Sex and outcomes of patients with microsatellite instability-high and BRAF V600E mutated metastatic colorectal cancer receiving immune checkpoint inhibitors

Abstract

Background: Immune checkpoint inhibitors (ICIs) are the gold standard therapy in patients with deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). A significant proportion of patients show resistance, making the identification of determinants of response crucial. Growing evidence supports the role of sex in determining susceptibility to anticancer therapies, but data is lacking for patients with MSI-H CRC.

Methods: In this real-world cohort comprising 624 patients with MSI-H mCRC receiving ICIs, we investigated the impact of sex on patients' outcomes, overall and according to RAS-BRAF mutational status or type of treatment (anti-PD-(L)1 with or without anti-CTLA-4 agents). We then investigated these associations also in two independent cohorts of patients with early-stage or advanced MSI-H CRC unexposed to ICIs. Finally, we explored two public microarray and RNA-seq datasets from patients with non-metastatic or metastatic MSI-H CRC to gain translational insights on the association between sex, BRAF status and immune contextures/ICI efficacy.

Results: Although no differences were observed between females and males either overall or in the BRAF wild-type cohort, male sex was associated with inferior progression-free survival (PFS) and overall survival (OS) in the BRAF mutated cohort (in multivariable models, HR for PFS: 1.79, 95% CI: 1.13 to 2.83, p=0.014, and for OS: 2.33, 95% CI: 1.36 to 3.98, p=0.002). Males receiving anti-PD-(L)1 monotherapy had the worst outcomes, with a 3-year PFS and 3-year OS of 23.9% and 41.8%, respectively, while the addition of anti-CTLA-4 agents rescued such a worse outcome. We also observed that females experienced a higher frequency of any-grade immune-related adverse events. Conversely, sex was not prognostic in the independent cohorts of patients with MSI-H CRCs not treated with ICIs. Exploratory transcriptomic analyses suggest that tumors of males with BRAF mutated MSI-H metastatic CRC are characterized by an enrichment of androgen receptor signature and an immune-depleted microenvironment, with a reduction in memory B cells, activated natural killer cells, and activated myeloid dendritic cells.

Conclusions: Overall, our findings suggest a complex interplay between sex and BRAF mutational status that may modulate the activity of ICIs in patients with MSI-H mCRC and pave the way to novel tailored strategies.

Keywords: Colorectal Cancer; Immune Checkpoint Inhibitor; Microsatellite; Mismatch repair - MMR.

Figure 1. Male sex is associated with worse outcomes in BRAF mutated deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) treated with ICIs. (a–d) Exploratory Kaplan-Meier analysis of the PFS and OS probability (with 95% CIs) and 3-year PFS and OS rates (as percentages) according to sex (a, b) and type of immune checkpoint inhibitors (ICIs) treatment (c, d) in patients with BRAF V600E mutated dMMR/MSI-H mCRC (n=161). (e, f) Multivariable Cox proportional hazards regression models of the PFS (e) and OS (f) in patients with BRAF V600E mutant dMMR/MSI-H mCRC (n=161). Covariates showing a statistically significant (p<0.20) association with PFS and OS at univariable models were further included in the multivariable models. At multivariable models, p values <0.05 were considered statistically significant. Abbreviations: aCTLA-4 combo, combination; aPD-1 mono, monotherapy; ECOG, Eastern Cooperative Oncology Group; histo, histology; inv, invasion; mets, metastasis; OS, overall survival; PFS, progression-free survival; PS, performance status; Ref, reference.

Figure 2. Sex is not associated with different prognosis in patients with metastatic deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H) and BRAF V600E mutated CRC treated in the pre-immunotherapy era. (a) Flow chart of collection of a large independent cohort of patients with metastatic dMMR/MSI-H CRC who were treated with chemotherapy with or without biologics in the pre-immunotherapy era (n=408), further selected for availability of BRAF mutational status (n=364). (b–e) Exploratory Kaplan-Meier analysis of PFS and OS according to sex in patients with dMMR/MSI-H CRC with (d, e) or without (b, c) BRAF V600E mutation. Abbreviations: mOS, median OS; mPFS, median PFS; mut, mutated; OS, overall survival; PFS, progression-free survival; Ref, reference; wt, wild-type.

Figure 3. Sex differences between BRAF mutated and BRAF wild-type deficient mismatch repair (dMMR)/microsatellite instability-high (MSI-H) CRC. (a) UMAP of participants from Gallois et al and GSE39582 stratified by BRAF mutational status. (b) Gene expression comparisons of PD-1 (PDCD1), PD-L1 (CD274), and CTLA4. P values were retrieved with an unpaired T-test. (c) GSEA pathway analysis with GO:BP gene signatures. Pathways were included if the comparisons in the BRAF-mut subgroup in the metastatic setting had an FDR q-value <0.05 and |NES| greater than 1.00. Raw GSEA results are provided in online supplemental material section 2. NES values were opaque if the FDR q-value was <0.05. (d) GSEA enrichment plots of the immune infiltration signature across gender/BRAF subgroups. (e) CIBERSORT deconvoluted immune cell type changes. T-statistic values were opaque if a two sided p value retrieved from an unpaired T-test was <0.05. Raw results are provided in online supplemental material section 2. (f) Boxplot of selected CIBERSORT deconvoluted immune cell type changes in the metastatic cohort. Abbreviations: FDR, false discovery rate; GO:BP, Gene Ontology:Biological Process; GSEA, gene set enrichment analysis; MUT, mutant; NES, normalized enrichments score; UMAP, Uniform Manifold Approximation and Projection; WT, wild-type.