Immune response to malignant cells is an incredibly complex phenomenon to characterize and varies drastically by cancer’s tissue of origin, site of metastasis, environmental factors, patients’ diet and lifestyle, and, perhaps most importantly, genetic makeup. Denoted as either “hot” or “cold” depending on the intensity, or lack thereof, of immune cell recruitment and immune-related pathway activation, the tumor microenvironment (TME) provides a significant area of study and one which can provide breakthroughs for cancer patients who, historically, may not have many therapeutic options with high efficacy. Colorectal cancer (CRC), traditionally characterized as “immune-cold” tumors, due to its unique TME and activated oncogenic pathways, remains a major barrier to effective immunotherapy, specifically in a subset of patients with microsatellite stable (MSS) tumors.¹

Typically, only CRC patients with dMMR/MSI-H tumors respond to immune checkpoint inhibitor (ICI) therapy, however, MSS CRC patients represent the largest patient subset of CRC and currently lack a repertoire of molecular biomarkers to combat immunosuppressive effects of the TME. Support for therapeutic targeting of the B-Raf (BRAF) gene, V-Ki-ras2 Kirsten Rat Sarcoma (KRAS) gene, and carcinoembryonic antigen (CEA) has been plentiful, but additional efforts must be focused into identifying molecular targets that may influence therapeutic response and augment the therapeutic armamentarium of CRC. Mesothelin (MSLN), a membrane-bound surface glycoprotein is a promising molecular target that is highly expressed in numerous cancer types, including MSS CRC. High expression of MSLN in CRC is correlated with higher rates of metastatic disease, upregulation of malignant cell proliferation, and a reduction in patient survival.^{1, 2} Additionally, MSLN has been show to play a significant role in encouraging a “cold” TME by enhancing expression of immunosuppressive genes and suppression of immune activating pathways.³ Our work further examines the role MSLN plays in CRC, specifically MSS CRC, the implications of MSLN expression and related immune/TME-regulation, and the possible relationship with CRC classification parameters including tumor sidedness and consensus molecular subtypes (CMS), both of which have massive prognostic and predictive implications.

Mesothelin Association with Tumor Sidedness, CMS Subtypes and Oncogenic Drivers

               Of 14,892 CRC patients in the Caris Life Sciences database, 7,466 were classified as “high” or “low” MSLN expressing by quartiles and 6,847 of those were MSS CRC patients. In general, MSLN expression correlated with right-sided CRC and CMS1 and CMS4 molecular subtypes. Our work supported existing data that a significant proportion of malignant CRC tissues demonstrated high MSLN expression compared to normal tissue. Furthermore, we showed that high MSLN expression was predominantly distributed across metastatic CRC compared to local CRC. Finally, high MSLN expression was more widely distributed across metastatic CRC in comparison to local CRC.

We reported enhanced mutation across a wide range of oncogenic drivers in MSS CRC including KRAS, FBXW7, RNF43, GNAS, SMAD2, and BMPR1A. Furthermore, MSS CRC with high MSLN expression appeared to enhance the enrichment of gene sets, as analyzed via GSEA, responsible for inflammatory responsiveness, IFNγ response, IFNα response, IL2-STAT5 signaling, and IL6-JAK-STAT3 signaling.  

Immune Regulation in MSLN High MSS CRC

               Our work describing the immune environment in relation to both MSLN expression and MSS CRC assists in identifying pivotal differences for MSS CRC patients that may be actionable in the clinic. Primarily, we have described significant differences between MSLN low and high MSS CRC patients in PD-L1 expression, where MSLN high patients had significantly higher PD-L1 IHC positivity than MSLN low. This patient population also had significantly higher T cell inflammation and IFNγ scores, corroborating our GSEA analysis on pathway enhancements in MSLN high MSS CSC patients. To refine this analysis in more detail, we performed immune cell fraction analysis using RNA deconvolution and immune-related gene expressions which uncovered large differences between MSLN high and low CRC patients, regardless of MSI status. MSLN high patients had higher prevalence of multiple immune cells including B cells, M1 and M2 macrophages, neutrophils, natural killer cells, and T-regulatory cells; And also had higher expression of immune checkpoint genes: CD274, HAVCR2, LAG3, CTLA4, PDCD1, PDCD1, LG2, etc. in the TME. This cell recruitment and upregulation in immune checkpoint genes signify a MSLN­-dependent transition from immune cold tumors to immune hot tumors that may be candidates for immunotherapeutic intervention.

Patient Survival in MSLN High MSS CRC

               Courtesy of insurance claims data and the Caris CodeAI™ clinico-genomic database, we generated Kaplan-Meier survival curves for patients with high and low MSLN expression stratified by median expression and for patients with the highest 25% and lowest 25% expression of MSLN in our patient cohort. Generally, patients with high MSLN expression displayed lower overall survival (OS) than patients with low MSLN survival, establishing MSLN as a marker of poor prognosis. Broken down into treatment-related OS, MSS CRC patients with high MSLN expression all had increased numerical median overall survival with pembrolizumab, nivolumab, and collective ICI therapies, though not statistically significant. Only nivolumab significantly enhanced median survival in all patients with MSLN high expression. This difference is further enhanced when analyzing the top and bottom 25% of MSLN expressing patients, where pembrolizumab, nivolumab, and collective ICI therapies again increased median survival in MSS CRC patients with highest 25% MSLN expression, while the entire cohort of patients with highest 25% MSLN expression exhibited lower median survival across all treatment groups.

Discussion

Our work sought to further clear the fog of MSLN expression and its relation to immune-related activity in a subset of CRC patients that urgently need additional therapeutic options. We demonstrated that high MSLN expression is correlated to specific CRC CMS subtypes that are associated with reduced immune activity, in addition to relational enhancements of immune-inhibitory gene expression. Also highlighting the importance of TME regulation is a recent phase 1 clinical trial that describes clinical response to the ICI drug combination botensilimab and balstilimab in MSS CRC patients.⁴ This trial, in compliment with our survival data showing enhanced survival trends in MSS CRC patients with high MSLN treated with ICI, provides support for growing the body of therapies available to these patients. By identifying additional therapy options and using MSLN as a therapeutic target or predictive indicator to therapeutic efficacy, MSS CRC patients have increasing hope for eliminating their cancers and leading long, healthy lives.

1.           Inoue S, Tsunoda T, Riku M, et al. Diffuse mesothelin expression leads to worse prognosis through enhanced cellular proliferation in colorectal cancer. Oncol Lett 2020;19:1741-1750.

2.           Malla M, Rozich N, Hassan A, et al. Correlation of mesothelin (MSLN) expression with future peritoneal metastases (PM) in colorectal cancer (CRC). Journal of Clinical Oncology 2020;38:176-176.

3.           Li Y, Tian W, Zhang H, et al. MSLN Correlates With Immune Infiltration and Chemoresistance as a Prognostic Biomarker in Ovarian Cancer. Front Oncol 2022;12:830570.

4.           Bullock AJ, Schlechter BL, Fakih MG, et al. Botensilimab plus balstilimab in relapsed/refractory microsatellite stable metastatic colorectal cancer: a phase 1 trial. Nature Medicine 2024.