Pan-cancer analysis of CDKN2A alterations identifies a subset of gastric cancer with a cold tumor immune microenvironment

Abstract

Background: Although CDKN2A alteration has been explored as a favorable factor for tumorigenesis in pan-cancers, the association between CDKN2A point mutation (MUT) and intragenic deletion (DEL) and response to immune checkpoint inhibitors (ICIs) is still disputed. This study aims to determine the associations of CDKN2A MUT and DEL with overall survival (OS) and response to immune checkpoint inhibitors treatment (ICIs) among pan-cancers and the clinical features of CDKN2A-altered gastric cancer.

Methods: This study included 45,000 tumor patients that underwent tumor sequencing across 33 cancer types from four cohorts, the MSK-MetTropism, MSK-IMPACT, OrigiMed2020 and TCGA cohorts. Clinical outcomes and genomic factors associated with response to ICIs, including tumor mutational burden, copy number alteration, neoantigen load, microsatellite instability, tumor immune microenvironment and immune-related gene signatures, were collected in pan-cancer. Clinicopathologic features and outcomes were assessed in gastric cancer. Patients were grouped based on the presence of CDKN2A wild type (WT), CDKN2A MUT, CDKN2A DEL and CDKN2A other alteration (ALT).

Results: Our research showed that CDKN2A-MUT patients had shorter survival times than CDKN2A-WT patients in the MSK MetTropism and TCGA cohorts, but longer OS in the MSK-IMPACT cohort with ICIs treatment, particularly in patients having metastatic disease. Similar results were observed among pan-cancer patients with CDKN2A DEL and other ALT. Notably, CDKN2A ALT frequency was positively related to tumor-specific objective response rates to ICIs in MSK MetTropism and OrigiMed 2020. Additionally, individuals with esophageal carcinoma or stomach adenocarcinoma who had CDKN2A MUT had poorer OS than patients from the MSK-IMPACT group, but not those with adenocarcinoma. We also found reduced levels of activated NK cells, T cells CD8 and M2 macrophages in tumor tissue from CDKN2A-MUT or DEL pan-cancer patients compared to CDKN2A-WT patients in TCGA cohort. Gastric cancer scRNA-seq data also showed that CDKN2A-ALT cancer contained less CD8 T cells but more exhausted T cells than CDKN2A-WT cancer. A crucial finding of the pathway analysis was the inhibition of three immune-related pathways in the CDKN2A ALT gastric cancer patients, including the interferon alpha response, inflammatory response, and interferon gamma response.

Conclusions: This study illustrates the CDKN2A MUT and DEL were associated with a poor outcome across cancers. CDKN2A ALT, on the other hand, have the potential to be used as a biomarker for choosing patients for ICI treatment, notably in esophageal carcinoma and stomach adenocarcinoma.

Keywords: CDKN2A; Immune checkpoint inhibitor; Immunotherapy; Pan-cancer.

Fig. 1

Overall schematic flow-chart of this study (A) and potential effects of CDKN2A alterations on the immune microenvironment and immunotherapy (B). MSK-MetTropism, Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets; MSK-IMPACT, Memorial Sloan Kettering-Integrated Mutational Profiling of Actionable Cancer Targets; ICI, immune checkpoint inhibitors; OS, overall survival; TCGA, the Cancer Genome Atlas

Fig. 2

CDKN2A alterations in the MSK-MetTropism cohort. (A) The distribution of CDKN2A-DEL, CDKN2A-MUT and CDKN2A-WT patients. CDKN2A-MUT: CDKN2A point mutation, CDKN2A-DEL: CDKN2A deletion, CDKN2A-WT: CDKN2A-wide type. (B) Frequency of CDKN2A DEL, CDKN2A MUT and CDKN2A other ALT in each cancer type. (C) Over survival (OS) in CDKN2A-WT and CDKN2A-alteration (ALT) patients. (D) OS in prespecified subgroups including CDKN2A-DEL, CDKN2A-MUT, CDKN2A-WT and CDKN2A other-ALT groups. HR: hazard ratio, CI: confidence interval. (E) Multivariable Cox regression analysis presented the association between CDKN2A DEL, CDKN2A MUT, CDKN2A other ALT, various types of cancer and OS after controlling for other factors including FGA, MSI score, TMB, age, and metastasis status. The median values for FGA, MSI Score, TMB, Age, and Mutation Count are 0.15, 0.24, 4.32, 63.9, and 5.0, respectively. Patients with values below the median were assigned to the reference group, while those equal to or above the median were assigned to the treatment group. Forest plot of association between CDKN2A ALT and OS of all patient (F) and patients diagnosed with primary cancer (G)

Fig. 3

Associations of CDKN2A alterations with OS and objective response rates to ICIs in the MSK-IMPACT cohort. (A) OS in prespecified subgroups including CDKN2A-DEL, CDKN2A-MUT, CDKN2A-WT and other ALT groups. (B) Multivariable Cox regression analysis presented the association between CDKN2A-DEL, CDKN2A-MUT and CDKN2A-other ALT and OS after controlling for other factors including drug type, sex, TMB, age, and metastasis status. Forest plot of association between CDKN2A-MUT (C) and CDKN2A-DEL (D) and OS. OS of CDKN2A-DEL, CDKN2A-MUT, CDKN2A-WT and other ALT groups with PD-1 (E) and CTLA-4 (F) inhibitors treatment, primary cancer (G) or metastasis (H)

Fig. 4

Associations of CDKN2A alterations with clinical outcomes in the TCGA cohort. OS (A) and PFS (B) in CDKN2A-WT and CDKN2A-ALT patients. OS (C) and PFS (D) in CDKN2A-DEL, CDKN2A-MUT, CDKN2A-WT and CDKN2A-other ALT patients. (E) Frequency of CDKN2A-DEL, CDKN2A-MUT and CDKN2A-other ALT in each cancer type. Forest plot of association between CDKN2A ALT and OS (F) and PFS (G). Forest plot of association between CDKN2A MUT(H) and DEL(I) and OS

Fig. 5

Clinical and CDKN2A alterations characteristics in the OrigiMed cohort. (A) Landscape of somatic CDKN2A alterations across 25 cancer types. (B) The alterations sites of CDKN2A in the OrigiMed cohort. (C) Heatmap showing the pattern of co-occurrence/mutual exclusivity of CDKN2A, APC, ATM, BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, and PALB2. (D) Correlation between CDKN2A ALT frequency and objective response rates to ICIs. (E) Frequency of CDKN2A-DEL, CDKN2A-MUT and CDKN2A-other ALT in each cancer type. Comparison of TMB (F), mutation count (G) and metastasis (H) among four subgroups

Fig. 6

Assessment of immune infiltration, immune signatures, and pathway enrichment in CDKN2A-mutated pan-cancer patients in the TCGA cohort. Associations of CDKN2A ALT with immune cells infiltration (A), immune-related genes (B), signaling pathways (C) in TCGA Cohort (D) OS in CDKN2A-DEL, CDKN2A-MUT, CDKN2A-WT and CDKN2A-other ALT patients from Smita Sihag esophagogastric cancer cohort. (E) Protein comparison of p14 ARF (p14) and p16INK4a (p16) in CDKN2A-mutated STAD patients from the TCGA cohort. (F) Western blotting showed the expression of p16 and p14 in stomach cancer cell lines after CDKN2A knockdown

Fig. 7

Single-cell transcriptional profiling of human GC. (A) scRNA-seq tSNE projection of 59,594 single cells from 21 primary GCs samples. (B) tSNE plot of 59,594 single cells from 21 primary GCs samples, color-coded by group. (C) Dot plot showing the gene expression patterns of cell-type marker genes in the scRNA-seq data. (D) Proportions of cell types in individual samples (above) and in different groups (bottom). scRNA-seq, single-cell RNA sequencing. (E-J) The GSEA plot shows enrichment of several pathways in CDKN2A ALT group. FDR < 0.05 is considered as significantly enriched. (K) tSNE plot of CD8 + T cells from 21 primary GCs samples. (L) The proportion of each CD8⁺ T cell cluster in CDKN2A-WT and CDKN2A- ALT patients