Association of TMB with regulatory T cell marker genes expression in human cancers
Regulatory T (Treg) cells play an important role in the maintenance of tumor immunosuppression [20]. We compared expression levels of 70 tumor-infiltrating Treg gene signatures [21] between the lower-TMB and the higher-TMB cancers in each of the 32 cancer types. We found that seven genes (ADPRH, IL1R1, KSR1, SOCS2, JAK1, NFAT5, and SSH1) were more highly expressed in the lower-TMB subtype than in the higher-TMB subtype of more than 10 cancer types (Additional file 1: Table S1). Of note, ADPRH had significantly higher expression levels in the lower-TMB subtype of 14 cancer types. The expression levels of the Treg gene-set were significantly higher in the lower-TMB subtype of 12 cancer types (HNSC, STAD, CHOL, UVM, PRAD, ACC, THCA, LUSC, ESCA, DLBC, KIRP, and LIHC) while were significantly higher in the higher-TMB subtype of 1 cancer type (THYM) (Wilcoxon rank-sum test, P < 0.05) (Fig. 1a). Interestingly, 27 Treg genes were more highly expressed in lower-TMB LIHC versus 1 more highly expressed in higher-TMB LIHC (Fisher’s exact test, P = 1.2*10− 8, OR = 42.42). In contrast, 14 Treg genes were more highly expressed in lower-TMB THYM versus 28 more highly expressed in higher-TMB THYM (Fisher’s exact test, P = 0.016, OR = 0.38). These results suggest that the relatedness between TMB and Treg cells infiltration degree depends on cancer types, whereas the lower-TMB subtype is likely to have stronger Treg cells infiltration than the higher-TMB subtype in diverse cancers.
Association of TMB with immune checkpoint genes expression in human cancers
Immune checkpoint molecules are important for tumor immune evasion [22]. We compared expression levels of 47 immune checkpoint genes [21] between the lower-TMB subtype and the higher-TMB subtype of cancers. We found that 12 genes had significantly higher expression levels in the higher-TMB subtype than in the lower-TMB subtype of at least 6 cancer types (Additional file 1: Table S2). The 12 genes included LAG3, CD80, TNFSF9, IDO1, CD70, KIR3DL1, CTLA4, PD-1, PD-L1, PD-L2, TIGIT, and TNFRSF9. Notably, LAG3 had higher expression levels in the higher-TMB subtype of 10 cancer types versus 2 cancer types of which LAG3 showed higher expression levels in the lower-TMB subtype. Interestingly, many immune checkpoint genes, which are established or promising targets for immune checkpoint blockade therapy, had significantly higher expression levels in the higher-TMB subtype of various cancers, such as CTLA4, PD-1, PD-L1, PD-L2, LAG3, IDO1 and TIGIT. In contrast, 16 immune checkpoint genes (C10orf54, CD200, CD40LG, ADORA2A, TNFSF14, BTLA, CD160, CD44, CD48, CD28, VTCN1, CD200R1, NRP1, TMIGD2, ICOS, and TNFSF15) had significantly higher expression levels in the lower-TMB subtype than in the higher-TMB subtype of at least 6 cancer types.
The expression levels of the immune checkpoint gene-set were significantly higher in the lower-TMB subtype than in the higher-TMB subtype of TGCT, KIRC, HNSC, ACC, THCA, LIHC, and THYM, while were significantly higher in the higher-TMB subtype of CESC, COAD, UCEC, and BLCA (Wilcoxon rank-sum test, P < 0.05) (Fig. 1b). Of the 47 immune checkpoint genes, 22 were more highly expressed in higher-TMB CESC versus 3 more highly expressed in lower-TMB CESC (Fisher’s exact test, P = 1.3*10− 5, OR = 12.55). In addition, 20 immune checkpoint genes were more highly expressed in higher-TMB COAD versus 1 more highly expressed in lower-TMB COAD (Fisher’s exact test, P = 1.98*10− 6, OR = 33). In contrast, no any immune checkpoint gene was more highly expressed in higher-TMB LIHC versus 22 more highly expressed in lower-TMB LIHC (Fisher’s exact test, P = 1.88*10− 8), and the similar result was observed in THCA. These data indicate that the association between TMB and the immune checkpoint activity is cancer type dependent, with in some cancers TMB being positively correlated with the immune checkpoint activity while in some other cancers they showing an inverse correlation.
Association of TMB with immune cell infiltration in human cancers
We compared the infiltration densities of 16 different immune cell subpopulations [23] between the lower-TMB subtype and the higher-TMB subtype of cancers. We found that 11 immune cell subpopulation marker genes had significantly higher expression levels in the lower-TMB subtype than in the higher-TMB subtype of at least 6 cancer types (Additional file 1: Table S3). The 11 genes included ENG (blood vessels), CD45RO (memory T cells), CD20 (B cells), CD1A (immature Dendritic Cells (iDCs)), TPSB2 (mast cells), PDPN (lymph vessels), CXCR5 (follicular helper T (Tfh cells)), IL3RA (plasmacytoid Dendritic Cells (pDCs)), CSF3 (neutrophils), CD3 (T cells), and CD57 (activated T or NK cells). In contrast, only CD68 (macrophages) had significantly higher expression levels in the higher-TMB subtype than in the lower-TMB subtype of at least 6 cancer types. It suggests that most of these immune cells have stronger infiltration in the lower-TMB subtype than in the higher-TMB subtype of cancers. Typically, 10 of the 16 immune cell subpopulation marker genes were more highly expressed in lower-TMB LIHC, but no one was more highly expressed in higher-TMB LIHC (Fig. 1c), indicating that heavy mutation load may inhibit immune cell infiltration in LIHC. Furthermore, we found 12 cancer types in which the expression levels of the immune cell subpopulation gene-set being significantly higher in the lower-TMB subtype, versus 1 cancer type in which the expression levels of this gene-set being significantly higher in the higher-TMB subtype (Fig. 1d). Again, this suggests that high TMB tends to inhibit immune cell infiltration in cancer.
Association of TMB with tumor-infiltrating lymphocytes (TILs) infiltration in human cancers
We compared expression levels of 120 TILs gene signatures [24] between the lower-TMB subtype and the higher-TMB subtype of cancers. We found that 90 genes had significantly higher expression levels in the lower-TMB subtype of at least 6 cancer types versus 5 having significantly higher expression levels in the higher-TMB subtype of at least 6 cancer types (Fisher’s exact test, P < 2.2*10− 16, OR = 67.11) (Additional file 1: Table S4). Notably, GIMAP6 had significantly higher expression levels in the lower-TMB subtype than in the higher-TMB subtype of 15 cancer types. GIMAP6 encodes the immunity-associated nucleotide 6 protein, a member of the GTPases of immunity-associated proteins family. This gene has been shown to be downregulated in several cancer types such as NSCLC [25] and LIHC [26]. Our results showed that this gene was more lowly expressed in higher-TMB LIHC than in lower-TMB LIHC. The expression levels of the TILs gene-set were significantly higher in the lower-TMB subtype of 9 cancer types (TGCT, KIRC, DLBC, HNSC, STAD, ACC, THCA, LIHC and THYM), while were significantly higher in the higher-TMB subtype of 2 cancer types (CESC and UCEC) (Fig. 2a). These results indicated that although the association between TMB and TILs infiltration was cancer type dependent, high TMB tended to inhibit TILs infiltration in various cancer types. Strikingly, 99 of the 120 TILs genes were more highly expressed in lower-TMB THYM compared to zero showing higher expression levels in higher-TMB THYM (Fisher’s exact test, P < 2.2*10− 16) (Fig. 2b). In addition, 88 TILs genes were more highly expressed in lower-TMB LIHC versus 3 more highly expressed in higher-TMB LIHC (Fisher’s exact test, P < 2.2*10− 16, OR = 104.46) (Fig. 2c).
Association of TMB with cancer-testis antigen genes expression in human cancers
Cancer-testis antigens (CTAs) are a group of immunogenic proteins that are aberrantly activated in a variety of cancer types, and thus are important targets for developing cancer immunotherapy [27]. We compared expression levels of 276 CTA genes [28] between the lower-TMB subtype and the higher-TMB subtype of cancers. We found that 28 CTA genes had significantly higher expression levels in the lower-TMB subtype of at least 6 cancer types, while 63 had significantly higher expression levels in the higher-TMB subtype of at least 6 cancer types (Fisher’s exact test, P = 8.23*10− 5, OR = 0.38) (Additional file 1: Table S5). Of note, 6 CTA genes CEP55, KIF2C, TTK, OIP5, CASC5, and NUF2 had higher expression levels in the higher-TMB subtype of at least 15 cancer types, while had higher expression levels in the lower-TMB subtype of at most 3 cancer types. Interestingly, a number of genes encoding CTAs that are potentially useful for developing cancer vaccines were in the list of 63 CTA genes with higher expression levels in the higher-TMB subtype, such as MAGEA (MAGEA-1, 2, 3, 4, 6, 8, 9B, 10, 11, 12), NY-ESO-1, and PRAME.
The expression levels of the CTA gene-set were significantly higher in the higher-TMB subtype of 13 cancer types, including BRCA, LUAD, LIHC, SKCM, CESC, BLCA, THYM, LAML, LGG, HNSC, LUSC, ACC, and SARC, and were significantly higher in the lower-TMB subtype of 4 cancer types (COAD, UVM, THCA, and UCEC) (Fig. 3a). In many cancer types, the number of CTA genes with higher expression levels in the higher-TMB subtype far exceeded that of CTA genes with higher expression levels in the lower-TMB subtype. For example, 111 CTA genes were more highly expressed in higher-TMB LUAD versus 20 more highly expressed in lower-TMB LUAD (Fisher’s exact test, P < 2.2*10− 16, OR = 8.58); 102 CTA genes were more highly expressed in higher-TMB BRCA versus 29 more highly expressed in lower-TMB BRCA (Fisher’s exact test, P = 1.83*10− 13, OR = 4.98). These results suggest that high TMB is associated with elevated expression of many CTAs in cancer.
Association of TMB with HLA genes expression in human cancers
HLA (human leukocyte antigen) plays an important role in regulating the immune system in humans [29]. We compared expression levels of 24 HLA genes (with expression values available in the TCGA RNA-Seq data) between the lower-TMB subtype and the higher-TMB subtype of cancers. We found that 6 genes (HLA-J, DOA, DOB, DPB1, DQA1, and DQB2) had significantly higher expression levels in the lower-TMB subtype of at least 5 cancer types, while no any HLA gene showed significantly higher expression levels in the higher-TMB subtype of at least 5 cancer types (Additional file 1: Table S6). The expression levels of the HLA gene-set were significantly higher in the lower-TMB subtype of LIHC, HNSC, ACC, PRAD, and LUAD, and were significantly higher in the higher-TMB subtype of CESC, COAD, LGG, and SKCM (Fig. 3b). In LIHC, HNSC, ACC, PRAD, and LUAD, there were much more HLA genes showing higher expression levels in the lower-TMB subtype than those showing higher expression levels in the higher-TMB subtype (10 versus 1, 5 versus 0, 14 versus 0, 10 versus 0, and 16 versus 0 for LIHC, HNSC, ACC, PRAD, and LUAD, respectively) (Fig. 3c). In contrast, in CESC, COAD, and SKCM, there were much more HLA genes with higher expression levels in the higher-TMB subtype than those with higher expression levels in the lower-TMB subtype (12 versus 0, 12 versus 0, and 6 versus 0 for CESC, COAD, and SKCM, respectively) (Fig. 3d). These results suggest that the association between TMB and HLA expression is cancer type dependent.
Association of TMB with cytokine-related genes expression in human cancers
Cytokines are important components of the tumor immune microenvironment (TIM) [30]. Of 261 cytokine and cytokine receptor (CCR) genes [31], 93 showed significantly higher expression levels in the lower-TMB subtype versus 31 showing significantly higher expression levels in the higher-TMB subtype of at least 6 cancer types (Fisher’s exact test, P = 1.77*10− 10, OR = 4.10) (Additional file 1: Table S7). Notably, TNFAIP8L3, CCL14, CX3CR1, CCL21, IL1R1, and IL33 had higher expression levels in the lower-TMB subtype of at least 13 cancer types, while had higher expression levels in the higher-TMB subtype of at most 2 cancer types. In contrast, ILF2 showed higher expression levels in the higher-TMB subtype of 13 cancer types, while showed higher expression levels in the lower-TMB subtype of 1 cancer type.
Interestingly, the expression levels of the CCR gene-set were significantly higher in the lower-TMB subtype of 12 cancer types (LUSC, DLBC, UVM, TGCT, PRAD, LUAD, KIRC, ACC, HNSC, THCA, STAD, and LIHC), while were significantly higher in the higher-TMB subtype of THYM solely (Fig. 4a). It suggests that high TMB may lead to depressed cytokine activity in diverse cancers. Indeed, 98 CCR genes were more highly expressed in lower-TMB LIHC versus 7 more highly expressed in higher-TMB LIHC (Fisher’s exact test, P < 2.2*10− 16, OR = 21.71). In addition, 86 CCR genes were more highly expressed in lower-TMB THCA versus 2 more highly expressed in higher-TMB THCA (Fisher’s exact test, P < 2.2*10− 16, OR = 63.35).
Association of TMB with pro-inflammatory genes expression in human cancers
Inflammatory responses play important roles in regulating the TIM [32]. We compared expression levels of 15 pro-inflammatory genes [33] between the lower-TMB subtype and the higher-TMB subtype of cancers. We found that 10 pro-inflammatory genes were more highly expressed in the lower-TMB subtype versus 4 more highly expressed in the higher-TMB subtype of at least 5 cancer types (Additional file 1: Table S8). Notably, CXCL9, CXCL10, and IFNG had significantly higher expression levels in the higher-TMB subtype of at least 10 cancer types, while had significantly higher expression levels in the lower-TMB subtype of at most 4 cancer types. The expression levels of the pro-inflammatory gene-set were significantly higher in the lower-TMB subtype of 6 cancer types (KIRC, TGCT, ACC, HNSC, KIRP, and THCA), and were significantly higher in the higher-TMB subtype of 10 cancer types (CESC, COAD, UCEC, BLCA, LUAD, BRCA, STAD, LGG, SKCM, and SARC) (Fig. 4b). Interestingly, 12 of the 15 pro-inflammatory genes were more highly expressed in lower-TMB THCA versus zero more highly expressed in higher-TMB THCA (Fig. 4c). In contrast, 11 pro-inflammatory genes were more highly expressed in higher-TMB COAD versus zero more highly expressed in lower-TMB COAD (Fig. 4d). These results indicate that the association between TMB and pro-inflammatory activity is cancer type dependent, with in some cancer types high TMB enhancing pro-inflammatory activity while in some other cancer types high TMB inhibiting pro-inflammatory activity in cancer.
Associations among TMB, immune signatures, and cancer prognosis
To explore the association among TMB, immune signatures, and cancer prognosis, we compared survival prognosis (overall survival (OS) and disease free survival (DFS)) between immune gene-set higher-expression-level and lower-expression-level lower-TMB cancers and higher-TMB cancers, respectively. We found that some cancer types showed a significant correlation between immune gene-set expression and survival prognosis in the lower-TMB subtype but not in the higher-TMB subtype. For example, higher expression levels of the Treg, immune checkpoint, immune cell infiltrate, TILs, and CCR gene-sets were consistently associated with worse DFS in lower-TMB GBM (log-rank test, P < 0.05), but there was no any immune gene-set whose expression was associated with survival prognosis in higher-TMB GBM (Fig. 5a). In contrast, some cancer types showed a significant correlation between immune gene-set expression and survival prognosis in the higher-TMB subtype but not in the lower-TMB subtype. For example, higher expression levels of the immune checkpoint, immune cell infiltrate, TILs, CCR, HLA, and pro-inflammatory gene-sets were consistently associated with better OS and/or DFS in higher-TMB SARC, but there was no any immune gene-set whose expression was associated with survival prognosis in lower-TMB SARC (Fig. 5b). These results indicate that there exist significant associations among TMB, immune signatures, and survival prognosis in some cancer types.