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PURPOSE Conventional wisdom has rendered patients with brain metastases ineligible for clinical trials for fear that poor survival could mask the benefit of otherwise promising treatments. Our group previously published the diagnosis-specific Graded Prognostic Assessment (GPA). Updates with larger contemporary cohorts using molecular markers and newly identified prognostic factors have been published. The purposes of this work are to present all the updated indices in a single report to guide treatment choice, stratify research, and define an eligibility quotient to expand eligibility. METHODS A multi-institutional database of 6,984 patients with newly diagnosed brain metastases underwent multivariable analyses of prognostic factors and treatments associated with survival for each primary site. Significant factors were used to define the updated GPA. GPAs of 4.0 and 0.0 correlate with the best and worst prognoses, respectively. RESULTS Significant prognostic factors varied by diagnosis and new prognostic factors were identified. Those factors were incorporated into the updated GPA with robust separation ( P < .01) between subgroups. Survival has improved, but varies widely by GPA for patients with non–small-cell lung, breast, melanoma, GI, and renal cancer with brain metastases from 7-47 months, 3-36 months, 5-34 months, 3-17 months, and 4-35 months, respectively. CONCLUSION Median survival varies widely and our ability to estimate survival for patients with brain metastases has improved. The updated GPA (available free at brainmetgpa.com) provides an accurate tool with which to estimate survival, individualize treatment, and stratify clinical trials. Instead of excluding patients with brain metastases, enrollment should be encouraged and those trials should be stratified by the GPA to ensure those trials make appropriate comparisons. Furthermore, we recommend the expansion of eligibility to allow for the enrollment of patients with previously treated brain metastases who have a 50% or greater probability of an additional year of survival (eligibility quotient > 0.50).

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.

684 Background: Management of non-metastatic pancreas cancer has been extensively studied in clinical trials over the past two decades, with a growing body of non-randomized data supporting the use of neoadjuvant chemotherapy in combination with surgery. Guidelines now recommend use of this treatment paradigm, but real-world follow-up is critical in understanding how these recommendations are translated into the clinic. Using real-world data, we aimed to evaluate the treatment modalities and subsequent survival outcomes for patients diagnosed with pancreatic adenocarcinoma within the MHealth/Fairview medical system. Methods: We identified 844 patients diagnosed with stage I-III pancreatic adenocarcinoma within the MHealth/Fairview system (Minneapolis, MN) from 2001-2021 with EHR data enriched by cancer registry abstracted data. Diagnoses were confirmed with ICD-10 coding and clinical staging. Patients were stratified by initial treatment with either surgery (N = 345) or neoadjuvant-intent chemotherapy (n = 499). Correlation between initial treatment modality, demographic identifiers, and time to treatment was calculated using both Pearson and Spearman method. Effect of initial treatment modality on overall survival was calculated using a Cox proportional Hazard (CPH) model controlling for age, sex, clinical stage, and time to treatment initiation (TTI). A sub-analysis with race was performed on samples where these data were available. Results: Relative to neoadjuvant chemotherapy, up-front surgery was associated with a significantly improved overall survival (Hazard Ratio (HR) 0.55 95% Confidence Interval (CI): 0.37 – 0.76; p < 0.001), when controlling for age, sex, clinical stage, and TTI. Surgical patients also had shorter mean TTI values (21 days; p < 0.001) relative to chemotherapy (28 days), and a slightly younger patient population (mean age 63, vs 66 for chemotherapy, p = < 0.001; rho = -0.14). We observed no significant correlation between age (p = 0.027) or TTI (p = 0.12) and survival within this population. We noted no significant correlation between race, sex, or clinical stage and initial treatment modality or overall survival. Conclusions: We observed that MHealth/Fairview system patients who received upfront surgery for stage I-III pancreas cancers demonstrated improved overall survival relative to other treatment modalities, even after controlling for the differences in age and TTI found between these patients and those who undergo initial chemotherapy. This is likely due to a multitude of factors not readily captured within this data set, including surgical patient selection and tumor biology. Further work is needed to understand why these findings diverge from study norms, and to optimize care for all patients with pancreatic adenocarcinoma.

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.

249 Background: An estimated 85% of CRC are microsatellite stable (MSS) and this population has limited response to immune checkpoint inhibitors (ICIs). Biomarkers are needed to identify responders. Although pembrolizumab received FDA universal approval for chemorefractory cancers with tissue TMB-H > 10 mut/Mb, it is not effective across all TMB-H cancers. We aim to identify TMB-H/MSS CRC patients that derive benefit from ICIs through analysis of circulating tumor DNA (ctDNA). Methods: We retrospectively queried Guardant Health database (2020-2022) for patients with advanced CRC who had ctDNA NGS (Guardant360, Redwood City, CA) as part of routine clinical care. The assay covers a 1Mb panel for TMB that counts synonymous and non-synonymous somatic SNVs and indels to calculate a TMB score. Co-mutations were evaluated for MSS cancers with plasma TMB-H; ≥20mut/Mb vs those with low TMB (TMB-L; < 20mut/Mb). RAS- mutated signature was defined as those harboring an activating somatic alteration in KRAS, NRAS, or BRAF. Statistical significance was calculated using Fisher’s exact test. Results: We identified 6412 CRC patients: 11% (711) TMB-H/MSS (median age 64), 69% (4426) TMB-L/MSS (median age 62), 3% MSI-H, and 17% unevaluable. When compared to TMB-L/MSS, patients with TMB-H/MSS had a significantly higher frequency of select genetic mutations, including TP53, APC, KRAS, EGFR, and PIK3CA (p < 0.001). Treatment-naïve TMB-H/MSS cancers had a higher frequency of ARID1A (p = 0.05) and TERT (p = 0.02) alterations, while those post therapy progression had higher frequency of KRAS (p = 0.02), EGFR (p < 0.001), and MAP2K1 (p = 0.002) alterations. A total of 556 (78%) CRC patients in the TMB-H/MSS cohort harbored a RAS-mutated genetic signature. Of those, 432 (78%) belonged to the post therapy progression group, likely representing adaptive clones post anti-EGFR therapy. While RAS-mutated tumors had a higher prevalence of PIK3CA (p = 0.001) and SMAD4 (p = 0.007) co-alterations, RAS-wildtype ones carried a higher frequency of ERBB2 (p = 0.068) alterations. Conclusions: We describe the genomic landscape of patients with plasma TMB-H/MSS CRC. A clinical cohort of TMB-H/MSS CRC patients identified by ctDNA and treated with ICIs will be presented.

240 Background: CD47 belongs to the immunoglobulin superfamily and is overexpressed in many tumor types. CD47 plays an important role in suppressing phagocytosis through binding to transmembrane protein SIRP-alpha on macrophages. Targeting CD47 is a novel strategy for cancer immunotherapy and is being evaluated in ongoing clinical trials. However, molecular characteristics of CD47-overexpressed colorectal cancer (CRC) are largely unknown. Methods: We retrospectively reviewed CRC patient samples (n = 14786) 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. Correlation of CD47 expression with danger-associated molecular pattern (DAMP)-related genes ( HMGB1, CALR, ANXA1, HSP90AA1, HSPA1A, and CXCL10) expressions was tested. DAMP signature calculated as composite z-score of the DAMP related genes was compared between CD47-high and -low patients classified according to the median level of CD47 expression. Distributions of KRAS and BRAF mutations, consensus molecular subtype (CMS), and signatures of oncogenic signaling pathways were compared between CD47-high and -low patients. In addition, overall survival (OS) was compared between CD47-high and -low patients available for survival data. Results: CD47 expression level was significantly higher in metastatic compared to primary lesions (1.07-fold, q < 0.05) and microsatellite instability high tumors compared to microsatellite stable tumors (1.15-fold, q < 0.05). CD47 expression was positively correlated with DAMP genes expression except for HSPA1A, and the DAMP signature (median score [MS]: 2.66 vs -2.29, q < 0.05) was significantly increased in CD47-high patients. KRAS mutations were less prevalent (45.87% vs 50.05%) and CMS1(17.72% vs 14.42%) and CMS4 (40.33% vs 27.28%) were more prevalent in CD47-high patients, while no difference was observed in the prevalence of BRAF mutations between CD47-high and -low patients. Signatures of EMT (MS:2.91 vs -3.19), TGF-beta (MS:3.72 vs -3.52), angiogenesis (MS:2.95 vs -2.78), MAPK (MS:4.99 vs -4.12), PI3K (MS:2.41 vs -2.03), and immune-related signaling pathways (MS:1.20 vs -2.51) were significantly enriched in CD47-high patients (all q < 0.05). CD47-high patients (n = 4873) showed significantly worse OS than CD47-low patients (n = 4898) (median OS, 32.4 vs 37.6 months; hazard ratio = 1.158, p< 0.01). Conclusions: Highly CD47-expressed CRC harbored activation of DAMPs and oncogenic signaling pathways that linked to aberrant tumor microenvironment and worse prognosis. Our results support intensive treatment strategies using CD47 inhibitors combined with cytotoxic agents and molecular targeted agents (such as anti-VEGF agents) in CD47-overexpressed CRC.

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

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]

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.

82 Background: Gastric/colorectal cancer (G&CRC) are among the most prevalent cancers. Efforts are ongoing to better assess their clinical/genomic factors. HER2 (human epidermal growth factor receptor 2) testing is strongly recommended in clinical practice to guide treatment. An accredited education program (GetSMART) was launched online (January 2021–April 2022) to equip healthcare providers (HCPs) with knowledge, skills, and confidence to accurately assess/apply molecular testing and therapy options for metastatic G&CRC patients. Methods: A knowledge/confidence-based assessment was embedded in four modules to measure learners’ knowledge mastery (% answering correctly with confidence) pre/post-activity. Self-reported confidence and anticipated performance in clinical practice were measured before/after each module. At the end of program, HCPs were asked about their commitment to change. McNemar’s test was applied to assess statistical significance in pre-post for paired items, and descriptive analysis for non-paired items. Results: Overall, 925 unique HCPs engaged in GetSMART, 31% (n=284) completed all modules, of which 57% (n=163) consented analysis of their data. A statistically significant increase (p

184 Background: SETD2, a key methyltransferase modulating histone 3 gene transcription, has been shown to act as a tumor suppressor gene by reducing oxidative stress, colonic inflammation and tumorigenesis in animal models. SETD2 gene expression has been reported to be significantly downregulated in CRC and linked with poorer patient survival. Additionally, SETD2 plays an important role in DNA repair and loss of function mutations have been associated with increased tumor mutational burden (TMB), mismatch repair (MMR) deficiency, and benefit from immunotherapy. Hence, we aimed to characterize the molecular features associated with SETD2 gene expression in CRC. Methods: 15,425 CRC tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or whole exome sequencing), RNA (whole transcriptome sequencing), and IHC were analyzed. SETD2-high and -low expression were defined as ≥ top and < bottom quartile of SETD2 transcripts per million (TPM), respectively. Cell infiltration in the tumor microenvironment (TME) was estimated by QuantiSEQ. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons ( q < .05). Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (T cell-inflamed) and MAPK pathway activation (MPAS). Real-world overall survival information was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: SETD2 expression was significantly increased in left-sided/rectal compared to right-sided tumors (median TPM [mTPM] 55.8 vs 51.1, q < .001). SETD2 mutations were associated with reduced SETD2 expression in pMMR/MSS CRC (mTPM 37.9 vs 54.3 in WT, q < .001), although not statistically significant in dMMR/MSI-H CRC (mTPM 43.6 vs 51.7 in WT, P = .17). Compared to SETD2-high tumors, SETD2-low was associated with increased rates of TMB-high (10.4% vs 8.2%, P = .009), dMMR/MSI-H (7.3% vs 5.6%, P = .02), and PD-L1 IHC levels (4.4% vs 2.5%, q < .002). Only minor differences in gene mutation and copy number rates were observed between SETD2-high and -low tumors , whereas SETD2-high TME was associated with increased immune cell infiltration, including neutrophils, B cells, NK cells, M2 macrophages, and dendritic cells ( q < .05). SETD2-high was also associated with increased T cell-inflamed and MPAS signatures (152% and 159% median increase, respectively, q < .001), regardless of tumor MMR status. Among dMMR/MSI-H CRC, SETD2-high tumors receiving immune checkpoint inhibitors had longer time-on-treatment (HR: 0.39, 95% CI: 0.2-0.76, P = .005) and a trend towards longer OS (HR: 0.29, 95% CI: 0.07-1.2, P = .069). Conclusions: Our data show distinct immune biomarkers and TME cell infiltration associated with SETD2 gene expression in CRC. These findings support a key role for SETD2 in modulating anti-tumor immunity and TME.

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

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.

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

4048 Background: Claudins are transmembrane proteins which maintain the tight junction between cells. The stomach specific isoform, CLDN18 isoform 2 (CLDN18.2), is emerging as a promising treatment target because of high expression in GC cells, including targeting via adoptive T-cell strategies. We characterized the molecular features associated with CLDN18 isoform 1 and 2 ( CLDN18.1/ 18.2) gene expression in GC. Methods: Tumor profiling was performed from 1967 samples by NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS) at Caris Life Sciences (Phoenix, AZ). EBER (Epstein Barr Virus) was tested by CISH. Top quartile transcripts per million for CLDN18.1/18.2 were considered high while bottom quartile low expression. X2, Fisher-exact, and Mann Whitney tests were used and significance adjusted for multiple testing by Benjamini-Hochberg (q .05) and EBER (2.15% vs 6.31%, p .05). There were more prevalent ARHGAP26 fusions in the CLDN18.1/18.2 high group (18.1: 9.5% vs 3.86%, p =.001; 18.2: 10.1% vs 0.9%, q .05) and TMB-H (19.6% vs 12.2%, p .05). Similarly, CLDN18.1/18.2 displayed an inverse relationship with M1 Macrophages, NK cells, CD4+ T cells, myeloid dendritic cells in the TME ( q

3129 Background: Disruption of the circadian clock has been linked to cancer risk, development and progression. Core clock proteins are emerging as novel therapeutic targets in cancer. We previously showed that polymorphisms in clock genes were associated with anti-VEGF treatment outcome in metastatic CRC. Here we further evaluated the molecular landscape of clock pathway alterations in CRC. Methods: 7591 CRC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Clock gene Score (CS) was determined using expression of core clock genes Z scores (positives of CLOCK, ARNTL, RORA/B/C and negatives of repressors CRY1/2, PER1/2/3, REVERBA/B) stratified by quartiles. xCell was used to quantify cell infiltration in the tumor microenvironment (TME). Consensus molecular subtypes (CMS) were assessed by RNAseq. Significance was determined as P-values adjusted for multiple testing ( q) of

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

3538 Background: PTPRT is a protein coding gene involved in signal transduction and cellular adhesion. It acts as a tumor suppressor gene and mutated PTPRT has been implicated in the early metastasis of CRC. PTPRT mutations have been reported as independent potential biomarkers for bevacizumab resistance in metastatic CRC and linked to improved response and survival of patients treated with checkpoint inhibitors in several tumors. Here we characterized the molecular features and clinical outcomes associated with PTPRT gene expression in CRC. Methods: 15025 CRC tested with NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS) by Caris Life Sciences (Phoenix, AZ) were analyzed. Top quartile transcripts per million for PTPRT expression were considered high (H) and bottom quartile low (L). Cell infiltration in the tumor microenvironment (TME) was estimated by QuantiSEQ. X2, Fisher-Exact, Mann-Whitney tests were used and significance determined as P-value adjusted for multiple comparisons ( Q

3128 Background: Macrophage Erythroblast Attacher (MAEA) plays an important role in actin cytoskeleton rearrangement in macrophages and erythroid cells. We previously reported that MAEA suppresses migration, invasion and enhances chemosensitivity in CRC cell lines. Here we aimed to characterize the molecular features associated with MAEA gene expression in CRC. Methods: 14416 CRC were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS). Top quartile transcripts per million (TPM) for MAEA expression were considered high (Q4) while bottom quartile low (Q1). Consensus molecular subtypes (CMS) were assessed using RNAseq. Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by QuantiSEQ. X2 and Fisher-Exact tests were used and significance was determined as P-value adjusted for multiple comparisons ( Q < 0.05). Results: MAEA expression was highest in rectal tumors (13.6 median TPM) followed by transverse and right-sided tumors (13.0 and 12.8, respectively) and lowest in left-sided tumors (12.5). Overall, MAEA TPM were associated with higher tumor mutational burden (≥ 10 Mut/Mb) (11.8% vs. 8.2%) and dMMR/MSI-H (8.7% vs. 5.1%) ( Q < 0.0001); however, the association with TMB was not observed in MSS tumors. In the MSS cohort, MAEA expression was the highest in CMS4 (14.9 median TPM) followed by CMS1 (12.5), CMS2 (11.9), and the lowest in CMS3 (10.3, all intergroup Q < 0.05). MAEA high was associated with lower mutation rates of APC and amplification of FLT1/ FLT3 while higher mutation rates of ASXL1, KMT2A/C/D, SMARCA4, FBXW7, PTEN, RNF43, BRCA2, HNF1A in the overall cohort ( Q < 0.05). In the MSS cohort, FBXW7 mutation significance with MAEA high expression held true ( Q < 0.05) while MAEA high expression trended to associate with higher mutation rates of KMT2D, SMARCA4, PTEN, BRCA2 mutations, and a lower frequency of FLT1/ FLT3 CNA ( P < 0.05 but Q > 0.05). High MAEA was associated with higher immune CI in the TME, including B cells, macrophages (M1 and M2), neutrophils, NK cells, Tregs, CD4+ T cells and myeloid dendritic cells both in the overall cohort and in MSS tumors (fold change: 1.11-1.33, all Q < 0.001). Conclusions: Our data show a strong association between MAEA gene expression and distinct molecular features (including CMS and immune biomarkers) and TME cell infiltration in CRC. These findings suggest that targeting MAEA may have relevant clinical applications in selected CRC subgroups and MAEA may be an important player in determining the composition of the TME.

2630 Background: MDM2/MDM4 are implicated in hyperprogression after immune checkpoint blockade (ICB). Our preclinical studies showed reduced T-cell killing of MDM2-amplified tumor cells that was overcome by an MDM2 antagonist or gene knockdown, and we observed additional tumor killing by T-cells with MDM2 inhibition plus anti-PD1. We hypothesized that MDM2/4 gene amplification/overexpression correlates with resistance to ICB and investigated the association of MDM2/4 alterations to overall survival (OS) following ICB across multiple solid tumors. Methods: Solid tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (NGS) were analyzed. MDM2/4 amplification (amp) was tested by NGS and determined as either amp4 (cutoff of >=4.0 copies) or amp6 (>=6.0) or amp8 (>=8.0). Real-world OS was obtained from insurance claims data and calculated from treatment start or tissue collection to last contact. Kaplan-Meier estimates were calculated for molecularly defined groups. X2/Fisher-Exact were used and significance determined as P-value adjusted for multiple comparisons (q

4127 Background: NENs can occur in many locations but have limited precision therapy options. DLL3 is a cell surface protein that is emerging as a promising therapeutic target in NENs including neuroendocrine prostate cancer (NEPC) and small cell lung cancer (SCLC). Our recent study indicated that ̃77% of NEPCs expressed DLL3, with expression in circulating tumor cells being highly concordant with matched biopsies. While there are ongoing clinical trials of drugs targeting DLL3, the repertoire of clinical and genomic features shared across other DLL3-expressing NEN cancers is ill-defined. Methods: We analyzed WES and WTS data from NENs identified across 29 different sites of origin using the Caris Life Sciences platform, excluding SCLC and including neuroendocrine carcinomas and neuroendocrine tumors. We used values above or below median DLL3 expression of all NEN samples to define DDL3-High/Low (H/L). Significance of molecular alterations in DLL3-H vs L was determined using Fisher’s-Exact/Mann Whitney/X2 test with Benjamini-Hochberg correction. Results: DLL3 expression across all 2672 NEN samples was observed in 27 of 29 NENs after excluding SCLC. NENs of anus, prostate, lung, bladder, and bile duct exhibited the greatest median DLL3 expression, whereas adrenal gland, small bowel, and nervous system displayed the lowest. Certain tissues of origin displayed more robust DLL3 expression, with 71% (50/66) of NEPC, 75% (87/122) of lung, and 77.3% (51/66) of bladder being DLL3-H compared to 14.4% (13/90) of adrenal and 7.9% (12/151) of small bowel NENs. DLL3-H NENs were associated with TMB-high status ( > 10 muts/Mb; 12.1% vs 4.5%, OR 2.7, q < 0.001) and more genomic alterations in several driver genes, including tumor suppressors TP53 (51% vs 23%, OR 2.3, q < 0.001) and RB1 (42% vs 10%, OR 4.2, q < 0.001), and oncogenes KRAS (14% vs 5.4%, OR 2.5, q < 0.001), MYC (5.7% vs 0.9%, OR 6.3, q < 0.001) and CCNE1 (5.3% vs 1.3%, OR 4.0, q = 0.001). Conversely, DLL3-L NENs exhibited more alterations in CTNNB1 (2.2% vs 5.2%, OR 0.42, q = 0.04), MEN1 (3.3% vs 11%, OR 0.30, q < 0.001), and BCOR (1.3% vs 4.1%, OR 0.32, q = 0.02). DLL3-H NENs also had significantly more alterations in PIK3CA (6.4% vs 3.0%, OR 2.1, q = 0.04), chromatin remodeling genes KMT2D (6.7% vs 2.6% OR 2.6, q = 0.005) and CREBBP (3.2% vs 0.9%, OR 3.6, q = 0.03), and WNT signaling gene APC (9.7% vs 5.2%, OR 1.9, q = 0.02). Conclusions: We confirmed DLL3 expression in NENs across different tissues of origin, with highest expression in poorly differentiated NENs. DLL3-H expression was associated with genomic features considered “undruggable” based on current precision therapy approaches. Therefore, DLL3-targeted therapies may serve as a promising strategy for NEN patients with functional loss of tumor suppressors TP53 and RB1, as well as increased activity of KRAS, WNT and MYC signaling.

3148 Background: Recent proteogenomic analyses of CRC revealed that driver gene alterations are enriched in the endocytosis pathway (Vasaikar S, et al. Cell 2019;177:1035-49). Endocytosis is a cellular system involving post-translational modification of plasma membrane proteins through internalization, intracellular trafficking, degradation, and recycling. Clathrin-mediated endocytosis (CME) is the main endocytic portal, and endosomal sorting complexes required for transport (ESCRT) play a critical role in the lysosomal degradation pathway. Besides the well-known function of endocytosis attenuating signaling pathways through receptor clearance from the cell surface, the opposite function contributing to signal maintenance has also been reported. However, the clinical implications of the endocytosis pathway alterations in CRC are largely unclear. Methods: We retrospectively reviewed CRC patient samples (n = 15025) submitted to a commercial CLIA-certified laboratory (Caris Life Sciences, Phoenix AZ). Next-generation sequencing of DNA and RNA (whole-transcriptome sequencing) and immunohistochemistry (IHC) were performed. CME-related (47 genes) and ESCRT-related (35 genes) expression signatures were calculated as composite z-scores and compared between subgroups stratified by RAS/ BRAF mutation status, MSS/MSI status, tumor sidedness, and consensus molecular subtype (CMS). VPS4A/ VPS4B expression correlation with major oncogenic pathway signatures (composite z-scores) and CMTM6/ CMTM4/ HIP1R expression association with PD-L1+ IHC were also assessed. Results: Among 17 endocytosis-related genes, no pathogenic/likely pathogenic mutations were identified. The CME-related signature was increased in RAS/ BRAF wild type vs. mutant (0.93 z-score difference, p= 0.04) and MSS vs. MSI-high (6.0 z-score difference, p< 0.01), while the ESCRT-related signature was higher in MSS compared to MSI-high (2.7 z-score difference; p< 0.01). No differences between tumor sidedness were observed in both CME- and ESCRT-related signatures (0.81 and 1.17 z-score differences, respectively). CMS4 had the highest expression of both signatures, while CMS3 had the lowest, of both CME- and ESCRT-related genes (each > 20 z-score difference, p< 0.01). VPS4A and VPS4B expression had a strong positive correlation with WNT, EGFR/MAPK, TGF-beta, and Notch pathway signatures (0.65-0.83 Spearman, all p< 0.01). CMTM6 expression was positively associated with PD-L1+ IHC (1.2-fold increase vs PD-L1-negative, p< 0.01), while CMTM4 and HIP1R expression showed a negative association (0.7- and 0.9-fold decrease, respectively, p< 0.01). Conclusions: This large study indicates endocytosis pathway expression is positively associated with oncogenic pathway signaling in CRC. Further analysis of RAS/ BRAF wild type, MSS, and CMS4 patient subgroups are warranted to determine the efficacy of targeting endocytosis pathways in CRC.

4571 Background: p53 target and cell cycle inhibitor CDKN1A/p21(WAF1) was initially not found to be mutated in cancer. TCGA analysis identified CDKN1A mutations are present but rare with frequencies of < 1%, but enrich in bladder cancer (̃8%). Truncating WAF1 mutations are associated with sensitivity to cisplatin and are associated with truncating Rb mutations in bladder cancer (RW+). We hypothesized RW+ bladder cancers may represent a unique subgroup with sensitivity to therapeutics. Methods: A total of 1104 urothelial tumors underwent molecular profiling at Caris Life Sciences (Phoenix, AZ) utilizing NGS of DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS). Wilcoxon, Fisher’s exact were used for statistical significance (p value without and q value with multi comparison correction). Immune cell fraction (QuanTIseq) and pathway analysis (ssGSEA) were assessed by mRNA analysis. Immune epitope prediction was performed using the NetMHCpan v4.0 method in the Immune Epitope Database. Results: Concurrent truncating mutation (frameshift, nonsense) for RB1 and WAF1 were detected in 47 tumors (RW+, 4.25%) and tumors with wild-type status for both RB1 and WAF1 genes were classified as RW- group (54.08%). Tumors harboring only one RB1 or WAF1 mutation were excluded for further analysis. When compared to RW- group, RW+ tumors showed lower mutation rate of TP53 (54.5% vs 80.9%, q < 0.05), ARID1A (23.5% vs 38.3%, p < 0.05), and PIK3CA (18.4% vs 31.9%, p < 0.05). Interestingly, RW+ was mutually exclusive with FGFR3 mutation (18.0% vs 0%, p < 0.05). We further evaluated RNA expression of DNA repair and checkpoint arrest pathways. Notably, E2F pathway (Normalized Enrichment Scores, NES: 0.89 vs 0.86, q < 0.01) and DNA G2M checkpoint (NES: 0.89 vs 0.86, q < 0.01) were found to be the most enriched in RW+ with respect to RW- group. In addition, mRNA levels of FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 were found to be overexpressed in RW+ group (q < 0.05). RW+ tumors also displayed a distinct immunological profile: They were associated with higher PD-L1 status (63.8% vs 37.3%, q < 0.01), higher median TMB (11 mut/Mb vs 8 mut/Mb, q < 0.01) and with less frequent loss of heterozygosity for HLA-DPA1 (51.1% vs 66.7%, p < 0.05), with more high-binding-affinity neoantigen load (4.78 vs 3.89, p < 0.05) to MHC proteins, consistent with the significantly more myeloid dendritic cells in in RW+ group (0.3 vs 0.04, q < 0.001). Conclusions: Concurrent truncating mutation in RB1 and WAF1 (RW+) bladder carcinomas have fewer p53, ARID1A, and PIK3CA mutations but are enriched for E2F targets, G2/M checkpoint genes, FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 and have a distinct immunological profile. The findings suggest therapeutic strategies for RW+ bladder cancers including Chk1/Wee1, PARP inhibitors, -/+ immunotherapy that may impact on clinical outcomes.

3547 Background: TIM-3 is an inhibitory checkpoint glycoprotein found on innate and adaptive immune cells and is highly expressed on tumor infiltrating lymphocytes. TIM-3 and its ligands, Galectin 9 (Gal9), HMGB1 and CEACAM1 play a critical role in immune regulation and preclinical data suggest a role in the pathogenesis of colorectal cancer (CRC). We aimed to characterize the molecular features and prognostic value of TIM3 and its ligands in CRC. Methods: Tumor molecular profiling was performed from 15,026 FFPE samples by NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS) and immunohistochemistry (IHC) at Caris Life Sciences (Phoenix, AZ). Top quartile transcripts per million (TPMs) for TIM-3, Gal9, HMGB1 and CEACAM1 were considered high (Q4) while bottom quartile low (Q1) expression (exp). X2/Fisher-exact tests were used for comparison and significance was determined as P-value adjusted for multiple comparison and this was found for the results reported here ( Q < 0.05). Cell infiltration in the tumor microenvironment (TME) was estimated by quanTIseq. Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined pts. Results: Gal 9/TIM3-high tumors had higher prevalence (prev) of high tumor mutational burden (TMB ≥ 10 Mut/Mb) (12% vs. 8%; 14% vs. 6%), deficiency in mismatch repair (dMMR) (9% vs. 5%; 10% vs. 4%), PD-L1 exp (5% vs. 3%; 7% vs. 2%), and was highest in transverse (Fold Change; FC: 1.05, 1.12) and right sided tumors (1.04, 1.10) compared to left sided tumors, and CMS4. In contrast, HMGB1 and CEACAM1-high tumors had lower prev of dMMR (5% vs. 8%, 3% vs. 12%), PD-L1 exp (3% vs. 5%, 3% vs. 6%) and TMB-H (8% vs. 11%, 6% vs. 16%), and was highest in CMS2. In MSS tumors, Gal9/TIM3-high tumors were associated (assoc) with lower frequency of TP53, amplifications (amp) of FLT1/3, CDX2, FOXO1, and CDK8 while CEACAM1 and HMGB1-high were assoc with higher mutation (mut) rates of TP53, APC, NRAS, amp of FLT1/3, CDX2, CDX8, and lower mut in GNAS, FBXW7 and RNF43. High Gal9 and TIM3 exp was assoc with higher infiltration of B cells, M1 and M2 macrophages, NK cells, CD4+ and C8+ T cells, and Tregs, while high HMGB1 and CEACAM1 exp was negatively assoc with Tregs, M1 macrophages, monocytes and CD8+ T cells. High exp of Gal9, TIM3, HMGB1, and CEACAM1 was assoc with worse OS in the entire cohort (HR 0.90, 95%CI, 0.84-0.97, P = 0.005, HR 0.81, 95% CI, 0.75-0.87, P< 0.00001; HR 0.88, 95% CI, 0.82-0.95, P < 0.001; HR 0.80; 95%CI, 0.74-0.86, P < 0.00003, respectively). Conclusions: Strong assoc were identified between Gal9/TIM3, HMGB1 and CEACAM1 gene exp and IO biomarkers, distinct molecular features, CMS, TME cell infiltration, and patient outcomes in CRC. Significantly different mut frequencies may signify unique subsets of CRC. These findings provide rationale for further evaluation of TIM3 and its ligands in CRC as prognostic biomarkers and potential therapeutic targets modifying the TME.

3625 Background: In colorectal cancer (CRC) and endometrial cancer (EC) patients (pts), preliminary data suggest a differential response to immune checkpoint inhibitors (ICIs) according to different MMR alterations. The drivers of this difference remain unknown and no reliable predictive biomarker has been found. We explored the genomic alterations, tumor mutation burden (TMB), immune-related gene expressions and signatures, tumor microenvironment (TME), neoantigen load and median overall survival (mOS) inCRC and EC pts treated with ICIs with different MMR alterations. Methods: 13,701 CRC and 3,315 EC specimens were tested at Caris Life Sciences (Phoenix, AZ) with Next Gen Sequencing (NGS) of DNA (592-gene or whole exome) and RNA (whole transcriptome). MMR/MSI status was determined by IHC of MMR protein and/or NGS. Immune cell abundance was quantified using quanTIseq. Gene expression profiles were analyzed for T cell-inflamed signature (TIS) and IFN-gamma scores. Immune epitope prediction was performed using the NetMHCpan v4.0 method in the Immune Epitope Database. Real-world mOS was obtained from insurance claims data and calculated from tissue collection or ICIs start to last contact. Statistical significance was determined using chi-square/Fisher-Exact and adjusted for multiple comparisons (adjusted p < 0.05). Results: In CRC, 84 (0.6%) pts had intact expression of MLH1 and PMS2 and co-loss of MSH2 and MSH6 (MutS) and 648 (4.7%) had co-loss of MLH1 and PMS2 and intact MSH2 and MSH6 (MutL). 117 (0.9%) had other MMR IHC loss. APC, KRAS, ERBB2, ERBB3 and MSH2 mutations rates were higher in MutS while BRAF mutation rate was higher in MutL. B cell, NK cell content and neoantigen load (high affinity epitopes: p < 0.05, intermediate: p < 0.01, low: p < 0.001) were higher in MutS. The mOS in MutS (N = 149) vs. MutL (N = 980) was 56 months (m) vs. 36 m (p = 0.003). In ICI-treated pts, the mOS in MutS (N = 28) vs. MutL (N = 149) was not reached (NR) vs. 32 m (p = 0.005). BRAF mutation didn’t impact survival in MutL. In EC, 48 (1.4%) pts had MutS and 915 (27.6%) had MutL. 81 (2.4%) had other MMR IHC loss. IHC-PD-L1, TMB, neoantigen load (high affinity epitopes: p < 0.01, intermediate: p < 0.0001, low: p < 0.0001), TIS, IFN-gamma scores, immune related gene expressions, TME (Macrophage M1, CD8+) were higher in MutS. The mOS in MutS (N = 94) vs. MutL (N = 1804) was NR vs. 47 m (p < 0.001). In ICI-treated pts, the mOS in MutS (N = 11) vs. MutL (N = 273) was NR vs. NR (p = 0.559). Conclusions: This is the largest study to explore differential response to ICIs in CRC and EC pts with different MMR alterations. In pts with CRC and EC, the mOS was longer in MutS compared to MutL. In ICI-treated pts, the mOS was longer in MutS compared to MutL in CRC but not in EC. Among the explored biomarkers, neoantigen load was higher in MutS compared to MutL in both CRC and EC and maybe the driving factor for differential response to ICIs.

4142 Background: The composition of the tumor microenvironment (TME) in PDACs is more heavily driven by mutant (mt) KRAS than any other cancer. How genomic alterations of PDAC including KRAS status affect the immune cell (IC) landscape remains unclear. Thus, we characterized IC types and the prevalence of immuno-oncologic (IO) biomarkers in PDAC by genomic and transcriptomic analysis, and investigated associations of mt KRAS with IC estimates in the TME. Our findings were compared to our previous study in CRC. Methods: A total of 4,142 PDAC and 3,727 CRC with KRAS- mts were analyzed using next-generation DNA sequencing (NextSeq, 592 gene panel or NovaSeq, WES), IHC, and whole transcriptome RNA sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by FA, IHC and NGS. TMB-H was classified based on a cut-off of >10 mutations per MB. ICs were estimated by QuantiSeq (Finotello 2019, Genome Medicine) or MCP counter (Betcht 2016, Genome Biology). Significance was determined by X2 and Fisher-Exact and p-adjusted for multiple comparisons (q+ & CD8+ T cells, T-reg, NK, myeloid dendritic and endothelial cells compared to KRAS wt. In CRC, a similar pattern was observed but more pronounced in PDAC. Immune-regulatory markers, were among multiple genes downregulated in KRAS-mt PDAC, including CTLA-4 and LAG3. Overall changes were most pronounced in cases harboring KRAS G12D, G12V, Q61, and rare KRAS variants. Conclusions: The TME of KRAS mt PDAC shows IC patterns similar to KRAS mt CRC. Actionable IO-targets, such as PDL1, are enriched in tumors harboring specific variants of KRAS mt PDAC including the targetable G12C variant. If G12D becomes druggable, it could be targetable in 35% patients with PDAC or 15% in CRC. These results demonstrate that the TME of PDAC and CRC shows immune-cold features. Tailored immunotherapeutic strategies would have to overcome these barriers in KRAS mt PDAC and CRC, possibly in combination with molecularly targeted treatment strategies.

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.

3546 Background: CXC-chemokine receptor 4 (CXCR4) is a ubiquitous chemokine receptor activated by the CXCL12 ligand and is implicated in tumor invasion, metastasis, and immune cell (IC) trafficking. High CXCR4 expression is associated with poor prognosis in colorectal cancer (CRC). < 10% of metastatic CRC cases harbor microsatellite instability (MSI-H) and demonstrate lower tumor mutation burden (TMB), decreased IC infiltration, and lack of response to current immunotherapy regimens. This study aims to interrogate the role of CXCR4 mRNA expression on the the tumor microenvironment (TME) and its prognostic and predictive value to tailor immunotherapeutic treatment strategies in CRC. Methods: A total of 15,026 CRC samples were analyzed using whole-exome sequencing, whole-transcriptome sequencing, and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Study cohort was stratified by CXCR4 mRNA expression levels in quartiles (Q1 (low) vs Q4 (high)). IC fraction was calculated by QuantiSeq, and real-world overall survival information was obtained from insurance claims data and calculated from tissue collection time to last day of contact. Statistical significance was determined using chi-square/Fisher-Exact and adjusted for multiple comparisons (q < 0.05). Results: Samples obtained from metastatic sites showed higher CXCR4 mRNA expression than those from primary tumors (22.7 vs 18.6 median transcripts per million (TPM), p < 0.001). CXCR4 mRNA expression was significantly lower in liver metastases than in non-liver metastases (21.2 vs 24.8 TPM, p < 0.001). Median CXCR4 mRNA expression was highest in the consensus molecular subtypes 4 (33.3 TPM) and lowest in 3 (13.0 TPM, p < 0.05). CXCR4 mRNA expression was positively associated with TMB-H, MSI-H/dMMR, and positive PD-L1 IHC status. In the TME, high CXCR4 mRNA expression was observed in tumors with a higher IC infiltration including B cells, M1/M2 macrophages, NK cells, CD8+ T cells and T-regs, regardless of MSI status. High CXCR4 mRNA expression in the primary tumor was associated with poor prognosis (HR 0.77, 95% CI 0.70-0.85; p < 0.001), regardless of MSI-status. In metastatic tumors, low mRNA expression was correlated with improved survival (HR 0.89, 95% CI 0.80-0.99; p = 0.34); however, this did not reach statistical significance in the MSS cohort (HR 0.90, 95% CI 0.80-1.0; p = 0.06). Of note, high CXCR4 mRNA expression was associated with improved survival in all patients with CRC who received pembrolizumab (HR 2.12, 95% CI 1.16-3.91; p = 0.013). Conclusions: This is the largest clinical dataset to date demonstrating high CXCR4 expression as a predictor for poor survival in CRC. Furthermore, high CXCR4 expression was associated with improved outcome after checkpoint inhibition immunotherapy, indicating its strong potential as a predictive biomarker that could inform immunotherapeutic strategies in CRC.

4018 Background: Small bowel adenocarcinoma (SBA) is a rare cancer with rising incidence and worse overall survival (OS) compared to other intestinal cancers. Clinical management of SBA is primarily extrapolated from colorectal cancer (CRC). Comprehensive genomic and transcriptomic profiling of SBA will facilitate development of disease-specific therapeutic strategies. We investigated molecular alterations and association with clinical outcomes in a large cohort of SBA pts. Methods: Tumors were analyzed using 592 gene next-generation sequencing of DNA (592 genes or WES), RNA (WTS) and IHC (Caris). Immune infiltration was calculated by QuantiSeq. OS was calculated from treatment start/tissue collection to last contact from insurance claims. Results: We analyzed 823 SBA tumors: 448 primary/312 metastases, 586 duodenal (DA)/95 jejunal (JA)/38 ileal (IA). Median age of JA pts was lower (57yo) than DA (67yo) and IA (68yo). Upon subsite comparison, HER2 overexpression (2.5%) and amplification (3.6%) was only seen in DA, while HER2 mutations (mts) were most common in JA (10%) and absent in IA. IA had the lowest rate of KRAS (30%) and APC (11%) mts and highest rate of DDR mts (37%). JAs were enriched in RSPO3 fusion (19%) and BRAF mts (21%). Among BRAF mts in SBA, class 3 comprised 53%, class 2 37% and class 1 10%. MSI/dMMR was seen in 8% SBA and TMB-H in 11% with no difference among subsites. Compared to 14000 CRC tumors, SBA had significantly higher immune infiltrates regardless of MSI status (p

4046 Background: Tumor specific antigens, such as NY-ESO-1 , are emerging as key tumor immune modulating factors with great potential to be used as therapeutic targets to enhance immunotherapy efficacy and expand treatment options for GC. We sought to compare GC tumors expressing high vs low levels of NY-ESO-1 in terms of immune cell abundance in the tumor microenvironment (TME) as well as distinct molecular features and immune biomarkers. Methods: 1967 tumor samples tested with NextGen Sequencing on DNA (592 genes or WES) and RNA (WTS) by Caris Life Sciences (Phoenix, AZ) were retrospectively reviewed. The top quartile of transcripts per million was considered high while the bottom quartile was considered low NY-ISO-1 expression. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous mutations. Mismatch repair deficiency/microsatellite instability (dMMR/MSI) status was evaluated by a combination of IHC, fragment analysis and NGS of known MSI loci. Gene fusions were detected based on WTS. X2, Fisher-exact, and Mann Whitney tests were used for comparison and significance adjusted for multiple testing by Benjamini-Hochberg correction ( q < 0.05). Cell infiltration in the TME was estimated by quanTIseq. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed signature: TIS). Results: The analysis was focused on primary tumors (N = 1323) in this initial study. Expression of NY-ESO-1 was lower in primary/local than metastases (Fold Change FC met vs primary: 1.60, q < 0.05). NY-ESO-1 expression did not appear to be strongly associated with distinct gene mutation profiles in GC. There were no significant differences between low and high expression of NY-ESO-1 with regards to well established immuno-oncology markers (dMMR/MSI, TMB, PD-L1). However, high NY-ESO-1 expression was positively associated with immune related gene expression including CD274, CD80, CD86, CTLA4, HAVCR2, IDO1, IFNG, LAG3, PDCD1, and PDCD1LG2 (FC low vs high: 0.56 to 0.79, q < 0.0001). High NY-ESO-1 expression was also positively associated with cell abundance in the TME including NK cells (FC = 0.87, q < 0.0001), monocytes (FC = 0.29, q < 0.05), myeloid dendritic cells (FC = 0.66, q < 0.0001), CD4+ non-reg T cells (FC = 0.54, q < 0.0001), and CD8+ T cells (FC = 0.73, q < 0.05). Similarly, tumors with high NY-ESO-1 expression were associated with higher TIS scores ( q < 0.0001). Conclusions: In our large cohort of GC, tumors expressing high NY-ESO-1 displayed a distinct landscape of immune cells in the TME and were associated with high expression of immune related genes, as well as high TIS score, which has been reported to predict benefit from anti-PD-1 treatment. The results of our analysis support an association between a more immunologically active tumor microenvironment and NY-ESO-1 gene expression which may have relevant implications on immunotherapy treatment for GC.

3523 Background: Defensins are antimicrobial peptides that play important roles in innate immune response. Deregulation of beta-defensin-1 ( DEFB1) gene expression has been implicated in several cancers and we previously showed that single nucleotide polymorphisms in DEFB1 are associated with clinical outcomes in patients with metastatic CRC. Hence, we aimed to further characterize the molecular features associated with DEFB1 gene expression in CRC. Methods: 14416 CRC tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or WES), RNA (WTS) and IHC were analyzed. Top quartile transcripts per million (TPM) for DEFB1 expression were considered high (Q4) while bottom quartile low (Q1). Consensus molecular subtypes (CMS) were assessed using RNAseq. Cell infiltration in the tumor microenvironment (TME) was estimated by QuantiSEQ. X2/Fisher-Exact were used and significance was determined as P-value adjusted for multiple comparison ( Q

177 Background: LRP1B is a member of the low-density lipoprotein receptor family and a tumor suppressor found to be downregulated in colon cancer (CRC). GRM3 is a receptor of glutamate, an amino acid and neurotransmitter. Inhibition of GRM3 reduces CRC cell growth. Recent data from CALGB/SWOG 80405 suggests that mutations (MT) of either LRP1B or GRM3 are associated with better and worse overall survival (OS) in patients treated with bevacizumab (Bev), respectively. We investigate the association of LRP1B or GRM3 mRNA levels with outcomes. Methods: A total of 13,780 CRC tumors (male 7,497, female 6,283) underwent comprehensive molecular profiling (Caris Life Sciences). Analyses included next-generation sequencing of DNA (592 genes, NextSeq, WES, NovaSEQ) and RNA (NovaSeq). Significance with multiple correction was indicated with q, otherwise p value. Gene Set Enrichment Analyses (GSEA) were performed (significance p

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.

477 Background: HCC is increasingly prevalent in older adults with rising incidence and an aging population worldwide. Retrospective studies show older patients with HCC may have an increased survival compared to younger patients. However, data is lacking regarding the genomic and biologic differences, that if identified, would potentially change how we treat this disease in younger vs. older patients. Hence, there is a need to better characterize the molecular landscape of the disease in an age-specific manner. We analyzed the association of age with genomic alterations and therapeutic response to sorafenib in a cohort of advanced HCC that had undergone comprehensive molecular profiling. Methods: 487 HCC samples (excluding variants) were analyzed using Next Generation Sequencing (592 gene panel, NextSeq), Whole Exome and Whole Transcriptome Sequencing (NovaSeq), and IHC at Caris Life Sciences (Phoenix, AZ). PD-L1 positivity was determined by IHC (SP-142 clone, cutoff ≥1, 1%). Tumor mutational burden (TMB) was a measure of total somatic mutations per Mb. Immune cell populations were determined by Microenvironment Cell Population (MCP) counter analysis of RNA expression data. Overall survival (OS) calculated from tissue collection to last contact and time on treatment (TOT) with sorafenib were extracted from insurance claims and calculated using Kaplan-Meier curves. Statistical analysis was done using Chi-square, Fisher Exact and Wilcoxon rank sum tests, with p values adjusted for multiple comparisons and q77: A3 (n=75)). With age, mutational frequencies in CTNNB1 (A1=13.04%, A2=33.43%, A3=38.24%) and TERT (A1=25%, A2=68.84%, A3=76.92%) increased, while ATM (A1=6.52%, A2=0.93%, A3=1.49%) decreased (p0.05). There were fold increases in median TMB (A2/A1=1.33, A3/A1=1.33, p65 (p=0.013). Conclusions: Increased alterations in oncogenic drivers and estimates of CD8+ T cells and B cells were observed in the elderly population with HCC. The enhanced presence of co-inhibitory molecules suggests potential immune evasion. While we observed reduced TOT with sorafenib, additional studies are needed to elucidate the impact of molecular alterations on outcomes with sorafenib and newer therapies (i.e. immunotherapy) in older adults.

534 Background: Incorporating PROs into CTs is critical for patients with pancreatic cancer, as these individuals often experience a high symptom burden and prioritize maximizing quality of life (QOL). Methods: We reviewed protocols of completed U.S. CTs listed on ClinicalTrials.gov that investigated curative/palliative interventions in pancreatic cancer from 1995-2020. We assessed up to August 2021 if CT publications reported PRO results through linked publications and an independent search through PubMed/MEDLINE. We extracted CT details (funding, eligibility, etc), and if PROs (outcome directly reported by patients) were listed as an endpoint (primary/secondary/exploratory). We classified interventions as cancer-directed (e.g. chemotherapy), supportive care (e.g. neurolysis), or other (e.g. curcumin). Results: We reviewed 619 protocols and included 379 in the analysis. Most CTs investigated cancer-directed interventions (317, 83.6%). Only 43 (11.4%) included PROs as endpoints (Table). In these, most of the PROs assessed QOL (34, 79.1%) and pain (15, 34.9%). For the 33/43 (76.7%) protocols that listed a specific PRO instrument, EORTC-QLQ C30 (11/33, 33.3%) was the most common. Only 6 (18.2%) protocols included pancreatic cancer-specific PROs, such as QLQ-PAN26. Supportive care CTs were more likely to assess PROs than cancer-directed CTs (odds ratio, OR= 62.6, 95% CI 16.7-234.3, p

283 Background: Doublet platinum or taxane-based therapies are the current standard backbone of treatment for advanced gastric/GEJ adenocarcinoma. Previously, anthracycline based triplets have been aborted due to lack of efficacy and toxicity. We hypothesize that the combination of FOLFOX and nab-paclitaxel (FOLFOX-A) will have a higher efficacy, a better tolerability, and enhanced patient reported outcomes. Methods: Phase II single arm trial of FOLFOX + nab-paclitaxel in chemotherapy–naïve unresectable, advanced gastric/GEJ adenocarcinoma. Nab-paclitaxel 150 mg/m2 · Oxaliplatin 85 mg/m2 · Leucovorin 400 mg/m2 · 5-FU 2400 mg/m2 over 46-48 hrs every 14 days. Primary objective was response rate.Evaluable disease according to RECIST v1.1 for solid tumors was required. Her-2 positive gastric tumor patients and those with clinically significant peripheral neuropathy were excluded. A Simon (1989), optimal two-stage design was employed. Response rates and Clopper-Pearson confidence intervals are reported. Progression free (PFS) and overall (OS) survival were estimated using the method of Kaplan-Meier. Results: A 29% response rate was observed in Stage 1 and therefore the study continued enrolling all planned 39 patients. Median age was 63 (range 20-80) years, 29 (74%) were male, 30 patients now are off treatment due to progression (17), initiation of alternative therapy (5) and side effects (3). 38 patients started treatment. Of the 35 patients evaluable for response, 14 (40.0%; 95%CI: 23.9% - 57.9%) had CR/PR as best response, with disease control rate of 80.0%. Progression-free (PFS) and overall survival (OS) were available for 36 patients, with median follow-up duration of 20 months (range: 1.9 - 34.45 months). Median PFS was 6.6 months (95%CI: 5.7 – 13.0), with 28.6% (95%CI: 15.2% - 53.9%) 12-month PFS rate. There were 21 deaths, median OS was 11.0 months (95%CI: 9.6 – 21.7), 12-month OS rate was 46.6% (95%CI: 30.9% - 70.3%). Treatment-related grade 3–4 toxicities included peripheral sensory neuropathy, anemia (18.4% each), decreased neutrophil count (15.8%), and diarrhea (7.9%). One patient died due to multi-organ failure, not treatment-related. 23 patients required dose reductions or discontinuation of at least 1 component of the 3-drug regimen due to toxicity. Median number of cycles completed was 4 (range: 0-30). Conclusions: FOLFOX-A has a significant response rate, expected toxicities, and should be considered for future investigation in combination with immunotherapy given the recent approvals.

598 Background: PDAC of the H and B/T differ in embryonic origin, cell composition, blood supply, lymphatic and venous drainage, and innervation. H tumors tend cause symptoms earlier and to present at earlier stages compared to B/T cancers. The impact of PDAC tumor location on patient presentation and survival has been shown in large national data-based analyses, although with conflicting results. We aimed to compare the molecular and tumor immune microenvironment (TIME) profiles of PDAC of the H vs. B/T. Methods: A total of 3499 PDAC samples were analyzed via next-generation sequencing (NGS) of RNA (whole transcriptome, NovaSeq), DNA (NextSeq, 592 genes or NovaSeq, whole exome sequencing) and immunohistochemistry (IHC, Caris Life Sciences, Phoenix, AZ). RNA deconvolution was performed using QuantiSeq (Finotello 2019, Genome Medicine) to quantify the immune cell infiltration. Pathway gene enrichment analyses were done using Gene Set Enrichment Analysis (GSEA, Subramaniam 2015, PNAS). Significance was determined as p values adjusted for multiple correction (q) of < 0.05. Results: Anatomic subsites of PDAC tumors were grouped by primary tumor sites into H (N = 2058) or B/T (N = 1384). There were significantly more metastatic tumors profiled from H vs. B/T (57% vs. 44%, p < 0.001). KRAS mutations (93.8% vs. 90.2%), genomic loss of heterozygosity (12.7% vs. 9.1%), and several copy number alterations ( FGF3, FGF4, FGF19, CCND1, ZNF703, FLT4, MUTYH, TNFRS14) trended higher in B/T when compared to H (p < 0.05 but q > 0.05). GNAS mutations (2.2% vs. 0.7%) trended higher in H vs. B/T (p < 0.05). No significant difference in immuno-oncology (IO) markers (TMB, PD-L1, MSI-H) were observed, but expression analysis of IO-related genes showed significantly higher expression of CTLA4 and PDCD1 in H (q < 0.05, fold change 1.2 and 1.3) and IDO1 and PDCD1LG2 expression trended higher in B/T (p < 0.05, fold change 0.95). When comparing median cell abundance values as part of TIME analysis, H had increased immune infiltration of B cells (0.045 vs. 0.043), M2 macrophages (0.035 vs. 0.032), neutrophils (0.056 vs. 0.052), NK cells (0.027 vs. 0.026), CD8+ T cells (% > 0: 48.2% vs. 43.2%), while B/T had increased infiltration of M1 macrophages (0.035 vs. 0.032) (all q < 0.05). GSEA showed enrichment of CTLA4 (normalized enrichment score (NES) 1.6, false discovery rate (FDR) 0.19) and primary immunodeficiency pathway enrichment (NES 1.7, FDR 0.11) in H. Conclusions: To our knowledge, this is one of the largest cohort of PDAC tumors subjected to broad molecular profiling. Differences in IO-related gene expression and TIME cell distribution suggest that response to IO therapies may differ in PDAC arising from H vs B/T. Subtle differences in the genomic profliles of H vs. B/T tumors were also observed.

156 Background: Emerging evidence suggests subsets of the Hippo pathway have multiple functions of tumor development and immune response regulation. Increased understanding of the molecular characteristics of its signaling pathways and the impact on immune cell trafficking will be critical to develop colorectal cancer (CRC) therapies. Methods: A total of 13,008 CRC tumors were analyzed at Caris Life Sciences (Phoenix, AZ) with whole transcriptome and whole exome sequencing (NovaSeq). MSI-H/dMMR was tested by NGS (next generation sequencing), immunohistochemistry (IHC), and fragment analysis. Tumor mutational burden (TMB)-High was determined with a 10-mt/MB cutoff. RNA-deconvolution using QuantiSEQ was used to assess immune cell infiltration in the tumor microenvironment. Consensus molecular subtypes (CMS) were developed using RNA expression data. Gene expression was reported as transcripts per million. Z-score totals of 6 core Hippo genes were calculated in groups: MST1+ STK3 (G1), LATS1+ LATS2 (G2), YAP1+ WWTR1 (G3), and all 6 genes together (G4). Tumors with bottom quartile (QL) z-scores were compared with the top quartile (QH) using χ2/Fisher-Exact test and adjusted with the Benjamini-Hochberg method: adjusted p + T cells were seen in QH than QL with a median fold change of 1.39. G1–4 z-scores all positively correlated with the expression of the analyzed immune-related genes. The highest Spearman rho averages were in HAVCR2 (0.54), CD86 (0.54), CD80 (0.53), PD-L2 (0.5), CD274 (0.46), and LAG3 (0.36). Significantly different mutation rates were seen in QH compared to QL in G1 ( TP53, KRAS, PIKCA, SMAD2, AMER1), G2 ( APC), G3 ( PIK3CA, APC), and G4 ( TP53, PIK3CA, KRAS). Conclusions: The Hippo pathway correlated with immune cell trafficking suggesting that YAP1/TAZ signaling may play a critical role in the immune responses. These findings may help develop novel therapeutic strategies targeting the Hippo pathway combined with immune therapies in CRC.

322 Background: Natural language processing (NLP) algorithms can be leveraged to better understand prevailing themes in healthcare conversations. Sentiment analysis, an NLP technique to analyze and interpret sentiments from text, has been validated on Twitter in tracking natural disasters and disease outbreaks. To establish its role in healthcare discourse, we sought to explore the feasibility and accuracy of sentiment analysis on Twitter posts (‘’tweets’’) related to prior authorizations (PAs), a common occurrence in oncology built to curb payer-concerns about costs of cancer care, but which can obstruct timely and appropriate care and increase administrative burden and clinician frustration. Methods: We identified tweets related to PAs between 03/09/2021-04/29/2021 using pre-specified keywords [e.g., #priorauth etc.] and used Twarc, a command-line tool and Python library for archiving Twitter JavaScript Object Notation data. We performed sentiment analysis using two NLP models: (1) TextBlob (trained on movie reviews); and (2) VADER (trained on social media). These models provide results as polarity, a score between 0-1, and a sentiment as ‘’positive’’ (>0), ‘’neutral’’ (exactly 0), or ‘’negative’’ (

3091 Background: Gene fusions often represent critical therapeutic targets across cancer subtypes. Fusions within the ErbB family of receptor tyrosine kinases, including EGFR, ERBB2 ( HER2) and ERBB4 ( HER4), have been previously described and represent potentially actionable alterations. Here, we report the relative incidence and functional characterization of these rare genomic events. Methods: Tumor samples (n = 64,354; representing > 40 tumors types) submitted to Caris Life Sciences (Phoenix, AZ) were molecularly profiled by next-generation sequencing of DNA (NextSeq, 592-gene panel; or NovaSeq, whole exome) and RNA (whole transcriptome). Gene fusion partners, in/out-of-frame status, retention of ERBB kinase domain, and topology of fusion breakpoints were characterized for each ERBB fusion transcript detected. Fusion prevalence was further examined in public data sets (TCGA, MSK-IMPACT and AACR GENIE). Results: From the Caris database, a total of 64 EGFR fusion isoforms were detected in 59 tumors (incidence 0.09%); 83% were in-frame and 91% retained the EGFR kinase domain. 206 ERBB2 fusion isoforms were detected in 114 tumors (0.18%); 37% were in-frame and 34% retained the ERBB2 kinase domain. 131 ERBB4 fusion isoforms were detected in 108 tumors (0.17%); 62% were in-frame and 51% retained the kinase domain. All fusions were detected at low incidence across all tumor types. EGFR fusions were most common in high grade glioma (1.7%, n = 35), largely driven by recurrent EGFR-SEPT14 fusions (n = 20). ERBB2 fusions were most common in esophageal/gastroesophageal junction carcinoma (1.1%, n = 20), with recurrent fusion to PGAP3 observed in multiple tumor types (n = 37). ERBB4 fusions were most common in ovarian (0.7%, n = 40) and bladder (0.7%, n = 15) cancers, which often resulted from recurrent fusion with IKZF2 (n = 36). EGFR and ERBB2 fusions were generated predominantly (44-48%) from inversion events, while ERBB4 fusions arose more frequently and at similar rates (27-32%) from deletions, duplications, or translocations. Mining of public data sets corroborated the prevalence of ERBB gene fusions: the frequency of EGFR fusions was 0.63%, ERBB2 was 0.14% and ERBB4 was 0.04%. TP53 mutations frequently co-occurred with ERBB2 and ERBB4 fusions ( > 60% average across public data sets), with higher co-mutation rates ( > 70%) observed for samples in the Caris database. Conclusions: ERBB gene fusions are detectable at low frequency in various tumor types and may represent a unique genomic subset of cancer. Identification of novel ERBB gene fusions warrants further investigation to determine the potential pathogenicity and actionability of these fusions.

3563 Background: The tumor microenvironment (TME) of colorectal cancers (CRC) is modulated by oncogenic drivers such as KRAS. The TME comprises a broad landscape of immune infiltration. How tumor genomics associates with the immune cell landscape is less known. We aim to characterize immune cell types in RAS wild-type (WT) and mutant (MT) CRC, and to examine the prevalence of immuno-oncologic (IO) biomarkers (e.g. tumor mutation burden (TMB), PD-L1, MSI-H/dMMR) in these tumors. We performed genomic and transcriptomic analysis to confirm associations of mutant RAS with immune infiltration of the TME conducive to metastasis vs. potential response to immunotherapies. Methods: A total of 7,801 CRC were analyzed using next-generation sequencing on DNA (NextSeq, 592 Genes and WES, NovaSEQ), RNA (NovaSeq, whole transcriptome equencing) and IHC (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by FA, IHC and NGS. TMB-H was based on a cut-off of > 10 mutations per MB). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons (q) was = than 50 yrs. In MSS tumors, there was a significantly higher neutrophil infiltration in KRAS MT (median cell fraction 6.6% vs. 5.9%) and NRAS MT (6.9%) overall and also when individual codons were studied. B cells, M2 macrophages, CD8+ T cells, dendritic cells and fibroblasts were lower in KRAS mutant tumors; B cells and M1 macrophages are lower in NRAS (q

3120 Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) are effective therapies for some patients with both germline and somatic BRCA1/2 mutations (MTs) or with homologous recombination repair deficiency (HRD). We aimed to characterize molecular differences between BRCA1 and BRCA2 MTs and their prognostic and/or predictive impact on PARPi outcomes in various cancer subtypes using real world data (RWD). Methods: Tumor samples obtained from patients with 35 types of cancer were analyzed by whole exome sequencing (WES, Novaseq) at Caris Life Sciences (Phoenix, AZ). High genomic loss of heterozygosity (gLOH-H) was defined as LOH-H in ≥16% of tested loci. MSI/MMR was tested by fragment analysis, IHC, and WES. Overall survival (OS) extracted from insurance claims was calculated from start of treatment or tissue collection until last contact or death using Kaplan-Meier curves. P-values adjusted for multiple comparisons (q-value of < 0.05 was considered to be significant). Results: In total, 17,640 tumors were included, of which 776 (4.3%) had tumor-based BRCA1/2 MTs. BRCA1/2 MTs were most commonly seen in ovarian (N = 221/2187, 10.1%), breast (138/2506, 5.5%), prostate (61/1131, 5.4%), pancreatic (48/1430, 3.4%), and non-small cell lung (100/4046, 2.5%) cancers. BRCA2 MTs were more frequent than BRCA1 except in ovarian cancers. BRCA1 MTs were more common in younger pts (median age, 61 vs 65 years, p

3092 Background: ADP-ribosylation factor 6 ( ARF6) is a small GTPase in the RAS superfamily, which regulates membrane trafficking, remodeling and tumor progression. Preclinical study shows that TP53 and KRAS cooperatively activate the ARF6-AMAP1 pathway which serves as a link by which pancreatic driver mutations promote tumor invasion, PD-L1 dynamics and immune evasion properties in pancreatic ductal adenocarcinoma (PDAC). The clinical impact of ARF6 on cancer progression and prognosis remains unclear. Methods: A total of 2,948 PDAC samples were analyzed using next-generation sequencing of RNA (whole transcriptome, NovaSeq) and DNA (NextSeq, 592 genes or NovaSeq, whole exome sequencing) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). QuantiSeq (Finotello 2019, Genome Medicine) was used to quantify immune cell infiltration. Overall survival (OS) was obtained from insurance claims, and Kaplan-Meier estimates were calculated for molecularly defined cohorts. Significance was determined as p values adjusted for multiple correction ( q) of

3527 Background: Globo H is a carbohydrate antigen that is highly expressed on the cell surface of epithelial cancers but not in normal tissue, and has been reported to correlate with poor prognosis. An attractive therapeutic target, Globo H-targeted agents are being tested in early clinical trials (e.g., OBI-833, a Globo H antigen conjugated to a mutated diphtheria toxin with potential antineoplastic activities, and OBI-999, an antibody-drug conjugate (ADC) consisting of a Globo H monoclonal antibody with a synthetic antineoplastic agent). We aim to describe the molecular features associated with Globo H expression in CRC. Methods: A total of 7,604 CRC tumors were tested by Caris Life Sciences (Phoenix, AZ) by NextGen DNA and RNA sequencing. The expression of β3GalT5, FUT-1 and FUT-2 were evaluated as surrogates for Globo H expression as they are the key enzymes in its biosynthesis. An average z-score of the 3 genes (GloboH) and of β3GalT5 (B3) alone were calculated; tumors with top quartile z-scores were considered expression-high (Q4) and bottom quartile, expression-low (Q1). QuantiSEQ was used to assess immune cell infiltration in the tumor microenvironment (TME). Statistical significance was determined using chi-square/Fisher-Exact and adjusted for multiple comparisons (q=10) (11.4% vs. 8.3%), PD-L1 positive (5.7% vs. 3.4%) and MSI-H/dMMR (8.3% vs. 5.5%). Strong positive associations were seen with mutations in BRAF, KRAS, RSPO3 fusion, and cMYC amplification with B3 alone and GloboH (all q

433 Background: The SLUG gene plays an important role in EMT by repressing E-cadherin and promotes metastasis. Previous data suggest that overexpressed SLUG gene in pancreatic cancer (PC) showing a high frequency of metastasis and poor prognosis. As SLUG contribution to characteristics or metastatic features remains elusive, we clarified its functional roles in PC progression. Methods: A total of 2958 pancreatic tumors were analyzed using Whole Transcriptome sequencing, NextGen Sequencing (NGS) (NextSeq, 592 gene panel) or Whole Exome Sequencing (WES) (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Microsatellite instability (MSI) status was tested by fragment analysis, immunohistochemistry (IHC) and NGS. PD-L1 expression was tested by IHC. Tumor mutational burden (TMB) was measured by counting all mutations found per tumor (a universal cutoff point of ≧10 mutations per MB). Immune cell fraction was calculated by quanTIseq (Finotello 2019, Genome Medicine). Results: A total of 1274 primary and 1684 metastatic pancreatic tumors were included for this study. They were divided equally into four classes in each group, according to their SLUG expression levels. Tumors in the highest quartile of SLUG expression (QH) showed significantly higher frequency in peritoneal-retroperitoneal-omentum metastasis (15.0%) compared to the lowest quartile (QL) (4.8%) (p = .0001). Similar trends were seen in the abdomen (6% vs 1%, p = .001) and bone (2.8% vs 0.0%, p = .005). However, liver (55.0% in QH vs 63.1% in QL) and lung (2.8% vs 14.1%) metastasis occurred most frequently in QL and the least frequently in QH (p = .0197 and p = .001, respectively). This data indicated that tumors with high SLUG gene expression levels tend to lead to disseminated metastasis, and with low expression levels, they tend to spread intravascularly. We detected significant differences among genetic mutations in ATM (5.7% in QL vs 1.8% in QH, p < 0.001) and APC (2.9% vs 0.5%, p < 0.001), and Wnt signaling expressions were higher in QL (4.6%) than QH (0.7%) (p < 0.001). Binary TMB-H and MSI-H tumors had higher frequencies in QL (2.7% and 2.1%) compared to QH (0.3% and 0.1%) (p < 0.001 in both). Contrastingly, PD-L1 expression levels were higher in QH (23.4%) compared to QL (11.0%) (p < 0.001) and had a linear relationship with the expression levels. The median values of the population of B cells, M1 and M2 macrophages were significantly higher in QH compared to QL, but those of myeloid dendritic and CD8+T cells conversely decrease as the SLUG expression increases. Conclusions: Our data indicated the SLUG expression level could determine the tumor characteristics in progression, especially the pattern of metastasis in PC, and it could possibly predict the prognosis and/or therapeutic effects. We also showed immune oncologic markers which have some relationships with SLUG expressions. Further investigation is warranted to better understand SLUG gene functions.

116 Background: Cachexia affects many cancer patients. Growth differentiation factor-15 (GDF15) is a protein that regulates weight and the stress response of cells. The GDF15 gene encodes a ligand of TGF-beta that triggers cachexia and modulates the progression from tumorigenesis to metastasis. Inhibition of GDF15 with an antibody restored muscle mass and fat in animal models. Serum levels rise in proportion to the progression of colon cancer, predict outcome, and have been correlated with CEA. Methods: We retrospectively reviewed 7607 CRC tumors profiled by Caris Life Sciences (Phoenix, AZ) from 2019 to 2020. Profiling included whole transcriptome sequencing (RNA-Seq by NovoSeq). Tumor mutational burden, mismatch repair status, and pathway genomic alterations were evaluated. QuantiSEQ was used to assess immune cell infiltration in the tumor microenvironment. Results: GDF15 expression ranged from 0 to 593 transcripts per million (TPM) with median of 30 (IQR = 15.02). There was no association with age, sex, or primary tumor sidedness. MSI-H/dMMR tumors had higher GDF15 expression (median 37 vs 30, p = 0.0004); TMB > = 17 tumors was seen in 5.9% of bottom quartile (Q1) GDF15 expressors and 8.3% of top quartile (Q4). PDL1 IHC positivity was inversely correlated with GDF15 expression (7.1% in Q1 vs. 2.6% in Q4, p < 0.0001). Genomic alterations associated with higher GDF15 expression (Q4 vs Q1) included genes on TGF-B (SMAD2/4), PI3K (PIK3CA, MTOR), chromatin remodeling (ARID1A, KMT2C), DDR (ATM) and Wnt pathway (APC); those inversely associated included MYC CNA and TP53. Q1 tumors had higher CNA of ERBB2 and FGFR1. Relative neutrophils and NK cells in the TME increased from Q1 to Q4 (p < 0.001). There was a decrease in CD8+ T-cells and Treg cells from Q1 to Q4. Conclusions: GDF15 expression correlates with increased dMMR/MSI-H and TMB, but not with PDL1 expression. Mutations and activated pathways associated with GDF15 expression may explain increased cachexia with more aggressive disease. The association with chromatin remodeling may warrant therapies targeting histone modification and epigenetics. The increase in NK cells but decrease in CD8+ T cells in the TME with increasing GDF15 suggests approaches to treatment. Higher CD8+ lymphocyte counts correlate with PFS with immunotherapy. Anti-PD-L1 therapy reinvigorates the killing function of CD8+ T cells. The decrease in CD8+ T cells and PDL1 positivity with rising GDF15 suggests worse outcome and a lack of response to anti-PDL1 therapy. NK cell checkpoint inhibitors, CARs, and an anti-GFRAL antibody are now in clinical trials and might be utilized in high GDF15 cancers. GDF15 is emerging as a target in the treatment of obesity and cachexia and as a prognostic marker in oncology. Understanding its expression in metastatic colon cancer may reveal which patients could benefit from developing anti-GDF15 targeted therapies against cancer progression.

4066 Background: NF1 encodes neurofibromin, which is a key GTPase-activating protein that downregulates RAS activation. Inactivating mutations in NF1 result in sustained activation of RAS signaling, a key driver for development of colorectal cancer (CRC), and have been suggested to be a potential mechanism of resistance to EGFR inhibition in RAS-wild type (WT) CRC. Little is known about molecular characteristics of NF1-mutated (MT) CRC. Methods: Tumor profiles from 8150 CRC patients (pts) with available NF1 mutation status were retrospectively reviewed. NextGen sequencing by a customized 592-gene panel was performed. Microsatellite instability (MSI) / mismatch repair (MMR) status, tumor mutational burden (TMB) and PD-L1 expression were tested. Molecular profiles between NF1-MT and NF1-WT pts were compared. Results: Out of 8150 pts, 176 (2.2%) had somatic NF1 mutations with pathogenic or presumed pathogenic function. A higher NF1-MT frequency was observed in MSI-H/dMMR vs MSS/pMMR (13.5% vs 1.4%, p < 0.0001), in right-sided vs left sided (2.9% vs 1.8%, p < 0.01), and in RAS-WT vs RAS-MT (3.0% vs 1.4%, p < 0.0001). In MSS/pMMR tumors, no association with sidedness was observed (right: 1.3% vs left: 1.2%, NS). The most prevalent co-mutations with NF-1 were APC (63.2%), ARID1A (57.5%), TP53 (51.5%), KMT2D (32.9%) and KRAS (32.4%) in all cases, and APC (76.2%), TP53 (69.5%), KRAS (38.8%), ARID1A (34.4%) and FBXW7 (21.5%) in MSS/pMMR cases. POLE mutation was observed in 18.4% of NF1-MT/MSS/pMMR pts. Compared to NF1-WT pts, NF1-MT pts had more frequent mutations in ARID1A (All: 57.5% vs 23.3%, p < 0.0001; MSS/pMMR: 34.4% vs 15.2%, p < 0.05), and less frequent mutations in KRAS (All: 32.4% vs 49.0%, p < 0.0001; MSS/pMMR: 38.8% vs 50.3%, p < 0.05). Also, NF1-MT pts had more frequent alterations in homologous recombination pathway compared to NF1-WT pts (All: 39.8% vs 7.5%, p < 0.0001; MSS/pMMR: 17.5% vs 4.4%, p < 0.0001). Mean TMB was significantly greater in NF1-MT than NF1-WT (All: 48.9/Mb vs 10.0/Mb, p < 0.0001; MSS/pMMR: 48.3/Mb vs 8.2/Mb, p < 0.0001). Also, PD-L1 positivity was higher in NF1-MT compared to NF1-WT (All: 12.9% vs 3.6%, p < 0.0001; MSS/pMMR: 7.1% vs 2.6%, p < 0.05). Conclusions: While more frequent than in RAS-MT pts, NF1-MT CRC was a small subset in RAS-WT pts. NF1-MT was associated with alterations in chromatin remodeling and DNA damage response pathways, as well as elevated TMB and PD-L1 expression, which may provide alternative therapeutic strategies beyond EGFR inhibition.