Molecular profiling METex14+ non-small cell lung cancer (NSCLC): Impact of histology.

OBJECTIVES: MET exon 14 skipping alterations (METex14+) represent a heterogeneous subgroup of non-small cell lung cancer (NSCLC) with distinct biological and genomic features. We characterized this heterogeneity in a large cohort, integrating genomic and transcriptomic profiling with clinical outcomes, to elucidate the histologic and molecular traits and survival patterns of METex14+ NSCLC. MATERIALS AND METHODS: NSCLC tissue samples (n = 28,739) underwent DNA-based next-generation sequencing (592 genes, NextSeq) or whole-exome sequencing (NovaSeq), RNA-sequencing including whole transcriptome sequencing (WTS, NovaSeq), and PD-L1 IHC (Dako 22C3) at Caris Life Sciences. Immune cell fractions were estimated from bulk RNA sequencing (quanTIseq). Real-world survival data (mOS) was calculated from insurance claims. Statistical analyses employed Chi-square, Fisher's exact, or Mann-Whitney U and log-rank tests and were corrected for hypothesis testing where applicable. RESULTS: A total of 711 METex14+ cases were detected. Of 575 cases of defined histology, 77 (13.6 %) were squamous (Sq), 474 (82.3 %) were nSq (non-squamous), and 24 (4.1 %) were adenosquamous. Mutations in POT1 and BRCA2 were enriched, and amplifications in MDM2, HMGA2, CDK4, and MET were common in METex14+ tumors. TMB-high and TP53 mutated tumors were reduced in METex14+ independent of histology. KEAP1 (2.1 vs 14.7 %) and STK11 mutations (0.8 vs 17.1 %) were reduced only in METex14+ nSq (vs METex14+ Sq, q < 0.05). While the prevalence of PD-L1 high tumors was enriched in METex14+ independent of histology, T-cell inflamed tumors were enriched only in nSq METex14+. B-cells and CD8+ T-cells (1.07-1.43-fold) were enriched in nSq METex14+, and dendritic cells (0.32 fold) were reduced only in METex14+ Sq. METex14+ tumors had a modest improvement in mOS compared to METex14- tumors (mOS = 22.9 m vs 18.6 m, HR = 0.914, p = 0.04). Moreover, METex14+ tumors who received immunotherapy (IO) had a modest improvement in survival (mOS = 27.5 m vs 21.8 m; HR = 0.803, p = 0.03) compared to those who did not receive IO. METex14+ nSq tumors were associated with improved mOS compared to METex14+ Sq tumors (mOS = 27.7 vs 8.9 m, HR = 0.493, p < 0.0001). CONCLUSION: METex14+ alterations are a heterogeneous subgroup of NSCLC. Our analysis reveals that METex14+ nSq exhibit improved survival compared to METex14+ Sq. The distinct genomic and transcriptomic variations across histologies warrant clinical consideration.

Large-scale analysis of CDH1 mutations defines a distinctive molecular subset with treatment implications in gastric cancer.

Although histological and molecular classifications have been extensively studied for gastric cancer (GC), targeted therapies for GC remain limited. CDH1 mutations (MT) are characteristic of genomically stable GC and are associated with poor prognosis, but lack effective or targeted therapies. Here, we showed the overall mutation frequency of CDH1 was 9.7% (155 of 1596). CDH1-MT GC showed significantly lower rates of PD-L1 positivity (CPS score ≥1) than CDH1-wildtype (WT) GC (56.7% vs. 73.3%, p < 0.05). Compared to CDH1-WT GC, mutations of ARID1A, WRN, POT1, CDK12, and FANCC were significantly higher, while TP53 and APC were significantly lower in CDH1-MT GC (p < 0.05); The rates of KRAS and HER2 amplifications were significantly lower, while CRKL and IGF1R amplifications were significantly higher in CDH1-MT GC, compared to CDH1-WT GC (p < 0.05). Frequently altered genes in CDH1-MT GC were especially enriched in DNA damage repair and cell cycle checkpoint pathways. Inhibition of E-cadherin sensitized GC cell lines to PARP and Wee1 inhibitors by disrupting DNA damage repair pathway and cell cycle checkpoint. This is the largest study to investigate the distinct genomic landscape of CDH1-MT GC. Our data indicated GC patients with CDH1 mutations could potentially benefit from agents targeting PARP and Wee1.

Selective loss of Y chromosomes in lung adenocarcinoma modulates the tumor immune environment through cancer/testis antigens.

There is increasing recognition that the sex chromosomes, X and Y, play an important role in health and disease that goes beyond the determination of biological sex. Loss of the Y chromosome (LOY) in blood, which occurs naturally in aging men, has been found to be a driver of cardiac fibrosis and heart failure mortality. LOY also occurs in most solid tumors in males and is often associated with worse survival, suggesting that LOY may give tumor cells a growth or survival advantage. We analyzed LOY in lung adenocarcinoma (LUAD) using both bulk and single-cell expression data and found evidence suggesting that LOY affects the tumor immune environment by altering cancer/testis antigen expression and consequently facilitating tumor immune evasion. Analyzing immunotherapy data, we show that LOY and changes in expression of particular cancer/testis antigens are associated with response to pembrolizumab treatment and outcome, providing a new and powerful biomarker for predicting immunotherapy response in LUAD tumors in males.

Consensus molecular subtyping of metastatic colorectal cancer expands biomarker-directed therapeutic benefit for patients with CMS1 and CMS2 tumors.

BACKGROUND: We developed a whole transcriptome sequencing (WTS)-based Consensus Molecular Subtypes (CMS) classifier using FFPE tissue and investigated its prognostic and predictive utility in a large clinico-genomic database of CRC patients (n = 24,939). METHODS: The classifier was trained against the original CMS datasets using an SVM model and validated in an independent blinded TCGA dataset (88.0% accuracy). Kaplan-Meier estimates of overall survival (OS) and time-on-treatment (TOT) were calculated for each CMS (p < 0.05 considered significant). RESULTS: CMS2 tumors were enriched on left-side of colon and conferred the longest median OS. In RAS-wildtype mCRC, left-sided tumors and CMS2 classification were associated with longer TOT with anti-EGFR antibodies (cetuximab and panitumumab). When restricting to only CMS2, there was no significant difference in TOT between right- versus left-sided tumors. CMS1 tumors were associated with a longer median TOT with pembrolizumab relative to other CMS groups, even when analyzing only microsatellite stable (MSS) tumors. DISCUSSION: A WTS-based CMS classifier allowed investigation of a large multi-institutional clinico-genomic mCRC cohort, suggesting anti-EGFR therapy benefit for right-sided RAS-WT CMS2 tumors and immune checkpoint inhibitor benefit for MSS CMS1. Routine CMS classification of CRC provides important treatment associations that should be further investigated.

Clinical and genomic features of classical and basal transcriptional subtypes in pancreatic cancer.

PURPOSE: Transcriptional profiling of pancreatic cancers (PC) has defined two main transcriptional subtypes, classical and basal. Initial data suggest shorter survival for patients with basal tumors and differing treatment sensitivity to FOLFIRINOX (FFX) and gemcitabine nab-Paclitaxel (GnP) by transcriptional subtype. EXPERIMENTAL DESIGN: We examined 8,743 patients with RNA sequencing from PCs performed at Caris Life Sciences (Phoenix, AZ). Classical and basal subtypes were identified using PurIST algorithm on RNA-sequencing and two cohorts were analyzed: (1) Biomarker cohort included patients with complete molecular profiling data (n = 7,250); (2) Outcomes cohort included patients with metastatic disease with available survival outcomes (n=5,335). RESULTS: In the biomarker cohort, 3,063 tumors (42.2%) were strongly classical (SC), and 2,015 tumors (27.8%) were strongly basal (SB). SC and SB tumors showed strong associations with histologic phenotypes and biopsy site. SB tumors had higher rates of KRAS, TP53, and ARID1A mutations, lower rates of SMAD4 mutation, and transcriptional evidence of epithelial mesenchymal transition. Sixty of 77 cases (78%) maintained their transcriptional subtype between temporally and/or spatially disparate lesions. In the outcomes cohort, SB subtype was associated with shorter overall survival time, regardless of whether they received FFX or GnP as first line chemotherapy. Mutant KRAS allele type was prognostic of outcomes, however this impact was restricted to SC tumors, whereas all mutant KRAS alleles had similarly poor outcomes in SB tumors. CONCLUSIONS: SB subtype is a strong independent predictor of worse outcomes, irrespective of upfront chemotherapy regimen. Clinical trials should investigate PC transcriptional subtypes as a biomarker.

Mesothelin expression correlates with elevated inhibitory immune activity in patients with colorectal cancer.

The expression of the protein Mesothelin (MSLN) is highly variable in several malignancies, including colorectal cancer (CRC), and high levels are associated with aggressive clinicopathological features and worse patient survival. Colorectal cancer is both a common and deadly cancer; being the third most common in incidence and second most common cause of cancer-related death. While systemic therapy remains the primary therapeutic option for most patients with stage IV (metastatic; m) CRC, their disease eventually becomes treatment refractory, and 85% succumb within 5 years. Microsatellite-stable (MSS) CRC tumors, which constitute more than 90% of patients with mCRC, are generally refractory to immunotherapeutic interventions. In our current work, we characterize MSLN levels in CRC, specifically correlating expression with clinical outcomes in relevant CRC subtypes, and explore how MSLN expression impacts the status of immune activation and suppression in the peritumoral microenvironment. Higher MSLN expression is prevalent in CMS1 and CMS4 CRC subtypes and correlates with higher gene mutation rates across the patient cohorts. Further, MSLN-high patients exhibit increased M1/M2 macrophage infiltration, PD-L1 staining, immune-inhibitory gene expression, enrichment in inflammatory, TGF-ß, IL6/JAK/STAT3, IL2/STAT5 signaling pathways, and mutation in KRAS and FBXW7. Together, these results suggest that MSLN protein is a potential target for antigen-specific therapy and supports investigation into its tumorigenic effects to identify possible therapeutic interventions for patients with high MSLN expressing MSS CRC.

VISTA Emerges as a Promising Target against Immune Evasion Mechanisms in Medulloblastoma.

BACKGROUND: Relapsed medulloblastoma (MB) poses a significant therapeutic challenge due to its highly immunosuppressive tumor microenvironment. Immune checkpoint inhibitors (ICIs) have struggled to mitigate this challenge, largely due to low T-cell infiltration and minimal PD-L1 expression. Identifying the mechanisms driving low T-cell infiltration is crucial for developing more effective immunotherapies. METHODS: We utilize a syngeneic mouse model to investigate the tumor immune microenvironment of MB and compare our findings to transcriptomic and proteomic data from human MB. RESULTS: Flow cytometry reveals a notable presence of CD45hi/CD11bhi macrophage-like and CD45int/CD11bint microglia-like tumor-associated macrophages (TAMs), alongside regulatory T-cells (Tregs), expressing high levels of the inhibitory checkpoint molecule VISTA. Compared to sham control mice, the CD45hi/CD11bhi compartment significantly expands in tumor-bearing mice and exhibits a myeloid-specific signature composed of VISTA, CD80, PD-L1, CTLA-4, MHCII, CD40, and CD68. These findings are corroborated by proteomic and transcriptomic analyses of human MB samples. Immunohistochemistry highlights an abundance of VISTA-expressing myeloid cells clustering at the tumor-cerebellar border, while T-cells are scarce and express FOXP3. Additionally, tumor cells exhibit immunosuppressive properties, inhibiting CD4 T-cell proliferation in vitro. Identification of VISTA's binding partner, VSIG8, on tumor cells, and its correlation with increased VISTA expression in human transcriptomic analyses suggests a potential therapeutic target. CONCLUSIONS: This study underscores the multifaceted mechanisms of immune evasion in MB and highlights the therapeutic potential of targeting the VISTA-VSIG axis to enhance anti-tumor responses.

Health Disparities among Patients with Cancer Who Received Molecular Testing for Biomarker-Directed Therapy.

Health disparities present a barrier to successful oncology treatment. The potential for precision oncology to reduce health disparities has not previously been analyzed. We performed a retrospective analysis of 12,627 patients from six major cancer centers whose tumors underwent molecular testing at Caris Life Sciences between 2010 and 2020. Kaplan-Meier and Cox regression were used to describe and analyze overall survival. The molecular and demographic features of the cohort were analyzed by χ2 and ANOVA tests. Black patients composed 25% of the cohort and White patients 63%. Among this molecularly-tested cohort, there were minimal outcome differences based on race, geographic location, or poverty level. When analyzing the interaction of age, race, and sex, racial-based disparities were noted primarily for young non-White women in the study cohort but were more pronounced for men and women of all ages in the broader patient population within the Surveillance, Epidemiology, and End Results database. Mutations in five genes-APC, EGFR, STK11, TP53, and KRAS-were found to affect overall survival among our cohort, and their prevalence varied by race in specific tumor types. Real-world outcomes data in mutation-defined cohorts also provided additional context to previously reported therapeutic response trends. Our study shows that patients who undergo molecular testing display reduced racial health disparities compared with the general population, whereas persistent racial disparities are influenced by age and sex. Genomic-driven racial disparities should be examined at a tumor lineage-specific level. Increased access to molecular testing for all eligible patients may play a role in improving health equity. Significance: This study is the largest of its kind to analyze health disparities and genomic features among a diverse multiinstitutional cohort of patients who underwent molecular testing. Continuing to increase awareness of and access to molecular testing approaches may help to reduce cancer health disparities and improve outcomes for all patients.

Pediatric glioma immune profiling identifies TIM3 as a therapeutic target in BRAF fusion pilocytic astrocytoma.

Despite being the leading cause of cancer-related childhood mortality, pediatric gliomas have been relatively understudied, and the repurposing of immunotherapies has not been successful. Whole-transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapeutic strategy that could be applied to multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher IFN signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia (MG) population designated MG-Act in BRAF-fused, MAPK-activated pilocytic astrocytoma (PA), but not in high-grade gliomas or normal brain. T cell immunoglobulin and mucin domain 3 (TIM3) was expressed on MG-Act and on the myeloid cells lining the tumor vasculature but not normal brain vasculature. TIM3 expression became upregulated on immune cells in the PA microenvironment, and anti-TIM3 reprogrammed ex vivo immune cells from human PAs to a proinflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven, low-grade gliomas, anti-TIM3 treatment increased median survival over IgG- and anti-PD-1-treated mice. Single-cell RNA-Seq data during the therapeutic window of anti-TIM3 revealed enrichment of the MG-Act population. The therapeutic activity of anti-TIM3 was abrogated in mice on the CX3CR1 MG-KO background. These data support the use of anti-TIM3 in clinical trials of pediatric low-grade, MAPK-driven gliomas.

Molecular Differences With Therapeutic Implications in Early-Onset Compared With Average-Onset Biliary Tract Cancers.

PURPOSE: Early-onset biliary tract cancer (eoBTC) is among the fast-growing subset of early-onset cancers, yet little is known about its biology. We sought to identify novel molecular characteristics of eoBTC in relation to average-onset BTC (aoBTC) using a real-world multiomics data set. METHODS: The study comprised patients with BTC whose tumors underwent molecular analyses at Caris Life Sciences and were categorized by age (<50 years for eoBTC, ≥50 years for aoBTC). P values were adjusted for multiple testing and considered significant at Q < 0.05 (molecular comparisons) or Q < 0.25 (Gene Set Enrichment Analysis [GSEA]). Insurance claims data were used for survival analysis. RESULTS: The study included 5,587 patients with BTC (453 eoBTC, median age = 44 years and 5,134 aoBTC, median age = 68 years). FGFR2 fusion (15.7% in eoBTC v 5.9% in aoBTC) and NIPBL fusion (1.1% v 0%) were significantly more prevalent in eoBTC (both Q < 0.001). The interferon gamma-IFG score (fold change [FC], 1.1; Q = 0.01) and T-cell inflammation score (FC, 17.3; Q = 0.03) were significantly higher in aoBTC. On GSEA, angiogenesis was enriched in eoBTC (normalized enrichment score [NES] = 1.51; Q = 0.16), whereas IFG (NES = -1.58; Q = 0.06) and inflammatory response (NES = -1.46; Q = 0.18) were enriched in aoBTC. The median overall survival (OS) was 16.5 (eoBTC) versus 13.3 months (aoBTC), hazard ratio = 0.86, P = .004. The median OS by FGFR2 fusion (with fusion v without) was 21.7 versus 15.0 months (P = .47) for eoBTC and 18.6 versus 12.2 months (P < .001) for aoBTC. CONCLUSION: We identified crucial differences including higher prevalence of FGFR2 fusions in eoBTC and variations in immunotherapy-related markers. Better outcomes in eoBTC were affected by the FGFR2 fusion status. Our findings underscore the need for ensuring access to next-generation sequencing testing, including prompt identification of actionable targets.

Correlative Analysis of ATM, RB1, ERCC2, and FANCC Mutations and Pathologic Complete Response After Neoadjuvant Chemotherapy in Patients with Muscle-invasive Bladder Cancer: Results from the SWOG S1314 Trial.

We previously reported that tumors harboring any one of four gene mutations (ATM, RB1, FANCC, or ERCC2) were likely to respond to neoadjuvant cisplatin-based chemotherapy (NAC), resulting in cancer-free surgical specimens at the time of cystectomy (pT0). Here, we report our validation of this finding. Using the CARIS 592 Gene Panel (Caris Life Sciences, Phoenix, AZ, USA), we analyzed 105 pre-NAC tumor specimens from a large multicenter trial (S1314) of either neoadjuvant gemcitabine and cisplatin (GC), or dose-dense methotrexate, vinblastine, Adriamycin, and cisplatin (DDMVAC). We found that a mutation in any one of these four genes predicted for pT0 at surgery (odds ratio = 5.36; 95% confidence interval [CI] 2.05, 14.02; two-sided p = 0.0006). The biomarker was better at predicting the presence of disease (negative predictive value for pT0 86%; 95% CI 73%, 94%) than the absence of disease (positive predictive value for pT0 48%; 95% CI 35%, 62%). There was no evidence of an interaction between the treatment arm (DDMVAC vs GC) and the genetic variant in terms of pT0. When combined with clinical assessment, these findings help inform patient selection for bladder preservation after cisplatin-based chemotherapy.

A paradigm shift in understanding vulvovaginal melanoma as a distinct tumor type compared with cutaneous melanoma.

OBJECTIVE: Our goal was to compare molecular and immune profiles of vulvovaginal melanoma (VVM) with cutaneous melanoma (CM) and explore the significance of immune checkpoint inhibitor (ICI) agents on survival. METHODS: Samples from VVM and CM tumors underwent comprehensive molecular and immune profiling. Treatment and survival data were extracted from insurance claims data and OS was calculated from time of ICI treatment to last contact. Statistical significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons. RESULTS: Molecular analysis was performed on 142 VVM and 3823 CM tumors. VVM demonstrated significantly (q < 0·01) less frequent BRAF and more frequent KIT, ATRX, and SF3B1 mutations. Alterations in pathways involving DNA damage and mRNA splicing were more common in VVM, while alterations in cell cycle and chromatin remodeling were less common. Immunogenicity of VVM was lower than CM, with an absence of high TMB (0% vs 46.9%) and lower PD-L1 positivity (18·0% vs 29·5%). Median immune checkpoint gene expression was lower in VVM, as were cell fractions for type I macrophages and CD8+ T-cells(q < 0·01). Myeloid dendritic cells were increased in VVM(q < 0·01). Median OS was shorter for VVM than for CM patients treated with ICIs (17·6 versus 37·9 months, HR:1·65 (95% CI 1·02-2·67) p = 0·04). CONCLUSIONS: VVM has a distinct molecular and immune profile compared to CM, which may contribute to the worse survival in VVM compared to CM patients treated with ICI therapy. Though ICIs have been a mainstay of treatment in recent years, our findings suggest that new therapeutic strategies are needed.

Molecular Landscape and Clinical Implication of CCNE1-amplified Esophagogastric Cancer.

Cyclin E overexpression as a result of CCNE1 amplification is a critical driver of genomic instability in gastric cancer, but its clinical implication is largely unknown. Thus, we integrated genomic, transcriptomic, and immune profiling analysis of 7,083 esophagogastric tumors and investigated the impact of CCNE1 amplification on molecular features and treatment outcomes. We identified CCNE1 amplification in 6.2% of esophageal adenocarcinoma samples, 7.0% of esophagogastric junction carcinoma, 4.2% of gastric adenocarcinoma samples, and 0.8% of esophageal squamous cell carcinoma. Metastatic sites such as lymph node and liver showed an increased frequency of CCNE1 amplification relative to primary tumors. Consistent with a chromosomal instability phenotype, CCNE1 amplification was associated with decreased CDH1 mutation and increased TP53 mutation and ERBB2 amplification. We observed no differences in immune biomarkers such as PD-L1 expression and tumor mutational burden comparing CCNE1-amplified and nonamplified tumors, although CCNE1 amplification was associated with changes in immune populations such as decreased B cells and increased M1 macrophages from transcriptional analysis. Real-world survival analysis demonstrated that patients with CCNE1-amplified gastric cancer had worse survival after trastuzumab for HER2-positive tumors, but better survival after immunotherapy. These data suggest that CCNE1-amplified gastric cancer has a distinct molecular and immune profile with important therapeutic implications, and therefore further investigation of CCNE1 amplification as a predictive biomarker is warranted. SIGNIFICANCE: Advanced gastric cancer has a relatively dismal outcome with a 5-year overall survival of less than 10%. Furthermore, while comprehensive molecular analyses have established molecular subtypes within gastric cancers, biomarkers of clinical relevance in this cancer type are lacking. Overall, this study demonstrates that CCNE1 amplification is associated with a distinct molecular profile in gastric cancer and may impact response to therapy, including targeted therapy and/or immunotherapy.

Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

Differential landscape of immune evasion in oncogenic RAS-driven primary and metastatic colorectal cancers.

Oncogenic drivers such as KRAS extensively modulate the tumor inflammatory microenvironment (TIME) of colorectal cancer (CRC). The influence of KRAS on modulating immune cell composition remains unclear. The objective of this study was to identify signatures of infiltrative immune cells and distinctive patterns that differ between RAS wild-type (WT) and oncogenic mutant (MT) CRC that explain immune evasion in MT tumors. A total of 7,801 CRC specimens were analyzed using next-generation DNA sequencing, whole-exome sequencing, and/or whole transcriptome sequencing. Deficiency of mismatch repair (dMMR)/microsatellite instability (MSI) and tumor mutation burden (TMB) were also assessed. KRAS mutations were present in 48% of CRC, similarly distributed in patients younger than vs. 50 years and older. In microsatellite stable (MSS) KRAS MT tumors, composition of the TIME included higher neutrophil infiltration and lower infiltration of B cells. MSI-H/dMMR was significantly more prevalent in RAS WT (9.1%) than in KRAS MT (2.9%) CRC. In MSS CRC, TMB-high cases were significantly higher in RAS MT (3.1%) than in RAS WT (2.1%) tumors. KRAS and NRAS mutations are associated with increased neutrophil infiltration, with codon-specific differences. These results demonstrate significant differences in the TIME of RAS mutant CRC that match previous reports of immunoevasive characteristics of such tumors.

Comparative molecular profiling of pancreatic ductal adenocarcinoma of the head versus body and tail.

Pancreatic ductal adenocarcinoma (PDAC) of the head (H) and body/tail (B/T) differ in embryonic origin, cell composition, blood supply, lymphatic and venous drainage, and innervation. We aimed to compare the molecular and tumor immune microenvironment (TIME) profiles of PDAC of the H vs. B/T. 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 (Caris Life Sciences, Phoenix, AZ). Significance was determined as p values adjusted for multiple corrections (q) of <0.05. 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 B/T vs. H (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). Expression analysis of immuno-oncology (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). To our knowledge, this is one of the largest cohorts 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 profiles of H vs. B/T tumors were observed.

Tissue-specific thresholds of mutation burden associated with anti-PD-1/L1 therapy benefit and prognosis in microsatellite-stable cancers.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 or its ligand (PD-1/L1) have expanded the treatment landscape against cancers but are effective in only a subset of patients. Tumor mutation burden (TMB) is postulated to be a generic determinant of ICI-dependent tumor rejection. Here we describe the association between TMB and survival outcomes among microsatellite-stable cancers in a real-world clinicogenomic cohort consisting of 70,698 patients distributed across 27 histologies. TMB was associated with survival benefit or detriment depending on tissue and treatment context, with eight cancer types demonstrating a specific association between TMB and improved outcomes upon treatment with anti-PD-1/L1 therapies. Survival benefits were noted over a broad range of TMB cutoffs across cancer types, and a dose-dependent relationship between TMB and outcomes was observed in a subset of cancers. These results have implications for the use of cancer-agnostic and universal TMB cutoffs to guide the use of anti-PD-1/L1 therapies, and they underline the importance of tissue context in the development of ICI biomarkers.