscape and analysis of ERBB2 amplification and short variant mutations in large-scale Chinese patients with colorectal 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 5536.

Genomic landscape of non-small-cell lung cancer with methylthioadenosine phosphorylase (MTAP) deficiency.

New treatment strategies for advanced non-small-cell lung carcinoma (NSCLC) include synthetic lethality targets focused on protein arginine methyl transferases such as PRMT5 that exploit the impact of genomic loss of methylthioadenosine phosphorylase (MTAP). Twenty nine thousand three hundred seventy nine advanced NSCLC cases underwent hybrid-capture based comprehensive genomic profiling between June 1, 2018 and May 31, 2020. PD-L1 expression was determined by immunohistochemistry (Dako 22C3 PharmDx assay). 13.4% (3928/29,379) NSCLC cases exhibited MTAP loss distributed in adenocarcinoma (59%), squamous cell carcinoma (22%), NSCLC not otherwise specified (16%), and 1% each for large-cell neuroendocrine, sarcomatoid, and adenosquamous carcinoma. Statistically significant differences in mitogenic driver alterations included more KRAS G12C mutations in MTAP-intact versus MTAP-lost (12% vs. 10%, p=0.0003) and fewer EGFR short variant mutations in MTAP-intact versus MTAP-lost NSCLC (10% vs. 13%, p < 0.0001). Statistically significant differences in currently untargetable genomic alterations included higher frequencies of TP53 (70% vs. 63%, p < 0.0001) and RB1 inactivation (10% vs. 2%, p < 0.0001) in MTAP-intact compared to MTAP-lost NSCLC. SMARCA4 inactivation (7% vs. 10%, p < 0.0001) was less frequent in MTAP-intact versus MTAP-lost NSCLC. Alterations in ERBB2, MET, ALK, ROS1, and NTRK1 did not significantly differ between the two groups. Predictors of immunotherapy efficacy were higher in MTAP-intact versus MTAP-lost NSCLC including tumor mutational burden (9.4 vs. 8.6 mut/Mb, p=0.001) and low (30% vs. 28%, p=0.01) and high PD-L1 (32% vs. 30%, p=0.01) expression. Alterations in biomarkers potentially predictive of immune checkpoint inhibitor resistance (STK11, KEAP1, and MDM2) were similar in the two groups. MTAP loss occurs in 13% of NSCLC, supporting the development of targeted therapies to exploit PRMT5 hyper-dependence. MTAP loss is accompanied by small differences in targeted and immunotherapy options which may impact future combination strategies.

Pan-cancer analysis of CD274 (PD-L1) mutations in 314,631 patient samples and subset correlation with PD-L1 protein expression

BackgroundThe effects of non-amplification short variant (SV) mutations in CD274 (programmed death-ligand 1 (PD-L1)) on PD-L1 protein expression and immune checkpoint inhibitors (ICPIs) therapy are unknown. Here, we present a...

Genomic landscape of MSH6-mutated clinically advanced castrate-resistant prostate cancer (mCRPC).

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 &lt;.0001) and was most frequently caused by biallelic co-deletion of both genes (73% of co-mutated cases). MSI-High status was present in 46% of MSH6mut mCRPC, which was significantly greater than the 2% seen in MSH6wt cases (P &lt;.0001). An MMR single nucleotide mutational signature was observed in 65% of MSH6mut cases, compared to 3% MSH6wt cases (P &lt;.0001). Among MSH6mut 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 &lt;.0001). Conclusions: MSH6mut 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]

Pan-cancer analysis of CD274 (PD-L1) mutations in 314,631 patient samples and subset correlation with PD-L1 protein expression.

2605 Background: The effects of non-amplification short variant (SV) mutations in CD274 (PD-L1) on PD-L1 protein expression and immune checkpoint inhibitor (ICPI) therapy are unknown. Here, we present a retrospective analysis of CD274 mutations detected by comprehensive genomic profiling (CGP) and correlate these results with tumor-cell PD-L1 immunohistochemistry (IHC)-based expression assessment to better understand the relationship between mutations and protein expression of PD-L1. Methods: FoundationOne CGP was performed on hybridization-captured, adaptor ligation-based libraries using DNA and/or RNA extracted from 314,631 tumor samples that were sequenced for up to 406 cancer related genes and select gene rearrangements. PD-L1 IHC was performed on a subset of cases (n = 213) using the DAKO 22C3 PD-L1 IHC assay and scored with the tumor proportion score (TPS). Results: Overall, the prevalence of CD274 SV mutations was low (0.3%, 1,081/314,631) with 577 unique variants. The most common CD274 SV mutations were R260H (n = 51), R260C (n = 18), R125Q (n = 12), C272fs*13 (n = 11), R86W (n = 10), and R113H (n = 10). The prevalence of CD274 mutations varied depending on tumor type with diffuse large B-cell lymphoma (1.9%, 19/997), cutaneous squamous cell carcinoma (1.6%, 14/868), endometrial adenocarcinoma (1.0%, 36/3740), unknown primary melanoma (0.9%, 33/3679), and cutaneous melanoma (0.8%, 32/3874) having the highest frequency of mutations. Ultraviolet exposure was likely a mechanism for CD274 SV mutations in cutaneous tumors with high frequencies of ultraviolet mutational signatures (cutaneous squamous cell carcinoma [84.6%, 11/13], cutaneous melanoma [93.8%, 30/32], and unknown primary melanoma [100%, 32/32]), and microsatellite instability (MSI) was likely a mechanism for development of CD274 mutations in non-serous endometrial adenocarcinoma. Of the R260H cases concurrently tested with PD-L1 IHC, most (81.8%, 9/11) had no PD-L1 expression, which contrasts to the five E237K cases where most (80%, 4/5) had PD-L1 expression. This difference in protein expression of these two mutations was significantly different (p = 0.036). It was notable that nearly all samples (88.9%, 16/18) with a clonal truncating variant (nonsense or frame shift indel) and PD-L1 testing showed a PD-L1 TPS score ≤1, whereas three of four samples with sub-clonal truncating variants had TPS scores ≥5. Conclusions: We defined the landscape of CD274 mutations in a large cohort of tumor types that can be used as a reference for examining CD274 mutations as potential resistance biomarkers for ICPI. Furthermore, we presented novel data on the correlation of CD274 mutations and PD-L1 protein expression, providing important new information on the potential functionality of these mutations and can serve as a basis for future research.

Genomic landscape of non-small cell lung cancer (NSCLC) with methylthioadenosine phosphorylase (MTAP) deletion.

9116 Background: NSCLC remains a major cause of cancer-associated mortality despite major advancements in treatments. In addition to immune checkpoint inhibitors (ICPI), new strategies for clinically advanced NSCLC now include the development of new synthetic lethality targets focused on protein arginine methyl transferases such as PRMT5 that exploit the impact of tumor cell genomic loss of MTAP. Methods: 29,379 advanced/metastatic NSCLC cases underwent hybrid-capture based comprehensive genomic profiling to evaluate all classes of genomic alterations (GA). 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 tumor cell expression was determined by DAKO 22C3 immunohistochemistry (IHC); low positive was a tumor proportion score (TPS) 1-49% and high positive was a TPS ≥50%. Results: 3,928 NSCLC exhibited MTAP homozygous loss. Cases had the following subtypes: adenocarcinoma (59%), squamous cell ca (22%), NSCLC NOS (16%), and large cell neuroendocrine, sarcomatoid, adenosquamous ca (all 1%). GA/tumor were similar when CDKN2A/B losses associated with 9p21 co-deletion with MTAP loss are excluded. Significant differences in currently targetable GA included KRAS G12C higher in MTAP-intact NSCLC (P =.0003) and EGFR short variant mutations higher in MTAP-deleted NSCLC (P &lt;.0001). MTAP-intact NSCLC had higher frequencies of GAs in TP53 (P &lt;.0001) and RB1 and a lower frequency of SMARCA4 (P &lt;.0001). GAs frequencies in ERBB2, MET, ALK, ROS1 and NTRK1 were similar. Biomarkers for potential ICPI efficacy were higher in MTAP-intact including TMB ≥10mut/Mb (P =.0002) and low and high PD-L1 IHC staining (P =.01). Biomarkers potentially predictive of ICPI resistance ( STK11 and KEAP1) were similar in both groups. Conclusions: MTAP loss occurs in 13% of NSCLC, supporting the development of novel targeted therapies designed to exploit PRMT5 hyper-dependence in these tumors. MTAP loss in NSCLC is accompanied by differences in targeted and ICPI options for these patients which may impact future combination strategies. Further study of anti-PRMT5 drugs that are enabled by MTAP loss in NSCLC appears warranted.[Table: see text]

Comprehensive genomic profiling (CGP) utilizing cell-free DNA (cfDNA) in patients (pts) with pancreatic ductal adenocarcinoma (PDAC).

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 &lt;0.001). 613/1009 (60.8%) of pts had at least one somatic alteration detected on cfDNA based CGP. TP53 (53.7%), KRAS (40.3%) and CDKN2A (6.5%) were the most frequently altered genes (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [somatic and/or germline BRCA2, ATM, and CHEK2 alterations], were detected in 12.3% (124/1009) pts – 11.4% (46/403) in KRAS mutated pts and 12.9% (78/606) in KRAS wildtype (WT) pts. Fusions were detected only in KRAS WT pts (1.5%, 6/403). Pts with cfDNA based CGP could be classified into 4 latent classes – class 1 ( KRAS - 100%, TP53 - 100% and CDKN2A – 33.3%), class 2 ( KRAS - 65.7%), class 3 ( KRAS – 68.2%, CDKN2A/B – 7.7% ) and class 4 ( KRAS – 38.5%, TP53 - 26.7%). Conclusions: The putative role of cfDNA based CGP in monitoring therapeutic response and clonal evolution in pts with PDAC may be predicated on cfDNA yield. In clinical scenarios where pre-treatment tissue availability is limited, cfDNA based CGP may be have utility in leveraging the predictive value of somatic and/or germline HR-DDR gene alterations. The prognostic and/or predictive relevance of the four latent classes needs validation in clinically annotated data sets. [Table: see text]

Predictive Biomarkers for Immune Checkpoint Inhibitors in Metastatic Breast Cancer.

We examined a large dataset of female metastatic breast cancers (MBCs) profiled with comprehensive genomic profiling (CGP) to identify the prevalence and distribution of immunotherapy responsiveness‐associated biomarkers. DNA was extracted from 3831 consecutive MBCs: 1237 (ERpos/HER2 neg), 1953 ERneg/HER2 amp, and 641 triple‐negative breast cancer (TNBC). CGP was performed using the FoundationOne® or FoundationOne®CDx NGS assay. Tumor mutational burden (TMB) and microsatellite instability (MSI) were determined in a subset of cases. PD‐L1 expression in immunocytes in a subset of cases was determined by immunohistochemistry using the companion diagnostic VENTANA PD‐L1 SP142 Assay. The median age of the cohort was 54 years (range 20–89). Genomic alterations (GAs)/tumor were similar (range: 5.9–7.3). Markers of potential immune checkpoint inhibitor (ICPI) benefit included: CD274 (PD‐L1) amplification (1%–3%), BRAF GA (1%–4%), TMB of ≥10 mutations/Mb (8%–12%), MSI‐high (0.1%–0.4%), PBRM1 GA (1%), and positive PD‐L1 staining of immunocytes ranging from 13% in ERpos/HER2 neg and 33% in ERneg/HER2 amp to 47% in the TNBC group. Potential markers of ICPI resistance included inactivating STK11 GA (1%–2%) and MDM2 amplification (3%–6%). MTOR pathway targets were common with lowest frequency in TNBC. ERBB2 short variant mutations were most frequent ERpos/HER2 neg and absent in TNBC. BRCA1/2 GA were least frequent in ERneg/HER2 amp. The demonstrations of clinical benefit of immunotherapy in MBC support the need for development and utilization of biomarkers to guide the use of ICPIs for these patients. In addition to guiding therapy selection, CGP shows potential to identify GA linked to response and resistance to ICPI in MBC.

Circulating Tumor DNA and Biomarker Analyses From the LOTUS Randomized Trial of First-Line Ipatasertib and Paclitaxel for Metastatic Triple-Negative Breast Cancer

Combining the oral AKT inhibitor ipatasertib with paclitaxel as first-line therapy for metastatic triple-negative breast cancer significantly improved progression-free survival (PFS) in the placebo-controlled, randomized, phase II LOTUS trial, with a more pronounced effect in patients with PIK3CA/AKT1/PTEN-altered tumors. We report findings from the extensive translational research program. Pretreatment plasma and tumor samples were evaluated for genetic alterations using FoundationACT and FoundationOne (Foundation Medicine, Cambridge, MA) hybrid capture next-generation sequencing assays, respectively. Prevalences of the most common mutations and PIK3CA/AKT1 mutation status were determined using both assays, and concordance was assessed. In longitudinal analyses, circulating tumor DNA (ctDNA) mutations were quantified in baseline and on-treatment (cycle 3, day 1 [C3D1]) samples. The relationship between outcomes and ctDNA fraction (CTF; highest variant allele frequency) and CTF ratio (C3D1 CTF to baseline CTF) was explored. Among 89 patients evaluable for ctDNA sequencing, 81 patients (91%) had 149 detectable mutations. There was high agreement between plasma- and tissue-based sequencing for known or likely short variant mutations but not amplifications. There was 100% concordance between ctDNA and tissue sequencing in patients with activating PIK3CA or AKT1 mutations. High baseline CTF was associated with shorter PFS in both treatment arms. Longitudinal analyses showed more favorable outcomes with lower absolute CTF at C3D1 and, to a lesser extent, greater CTF decreases. These results suggest that plasma ctDNA sequencing may allow reliable and convenient assessment of prognosis and identification of genetic markers associated with increased benefit from ipatasertib. On-treatment CTF showed a meaningful association with objective response and PFS.

Patient-matched tissue and liquid biopsies identify shared and acquired genomic alterations in breast cancer.

1050 Background: Liquid biopsy-based comprehensive genomic profiling (CGP) is a minimally invasive approach to potentially identify truncal driver alterations and mechanisms of acquired resistance. However, there are limited data comparing solid tissue and plasma biopsies from the same patient in breast cancer. Methods: CGP was performed on matched tumor tissue and plasma samples from 444 patients with breast cancer (Foundation Medicine, Inc.) to identify short variant mutations and rearrangements in at least 62 genes. ER/PR/HER2 status was abstracted for a subset of patients (n = 273). Patients were primarily ER+/HER2- (58%) with fewer HER2+ (16%) and TNBC (25%). Results: Samples were temporally heterogeneous with a median time between matched tissue-plasma collection of 276 days (interquartile range 45 - 650). Positive percent agreement (PPA) to tissue biopsy was 81.5% with higher PPA for collection interval ≤1y v &gt; 1y (82.5% v 76.5%). PPA was highest in known truncal driver genes ( AKT1, PTEN, BRCA2, BRCA1, TP53, PIK3CA: 85-89% PPA). PPA of tissue to liquid biopsy was 50.5% with strong time dependence (62.4% ≤1y, 37.9% &gt; 1y), presumably due to intervening therapies. Acquired alterations in liquid biopsy were primarily known resistance mechanisms ( ESR1, NF1, ERBB2, PIK3CA, PTEN, TP53, BRCA1, and BRCA2), were often polyclonal, associated with longer collection intervals, and exhibited higher prevalence in the ER+/HER2- subtype (62% ER+/HER2-, 33% HER2+, 42% TNBC). Acquired alterations significantly enriched in ER+ cases included ESR1, NF1, ERBB2 and TP53. For PIK3CA, a PPA of 84.6% was observed with solid as baseline and 79.1% with liquid as baseline; prevalence was similar (36.5% solid, 35.6% liquid). Alpelisib companion diagnostic alterations (CDx) had a similar PPA (84.0% solid, 83.3% liquid). Overall percent agreement (OPA) for PIK3CA CDx in paired samples was 91.4% (406/444). When acquired PIK3CA alterations are observed they tend to be subclonal, co-occur with shared PIK3CA alterations, and consist of rare alterations (E726K, E453K, M1043I). Conclusions: While high PPA to tissue was observed for alterations in truncal driver genes, acquired alterations observed in plasma were frequently polyclonal, often identify potential mechanisms of therapeutic resistance and are, in part, a function of time and intervening treatments relative to tissue CGP. These results demonstrate high PPA to tissue suggesting that liquid biopsy has clinical validity in identifying truncal alterations and can also identify acquired alterations in breast cancer.

Abstract P3-06-09: Incidence of ROS1 genomic alterations in breast cancer

Abstract Background: ROS1 is an important proto-oncogene involved in the development of various cancers and increased expression is a poor prognostic feature in breast cancer. Activation of the ROS1 tyrosine kinase receptor has been reported and is being targeted with several FDA approved small molecule inhibitors, primarily in lung cancer. However, the frequency and type of ROS1 alterations in breast cancer have not been fully explored. The purpose of this study was to identify the incidence and type of ROS1 genomic alterations occurring in breast cancer as a prelude to potentially targeting this protein in the setting of metastatic breast cancers that harbor these changes. Methods: We queried 21,125 breast cancer samples (~50% from distant metastatic sites) from the Foundation Medicine breast cancer database, 5,964 breast cancer samples from five publicly available breast cancer databases (MBC Project, INSERM, METABRIC, TCGA, MSK-IMPACT), and 217 breast cancer samples from Project GENIE v6.5 to identify the incidence of ROS1 alterations in breast cancer. We identified fusion partner genes and classified each alteration type into the following categories: fusion, rearrangement, short variant (missense or indel), or copy number variation. A computational approach was used to distinguish somatic from germline single nucleotide polymorphisms in the Foundation Medicine database (Sun et. al., 2018). PolyPhen2 and in vitro analyses were used to investigate the effect of ROS1 nonsynonymous mutations on the ROS1 protein. Using Foundation Medicine, we made associations with ROS1 alterations and receptor status, histologic subtype, metastatic site, tumor mutational burden, and co-occurring mutated genes. Estrogen receptor (ER) status was available for 2,371 samples. HER2 status and all other analyses were identified from the entire cohort. Results: The incidence of any ROS1 alteration across the Foundation Medicine breast cancer database, including variants of unknown significance, was 4.6% and ranged from 1.67% to 6.9% from the five publicly available breast cancer databases and Project GENIE. Foundation Medicine samples revealed 5 (0.024%) breast cancers with ROS1 fusions, 86 (0.41%) with rearrangements, 733 (3.47%) with short nucleotide variants, and 182 (0.86%) with copy number variations. ROS1 alterations occurred similarly in HER2, TNBC, and ER+ breast cancers, 5.24%, 4.5%, and 4.37%, respectively (ns, all p&amp;gt;0.2). Invasive ductal carcinoma, invasive lobular carcinoma, and metaplastic carcinoma harbored ROS1 alterations at 4.35%, 3.65%, and 4.36%, respectively (ns, all p&amp;gt;0.3). ROS1 alterations were significantly associated with metastatic breast cancer spread to the brain (p=0.0005) and lymph nodes (p=0.017). There were 18 gene mutations significantly co-occurring with ROS1 alterations when controlling for multiple comparisons. The top 5 were TP53 (OR 1.55, p=3.12 × 10−8), RB1 (OR 1.66, p=0.0001), NOTCH3 (2.27, p=0.0001), TAF1 (OR 4.82, p=0.0002), and ARID1A (OR 1.71, p=0.0003). Conclusions: A modest incidence of genomic alterations in ROS1 occurs across all breast cancer subtypes and histologies and may be more actionable than currently recognized. New somatic alterations in the ROS1 gene were identified from Foundation Medicine that were not detected in publicly available databases or Project GENIE. Investigations using PolyPhen2 and in vitro analyses of ROS1 gene activation from these newly discovered somatic alterations and other short variant mutations are currently being investigated in our lab with results to be reported. Co-occurring mutations reveal a unique genotype associated with ROS1 alterations that may play a biologic role in ROS1-mediated pathogenesis and should be explored further. ROS1 alterations occurring in breast cancers might be leveraged in an innovative personalized medicine treatment approach for breast cancer patients. Citation Format: Jeremy Force, Ethan S Sokol, Mary Love Taylor, Dale Huang, Paul K Marcom, Monika Davare, Jeffrey Marks. Incidence of ROS1 genomic alterations in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-09.

KRAS amplification and mutation as independent events in gastroesophageal adenocarcinomas (GEA).

e15565 Background: KRAS mutations are common oncogenic events across cancers, but effective RAS-directed therapies are lacking. However, recent studies support use of PD-1 blockade in most subsets of lung cancer with KRAS short variant mutations (KRASSV) (PMID: 28039262), and preclinical data supports combined MEK and SHP2 inhibition in KRAS amplified ( KRASa) GEA (PMID: 30093730). We sought to explore the landscape of KRAS altered GEA and compare genomic profiles of KRAS-altered and KRAS wild-type (WT) cases for biomarkers of response to targeted therapies and immune checkpoint inhibitors. Methods: 6,667 tissue specimens from patients with advanced GEA were assayed using hybrid capture-based comprehensive genomic profiling. Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 or 114 loci. Descriptive statistics were used to compare among subgroups. Results: KRASSV and KRASa were identified in 11% and 5.8% of gastric adenocarcinoma (GA), respectively, and in 7.2% and 17% of esophageal adenocarcinoma (EA), respectively. KRASa and KRASSV were nearly mutually exclusive, co-occurring in only 4.4% of KRAS altered cases. ERBB2 alterations were less common in KRASSV and KRASa GEA (both 9%) as compared with KRAS WT GEA (19%) (p = 1.9E-16). EGFRa was less common in KRASSV vs KRASa GEA (1.9% vs 9.3%, p = 2.6E-8), whereas PIK3CASV was more common in KRASSV vs KRASa (16% vs 5.0%, p = 1.5E-11). Median TMB for all groups was similar; however, KRASSV GEA had a higher mean TMB (9.7 mut/Mb) as compared to KRASa (5.1 mut/Mb, p = 5.0E-12) and KRAS WT cases (5.8 mut/Mb, p = 2.2E-07). KRAS codon 12/13 accounted for &gt; 80% of predicted pathogenic mutations. MSI-high was also more prevalent in KRASSV (11.3%) vs KRASa (0.9%, p = 4.8E-15) and KRAS WT GEA (2.4%, p = 1.8E-25). MSI-high KRASSV GEA was associated with older patient age (median 72 years) and with high TMB (median 40.9 mut/mb). Conclusions: GA was enriched for KRAS mutation whereas EA was enriched for KRAS amplification. KRAS WT vs KRASSV vs KRASa each presented distinct genomic profiles. KRASa in the absence of KRAS mutation exists in 11% of GEA and warrants further exploration to inform combination treatment strategies.

RAS-amplified colorectal cancers: Microsatellite stability status, RAS/BRAF mutations, and prediction of anti-EGFR resistance.

3533 Background: Pre-clinical models implicate RAS amplifications ( RASa) as a mechanism of resistance to anti-Epidermal Growth Factor Receptor (EGFR) therapies. Yet, there is little guidance on the impact of RASa, as identified by next generation sequencing (NGS), on anti-EGFR response. Methods: We investigated the Foundation Medicine (FM) database for RASa in CRC and characterized this population based on patient (pt) characteristics and other concurrent genomic alterations. We subsequently investigated City of Hope (COH) mCRC molecular data set (using FoundationOne) and described the RASa population characteristics and response to anti-EGFR. Results: FM cohort included 21,315 CRC unique pt cases. 365 (1.5%) pts had RASa, of which 123 (0.6%) had ≥ 20 copy number (CN) ( RASa≥20). The incidence of MSI-H, RAS and BRAF short variant mutations in the overall, RASa, and RASa≥20 populations were (MSI-H: 5%, 0%, 0%), ( RAS: 54%, 32%, 2%), and ( BRAF: 6%, 1%, 0%), respectively. Copy number variation inversely correlated with likelihood of RAS mutation. COH cohort included 396 mCRC. 13 pts (3.3%) had RASa. The median RAS CN was 25 (7 - 79); 8/13 pts had RASa≥20. All but 3 COH pts with RASa had a RAS/BRAF-WT tumors. Pts with concurrent RAS mutations had relatively low RAS CN (7, 8, &amp;13). Tumors with RASa were predominantly left-sided (12/13). 7/13 pts were treated with anti-EGFR therapy. All 7 pts were RAS/ BRAF wild-type: 6 left-sided; 3 pts 1st/2nd lines (1 pt oxaliplatin-based and 2 pts irinotecan-based) and 4 pts chemo-refractory (all irinotecan-based). All 4 chemo-refractory pts had RASa≥20and progressed on anti-EGFR on 1st post-treatment CT (PFS in all pts ≤ 3 mo). One 1st line pt had SD (PFS = 4 mo). One 2nd line pt had SD (PFS = 7 mo) and one had PD as best response. Conclusions: NGS identifies RASa and RASa≥20in 1.5% and 0.6% of CRC, respectively. RASa≥20 tumors are MSS and, generally lack RAS/ BRAF mutations, and predict for resistance to anti-EGFR. This supports the potential relevance of 20-CN cut-point for the exclusion of pts from anti-EGFR. These findings would benefit from additional validation from retrospective analyses of completed prospective anti-EGFR clinical trials.

Extra-mammary Paget’s disease (EMPD) of the skin: A comprehensive genomic profiling (CGP) study.

9591 Background: EMPD is an intraepidermal adenocarcinoma which frequently progresses into invasive/metastatic disease. Methods: 29 EMPD underwent hybrid-capture based CGP to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and reported as mut/Mb. Microsatellite instability (MSI) was determined on 114 loci. PD-L1 expression was determined by IHC (Dako 22C3 antibody). Results: All patients had locally recurrent and/or metastatic disease. The 20 (69%) male and 9 (31%) female patients had a median age of 70 years (range 45 to 101 years). The primary EMPD site, known in 90% of cases and involved the scrotum (6 cases), groin (6 cases), vulva (5 cases), anus (3 cases), perineum (2 cases), abdomen (2 cases), penis (1 case) and buttock (1 case). was used for sequencing in 15 (52%) cases and a metastatic site 14 (48%) cases. The GA/tumor frequency was 5.0. The most frequent currently untargetable GA involved TP53 (48%), CDKN2A (38%), CDKN2B (31%), KMT2C (28%), and MYC (14%). The most frequent potentially targetable GA were identified in ERBB2 (35%), ERBB3 (17%) and PTEN (14%) and rarely in PDGFRA, CDK4/6, NF1/2, and ROS1 (fusion) all at 3%. The ERBB2 GA included short variant (SV) activating mutations (21%), amplifications (10%) and multiple ERBB2 GA in 3%. This finding is in contrast with mammary Paget’s disease which classically features &gt; 90% of cases with ERBB2 amplification. No EMPD cases were either MSI-High or stained positively for PD-L1 expression. The median and mean TMB were 5.2 mut/Mb and 6.3 mut/Mb, respectively; 4 (14%) had TMB ≥ 10 mut/Mb and 1 (3%) had TMB ≥ 20 mut/Mb. Conclusions: EMPD is a rare source of relapsed/metastatic adenocarcinoma which features GA that are distinct from the more common disease originating on the nipple of the breast including a lower ERBB2 GA frequency and lower relative frequency of ERBB2 amplification versus SV mutations. A variety of MTOR pathway, cell cycle and kinase targets are also identified when EMPD undergoes CGP. However, the low TMB and absence of both MSI-High status and PD-L1 expression in EMPD cohort indicates a likely lack of benefit for immunotherapies for patients suffering from this rare form of malignancy.

KRAS amplification and mutation are independent events in gastroesophageal adenocarcinomas (GEA).

70 Background: KRAS mutations are common oncogenic events across cancers, but effective RAS-directed therapies are lacking. However, recent studies support use of PD-1 blockade in most subsets of lung cancer with KRAS short variant mutations (KRASSV) (PMID: 28039262), and preclinical data supports combined MEK and SHP2 inhibition in KRAS amplified ( KRASa) GEA (PMID: 30093730). We sought to explore the landscape of KRAS altered GEA and compare genomic profiles of KRAS-altered and KRAS wild-type (WT) cases for biomarkers of response to targeted therapies and immune checkpoint inhibitors. Methods: 6,667 tissue specimens from patients with advanced GEA were assayed using hybrid capture-based comprehensive genomic profiling. Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 or 114 loci. Descriptive statistics were used to compare among subgroups. Results: KRASSV and KRASa were identified in 11% and 5.8% of gastric adenocarcinoma (GA), respectively, and in 7.2% and 17% of esophageal adenocarcinoma (EA), respectively. KRASa and KRASSV were nearly mutually exclusive, co-occurring in only 4.4% of KRAS altered cases. ERBB2 alterations were less common in KRASSV and KRASa GEA (both 9%) as compared with KRAS WT GEA (19%) (p = 1.9E-16). EGFRa was less common in KRASSV versus KRASa GEA (1.9% vs. 9.3%, p = 2.6E-8), whereas PIK3CASV was more common in KRASSV versus KRASa (16% vs 5.0%, p = 1.5E-11). Median TMB for all groups was similar; however, KRASSV GEA had a higher mean TMB (9.7 mut/Mb) as compared to KRASa (5.1 mut/Mb, p = 5.0E-12) and KRAS WT cases (5.8 mut/Mb, p = 2.2E-07). KRAS codon 12/13 accounted for &gt; 80% of predicted pathogenic mutations. MSI-high was also more prevalent in KRASSV (11.4%) versus KRASa (0.9%, p = 4.8E-15) and KRAS WT GEA (3.0%, p = 1.8E-25). MSI-high KRASSV GEA was associated with older patient age (median 72 years) and with high TMB (median 40.9 mut/mb). Conclusions: GA was enriched for KRAS mutation whereas EA was enriched for KRAS amplification. KRAS WT versus KRASSV versus KRASa each presented distinct genomic profiles. KRASa in the absence of KRAS mutation exists in 11% of GEA and warrants further exploration to inform combination treatment strategies.

RAS amplified colorectal cancers are enriched in RAS-WT, BRAF-WT, MSS tumors and may predict for anti-EGFR resistance.

547 Background: Pre-clinical models implicate RAS amplifications ( RASa) as a mechanism of resistance to anti-Epidermal Growth Factor Receptor (EGFR) therapies. Yet, there is little guidance on the impact of RASa, as identified by next generation sequencing (NGS), on anti-EGFR response. Methods: We investigated the Foundation Medicine (FM) database for RASa in CRC and characterized this population based on patient (pt) characteristics and other concurrent genomic alterations. We subsequently investigated City of Hope (COH) mCRC molecular data set (using FoundationOne) and described the RASa population characteristics and response to anti-EGFR. Results: FM cohort included 21,315 CRC unique pt cases. 365 (1.5%) pts had RASa, of which 123 (0.6%) had ≥ 20 copy number (CN) ( RASa≥20). The incidence of MSI-H, RAS and BRAF short variant mutations in the overall, RASa, and RASa≥20 populations were (MSI-H: 5%, 0%, 0%), ( RAS: 54%, 32%, 2%), and ( BRAF: 6%, 1%, 0%), respectively. RASa≥20 tumors had higher level of genomic amplification than the overall population (29% vs. 5% had ≥ 5 genes amplified). COH cohort included 338 mCRC. 12 pts (3.6%) had RASa. The median RAS CN was 27 (7 - 72); 8/12 pts had RASa≥20. All but 2 COH pts with RASa had a RAS/BRAF-WT tumors. Both pts with concurrent RAS mutations had relatively low RAS CN (7&amp;13). Tumors with RASa were predominantly left-sided (11/12). 7/12 pts were treated with anti-EGFR therapy. All 7 pts were RAS/BRAF wild-type: 6 left-sided; 3 pts 1st/2nd lines (1 pt oxaliplatin-based and 2 pts irinotecan-based) and 4 pts chemo-refractory (all irinotecan-based). All 4 chemo-refractory pts had RASa≥20 and progressed on anti-EGFR on 1st post-treatment CT (PFS in all pts ≤ 3 mo). One 1st line pt had SD (PFS = 4 mo). One 2nd line pt had SD (PFS = 7 mo) and one had PD as best response. Conclusions: NGS identifies RASa and RASa≥20in 1.5% and 0.6% of CRC, respectively. RASa≥20 tumors are MSS and, generally lack RAS/ BRAF mutations, and predict for resistance to anti-EGFR. This supports the potential relevance of 20-CN cut-point for the exclusion of pts from anti-EGFR. These findings would benefit from additional validation from retrospective analyses of completed prospective anti-EGFR clinical trials.

Abstract 4003: Comprehensive genomic profiling of Merkel cell carcinoma samples reveals bimodal distribution of tumor mutational burden and two mutually exclusive candidate mechanisms of malignant transformation

Abstract Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer. Etiologic risk factors include long-term UV exposure, Merkel cell polyomavirus (MCV) infection, age, and immunosuppression. Large-scale investigations of MCC are hindered by its rarity and comprehensive understanding of this disease is currently lacking. In a study of 275 patients, representing the largest sequenced cohort of MCC samples to date, we sought to characterize the genomic landscape of MCC as well as identify whether causality could be attributed to MCV infection, UV exposure, or a synergistic effect between the two. Comprehensive genomic profiling of 395 cancer-related genes, including evaluation of tumor mutational burden (TMB) and mutational signatures, was performed on MCC tumors from 275 patients. The TMB of MCC exhibited a stark bimodal distribution between TMB high (≥20 mutations/mb) and TMB low (&amp;lt;6 mutations/mb) specimens with only 22 specimens having an intermediate TMB. Overall, 101/275 (37%) of MCC samples were TMB high (median 53.1 mutations/mb) with the majority carrying short variant mutations in TP53, RB1, or NOTCH1. Analysis of mutational signatures found that 94/101 (93%) of TMB high samples exhibited signs of UV damage. No signatures indicative of APOBEC activity or of intrinsic DNA repair defects such as microsatellite instability were observed in the TMB high group. In contrast, 152/275 (55%) of MCC specimens were TMB low (median 1.2 mutations/mb) with 39% of these samples having no known or likely genomic driver. We hypothesized that the primary driver in the TMB low group was MCV integration and examined MCV infection through de novo assembly of non-human sequencing reads in all specimens of the MCC cohort. We found 99/152 (65%) of TMB low specimens to be positive for MCV integration as opposed to 0/101 (0%) of TMB high specimens, which was a statistically significant difference (P = 2.20e-31, Fisher's exact test). Overall, 3 of the 22 TMB intermediate specimens were MCV-positive. In a large cohort of 275 patient samples, we have described two genomically distinct subsets of MCC: a TMB high group defined by UV damage with mutations in well-known tumor suppressors and a TMB low group defined by MCV integration. We propose that MCV integration is the dominant driver in the TMB low cohort. Indeed, most MCV sequences that we observed appear to be derived from the large or small T antigens, two established viral oncogenes. These results are suggestive of two distinct etiologies of MCC that appear to be mutually exclusive, namely UV exposure vs. MCV integration. These etiologies may have significant impact on targeted and immuno-therapy options for MCC patients. Citation Format: Meagan Montesion, Ethan S. Sokol, Todd C. Knepper, Andrew S. Brohl, Garrett M. Frampton, Phil J. Stephens, James A. DeCaprio, Kenneth Y. Tsai, Lee A. Albacker. Comprehensive genomic profiling of Merkel cell carcinoma samples reveals bimodal distribution of tumor mutational burden and two mutually exclusive candidate mechanisms of malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4003.