Purpose: Oncogenic mutations in KRAS have been identified in >85% of pancreatic ductal adenocarcinoma (PDAC) cases, with G12D, G12V, and G12R being the most frequent variants. Using large clinical and genomic databases, this study characterizes prognostic and molecular differences between KRAS variants, focusing on KRASG12D and KRASG12R. Experimental Design: PDAC samples were tested using DNA and RNA sequencing. The MAPK activation score and tumor microenvironment were analyzed from RNA expression data. Real-world overall survival (OS) obtained from insurance claims data was calculated from tissue collection to last contact. Significance was determined by χ2 and Fisher exact tests. Results: A total of 3,755 patients with PDAC harboring KRASG12D (n = 1,766), KRASG12V (n = 1,294), KRASG12R (n = 621), or KRASG12C (n = 74) variants were identified. Patients with G12R mutations had longer OS compared with those with G12D overall (12.7 vs. 10.1 months; P value = 0.0001), with similar trends in patients treated with gemcitabine/nab-paclitaxel (13.5 vs. 10.4 months; P value = 0.0002) or FOLFIRINOX (18.3 vs. 14.0 months; P value<0.001). ARID1A and KMT2D mutations were more frequent in the G12D subgroup. Several genes involved in glucose and glutamine metabolism were less expressed in G12R compared with G12D. PD-L1 expression was lower in G12R compared with G12D (13% vs. 19%). Conclusions: KRAS G12D tumors exhibited a distinct molecular profile compared with G12R tumors, including genes involved in the MAPK pathway, immune activation, and glucose and glutamine metabolism. Patients with G12D mutations had lower OS compared with those with G12R. Based on these data, future studies should address the KRAS mutation status and explore distinct therapeutic vulnerabilities.

Objective: Expression of CXC-chemokine receptor 4 (CXCR4) is associated with poor prognosis in colorectal cancer (CRC), metastasis and immune cell infiltration. Currently, only patients with metastatic CRC with mismatch repair deficient (dMMR) tumours (<10%) are eligible for immune checkpoint blockade (ICB). Methods and analysis: 15 026 CRC tumour samples were analysed at Caris Life Sciences using whole-exome sequencing, whole-transcriptome sequencing and immunohistochemistry (IHC). This was a retrospective analysis in which all consecutive CRC cases between 2008 and 2023 with available molecular profiling and survival data were included. To elucidate the role of CXCR4 mRNA expression in the tumour microenvironment (TME) and response to ICB, CRC samples were stratified by high and low quartiles of CXCR4 mRNA expression levels and compared for molecular and clinical characteristics. Tumour mutational burden [TMB) and dMMR status were determined from DNA sequencing data. Immune cell infiltration was calculated from deconvolution of bulk RNA sequencing data. PD-L1 expression was determined by IHC stain intensity. Information for real-world survival and pembrolizumab treatment was obtained from the CODEai database using insurance claims-based data. Overall survival was calculated from date of either first specimen collection or treatment to last of healthcare contact with HR and p value calculated using the Cox proportional hazards model and log-rank test, respectively. Results: Metastatic sites showed higher CXCR4 expression than primary tumours (22.7 vs 18.6 median transcripts per million [TPM], p<0.001). CXCR4 expression was lower in liver than non-liver metastases (21.2 vs 24.8 TPM, p<0.001). Median CXCR4 mRNA expression was positively associated with high TMB, dMMR and positive PD-L1 status. High CXCR4 expression was associated with tumours with higher infiltration of B cells and M1/M2 macrophages. High CXCR4 expression in the primary tumour was associated with worse prognosis (HR 0.92, 95% CI 0.86 to 0.99, p<0.02), regardless of dMMR status. In metastatic tumours, high CXCR4 mRNA expression correlated with improved survival (HR 1.12, 95% CI 1.04 to 1.20, p=0.003). Remarkably, high CXCR4 expression was associated with improved survival in all ICB-treated patients. Conclusion: Correlation of high CXCR4 expression with changes in the TME and improved outcomes following ICB suggests its potential for selecting immunotherapeutic strategies in CRC.

Responses to immune checkpoint inhibitors (ICIs) differ significantly between uveal melanoma (UM) and cutaneous melanoma (CM) patients. We investigated the immune profile of metastatic UM(mUM) patients and identified markers that are predictive of improved survival. Metastatic liver samples from 189 UM patients and 48 CM patients were analyzed at genomic and transcriptional levels, and survival analysis was performed on a subgroup of 76 ICI-treated mUM patients. UM liver metastases seem to preserve the genomic and immune characteristics of primary UM (pUM), with a low prevalence of ICI markers and high mutation rates of GNA11, GNAQ, BAP1, and SF3B. Compared with mCM, UM liver metastasis showed lower infiltration of several immune cells, but a greater proportion of M2 macrophages. Compared with UM liver metastases, CM liver metastases showed higher expression of G2M checkpoint and EF2 target genes. Among the mUM and mCM samples, expression of G2M and E2F pathway genes was highest in tumors with high TMB values and T-cell inflamed scores. A longer median overall survival (OS) was observed in mUM patients with higher expression of LAG3, HLA class I, or HLA class II; which may represent a small proportion of immune hot tumors. Expression patterns of G2M checkpoint and E2F targets suggest a possible contribution to immunotherapy response.

Background/Objectives: Colorectal cancer (CRC) is the second leading cause of cancer-related death in the US. The presence of deficient DNA mismatch repair and microsatellite instability (dMMR/MSI) in CRC is linked to responses to immune checkpoint inhibitors (ICIs). This study investigates the impact of metformin on the tumor microenvironment (TME) and clinical outcomes of patients with dMMR/MSI CRC treated with ICIs, aiming to better understand its potential role in enhancing ICI efficacy. Methods: Of 25,011 CRC patients in Caris database, 47 received both metformin and ICI therapy (Met-ICI group), and 475 patients received ICI therapy alone (ICI group). Samples underwent genomic or transcriptome sequencing at Caris Life Sciences. Immune cell fractions were estimated using quanTIseq. Univariate and multivariate survival analyses were conducted using the Cox proportional model. Results: The TME analysis of CRC patient samples revealed that TMB-High (≥10 mut/Mb) was more prevalent in the “ICI” group compared to the “Met-ICI” group (99.1% vs. 95.6%, p = 0.036). Mutation rates for most genes between the two groups were similar, but CIC gene mutations were more common in the “ICI” group than in the “Met-ICI” group (23.2% vs. 4.8%, p = 0.006). No significant differences were observed in the PD-L1 positivity rate or immune checkpoint gene expression (including IDO1, IFNG, TIM3, and CTLA4). M1 macrophages and neutrophils showed the highest infiltration among immune cells. However, the fractions of infiltrated immune cells were similar between the two cohorts. Univariate and multivariate analyses showed that there was no significant difference in patient survival between “ICI” and “Met-ICI” cohorts. Conclusions: In this retrospective analysis of real-world clinical outcomes, the concurrent use of metformin with ICIs in patients with dMMR/MSI CRC did not reveal an impact on clinical outcomes.

Purpose:Oncogenic mutations in KRAS have been identified in >85% of pancreatic ductal adenocarcinoma (PDAC) cases, with G12D, G12V, and G12R being the most frequent variants. Using large clinical and genomic databases, this study characterizes prognostic and molecular differences between KRAS variants, focusing on KRASG12D and KRASG12R.Experimental Design:PDAC samples were tested using DNA and RNA sequencing. The MAPK activation score and tumor microenvironment were analyzed from RNA expression data. Real-world overall survival (OS) obtained from insurance claims data was calculated from tissue collection to last contact. Significance was determined by χ2 and Fisher exact tests.Results:A total of 3,755 patients with PDAC harboring KRASG12D (n = 1,766), KRASG12V (n = 1,294), KRASG12R (n = 621), or KRASG12C (n = 74) variants were identified. Patients with G12R mutations had longer OS compared with those with G12D overall (12.7 vs. 10.1 months; P value = 0.0001), with similar trends in patients treated with gemcitabine/nab-paclitaxel (13.5 vs. 10.4 months; P value = 0.0002) or FOLFIRINOX (18.3 vs. 14.0 months; P value<0.001). ARID1A and KMT2D mutations were more frequent in the G12D subgroup. Several genes involved in glucose and glutamine metabolism were less expressed in G12R compared with G12D. PD-L1 expression was lower in G12R compared with G12D (13% vs. 19%).Conclusions:KRASG12D tumors exhibited a distinct molecular profile compared with G12R tumors, including genes involved in the MAPK pathway, immune activation, and glucose and glutamine metabolism. Patients with G12D mutations had lower OS compared with those with G12R. Based on these data, future studies should address the KRAS mutation status and explore distinct therapeutic vulnerabilities.

Supplementary Table S1: Comparison of genomic alterations between KRAS G12R and G12D subgroups related to the PI3K/AKT/MTOR pathway.