Cell-inflamed Signature Research Articles

Abstract 7037: Comprehensive molecular and immune profiling of triple negative invasive lobular carcinoma

Abstract Background: Triple-negative invasive lobular carcinoma (TN-ILC) is a rare (0.1-1.4%) breast cancer with prognosis worse than ER positive ILC. Currently there are no targeted therapies or clinical trials specifically for TN-ILC. A comprehensive analysis of the molecular and immune landscape can help identify novel targets and pathways for TN-ILC to improve patient outcomes. Here, we characterized the molecular and immune signature of TN-ILC. Methods: 11,760 breast cancer samples (Invasive ductal (ID) TNBC, n=364; TN-ILC, n=31) were analyzed (592, NextSeq; WES, NovaSeq), (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) totaled somatic mutations per tumor (high ≥10 mt/MB) was tested by NGS. Microsatellite-instability (MSI) was tested by NGS. Immune cell fractions were calculated by deconvolution of WTS. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: TN-ILC had higher frequency of CDH1 (0.83% vs. 66.67%), ERBB2 (0.84% vs.28.57%), ARID1A (1.65% vs. 23.33%), CBFB (0.55% vs.16.67%), AKT1 (2.22% vs.16.13%) and KMT2C (3.83% vs.13.79%), but lower frequency of TP53 (89.7%5 vs. 38.71%) mutations (all p<0.05) compared to ID-TNBC. TN-ILC had higher frequency of TMB high (23.3% vs. 5.2%, p<0.05) but there was no difference in dMMR/MSI-H (0% vs 1.1%, p=1). TN-ILC had higher AR RNA (FC: 14.2), protein expression (80.6% vs. 24.8%) and higher frequency of fusion variant-AR (16.1% vs 4.9%) (all p < 0.05) compared to ID-TNBC. Analysis of inferred immune cell infiltrates showed that TN-ILC had higher infiltration of M2 macrophages (5.34% vs. 2.94%) and neutrophils (4.83% vs. 2.65%) but lower infiltration of M1 macrophages (3.46% vs 2.17%) and CD8 T cells (0.58% vs. 0.11%) (all p<0.05). TN-ILC had lower T cell inflamed signature (16.1% vs 35.7%, p<0.05), decreased immune checkpoint genes (CD274, CTLA4, FOXP3, LAG3, IDO, FC: 1.2-2.7, all p < 0.05), PD-L1 protein expression (SP142: 30.4% vs. 50.2%, p=0.06; 22c3: 15.4% vs. 42.6%, p = 0.05) and differential expression of BCL2 family genes (upregulation: BIK, FC: 2.2; downregulation: BAX, BAK1, BID, PMAIP1, MCL1, BCL2A1, BCL2L10, FC: 1.0-6.3, all p < 0.05) compared to ID-TNBC. Conclusion: These data suggest that TN-ILC had higher frequency of CDH1, ERBB2, AKT1, ARID1A mutations, higher M2 macrophages and neutrophils and lower M1 macrophages and CD8 T cells infiltration and, lower T cell inflamed signature. High TMB and AR expression can translate into use of immunotherapy (ICI) and AR antagonists in these patients. Additonal analysis to determine the optimal biomarker for ICI response in TN-ILC is needed. Results of our study need to be validated in larger studies with survival correlation. Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Jose Leone, Priya Jayachandran, Stephanie Graff, Mathew Oberley, George Sledge, Asher Chanan-Khan. Comprehensive molecular and immune profiling of triple negative invasive lobular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7037.

Abstract 3890: Comprehensive molecular and immunological characterization of early-onset esophagogastric cancer

Abstract Background: The incidence of early-onset esophagogastric cancer (EOEGC), defined as age of diagnosis <50, has increased in recent decades. Compared to average-onset esophagogastric cancer (AOEGC), EOEGC are more likely to be genomically stable, have diffuse histology, and present with more advanced disease. Here, we utilized a large real-world data cohort to characterize EOEGC with molecular and immune signatures. Methods: 5,175 esophagogastric cancer (EGC) samples (<50 yrs, n=530; ≥50 yrs, n=4645) were tested by next generation sequencing (NGS) (592, NextSeq; WES, NovaSeq) and whole transcriptome sequencing (WTS) (NovaSeq) (Caris Life Sciences, Phoenix, AZ). EGC patients with age <50 and ≥50 were classified as EOEGC and AOEGC, respectively. Deficient mismatch repair (dMMR)/microsatellite-instability (MSI) was tested by IHC and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high>10 mt/MB). Pathway enrichment determined by Gene Set Enrichment Analysis (Broad Institute, Cambridge, MA). Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: EOEGC had higher frequency of CDH1 (18.9% vs 6.5%) but lower frequency of TP53 (65.9% vs 74.4%) mutations compared to AOEGC (all q<0.05). EOEGC had higher frequency of CCNE1 (6.8% vs 4.4%), MYC (6.1% vs 4.1%) and FGFR2 (3.45% vs 1.87%) copy number alterations and ARHGAP26 (5.67% vs 2.2%) fusion. EOEGC had lower frequency of TMB high (3.8 vs 10.1%) and dMMR/MSI-H (1.3% vs 5.7%) (all p<0.05). EOEGC had pathway enrichment of epithelial mesenchymal transition (EMT) (NES: 1.8, FDR: 0.0) and angiogenesis (NES: 1.3, FDR: 0.24). EOEGC had lower median MAPK pathway activity score (MPAS) (-0.16 vs 0.13, p<0.05). Analysis of inferred immune cell infiltrates showed that EOEGC had increased infiltration of B cells (4.7% vs 3.9%) and M2 macrophages (3.6% vs 3%) but decreased infiltration of M1 macrophages (3.7% vs 4%) (all q<0.05). EOEGC had increased T cell inflamed signature (31.3% vs 26.1%, p<0.05) and increased expression of HAVCR2 (TIM3) but decreased expression of FOXP3 and CD274 (PD-L1) (FC: 0.84-1.09, all p<0.05). There was no difference in LAG3, CTLA4, and IDO1 gene expression. Conclusion: EOEGC has several unique molecular and tumor immune microenvironment features. In this real world cohort, EOEGC has increased frequency of CDH1 mutations, ARHGAP26 fusions, enrichment of EMT and angiogenesis pathways, decreased MAPK pathway activity, decreased frequency of TMB high and dMMR/MSI-H, and a unique immune cell infiltrate with decreased M1 macrophages and increased M2 macrophages. These unique differential characteristics present therapeutic opportunities but also demonstrate the limitations of currently approved therapies in this subset of patients. Citation Format: Lawrence W. Wu, Sachin K. Deshmukh, Sharon Wu, Joanne Xiu, Alex Farrell, Vincent Lam, Emil Lou, Sanjay Goel, Chadi Nabhan, Ryan H. Moy. Comprehensive molecular and immunological characterization of early-onset esophagogastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3890.

Identification and characterization of immunogenic neoantigens in colorectal cancer (CRC).

161 Background: Presentation of neoantigens, short peptides derived from tumor-specific somatic mutations, by HLA molecules triggers the activation of anti-tumor immune response. Recognition of neoantigens by autologous T cells promotes sensitivity to immune checkpoint inhibitors (ICI) in mismatch repair deficient (MMRd)/microsatellite instability high (MSI-H) tumors. Neoantigen-targeted reactivity has also been reported in microsatellite stable (MSS) tumors, and neoantigens are emerging targets for tumor vaccines and adoptive cell therapy. Here, we aimed to comprehensively assess the spectrum of immunogenic neoantigens in CRC to identify new subpopulations for these therapeutic strategies. Methods: 7,053 CRC tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (720-gene panel) and RNA (whole transcriptome) were analyzed. MSI status was determined by immunohistochemistry of MMR proteins 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 in the tumor microenvironment (TME) was estimated from RNA expression profiles using MCP-Counter. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed signature, TIS). Results: 6,600,005 unique peptide:allele interactions were tested, of which 368,468 (5.6%) were predicted neoantigens with patient-specific HLA alleles, including 16,184 (0.25%) with high affinity. MMRd/MSI-H tumors (6.9%) had higher neoantigen load at all affinity levels compared to MSS, with 10,243 (63.3%) and 5,621 (34.7%) of the predicted high affinity neoantigens exclusively associated with MSS and MMRd/MSI-H, respectively. 32 recurrent high affinity neoantigens were identified in > 10 samples, most frequently derived from APC mutations in MSS tumors (78% of recurrent neoantigens), and from RNF43 (25%), ASXL1 (20%), and KMT2D (15%) in MMRd/MSI-H. TIS scores positively correlated with the abundance of various immune cell populations in the TME, notably cytotoxic lymphocytes (r = 0.46). Among the recurrent high affinity neoantigens, those with the highest mean TIS scores (> 90th percentile) resulted from mutation of MSH3 (K383fs, 5.1%) and KMT2D (multiple variants, 2.8%) in MMRd/MSI-H CRC, while those in MSS CRC derived from SOX9 (multiple variants, 0.2%) and APC (multiple variants, 0.7%) but were not associated with increased mean TIS scores (~55th percentile). Conclusions: This is one of the largest studies to investigate the landscape of immunogenic neoantigens in CRC. We were able to identify candidate recurrent peptides with high HLA binding affinity and an association with a positive TIS signature supporting the role of neoantigens as potential cancer immunotherapy targets.

Identification and characterization of immunogenic neoantigens in biliary cancer (BC) and pancreatic cancer (PC).

552 Background: Recognition of tumor neoantigens by autologous T cells activates immune surveillance and has been reported to promote sensitivity to immune checkpoint inhibitors (ICI) in mismatch repair deficient (MMRd)/microsatellite instability high (MSI-H) tumors. Neoantigen-targeted reactivity has also been reported in microsatellite stable (MSS) tumors. Neoantigens are emerging targets for novel immunotherapy strategies, including tumor vaccines, in BC and PC. We aimed to comprehensively assess the spectrum of immunogenic neoantigens in BC and PC. Methods: 3728 tumor specimens (1389 BC; 2339 PC) tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (720-gene panel) 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 in the tumor microenvironment (TME) was quantified using the MCP Counter method. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed signature, TIS). Results: MMRd/MSI-H rate was 1.8% in BC and 1.4% in PC. 117219 unique peptide:allele interactions with predicted binding-level affinity for patient-specific HLA alleles were identified (48781 in BC; 71182 in PC). MMRd/MSI-H tumors had higher neoantigen load at all affinity levels compared to MSS. Only 4 recurrent neoantigens with binding affinity (observed in >10 samples) were identified in BC and derived from mutations in KRAS (3/4) and IDH1 (1/4), while 78 were found in PC, mostly associated with mutations in KRAS (53/78) and TP53 (16/78). The frequency of individual neoantigens was particularly low in MSS BC (< 2%). Across both cancer types, TIS scores positively correlated with the abundance of immune cell populations in the TME, notably cytotoxic lymphocytes (r > 0.40). Recurrent neoantigens associated with highest average TIS scores resulted from mutation of KRAS (G12D/V, 1.8%/1.5% of samples, respectively) in BC yet mean TIS scores were low (~60th percentile overall). Similarly, TP53 (Y220C/R273C, 0.6%/0.7%, respectively) and CDKN2A (multiple variants, 0.5%) were associated with the highest yet relatively low mean TIS scores in MSS PC (~73th percentile) compared to mean TIS scores associated with MSH3 (K383fs, 12%) and KRAS (G12D, 12%) in MMRd/MSI-H PC (~90th percentile). Conclusions: This is the largest study to investigate the landscape of immunogenic neoantigens in BC and PC. The frequency of high-level binding affinity neoantigens was relatively low and associated with relatively lower TIS scores in MSS tumors, which may contribute to the immunogenic cold TME characterizing these tumor types.

Comprehensive Immune Profiling Unveils a Subset of Leiomyosarcoma with "Hot" Tumor Immune Microenvironment.

Purpose: To investigate the immune biomarker in Leiomyosarcoma (LMS), which is rare and recognized as an immune cold cancer showing a poor response rate (<10%) to immune checkpoint inhibitors (ICIs). However, durable response and clinical benefit to ICIs has been observed in a few cases of LMS, including, but not only, LMS with tertiary lymphoid structure (TLS) structures. Patients and methods: We used comprehensive transcriptomic profiling and a deconvolution method extracted from RNA-sequencing gene expression data in two independent LMS cohorts, the International Cancer Genome Consortium (ICGC, N = 146) and The Cancer Genome Atlas (TCGA, N = 75), to explore tumor immune microenvironment (TIME) in LMS. Results: Unsupervised clustering analysis using the previously validated two methods, 90-gene signature and Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), identified immune hot (I-H) and immune high (I-Hi) LMS, respectively, in the ICGC cohort. Similarly, immune active groups (T-H, T-Hi) were identified in the TCGA cohort using these two methods. These immune active ("hot") clusters were significantly associated, but not completely overlapping, with several validated immune signatures such as sarcoma immune class (SIC) classification and TLS score, T cell inflamed signature (TIS) score, immune infiltration score (IIS), and macrophage score (M1/M2), with more patients identified by our clustering as potentially immune hot. Conclusions: Comprehensive immune profiling revealed a subset of LMS with a distinct active ("hot") TIME, consistently associated with several validated immune signatures in other cancers. This suggests that the methodologies that we used in this study warrant further validation and development, which can potentially help refine our current immune biomarkers to select the right LMS patients for ICIs in clinical trials.

Molecular profiling and characterization of the tumor immune microenvironment (TME) in appendiceal carcinoma (AC).

3622 Background: The rarity of AC presents challenges in understanding disease pathogenesis. We previously showed that AC has higher rates of mutations in KRAS and GNAS and lower rates of TP53, APC, and PIK3CA than CRC. The appendix also has many lymphoid clusters and regulates IgA production in the large bowel, suggesting that AC may be subject to more lymphocytic regulation than CRC. We sought to characterize the molecular profile and TME across AC histopathological types. Methods: AC samples were analyzed by DNA sequencing (592 genes, NextSeq, or whole exome sequencing, NovaSeq), whole transcriptome sequencing (WTS, NovaSeq), and immunohistochemistry (IHC) for molecular profiling, including microsatellite instability (MSI), mismatch repair (MMR), PD-L1 (SP142), and tumor mutational burden (TMB). Microenvironment Cell Population-counter Quantiseq was used to quantify tumor immune contexture using WTS. AC histopathology was derived from pathology reports. Mann-Whitney U and ChiSquare tests were applied as appropriate, with P-values adjusted for multiple comparisons using Benjamini-Hochberg. Results: AC (N = 731) were grouped by histology: 5% goblet cell (GC), 6% high-grade adenocarcinoma (HGA), 14% low grade mucinous (LGM), 33% mucinous adenocarcinoma (MA), 1% pseudomyxoma peritonei (PMP), 6% signet ring cell carcinoma (SRC), and 35% adenocarcinoma not otherwise specified (NOS) (see Table). Age at collection and PD-L1+ (0-5.2%) did not differ by histopathology. Seven patients had dMMR (4 NOS and 3 MA). Median TMB was significantly higher in NOS vs. GC (4 mutations/megabase vs 2, q &lt; 0.001), NOS vs. LGM (4 vs 3, q = 0.048), MA vs. GC (4 vs 2, q &lt; 0.001), and MA vs. LGM (4 vs 3, q = 0.037). Distinct TME patterns were observed in NOS vs. MA (median cell fraction: dendritic cells 0.07 vs 0, q &lt; 0.01; M2 macrophages 0.055 vs 0.042, q = 0.030; natural killer cells 0.034 vs 0.031, q = 0.011; CD4 T cells 0.006 vs 0, q = 0.044) and GC vs. MA (M1 macrophages 0.039 vs 0.057, q = 0.021; dendritic cells 0.012 vs 0, q &lt; 0.01; B cells 0.061 vs 0.050, q = 0.013; M2 macrophages 0.074 vs 0.042, q &lt; 0.01; natural killer cells 0.041 vs 0.031, q = 0.01; and neutrophils 0.017 vs 0.038 q = 0.045). T cell inflamed signature (TIS) and immune-oncology (IO) marker gene expression were higher in HGA and lower in LGM and MA. Somatic mutation rates differed by histopathology (see Table). Conclusions: There is significant heterogeneity in TMB, TME, and mutational profiles across AC histologies. MA has a particularly immune-cold TME shown by lower infiltration of lymphocytes, TIS, and IO gene expression. These findings are critical to identify novel biomarkers and develop new therapeutic strategies for AC. [Table: see text]

Large-scale transcriptomic profiling of the tumor immune microenvironment in ALK+ lung cancer.

9020 Background: Patients (pts) with ALK+ Non-Small Cell Lung Cancer (NSCLC) do not derive significant clinical benefit from immune checkpoint inhibitors. To better understand this lack of immunotherapy sensitivity, we aimed to characterize major immune components of the tumor microenvironment (TME) by comprehensive transcriptomic and immunohistochemistry (IHC) analyses. Methods: We analyzed NGS data from 5490 NSCLC pts that underwent DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS) sequencing at Caris Life Sciences (Phoenix, AZ). 374 ALK-rearranged cases were evaluated, along with 3169 KRAS-mut ( STK11/ KEAP1-wt) and 1947 EGFR-mut cases serving as comparators with known heterogenous and inert immune TMEs, respectively. PD-L1 (22C3) was evaluated by IHC. Immune cell fractions were inferred using quanTIseq. Gene expression profiles were analyzed for a T cell-inflamed signature (TIS) predictive of response to immunotherapy and for other immune modulatory genes such as IFNG, GZMB, TGFB1, and those of the adenosine pathway ( CD73/NT5E, CD39/ENTPD1, ADORA1, ADORA2A/B). A significant difference between genomic subgroups was defined as fold-change &gt; 1.2. In an independent cohort of 14 ALK+ NSCLC pts, density and spatial organization of CD4+ and CD8+ T cells, Tregs, major myeloid lineage cells, PDL1, and CD73 were assessed by quantitative IHC. Results: ALK+ tumors were associated with high PD-L1 (≥50%) expression (40% vs 47% for KRAS-mut vs 18% for EGFR-mut, p &lt; 0.001) and low TMB (median 3 mut/MB vs 9 for KRAS-mut vs 4 for EGFR-mut, p &lt; 0.001). The abundance of CD8+ T cells (fold-change -1.3, p &lt; 0.001), Tregs (fold-change -1.2, p &lt; 0.001), M2 macrophages (fold-change 1.2, p &lt; 0.001), and CD4+ T cells (fold-change 1.9, p &lt; 0.001) differed from KRAS-mut; notably, similar to EGFR-mut. In ALK+ tumors, IFNG (fold-change -1.5, p &lt; 0.001), GZMB (fold-change -1.6, p &lt; 0.001), TGFB (fold-change -1.3, p &lt; 0.001), LAG-3 (fold-change -1.4 p &lt; 0.001), CD73/NT53 (fold-change -1.7 p &lt; 0.001) , and ADORA2A (fold-change -1.4, p &lt; 0.001) were decreased while ADORA1 (fold-change 1.3, p &lt; 0.001) was increased compared to KRAS-mut. EML4-ALK comprised 94.7% of the ALK+ tumors and distribution of EML4-ALK variants was consistent with prior reports (e.g. v1 35.6%, v3 35.1%). Immune cell fractions and immune-related gene expression did not vary significantly between major variant subgroups (v1 vs non-v1, and v3 vs non-v3, p &gt; 0.05). Conclusions: To our knowledge this is the largest transcriptomic analysis of the ALK+ NSCLC TME. Despite high levels of PD-L1, ALK+ tumors exhibit multiple features of an inert immune TME, primarily characterized by low TMB and decreased CD8+ T cells and immune activation markers. Our findings suggest that, while immunosuppressive factors such as M2 macrophages and adenosine signaling may also be targeted, strategies to enhance immunogenicity are critical for an effective immune response in ALK+ NSCLC.

A Novel Computational Framework for Predicting the Survival of Cancer Patients With PD-1/PD-L1 Checkpoint Blockade Therapy.

BackgroundImmune checkpoint inhibitors (ICIs) induce durable responses, but only a minority of patients achieve clinical benefits. The development of gene expression profiling of tumor transcriptomes has enabled identifying prognostic gene expression signatures and patient selection with targeted therapies.MethodsImmune exclusion score (IES) was built by elastic net-penalized Cox proportional hazards (PHs) model in the discovery cohort and validated via four independent cohorts. The survival differences between the two groups were compared using Kaplan-Meier analysis. Both GO and KEGG analyses were performed for functional annotation. CIBERSORTx was also performed to estimate the relative proportion of immune-cell types.ResultsA fifteen-genes immune exclusion score (IES) was developed in the discovery cohort of 65 patients treated with anti-PD-(L)1 therapy. The ROC efficiencies of 1- and 3- year prognosis were 0.842 and 0.82, respectively. Patients with low IES showed a longer PFS (p=0.003) and better response rate (ORR: 43.8% vs 18.2%, p=0.03). We found that patients with low IES enriched with high expression of immune eliminated cell genes, such as CD8+ T cells, CD4+ T cells, NK cells and B cells. IES was positively correlated with other immune exclusion signatures. Furthermore, IES was successfully validated in four independent cohorts (Riaz’s SKCM, Liu’s SKCM, Nathanson’s SKCM and Braun’s ccRCC, n = 367). IES was also negatively correlated with T cell–inflamed signature and independent of TMB.ConclusionsThis novel IES model encompassing immune-related biomarkers might serve as a promising tool for the prognostic prediction of immunotherapy.

The differential response to immune checkpoint inhibitors in colorectal and endometrial cancer patients according to different mismatch repair alterations.

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 &lt; 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 &lt; 0.05, intermediate: p &lt; 0.01, low: p &lt; 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 &lt; 0.01, intermediate: p &lt; 0.0001, low: p &lt; 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 &lt; 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.

Claudin 18 (CLDN18) gene expression and related molecular profile in gastric cancer (GC).

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 &lt;.05). Cell infiltration in the tumor microenvironment (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: CLDN18.2 expression was detected in 97% of the samples and CLDN18.1 in 63%. Primary tumors had significantly higher expression levels of both CLDN18.1/18.2 (Fold Change 18.1: 0.50; 18.2: 0.65), compared to metastatic tumors ( p &lt;.05), thus we focused on the comparison of CLDN18 high and low in primary tumors. CLDN18.2 high expression group had higher CLDN18: ARHGAP26 fusion positive rate (low vs high: 0.91% vs 5.5%, q &lt;.0001), and a trending association with CDH1 mutation (11.7% vs 20.7%, p &lt;.01, q &gt;.05) and EBER (2.15% vs 6.31%, p &lt;.05, q &gt;.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 &lt;.0001), with the most common fusion between CLDN18 exon 5 and ARHGAP26 exon 12. CLDN18.2 high expression demonstrated an inverse trending correlation with PD-L1 (24.9% vs 18.3%, p &lt;.05; q &gt;.05) and TMB-H (19.6% vs 12.2%, p &lt;.05; q &gt;.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 &lt;.05); with higher CLDN18 expression associated with fewer immune cells and a colder TME, especially in isoform 2. The TIS score was significantly higher in the CLDN18.2 high expression group ( q &lt;.05), but lower in CLDN18.1 high expression group ( q &lt;.0001), respectively. Conclusions: This is one of the most comprehensive dedicated analyses on CLDN18 related to tumor molecular features, TME and immunotherapy response in GC. Tumors expressing high CLDN18, especially 18.2, displayed distinct genomic and transcriptomic alterations in immune biomarkers and immune cell infiltration in the TME. Anti-CLDN18.2 monoclonal antibodies are being tested in GC and CLDN18 is a target for ADC and CAR-T therapies. Our data suggest that expression may play a role in guiding patient selection and treatment combinations.

Characterization of NY-ESO-1 gene expression in gastric cancer (GC).

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 &lt; 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 &lt; 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 &lt; 0.0001). High NY-ESO-1 expression was also positively associated with cell abundance in the TME including NK cells (FC = 0.87, q &lt; 0.0001), monocytes (FC = 0.29, q &lt; 0.05), myeloid dendritic cells (FC = 0.66, q &lt; 0.0001), CD4+ non-reg T cells (FC = 0.54, q &lt; 0.0001), and CD8+ T cells (FC = 0.73, q &lt; 0.05). Similarly, tumors with high NY-ESO-1 expression were associated with higher TIS scores ( q &lt; 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.

TcellInflamedDetector: an R package to distinguish T cell inflamed tumor types from non-T cell inflamed tumor types.

A major issue in the use of immune checkpoint inhibitors is their lack of efficacy in many patients. Previous studies have reported that the T cell inflamed signature can help predict the response to immunotherapy. Thus, many studies have investigated mechanisms of immunotherapy resistance by defining the tumor microenvironment based on T cell inflamed and non–T cell inflamed subsets. Although methods of calculating T cell inflamed subsets have been developed, valid screening tools for distinguishing T cell inflamed from non–T cell inflamed subsets using gene expression data are still needed, since general researchers who are unfamiliar with the details of the equations can experience difficulties using extant scoring formulas to conduct analyses. Thus, we introduce TcellInflamedDetector, an R package for distinguishing T cell inflamed from non–T cell inflamed samples using cancer gene expression data via bulk RNA sequencing.

Identification and characterization of recurrent neoantigens in upper gastrointestinal (GI) cancers.

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 &gt; 0.80, P &lt; 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.

Abstract PO-018: Inflaming advanced solid tumors including pancreatic cancer using LOAd703, a TMZ-CD40L/4-1BBL-armed oncolytic virus

Abstract Pancreatic ductal adenocarcinoma (PDAC) is resistant to PD1/PDL1 blocking antibodies. The purpose of this study was to evaluate if immunotherapy with LOAd703, an oncolytic adenovirus armed with trimerized CD40L and 4-1BBL, can sensitize PDAC to mAbs targeting PD1/PDL1. The high content of myeloid-derived suppressor cells (MDCSs) in the tumor microenvironment may in part explain the limited anti-tumor T cell response in PDAC. We have previously shown that gemcitabine can reduce MDSCs in patients with PDAC, with the T cell proliferative capacity remaining intact. Further, LOAd703 has been demonstrated to efficiently and robustly kill PDAC cells and stimulate the maturation of dendritic cells (DCs), which, in turn, induces T cell activation. Patients with advanced PDAC, colorectal, biliary, and ovarian cancer (NCT03225989) were treated with intratumoral injections of LOAd703 combined with appropriate chemotherapy such as gemcitabine ± nab-paclitaxel for PDAC patients, or combined with gemcitabine if there was no standard of care. Blood sampling was performed for immune cell profiling and anti-adenovirus antibodies (flow cytometry, ELISA). Tumor biopsies were also analyzed for mRNA expression (NanoString; PanCancer Immune Profiling Panel). The study was approved by the ethical review board and the Swedish Medical Products Agency. The dose of LOAd703 was escalated (5 × 10e10 VP, 1 × 10e11 VP, 5 × 10e11VP) in separate cohorts of new patients and administered every other week. To date, blood samples from 23 patients have been analyzed by flow cytometry to profile immune cells. The mean percentages of monocytic- and granulocytic MDSCs, Tregs and M2-like myeloid cells were significantly decreased after treatment initiation. The effector memory (EM) and central memory (CM) CD8+ T cells were significantly increased, while naïve and CM cells were increased among CD4 T cells. Both CD4 and CD8 T cells expressing ICOS were present post-treatment but only CD8 T cells showed significant elevation of PD1. The NanoString data were analyzed using published mRNA immune signatures (bioinformatics). LOAd703 in combination with chemotherapy inflamed tumor lesions as shown by increased markers of the T cell inflamed signature (16 genes), T effector cell signature (19 genes), expanded immune signature (25 genes) and IFNg-related gene signature (12 genes) (week 1 versus week 13). Anti-adenovirus antibody titers increased in all patients after treatment initiation. The antibody levels did not correlate with the dose of virus, radiological response to treatment, nor overall survival. The results presented herein show that LOAd703 combined with chemotherapy enhances immune reactivity in patients with immune cold tumors as demonstrated by increases in EM and CM T cells in the peripheral blood, while suppressive immune cells were decreased. Further, the tumor milieu was inflamed post treatment as shown by mRNA profiling. Hence, LOAd703 may sensitize immune cold tumors to mAbs targeting PD1/PDL1. Citation Format: Jessica Wenthe, Emma Eriksson, Linda Sandin, Tanja Lövgren, Justyna Leja Jarblad, Hanna Dahlstrand, Ulla Olsson-Strömberg, Aglaia Schiza, Anders Sundin, Sandra Irenaeus, Eric Rowinsky, Gustav Ullenhag, Angelica Loskog. Inflaming advanced solid tumors including pancreatic cancer using LOAd703, a TMZ-CD40L/4-1BBL-armed oncolytic virus [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-018.

Immunogenomic determinants of tumor microenvironment correlate with superior survival in high-risk neuroblastoma

BackgroundTumor-infiltrating CD8+ T cells and neoantigens are predictors of a favorable prognosis and response to immunotherapy with checkpoint inhibitors in many types of adult cancer, but little is known about...

A First in Class APRIL Fully Blocking Antibody Targets Novel Immune Regulation of APRIL in Multiple Myeloma: Further Therapeutic Implication

A proliferation inducing ligand (APRIL), a critical paracine factor for human multiple myeloma (MM), is mainly secreted by myeloid cells, including macrophages and osteoclasts in the bone marrow (BM) microenvironment. Recently, an antagonistic APRIL monoclonal antibody (mAb) 01A significantly reduced myeloma burden in SCID-hu mice (Blood. 2016;127:3225-3236). This data suggests that the humanized 01A named BION-1301, which also potently (IC50 <1 nM) blocks APRIL binding to its receptors B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI) is likely to exert anti-MM activity in vivo. Here, we further characterize the novel inhibition by 01A and a hIgG4 chimeric version C4 on multiple immune cells affected by APRIL in the immunosuppressive MM BM milieu. First, using gene-modified RPMI8226 MM cells, 01A potently and selectively blocks growth and viability of APRIL-overexpressing but not BCMA-overexpressing RPMI8226 transfectants. Significantly, 01A inhibits MM cell growth and survival induced by osteoclasts, as a physiologically relevant endogenous source of APRIL. Next, we demonstrate that pretreatment of APRIL decreases MM cell lysis induced by Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, APRIL prevents anti-BCMA (J6M0)-induced ADCC against BCMA-expressing MM cell lines in a dose-dependent manner. Conversely, 01A or C4 (a surrogate of BION-1301) reverts APRIL-reduced MM cell lysis induced by J6M0 in the presence of PBMC or NK effector cells. These data indicate that targeting CD38 and BCMA may not be sufficient in MM. To further define the role of APRIL mediating anti-MM immune responses, we show that T cells do not express BCMA while natural T regulatory (Treg) cells significantly express higher TACI when compared with corresponding conventional T cells (Tcon) from the same individual (Abstract #102467). In parallel, detailed gene expression profiling in multiple solid tumors show that the tumor-resident Treg markers are significantly correlated with TACI, along with a strong association of APRIL expression levels with T cell inflamed signature. In addition to natural Treg, MM cell-induced Tregs (iTregs) also showed significantly increased TACI levels when compared with paired conventional T cells (Tcon) from the same individual in ex vivo cocultures. Importantly, APRIL further enhances MM cell-induced iTregs with IL-10-producing and CD15s+[JD2] FOXP3high fractions; conversely, 01A blocks APRIL-induced upregulation of iTreg. Osteoclasts also upregulated iTreg generation induced by MM cells. On the contrary, 01A or C4 partially blocks such induction. Importantly, APRIL directly promotes proliferation and viability of Treg via the induction of CCND1/2, BCL2, and BCL2L1/BCL-xL, which is blocked by 01A or C4. Finally, APRIL enhances the suppressive function of Treg on the proliferation of autologous conventional T cells, which could also be overcome by 01A or C4. Collectively, our data therefore demonstrate that APRIL, via TACI but not BCMA, critically modulates regulatory T cell function; and, importantly, 01A or C4 targets these immune regulatory subsets that have been linked to MM progression via APRIL-dependent manner. Moreover, since patients with active diseases have severe bone lesions induced by hyperactive osteoclasts, inhibiting the APRIL-TACI interaction may also overcome osteoclast-inhibited T cell killing on MM cells. Anti-APRIL Ab (BION-1301) will be entering clinical trial in late 2017 to target the immunosuppressive BM microenvironment and improve patient outcome in MM. DisclosuresDulos:Aduro Biotech Europe: Employment, Equity Ownership. Van Elsas:Aduro Biotech Europe: Employment, Equity Ownership. Richardson:Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

RB1 and TP53 co-mutations correlate strongly with genomic biomarkers of response to immunity checkpoint inhibitors in urothelial bladder cancer

BackgroundMuscle invasive urothelial bladder carcinoma (MIBC) present RB1 and TP53 somatic alterations in a variable percentage of tumors throughout all molecular subtypes. MIBCs with neuroendocrine features have a high response rate to immunity checkpoint inhibitors (ICIs). Whether the presence of somatic co-alterations in these 2 genes in MIBCs is relevant to their responsiveness to ICIs is not known.MethodsThe potential correlation of different genomic biomarkers of response to ICIs like tumor mutational burden (TMB), single nucleotide variants (SNV) predicted neoantigens, DNA damage response (DDR) genes, DNA somatic signatures and TILs infiltrate was explored in patients with somatic co-alterations in RB1 and TP53 (RB1&TP53) as compared with patients with no alterations in any (double wild type, DWT) or with alterations in just one of the 2 genes. The Cancer Genome Atlas (TCGA) pancancer BLCA dataset of cystectomy specimens (n = 407) with mutation, copy number alterations and transcriptomic (RNA sequencing) data as well as the IMVigor 210 study (n = 348) of metastatic urothelial bladder cancers treated with atezolizumab (PD-L1 inhibitor) with clinical response data containing transcriptomic (RNA sequencing), along with a subset (n = 274) with mutation and copy number data were used for this purpose. A novel tumor microenvironment metascore (TMM) was developed based in a LASSO regularized Cox model with predictive and prognostic ability.ResultsSamples with co-altered RB1&TP53: a) were enriched in immunity effectors (CD8 cytotoxic lymphocytes, NK cells) and display higher scores of a T cell inflamed signature; b) have a higher TMB, higher number of SNV predicted neoantigens and higher TILs fractions; c) have a higher number of DDR mutated and deep deleted DDR genes; d) have DNA somatic signatures 2 and 13 related to APOBEC mutagenesis. Using the IMVigor 210 dataset, RB1&TP53 samples had the highest response rate to atezolizumab and a strong correlation with TMB and TMM. The consensus molecular subtype classification in the IMVigor 210 dataset showed a significant correlation with both the response to treatment (p = 0.001, Chisquare) and the presence of RB1 and TP53 genomic alterations (p < 0.001, Chisquare).ConclusionsRB1&TP53 co-alterations are strongly associated with genomic biomarkers of response to ICIs in MIBCs.

Immune landscape of inflammatory breast cancer suggests vulnerability to immune checkpoint inhibitors

ABSTRACT Background. Anti-PD1/PDL1 immune checkpoint inhibitors (ICIs) showed promising results in breast cancer, and exploration of additional actionable immune checkpoints is ongoing. Inflammatory breast cancer (IBC) is an aggressive form of disease, the immune tumor microenvironment (TME) of which is poorly known. We aimed at providing the first comprehensive immune portrait of IBCs. Methods. From the gene expression profiles of 137 IBC and 252 non-IBC clinical samples, we measured the fractions of 22 immune cell types, expression of signatures associated with tertiary lymphoid structures (TLS) and with the response to ICIs (T cell-inflamed signature: TIS) and of 18 genes coding for major actionable immune checkpoints. The IBC/non-IBC comparison was adjusted upon the clinicopathological variables. Results. The immune profiles of IBCs were heterogeneous. CIBERSORT analysis showed profiles rich in macrophages, CD8+ and CD4 + T-cells, with remarkable similarity with melanoma TME. The comparison with non-IBCs showed significant enrichment in M1 macrophages, γδ T-cells, and memory B-cells. IBCs showed higher expression of TLS and TIS signatures. The TIS signature displayed values in IBCs close to those observed in other cancers sensitive to ICIs. Two-thirds of actionable immune genes (HAVCR2/TIM3, CD27, CD70, CTLA4, ICOS, IDO1, LAG3, PDCD1, TNFRSF9, PVRIG, CD274/PDL1, and TIGIT) were overexpressed in IBCs as compared to normal breast and two-thirds were overexpressed in IBCs versus non-IBCs, with very frequent co-overexpression. For most of them, the overexpression was associated with better pathological response to chemotherapy. Conclusion. Our results suggest the potential higher vulnerability of IBC to ICIs. Clinical trials.

Abstract B04: Molecular correlates of the non-T cell-inflamed tumor microenvironment in head and neck squamous cell carcinoma

Abstract Background: Immunotherapy confers clinical benefit in head and neck squamous cell carcinoma (HNSCC); however, only a fraction of patients respond. The non-T cell-inflamed tumor microenvironment is associated with lack of response, and tumor-intrinsic molecular signaling pathways may be critical drivers of immune exclusion. Therefore, understanding correlations between T-cell inflammation and mutated or activated oncogenic pathways may inform combination immunotherapy approaches. Methods: RNAseq, somatic mutations, and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database for 484 HNSCC patients (88 HPV-positive, 396 HPV-negative). Gene expression was used to segregate tumors into non-T cell-inflamed, T cell-inflamed, or intermediate groups and genes more frequently mutated in non-T cell-inflamed versus inflamed (or vice versa) were identified. Differentially expressed genes (DEGs) between the two groups were detected by limma voom with precision weights. Pathway activation was identified by Ingenuity Pathway Analysis® (IPA). Association between molecular markers and patients’ overall survival (OS) was tested using Cox proportional-hazards regression models. Results: 132 (27%) and 183 (38%) tumors were categorized into the non-T cell-inflamed and inflamed group, respectively, with the rest intermediate. Survival analysis revealed that among age, sex, tumor grade, and HPV status, age was the strongest prognostic factor (p=0.003), with older patients showing a decreased 5-year survival. The absence of a T cell-inflamed signature was significantly associated with poorer prognosis within patients ≤65 years of age (p=0.03, HR=1.18) and remained significant after adjusting for covariates such as HPV status. This association was not observed in older patients. Comparison of somatic mutational profiles between the non-T cell-inflamed and inflamed groups identified top mutated genes enriched (TP53, COL11A1, NSD1) and depleted (CASP8) in noninflamed relative to inflamed (FDR-corrected p&amp;lt;0.05). Comparing gene expression profiles of non-T cell-inflamed to inflamed patient groups identified 4092 DEGs (FDR-corrected p&amp;lt;0.05, fold change ≥1.5 or ≤-1.5). Utilizing these DEGs as downstream target molecules in IPA causal network analysis, activation of MYC, SOX2, NFE2L2, and CTNNB1 transcriptional programs correlated with the non-T cell-inflamed tumor microenvironment (z-score ≥2.0, p&amp;lt;0.05). Of note, NFE2L2 (nuclear factor erythroid-2-related factor 2; NRF2) has been described as a context-dependent regulator of innate immune responses and modulator of checkpoint inhibitor response in murine models. Conclusions: A discrete list of oncogenes, some with known targeted therapies, associates with immune exclusion, suggesting rational immunotherapy combinations to overcome non-T cell-inflamed tumors in HNSCC. Citation Format: Riyue Bao, Jason J. Luke. Molecular correlates of the non-T cell-inflamed tumor microenvironment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B04.

Gene Expression Profiling of PDGFRA Mutant GIST Reveals Immune Signatures as a Specific Fingerprint of D842V Exon 18 Mutation.

Platelet Derived Growth Factor Receptor Alpha (PDGFRA) mutations occur in only about 5–7% of gastrointestinal stromal tumors (GIST), notably with alterations on exons 12/14/18. The most frequent PDGFRA mutation is the exon 18 D842V, which is correlated to specific clinico-pathological features, such as primary imatinib resistance and higher indolence. Here, we present a gene expression profile (GEP) comparison of D842V vs. PDGFRA with mutations other than D842V (non-D842V). GEP was followed by in silico bioinformatic analysis aimed at evaluating differential expression, tumor microenvironment composition and pathway enrichment. We found a large set of oncogenes, transcription factors and nuclear receptors downregulated in the D842V mutant. Conversely, D842V showed a significant enrichment of immune- and interferon- related gene signatures. Differences in tumor microenvironment composition were also highlighted, including a higher abundance of CD8+ T-cells and an overexpression of the T cell-inflamed signature in the D842V mutant subgroup, which is predictive of immunotherapy response. PDGFRA D842V vs. non-D842V GIST display a different expression profile, with a prominent immunological signature, that could represent a proof of principle for testing immunotherapeutic strategies in this drug-orphan subset of GIST.