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560 Background: Active Wnt signaling via WNT5A through ROR1 and its partner, ROR2, or WNT5A/frizzled 2 (FZD2) is linked to processes driving tumorigenesis, disease progression and therapy resistance. The role of this pathway in the pathogenesis of urothelial carcinoma (UC) is not fully elucidated. In adult tissue, ROR1 is largely absent, which makes it ideal for targeted therapies, with several ROR1 targeting agents in early clinical development. We utilized a large dataset of molecularly characterized UC tumors to investigate the significance of Wnt5a/ ROR1, ROR2 or FZD2 transcriptional expression. Methods: NextGen Sequencing of DNA (592 genes or WES)/RNA (WTS) was performed for 4743 UC tumors submitted to Caris Life Sciences (Phoenix, AZ). PD-L1 expression (SP142; Positive (+): ³2+, ³%5) was tested by IHC. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed; Bao, 2020). WNT5a, ROR1, ROR2, and FZD2-high and -low expression were defined as ³ top and < bottom quartile of transcripts per million (TPM), respectively. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons ( q < .05). Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan Meier estimates were calculated for molecularly defined cohorts. Results: We observed similar expression of these WNT5A signaling pathway genes between upper (N=795) and lower tract UC (N=3,204): WNT5A (22.7 v. 22.2 median TMP (mTPM), q = .18), FZD2 (3.4 v 3.5, q = .93), ROR1 (2.0 v. 1.7, q = .05), and ROR2 (2.1 v 2.5, q < .01). WNT family gene expression varied significantly between primary (N=2,756) and metastatic sites (N=1,361): WTN5A (25.2 v 16.8 mTPM), FZD2 (3.2 v 4.05), ROR1 (1.7 v 2.1), and ROR2 (2.4 v 2.6) for primary vs. metastatic sites respectively ( q < .05 for all). Comparison of high- and low-expression subgroups revealed variation in the prevalence of TP53, FGFR3 and RB1 mutations, as well as PDL1+ staining and T cell-inflamed scores (Table). High gene expression for ROR2 (HR 0.66, 95% CI 0.56-0.78, p < .001) and FZD2 (HR 0.75, 95% CI 0.63-0.89, p < .001) was associated with worse OS compared to low gene expression. No significant difference in OS was observed for WNT5A (HR 0.97, 95% CI 0.82-1.15, p < .76) and ROR1 (HR 0.86, 95% CI 0.72-1.01, p < .068). Conclusions: Distinct genomic and immune landscapes for the four investigated WNT pathway components were observed and should be leveraged to identify combination therapies that complement the current pipeline of WNT pathway-targeting drugs. [Table: see text]

741 Background: While pancreatic adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths, the highly aggressive PDAC subtype of undifferentiated sarcomatoid carcinoma (USC) remains poorly characterized as it comprises only 2-3% of all PDAC histology. Previous case reports suggest that immune checkpoint inhibitors could be a promising treatment strategy for USC, but the prevalence of established predictive biomarkers of response are largely unknown in this unique subpopulation. We identified PDAC USC patient samples from a large dataset and performed comprehensive genomic profiling to determine the prevalence of biomarkers associated with response to immunotherapy. Methods: PDAC USC patient samples (N=43) underwent central pathology review to confirm this diagnosis and were compared to non-USC PDAC patient samples (N=5562). Next-generation sequencing of DNA and RNA was performed at Caris Life Sciences (Phoenix, AZ). PD-L1 expression was tested by IHC (SP142; Positive (+): ≥ 2+, ≥%5). A combination of IHC, NGS, and fragment analysis was used to assess deficient mismatch repair/microsatellite instability high (dMMR/MSI). High tumor mutational burden (TMB-High) was defined as ≥10 mutations/MB. Immune cell fractions of the tumor microenvironment were estimated by RNA deconvolution analysis using quanTIseq. Chi-square tests with Bonferoni correction for multiple comparisons were used to determine significance. Results: Among PDAC USC samples, KRAS (86% USC vs 90% non-USC, p = 0.31, q = 1), TP53 (86% vs 77%, p = 0.16, q = 1), and CDKN2A (18% vs 23%, p = 0.45, q = 1) were the most commonly mutated genes with a similar prevalence compared to non-USC histologies; however, KRAS was amplified more frequently (7% USC vs 5%) with neutrophils (85% vs 57%, q = 0.03) or M2 macrophages (52% vs 28%, p = 0.006, q = 0.06). Conclusions: This work represents the largest molecular analysis of PDAC USC samples to date. Our analysis uncovered a different prevalence of amplified KRAS and PD-L1 expression in USC as compared to other PDAC histologies amidst an immune desert lacking lymphocytes in the USC tumor microenvironment. These findings provide evidence for further investigation into combination therapy of KRAS inhibitors with immune checkpoint inhibitors to target these immune-imbalanced microenvironment landscapes.

655 Background: Demographic variables such as age and sex have long been associated with risk of cancer incidence, therapeutic response, and mortality. In NENs, specifically, studies have shown differences in overall survival based on age at diagnosis and sex. However, studies investigating the molecular determinants associated with age and gender have been lacking. To delineate the mechanisms of age, gender, and outcomes, we queried a large real-world dataset of NENs originating from the GI-tract and pancreas and characterized their molecular and immune profiles. Methods: 1935 cases (GI: n=1431, Pancreas: n=504) of NENs were analyzed using Next Generation Sequencing or Whole Exome Sequencing of DNA, and 1211/1935 cases (GI: n=917, Pancreas: n=294) underwent Whole Transcriptome Sequencing at Caris Life Sciences (Phoenix, AZ). To study the age-associated effect on the molecular and immune landscapes, we applied two age cutoffs: below 50 and 70 years of age at the time of tissue collection, respectively. Significance was determined using chi-square, Fisher-Exact or Mann-Whitney U, and p-values were adjusted for multiple comparisons (q=10 Mut/Mb) were more prevalent and KRAS mutations were less prevalent with increasing age. Interestingly, in pancreatic NENs (P-NENs), KRAS mutations were more prevalent and TMB_H were less prevalent with increasing age. Mutations specific to the sites of origin such as APC (GI) and MUTYH (pancreas) were negatively associated with age while SMAD4 (pancreas) was positively associated with age. While no differences in immune checkpoint gene (ICG) expression were associated with age, immune cell estimates of M1 macrophages (anti-tumorigenic) in GI-NENs were higher in patients above 70 and M2 macrophages (pro-tumorigenic) were higher in patients below 50 (p0.05). For the analysis with gender, mutations in p53 were more prevalent among males with GI-NEN while dMMR/MSI-H were more prevalent in females with P-NENs. No significant differences in ICG expression and immune cell estimates were associated with gender. Conclusions: Our study queries one of the largest datasets of GI and P-NENs to date. We observe distinct molecular profiles associated with age and gender in both GI- and P-NENs as well as a distinct immune profile associated with age in GI-NENs. While these results are only hypothesis generating, they are suggestive of how patient demographics may be utilized for rational therapy design. [Table: see text]

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.

649 Background: The mTOR inhibitor, Everolimus (EVE), is FDA-approved for the treatment of advanced PNENs on the basis of delay of progression. The RADIANT-3 trial showed an increase in PFS from 4.6 to 11 months compared to placebo with an ORR of only 5%. Prior studies suggest that targeting the aberrant expression of mTOR regulators p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) in PNENs sensitizes these tumors to EVE. To further qualify these observations, we queried a large real-world dataset of PNENs, characterizing the molecular and immune landscapes, as well as the clinical outcomes associated with aberrant PAK4 and NAMPT expression. Methods: 294 cases of PNENs were analyzed using Next Generation Sequencing (NextSeq) and Whole Exome and Whole Transcriptome Sequencing (NovaSeq) at Caris Life Sciences (Phoenix, AZ). For our analyses, we stratified our study cohort into four groups based on the median expression of PAK4 and NAMPT: PAK4-low/NAMPT-low, PAK4-low/NAMPT-high, PAK4-high/NAMPT-low and PAK4-high/NAMPT-high. Significance was determined using chi-square, Fisher-Exact or Mann-Whitney U, and p-values were adjusted for multiple comparisons (q < 0.05). Results: High prevalence of mutations in PTEN (10.71% vs 1.18%; p < 0.05, q > 0.05), a tumor suppressor of the mTOR pathway and high expression of genes activated in response to mTOR activation such as SLC2A1 (3.07-fold), PFKP (3.32-fold), SCD (2.70-fold), MVK (2.92-fold) and G6PD (2.58-fold) were observed in PAK4-high/NAMPT-high compared to the PAK4-low/NAMPT-low tumors (all q < 0.05). A congruent enrichment of PI3K/AKT/mTOR and glycolysis pathways by single-sample gene set enrichment analysis was observed in these tumors (all q < 0.05). When querying the immune landscape, we observed enrichment in inflammatory response, IL6/JAK/STAT3, IL2/STAT5 signaling pathways and immune checkpoint genes such as PDCD1 (5.14-fold), CD274 (2.84-fold), PDCD1LG2 (2.44-fold), CD80 (3.00-fold), CD86 (2.82-fold), IDO1 (1.92-fold), LAG3 (3.09-fold), HAVCR2 (2.66-fold) and CTLA4 (4.49-fold) in the PAK4-high/NAMPT-high tumors (all q < 0.05). Immune cell infiltration estimates revealed an increase in Neutrophils, NK cells and Tregs in the PAK4-high/NAMPT-high tumors (p < 0.05, q > 0.05). Conclusions: Our study demonstrates that PAK4-high/NAMPT-high PNENs are associated with distinct molecular and immune profiles. While the dual blockade of PAK4 and NAMPT has been reported to enhance the efficacy of EVE in PNENs, whether such a blockade would enhance the efficacy of immunotherapeutics warrants further investigation.

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

3071 Background: Targeted therapy of NENs based on the presence of SSTRs fills a unique niche in tumor biology and clinical treatment of patients with solid tumors. SSTRs have multiple isoforms and are collectively expressed in the majority of NENs. However, subtypes are still not routinely tested and thus not assessed for clinical decision-making, especially for patients meriting consideration of targeted radionucleotide therapy. Clarifying the landscape of SSTR subtypes using molecular techniques more sensitive than immunohistochemistry (IHC)-the standard of testing, and identifying associated genomic biomarkers that differ between them, will pave the way for more sophisticated decision-making in the future. Additionally, leveraging transcriptomics to better assess mitotic markers such as Ki-67 to assess tumor grade, would increase diagnostic accuracy. Here we provide initial validation across a spectrum of NENs. Methods:1595 NENs were analyzed using Next Generation Sequencing (592 gene panel, NextSeq), Whole Exome and Transcriptome Sequencing (NovaSeq), and IHC at Caris Life Sciences (Phoenix, AZ). Significance was determined using chi-square, Fisher-Exact or Mann-Whitney U and p-adjusted for multiple comparisons (qs= 0.63) and SSTRs 1 and 2 in LG-NENs (rs= 0.64) were positively correlated. Overall, the prevalence of altered TP53, RB1, PIK3CA, APC, KRAS was higher and MEN1 was lower in HG-vs LG-NENs (q

5588 Background: Immunohistochemistry (IHC) for HER2 in E-EMCA is not standard of care. We aimed to establish the correlation of HER2 transcript to IHC expression in the much more frequently tested uterine serous carcinoma (USC). We applied the threshold calculated in USC to E-EMCA and compared molecular and immune profiles among HER2+ and HER2- E-EMCA tumors, which may affect response to targeted therapy. Methods: 1462 E-EMCA tumors were analyzed using next-generation sequencing (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 was tested by IHC (SP142, >1%). Microsatellite instability (MSI) was tested by FA, IHC and NGS. TMB was measured by totaling somatic mutations per tumor (TMB-H: >10 mutations/MB). LOH cut-off was > 16%. HER2+ cut-off by WTS was determined by Receiver Operator Characteristic (ROC) analysis in USC tumors by comparing to HER2 IHC/CISH results using 2018 Breast Cancer ASCO/CAP Guidelines. Immune cell infiltrates were calculated by Quantiseq. Real world overall survival (OS) was extracted from insurance claims data and calculated using Kaplan-Meier survival curves for molecularly defined cohorts. Significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons (q-value < 0.05), p < 0.05 but q > 0.05 was considered a trend. Results: We determined a cut-off of > 62.99 TPM for HER2+ with a sensitivity of 81.5%, specificity of 87.6% and AUC of 0.92 in Uterine Serous. When this cut-off is applied to E-EMCA, 76 of 1462 (5.2%) E-EMCA tumors were HER2+. HER2+ tumors had fewer mutations (mt) in PI3KR1, PTEN and CTNNB1 but higher mts in TP53 and more frequent LOH (q < 0.05). HER2+ tumors had a trend towards decreased MSI-H status (22.4% vs 39.1%; p = 0.003, q = 0.058) and TMB-H (25.4% vs 41.5%; p = 0.007, q = 0.084). MSS HER2+ E-EMCA had a similar mutational profile compared to all HER2+ tumors; MSI-H HER2+ E-EMCA had a trend towards higher DDR pathway gene mts compared to MSI-H HER2- EMCA tumors. HER2+ tumors had increased Dendritic cell (3.84% vs 2.97%) but decreased Neutrophil (2.66% vs 5.20%) and T-reg (1.38% vs 2.07%) infiltration (q < 0.01). HER2+ tumors had higher immune checkpoint gene expression of CD80, HAVCR2 and PDCD1LG2 (q < 0.01), and increased T-cell inflamed and MAPK activation score (q < 0.01). MSS HER2+ E-EMCA tumors had a similar immune profile when compared to all HER2+ tumors; MSI-H HER2+ E-EMCA tumors had increased Treg infiltration and MAPK activation score. Median OS was significantly worse for HER2+ pts compared to HER2- (64.3 vs. 23.6 months, HR: 1.93(1.32-2.80), p < 0.001). Conclusions: Using a WTS cutoff from USC, we found 5% of E-EMCA are HER2+ and showed distinct molecular and immune profile compared to HER2- tumors. HER2+ confers a worse OS compared to HER2- tumors. Furthermore, HER2+ tumors demonstrate an immune hot phenotype suggesting that immunotherapy may be a potential therapeutic option.

9119 Background: MET exon 14 skipping mutations (METex14) join a growing list of viable therapeutic targets in advanced NSCLC. Several unique features distinguish METex14 from other established targets. METex14 has been characterized as a tumor-agnostic genomic alteration, though most frequently reported in lung adenocarcinoma. However, METex14 represents a family of mutations (mt), not a single alteration, and there is notable heterogeneity in histology. The degree and significance of heterogeneity within METex14 have not been well characterized. Methods: NSCLC tissue samples were analyzed with DNA-based next-generation sequencing (NGS; 592 genes, NextSeq) or whole-exome sequencing (NovaSeq), RNA-based whole transcriptome sequencing (WTS, NovaSeq), and immunohistochemistry (IHC) at Caris Life Sciences (Phoenix, AZ). PD-L1 expression utilized the 22C3 clone (Dako); TMB-high was defined as ≥ 10 mt/Mb. Wilcoxon or Fisher’s exact were used to determine statistical significance (p without and q with multi comparison correction). Immune cell fraction (QuanTiseq) and pathway analysis (ssGSEA) were informed by WTS analysis. Results: A total of 440 METex14 cases were identified: 49 (11.1%) with squamous histology, 381 (86.6%) with non-squamous histology, and 10 (0.2%) with adenosquamous histology. A total of 147 distinct METex14 mutations were detected. The most common METex14 mutations were D1028H (8.4%), D1028N (7.0%), c.3082+2T > C (5.7%), D1028Y (5.2%), and c.3082+1G > A (4.5%). Co-mutations in TP53 were common (43.9%) but varied by specific METex14 mutation; TP53 co-mutations were observed in 53.9% of c.3082+3A > T but only 21.1% of c.3082+1G > T. Among all METex14 cases, 8.6% were TMB-high, but this varied by specific mutation with a median TMB of 2 mt/Mb in MET c.3082+2T > A and a median of 7 mt/Mb in MET c.3082+1G > C (q < 0.05). PD-L1 expression ≥ 1% was present in 82.2% of METex14 samples but also varied by specific METex14 mutation with a median PD-L1 tumor proportion score (TPS) of 97.5% in MET c.3082+1G > C and a median TPS of 0% in MET c.3082+3A > G (q < 0.05). Co-mutations varied by histology: in squamous METex14, 90.4% had TP53 mt (p < 0.001), 17.9% had KMT2D mt (p < 0.05), and 10.7% had PIK3CA mt (p < 0.05), while in non-squamous METex14, 60.7% had TP53 mt, 2.7% had KMT2D mt, and 4.3% had PIK3CA mt. Survival was numerically shorter in squamous METex14 NSCLC compared to non-squamous (HR 1.22, p = 0.47, mOS 336 vs.1106 days). Conclusions: There is significant heterogeneity within METex14 NSCLC with differences in co-mutations, TMB, and PD-L1 expression noted among different METex14 mutations. While METex14 is detected in both squamous and non-squamous NSCLC, there are differences in enrichment of oncogenic pathways. The clinical impact of these differences warrants further investigation.

5590 Background: Uterine carcinosarcomas (UCS) are rare, aggressive tumors accounting for 5% of all uterine cancers. Recurrence rates are high and 5-year survival is only 18-39%. HER2 is an emerging prognostic and therapeutic target in uterine cancer including UCS. Testing algorithms and platforms in breast and gastric cancers are well studied and validated, but optimal HER2 testing in uterine cancer is not yet established. We aimed to determine HER2 prevalence in UCS and the concordance of chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and next generation sequencing (NGS) platforms to aid in the development of UCS specific testing guidelines. We also evaluated the rate of downstream mutations and immune biomarkers that may affect response to HER2 directed therapy. Methods: Eight hundred and seventy-five UCS tumor samples (primary 81.4%; metastatic 17.1%; unknown 1.5%) were analyzed using NGS (592, NextSeq; WES, NovaSeq, Caris Life Sciences, Phoenix, AZ). A subset of tumors with HER2 positivity were tested with IHC (4B5, Ventana) and/or CISH (INFORM DUAL HER2 ISH Assay, Ventana) based on 2018 ASCO/CAP HER2 Breast Cancer guidelines. Amplification of ERBB2/HER2 by NGS used a copy number cut-off >6. PD-L1 expression was analyzed by IHC (SP142, positive cut-off ≥1%, Ventana). Tumor mutational burden (TMB) was measured by counting all somatic mutations found per tumor (TMB high cut-off > 10 mutations per Mb). Microsatellite instability (MSI) was tested by fragment analysis (FA), IHC and NGS. Results: Rates of HER2 positivity were 3.4% (28/820) by NGS. Among the IHC/CISH-tested cohort, 7.2% was positive (10/139) by IHC, and 19.5% (27/133) by CISH. In the 105 samples tested with both IHC and CISH, the concordance was 100%. Specifically, 9/105 patients (8.6%) were IHC+/CISH+ and 96 patients (91.4%) were IHC-/CISH-. The concordance between CISH and NGS (N = 127) was 90.6% (sensitivity 100% and specificity 89.3%). Common gene alterations in CISH HER2+ UCS tumors that may implicate resistance to HER2 targeted therapy included mutations in TP53 (100%), FBXW7 (22.2%), PIK3CA (33.3%), PIK3R1 (18.5%), PTEN (3.7%) and KRAS (3.7%) and gene amplification of KRAS (11.1%). CISH+ HER2+ UCS tumors had low immunotherapy biomarker prevalence (0% MSI-H, 0% TMB high, 7.7% PD-L1+). Conclusions: Increased HER2 positivity was detected via CISH testing compared to IHC and NGS, which may reflect the heterogeneity of HER2 amplification due to mixed histology between the sarcoma and carcinoma portion of the tumor. High concordance rates were observed between CISH and IHC. These testing platforms need to be validated by response to HER2 targeted therapies in order to develop UCS specific testing guidelines.

2633 Background: Older patients (pts) with cancer are underrepresented in registrational clinical trials for immune checkpoint inhibitor (ICI) therapies. There may be relevant differences in the makeup of the tumor microenvironment (TME) and in genomic signatures of cancer in older pts. This analysis explores differences in the genomic makeup of common cancers and their TME in pts ≥ 80 years (yr) of age, compared to younger pts. Methods: Next-generation sequencing of DNA (592 gene panel, NextSeq or whole-exome sequencing, NovaSeq) and RNA (whole transcriptome sequencing, NovaSeq) was performed for non-small cell lung carcinoma (NSCLC; n = 19,891), melanoma (MEL n = 2,899), and renal cell carcinoma (RCC; n = 1,333) pt samples submitted to a CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was assessed by immunohistochemistry (IHC), and high tumor mutational burden (TMB-H) was defined as ≥10 mut/Mb. Pts were stratified into age subgroups of ≥80 and < 80 yr for comparison of DNA damage response (DDR) gene alterations, gene expression profiling, and TME analysis (MCP-counter; Becht, 2016). P-values were adjusted for multiple hypotheses testing (Benjamini-Hochberg) unless noted as exploratory. Results: Pts ≥80 yr accounted for 16.0%, 19.9% and 5.3% of NSCLC, MEL and RCC pts, respectively. Compared to pts < 80 yr, NSCLC and MEL pts ≥80 yr had similar DDR gene mutation rates, while BRCA1 mutations were more common in MEL pts ≥80 yr (2.1 vs 0.8%; exploratory- p < 0.05). NSCLC ≥80 yr TMEs had increased abundance of fibroblasts (1.09-fold, p < 0.01), dendritic cells (1.07-fold, p < 0.01) and macrophages (1.04-fold, p < 0.01), and MEL≥80 yr TMEs had fewer infiltrating T-lymphocytes (0.87-fold, p = 0.02). Increased expression of immune checkpoint (IC) genes PDCDL1G2 (PD-L2; 1.11-fold), HAVCR2 (TIM-3; 1.11-fold ), and CD80/86 (1.07/1.08-fold, p < 0.05) was seen in NSCLC pts ≥80 yr, while IL-6 expression was decreased (0.88-fold; p < 0.05). The largest change in IC gene expression was for IL-6 (1.24-fold, p = 0.78) in MEL, and GZMB (0.56-fold ; p = 0.17) in RCC ≥80 yr. TMB-H was less common in NSCLC (29.7 vs 36.5%, p < 0.001) and more common in MEL pts ≥80 yr (65.7 vs 49.0%, p < 0.01), and PD-L1 (IHC-SP142, ≥2+|5%) expression was less frequent in RCC pts ≥80 yr (9.1 vs 19.4%, exploratory p < 0.05). Profiling of glutamine and glucose metabolism-related genes revealed increased SLC38A5 (1.17-fold; p < 0.0001) and decreased G6PC (0.65-fold, p < 0.01) expression in NSCLC ≥80 yr. While not statistically significant, MEL and RCC pts ≥80 yr had opposite trends for SLC38A5 and G6PC expression. Conclusions: Our analysis provides new insights to immune landscape of NSCLC, MEL, and RCC pts ≥80 yr. Differential gene expression and TME composition changes in this population suggest unique, cancer-specific therapeutic opportunities, and a potential to explore biomarkers of response to ICIs.

3589 Background: In patients with colorectal cancers (CRCs), prior studies have reported that various TP53 mutations have prognostic significance. The anatomic location of the primary CRC and the TP53 mutation subtype influence patient survival. In this study, we explored the prognostic significance of TP53 mutations (mTP53) classified as gain-of-function (GOF) or non-GOF in patients with right-sided (RCC) and left-sided CRCs (LCC). Methods: CRC specimens (6,248 RCCs and 14,215 LCCs) were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing (NGS) of DNA (592-gene panel or whole- exome sequencing). R175H, R248W, R248Q, R249S, R273H, R273L, and R282W were defined as GOF mTP53 and all other mTP53 were defined as non-GOF mTP53. MSI-H/dMMR status was determined by immunohistochemistry (IHC) of MMR proteins and/or NGS. Real-world median overall survival (mOS) was obtained from insurance claims data and calculated from tissue collection to last contact using Kaplan-Meier estimates. Results: GOF mTP53 and non-GOF mTP53 were identified in 15% and 39% respectively, in RCC and 17% and 46% respectively, in LCC. In RCC, the mOS for patients with wild-type TP53 ( wtTP53) vs. GOF mTP53 was 34 months (m) vs. 23m (p < 0.00001), and the mOS for patients with wtTP53 vs. non-GOF mTP53 was 34m vs. 27m (p < 0.001). In LCC, the mOS for patients with wtTP53 vs. GOF mTP53 was 35m vs. 33m (p = 0.056), and the mOS for patients with wtTP53 vs. non-GOF mTP53 was 35m vs. 35m (p = 0.32). The mOS for patients with non-GOF mTP53 vs. GOF mTP53 in RCC and LCC was 28m vs. 24m (p = 0.096), and 35m vs. 34m (p = 0.175), respectively. The prognostic value of GOF mTP53 and non-GOF mTP53 was further explored in relation to MSI-H/dMMR, RAS, BRAF, and PIK3CA mutation status. The worse prognosis associated with mTP53 in RCC was seen in all comparisons, except in GOF mTP53/MSI-H/dMMR, and non-GOF mTP53/ wtKRAS subgroups. In patients with LCC, worse prognosis associated with GOF mTP53 and non-GOF mTP53 was only noticeable in KRAS and PIK3CA mutant subgroups. Conclusions: This is the largest study to explore TP53 mutations and their prognostic significance in patients with RCC and LCC. The prevalence of GOF mTP53 and non-GOF mTP53 was higher in LCC compared to RCC. However, both GOF mTP53 and non-GOF mTP53 were associated with worse mOS for patients with RCC, but not LCC. Our study validates the sidedness-dependent prognostic significance of TP53 mutations. It also shows that the worse prognosis of mTP53 is independent of the approach of collectively classifying TP53 mutations into GOF vs non-GOF. Given the sheer extent and diversity of TP53 mutations, a more nuanced approach towards re-classification of GOF mTP53 is warranted. Detailed information on p53 mutations will be crucial for the interpretation of future clinical trials and for the design of novel therapeutic strategies.

e17622 Background: 50% of endometrial atypical hyperplasia (AH) cases on endometrial sampling have concurrent endometrial cancer (EC) on hysterectomy. Little data exists regarding molecular signatures that can predict the presence of concurrent endometrial cancer in patients with AH on biopsy. Therefore, we set to characterize molecular landscapes of endometrial samples diagnosed with AH with EC on the final hysterectomy specimen, using matched controls. Methods: Karmanos Cancer Institute pathology database was searched for patients with AH on endometrial curettage or biopsy who had a hysterectomy within 6 months. Only samples with adequate tissue for next generation sequencing were included. In total, 59 tissue samples from 34 patients were included: there were 15 AH endometrial biopsy samples, 18 AH final hysterectomy samples (13 matched pairs), 13 EC endometrial biopsies and 13 samples that were EC on final hysterectomy samples (12 matched pairs) that were analyzed using next-generation sequencing (WES) and/or whole transcriptome sequencing (WTS) (NovaSeq). TMB was measured by counting all somatic mutations found per tumor (TMB-high: >10 mutations per MB). Immune cell infiltrates were calculated by Quantiseq. Significance was determined using Fisher exact, Chi-square and Mann-Whitney U test and adjusted for multiple comparisons: p < 0.05 but q > 0.05 was considered a trend. Results: Clinical analysis demonstrated 15/34 patients with endometrial AH on initial sampling and EC on the final hysterectomy. 2 out of 15 patients were > stage II at time of surgery. Molecularly, fewer PTEN mutations were found between AH (2/8) and EC (14/17) on final hysterectomy (25% vs 82.4%, p = 0.01). Samples of EC origin were MSI-H by NGS-MSI (3/18) and had no mutations in PPP2R1A (0/16). Comparing hysterectomy samples, EC trended toward increased CTLA4 (FC: 6.97-fold) expression and immune cell infiltration of Macrophage M1 (+1.19%), NK cells (+2.13%), CD8+ T Cells (+1.27%), regulatory T cells (+2.17%) and Dendritic cells (+2.87%) compared to AH samples (all p < 0.05). Similarly, when comparing endometrial biopsies, EC samples trended toward increased expression of CTLA4 (12.6-fold), HAVCR2/TIM3 (FC: 2.59-fold) and IFNG (FC: 17.8-fold) immune checkpoint genes, as well as increased immune cell infiltration of Neutrophils (+11.7%), CD8+ T cells (+2.39%) and regulatory T cells (+2.43%) compared to AH samples (all p < 0.05). Conclusions: There are molecular and tumor microenvironment differences seen between AH endometrial biopsies that have concurrent EC compared to those that do not on the final hysterectomy specimens. These differences may lead to advances in identifying appropriate patients for fertility sparing treatments, versus those that can be managed surgically by a benign gynecologist or a gynecologic oncologist. Analyses with larger sample sizes are needed and are ongoing.

3120 Background: The cell-surface transferrin receptor TFR1 imports iron-bound transferrin into cells via clathrin-mediated endocytosis. Tumors require constitutive iron import to drive proliferation, and several studies establish TFR1 as a target able to facilitate intracellular delivery of cytotoxic therapeutic molecules. Our own work previously revealed association between high expression of TFRC, the gene encoding TFR1, and high risk for poor outcome in diffuse large B-cell lymphoma (DLBCL). We showed therapetuic targeting of TFR1 in DLBCL results in significant anti-tumor benefit. Systematic analysis of TFRC expression as a prognostic marker across tumor types, however, has not been investigated. Methods: Tissue 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. Overall survival (OS) was calculated from date of tissue collection to last contact from insurance claims data and employed Kaplan-Meier analysis by Wilcoxon statistics, with p < 0.05 defined as significant. Results: Amongst 47 cancer types included, colorectal cancer (CRC) displayed the highest level of TFRC mRNA, followed by gastric cancer. In an all-tumor cohort (n = 93248), patients with higher TFRC expression (cutoff = median) had significantly worse OS (HR = 1.348, 95% CI [1.317-1.38], p < 0.00001). This was statistically significant in 23 individual tumor types. Drilling down further, TFRC adverse prognostic value was mainly driven by cohorts with larger number of samples in the database, including non-small cell lung cancer (n = 17309), CRC (n = 12860), breast cancer (n = 8632), ovarian carcinoma (n = 7998), uterine neoplasms (n = 6097), prostate adenocarcinoma (n = 3411), glioblastoma (n = 2821), gastric cancer (n = 1579), and others. Surprisingly, TFRC overexpression correlated with improved outcome in vulvar squamous cell carcinoma (VSCC, n = 297). TFRC was found to be most prognostic in prostate adenocarcinoma with median OS 1139 days in pts with high vs 3230 days in pts with low TFRC (HR = 2.556, 95% CI [2.213-2.951], p < 0.00001). Conclusions: Our study is the first to combine modern molecular profiling with a large cohort of clinical tissue samples to reveal a prognostic role for TFRC expression in a variety of solid tumor types. We found TFRC overexpression to be prognostic in a large proportion of histologies, though surprisingly association with improved OS in VSCC. Highest expression occured in CRC and gastric cancer, diseases with needs for new therapies. A number of TFR1-targeting therapeutics are currently at various stages of development, and warrant further investigation in disease cohorts identified from our study.

11525 Background: Osteosarcoma (OS) incidence is characterized by a bimodal age distribution, with peaks in early adolescence and in adults > 65 years of age. In contrast to adolescents, OS in adults is frequently considered as a secondary neoplasm (i.e., transformation of Paget´s disease of the bone, radiation induced). Yet, the literature is scarce regarding the impact of age on the molecular landscape of OS. Herein, we sought to explore the association between age and the genomic profile as well as the tumor immune microenvironment (TME) in a large cohort of OS patients. Methods: 208 specimens were centrally analysed at the Caris Life Sciences laboratory with DNA seq (NextSeq, 592 gene panel or NovaSeq, whole-exome sequencing), RNA seq (Archer fusion panel or whole-transcriptome sequencing) and immunohistochemistry (IHC). RNA deconvolution and differential expression analyses were performed using the Microenvironment Cell Populations counter method for quantification of immune cell populations and gene expression profiling. The cohort was stratified into three distinct age groups (< 25 years [n = 83], 25-45 years [n = 58], > 45 years [67]). Results: Overall, the most frequently detected mutations were in TP53 (37%), RB1 (13%), ATRX (9%), TERT (6%), PTEN (5%), PIK3CA (4%) and KMT2D (3%). Copy number alterations were most frequently detected in CDK4 (12%), LRIG3 (11%), FLCN (11%), MDM2 (9%), CCND3 (9%), VEGFA (8%), TFEB (8%). Interestingly, age-based stratification revealed an increased frequency of FLCN (19.7 vs 4.7%, p < 0.01), CCND3 (13.9 vs 3.1%, p < 0.05), and HSP90AB1 (11.3 vs 0.0%, p < 0.01), alterations in patients < 25 years compared to > 45 years. TME analysis revealed that patients > 45 years have decreased B-cell abundance compared to patients < 25 years (2.9-fold decrease, p < 0.05) and 25-45 years (4.8-fold decrease, p < 0.05). Although not statistically significant, median transcriptional expression of PD-L1 was numerically increased in patients > 45 years (1.8-fold compared to 25-45 years, p = 0.17; 2.0-fold compared to < 25 years, p = 0.27), which was consistent with increasing rates of IHC PD-L1 expression with age (5.3%, 9.4%, and 17.5%, respectively, p = 0.06). Conclusions: To the best of our knowledge, this study represents the largest cohort of molecularly characterized OS. Age-associated differences in the genetic landscape and TME composition, including increased gene amplifications observed in younger patients and decreased B-cell abundance in older patients, might suggest fundamental underlying molecular and biological differences.

246 Background: Neoantigens are short peptides derived from tumor-specific somatic mutations that can bind to HLA molecules and be presented on the cell surface to activate the immune system. Recognition of neoantigens by autologous T cells promotes sensitivity to immune checkpoint blockade of mismatch repair deficient (MMRd)/microsatellite instability high (MSI-H) tumors. Neoantigen-targeted reactivity by autologous T cells has also been reported in microsatellite stable (MSS) tumors. We aimed to comprehensively assess the spectrum of neoantigens in upper GI cancers and identify recurrent immunogenic candidate neoantigens. Methods: 600 tumor specimens including 268 esophageal (EC), 211 gastric (GC), and 121 gastroesophageal junction (GEJ) cancers tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing (NGS) on DNA (whole exome) and RNA (whole transcriptome) were analyzed. MSI status was determined by immunohistochemistry of MMR protein and/or NGS. Immune epitope prediction was performed on translated peptide sequences harboring detected mutations using the NetMHCpan v4.0 method in the Immune Epitope Database, with HLA genotyping performed using arcasHLA. Immune/stromal cell abundance was quantified using the Microenvironment Cell Populations Counter method. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed signature, TIS). Results: Overall, the median patient age was 66 years (range 22-90), 72.8% were male, and 33.7% of samples were metastatic site biopsies. MMRd/MSI-H rate was 2.2% in EC, 12.4% in GC, and 2.5% in GEJ. A total of 1172 unique recurrent neoantigens with predicted binding-level affinity for patient specific HLA alleles were identified (317 in EC, 786 in GC, 157 in GEJ), with 442 and 552 neoantigens exclusively associated with MSS and MSI-H tumors, respectively. Across each cancer type, a higher positive TIS score correlated with both immune and stromal cell population abundance in the tumor microenvironment, most notably cytotoxic lymphocyte and monocytic cell types (r > 0.80, P < 0.0001). Recurrent peptides associated with highest average TIS scores resulted from mutation of TP53 (R248W, 2 peptides: 2.8%/3.2% of samples, respectively) and CUX1 (L162F, 1.6%) in MSS EC; REL (D318A, 2.3%) and ERBB2 (V842I, 1.2%) in MSS GC; MSH3 (K383fs, 36%) and ASXL1 (G645fs, 24%) in MMRd/MSI-H GC; and HOXD12 (A70-A71dup , 2.8%) and TP53 (R248Q, 3.7%) in MSS GEJ. Conclusions: This is one of the largest studies to investigate the landscape of recurrent neoantigens in upper GI cancers. We were able to identify candidate recurrent peptides with high HLA binding affinity and an association with a positive TIS signature in both MSI and MSS tumors, supporting the role of recurrent neoantigens as potential cancer immunotherapy targets.

594 Background: Young-onset pancreatic cancer (YOPC; < 50 years at diagnosis) has been associated with male preponderance, extensive smoking history, and a trend towards improved survival compared with average-onset pancreatic cancer (AOPC; ≥70 years). However, the genomic and transcriptomic correlates underlying these clinical differences are incompletely understood. Using a large matched genomic-transcriptomic next-generation sequencing (NGS) dataset, we sought to characterize the distinct molecular landscape associated with YOPC compared with AOPC. Methods: A total of 2430 pancreatic ductal adenocarcinoma NGS samples (YOPC n = 292; AOPC n = 2138) with matched whole-transcriptome (NovaSeq) and DNA (NextSeq, 592-gene or NovaSeq, whole-exome) sequencing data were analyzed (Caris Life Sciences, Phoenix, AZ). Immune deconvolution was performed using the QuantiSeq pipeline. Limited clinical data precluded stage- and treatment-stratified comparisons between cohorts. Overall survival (OS) was obtained from insurance claims, and Kaplan-Meier estimates were calculated for age- and molecularly-defined cohorts. Significance was determined as FDR-corrected P-values (Q) < 0.05. Results: Of 2430 PDAC patients undergoing NGS, YOPC patients (median age 46 years) were more likely to be male (65% vs. 52%; P < 0.001) and current smokers (32% vs. 11%; P = 0.02) compared with AOPC patients (median age 75 years). YOPC patients had higher proportions of mismatch repair-deficient (MMR)/MSI-H (2.8% vs. 0.8%, P = 0.001), BRCA2-mutant (4.7% vs 2.1%, P = 0.009), and PALB2-mutant (1.4% vs 0.5%, P = 0.04) tumors compared with AOPC patients, while tumors in AOPC patients had more frequent SMAD4 (20.1% vs. 14.7%, P = 0.03), RNF43 (6.3% vs. 2.5%, P = 0.012), CDKN2A (24.8% vs. 19.2%, P = 0.04), and SF3B1 (2.7% vs. 0.7%, P = 0.04) mutations. YOPC patients also demonstrated lower HLA-DPA1 homozygosity (55.2% vs. 64.1%, Q < 0.05) vs. AOPC patients. Notably, YOPC patients demonstrated significantly lower incidence of KRAS-mutant (81.3% vs. 90.9%, Q < 0.01) tumors compared with AOPC patients. In the KRAS-wildtype subset (n = 225), YOPC tumors were more likely to be driven by NRG1 and MET fusions, while BRAF fusions were exclusively observed in AOPC patients. Computationally inferred immune deconvolution revealed enrichment of NK cell (Q = 0.04) and M2 macrophages (Q = 0.01) populations in YOPC tumors. There was an association with improved OS in YOPC patients with KRAS-wildtype (median 22.4 [YOPC- KRASWT] vs. 15.1 [AOPC- KRASWT] months, P = 0.02) but not KRAS-mutant (P = 0.28), tumors compared with AOPC patients. Conclusions: In this large real-world multi-omic characterization of age-stratified molecular differences in PDAC, YOPC is associated with a distinct molecular landscape compared with AOPC. These data reveal molecular features of YOPC with prognostic and therapeutic implications.