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. CRC 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 affect 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. High MSLN expression is highly prevalent in CMS1 and CMS4 CRC subtypes as well as in mCRC tissue 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-β, IL6/JAK/STAT3, IL2/STAT5 signaling pathways and mutation in KRAS and FBXW7. Together, these results suggest 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.


Introduction
In the United States, approximately 4% of people will develop colorectal cancer (CRC) at some point in their lifetime. 1,2In 2023, it is predicted that the incidence of CRC will be 153,000 new cases and over 52,500 deaths will occur in the US. 3 Recently, the epidemiology of the disease has shifted with an increased incidence observed in individuals younger than 50 years.The average age of diagnosis was 72 in the early 2000s but has decreased to age 66 in the 2020s. 4As such, there are signi cant efforts to identify novel therapeutic targets and implement immunotherapies in CRC.Patients with CRC that has metastasized eventually become refractory to traditional systemic therapeutic approaches and succumb to the disease. 5though there is a relatively high mortality rate associated with CRC, improvements in molecular characterization of tumors have permitted the development of novel treatment options for a subset of patients.These improvements have extended the median overall survival (OS) of patients with CRC from approximately 12 months to 25 to 30 months over the past 5 decades. 6,7Of note, molecular targets with therapeutic implications include adenomatous polyposis coli (APC), β-catenin, epithelial growth factor receptor (EGFR), V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), and TP53, among others. 8Targeting known drivers of cancer progression is critical to expanding the fraction of patients who may bene t from precision treatment of their CRC. 8Most recently, other genetic abnormalities, including the high frequency of FBXW7 mutations, a nding that portends a poor prognosis, are under active investigation as additional potential molecular targets in CRC. 9 However, a majority of patients with CRC have tumors that are microsatellite stable (MSS), a genotype which globally tends to be resistant to immunotherapies. 10With continued advancements in molecular characterization of MSS CRC tumors, it is hoped that methods of immunological targeting will be developed to improve upon current immunotherapy success levels in patients presenting with a MSS genotype.
A promising cellular therapeutic target under investigation is mesothelin (MSLN).2][13][14][15] Both preclinical and clinical studies examining MSLN expression in mesothelioma, pancreatic cancer, and lung adenocarcinoma show evidence that enhanced MSLN expression portends a reduction in survival in animals and humans. 13,14,16,17Speci cally, in CRC, increased MSLN expression is correlated with the development of metastatic disease, a reduction in patient survival, and an upregulation of CRC cell proliferation.11 Numerous efforts to develop MSLN targeted therapies have been underway based on these preclinical data, speci cally in mesothelioma, lung, and pancreatic cancers. 11-13,15−18 However, work is still needed to exploit the therapeutic potential of high levels of MSLN expression in CRC.
This study seeks to correlate MSLN expression with known CRC prognostic signatures including tumor sidedness, metastatic sites, and the CRC consensus molecular subtype (CMS).Additionally, we seek to uncover associations of MSLN expression with speci c CRC and immune-related molecular markers through examination of these relationships in all CRC patients and in the subset of MSS CRC patients.
These interactions could identify new therapeutic options for MSS CRC patients with high levels of MSLN expression and lead to further investigations into therapeutics with clinical relevance to MSLN expression patterns.

Next Generation Sequencing (NGS)
NGS was performed on genomic DNA isolated from 14,892 formalin-xed para n-embedded (FFPE) CRC tumor samples by a commercial CAP/CLIA lab (Caris Life Sciences, Phoenix, AZ) using the NextSeq or NovaSeq 6000 platforms (Illumina, Inc., San Diego, CA).The Caris platform uses a 592-gene panel and 700 gene panel at high depth and coverage as previously described. 19Tumor enrichment was performed using manual microdissection techniques.Genetic variants identi ed were interpreted by board-certi ed molecular geneticists and categorized as 'pathogenic,' 'likely pathogenic,' 'variant of unknown signi cance,' 'likely benign,' or 'benign,' according to the American College of Medical Genetics and Genomics (ACMG) standards.When assessing mutation frequencies of individual genes, 'pathogenic,' and 'likely pathogenic' were counted as mutations. 20The copy number alteration (CNA) of each exon was determined by the average depth of the sample along with the sequencing depth of each exon and comparing to a pre-calibrated value.
Tumor Mutation Burden (TMB) TMB was measured by counting all mutations found per tumor that had not been previously denoted as germline alterations in dbSNP151, Genome Aggregation Database (gnomAD) databases or benign variants identi ed by Caris geneticists as previously described. 21A cutoff level of ≥ 10 mutations per MB was used to characterize tumors as TMB high based on evidence from the KEYNOTE-158 pembrolizumab trial which showed that patients with a TMB of ≥ 10 mt/MB across several tumor types had higher response rates than patients with a TMB of < 10 mt/MB. 22ole Transcriptome Sequencing (WTS) A Qiagen RNA FFPE tissue extraction kit (Germantown, MD) was used for extraction, and the RNA quality and quantity were determined using the Agilent TapeStation (Santa Clara, CA) prior to WTS on the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) as previously described. 23For transcript counting, transcripts per million numbers were generated using the Salmon expression pipeline. 24RNAdeconvolution was performed via quanTIseq to assess immune cell in ltration within the tumor microenvironment (TME). 25Tumors were characterized as MSLN-high(H) and MSLN-low(L) based upon the top and bottom quartile of transcripts per million (TPM) expression, respectively.Gene set enrichment analysis (GSEA) was conducted in order to examine the enrichment or depletion of groups of genes associated with different biological pathways.This analysis was performed using Broad Institute software. 26munohistochemistry Immunohistochemistry (IHC) of PD-L1 (SP142 clone), MLH1 (M1 clone), MSH2 (G2191129 clone), MSH6 (44 clone), and PMS2 (EPR3947 clone) was completed on FFPE tissue slides.Slides were stained as per the manufacturer's instructions (Ventana Medical Systems, Inc. Tucson, AZ), and were optimized and validated per CLIA/CAP and ISO requirements.Staining was scored for intensity (0 = no staining; 1 + = weak staining; 2 + = moderate staining; 3 + = strong staining) and staining percentage (0-100%).The complete absence of protein expression of any of the 4 proteins tested (0 + in 100% of cells) was considered de cient MMR.A board-certi ed pathologist evaluated all IHC results independently.

MSI/MMR Status
Multiple test platforms were used to determine the MSI or MMR status of the tumors pro led, including fragment analysis (FA, Promega, Madison, WI), IHC (see IHC method) and NGS (7,000 target microsatellite loci were examined and compared to the reference genome hg19 from the University of California).The three platforms generated highly concordant results, as previously reported.In the rare cases of discordant results, the MSI or MMR status of the tumor was determined in the order of IHC, FA and NGS. 27

CODEai™
Insurance claims data were used to calculate real-world overall survival (rwOS) via ' rst of treatment' to 'last contact' patient records.Kaplan-Meier estimates were calculated for molecularly de ned patient cohorts.

Statistical analysis
Statistical signi cance was determined using Chi-square and Mann-Whitney U tests, with p values adjusted for multiple comparisons (q ≤ 0.05).rwOS signi cance was determined with p ≤ 0.05.

MSLN Cohort Demographics and Expression Distribution in Primary Tumors, Metastatic Site, Tumor Side, and CMS Subtypes
The study population comprised 7,446 patients, of which 6,847 patients were assigned to the "MSS cohort" according to MSI status.Both the entire patient cohort and MSS cohort were dichotomized as MSLN Low or MSLN High.Shown in Table 1A, the MSLN low group included 3,723 patients with 54.9% being male and 45.1% female.The MSLN high group included 3,723 patients with 52.9% being male and 47.1% female.The median age was 63 years and 62 years respectively.In Table 1B, the MSLN low cohort included 3,377 patients, with a median age of 62 years with 56.5% being male and 43.5% female.The MSLN high group was comprised of 3,470 patients, 53.7% of whom were male and 46.3% female.The median age was 62 years for both MSLN expression levels in the MSS cohort.
MSLN expression patterns were compared across primary and metastatic sites, metastatic location, CRC side of origin, and CMS subtypes (Fig. 1). Figure 1A and 1B show MSLN expression in the primary tumor versus metastatic sites in all patients.Overall, tumor samples from metastatic sites expressed MSLN at signi cantly higher levels compared to the primary tumor site (q ≤ 0.01) (Fig. 1A).Samples from metastases to the skin (39.4 TPM), connective/soft tissue (11.9 TPM), and the peritoneum/retroperitoneum (11.7 TPM) exhibit the highest MSLN expression levels amongst metastatic sites (Fig. 1B).Shown in Fig. 1C, MSLN expression was highest in right sided CRC tumors in both the entire cohort (6.1 TPM), and MSS (6.2 TPM), cohorts (q ≤ 0.01).Additionally, MSLN expression was high in transverse tumor locations in both patient cohorts.Left sided tumors exhibited the lowest median MSLN expression levels at 4.8 TPM.Similar expression patterns between the entire and MSS cohorts were observed when comparing CMS subtypes (Fig. 1D).Both cohorts exhibited the highest MSLN expression in CMS1 (8.3 and 10.4 TPM respectively, q ≤ 0.001) and CMS4 (10.1 and 10.0 TPM respectively, q ≤ 0.001).

Mesothelin High Tumors Exhibit Greater Expression of PD-L1 and Higher T-cell In amed Score, Immune Cell In ltration, and Expression of Immunosuppressive Genes
Figure 3 examines the prevalence of immune markers across MSLN high and low tissue expression split into the two cohorts as mentioned previously.Across the entire cohort (Fig. 3A), MSLN low expression had a signi cant association with higher tumor mutation burden (TMB) and DNA mismatch repair (dMMR)/microsatellite instability-high (MSI-H) positivity in comparison to MSLN high expression.However, MSLN high expression exhibited an association with high PD-L1 expression via IHC (IHC-PD-L1) compared to MSLN low expression.In the MSS cohort, MSLN high tumor samples yielded signi cantly higher IHC-PD-L1 positivity compared to MSLN low expression (Fig. 3B). Figure 3C and 3D show similar results regarding T-cell in ammation quanti cation and IFN-γ scores.Across both cohorts it was found that MSLN high expression signi cantly correlated with both markers of T-cell in ammation and low IFNγ scores.Speci c immune cell in ltration into the TME (Fig. 4A) and immune gene expression (Fig. 4B) was examined within each cohort in addition to the immune marker results presented in Fig. 3. Several immune cell types including B cells, M1 and M2 macrophages, neutrophils, natural killer (NK) cells, and regulatory T-cells (Tregs) were more prevalent in MSLN high expressing tumors across both the entire cohort and MSS cohort.Speci cally, M1 and M2 macrophages were most signi cant within these groups.Only dendritic cells (DC) were more prevalent in MSLN low tumors.Full immune cell fraction data values are presented in supplementary table 2. Immune marker gene expression levels, shown in Fig. 4B, were all higher in MSLN high tumors across both cohorts except for IL12A.Particularly, HAVCR2 (TIM-3), CD80, CD86, and IL1B expression were of highest magnitude change between MSLN high and low expressing tumors.In the entire cohort of patients, comparing MSLN high and MSLN low groups, HAVCR2 (15.33 TPM vs. 8.39 TPM, q ≤ 0.05), CD80 (4.14 TPM vs. 2.73 TPM, q ≤ 0.05), CD86 (7.07 TPM vs. 4.12 TPM, q ≤ 0.05), and IL1B (9.00 TPM vs. 8.76 TPM, q ≤ 0.05) were all signi cantly higher in the MSLN high group.A relationship in HAVCR2 expression was seen in the MSS cohort between MSLN high and low tissue (15.00 TPM vs 8.03 TPM, q ≤ 0.05).In the MSS cohort, MSLN high tissue again yielded increased gene expression in HAVCR2 (15.00 TPM vs. 8.03 TPM, q ≤ 0.05), CD80 (4.08 TPM vs. 2.66 TPM, q ≤ 0.05), CD86 (8.76 TPM vs. 3.97 TPM, q ≤ 0.05), and IL1B (8.76 TPM vs. 6.71TPM, q ≤ 0.05).

Gene Set Enrichment Analysis of MLSN High Tumors
Association of immune cell recruitment, tumor microenvironment, and corresponding gene sets related to immune response can support the possibility of a gene target being a marker for tumor susceptibility to antigen-speci c immunotherapy.Complementing the immune marker and microenvironment analyses, a Gene Set Enrichment Analysis (GSEA) was performed to evaluate the differences in MSLN-associated gene expression between patients with MSLN low and MSLN high tumors within each cohort.For all gene sets shown (Fig. 5A, B), a positive normalized enrichment score (NES) was observed, indicating higher gene enrichment for patients with MSLN high tumors.Each cohort of patients exhibited signi cantly high NES for gene sets related to immune response related to MSLN high expression, shown using red bars.Speci cally, TNFα signaling via NFKβ (NES = 1.33,False Discovery Rate (FDR) = 0.09), IL2 STAT5 signaling (NES = 1.32,FDR = 0.08), IFN-γ response (NES = 1.33,FDR = 0.09), and IL6 JAK STAT3 signaling (NES = 1.29,FDR = 0.12) were three such pathway enrichments related to immune response that were signi cantly enriched in patients with MSLN high tumors in contract with those who had MSLN low tumors.In the MSS cohort, it must rst be noted that one of the most highly enriched pathways was that of the in ammatory response (NES = 1.44,FDR = 0.01).Additionally, immune-related pathways including IFN-γ Response (NES = 1.44,FDR = 0.02), IL2 STAT 5 Signaling (NES = 1.37,FDR = 0.06), IFNα Response (NES = 1.37,FDR = 0.07), IL6 JAK STAT3 Signaling (NES = 1.36,FDR = 0.07), and TNFα Signaling via NFKβ (NES = 1.39,FDR = 0.09) were all highly signi cant in the MSS cohort.These pathways were more signi cant based on FDR in the MSS cohort than in the entire cohort, possibly suggesting a higher role of immune modulation in patients with MSS tumors.

Discussion
Colorectal cancer is the third most common cause of cancer and second highest cause of cancer related mortality in the United States. 29There is signi cant heterogeneity among patients with CRC in terms of genomic abnormalities that drive tumor progression and these differences correlate with prognosis.By establishing connections between novel biomarkers and prognostic signatures of CRC including tumor sidedness, metastatic sites, CMS category, and immune cell recruitment, we may be able to preferentially select treatments for patients with enhanced expression patterns of these biomarkers, possibly leading to an improvement in the survival outcomes.Mesothelin has been shown to play a role in promoting cancer cell survival, proliferation, and invasion of cancer cell lines enabling cancer cells to thrive in an in ammatory milieu. 17,30,31Our work begins to uncover the relevance of MSLN overexpression in CRC patients as well as the implications of MSLN expression levels on pivotal CRC prognostic information including CRC tumor sidedness, CMS subtype, genetic mutation landscape, and immune response modulation.We have shown that increased MSLN expression is positively correlated with right-sided CRC, CMS1, and CMS4 molecular subtypes, possibly playing a role in the poor prognosis associated with rightsided CRC and CMS1/4 molecular subtypes. 32,33Currently, it is understood that CMS1 is characterized by increased immune-related gene expression, including activation of immune evasion that is commonly observed in MSI CRC, with high rates of right-sided tumors and poor survival rates with disease relapse. 34MS2 is typically categorized by upregulation of WNT and MYC, leading to carcinogenesis, and enhanced epithelial cell differentiation. 34CMS3 often is seen with mixed MSI status but high rates of KRAS mutation and CMS4 is associated with high rates of epithelial-to-mesenchymal transition (EMT) gene expression in addition to activation of TGF-β and in ammatory pathways, contributing to the poorest overall survival among CMS subtypes. 34Regarding tumor sidedness, previous work has shown that right-sided tumors are typically associated with a poorer 5-year survival (55% vs 59%) and an increased risk of CRC recurrence versus left-sided tumors. 35,36 stated, our results show an increased association of CMS1, CMS4, and right-sided CRC with high MSLN expression.Due to this relationship, it can be posited that this increase in MSLN gene expression does also play a role in the associated features of these molecular subtypes and tumor location.The immune and in ammatory enhancements seen with CMS1 and CMS4 CRC are mirrored in the increased immune cell recruitment, immune-related gene expression, and enhancement of immune and in ammatory pathways shown with high MSLN expression (Figs. 4, 5).Further, CMS4 has speci cally been linked to EMT enhancements creating desirable conditions for rapid tumor growth and metastatic activity.37 It is plausible that with increased MSLN expression, speci cally in CMS4, there may be a link between MSLN expression and EMT.Bringing focused attention to the MSS cohort of patients and the relationship between MSLN and EMT activation, we have shown that high MSLN expression in this subset of patients exhibit increased TGF-β signaling (Fig. 5B), KRAS signaling (Fig. 5B), MAPK activation (Supp.Figure 1), and signi cant enrichment of the EMT gene set (Fig. 5B). 38,39Each of these factors, in addition to associated increased CA125/MUC16 expression (Supp.Figure 2), an MSLN binding domain, which has also been shown to play a role in tumorigenesis and STAT3 phosphorylation 40 , support the consideration of MSLN as a prospective biomarker in CRC.
Directly related to the potential clinical relevance of MSLN as a biomarker is the difference in survival time for patients with tumors exhibiting high MSLN expression versus low MSLN expression across multiple therapeutic regimens.Illustrated in Fig. 6, CODEai™ analysis revealed some signi cant differences across these patient cohorts, speci cally in patients with tumors classi ed as MSS.MSS tumors that had high MSLN expression had a worse associated median survival time when treated with 5-FU-chemotherapy and ipilimumab alone (Fig. 6B).Notably, there was a shift in this pattern when patients with MSLN high tumors that were also MSS exhibited a higher median survival time than the MSLN low cohort when treated with nivolumab, pembrolizumab, Ipi-Nivo combination, or the ICI combination.By determining MSLN expression oncologists may choose to recommend treatment with ICI and that strategy may enhance survival outcomes.The patients with highest and lowest quartiles of MSLN expression were also investigated using CODEai™ analysis (Fig. 7).The lowest 25% of MSLN expressing tumors in the entire cohort showed longer survival times than the tumors with the highest 25% of MSLN expression when treated with different forms of ICI (Fig. 7A).What is most noteworthy, is that when we analyzed the cohort of patients with tumors classi ed as MSS, once again, the survival patterns shifted, with the MSLN high cohorts surviving longer than the MSLN low cohort across all ICI treatment groups (consisting of nivolumab, pembrolizumab, Ipi-Nivo, and combination ICI treatment) (Fig. 7B).It is this combination of high MSLN expression and MSS status that yields an improved survival with ICI treatment that implicates MSLN as a possible biomarker in MSS CRCs.Moreover, it may be suggested that this improved survival outcome with ICI in patients with MSLN high MSS CRC is due to the immunosuppressive TME induced by increased MSLN expression.
Our study has demonstrated a strong association between MSLN expression and a differentially modulated immune microenvironment, highlighted by an increase in M1/M2 macrophage recruitment, but a lowered IFN-γ score and general immunosuppressive effect, though pathway activation is shown in GSEA analysis.It should also be emphasized that high MSLN expression, especially in MSS patients, was associated with signi cantly increased IHC-PD-L1 staining (Fig. 3B) and enhanced gene expression of PD-1, CTLA4, FOXP3, LAG3, and TIM3 (Fig. 4B).Further, total gene set enrichment analysis revealed signi cant activation of gene sets associated with TGF-β signaling, IL6/JAK2/STAT pathway signaling, IL2/STAT5 pathway signaling, and PIK3/AKT/mTOR signaling (Fig. 5B).These ndings are supported by other studies that have described the effects of MSLN-related immunosuppression and tumor immune escape in other cancers via upregulation of immunosuppressive genes and cytokine production. 41,42mmune cell response, relative 'heat' of the tumor microenvironment, immune/in ammatory pathway activations, and genetic mutation pro les may predict the likelihood of bene t from immunotherapy in relation to MSLN expression, speci cally in patients with MSS tumors.Regarding gene mutation patterns, recent studies have determined that increased mutation in KRAS and FBXW7 have been implicated as immunosuppressive biomarkers that play a role in poor immunotherapeutic responses in CRC. 43,44dditionally, high co-expression of HAVCR2 and MSLN have been shown to be prevalent in several cancers and may also be vulnerabole to the use of CAR-T therapies.Pre-clinical studies have shown the utility of CAR-T therapy in an MSLN high ovarian cancer model. 45Taking this deeper into the HAVCR2-MSLN relationship, RNA-interference therapies have also been used in combination with CAR-T approaches to rst knock down HAVCR2/TIM3 expression and enhance the cytotoxic functioning and proliferation capacity of therapeutic CAR-T cells in numerous in vitro cancer models. 46TIM-3 has been shown to be a candidate for gene knockdown to enhance cell-based immunotherapy previously. 47Taking these studies together, in combination with our conclusions of MSLN high implications on patient response to immunotherapies, may yield a prospective combination therapy approach.In MSLN high expressing tumors that are MSS, we have reported signi cantly increased mutation rates in KRAS and FBXW7 as opposed to our ndings in MSLN low expressing patient cohorts, suggesting that MSLN status may also be relevant to the modulation of the TME and immune response in CRC.Taking this one step further, the top quartile of MSLN expressing tumor cohorts exhibited signi cantly longer survival times, again supporting MSLN as a potential biomarker.We have also demonstrated (Figs. 6, 7) that a subset of patients, particularly the MSS subset, show an MSLN expression-dependent response to ICI therapies, highlighting the prospect of MSLN as a clinically relevant biomarker for therapeutic selection.However, it is recommended that additional pre-clinical testing using genetic strategies to modulate levels of MSLN to be completed to determine if any causative relationship exists between MSLN expression and modulation of the TME towards immune suppression in MSS CRC.Improved survival in MSLN high CRCs post-ICI could be attributed to increased PD-L1 protein, CD274 (PD-L1), PDCD1 (PD-1), and CTLA4 gene expression, and enrichment of in ammatory response pathways in these tumors.However, the small size of the ICI therapy cohorts for survival analysis is one of the limitations of this study, which will require further validation in larger studies.In addition, data on tumor stage and grade were available in a limited number of patients included in our analysis and, therefore, we were also unable to determine the relationship of these prognostic variables with treatment outcomes.Future prospective analysis will be required to answer such important questions.Further studies should be performed to fully uncover the implication of MSLN expression in CRC patients, speci cally MSS patients, to improve the e cacy and availability of therapeutic options to a patient population that currently sees limited bene t beyond progression on the current standard of care treatment options.

Declarations
Competing Interests -Midhun Malla: In the last 24 months, Dr. Midhun Malla has received grant funding from National Institute of General Medical Services-NIH (Grant # 5U54GM104942-05).The content is solely the responsibility of the author and does not necessarily represent the o cial views of the National Institutes of Health.This work was supported by Caris Life Sciences Inc. and all other authors have no competing interests to declare that are relevant to the content of this article.