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e18050 Background: Adenoid cystic carcinoma (ACC) is a rare malignancy of glandular tissue with a high rate of local recurrence and metastatic disease for which clinical behavior varies widely. Currently, no standard therapy exists, and available therapies have low response rates. Therefore, prognostic biomarkers to determine optimal treatment strategies are urgently needed. Here we investigate the molecular landscape and therapeutic targets of ACC. Methods: ACC samples were analyzed using next generation sequencing (NGS) (NextSeq, 592 gene panel) or whole exome sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Pathogenic fusion events were detected using whole transcriptome sequencing (NovaSeq). Statistical significance was determined using the Chi-square test and adjusted for multiple comparisons. Tumor infiltrating Immune cell fractions were determined using the QuanTIseq deconvolution algorithm and WTS data. Results: 327 ACC patients were identified, median age of 58 years, 62% female and 62% metastatic disease. The most frequent mutations (excluding indeterminate results) involved genes in the NOTCH pathway ( NOTCH1 16% [41 of 298 cases]), chromatin remodeling ( ARID1A 24.3% [27 of 138 cases], KDM6A 12.5% [28 of 224 cases], KMT2C 10.7% [21 of 197 cases]) and TP53 10.2% (25 of 246 cases). Chromatin remodeling genes were co-mutated with NOTCH1. NOTCH1 mutations were found in 41 cases, 18 primary and 23 metastatic were associated with reduced median overall survival (mOS 1.8 years vs 3.8 years, p = 0.01). Mutations were most frequent in the PEST (n = 35), NRR (n = 18), PEST+NRR (n = 16) and EGF-like domains (n = 11). PEST domain mutations were found exclusively in ACC originating from the head and neck (H&N). GSEA showed that MYC, E2F and G2M target pathways were enriched in NOTCH1 mutated ACC regardless of the presence of MYB fusions. All three pathways were enriched in PEST domain mutations whereas only MYC targets were enriched in NRR and NRR+PEST domain mutations indicating that PEST domain mutations drive transcriptomic changes in NOTCH1 mutated ACC. MYC gene expression was significantly upregulated in NOTCH1 mutated ACC. Fusion events were detected in 45% (104/231). MYB:NFIB was the most common fusion (40%) and was more frequent in H&N origin than other sites. There was no difference in the mOS between MYB fusion positive and negative ACC (4.4 years vs 5.5 years, p = 0.14). M2-polarized macrophages, myeloid dendritic cells and neutrophils were significantly higher in metastatic compared to primary ACC while NK cells were more abundant in primary tumor. Conclusions: ACC is a genetically heterogenous disease with an immune-excluded microenvironment. NOTCH1 mutations were associated with worse, while MYB:NFIB fusion was not associated with prognosis. Our study shows that M2 macrophages and MYC are potential novel therapeutic targets in ACC.

e18024 Background: Multiple studies investigating immune checkpoint inhibitors (ICI) in head and neck squamous cell cancer (HNSCC) patients have demonstrated prolonged survival of p16+ versus p16- tumors. However, the data has been conflicting. Methods: We queried the Caris Life Sciences CODEai database to assess survival outcomes of HNSCC patients who received ICIs comparing p16+ and p16- subgroups. A standard cut-off of 2+, >70% p16 staining was used. Presence of HPV16/18 genomes was tested using whole exome sequencing. Patients were considered smokers if they had >15 pack-years of tobacco use. PD-L1 expression was assessed by the 22c3 antibody, ≥1 being positive. Gene mutations were evaluated using Next-Generation Sequencing (NGS) (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was assessed by somatic nonsynonymous missense mutations. Real world overall survival (rwOS) was obtained from insurance claims data and Kaplan-Meier estimates were calculated from the date of treatment start to the date of last contact. Time on treatment (TOT) was calculated from date of start to completion of ICIs. Results: 2905 patients with HNSCC were identified in the Caris database. 41% (215/525) were smokers. Among patients who were tested for p16 and/or HPV, 32% (251/791) expressed p16 and 28% (91/236) were HPV+. The majority of p16+ tumors were oropharynx (OP) in origin (68%, 171/251). 87% (970/1111) of tumors expressed PD-L1 and 16% (216/1362) had TMB ≥10/Mb. TP53 (54%, n=1115/2076) and CDKN2A (17%, n=281/1649) were the most common mutations. When compared to p16-, the p16+ group had a higher prevalence of TMB ≥10/Mb (26% vs 17%) and RB1 mutations (21% vs 4%) but lower number of smokers (15% vs 34%) and TP53 mutations (32% vs 58%). Similar to previous reports, p16+ oropharynx squamous cell carcinoma (OPSCC) patients survived longer than p16- patients, rwOS, 47 vs. 20 months (HR=0.55, p = 0.014) respectively. Among patients who were treated with ICIs, p16+ and p16- non-OP HNSCC, rwOS was not reached (NR) for both groups at follow up of 22 months while TOT for p16+ and p16- respectively, was 3.5 vs. 2.7 months, HR 0.507, p=0.039. No statistically significant difference was found in rwOS (19.3 vs. 24 months, HR 1.8, p=0.27) or TOT (3.7 vs. 4.1 months, HR 0.78, p=0.38) between p16+ and p16- OPSCC groups treated with ICIs, respectively. Conclusions: P16+ non-OP HNSCC patients receiving ICIs were found to remain on treatment longer compared to p16- patients which was not reproduced in the OPSCC subgroup. Randomized controlled trials are needed to verify p16 as a prognostic marker for ICI therapy.[Table: see text]

5063 Background: ATM mutations, one of a family of DNA repair defects prevalent in prostate cancer, have been included in a list of actionable mutations for PARP inhibitor (PARPi) therapeutic trials. Despite preclinical evidence, PARPi have shown minimal clinical activity in ATM mutant prostate cancer (ATMmPCa). The present analysis explores co-occurring genomic alterations that may drive outcomes of metastatic PCa (mPCa) patients with tumors harboring ATM mutations and provide clues for understanding therapy resistance and potential targets. Methods: This study included molecular profiling analysis of 1375 cases of mPCa. Tumors were analyzed using next-generation sequencing (NGS), whole transcriptome sequencing (WTS), and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). dMMR/MSI-H status was determined by IHC, NGS, and fragment analysis and tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. We performed differential gene expression analysis of HR-associated transcripts such as ATR, PARP1-3, RAD50, RAD51A/B/C/D and RAD54. Significance was determined using the ꭓ2 test and Benjamini-Hochberg method. Results: Fifty-nine (4.2%) cases harbored pathogenic ATM mutations, 84 (6.2%) harbored BRCA2 mutations. 1018 tumors (74%) were deemed homologous recombination proficient (HRP) and 155 tumors (11.3%) were HR Deficient (HRD); harboring one or more mutation in HR-related genes excluding ATM and BRCA2. The mutation rate of TP53 was significantly lower in ATMmPCa (12.0%) compared to BRCA2mPCa (35%), HRD (35%) and HRP (46.6%) tumors. ATMmPCa showed higher rates of SMAD2 (3.7%/1%) and FLCN (5.2%/0.3%) alterations compared to HRP cases. PARP1 and RAD51D gene expression was reduced in ATMmPCa compared to HRP (p < 0.05) and BRCA2mPCa ( p < 0.05) tumors, respectively. No differences in gene expression levels were detected for ATR, PARP2, PARP3, RAD50, and RAD54. Chromosomal segments demonstrating differential CNA in ATMmPCa vs HRP, HRD, or BRCA2mPCa included FGF19, FGF4, PTPN11, ALDH2, DAXX, BCL7A, CCND1, BMPR1A and MEF2B (Q-value < 0.05 determined by ꭓ2). The most common CNA in ATMmPCa was CCND1, present in approximately 13% (7/55) of cases. Compared to BRCA2mPCa and HRD cases, ATMmPCa cases are less likely to display markers of immunotherapy response such as dMMR/MSI-H or TMB ≥10 mutations/MB. Conclusions: ATMmPCa demonstrated several differences in co-occurring alterations compared to BRCA2mPCa, HRD and HRP mPCa. ATMmPCa tumors were less likely to harbor alterations in TP53 compared to BRCA2, HRD or HRP tumors. CNA in ATMmPCa occurred in 9 genes across distinct mPCa molecular subtypes and were enriched for those associated with the 11q13 amplicon harboring Cyclin D1. The FGF and PTPN11 related pathways are potentially targetable pathways in ATMmPC and may merit further study.

e18026 Background: Previous studies have consistently shown that p16+ oropharynx squamous cell cancer (OPSCC) patients have better outcomes. The significance of molecular and transcriptional signatures and its correlation with p16 expression remains unclear. Methods: We queried the Caris Life Sciences database to assess the molecular and transcriptional signatures related to p16+ and p16- head and neck squamous cell cancer (HNSCC) patients. Comprehensive molecular profiling including whole exome sequencing (WES), targeted Next-Generation Sequencing (NGS), and immunohistochemistry (IHC) 22c3 for PD-L1 was performed (CPS ≥1 considered positive) (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. A standard cut-off of 2+, > 70% p16 staining was used. HPV16/18 was tested using WES. Patients were considered smokers if they had > 15 pack-years. Real world overall survival (rwOS) was obtained from insurance claims data and Kaplan-Meier estimates were calculated from the date of start of treatment to the date of last contact. Results: 948 cases of OPSCC were identified in the Caris database. 41% (82/199) were smokers. Where p16 and HPV data was available, 41% were p16+ (171/420) while HPV positivity rate was 52% (71/148). We noted a small number of patients with discordant p16 and HPV status (7 p16+/HPV-, 8 p16-/HPV+) in 327 patients who had HPV status available. Most common mutations were TP53 (33%), PIK3CA (17%) and KMT2D (10.6%). 87% were PD-L1 positive (342/394), with high expression in both p16+ and p16- subgroups, 90% and 85% respectively. 10% had TMB≥10/Mb (48/463). TP53 mutations were more common in p16- (49%) tumors in contrast to p16+ (10%) (p < 0.0005), while no statistical difference was detected in TMB≥10/Mb between the groups. CDKN2A, TERT and NOTCH1 mutations were more prevalent in tumors that were p16- or HPV- in contrast to tumors that were p16+ or HPV+ (p < 0.05). FGF3, CCND1, FGF4, FGF19 copy number alterations (CNA) were less common in p16+ OPSCC compared to p16- or HPV16- OPSCC (p < 0.0005). P16+ patients had longer rwOS when compared to p16-, 47 months versus 20 months (HR = 0.55, p = 0.014) respectively. Conclusions: Higher frequency of CDKN2A, TERT and NOTCH1 mutations among p16- (versus p16+) raise the possibility of potential targets for treatment in this group with poor prognosis. However, it remains unclear if these can serve as independent predictors of survival.[Table: see text]

e18056 Background: Adenoid cystic carcinoma (ACC) is a rare malignancy of glandular tissue with a high rate of local recurrence and metastatic disease. Despite being regarded as an indolent disease, the clinical course of recurrent and metastatic ACC (R/M ACC) is highly variable. Responses to chemotherapy (chemo) are uniformly poor. Several multi-targeted tyrosine-kinase inhibitors (mTKIs), EGFR inhibitors (EGFRi) and other targeted agents have been studied in single-arm early phase trials with response rates ranging from 0-16% and progression free survival ranging from 2.5-17 months. However, there have been no comparative clinical trials and it is not known if one treatment strategy is superior. We undertook this retrospective study to assess the real-world clinical outcomes in patients with adenoid cystic carcinoma using the Caris Life Sciences database. Methods: Real world overall survival (rwOS) for cases of ACC was obtained from insurance claims data and Kaplan-Meier estimates were calculated from the date of collection to the date of last contact. Cases were divided into subgroups based on treatment received – chemo (including platinum agents, taxanes, 5FU, topoisomerase inhibitors, anthracyclines), EGFRi (cetuximab, erlotinib, lapatinib), mTKIs (pazopanib, axitinib, sunitinib, cabozantinib, lenvatinib, sorafenib) and immune checkpoint inhibitors (ICIs) (atezolizumab, ipilimumab, pembrolizumab, nivolumab). Results: 368 patients (pts) were identified with ACC, 16 were locally recurrent and 216 tumors were taken from metastatic sites. 50 pts received chemo, 6 were treated with EGFRi and 15 with mTKIs. Pts who received combination EGFRi and chemo or mTKI and chemo were excluded. The median overall survival (mOS) all patients with metastatic ACC was 2.8 years (yrs). The mOS of pts with R/M ACC was 3 yrs for chemo, 2.9 yrs for EGFRi and 1.5 yrs for mTKIs. There was no significance in mOS between chemo vs mTKIs (HR 0.85, 95% CI 0.3 - 2, p = 0.72) and chemo vs EGFRi (HR = 0.88, 95% CI 0.3 - 2.5, p = 0.78). We further compared the outcomes of those treated with EGFRi (n = 8) with mTKIs (n = 19) in the entire cohort. For most pts, these agents were given as front line therapy. 25% (2/8) of patients had received treatment prior to EGFRi and 20% (4/9) prior to mTKIs (p = 1). There was no significant difference in mOS with HR 0.6 (95% CI 0.16 - 2.6), p = 0.6. We also compared the mOS of patients who received ICIs (n = 22) with those who did not (n = 346) but there was no significant difference (mOS 3.19 vs 3.17 yrs respectively, HR 0.87, 95% CI 0.47- 1.61, p = 0.65). Conclusions: There was no significant difference in the mOS between pts with R/M ACC who were treated with chemo, EGFRi or TKIs and in those who received ICIs compared to those who did not in our limited patient population. This highlights the need for predictive biomarkers for better patient selection with the goal of personalizing treatment strategies for this disease.

465 Background: UTUC is a rare genitourinary malignancy and a number of studies, limited by small sample sizes, have attempted to characterize its mutational landscape. Because immunotherapy is commonly used for this disease type, we evaluated the prevalence of microsatellite instability and characterized the mutational landscapes of UTUC in a large contemporary patient cohort. Methods: UTUC tumor samples were analyzed using next generation sequencing (NGS) (NextSeq, 592 gene panel) or whole exome sequencing (WES) (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Mismatch repair status (deficient [dMMR] or proficient [pMMR]) and microsatellite instability status (MSI-high or stable [MSS]) were detected by immunohistochemistry (IHC), fragment analysis, and NGS. Tumor mutational burden (TMB) was measured by counting all somatic mutations found per tumor (high cutoff ≥ 10 mutations per MB). PD-L1 expression was tested by IHC using PD-L1 antibody clones 22c3 (Agilent; positive cutoff CPS ≥ 10) and SP142 (Ventana; positive cutoff ≥ 5% IC). Pathogenic fusion events were detected using whole transcriptome sequencing (NovaSeq). Statistical significance was determined using the Chi-square test and adjusted for multiple comparison. Results: 538 patients with included – median (range) age 71.5 (30-89) years and 37.5% female/62.5% male. Prevalence of dMMR/MSI-H was 3.9% (21/538) and TMB-high was 22.7% (96/423). Significant molecular differences were not detected in primary vs metastatic disease or in male vs female cases. dMMR/MSI-H tumors had higher frequency of TMB-high compared to MSS tumors (100% vs. 19%, p = 0.00003). dMMR/MSI-H tumors also had a higher frequency than MSS tumors for mutations in genes involved in chromatin remodeling (ASXL 82.4%, CREBBP 60%, SMARCA4 40%, KMT2D 95%, ARIDIA 100%, KMT2A 20%, KMT2C 35.3%, NSD1 20%), DNA-damage repair (FANCG 10%, ATM 45%, ATRX 40%) and other biological pathways (RNF43 10%, PTCH1 21.4%, ERBB3 30%, CDKN2A 25%, TSC2 15%, FLNC 15%, HNF1A 20%, CIC 15%, DNMT3A 17.6%); all adjusted p < 0.05. Pathogenic fusions were detected in 3.8% (17/443) cases, with FGFR3 fusion being the most common, occurring in 2.7% (12/443) cases. PD-L1 positivity was identified in 33.2% (133/400) cases tested by 22c3 antibody and 28.4% (89/313) cases tested by SP142 antibody. No difference was seen in PD-L1 positivity between MSI-H/dMMR vs. MSS tumors. Conclusions: In the largest analysis to date, we found a 3.9% prevalence of dMMR/MSI-high rate in UTUC. All dMMR/MSI-H tumors displayed TMB-high. PD-L1 positivity was comparable between dMMR/MSI-H and MSS tumors. dMMR/MSI-H tumors had a significantly higher rate of mutations in genes involved in chromatin remodeling and DDR biological pathways. These results could inform design of targeted therapy trials in UTUC.