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Purpose: In patients with metastatic prostate cancer (mPC), ATM and BRCA2 mutations dictate differences in PARPi inhibitor response and other therapies. We interrogated the molecular features of ATM- and BRCA2-mutated mPC to explain the divergent clinical outcomes and inform future treatment decisions. Experimental design: We examined a novel set of 1,187 mPCs after excluding microsatellite-instable (MSI) tumors. We stratified these based on ATM (n = 88) or BRCA2 (n = 98) mutations. As control groups, mPCs with mutations in 12 other homologous recombination repair (HRR) genes were considered non-BRCA2/ATM HRR-deficient (HRDother, n = 193), whereas lack of any HRR mutations were considered HRR-proficient (HRP; n = 808). Gene expression analyses were performed using Limma. Real-world overall survival was determined from insurance claims data. Results: In noncastrate mPCs, only BRCA2-mutated mPCs exhibited worse clinical outcomes to AR-targeted therapies. In castrate mPCs, both ATM and BRCA2 mutations exhibited worse clinical outcomes to AR-targeted therapies. ATM-mutated mPCs had reduced TP53 mutations and harbored coamplification of 11q13 genes, including CCND1 and genes in the FGF family. BRCA2-mutated tumors showed elevated genomic loss-of-heterozygosity scores and were often tumor mutational burden high. BRCA2-mutated mPCs had upregulation of cell-cycle genes and were enriched in cell-cycle signaling programs. This was distinct from ATM-mutated tumors. Conclusions: Tumoral ATM and BRCA2 mutations are associated with differential clinical outcomes when patients are stratified by treatments, including hormonal or taxane therapies. ATM- and BRCA2-mutated tumors exhibited differences in co-occurring molecular features. These unique molecular features may inform therapeutic decisions and development of novel therapies.

Background: Disruption of circadian processes has been linked to cancer initiation, progression, metastasis, resistance, and mortality. Clock proteins are an emerging target for therapy in breast cancer. Circadian rhythms are controlled by a network of transcription/translation feedback loops primarily driven by BMAL and CLOCK and the transcriptional repressors period (PER1-3) and cryptochrome (CRY1-2). We investigated the molecular and clinical associations of clock genes in breast cancer. Methods: A total of 9563 breast tumors underwent molecular profiling (Caris Life Sciences). Analyses included next-generation sequencing of DNA (592 genes-NextSeq, WES-NovaSeq) and RNA (NovaSeq). Clock gene Score (CS) was determined using expression of clock pathway gene Z scores (positives of BMAL, CLOCK and negatives of PER1/2 and CRY1/2) and then stratified into quartiles. xCell was used to quantify immune cell infiltration in the tumor microenvironment (TME). ER/PR was tested by IHC and HER2 was tested by either IHC or CISH. Significance was determined as P values adjusted for multiple comparison (Q) of < 0.05. Real-world survival information was obtained from insurance claims data and was calculated from either tissue collection to last contact or time on treatment (TOT); comparison was done by Kaplan-Meier test. Results: TNBC had the highest median CS score, while HR+/HER2- had the lowest CS (0.96 vs 0.26 q Citation Format: Priya Jayachandran, Yasmine Baca, Joanne Xiu, Yuanzhong Pan, Phil Walker, Francesca Battaglin, Hiroyuki Arai, Moh’d Khushman, Janice Lu, Darcy Spicer, Shannon Mumenthaler, Richard Goldberg, Benjamin Weinberg, Emil Lou, Michael Hall, Arielle L. Heeke, W. Michael Korn, Steve A. Kay, Heinz-Josef Lenz, Evanthia T. Roussos Torres. Clock Genes in Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-06.

Introduction: Estrogen receptor (ER) loss occurs in about 20% of recurrent breast cancers (BC) and is associated with unresponsiveness to endocrine therapy (ET) and poor prognosis. Prior studies evaluating ER-loss included predominately patients with invasive ductal carcinoma (IDC), and therefore the impact of ER-loss in invasive lobular carcinoma (ILC) is unknown. In this retrospective analysis, using real-world data, we aimed to determine the prevalence and clinical significance of ER-loss in ILC. Methods: Advanced BC were molecularly profiled at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing of DNA (592-gene panel or whole-exome sequencing), RNA (whole transcriptome sequencing, WTS) and immunohistochemistry (IHC) of select markers. A large real-world evidence (RWE) database combining Caris’ molecular data with clinical information obtained from insurance claims data (CODEai) was interrogated and overall survival (OS) was calculated from time of tissue collection to last patient contact. A tumor was considered to have ER-loss if therapies approved only for ER-positive BC were prescribed prior to obtaining a negative ER IHC result. OS was compared using Kaplan-Meier estimates for defined patient cohorts; significance was determined as p values < 0.05. For molecular analyses, Fisher-Exact or Chi-Square tests were used to determine p values. Correction for multiple comparisons was performed using Benjamini-Hochberg to calculate q values. Results: The RWE database included 24,824 patients with advanced BC. At the time of tissue collection for molecular profiling, 6,786 advanced BC patients had been previously treated with ET (with or without mTOR or CDK4/6 inhibitors), of whom 1,338 had data available on histologic classification and ER IHC. The final analytical cohort included 263 patients with ILC and 1,075 with IDC. ER-loss was identified in 11.4% of ILC (n=30/263) and 19.6% (n=210/1075) of IDC (p=0.0017). In ILC, ER-loss was associated with significantly worse OS (HR: 1.75, 95%CI: 1.10-2.79, p=0.016) compared with no ER-loss. In the cohort of patients with ER-loss, patients with ILC had significantly worse OS compared with IDC (HR=2.03, 95% CI: 1.267-3.251, p=0.003). Further, when 1,016 tumors with ER-loss (regardless of histology) were stratified by the median OS (mOS=11mo), positive PD-L1 expression (34% vs. 22%, p=0.04, q=0.22), HER2 IHC positivity (16% vs. 7.8%, p=0.003, q=0.08) and HER2 amplification (16% vs. 4.7%, p=0.0006, q=0.04) were enriched in patients with longer mOS; while amplification of TEFB (0.38% vs. 2.6%, p=0.047, q=0.23) and MYB (0.38% vs. 2.6%, p=0.047, q=0.23) were enriched in patients with shorter mOS. WTS identified 197 differentially expressed genes, the majority of which were enriched in patients with longer mOS (q< 0.05). Conclusions: In this large real-word dataset, ER-loss likely occurred in 11.4% of ILC and was associated with worse OS compared to both patients with IDC and ER-loss and ILC without ER-loss. Our analysis had several limitations; notably, our definition of ER-loss was based on prior treatment, we could not distinguish between de novo or recurrent metastatic disease and time of tissue collection was not standardized during the course of treatment. Thus, additional studies are needed to confirm these findings. However, this study does suggest that ER-loss occurs in a subset of patients with ILC and has poor prognostic implications. Citation Format: Whitney L. Hensing, Joanne Xiu, W. Michael Korn, Stephanie L. Graff, Irene Kang, Evanthia T. Roussos Torres, Arielle L. Heeke, Andrew A. Davis, Nusayba A. Bagegni, Katherine K. Clifton, Ron Bose, Cynthia Ma, Foluso O. Ademuyiwa. Prevalence and prognosis of ER-loss in advanced invasive lobular carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-08.

The prevalence of homologous recombination–DNA damage response (HR-DDR) genetic alterations is of therapeutic interest in gastroesophageal cancers. This study is a comprehensive assessment of HR-DDR mutation prevalence across gastroesophageal adenocarcinomas and squamous cell carcinomas. Here we investigate the association of HR-DDR mutations with known predictors for immune-checkpoint inhibition [deficiency in mismatch-repair (dMMRP), tumor mutational burden (TMB), and programmed death ligand 1 (PD-L1)]. We confirmed HR-DDR mutations are present in a subset of gastroesophageal adenocarcinomas (23%) and gastroesophageal squamous cell carcinomas (20%). Biomarker expression of dMMRP (18% vs. 1%) and TMB-high with a cutoff of ≥10 mt/MB (27% vs. 9%) was significantly more prevalent in the DDR-mutated cohort compared with the non-DDR-mutated cohort. Mean combined positive score for PD-L1 in the total adenocarcinoma cohort was significantly higher in the DDR-mutated cohort compared with the non–DDR-mutated cohort (10.1 vs. 5.8). We demonstrated that alterations in ARID1A, BRCA2, PTEN, and ATM are correlated with dMMRP, TMB-high, and increased PD-L1 expression in gastroesophageal adenocarcinomas. Our findings show that a subset of gastroesophageal tumors harbor HR-DDR mutations correlated with established immune biomarkers. By better understanding the relationship between HR-DDR mutations and immune biomarkers, we may be able to develop better immunotherapy combination strategies to target these tumors.

Background HER2 alteration (mutation and/or amplification) is associated with poor survival in NSCLC and can mediate resistance to EGFR tyrosine kinase inhibitors. Methods We retrospectively analyzed de-identified molecular information from 12,946 NSCLC samples that underwent next-generation sequencing (NGS) with Caris Life Sciences. The objectives were to determine the prevalence and type of HER2 alterations with and without EGFR as a co-mutation. Insurance claims were utilized to obtain outcomes data. Results Three hundred and twenty-one patients (2.5%) had HER2 alteration: mutation in 197 patients and amplification in 134. Median age was 65 years and 62% were female. A total of 84% were adenocarcinoma. HER2 exon 20 insertion was most common (69%). A total of 1551 (12%) patients had EGFR mutations. Among samples with EGFR mutations, 24 (1.5%) had concurrent HER2 alteration (8 with HER2 mutation and 16 with amplification). Among 8 patients who had both EGFR and HER2 mutations, 3 had EGFR exon 19 deletions and exon 8 HER2 mutation (S310F). One-third of the patients (7/21) with HER2 extracellular domain (ECD) mutation had co-occurring EGFR mutations. All 7 were S310. Patients with concurrent EGFR mutation and HER2 amplification had longer median time on treatment with EGFR TKI(s) than those with EGFR mutation without HER2 amplification (HR 2.284, P =.004). Conclusion A minority of NSCLC samples with EGFR mutations had HER2 alterations. In patients with both mutations, exon 21 mutations for EGFR and exon 8 mutations for HER2 were common. It will be critical to continue to accumulate valuable clinical data for further real-world outcomes analysis.

e21000 Background: Cancer/testis antigens (CTAs) are strongly expressed in some solid tumors but minimally expressed in normal tissue, making them appealing therapeutic targets. KK-LC-1 (CXorf61) has cytoplasmic expression in some types of gastric and breast cancer and reports of expression in one-third of lung cancer tumors. Here, we characterize the molecular subtype of lung cancers expressing KK-LC-1 to plan rational clinical trials of T-cell receptor therapy (TCR-T) targeting KK-LC-1. Methods: A total of 9790 non-small cell lung cancer (NSCLC) tumors that underwent whole transcriptome sequencing (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ) were analyzed. Tumors were split into quartiles based on KK-LC-1 expression and pathological and molecular differences were investigated. PD-L1 expression was tested by IHC using 22c3 (Dako) and TPS scores were reported. Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). Statistical significance was determined using chi-square/Fisher-Exact and adjusted for multiple comparisons (adjusted p < 0.05). Results: Adenocarcinoma had significantly higher KK-LC-1 expression than squamous cell carcinoma (median 3.25 vs. 1.17 transcripts per million (TPM), p < 0.0001). There is statistically higher expression of KK-LC-1 in pan wild type (3.95 TPM) compared to tumors with EGFR mutation (1.95 TPM), ALK fusion (0.6 TPM), MET exon-14-skip mutation (1.22 TPM), RET fusion (1.42 TPM), and ROS1 fusion (1.78 TPM). Tumors within the highest quartile of KK-LC-1 expression (Q4) had a greater proportion of TMB > 10 mutations per megabase (mt/MB) (44% vs. 28%) compared to Q1. No difference was seen in PD-L1 expression. In adenocarcinoma, Q4 had a higher TMB compared to Q1 (9 mt/MB vs. 5 mt/MB). There was a higher KRAS mutation prevalence in Q3/Q4 (34.8%/35.0%) than Q1/Q2 (22%/29%) but a lower ALK fusion prevalence in Q3/Q4 (1.0%/0.5%) compared to Q1/Q2 (3.3%/2.6%). Increased KK-LC-1 expression is associated with increased M1 Macrophage abundance. Conclusions: In our population, KK-LC-1 expression was higher in adenocarcinoma. Higher levels of KK-LC-1 expression were seen in pan-wild type and KRAS mutated tumors and associated with higher TMB while lower levels of expression were seen in driver positive cancers including EGFR, ALK, MET, RET and ROS1. TCR-T therapy directed against KK-LC-1 should be explored in patients whose clinical features reflect these characteristics.

Background Nuclear protein transport is essential in guiding the traffic of important proteins and RNAs between the nucleus and cytoplasm. Export of proteins from the nucleus is mostly regulated by Exportin 1 (XPO1). In cancer, XPO1 is almost universally hyperactive and can promote the export of important tumor suppressors to the cytoplasm. Currently, there are no studies evaluating XPO1 amplifications and mutations in NSCLC and the impact on outcomes. Methods Tumor samples were analyzed using next-generation sequencing (NGS) (NextSeq, 592 Genes), immunohistochemistry (IHC), and whole transcriptome sequencing (WTS, NovaSeq) (Caris Life Sciences, Phoenix, AZ). Survival was extracted from insurance claims data and calculated from time of tissue collection to last contact using Kaplan-Meier estimate. Results Among 18,218 NSCLC tumors sequenced, 26 harbored XPO1 mutations and 24 had amplifications. XPO1 mutant tumors were more likely to have high TMB (79% vs. 52%, p = 0.007) and less likely to have high PD-L1 (32% vs. 68%, p = 0.03). KRAS co-mutations were seen in 19% (n = 5) and EGFR co-mutations were rare (n = 2). Among the 17,449 NSCLC tumors with clinical data, there were 24 XPO1 mutant. Comparison of survival between XPO1 mutant and WT showed a negative association with a hazard ratio (HR) of 1.932 (95% CI: 1.144–3.264 p = 0.012). XPO1 amplification was not associated with survival. Conclusions XPO1 pathogenic mutations were associated with a poor survival in NSCLC. Although XPO1 mutations are rare in NSCLC, further studies to assess its associations with treatment responses are warranted.

Background Squamous cell carcinoma of the anal canal (SCCA) is an uncommon malignancy with limited therapeutic options. Nivolumab and pembrolizumab show promising results in patients with SCCA. Human papillomavirus (HPV)-negative tumors are frequently TP53-mutated (TP53-MT) and often resistant to therapy. Methods We present a large molecularly-profiled cohort of SCCA, exploring the underlying biology of SCCA, differences between TP53-wild type (TP53-WT) and TP53-MT tumors, and differences between local and metastatic tumors. SCCA specimens (n=311) underwent multiplatform testing with immunohistochemistry (IHC), in situ hybridization (ISH) and next-generation sequencing (NGS). Tumor mutational burden (TMB) was calculated using only somatic nonsynonymous missense mutations. Chi-square testing was used for comparative analyses. Results The most frequently mutated genes included PIK3CA (28.1%), KMT2D (19.5%), FBXW7 (12%), TP53 (12%) and PTEN (10.8%). The expression of PD-1 was seen in 68.8% and PD-L1 in 40.5% of tumors. High TMB was present in 6.7% of specimens. HER2 IHC was positive in 0.9%, amplification by chromogenic in situ hybridization (CISH) was seen 1.3%, and mutations in ERBB2 were present in 1.8% of tumors. The latter mutation has not been previously described in SCCA. When compared with TP53-WT tumors, TP53-MT tumors had higher rates of CDKN2A, EWSR1, JAK1, FGFR1 and BRAF mutations. PD-1 and PD-L1 expression were similar, and high TMB did not correlate with PD-1 (P=0.50) or PD-L1 (P=0.52) expression. Conclusions Molecular profiling differences between TP53-MT and TP53-WT SCCA indicate different carcinogenic pathways which may influence response to therapy. Low frequency mutations in several druggable genes may provide therapeutic opportunities for patients with SCCA.

Frequent mutations of genes in the histone-lysine N-methyltransferase 2 (KMT2) family members were identified in gastric cancers (GCs). Understanding how gene mutations of KMT2 family affect cancer progression and tumor immune microenvironment may provide new treatment strategies. A total of 1245 GCs were analyzed using next-generation sequencing, whole transcriptome sequencing, immunohistochemistry (Caris Life Sciences, Phoenix, AZ). The overall mutation rate of genes in the KMT2 family was 10.6%. Compared to KMT2-wild-type GCs, genes involved in epigenetic modification, receptor tyrosine kinases/MAPK/PI3K, and DNA damage repair (DDR) pathways had higher mutation rates in KMT2-mutant GCs (p

Purpose: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. Experimental Design: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. Results: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24–6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. Conclusions: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

BACKGROUND Older age is a poor prognostic factor for glioblastoma (GBM) patients. We tested whether the intrinsic molecular landscape of the tumor may contribute to this poor prognosis. METHODS In accordance with the 2021 WHO classification scheme, we included only isocitrate dehydrogenase (IDH) wild type GBM. Based on published literature, we defined older as age > 65. RNA expression, gene amplification, tumor mutational burden (TMB) and mutational profiles were analyzed in three unique datasets: Tempus (n = 1,410), Caris (n = 1,432), and TCGA (n = 557). Comparison were made between < 65 and ³ 65 year olds using Pearson’s Chi-squared tests, Fisher’s exact tests, or Wilcoxon rank-sum where appropriate. RESULTS From our evaluable gene sets, TERT promoter mutations were more prevalent in patients ³ 65 years old (Caris 82.64 vs 77.27%, p = 0.016; Tempus 58.0 vs 49.0%, p = 0.002). There were no significant differences in PDCD1, CD274, CD3E, TNFRSF18, CD40, CD8A, TNFRSF4, CTLA4, HAVCR2, TNFSF9, CD274, or CDKN2A; PDL-1 (by IHC); dMMR/MSI-H, TMB; CDK6 amplification, EGFR amplification, EGFR, EGFRvIII, EGFR fusions, MET fusions, PTEN, TP53, or NF-1. MGMT promoter methylation (Caris data) was more common in the older group (49.73 v 34.14%, p < 0.001). TGCA data demonstrated that gene expression, TMB, and methylation did not change significantly with age. Additionally, PCOLCE2 and SLC10A4 were differentially methylated, and missense mutations, of any type, were more common in the older group (p=0.006). CONCLUSION Despite worse survival outcomes for older patients with IDHwt GBM as compared to younger counterparts, the molecular landscape is similar at the genomic, transcriptomic and epigenomic levels. The key exception is TERT promoter mutations that are more common in older GBM patients. Poorer survival is therefore not likely to be attributable solely to intratumoral factors.

INTRODUCTION Leptomeningeal disease (LMD) is a challenging complication of high grade glioma (HGG) and critical questions remain unanswered regarding clinicopathologic risk factors, molecular associations, and optimal treatment. METHODS Patients with molecularly-profiled HGG (Caris Life Sciences; Phoenix, AZ) with LMD at two institutions were included. Medical records were reviewed for clinicopathological characteristics, treatment, and outcome. Kaplan-Meier estimates of patient survival were performed on censored data using Cox’s proportional hazard model. RESULTS 43 patients (male: 33, female: 10; median age: 56 years) were identified, comprising 41 grade 4 (glioblastoma: 38; gliosarcoma: 2; H3K27M diffuse midline glioma: 1) and 2 grade 3 tumors (astrocytoma: 1; pleomorphic xanthoastrocytoma: 1). LMD diagnosed at HGG diagnosis (n=18) versus recurrence (n=22) was associated with longer post-LMD survival [pLMD-OS: 15.3m vs. 4.8m, HR: 0.07, 95% CI: 0.02-0.29, p=0.0004] but similar overall survival [mOS: 15.3m vs. 12.3m; HR: 0.82; 95% CI: 0.36-1.85; p=0.63]. Pathology-diagnosed LMD (n=15) versus MRI-diagnosed LMD (n=26) was associated with longer post-LMD survival [pLMD-OS: 15.4m vs. 5.2m, HR: 14.9, 95% CI: 0.01-0.30, p=0.0004] but similar overall survival [mOS: 17.1m vs. 12.3m; HR: 0.66; 95% CI: 0.3-1.58; p=0.38]. Post-LMD survival was significantly prolonged for supratentorial (n=28) versus infratentorial/spinal (n=4) locations regardless of the diagnostic modality [pLMD-OS: 2.6m vs. 11.3m, HR: 14.4, 95% CI: 2.73-75.7, p=0.0017], and did not significantly differ between symptomatic (n=20) and asymptomatic (n=23) patients [pLMD-OS: 4.8m vs. 11.2m, HR: 1.75, 95% CI: 0.82-3.77, p=0.15). pTERT mutation (81%), EGFR amplification (43%), and MGMT methylation (33%) were prevalent but IDH1 mutation was rare (2.8%). Comparison with a separate glioblastoma cohort (n=1400) suggested more frequent amplification of CHIC2, MDM4, and KDR, higher mutation rates of RUNX1, APC, and RAD51C, colder tumor microenvironment (TME), and lower expression of immune checkpoint-related genes. CONCLUSIONS Clinicopathological characteristics affect post-LMD survival, and cohort comparison suggests molecular and TME differences in LMD-HGG tumors.

Purpose: Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies. Experimental Design: A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings. Results: The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1–RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings. Conclusions: This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.

Purpose:Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC.Experimental Design:To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations.Results:Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation.Conclusions:These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

Purpose: FOLFOX, FOLFIRI, or FOLFOXIRI chemotherapy with bevacizumab is considered standard first-line treatment option for patients with metastatic colorectal cancer (mCRC). We developed and validated a molecular signature predictive of efficacy of oxaliplatin-based chemotherapy combined with bevacizumab in patients with mCRC. Experimental Design: A machine-learning approach was applied and tested on clinical and next-generation sequencing data from a real-world evidence (RWE) dataset and samples from the prospective TRIBE2 study resulting in identification of a molecular signature, FOLFOXai. Algorithm training considered time-to-next treatment (TTNT). Validation studies used TTNT, progression-free survival, and overall survival (OS) as the primary endpoints. Results: A 67-gene signature was cross-validated in a training cohort (N = 105) which demonstrated the ability of FOLFOXai to distinguish FOLFOX-treated patients with mCRC with increased benefit from those with decreased benefit. The signature was predictive of TTNT and OS in an independent RWE dataset of 412 patients who had received FOLFOX/bevacizumab in first line and inversely predictive of survival in RWE data from 55 patients who had received first-line FOLFIRI. Blinded analysis of TRIBE2 samples confirmed that FOLFOXai was predictive of OS in both oxaliplatin-containing arms (FOLFOX HR, 0.629; P = 0.04 and FOLFOXIRI HR, 0.483; P = 0.02). FOLFOXai was also predictive of treatment benefit from oxaliplatin-containing regimens in advanced esophageal/gastro-esophageal junction cancers, as well as pancreatic ductal adenocarcinoma. Conclusions: Application of FOLFOXai could lead to improvements of treatment outcomes for patients with mCRC and other cancers because patients predicted to have less benefit from oxaliplatin-containing regimens might benefit from alternative regimens.

Background: Invasive lobular carcinoma (ILC) is the second most common type of invasive breast cancer and accounts for 10-15% of all cases. Though ILC has distinct clinical, prognostic and molecular features, studies are limited and include few patients. ILCs show a decreased response to neoadjuvant chemotherapy and an increased resistance to endocrine therapy. Thus, there is a great need to identify alternative therapies, such as immunotherapy, that could improve overall survival. Success of immunotherapy largely depends on tumor immunogenicity which varies with histologic type. Determination of predictive and prognostic biomarkers for ILC will help determine who can benefit the most. Our study investigates canonical markers of immunogenicity - PD-L1 expression and Tumor Mutational Burden (TMB) - in patients with ILC compared to invasive ductal carcinoma (IDC). We further correlate these markers in different subtypes i.e. hormone receptor positive (HR+), HER2 positive (HER2+), and in triple negative breast cancers (TNBC). We also analyze differences in immune cell profiles constituting the tumor microenvironment (TME) and differences in genomic alterations between ILC and IDC. Methods: A retrospective data analysis was performed to identify breast cancer tumors with ILC or IDC histology profiled at Caris Life Sciences (Phoenix, AZ) that were tested for PD-L1 by SP142 assay and had whole transcriptome sequencing data available. ILC and IDC cases were further subtyped as HR+ and HER2+ and TNBC. PD-L1 expression in immune cell was assessed using Ventana PD-L1 (SP142) histochemical assay and PD-L1 expression in tumor cell was assessed by laboratory developed test using SP142 clone with staining higher than 2+ considered positive. TMB was measured by counting somatic non-synonymous missense mutations on the 592 gene panel (Nextseq) next generation sequencing (NGS) assay, and ≥ 10 mutations/megabase (mut/Mb) was considered high. Using the whole transcriptome RNA sequencing (NovaSeq) data we analyzed the difference in immune cell profiles constituting the TME using a computational RNA deconvolution approach. NGS was used to identify significant differences in genomic alterations between ILC and IDC. Results: We identified 1,359 breast cancer patients (ILC: 194 vs IDC: 1,165). Among ILC, 79% were HR +, 11% TNBC, 10% ILC: 1% vs. IDC: 5% p=0.017). PD-L1 expression in tumor cells was also lower in ILC (ILC 1% vs IDC 5%; p =0.0136). All subtypes of ILC had lower PD-L1 expression in immune and tumor cells when compared to IDC. TMB was similar and comparable in IDC and ILC (median TMB 7 mut/Mb). Assessment of TME showed a significantly increased abundance of cytotoxic lymphocytes and monocytic cells in IDC, and stromal endothelial cells in ILC. Androgen receptor (AR) expression, and mutations in CDH1 (ILC 76% vs IDC 2%) and PIK3CA (ILC 44% vs IDC 30%) were more common in ILC. CDH1 mutation was significantly higher in both TN ILC (ILC 60% vs IDC 1%; p Citation Format: Upama Giri, Joanne Xiu, Tatiana Karadimitriou, Sofi Castanon, Foluso Ademuyiwa, Paula Pohlmann, Elias Obeid, Jasgit Sachdev, Elisa Krill-Jackson, Michael Simon, Antoinette Tan, Neelima Denduluri, Lee Schwartzberg, Michael Korn, Evanthia T Roussos Torres. Molecular evaluation of immunogenicity and genomic alterations in invasive lobular breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-27.

Recurrent mutations in the gene encoding the Speckle Type POZ Protein, SPOP, the substrate-binding components of E3-ubiquitin ligase, have been identified as common alterations in prostate cancer (PCa) and endometrial cancer (EC). SPOP mutations are associated with PCa and EC growth and progression. Here, we characterized SPOP mutations in PCa and EC, and their association with molecular and immune signatures. 7,398 PCa and 19,188 EC samples were analyzed by next-generation sequencing (592, NextSeq; WES, NovaSeq), (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Real world overall survival (rwOS) was extracted from insurance claims and depicted using Kaplan-Meier survival curves for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U tests, with p-values adjusted for multiple comparisons (q Citation Format: Ludimila Cavalcante, Sachin K. Desmukh, Benedito A. Carneiro, Kartik Angara, Tyler E. Mattox, Sharon Wu, Joanne Xiu, Philip Walker, Matthew Oberley, Chadi Nabhan, Emmanuel S. Antonarakis. Opposing roles of SPOP mutations in human prostate and endometrial cancers [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 937.

Background: Although there is increased awareness leading to early detection and prevention, Hispanic American (HA) men with prostate cancer (PC) continue to have greater mortality rates compared to their Non-Hispanic White (NHW) counterparts. Moreover, HA men with PC are more likely to have more advanced disease compared to NHW men. Here, we characterized the molecular and immune differences in HA and NHW tumors and their association with PC ethnic disparity. Methods: 88 PC samples (HA=34, NHW=54) obtained from treatment-naive metastatic hormone-sensitive PC (mHSPC) were analyzed by next-generation sequencing (592, NextSeq; WES, NovaSeq), Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). AR signature and NEPC score were calculated based on the expression level of previously defined genes (Hieronymus et al. 2006, Beltran et al. 2016). Medical record was reviewed in a deidentified fashion for clinal features. Statistical significance was determined using chi-square and Mann-Whitney U (p Results: HA patients with PC had higher level of PSA (111.5 vs 52.6 ng/ml, p=0.12) and stage (T4: 41% vs 14% p=0.01, M1c: 41% vs 16% p=0.04) but no significant difference in frequency of Gleason score > 4 (79% vs 73%, p=0.61) or stage N1 (69% vs 61%, p=0.85) compared to NHW. HA PC had a significantly higher frequency of TMPRSS2-fusion (46.4% vs 20.0%, p=0.04) compared to NHW PC. By Gene Set Enrichment Analysis, HA PC had enrichment of KRAS signaling (NES: 1.44, FDR=0.02), Hedgehog signaling (NES: 1.45, FDR=0.04), NOTCH signaling (NES: 1.41, FDR=0.07), Hypoxia (NES: 1.38, FDR=0.10) and IL2-STAT5 signaling (NES: 1.35, FDR=0.10) pathways compared to NHW tumors. There was no difference in median Androgen Receptor signature (p=0.78) or Neuroendocrine Prostate Cancer (NEPC) score (p=0.79). Also, no significant difference in AR positivity by IHC (100% vs 95.7%) between HA and NHW PC tumors (p=0.51). HA PC had significantly higher expression of stem cell markers ALDH1A1 (2.1-fold), ALDH1A2 (3-fold), and PROM1 (1.6-fold) and immunoinhibitory genes PDCD1LG2 (1.68-fold) and FOXP3 (1.45-fold) compared to NHW tumors (all p Conclusion: Our data suggest that HA mHSPC is associated with higher levels of PSA, stage, TMPRSS2-fusions, stemness marker expression, immunoinhibitory gene expression, and increased M1 Macrophage and NK cell infiltration. Together, these findings suggest a crucial role of differential molecular and tumor immune microenvironment in PC ethnic disparity. A better understanding of these differences with additional research may help in designing the approaches for reducing the ethnic disparities gaps in PC patients. Citation Format: Alejandro Recio-Boiles, Sachin Kumar Deshmukh, Juan Chipollini, Ken Batai, Sharon Wu, Joanne Xiu, Alex Farrell, Milan Radovich, Elisabeth Heath, Rana McKay, Chadi Nabhan. Molecular characterization of prostate cancer between Hispanic American and Non-Hispanic Whites: Implications for cancer ethnic health disparities [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 1895.

562 Background: Recent data suggest that patients with liver metastases (mets) are resistant to immune checkpoint inhibition (CPI) independent of historical biomarkers of CPI efficacy, raising the question of whether liver mets may be associated with an immunosuppressive tumor microenvironment (TME). We investigated the immune TME of hepatic and non-hepatic mets compared to primary tumors in advanced urothelial carcinoma (UC) tissue samples. Methods: NextGen sequencing (NGS) of DNA (592-gene/whole exome) and RNA (whole transcriptome) from UC tissue samples (N=4746) was performed at Caris Life Sciences (Phoenix, AZ). Immune cell infiltration was estimated by RNA expression deconvolution (MCP-counter). PD-L1 expression (SP142: immune cell stain ≥ 5%; 22c3: CPS ≥ 10) was assessed by immunohistochemistry (IHC). Deficient mismatch repair/high microsatellite instability (dMMR/MSI-H) was tested by IHC/NGS. Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan-Meier estimates were calculated. Mann–Whitney U and X2/Fischer-Exact tests were applied where appropriate, with p-values adjusted for multiple comparisons (Benjamini-Hochberg). *P+ T and B cells (0.55* and 0.29-fold*) but increased monocytic lineage cells (1.23-fold*), while non-hepatic mets had increased CD8+ T, NK, and monocytic lineage cells (1.28*, 1.27*, 1.31-fold*) with no difference in B cells (1.05-fold). Hepatic mets had decreased expression of integrin LFA-1/ ITGAL (0.77-fold*), as well as hyaluronic acid (HA) receptor CD44 and synthase HAS2 (0.61 and 0.61-fold*), compared to primary tumors, whereas expression of these genes and LFA-1 ligand ICAM1 was increased in non-hepatic mets (1.08 to 1.30-fold*). Hepatic mets had increased expression of immunosuppressive cytokines CCL2 and CXCL2 (1.72* and 2.32-fold*) and decreased expression of pro-inflammatory cytokines CCL5 and CXCL10 (0.63* and 0.79-fold*) compared to primary tumors. PD-L1+ IHC was less frequent in hepatic mets compared to primary tumors and non-hepatic mets. TMB-High (≥10 mut/MB) and dMMR/MSI-H rates were similar across tumor sites. Hepatic mets (N=40) were associated with worse OS from the start of pembrolizumab compared to non-hepatic mets (N=177) (19.6 vs 4.4 months, HR 3.01, 95% CI 1.91-4.75, p

237 Background: CDC37-HSP90 axis is an essential chaperoning system for stabilization of kinases. CDC37 determines selectivity of client kinases recognized by HSP90. We previously showed patients with CDC37-dependent ( CDC37 high expression) colorectal cancer (CRC) derived more benefit from regorafenib and bevacizumab both of which target HSP90 client kinases or signaling pathway, but not from cetuximab which targets HSP90 non-client kinase. However, molecular characteristics and interaction with relevant signaling pathways in CDC37-dependent CRC are largely unknown. Methods: We retrospectively reviewed CRC patient samples submitted to a commercial CLIA-certified laboratory (Caris Life Sciences, Phoenix AZ). Next-generation sequencing of DNA and RNA (whole-transcriptome sequencing) and immunohistochemistry were performed. Molecular profiles between top quartile transcripts per million for CDC37 expression (Q4, CDC37-high: n = 5056) and bottom quartile (Q1, CDC37-low: n = 5056) were compared for gene mutations, microsatellite instability (MSI) status, PD-L1 expression, co-expression of HSP90 genes ( HSP90AA1 and HSP90AB1), MAPK pathway activity score (MPAS), T-cell inflamed signature, and cell infiltration in the tumor microenvironment (TME) assessed by QuantiSEQ. Gene set enrichment analysis (GSEA) was performed between CDC37-high (Q4) and CDC37-low (Q1) tumors. Results: KRAS mutations trended higher in CDC37-high tumors compared to CDC37-low tumors (49.5% vs 47.1%) while APC (76.9% vs 72.3%), TP53 (75.5% vs 71.8%) and TCF7L2 (5.0% vs 3.5%) mutations were significantly higher in CDC37-high tumors compared to CDC37-low tumors (q < 0.05). No significance in expression quartiles were observed for dMMR/MSI-H or PD-L1 expression. HSP90AA1 and HSP90AB1 expression levels were significantly higher in CDC37-high tumors (2.9-fold and 2.6-fold in median, respectively; both q< 0.05). Both MPAS and T-cell inflamed signatures were significantly higher in CDC37-high tumors ( q< 0.05). Infiltration of NK cells, Tregs, neutrophils, M1 macrophages, and B-cells were greater in CDC37-high tumors (q < 0.05). GSEA showed several signaling pathways, such as KRAS, TGF-beta, hypoxia, WNT-beta catenin, and PI3K-AKT-MTOR, were enriched in CDC37-high tumors. Conclusions: Highly CDC37-expressed (CDC37-dependent) CRC was associated with aberrant TME where abundance of immune cell infiltrate and broad activation of kinase-related signaling pathways were evident. Our results support the kinase-stabilizing function of CDC37 in CRC. Further investigations combined with survival data are warranted to address the prognostic and predictive values of CDC37 expression in targeted therapies of CRC.

185 Background: Down syndrome (DS), a genetic disorder caused by trisomy of chr 21, is associated with a considerably lower risk for solid tumors and other angiogenesis related diseases. DSCR1 belongs to a family of evolutionary conserved protein-coding genes located on chr 21 and is highly upregulated in DS patients. Its product, calcipressin-1, has been shown to reduce cancer risk by suppressing angiogenesis. We previously reported that a germline polymorphism in DSCR1 was associated with time to recurrence in resected CRC and anti-VEGF treatment outcomes in metastatic CRC. Here, we analyzed the molecular landscape of CRC according to DSCR1 expression levels. Methods: 20,237 samples from CRC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Top quartile transcripts per million (TPM) for DSCR1 expression were considered high (Q4) while bottom quartile low (Q1). Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by RNA deconvolution analysis using QuantiSEQ. Interferon-gamma and T-cell inflamed signatures were also calculated from RNA data. X2 and Fisher-Exact tests were used and statistical significance was determined as P-value adjusted for multiple comparisons ( q < 0.05). Results: DSCR1 expression was higher in primary tumors than metastases (10.5 vs 8.1 median TPM, q < 0.05). No significant difference was observed in right- versus left-sided tumors, however rectal tumors showed the highest DSCR1 expression ( P < 0.05). Overall, high DSCR1 TPM were associated with TMB-high (11.3% vs 9.3%), dMMR/MSI-H (7.9% vs 5.9%), and PD-L1 (4.7% vs 3.1%) ( q < 0.01); the association with TMB-H was not significant in pMMR/MSS. DSCR1 high was associated with lower mutation rates of APC, KRAS, TP53 and amplification of FLT1/ FLT3, while higher mutation rates of KMT2C/D, BRAF, PTEN, RNF43, and RSPO3 fusions ( q < 0.0001). Gene set enrichment analysis showed that high DSCR1 expressing tumors were enriched in alterations of several pathways including hypoxia, apoptosis, DNA repair, KRAS signaling, inflammatory response, and oxidative stress-related pathways (all P < 0.05, FDR < 0.25). B cells, macrophages, neutrophils, NK cells, Tregs, cancer associated fibroblasts and endothelial cells were more abundant in the TME of tumors with high DSCR1 while myeloid dendritic cells were lower, regardless of MMR status (all q < 0.001). DSCR1 expression was associated with a higher T-cell inflamed signature and IFN score ( q < 0.05). Conclusions: This is the first and most extensive profiling study to investigate DSCR1 expression in CRC. Our data show a strong association between tumor DSCR1 gene expression and distinct molecular features and TME cell infiltration. These findings suggest that DSCR1 holds potential as a novel therapeutic target for CRC and may be an important player in TME modulation.

592 Background: Tumor-agnostic approvals of immune checkpoint inhibitors (ICI) include deficient mismatch repair/microsatellite instability high (dMMR/MSI-H) and high tumor mutational burden (TMB) while in cancer like metastatic gastroesophageal cancers, ICI use has been predicated on PD-L1 expression. ICI are increasingly used for metastatic HCC, but without required markers. We aimed to examine the genomic landscape of HCC in context of PD-L1 expression, and to determine clinical responses to ICI in this setting. Methods: Next-generation sequencing of DNA (592 or WES) and RNA (WTS) was tested at Caris Life Sciences (Phoenix, AZ). PD-L1 expression was tested by IHC (SP142) and compared as high (2+,5%), low (1-2,1%) and negative (0). dMMR/MSI-H was tested by IHC/NGS and TMB-High was defined as ³10 mutations/MB. QuantiSEQ was used to estimate the tumor microenvironment. X2/Fisher-Exact were used and significance was determined as P-value adjusted for multiple comparison ( Q < 0.05). Real-world overall survival (rwOS) was obtained from insurance claims and calculated from tissue collection to last contact; time-on-treatment (TOT) was calculated from start to finish of ICI treatments. Results: Overall, 17.7% of HCC expressed PD-L1 by IHC; 79/1306 (6.1%) had high expression. The overall prevalence of dMMR/MSI-H was 0.2%; TMB was high in 5.1%. PD-L1 expression was not associated with MSI-H or high TMB. When comparing tumors that are PD-L1 high vs. negative vs. low, expression of several immuno-oncologic (IO) markers LAG3 (median TPM: 2.4, 0.8, 0.5), CTLA-4 (2.9, 1.2, 0.5), IDO1 (4.2, 1.3, 0.6) and others, as well as T-cell inflamed and IFNg scores all decreased significantly with PD-L1 (all q

735 Background: We have studied the role of KRAS mutations in relation to the prognosis in patients with advanced pancreatic ductal adenocarcinoma (PDAC). KRAS is a well-described oncogenic driver in PDAC, with mutations identified in over 90% of cases, typically involving codon 12. The three predominant missense variants include G12D, G12V and G12R. PDAC has the highest rate of G12R mutations compared to other malignancies, comprising 15-20% of KRAS-mutated tumors. This study presents a new finding in the progression of advanced PDAC utilizing a large clinical and genomic database to further characterize the clinical features of pathogenic KRAS variants in PDAC with a focus on G12R. Methods: PDAC samples were tested using whole transcriptome sequencing (WTS; Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ). Transcriptomic signatures including MPAS (MAPK activation score), T-cell inflamed score and tumor micro environment (TME) characterization were calculated on WTS data. Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons (q) was < 0.05 (Benjamini-Hochberg). Real-world overall survival (rwOS) was obtained from insurance claims data and calculated from tissue collection to last contact; time-on-treatment (TOT) was calculated from start to finish of specific treatments; comparison was done by Kaplan-Meier test. Results: A total of 5,555 patients with PDAC harboring either KRAS G12D (n = 2,671), G12V (n = 1,871) G12R (n = 904) or G12C (n = 109) variants were identified. The patients with KRAS G12R mutant tumors had significantly longer median real-world overall survival (mRWOS) compared to G12D (452 vs 358 days, HR 0.82, CI 0.74 – 0.9, p < 0.0001). This difference persisted regardless of treatment with FOLFIRINOX or gemcitabine plus nab-paclitaxel. There was no difference in outcomesbetween patients with KRAS G12R, G12V or G12C. PD-L1 expression was significantly lower in G12R than in G12C or G12D (13% vs 27% vs 19%,) while the prevalence of TMB-H and dMMR was comparable across isoforms. Conclusions: Patients with KRAS G12R variants has improved rwOS compared to G12D irrespective of the chemotherapy regimen administered. Immune profiling suggested that the immune contexture in G12R-driven tumors are distinct from G12D as reflected by reduced PDL1 staining, decreased levels of multiple checkpoint receptors. We aim to further explore the molecular basis for these differences with a focus on PI3K and MAPK pathways. Based on this data, survivorship studies in patients with advanced PDAC should consider reporting KRAS mutational status.

202 Background: Large studies have identified immune checkpoint inhibitors (ICI) as an effective therapy for deficient mismatch repair/microsatellite instability high (dMMR/MSI-H) colorectal cancer (CRC). However, a subset of dMMR/MSI-H CRC patients exist that do not benefit from ICI and show rapid cancer progression within the first 6 months of therapy. Genetic alterations of the host immune system, including loss of β2M and single copy loss of HLA molecules, can contribute to innate resistance to ICI. In this study, we sought to analyze the role of expression of HLA genes and β2M as determinants of innate resistance to ICI by analyzing an extensive clinico-genomic database. Methods: Next-generation sequencing (NGS) of DNA (592-gene or whole exome) and RNA (whole transcriptome) was performed on CRC patient samples (n = 24,394) submitted to a CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ). dMMR/MSI-H was assessed by IHC/NGS. PD-L1 expression was tested by IHC (SP142; positive (+): ≥2+, ≥%5). Real world overall survival and treatment data were obtained from insurance claims data. Time-To-Next-treatment (TTNT) was calculated from start of ICI monotherapy to the start of next therapy, or death. Kaplan-Meier estimates were used for comparison. A composite signature of MHC II gene expression was tested for molecular associations. Immune cell infiltration was estimated by RNA deconvolution using quanTIseq. Statistical significance was determined using Fisher’s Exact/Mann Whitney/X2 tests. Results: We identified 1549 patients with dMMR/MSI-H CRC; 242 patients of these had received pembrolizumab or nivolumab. Using TTNT as a proxy for progression on treatment, we divided the patients into two cohorts: >180 days TTNT and 180 days TTNT; good responders) and 34 patients (

225 Background: HR deficient (HRD) CRC has improved outcomes following exposure to DNA damaging agent (DDA) (irinotecan, IR; oxaliplatin, OX) compared to HRP CRC. Low expression of wild type (WT) BRCA1 mRNA is associated with prolonged OS in ovarian cancer; however, this finding has not been investigated in CRC or outside of BRCA. Here, we examine the effect of low expression of HR genes in HRP CRC on post-DDA survival. Methods: 12,860 CRC samples were analyzed by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq) at Caris Life Sciences (Phoenix, AZ). Samples were classified by RNA expression percentiles. Real world OS was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from first of OX or IR to last contact. Results: Post-IR survival was prolonged with low expression of ATM, CHEK2 and PALB2 in WT ATM, PALB2, and CHEK2 WT CRC, respectively (bottom vs. top 10%, bottom vs. top 25%; p

30 Background: The use of immune checkpoint inhibitors (ICI) in MSI-H/MMRd CRC patients has profoundly changed the treatment landscape in the metastatic setting; the most recent NICHE-2 trial showed 100% response rate in MSI-H/MMRd locally advanced CRC patients treated with neoadjuvant ICI, further highlighting the importance of identifying these patients accurately to enhance patient care. Here we report the concordance of NGS-MSI and IHC-MMR from a very large cohort of CRC tumors and study the molecular characteristics and clinical outcomes of these patients. Methods: A total of 28,105 CRC tumors were analyzed by NGS (592 genes, NextSeq or WES, NovaSeq) and IHC. MMRd was defined as complete loss of ≥1 IHC stains (MLH1, MSH2, MSH6, or PMS2) and proficient (MMRp) as any positive staining for all four proteins. MSI status was determined from over 7,000 target microsatellite loci covered by NGS. Central pathology review (CPR) of MMR IHC was done on 73 cases with discordant MMR/MSI results. Real-world overall survival was obtained from insurance claims and calculated from either tissue collection or treatment start to last contact; Kaplan-Meier estimates were calculated for molecularly defined patients. Results: For CRC tumors with NGS and IHC, 28031/28105 were concordant (99.74%), 0.09% were MMRd/MSS, and 0.2% were MMRp/MSI-H. After CPR, 23/24 (96%) of MMRd/MSS cases and 46/49 (94%) of MMRp/MSI-H were confirmed. Of the 46 post-CPR MMRp/MSI-H CRC samples, 52% had ≥1 pathogenic missense mutation in including MLH1/PMS2/MSH2/6 or MLH3, MSH3, PMS1 or POLE. Of the 23 post-CPR MMRd/MSS CRC samples, 38% had MLH1 loss, 91% PMS2 loss, no MSH2 loss and 9% MSH6 loss. When comparing clinical outcomes with concordant MSS/MMRp tumors, concordant MSI-H/MMRd patients had significantly longer OS (HR=1.131 [95% CI: 1.02-1.254], p28,000 CRC tumors that the concordance of IHC-MMR/NGS-MSI is 99.74%. Clinical outcome from a large cohort of patients show NGS is not inferior compared to IHC in identifying patients with MSI-H/MMRd. The additional lens that NGS-MSI offers is of value in identifying CRC patients who may benefit from ICI therapy. [Table: see text]

287 Background: Addition of immune checkpoint blockade to anti-HER2 therapy has improved outcomes in HER2-positive GEC. Anti-HER2 antibody-drug conjugates have shown activity in some HER2-L tumors in other tumor types. We aimed to compare the molecular profile and TIME of HER2-L and HER-H GEC. Methods: 8678 GEC (gastric, GE junction, and esophageal) adenocarcinoma and squamous cell carcinoma samples were analyzed by next-generation sequencing (NGS) of RNA (whole transcriptome, NovaSeq), DNA (592 genes, NextSeq, or whole exome sequencing, NovaSeq), and immunohistochemistry (IHC, Caris Life Sciences, Phoenix, AZ). Cohorts were stratified by IHC HER2 values of 0 (non-expressors), 1-2+ (HER2-L), or 3+ (HER2-H) and compared using X2 or Fisher-Exact. Statistical significance was determined as P-value adjusted for multiple comparisons (q < 0.05). Microenvironment cell population (MCP) counter was used to quantify immune cell infiltration. Results: Tumor were grouped into HER2 non-expressors (N = 5217), L (N = 2660), and H (N = 801). Mutations of TP53 (72% vs 92%) and amplification of MYC (4% vs 7%), CCNE1 (7% vs 12%), CCND3 (2% vs 4%), CDK6 (3% vs 6%), SMARCE1 (2% vs 25%) and RARA (3% vs 24%) were significantly lower in HER2-L compared to HER2-H (q < 0.05). ARID1A (14% vs 9%), PIK3CA (8% vs 3%), KRAS (9% vs 2%), GNAS (2% vs 0.3%), KMT2D (6% vs 1%), CDH1 (5% vs 1%), and ATM (4% vs 1%) mutations were significantly higher in HER2-L compared to HER2-H (q < 0.05). HER2-L was associated with more TMB-H (9.3% vs. 5%; q 0.05). Immuno-oncology (IO)-related gene expression inversely correlated with HER2 expression with lowest expression of PDCD1LG2, CD274, CTLA4, PDCD1, HAVCR2, CD80, IFNG, LAG3, and CD86 in HER2-H (q < 0.05). HER2-L had significantly higher median immune infiltration of B cells (fold change [FC]: 1.22), T cells (FC: 1.16), CD8+ T cells (FC: 1.56), NK cells (FC: 1.12), neutrophils (FC: 1.10), cytotoxic lymphocytes (FC: 1.36), and myeloid dendritic cells (FC: 1.34), compared to HER2-H (q < 0.05). HER2 non-expressors showed similar immune cell infiltrates compared to HER2-H. HER2-H was associated with lower T-cell inflamed scores and IFN gamma signature when compared to HER2-L and non-expressors (q < 0.05). Conclusions: To our knowledge, this study is the first and largest comparison of molecular profile and TIME in HER2-expressing GEC. We demonstrated distinct molecular and TIME profiles with higher immunogenic profiles in HER-L as compared to HER2-H. IO-related gene expression and TIME cell distribution differences in HER2-H GEC suggest that response to IO and HER2 therapy combinations is likely related to HER2-targeted treatment effect on TIME rather than baseline immunogenicity of the tumor.

224 Background: Driver mutations in the exonuclease domain (ExoD) of DNA polymerase epsilon ( POLE) have been associated with high tumor mutation burden (TMB). High TMB is an important marker for immunotherapy, and thus understanding the mechanisms that lead to high TMB is an area of active investigation. Many tumors with POLE ExoD driver mutations have additional POLE variants of uncertain significance (VUS). Some of these VUS lie within the ExoD, while most lie outside the ExoD. This study investigates the role of such POLE VUS on TMB and POLE functionality of ExoD-mutated tumors. Methods: Retrospective colorectal cancer (CRC) genomic profiles from Caris Life Sciences (n=1,870) were separated into groups based on the presence of POLE ExoD mutations and the TMB status. Groups were defined as: ‘TMB-high (TMB-H) POLE ExoD driver without additional POLE VUS’, ‘TMB-H POLE ExoD driver plus POLE VUS’, and ‘TMB-low (TMB-L) POLE variant(s) without ExoD driver’. An additional group with ‘TMB-H POLE variant(s) without ExoD driver’ was identified as potentially novel drivers for future study. The microsatellite instability status (MSI or MSS) was also considered. The level of TMB, the presence of polymerase proofreading-associated mutational signatures and POLE stability/functionality analyzed using AlphaFold2 models and Rosetta Molecular Modeling Suite, were evaluated. Results: POLE variants were identified in 4.9% of the CRCs studied (92/1870). In all, 52.2% (48/92) of those tumors were TMB-H, and 38.0% (35/92) of them contained POLE ExoD drivers. The highest median TMB (mTMB) was found in the ‘TMB-H POLE ExoD driver plus POLE VUS’ group, and this was significantly different from the ‘TMB-H POLE ExoD driver without additional POLE VUS’ and the ‘TMB-L POLE variant without ExoD driver’ group, even when MSI tumors were excluded (p

245 Background: Mesothelin (MSLN) is highly expressed in several malignancies including colorectal cancer (CRC) compared to normal tissue and is associated with tumor cell proliferation and worse patient survival. MSLN could be a potential target for antigen-specific therapy. Here, we characterized the MSLN expression in CRC and its association with molecular and immune-related markers. Methods: 15,673 CRC samples were tested by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Microsatellite-instability (MSI) was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high>10 mt/MB). Tumors with MSLN-high(H) and MSLN-low(L) expression were classified by top and bottom quartile, respectively. Real world overall survival (OS) was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, has been implicated in the pathogenesis of many cancers, with involvement in cell-cycle regulation, apoptosis, and immune response. Small-molecule GSK-3β inhibitors are currently undergoing clinical investigation. Tumor sequencing has revealed genomic alterations in GSK-3β, yet an assessment of the genomic landscape in malignancies is lacking. This study assessed >100,000 tumors from two databases to analyze GSK-3β alterations. GSK-3β expression and immune cell infiltrate data were analyzed across cancer types, and programmed death-ligand 1 (PD-L1) expression was compared between GSK-3β–mutated and wild-type tumors. GSK-3β was mutated at a rate of 1%. The majority of mutated residues were in the kinase domain, with frequent mutations occurring in a GSK-3β substrate binding pocket. Uterine endometrioid carcinoma was the most commonly mutated (4%) tumor, and copy-number variations were most commonly observed in squamous histologies. Significant differences across cancer types for GSK-3β–mutated tumors were observed for B cells (P = 0.018), monocytes (P = 0.002), dendritic cells (P = 0.005), neutrophils (P = 0.0003), and endothelial cells (P = 0.014). GSK-3β mRNA expression was highest in melanoma. The frequency of PD-L1 expression was higher among GSK-3β–mutated tumors compared with wild type in colorectal cancer (P = 0.03), endometrial cancer (P = 0.05), melanoma (P = 0.02), ovarian carcinoma (P = 0.0001), and uterine sarcoma (P = 0.002). Overall, GSK-3β molecular alterations were detected in approximately 1% of solid tumors, tumors with GSK-3β mutations displayed a microenvironment with increased infiltration of B cells, and GSK-3β mutations were associated with increased PD-L1 expression in selected histologies. These results advance the understanding of GSK-3β complex signaling network interfacing with key pathways involved in carcinogenesis and immune response.

Goblet cell carcinoid (GCC) is a distinct subtype of appendiceal neoplasm that exhibits unique clinical and pathologic features. We aimed to reveal the molecular profiles of GCC compared with other appendiceal tumors, such as adenocarcinomas and neuroendocrine tumors. A total of 495 appendiceal tumor samples (53 GCCs, 428 adenocarcinomas, and 14 neuroendocrine tumors) were tested with next-generation sequencing (NGS) on a 592-gene panel and IHC. Microsatellite instability (MSI)/mismatch repair (MMR) status was tested with a combination of NGS, IHC, and fragment analyses. Tumor mutational burden (TMB) was evaluated by NGS, and PD-L1 expression was tested by IHC (SP142). The most prevalent mutated genes within GCCs were TP53 (24.0%), ARID1A (15.4%), SMAD4 (9.4%), and KRAS (7.5%). Pathway-specific alterations were dominantly observed in cell cycle, MAPK, epigenetic, and TGFβ signaling pathways. GCCs as compared with adenocarcinomas exhibited significantly lower mutation rates in KRAS, GNAS, and APC, and significantly higher mutation rates in CDH1, CHEK2, CDC73, ERCC2, and FGFR2. GCCs as compared with neuroendocrine tumors showed significantly lower mutation rates in KRAS, APC, BRCA2, and FANCA. In GCCs, MSI high/MMR deficient, TMB high (≥17 mutations/Mb), and PD-L1 expression were seen in 0.0%, 0.0%, and 2.0% of tumors, respectively. No significant differences were observed in any immunotherapy-related markers examined when compared with adenocarcinomas and neuroendocrine tumors. In conclusion, GCCs had considerably distinct mutational profiles compared with appendiceal adenocarcinomas and neuroendocrine tumors. Understanding these molecular characteristics may be critical for the development of novel and more effective treatment strategies for GCC.

Introduction Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of non–small-cell lung cancer (NSCLC) harboring mutations in many canonical NSCLC-driver genes (eg, TP53, KRAS, MET). Protection of telomeres 1 (POT1) mutations are observed in angiosarcoma and chronic lymphocytic leukemia, but their frequency in other solid tumors, including NSCLC subtypes, has not been rigorously explored. Materials and Methods We analyzed next-generation sequencing data from 62,368 tumors, including 11,134 NSCLCs and 100 PSCs. We performed logistic regression to identify associations between POT1 mutation frequency and tumor histology across 184 tumor categories, adjusting for tumor mutational burden. We further explored co-occurring gene mutations in genes previously reported to underlie PSC tumorigenesis. Results Across 184 tumor categories, POT1 mutations were most frequent in PSC and were 14 times more common in PSC (28%) than in other tumor types (P = 1.23 × 10-31) and 6.7 times more common in PSC than other NSCLCs (P = 5.1 × 10-17). PSCs harboring KRAS mutations were significantly more likely to harbor POT1 mutations (P = 1.3 × 10-3), whereas those with TP53 mutations were less likely to harbor POT1 mutations (P = .037). One-fourth of POT1-mutated PSCs harbored a second POT1 mutation. Across all PSCs, 83% of POT1 mutations were in the OB1/OB2 (DNA-binding) domain (P = 1.5 × 10-5), an enrichment not observed in other tumor types. Conclusion We report an unanticipated association between POT1 mutation and PSC. Unlike other molecular alterations that are frequent across NSCLC subtypes, POT1 mutations are largely unique to PSC. This finding may help to develop disease-defining molecular subgroups within PSC and presents opportunities for molecularly stratified prognostication and therapy.

Background ARID1A is a key subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex which regulates dynamic repositioning of nucleosomes to repair DNA damage. Only small pilot studies have evaluated the role of ARID1A mutation in colorectal cancer (CRC). The aim of the present study was to explore the potential impact of ARID1A mutation on clinicopathological and molecular characteristics in CRC. Methods We used integrated data sets of 7978 CRC cases (one data set from a clinical laboratory improvement amendments [CLIA]-certified laboratory and three independent published data sets). The associations of ARID1A mutation with molecular characteristics including immune profile (the status of microsatellite instability [MSI], tumour mutational burden [TMB], programmed death ligand 1 [PD-L1] and estimated infiltrating immune cells), clinicopathological features and related pathways were analysed using next-generation sequencing, RNA sequencing and immunohistochemistry. Results ARID1A mutant samples had more genomically unstable tumour features (MSI-high and TMB-high) and exhibited more characteristics of a T-cell–inflamed microenvironment (PD-L1 expression and high estimated infiltrating cytotoxic T lymphocytes [CTLs]) than ARID1A wild-type samples in the discovery and validation cohorts. Even ARID1A mutant samples without MSI-high status were TMB-high, had high levels of PD-L1 expression and high estimated infiltrating CTLs. ARID1A mutations were more common with right-sided primary and earlier stage tumours. ARID1A mutant tumours mainly had co-occurring gene mutations related to chromatin modifying, DNA repair, WNT signalling and epidermal growth factor receptor inhibitor resistance pathways, and ARID1A mutations strongly regulated DNA repair pathways. Key genes for chemotherapy/radiotherapy sensitivity were suppressed in ARID1A mutant samples. Conclusions Our findings may provide novel insights to develop individualised approaches for treatment of CRC based on ARID1A mutation status.

Homologous recombination (HR)-deficient breast tumors may have genomic alterations that predict response to treatment with PARP inhibitors and other targeted therapies. Comprehensive molecular profiles of 4647 breast tumors performed at Caris Life Sciences using 592-gene NGS were reviewed to identify somatic pathogenic mutations in HR genes ARID1A, ATM, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CHEK1/2, FANCA/C/D2/E/F/G/L, KMT2D, MRE11, NBN, PALB2, RAD50/51/51B, and WRN, as well as 41 markers that may be associated with treatment response to targeted anticancer therapies. 17.9% of breast tumors had HR mutations (HR-MT, 831/4647) [ER/PR+ , HER2− 18.3%, n = 2183; TNBC 18.2%, n = 1568; ER/PR+ , HER2+ 15.6%, n = 237; ER/PR−, HER2+ 12.9%, n = 217; unknown n = 442]. Mean TMB was higher for HR-MT tumors across subtypes (9.2 mut/Mb vs 7.6 h-wild type (HR-WT), p ≤ 0.0001) and independent of microsatellite status. MSI-H/dMMR was more frequent among HR-MT tumors (2.1% HR-MT vs 0.2% HR-WT, p ≤ 0.0001), as was tumor PD-L1 overexpression (13.2% HR-MT vs 11.0% HR-WT, p = 0.08). Additional co-alterations were similar between HR-MT and HR-WT, with the exception of PIK3CA (30.3% HR-WT vs 26.4% HR-MT, p = 0.024) and AKT1 (3.7% HR-WT vs 2.1% HR-MT, p = 0.021). AR overexpression and PIK3CA mutations were more common among ER/PR+ tumors. ERBB2 mutations were seen in both HER2+ and HER2− tumors. HR-MT was common across breast cancer subtypes and co-occurred more frequently with markers of response to immunotherapy (MSI-H/dMMR, TMB) compared to HR-WT tumors. Mutations were identified in both HR-MT and HR-WT tumors that suggest other targets for treatment. Clinical trials combining HRD-targeted agents and immunotherapy are underway and could be enriched through comprehensive molecular profiling.

Microsatellite instability-high (MSI-H) and tumor mutational burden (TMB) are predictive biomarkers for immune-checkpoint inhibitors (ICIs). Still, the relationship between the underlying cause(s) of MSI and TMB in tumors remains poorly defined. We investigated associations of TMB to mismatch repair (MMR) protein expression patterns by immunohistochemistry (IHC) and MMR mutations in a diverse sample of tumors. Hypothesized differences were identified by the protein/gene affected/mutated and the tumor histology/primary site. Overall, 1057 MSI-H tumors were identified from the 32 932 tested. MSI was examined by NGS using 7000+ target microsatellite loci. TMB was calculated using only nonsynonymous missense mutations sequenced with a 592-gene panel; a subset of MSI-H tumors also had MMR IHC performed. Analyses examined TMB by MMR protein heterodimer impacted (loss of MLH1/PMS2 vs. MSH2/MSH6 expression) and gene-specific mutations. The sample was 54.6% female; mean age was 63.5 years. Among IHC tested tumors, loss of co-expression of MLH1/PMS2 was more common (n = 544/705, 77.2%) than loss of MSH2/MSH6 (n = 81/705, 11.5%; P < .0001), and was associated with lower mean TMB (MLH1/PMS2: 25.03 mut/Mb vs MSH2/MSH6 46.83 mut/Mb; P < .0001). TMB also varied by tumor histology: colorectal cancers demonstrating MLH1/PMS2 loss had higher TMBs (33.14 mut/Mb) than endometrial cancers (20.60 mut/Mb) and other tumors (25.59 mut/Mb; P < .0001). MMR gene mutations were detected in 42.0% of tumors; among these, MSH6 mutations were most common (25.7%). MSH6 mutation patterns showed variability by tumor histology and TMB. TMB varies by underlying cause(s) of MSI and tumor histology; this heterogeneity may contribute to differences in response to ICI.

Background: PI3K/AKT signaling pathway is often activated in breast cancer (BC), through mutations in PIK3CA or AKT1, and alterations in PTEN, thus promoting cell survival. PI3K and AKT inhibitors have been shown to have significant activity against tumor progression and to overcome resistance in BC. We aim to explore the prevalence of PI3K pathway alterations and co-expression with other markers in different BC subtypes. This approach could allow identification of novel drug combinations that have the potential to elicit synergistic growth inhibition and be further explored in the clinical trial setting. Methods: Molecular profiles of 4895 female and male BC cases submitted to Caris Life Sciences were reviewed to identify pathogenic or presumed pathogenic mutations in PIK3CA, AKT1, PTEN, PIK3R1 and PIK3R2 using 592-gene next generation sequencing (NGS; average read depth 500 ×). Cases with loss of PTEN by IHC were also included. Co-mutation frequency with PIK3CA-AKT1-PTEN alterations were examined, including homologous recombination deficiency (HRD) genes and DNA damage response (DDR) pathways, markers of response to immune-oncology (IO) agents and RAS signaling pathway. Cases were classified in BC subtypes according to the ASCO-CAP guidelines. Results: The median age was 58 (range: 17-90). Gender frequency and primary/ metastatic site frequency were similar across BC subtypes. 3558 (72.7%) cases had at least one alteration in the PIK3CA-AKT1-PTEN pathway: 1472 (30.1%) cases harbored a PIK3CA mutation, 174 (3.6%) harbored an AKT1 mutation, and 2682 (54.8%) had PTEN alterations (PTEN mutation in 344, 7.0% and/or PTEN loss by IHC in 2516, 51.4% of cases). The most common hotspot mutations in PIK3CA were in the kinase domain (H1047R in 567 cases, 38.5% of all PIK3CA alterations) and in the helical domain (E545K in 304 cases, 20.7% of all PIK3CA alterations). Uncommon activating PIK3CA mutations were seen in 8.4% of breast tumors. The single hotspot mutation, E17K, was the most common AKT1 mutation (n=164, 94.3% of all AKT1 mutations). 81 (1.7%) tumors harbored a PIK3R1 mutation, with 66 unique alterations identified (4 pathogenic, 55 presumed pathogenic), and 4 (0.08%) cases harbored a PIK3R2 mutation (G373R mutation). With respect to the different BC subtypes, PIK3CA was the most frequent alteration in HER2 positive BC (present in 96.2% of mutated cases). Within HER2 negative subtypes, PTEN was most frequently altered and PTEN mutation or PTEN loss by IHC was present in 79.3% of mutated cases. Triple negative breast cancer (TNBC) was the subtype with the lowest frequency of PIK3CA mutations (18.0% in TNBC vs. 37% in other subtypes). The frequency of selected co-mutations with PIK3CA-AKT1-PTEN alterations is illustrated in table 1. Notable co-alterations in this cohort include increased PD-L1 expression and high tumor mutational burden (TMB). There were no significant increases in the frequency of mutations in DDR pathway mutations (not shown) in the PIK3CA-AKT1-PTEN altered cohort. Conclusions: We observed a high prevalence of expected hotspot mutations in PIK3CA and AKT1 across BC subtypes. There was a significant increase in PD-L1 expression in tumor cells, and high TMB in PIK3CA-AKT1-PTEN mutated cohorts. A similar association was seen with RAS signaling pathways. Further development of drugs that affect the PIK3CA-AKT1-PTEN pathway in all BC subtypes and combination with drugs that target the immune system may be of interest. Table 1. Selected co-alterations based on statistical significancePathwayGene/ ProteinAll subtypes (%)HR+ HER2+ (%)HR- HER2+ (%)HR+HER2- (%)TNBC (%)MTWTMTWTMTWTMTWTMTWTHomologous recombinationBRCA13.02.71.00.60.00.80.8*1.8*6.16.0BRCA24.15.41.02.53.62.34.1*7.9*4.43.0PALB20.7*1.3*0.00.00.00.00.5*2.5*0.90.3Possible predictors of IO benefitPD-149.850.038.552.266.766.737.541.766.058.1PD-L1 (SP142)6.9*4.2*3.30.78.14.93.33.011.68.1(Tumor Cells)PD-L1 (SP142)29.226.650.018.825.027.312.916.742.140.4(Immune Cells)MSI0.70.60.00.01.20.80.80.40.71.2TMB-High (≥ 10 mut/Mb)22.9*18.8*36.0*17.2*29.825.921.2*16.6*23.621.3Chromatin remodelingARID1A12.4*18.6*19.423.87.118.917.121.55.89.6ARID20.70.80.01.90.00.70.80.60.60.9RAS-RAF-MEK-ERK HRAS0.7*0.1*0.00.00.00.00.20.11.40.3KRAS2.0*1.1*1.00.00.00.01.91.22.22.0NRAS0.20.00.00.00.00.00.20.00.30.0BRAF0.50.11.00.01.20.00.60.30.40.0OthersTP5360.9*53.1*66.360.885.586.738.4*25.6*84.786.4CDH110.3*6.1*9.1*2.5*4.82.215.1*9.6*4.9*2.3*NF16.2*2.1*9.5*0.8*2.73.65.8*1.8*6.7*2.8*RB15.5*2.6*2.21.33.63.03.9*1.7*7.85.0ERBB22.3*3.4*3.03.13.56.73.23.51.0*2.3*Notes: MT = at least 1 pathogenic mutation in PIK3CA, AKT1, or PTEN or PTEN loss by IHC. WT = no pathogenic mutations in PIK3CA, AKT1, and PTEN.*statistically significant difference between MT and WT Citation Format: Katia Khoury, Antoinette Tan, Andrew Elliott, Joanne Xiu, Zoran Gatalica, Arielle L Heeke, Claudine Isaacs, Paula R Pohlmann, Lee S Schwartzberg, Michael Simon, Michael W Korn, Sandra M Swain, Filipa Lynce. Prevalence of phosphatidylinositol-3-kinase (PI3K) pathway alterations and co-alteration of other markers 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 P4-09-04.

Background: Among women with invasive breast cancer, the clinical significance of BRCA1 and BRCA2 variants of unknown significance (VUS) remain uncertain. A closer analysis of the somatic mutational profiles of women with invasive breast cancer and BRCA1 or 2 VUS could provide a better understanding of the pathogenesis of the disease and potentially allow for the development of targeted treatment regimens. We hypothesized that breast cancers with BRCA1/2 VUS will have different somatic genomic profiling patterns and tumor mutational burden (TMB) compared with BRCA1/2 wildtype (WT). Methods: We compared the tumor molecular profiling patterns for breast cancer with somatic BRCA1/2 VUS to BRCA1/2 WT using a multiplatform profiling service (Caris Life Sciences) consisting of next generation sequencing (NGS), protein expression (immunohistochemistry [IHC]) and gene amplification (fluorescence or chromogenic in situ hybridization [FISH or CISH]). We also determined the differences in TMB between VUS and WT. We defined ER/PR/Her2 status based on IHC and/or CISH results. DNA or mutation results with >1% frequency were analyzed using Chi Square test; p Citation Format: Hadeel Assad, Isabel Hart, Kristen Purrington, Joanne Xiu, Paula R Pohlmann, Sandra Swain, Gregory Vidal, Michael Simon. Somatic genomic profiles of breast tumors differ by BRCA1 and BRCA2 variants of unknown significance status [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 P4-09-05.

BackgroundThe shelterin complex is composed of six proteins that protect and regulate telomere length, including protection of telomeres 1 (POT1). Germline POT1 mutations are associated with an autosomal dominant familial cancer syndrome presenting with diverse malignancies, including glioma, angiosarcoma, colorectal cancer and melanoma. Although somatic POT1 mutations promote telomere elongation and genome instability in chronic lymphocytic leukaemia, the contribution of POT1 mutations to development of other sporadic cancers is largely unexplored.MethodsWe performed logistic regression, adjusted for tumour mutational burden, to identify associations between POT1 mutation frequency and tumour type in 62 368 tumours undergoing next-generation sequencing.ResultsA total of 1834 tumours harboured a non-benign mutation of POT1 (2.94%), of which 128 harboured a mutation previously reported to confer familial cancer risk in the setting of germline POT1 deficiency. Angiosarcoma was 11 times more likely than other tumours to harbour a POT1 mutation (p=1.4×10−20), and 65% of POT1-mutated angiosarcoma had >1 mutations in POT1. Malignant gliomas were 1.7 times less likely to harbour a POT1 mutation (p=1.2×10−3) than other tumour types. Colorectal cancer was 1.2 times less likely to harbour a POT1 mutation (p=0.012), while melanoma showed no differences in POT1 mutation frequency versus other tumours (p=0.67).ConclusionsThese results confirm a role for shelterin dysfunction in angiosarcoma development but suggest that gliomas arising in the context of germline POT1 deficiency activate a telomere-lengthening mechanism that is uncommon in gliomagenesis.

Purpose: Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels. Experimental Design: The Cancer Genome Atlas database was used to explore molecular and immunologic features associated with CXCR4 mRNA expression in PDAC. A large real-word dataset (n = 3,647) served for validation and further exploratory analyses. Single-cell RNA analyses on a publicly available dataset and in-house multiplex immunofluorescence (mIF) experiments were performed to elaborate cellular localization of CXCR4. Results: High CXCR4 mRNA expression (CXCR4high) was associated with increased infiltration of regulatory T cells, CD8+ T cells, and macrophages, and upregulation of several immune-related genes, including immune checkpoint transcripts (e.g., TIGIT, CD274, PDCD1). Analysis of the validation cohort confirmed the CXCR4-dependent immunologic TME composition in PDAC irrespective of microsatellite instability–high/mismatch repair–deficient or tumor mutational burden. Single-cell RNA analysis and mIF revealed that CXCR4 was mainly expressed by macrophages and T-cell subsets. Clinical relevance of our finding is supported by an improved survival of CXCR4high PDAC. Conclusions: High intratumoral CXCR4 mRNA expression is linked to a T cell– and macrophage-rich PDAC phenotype with high expression of inhibitory immune checkpoints. Thus, our findings might serve as a rationale to investigate CXCR4 as a predictive biomarker in patients with PDAC undergoing immune checkpoint inhibition.

PURPOSE Tumor mutational burden (TMB) is a developing biomarker in non–small-cell lung cancer (NSCLC). Little is known regarding differences between TMB and sample location, histology, or other biomarkers. METHODS A total of 3,424 unmatched NSCLC samples, including 2,351 lung adenocarcinomas (LUADs) and 1,073 lung squamous cell carcinomas (LUSCs), underwent profiling, including next-generation sequencing of 592 cancer-related genes, programmed death ligand 1 immunohistochemistry, and TMB. The rate TMB of 10 mutations per megabase (Mb) or greater was compared between primary and metastatic LUAD and LUSC. Molecular alteration frequency was compared at a cutoff of 10 mutations/Mb. RESULTS LUAD metastases were more likely to have a TMB of 10 mutations/Mb or greater compared with primary LUADs (38% v 25%; P < .001), and this difference was most pronounced with brain metastases (61% v 35% for other metastases; P < .001). The median TMB for LUAD brain metastases was 13 mutations/Mb compared with six mutations/Mb for primary LUADs. Variability existed for other LUAD metastasis sites, with adrenal metastases most likely to meet the cutoff of 10 mutations/Mb (51%) and bone metastases least likely to meet the cutoff (19%). TMB was more commonly 10 mutations/Mb or greater for LUSC primary tumors than for LUAD primary tumors (35% v 25%, respectively; P < .001). LUSC metastases were more likely to have a TMB of 10 mutations/Mb or greater than LUSC primary tumors. Poorly differentiated disease was more likely have a TMB of 10 mutations/Mb or greater when stratified by histology and primary tumor or metastasis. Site-specific molecular differences existed at this TMB cutoff including programmed death ligand 1 positivity and STK11 and KRAS mutation rate. CONCLUSION TMB is a site-specific biomarker in NSCLC with important spatial and histologic differences. TMB is more frequently 10 mutations/Mb or greater in LUAD and LUSC metastases and highest in LUAD brain metastases. Along this TMB cutoff, clinically informative distinctions exist in other tumor profiling characteristics. Further investigation is needed to expand on these findings.

Simple Summary The prognosis of recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) remains poor. However, human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) patients live longer than those that are negative for HPV infection. In addition, some R/M HNSCC patients respond well to immune checkpoint blockade (ICB) therapies including pembrolizumab and nivolumab, but whether HPV infection is correlated with a good response to ICB is unclear. Here we attempt to understand if ICB treatment improves survival outcomes of HPV and/or surrogate marker p16−positive OPSCC and non-OP HNSCC. We also investigate other potential biomarkers and mutations that may predict improved response to ICB in both HPV−positive and -negative HNSCC patients. With better biomarkers, future treatment can be better tailored to individual patients to improve survival. Abstract Recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients overall have a poor prognosis. However, human papillomavirus (HPV)-associated R/M oropharyngeal squamous cell carcinoma (OPSCC) is associated with a better prognosis compared to HPV−negative disease. Immune checkpoint blockade (ICB) is the standard of care for R/M HNSCC. However, whether HPV and its surrogate marker, p16, portend an improved response to ICB remains controversial. We queried the Caris Life Sciences CODEai database for p16+ and p16− HNSCC patients using p16 as a surrogate for HPV. A total of 2905 HNSCC (OPSCC, n = 948) cases were identified. Of those tested for both HPV directly and p16, 32% (251/791) were p16+ and 28% (91/326) were HPV+. The most common mutation in the OPSCC cohort was TP53 (33%), followed by PIK3CA (17%) and KMT2D (10.6%). TP53 mutations were more common in p16− (49%) versus the p16+ group (10%, p < 0.0005). Real-world overall survival (rwOS) was longer in p16+ compared to p16− OPSCC patients, 33.3 vs. 19.1 months (HR = 0.597, p = 0.001), as well as non-oropharyngeal (non-OP) HNSCC patients (34 vs. 17 months, HR 0.551, p = 0.0001). There was no difference in the time on treatment (TOT) (4.2 vs. 2.8 months, HR 0.796, p = 0.221) in ICB-treated p16+ vs. p16− OPSCC groups. However, p16+ non-OP HNSCC patients treated with ICB had higher TOT compared to the p16− group (4.3 vs. 3.3 months, HR 0.632, p = 0.016), suggesting that p16 may be used as a prognostic biomarker in non-OP HNSCC, and further investigation through prospective clinical trials is warranted.

Lymph nodes (LNs) and distant metastases can arise from independent subclones of the primary tumor. Herein, we characterized the molecular landscape and the differences between LNs, distant metastases and primary colorectal cancers (CRCs). Samples were analyzed using next generation sequencing (NGS, MiSeq on 47 genes, NextSeq on 592 genes) and immunohistochemistry. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. In total, 11,871 samples were examined, comprising primaries (N = 5862), distant (N = 5605) and LNs metastases (N = 404). The most frequently mutated genes in LNs were TP53 (72%), APC (61%), KRAS (39%), ARID1A (20%), PIK3CA (12%). LNs showed a higher mean TMB (13 mut/MB) vs distant metastases (9 mut/MB, p p p = 0.017, respectively) classifications were more frequent in primaries and LNs vs distant metastases (9.5% and 8.8% vs 4.2%, p p = 0.001, respectively). TMB-high is significantly more common in LNs vs distant metastases and primaries (P APC, KRAS, PI3KCA, KDM6A, and BRIP1 (p

Loss-of-function alterations of Neurofibromin 1 (NF1) activate RAS, a driver of colorectal cancer. However, the clinical implications of NF1 alterations are largely unknown. We performed a comprehensive molecular profiling of NF1-mutant colorectal cancer using data from 8150 patients included in a dataset of commercial CLIA-certified laboratory (Caris Life Sciences). In addition, NF1 expression levels were tested for associations with clinical outcomes using data from 431 patients in the CALGB/SWOG 80405 trial. In the Caris dataset, 2.2% of patients had pathogenic or presumed pathogenic NF1 mutations. NF1-mutant tumors more frequently harbored PIK3CA (25.0% vs. 16.7%) and PTEN mutations (24.0% vs. 4.2%) than wild type tumors. Gene set enrichment analysis revealed that MAPK and PI3K pathway signatures were enriched in NF1-mutant tumors. In the CALGB/SWOG 80405 cohort, low NF1 expression was associated with poor prognosis, and high NF1 expression was associated with better efficacy of cetuximab than bevacizumab. Together, we revealed concurrent genetic alterations in the PI3K pathways in NF1-mutant tumors, suggesting the need to simultaneously block MAPK and PI3K pathways in treatment. The potential of NF1 alteration as a novel biomarker for targeted therapy was highlighted, warranting further investigations in clinical settings.

Background Gliosarcoma (GS) refers to the presence of mesenchymal differentiation (as seen using light microscopy) in the setting of glioblastoma (GB, an astrocytoma, WHO Grade 4). Although the same approach to treatment is typically adopted for GS and GB, there remains some debate as to whether GS should be considered a discrete pathological entity. Differences between these tumors have not been clearly established at the molecular level. Methods Patients with GS (n=48) or GB (n=1229) underwent molecular profiling (MP) with a pan-cancer panel of tests as part of their clinical care. The methods employed included next-generation sequencing (NGS) of DNA and RNA, copy number variation (CNV) of DNA and immunohistochemistry (IHC). The MP comprised 1153 tests in total, although results for each test were not available for every tumor profiled. We analyzed this data retrospectively in order to determine if our results were in keeping with what is known about the pathogenesis of GS by contrast with GB. We also sought novel associations between the MP and GS vs. GB which might improve our understanding of pathogenesis of GS. Results Potentially meaningful associations (pAll of the differences we observed have been associated with epithelial-to-mesenchymal transition (EMT) in other tumor types. Many of the changes we saw in GS are novel in the setting of glial tumors, including copy number amplification in LYL1 and mutations in PTPN11. Conclusions GS shows certain characteristics of EMT, by contrast with GB. Treatments targeting immuno-evasion may be of greater therapeutic value in GS relative to GB.

Purpose: The natural history and prognosis of appendiceal adenocarcinomas differ from those of adenocarcinomas arising in other large bowel sites. We aimed to compare the molecular profiles exhibited by appendiceal adenocarcinomas and colorectal cancers, or between the histopathologic subtypes of appendiceal adenocarcinoma. Experimental Design: A total of 183 samples from appendiceal adenocarcinoma [46 adenocarcinoma, not otherwise specified (NOS), 66 pseudomyxoma peritonei (PMP), 44 mucinous adenocarcinoma (MU), and 27 signet ring cell carcinoma (SR)], 994 from right-sided colorectal cancer (R-CRC), and 1,080 from left-sided CRC (L-CRC) were analyzed by next-generation sequencing (NGS) and IHC markers. Microsatellite instability (MSI) and tumor mutational burden (TMB) were tested by NGS, and programmed death ligand 1 (PD-L1) by IHC. Results: We observed high mutation rates in appendiceal adenocarcinoma samples for KRAS (55%), TP53 (40%), GNAS (31%), SMAD4 (16%), and APC (10%). Appendiceal adenocarcinoma exhibited higher mutation rates in KRAS and GNAS, and lower mutation rates in TP53, APC, and PIK3CA (6%) than colorectal cancers. PMP exhibited much higher mutation rates in KRAS (74%) and GNAS (63%), and much lower mutation rates in TP53 (23%), APC (2%), and PIK3CA (2%) than NOS. Alterations associated with immune checkpoint inhibitor response (MSI-high, TMB-high, PD-L1 expression) showed similar frequency in appendiceal adenocarcinoma compared with L-CRC, but not R-CRC, and those of NOS were higher than other subtypes of appendiceal adenocarcinoma and L-CRC. Conclusions: Molecular profiling of appendiceal adenocarcinoma revealed different molecular characteristics than noted in R-CRC and L-CRC, and molecular heterogeneity among the histopathologic subtypes of appendiceal adenocarcinoma. Our findings may be critical to developing an individualized approach to appendiceal adenocarcinoma treatment.

INTRODUCTION IDH mutations are a defining feature of lower-grade glioma and secondary glioblastoma. Approximately 95% of glioma-associated IDH mutations are in codon 132 of IDH1, but a small proportion are in IDH2. IDH mutations produce the oncometabolite 2-hydroxyglutarate, which induces global DNA hypermethylation and is associated with an immunosuppressive tumor microenvironment. IDH1 is localized in the cytosol while IDH2 is found in the mitochondrial matrix, and mutations in these genes may have differing effects on the tumor microenvironment. METHODS Formalin-fixed, paraffin-embedded tissue from 633 IDH-mutant gliomas (615 IDH1-mutant, 18 IDH2-mutant) underwent whole-exome and whole-transcriptome sequencing at Caris Life Sciences (236 grade 2/3 astrocytoma, 158 grade 2/3 oligodendroglioma, 202 IDH-mutant glioblastoma, 37 glioma, NOS). QuantiSEQ was used to infer tumor-infiltrating immune cell populations from RNAseq data, and gene-set enrichment analyses (GSEA) were performed using Wikipathway. RESULTS IDH1-mutant gliomas had higher levels of pro-inflammatory M1 macrophages (P=0.04), modestly higher levels of monocytes (P=0.08), and lower levels of neutrophils (P=0.04) – typically considered immunosuppressive – compared with IDH2-mutant gliomas. No differences were observed in levels of B cells, dendritic cells, NK cells, or T cell subsets (Treg, CD4+, CD8+). IDH2-mutant gliomas were enriched for hallmark oligodendroglioma mutations (TERT promoter, CIC, FUBP1), while IDH1-mutant gliomas were enriched for hallmark astrocytoma mutations (ATRX, TP53). However, associations with tumor-infiltrating immune cells persisted after excluding 1p/19q co-deleted oligodendroglioma from analyses. GSEA revealed upregulation of the microglial TYROBP signaling pathway, the microglial phagocytic pathway, and of Type II Interferon signaling in IDH1-mutant gliomas versus IDH2-mutant gliomas. CONCLUSIONS Although IDH2 mutations are generally thought to function similarly to IDH1 mutations, we observe differences in tumor-infiltrating immune cells across groups. IDH2-mutant gliomas appeared to have a more immunosuppressive tumor microenvironment than their IDH1-mutant counterparts. Early-phase immunotherapy trials should consider covariate-adaptive randomization approaches to equally allocate IDH2-mutant gliomas across treatment arms.

Background: While most EGFR-mutant NSCLC tumors are resistant to checkpoint inhibitors, a small subset has durable responses. Certain molecular subtypes of EGFR may be more responsive. There is a lack of clarity on the genomic and immune profiles of EGFR subtypes, and further elucidation of this may assist in optimally identifying those likely to respond to immune-based therapies. Methods: Profiles of 5,510 lung adenocarcinoma specimens were obtained utilizing next-generation sequencing of 592 genes (Caris Life Sciences) and categorized by EGFR subtype. PD-L1 expression (Ab: 22c3), tumor mutational burden (TMB), immune cell type fractions, and co-mutations in TP53 were assessed by EGFR subtype and compared to wild-type (WT) tumors. Results: Of the total cohort, 791 (14.4%) were EGFR-mutant (Table). Among EGFR-mutant tumors, exon 19 deletions were most common (49.7%), followed by L858R, (32.9%), exon 20 insertions (13.0%), L861Q (2.9%), and G719X (1.5%). PD-L1 positivity of each EGFR subtype as compared to WT did not vary, except for L858R tumors which had a significantly lower percentage of PD-L1 positive cases. Exon 19 deletion, L858R, and exon 20 insertion tumors were significantly less likely to have high PD-L1 or high TMB compared to WT. Among the EGFR subtypes, L861Q and G719X tumors had the greatest percentage with high TMB. TP53 co-mutations were frequent in EGFR tumors, especially among L861Q and G719X tumors. Exon 19 deletion and L858R tumors had significantly less CD8+ and greater CD4+ T cell fractions compared to WT. Neutrophil and M2 macrophage cell fractions were significantly higher in exon 19 deletion, L858R, and exon 20 insertion tumors compared to WT. Conclusions: Most subtypes of EGFR have profiles consistent with decreased immunogenicity; however L861Q and C719X tumors have a greater percentage with high TMB or TP53 co-mutations. Such features in uncommon EGFR subtypes may correlate with responsiveness to immune-based therapies and warrant further study. Tumor Characteristic EGFR WT(n=4719) Exon 19 del(n=393) L858R(n=260) Exon 20 ins(n=103) L861Q(n=23) G719X(n=12) PD-L1 positive (≥1%) 57.7% 51.0% 49.0% a 49.5% 47.8% 50.0% PD-L1 high (≥50%) 30.6% 19.7% a 18.8% a 14.4% a 21.7% 16.7% TMB high (≥10 mut/Mb) 35.3% 1.0% a 3.7% a 1.0% a 9.5% 18.2% TP53 co-mutation 53.9% 61.7% 63.6% a 59.2% a 69.6% a 83.3% a Median % cell fraction CD8+ 0.7 0.3 a 0.4 a 0.3 0.6 0.2 CD4+ 0.0 0.6 a 0.8 a 1.3 a 1.7 1.1 Neutrophils 5.5 8.0 a 7.3 a 7.8 a 7.4 6.4 M2 macrophages 5.5 6.4a 6.9a 6.7a 5.8 4.3 aq-value < 0.05 when compared to EGFR WT (corrected for multiple comparisons) Citation Format: J. Nicholas Bodor, Joanne Xiu, Vinicius Ernani, Supreet Kaur, Hirva Mamdani, Pavel Brodskiy, Sai-Hong I. Ou, Patrick C. Ma, Margie L. Clapper, W. Michael Korn, Joseph Treat. Genomic and immune characteristics of EGFR subtypes in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4119.

Background: Alterations in the RAS pathway have been linked to tumorigenesis, apoptosis, metabolism and angiogenesis. Mutations of KRAS in NSCLC are more frequent and better characterized, however, other family members such as HRAS remain under investigated and RAS remains a challenging therapeutic target. HRAS has been indirectly targeted with tipifarnib, a farnesyltransferase inhibitor rendering HRAS inactive in head and neck tumors. Here, we characterize the incidence, genomic landscape, and clinical context of HRAS alterations in NSCLC. Methods: A total of 29,767 NSCLC tumor samples underwent comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq), RNA (NovaSeq, whole transcriptome sequencing, WTS) and immunohistochemistry. MAPK activation was assessed using the MPAS gene expression signature. Wilcoxon, Fisher’s exact, or Dunnett’s tests were used to determined statistical significance (displayed as p value without and q value with multi-comparison correction). Overall survival was calculated from date of tissue collection to last contact from insurance claims data and used for Kaplan-Meier analysis. Comparisons were conducted between HRAS mutated tumors and the entire NSCLC general cohort (GC). Results: HRAS mutations (Hm) were detected in 128 of 29767 NSCLC samples (0.4%) and were significantly enriched in older patients (median age, 71 vs. 69 years; q Conclusions: HRAS mutations are detectable but uncommon events in NSCLC and significantly enriched in squamous histology. HRAS mutations often occur with PIK3CA co-mutations and trended towards higher activation of MAPK pathway and MSI-H frequency. This warrants further investigation on possible clinical applications of HRAS pathway inhibitors and utility of immune checkpoint inhibitors for this subset of NSCLC. Citation Format: Asaad Trabolsi, Estelamari Rodriguez, Samuel Alexander Kareff, Michael Korn, Joanne Xiu, Stephen Liu, Philip walker, Patrick Ma, Hirva Mamdani, Jorge Nieva, Hossein Borghaei, Chadi Nabhan, Misako Nagasaka, Sonam puri, Gilberto Lopes. Molecular characteristics of HRAS mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 48.

BACKGROUND FGFR signaling is central for cancer cell proliferation, migration, angiogenesis, and survival. The frequency and type of FGF/FGFR aberrations are not well-characterized across invasive breast cancer subtypes and metastatic sites of disease. In hormone receptor positive breast cancers, FGFR1 amplification correlates with early progression on endocrine therapy and promotes resistance to CDK4/6 inhibition. Our study evaluated the incidence and characterization of FGF alterations in invasive breast cancers and examined differences based on histologic subtype, molecular subtype and site. METHODS Breast cancer samples underwent molecular profiling by Caris Life Sciences. Analyses included NGS of DNA (592 Gene Panel, NextSeq, and WES, NovaSEQ), and IHC (Caris Life Sciences, Phoenix, AZ). Biomarker results were compiled from cases within the breast cohort including data from any sequencing panel, gene expression/RNA seq panel, and IHC. All results listed below were statistically significant (p < 0.05) as determined by chi-square test and Benjamini Hochberg correction. RESULTS 12058 breast cancer tumors were analyzed: 4189 from primary breast specimens (35%) and 7869 from metastatic sites (65%). 4305 (36%) were ductal and 671 (6%) were lobular histology with remaining other/unknown. 6413 (53%) were HR+HER2-, 3504 (29%) were triple negative, 403 (3%) were HR+HER2+ and 363 (3%) were HR-HER2+ with 1375 (13%) of unknown subtype. In the entire cohort, the most commonly amplified genes were FGF19 (11.49%), FGF3 (10.75%), FGF4 (9.98%), CCND1 (12.36%), and FGFR1 (9.08%). FGFR1-4 amplification was present in 11.01% of all cases. FGF19 amplification was more prevalent in lobular breast cancer compared to ductal (12.1% vs 9.1%). FGF19, FGF23, FGF3, FGF4, FGF6 amplification varied across molecular subtypes being most prevalent in HR+/HER2- (15.7%), HR+/HER2+ (12.1%), and least prevalent in HR-/HER+ tumors (3.4%). FGFR1 amplification was most common among FGFR1-4 amplifications and most prevalent in HR+HER2- (12%). Across metastatic sites, FGF ligands displayed different patterns of amplification with FGF19, FGF3 and FGF4 amplification most prevalent in liver and bone metastases. FGFR1 amplification was statistically different across metastatic sites and most prevalent in liver metastases (16%) followed by bone (10%). Patients receiving CDK4/6 inhibitors with FGFR1 amplification had shorter time on treatment than FGFR1 wild type (HR = 0.892, p = 0.03). CONCLUSION FGF alterations in invasive breast cancers vary by molecular subtype and site of disease. FGFR1, FGF19, FGF23, FGF3, FGF4, and FGF6 amplifications were statistically different across subtypes (most prevalent in HR+/HER2- and HR+/HER2+) and metastatic sites (most prevalent in liver and bone metastases). The prevalence in HR+ subtypes lend support to the role of FGF in endocrine resistance. Citation Format: Kush C. Gaur, Justin Tiu-Lim, Julie McGrath, Phil Walker, Michael Korn, Joanne Xiu, Arielle Heeke, Virginia Kaklamani, Antoinette Tan, Janice Lu, Darcy Spicer, Evanthia Roussos-Torres, Priya Jayachandran, Audrey Saghian, Amir Goldkorn, Irene Kang. Comprehensive characterization of FGF/FGFR alterations in invasive breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5793.