Identification of the CIMP in PC patients
To identify the CIMP in PC patients, we first screened out the differential CpGs between PC samples and normal samples with the DNA methylation data of 185 PC patients and 10 normal controls downloaded from TCGA. In total, 22,450 differential CpGs were identified, and among these CpGs, 12,937 were hypermethylated CpGs and 9,513 were hypomethylated CpGs. Next, univariate Cox analysis was performed to identify OS- related CpGs and 3102 CpGs were selected with p <0.05. Then, 1037 out of 3102 CpGs with p <0.01 were further used to identify the most OS-related CpGs by multivariate Cox analysis. Seventy-two CpGs were finally selected for unsupervised consensus clustering analysis with p<0.05 (Supplementary materials). Based on the unsupervised consensus clustering analysis, 184 PC patients were clustered into three distinct groups (Figure 2A-2C), namely CIMP-L subgroup (n=46), CIMP-M subgroup (n=82) and CIMP-H subgroup (n=56). The methylation level of CIMP-L subgroup was low while patients of the CIMP-H subgroup had widespread hypermethylated CpGs.
Next, the associations between CIMP status and clinical characteristics were analyzed. As were shown at Table 1, there were more patients with advanced T stage and TNM stage in the CIMP-M subgroup and CIMP-H subgroup compared to that in CIMP-L subgroup (all p<0.05, Table 1). Besides, Kaplan-Meier analysis showed that there were significant differences in OS and PFS among PC patients from different CIMP status. Patients of the CIMP-H subgroup had poorer OS and RFS while the patients of CIMP-L subgroup had better OS and RFS (all p<0.05, Figure 2D, 2E). Moreover, univariate Cox analysis indicated that CIMP status were significantly related with OS and PFS, and multivariate Cox analysis also suggested that CIMP status were independent prognostic factors for OS and PFS of PC patients after adjusting for gender, age, T state, N stage, M stage and TNM stage (all p<0.05, Table 2).
Mutational landscapes of PC patients with different CIMP status
A number of mutated genes with deregulated DNA methylation had been identified to be played important roles in the development and progression of PC (8). The association of CIMP status with gene mutations was analyzed with somatic mutation data of 177 PC patients downloaded from TCGA. As were shown at Figure 3, there were significantly higher somatic mutation burdens among patients with different CIMP status. All the PC patients of the CIMP-H subgroup (n=54) had gene mutation, and 79 out of 81 PC patients of the CIMP-M subgroup had gene mutation, while only 31 out of 41 PC patients of the CIMP-L subgroup had gene mutation. Obviously, significantly higher somatic mutation burdens in KRAS, TP53, SMAD4, CDKN2A and TTN were observed in patients of CIMP-H group (Figure 3A-3C), which had been shown to be major driver genes in PC (23). Similarly, significant difference of tumor mutational burden (TMB), served as a biomarker of immunotherapy responses, were also found among patients with different CIMP status. Higher TMB were found at patients of CIMP-H subgroup, while lower TMB were observed at patients with CIMP-L status (Figure 3D).
Landscape of TME in PC patients with distinct CIMP status
Consisted of cancer cells, stromal cells and extracellular components, the TME had been demonstrated to play indispensable roles in tumorigenesis, progression, metastasis, recurrence, and drug resistance of PC (24). The difference of TME in patients with distinct CIMP status was also analyzed. As were shown at Figure 4, significantly lower stromal score, immune score and estimate score were found at patients of CIMP-H subgroup, while significantly higher stromal score, immune score and estimate score were found at patients of CIMP-L subgroup (Figure 4A-4C). Similarly, significantly higher tumor purity score was also observed in patients of CIMP-H subgroup (Figure 4D).
Cancer stem cells (CSCs) were cancer cells that possessed the ability to give rise to all tumor cell types and CSCs were considered to be responsible for tumor growth, metastasis and recurrence, and resistant to chemotherapy and radiation therapy. The association of CIMP status with cancer stemness indices was explored. As expected, significantly higher tumor stemness indices, including mRNAsi score, mDNAsi score EREG- mDNAsi score were found at patients of CIMP-H subgroup compared to that in patients of CIMP-L subgroup (Figure 5A, 5C, 5D).
Next, we analyzed the difference of tumor-infiltrating immune cells among patients with distinct CIMP status. As were shown at Figure 6, significantly lower total T cells, total B cells, naive B cells, CD8 T cells, CD4 T cells, resting memory CD4 T cells and activated memory CD4 T cells were found in patients of CIMP-H subgroup compared to that in patients of CIMP-L subgroup (Figure 6A-6F). Besides, significantly higher M0 macrophages was found in patients of CIMP-H subgroup and significantly higher regulatory T cells was found at patients of CIMP-M subgroup compared to that in patients of CIMP-L subgroup (Figure 6G-6H). It had been reported that different kinds of immune cell subsets were recruited into the TME via interactions between chemokines and their chemokine receptors (25). We furthered analyzed the difference of expression of 58 kinds of chemokines among patients with different CIMP status. As were summarized at Table 3, in line with the results of tumor-infiltrating immune cells, 31 kinds of chemokines, such as CCL2, XCL2, CCR2, CCL5 and CCR5, were found to be overexpressed in patients of CIMP-L subgroup, while only 7 kinds of chemokines, such as CXCL14 and CXCL16, were found to be increased in patients of CIMP-M and CIMP-H subgroup. Taken together, these results suggested that patients in CIMP-H subgroup have a distinct TME, characterized by higher tumor purity and tumor stemness, and lower immune activation and immune infiltration.
Expression of immune checkpoint genes in PC patients with different CIMP status
The advent of immunotherapy, especially checkpoint inhibitor-based immunotherapy, had revolutionized cancer treatments, especially for patients with advanced tumor. These treatments functioned through blockade of immunosuppressive checkpoints, so the expression of these immune checkpoint genes were necessary for checkpoint inhibitor immunotherapy (26). The difference of expression in10 immune checkpoint genes (including PD-1, PD-L1, CTLA4, PD-L2, CD86, CD80, CD276, VTCN1, Tim-3 and LAG-3) in PC patients with different CIMP status were further analyzed. As were shown at Figure 7, the expression of PD-1, CTLA4, CD86, VTCN1 and LAG-3 of PC patients of CIMP-H subgroup were significantly lower than that of patients of CIMP-L subgroup (Figure 7A, 7C, 7E, 7H-J). These results may indicate that checkpoint inhibitor immunotherapy to be less effective in patients of CIMP-H subgroup as they showed less expression of immune checkpoint genes.
Potential mechanism by which CIMP status to influence the prognosis of PC patients
GSEA analysis was performed to explore the underlying biological mechanism by which CIMP influenced prognosis of PC patients. As was shown at Figure 8, KEGG pathways, such as “P53 signaling pathway”, “notch signaling pathway”, “calcium signaling pathway”, “DNA replication” and “base excision repair” were found to be significantly enriched in patients of CIMP-H subgroup compared that in patients of CIMP-L patients (Figure 8A). Similarly, “P53 signaling pathway”, “base excision repair” and “proteasome” were found to be significantly enriched in patients of CIMP-M subgroup compared that in patients of CIMP-L subgroup (Figure 8B). These results may suggest that the CIMP status may influence the prognosis of PC patients by regulation of the aforementioned biological process.
Validation of CIMP status in PC patients of ICGC dataset
To independently test the CIMP status in PC patients, DNA methylation data of 264 PC patients, somatic mutation data of 264 PC patients, mRNA expression of 175 PC patients and clinical-pathological parameter of 264 PC patients were downloaded from ICGC (https://dcc.icgc.org/). Unsupervised consensus clustering analysis was also performed for 264 PC patients based on the expression of these 72 OS-related CpGs. Similarly, these 264 PC patients were also clustered into three distinct groups (Figure 9A), there were 58 PC patients in CIMP-L subgroup, 171 PC patients in CIMP-M subgroup, and 35 PC patients in the CIMP-H subgroup. Besides, significant associations between CIMP status and clinical-pathological characteristics, including age, T state, N stage, M stage and TNM stage, were also observed (Table 4). Moreover, Kaplan-Meier analysis showed that there were significant differences in OS among PC patients with different CIMP status. Patients of the CIMP-H subgroup had poorer OS while the patients of CIMP-L subgroup had better OS (p=0.003, Figure 9A). Univariate and multivariate Cox analysis also suggested that the CIMP status were significantly related with OS, and was also an independent prognostic factor for OS of PC patients after adjusting for gender, age, T state, N stage, M stage and TNM stage (p=0.012, Table 5).
The association of CIMP status with gene mutations was further analyzed. In line with the results in TCGA, all the 35 PC patients of the CIMP-H subgroup (100%) had gene mutation; 168 out of 171 PC patients of the CIMP-M subgroup (98%) had gene mutation, while only 35 out of 58 PC patients of the CIMP-L subgroup (58.66%) had gene mutation. Obviously, higher somatic mutation burdens in KRAS, TP53, CDKN2A and TTN were found at patients of CIMP-H group compared to that in patients of CIMP-L group (Figure 9B). Besides, higher TMB were also found at patients of CIMP-H subgroup, while lower TMB were observed at patients with CIMP-L status (Figure 9B).
The landscape of TME among PC patients with distinct CIMP status was also explored. As expected, significantly lower stromal score, immune score and estimate score were found at patients of CIMP-L subgroup. Meanwhile, significantly lower total T cells, total B cells, CD8 T cells, memory CD4 T cells were found in patients of CIMP-H subgroup, but significantly higher follicular helper T cells were found in patients of CIMP-H subgroup (Figure 9C).
Finally, the association of CIMP status with immune checkpoint genes was also analyzed. Only expression of 4 immune checkpoint genes, including PD-1, PD-L1, CD86 and CD276 were available. Similarly, lower expression of PD-1, PD-L1 and CD86 were found at patients of CIMP-H subgroup, but the difference was not statistically significant (Figure 9D).