Molecular features and clinicopathological assessment of CRC patients
There are 462 COAD patient data in TCGA database, including 390 cases with SNP and transcriptome data. The median age at diagnosis is 68 years. There are 52 patients with MSI/dMMR, and 338 with MSS/pMMR, including 261 with APC-mt and 77 with APC-wt. There are 152 patients with KRAS-mt and 186 patients with KRAS-wt. There are 194 patients with TP53-mt and 144 patients with TP53-wt. There were 45 patients with KRAS-/P53-/APC+/MSS, 55 patients with KRAS+/P53-/APC+/MSS, 71 patients with KRAS+/P53+/APC+/MSS, 11 patients with KRAS+/P53+/APC-/MSS, 15 patients with KRAS+/P53-/APC-/MSS, 90 patients with KRAS-/P53+/APC+/MSS, 22 patients with KRAS-/P53+/APC-/MSS, and 29 patients with KRAS-/P53-/APC-/MSS.
Of the 390 colon cancer patients with SNP and transcriptome data, 14.6% (57/ 390) were in stage I, 23.3% (91/390) were in stage II, 34.9% (136/ 390) were in stage III, and 13.9% (54/390) were in stage IV. The 52 MSI/dMMR colon cancer patients accounted for 2.3% (9/390) in stage I, 4.1% (16/390) in stage II, 5.4% (21/390) in stage III, and 1.5% (6/390) in stage IV. MSS/pMMR colon cancer patients in the database are mainly in stage II, while MSI/dMMR colon cancer patients are mainly in stage III. APC mutations were detected in 75% (293 of 390) of the tumors. In the APC-mt cases, the frequencies of KRAS and TP53 mutations were 48.0% and 60%, respectively. In the APC-wt cases, the KRAS and TP53 mutation rates were 33% and 42%, respectively. Moreover, the BRAF mutation rate was significantly increased by 36% in this group compared to the APC-mt group.
We found that APC mutations were strongly and negatively correlated with BRAF mutations and positively correlated with KRAS and TP53 mutations (P≤0.05).
Comparison of gene expression profile and different gene subtypes in colon cancer based on ImmuneScores, StromalScores, and tumor mutation burden (TMB)
To reveal the relationship between MSS/pMMR colon cancer overall gene expression profile and APC mutation, we compared the Affymetrix microarray data of all 338 colon cancer cases obtained from TCGA database. The heat map in Figure 2A shows the different gene expression profiles of APC-wt/MSS and APC-mt/MSS cases. In the APC gene mutation group, 379 genes were down-regulated and 117 genes were up-regulated (fold change > 1.5, P < 0.05; Supplementary Table 1). In MSS/pMMR colon cancer, the expression of immune checkpoint genes such as PD-1, PD-L1, and CTLA4 in the APC-mt/MSS group was significantly lower than in the APC-wt/MSS group. Compared with KRAS-wt/MSS group, the expression of CTLA4 and PD-L1 in the KRAS-mt/MSS group was down-regulated. The difference, however, was smaller than that between the APC-wt and APC-mt groups. The expression of PD-1, PD-L1, and CTLA4 did not differ between TP53-wt/MSS and TP53-mt/MSS groups. In addition, the ImmuneScore and StromalScore calculated by the ESTIMATE were significantly lower in the APC-mt/MSS group than in the APC-wt/MSS group. The ImmuneScore and StromalScore calculated for the KRAS-mt/MSS group were also lower than those in the KRAS-wt/MSS group. The range of the calculated scores was, however, smaller than that between the APC groups. The TP53-mt/MSS group had a lower ImmuneScore than that of the KRAS-wt/MSS group, but the difference was not so obvious. Compared with APC-wt/MSS group, the TMB was significantly lower in the APC-mt/MSS group than in the APC-wt/MSS group. Mutations in KRAS or TP53 did not affect the TMB. These results suggest that the proportion of immune-related components and expression of immune checkpoint are higher in the APC-wt/MSS colon cancer tumor microenvironment. The presence of APC-wt/MSS, combined with TMB, is consistent with this increase. It can, therefore, be presumed that APC-wt/MSS colon cancer could become a beneficiary of immune checkpoint inhibitors. A follow-up research will need to focus on further exploration in this direction.
Composition of immune cells in MSS/pMMR colon cancer with different genetic subtypes
We studied the proportion of immune cells infiltrating between different genetic subtypes in the colon cancer cohort retrieved from TCGA. All 338 MSS/pMMR colon cancer samples met CIBERSORT requirements at P < 0.05. The proportion of CD8+ T cells is significantly lower, and the proportion of M0 macrophages is significantly higher in APC-mt/MSS colon cancer in comparison to APC-wt/MSS. However, neither mutations in KRAS nor TP53 could affect the proportion of infiltrating immune cell types in MSS/pMMR colon cancer. Considering the results shown in Figure 3, APC-wt/MSS has a higher percentage of immune-related components infiltration in the tumor microenvironment compared to APC-mt/MSS colon cancer. Furthermore, through the application of univariate and multiple Cox regression analyses, we demonstrate that immune cell infiltration and APC gene status in CRC patients are related to PD-1 expression. In addition, assessment of TILs in the tumor microenvironment is challenging, immune cell cytolytic activity (CYT) might be used to assess TILs including CD8+ CTL and other immune cells (e.g., natural killer T cells). And CYT was measured by the mRNA expression levels of granzyme A (GZMA) and perforin 1 (PRF1) [15]. Figure3J and K showed that GZMA and PRF1 were significantly higher expression in APC-wt/MSS than APC-mt/MSS colon cancer. The immune-promoting lymphocyte infiltration ratio, such as CD8+ T cells, and the expression ratio of PD-1, the immune checkpoints, have significantly increased. It is speculated that APC-wt colon cancer is more likely to be a "hot tumor," and is more likely to benefit from anti-tumor immunotherapy.
Relationship between immune status and APC mutations in CRC patients
We divided the CRC samples in the cohort retrieved from TCGA into APC-wt (77 samples) and APC-mt (261 samples) groups and performed gene set enrichment analysis (GSEA). The results show that APC-wt colon cancer was significantly enriched in 115 KEGG pathways (P < 0.05; Supplementary Table S2-S3). These include pathways related to immune signaling, such as natural killer cell-mediated cytotoxicity, leukocyte transendothelial migration, NOD-like receptor signaling, TOLL-like receptor signaling, TGF-β signaling, and other immune-related signaling pathways (Figure 4A). We then performed Gene Ontology (GO) analysis of the immune-related DEGs in APC-wt/MSS colon cancer. Circular plot of GO pathways was enriched by processes regulating leukocyte and T cell activation, and leukocyte cell−cell adhesion. These findings again indicate that APC mutations play a role in the immune response of colon cancer.
Calculation and validation of the ImmunoScore, and evaluation of its prognostic ability in the CRC cohort retrieved from TCGA
We have identified 65 overlapping genes (shown in Table S4) among the DEGs (496 genes related to APC status, shown in Supplementary Table S1) and the DEGs related to immunophenotypes (1297 genes shown in Supplementary Table S5). Using Lasso and Cox regression analyses, the eight genes with the highest prognostic value were identified (Figure 5A, B). We then selected these genes to establish an immune scoring model, which was assessed for its ability to predict the prognosis of CRC patients. The formula of the immune scoring model is described in the Materials and methods section. Next, we divided the CRC patients into a high score and low score groups based on an optimal cutoff value of the immune score obtained by the survminer R package. The results show that patients with a high score had a worse OS than those with a low score (Figure 5C). Figure 3b shows that the area under the ROC curve (AUC) of the 5-years OS prognostic model is 0.614. Figure 3c shows the immune score distribution and selected gene expression data.
Immunohistochemical expression of markers for TIMTs
The tumors of all 42 patients were immunopositive for markers of TIMT. 8/22 APC-mt/MSS colon cancer (36.3%) was immunopositive for CD8, and 12/20 APC-wt/MSS colon cancer (60%) was immunopositive for CD8. 5/22 APC-mt/MSS colon cancer (22.5%) was immunopositive for PD-L1, and 11/20 APC-wt/MSS colon cancer (55%) was immunopositive for PD-L1. In addition to the positive rate, the degrees of positive expression for CD8 and PD-L1, APC-wt/MSS is significantly higher than APC-mt/MSS with immunostaining. TIMTs divide tumors into four categories based on the presence or absence of TILs and PD-L1 expression levels (type I: TILs+ and PD- L1+; type II: TILs- and PD-L1-; type III: TILs+ and PD- L1-; type IV: TILs- and PD-L1+). The 42 patients, containing APC-mt/MSS and APC-wt/MSS two groups, were divided into one of the above four types according to expression patterns of markers CD8 and PD-L1. 2/22 APC-mt/MSS colon cancer (9.1%) was immunopositive for both CD8 and PD-L1 (TIMT I). 6/22 APC-mt/MSS colon cancer (27.2%) was immunopositive for only CD8 (TIMT III). 3/22 APC-mt/MSS colon cancer (13.6%) was immunopositive for only PD-L1 (TIMT IV). 11/22 APC-mt/MSS colon cancer (50%) was immunonagtive for either CD8 or PD-L1 (TIMT II). 10/20 APC-wt/MSS colon cancer (50%) was immunopositive for both CD8 and PD-L1 (TIMT I). 2/20 APC-wt/MSS colon cancer (10%) was immunopositive for only CD8 (TIMT III). 1/20 APC-wt/MSS colon cancer (5%) was immunopositive for only PD-L1 (TIMT IV). 7/20 APC-wt/MSS colon cancer (35%) was immunonagtive for either CD8 or PD-L1 (TIMT II). Therefore, APC-wt/MSS mainly includes TIMT I type colon cancer, which was consistent with the statistical result in the TCGA database. No difference in clinical characteristics between the two groups. (Table 1)
Table 1. Clinical characteristics of patients with APC-mt/MSS and APC-wt/MSS colon cancer
Characteristics
|
APC-mt/MSS Patients
|
APC-wt/MSS Patients
|
Patients (n)
|
22
|
20
|
Age (years), median (range)
|
67.5(42-89)
|
66.4(43-89)
|
>60 years (n)
|
14
|
12
|
≤60 years (n)
|
8
|
8
|
Sex (Male/Female) (n/n)
|
10/12
|
9/11
|
KPS score (%), median (range)
|
80(30-100)
|
80(20-100)
|
≥60% (n (%))
|
18
|
17
|
<60% (n (%))
|
4
|
3
|
Stage I (n)
|
0
|
0
|
Stage II (n)
|
4
|
3
|
Stage III (n)
|
10
|
10
|
Stage IV (n)
|
8
|
7
|