CMTM6 and PD-L1 coexpression is associated with an active immune microenvironment and a favorable prognosis in colorectal cancer

Abstract

Background: CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6), a programmed death-ligand 1 (PD-L1) regulator, is widely expressed in various tumors and regulates the immune microenvironment. However, its prognostic value remains controversial, and the roles of CMTM6 in colorectal cancer (CRC) are still unknown. In this study, we aimed to elaborate the expression patterns of CMTM6 and PD-L1 in CRC and investigate their relationship with the infiltration of T cells and the prognosis of patients with CRC.

Methods: Analysis of CMTM6 mRNA levels, gene ontology enrichment analysis and single-sample gene set enrichment analysis were performed in a The Cancer Genome Atlas colon cancer cohort. The expression of CMTM6 and PD-L1 and the infiltration of T cells in tumor tissues from our cohort containing 156 patients with CRC receiving adjuvant chemotherapy and 77 patients with CRC without chemotherapy were examined by immunohistochemistry assay.

Results: CMTM6 expression was upregulated in CRC compared with normal colon tissues, and CMTM6 levels were lower in advanced tumors than in early-stage tumors. High expression of CMTM6 correlated with lower pT stage and more CD4⁺/CD8⁺ tumor-infiltrating lymphocytes (TILs) and predicted a favorable prognosis in CRC. PD-L1 was expressed in CRC tissues at a low level, and PD-L1 positivity in tumor stroma (PD-L1(TS)), but not PD-L1 positivity in cancer cells (PD-L1(CC)), was associated with an increased density of CD4⁺ TILs and a favorable prognosis. The coexpression status of CMTM6 and PD-L1(TS) divided patients with CRC into three groups with low, moderate and high risks of progression and death, and patients with CMTM6^High/PD-L1(TS)⁺ status had the longest survival. Moreover, the prognostic value of CMTM6/PD-L1 expression was more significant in patients with CRC treated with adjuvant chemotherapy than in those not treated with chemotherapy.

Conclusion: CMTM6 has a critical impact on the immune microenvironment and can be used as an independent prognostic factor for CRC. The coexpression status of CMTM6 and PD-L1 can be used as a new classification to stratify the risk of progression and death for patients with CRC, especially for patients receiving adjuvant chemotherapy. These findings may provide insights into improving responses to immunotherapy-included comprehensive treatment for CRC in the future.

Keywords: B7-H1 antigen; biomarkers; gastrointestinal neoplasms; immunohistochemistry; lymphocytes; tumor; tumor-infiltrating.

Figure 1

The expression pattern of CMTM6 in CRC and the association of CMTM6 expression with the prognosis of patients with CRC. (A–C) Relative CMTM6 mRNA levels (shown as log2(x+1) values) (A) in CRC tissues and adjacent normal tissues from the TCGA cohort or (B) in tumor tissues with various consensus molecular subtypes (CMS1–4) or (C) clinical stages (I/II and III/IV). (D) CMTM6 protein levels (IHC Scores) in CRC tissues at early (I/II) or advanced stages (III/IV). (E) Kaplan-Meier survival curves for progression-free survival (PFS) and overall survival (OS) of patients with CRC stratified by CMTM6 levels. CMTM6, CKLF-like MARVEL transmembrane domain-containing 6; CRC, colorectal cancer; IHC, immunohistochemistry; TCGA, The Cancer Genome Atlas.

Figure 2

A comparison of immune cell infiltration in CRC samples with high or low levels of CMTM6 expression. (A) ssGSEA analysis of RNA-Seq data from the TCGA cohort. Immune cell types were defined by specific gene markers. (B–D) IHC analysis of (B) CD4⁺, (C) CD8⁺ and (D) FoxP3⁺ T cell infiltration in CRC tissues with high or low CMTM6 expression from the SYSUCC cohort. *p<0.05; **p<0.01; ***p<0.001; n.s, no significant difference. CMTM6, CKLF-like MARVEL transmembrane domain-containing 6; CRC, colorectal cancer; IHC, immunohistochemistry; ss-GSEA, gene set enrichment analysis; SYSUCC, Sun Yat-sen University Cancer Center; TCGA, The Cancer Genome Atlas.

Figure 3

The association of PD-L1 expression with the infiltration of T cells and prognosis of patients with CRC. (A–C) Kaplan-Meier survival curves for PFS and OS of patients with CRC from the SYSUCC cohort based on the expression status of (A) PD-L1(whole), (B) PD-L1(TS) or (C) PD-L1(CC). (D–F) The infiltration of (D) CD4⁺, (E) CD8⁺ or (F) FoxP3⁺ T cells in CRC tissues with PD-L1(TS)⁺ or PD-L1(TS)⁻ from the SYSUCC cohort. (+), PD-L1(TS)⁺; (−), PD-L1(TS)⁻. *p<0.05; n.s, no significant difference. CC, cancer cells; CRC, colorectal cancer; IM, invasive margin; OS, overall survival; PD-L1, programmed death-ligand 1; PFS, progression-free survival; SYSUCC, Sun Yat-sen University Cancer Center; TS, tumor stroma.

Figure 4

The association of coexpression of CMTM6/PD-L1 with the infiltration of T cells and the prognosis of patients with CRC. (A) Kaplan-Meier survival curves for progression-free survival (PFS) and overall survival (OS) of patients with CRC from the SYSUCC cohort based on the coexpression status of CMTM6/PD-L1(TS). (B) Kaplan-Meier survival curves for PFS and OS of patients with CRC in three groups: group 1, CMTM6^High/PD-L1(TS)⁺; group 2, CMTM6^High/PD-L1(TS)⁻ or CMTM6^Low/PD-L1(TS)⁺ and group 3, CMTM6^Low/PD-L1(TS)⁻. (C) The infiltration of CD4⁺ (upper), CD8⁺ (middle) or FoxP3⁺ (lower) in CRC tissues from groups 1, 2 and 3. *p<0.05; **p<0.01; ***p<0.001; n.s, no significant difference. CMTM6, CKLF-like MARVEL transmembrane domain-containing 6; CRC, colorectal cancer; PD-L1, programmed death-ligand 1; SYSUCC, Sun Yatsen University Cancer Center; TS, tumor stroma.

Figure 5

Subgroup survival analysis for patients with CRC receiving or not receiving adjuvant chemotherapy. Kaplan-Meier survival curves for progression-free survival and overall survival of patients treated with (A, C, E) or not treated with (B, D, F) adjuvant chemotherapy based on (A, B) CMTM6 expression, (C, D) PD-L1(TS) expression, or (E, F) the coexpression status of CMTM6/PD-L1(TS) (group 1, CMTM6^High/PD-L1(TS)⁺; group 2, CMTM6^High/PD-L1(TS)⁻ or CMTM6^Low/PD-L1(TS)⁺; and group 3, CMTM6^Low/PD-L1(TS)⁻). p<0.05 indicating significant difference. CMTM6, CKLF-like MARVEL transmembrane domain-containing 6; CRC, colorectal cancer; PD-L1, programmed death-ligand 1; PD-L1(TS), PD-L1 in the tumor stroma; TS, tumor stroma.