Prognostic value of immune scores in the microenvironment of colorectal cancer
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- Published online on: September 18, 2020 https://doi.org/10.3892/ol.2020.12119
- Article Number: 256
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Copyright: © Yuan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Colorectal cancer (CRC) has the second-highest mortality rate among all cancers, and its associated mortality ranks fourth (9.0%) and third (9.5%) among all cancers in male and female patients with cancer, respectively (1). In clinical practice, surgical resection is a common treatment for CRC (2). However, approximately 50–60% of patients diagnosed with CRC have metastasis and 80–90% of them are unresectable liver metastases (3,4). In recent years, cancer immunotherapy has become an important means of cancer treatment (5), and it is one of the hotspots in the field of cancer research. However, the complex components and heterogeneity of the tumor microenvironment pose a huge challenge to cancer immunotherapy.
The tumor microenvironment, which is composed of molecules such as immune cells and mesenchymal cells, is the cell environment in which the tumor is located (6). A pan-immune immunogenomic analysis revealed that numerous tumor-infiltrating lymphocytes associated with adaptive immunity are associated with a good prognosis, including activated CD8+ T cells, resting memory CD4+ T cells and effector memory CD4+ T cells (7). To achieve precise immunotherapy, studies have defined the tumor mutation burden (TMB) by the number of mutations per megabase of DNA, as a predictive biomarker for evaluation (8). The higher the TMB is associated with the likelihood of the tumor being sensitive to immunotherapies (9). Immune cells are the major non-tumor component of a tumor microenvironment and have been indicated to be valuable for the diagnosis and prognostic assessment of patients with CRC (10). Recently, an algorithm called ESTIMATE, which calculates an immune score based on specific gene expression characteristics of immune cells, was used to predict the infiltration of non-tumor cells (11). Subsequent studies have demonstrated the effectiveness of this big data-based algorithm (12–14).
Using the CRC cohort data from The Cancer Genome Atlas (TCGA) database and immune scores derived from the ESTIMATE algorithm, the present study investigated whether tumors with a higher mutation burden, neoantigen burden and a greater infiltration degree of 22 immune cell subsets are associated with higher immune scores.
Materials and methods
Data sources and preprocessing
All genomic, clinical and mutation annotation format (MAF) data were obtained from TCGA CRC cohorts in February 2019 according to the following specific parameters: The major site was the colon or rectum and the experimental strategy was RNA sequencing. First, the samples with survival times of <30 days were discarded and the samples without complete clinical information or MAF data were removed. Finally, the data of 432 tumor samples with 18 matched normal samples were retained.
The gene expression data in fragments per kilobase of transcript per million mapped reads format were also retrieved from TCGA and converted to transcripts per million (TPM). The immune scores and stromal scores were calculated by using the ESTIMATE algorithm based on the expression values in TPM (11). By comparing the differences in overall survival (OS), the optimal threshold for immune score grouping was determined. When the patient's immune score was above this threshold, the sample was assigned to the high immune score (HIM) group and otherwise to the low immune score (LIM) group.
The data used for verification were downloaded from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and relevant datasets were identified using the following key words: (‘colorectal cancer’ OR ‘colon cancer’) AND (‘prognosis’ OR ‘prognostic’ OR ‘survival’) AND (‘Homo sapiens’). Datasets obtained by sequencing using the GPL570 platform were selected and certain datasets were excluded. Duplicates and datasets with small sample sizes (n<50) were also excluded. The datasets GSE17536 and GSE21510 associated with CRC were finally selected and merged into a combined dataset. Gene probe names were transformed into gene names based on platform annotation files. Subsequently, the gene expression data for each sample and the corresponding clinical information were organized for further analysis.
Functional analysis in silico
The immune cytolytic activity scores were obtained by the geometric mean of the Granzyme A (GZMA) and Perforin 1 (PRF1) expression values in TPM (15). The data regarding microsatellite stable (MSS) or microsatellite instability-high (MSIH) status for 291 TCGA CRC samples were also obtained (16). The consensus molecular subtypes (CMSs) classification classifies CRC into four molecular subtypes (CMS1, CMS2, CMS3 and CMS4) with distinct biological characteristics. The samples in the HIM and LIM groups were classified according to the CMS system using the R package CMScaller (17).
The immune-associated genes were downloaded from the immunology database and analysis portal (ImmPort; http://immport.niaid.nih.gov) (18). These immune-associated genes have a variety of roles in immune pathways. After filtering out the synonymous variants and variants in intergenic or noncoding regions, the maftools package (19) was used for mutation burden analyses and mutational spectral visualization. The neoantigens for each sample and neoantigen origin protein information were downloaded from The Cancer Immune Atlas (TCIA; http://tcia.at/home).
To evaluate tumor-infiltrating immune cell (TIIC) composition in CRC, the CIBERSORT deconvolution algorithm (20) was used to estimate the proportion of 22 immune cell types in HIM and LIM.
Differentially expressed mRNAs between samples from the HIM and LIM groups were screened using edgeR (21) with the criteria of |log2fold change| >1.5 and a false discovery rate (FDR) <0.01. The TIMER online database (22) was used to analyze and visualize the abundance of TIICs according to differentially expressed genes. Cytoscape software (23) (two plugins: ClueGO and CluePedia) was used for the Kyoto Encyclopedia of Genes and Genomes analyses and only pathways with P<0.05 were considered. The GeneMANIA (24) plugin was also employed to investigate the functional association of the differentially expressed mRNAs between samples from the HIM and LIM groups.
Statistical analysis
All statistical analyses were performed using R software (version 3.5.0) and Bioconductor (https://www.bioconductor.org/). An unpaired t-test was used to compare differences in various parameters (including stromal scores, cytolytic activity scores, and MSS vs. MSIH status) between samples from the HIM and LIM groups, and to compare the non-synonymous mutation burden in the HIM. A Wilcoxon rank sum test was used to examine differences in medians. Kaplan-Meier curve analyses using the survival package version 3.2 (25) were performed to analyze the association between the mRNA expression profiles and OS. P<0.05 was considered to indicate statistical significance.
Results
Immune scores, sample grouping and demographic statistics
The gene expression profiles and clinical information of all 432 patients with CRC from the TCGA database were retrieved. Based on the ESTIMATE algorithm, the immune scores were distributed between −899.56 and 2,999.28 (Fig. 1A). The optimal threshold for dividing samples into the HIM and LIM groups was determined using the function surv_cutpoint of the survival package in R (cutoff=3.58). The detailed clinical and pathological characteristics of the study population in HIM and LIM, including age, sex, ethnicity, pathological stage, tumor (T) stage, nodal (N) stage and metastasis (M) stage, are summarized in Table I. The median age for all patients was 60 years (interquartile range, 31–90 years). Of the total patients, the HIM group contained 171 (52.6%) male and 154 (47.4%) female and the LIM group contained 24 (22.4%) male and 83 (77.6%) female.
Comparison of characteristics between the HIM and LIM groups
Immune scores represent the infiltration of immune cells in tumor tissues. It was indicated that MSIH tumors were associated with higher immune scores compared with MSS tumors (P<0.001; Fig. 1B). Furthermore, samples in the HIM group had higher stromal scores than those in the LIM group (P<0.001; Fig. 1C), indicating a positive association between immune and stromal scores. In addition, the association of the immune score and cytolytic activity score in CRC was examined. Of note, the distribution of cytolytic activity scores in the HIM and LIM groups was similar to that of the stromal scores, with a significantly higher score in the HIM group (P<0.001; Fig. 1D).
A Kaplan-Meier survival curve analysis was also performed, indicating that OS in the HIM group was longer than that in the LIM group (P=0.0313 according to the log-rank test; Fig. 1E). CMSs based on gene expression profiles provide a biological stratification framework with great potential for biomarker development. The distribution of CMS subtypes in the HIM and LIM groups is presented in Fig. 1F. In the LIM group, CMS2 accounted for almost half of the cases (48.6%), while in the HIM group, the other CMSs were comparatively more prevalent, particularly CMS4 (Fig. 1F).
Landscape of immune infiltration in HIM CRC
To further analyze the differences in immune cell invasion between the HIM and LIM groups, the CIBERSORT algorithm was used to investigate the differences in the proportion of 22 immune cell subsets between samples from the two groups. The differences in the proportion of 22 immune cell types detected in all LIM samples were not significant (P>0.05). Fig. 2A presents the immune cell distribution in the HIM samples. Of note, the analysis indicated that gamma delta T cells were not present in any of the samples.
Differences in the proportion of TIICs between the normal tissue samples and HIM samples were then analyzed. Compared with the normal tissues, there were significant changes in the proportion of 15 TIICs in the HIM samples (Fig. 2B). In the HIM samples, naïve B cells, memory B cells, plasma cells, activated natural killer (NK) cells, monocytes, M2 macrophages, resting mast cells and eosinophils decreased significantly, while activated memory CD4+, follicular helper T cells, regulatory T cells, resting NK cells, M0 macrophages, M1 macrophages and activated mast cells were significantly increased.
Prognostic value of immune infiltration in HIM
To determine the prognostic capacity of TIICs in CRC, a survival analysis was performed based on the proportion of immune cells in HIM samples. It was indicated that resting memory CD4+ T cells and regulatory T cell levels were associated with survival in patients with CRC (Fig. 2C). Of note, patients with CRC and low proportions of resting memory CD4+ T cells had significantly longer survival times than the patients with high proportions.
To further investigate whether TIICs are involved in the development and progression of CRC, the HIM samples were divided into several subgroups based on the pathological TNM stage (T3+T4 vs. T1+T2, N2+N3 vs. N0+N1, M1 vs. M0) and pathological stage (I–II vs. III–IV). Comparative analyses of the TIIC proportions indicated that plasma cells demonstrated a statistically significant association with the pathological T stage (P=0.037). Furthermore, M1 macrophages were significantly associated with multiple types of clinical stage (Fig. 2D).
Mutation burden in relation to immune scores
A high mutation burden is one of the characteristics of malignant tumors. To obtain the burden of non-synonymous mutations in HIM and LIM samples, the somatic mutations detected by the mutect2 software in the two groups were analyzed. The median non-synonymous mutation burden was 111.5 in HIM samples and 96 in LIM samples (Fig. S1). The mutational patterns of the highly mutated genes were distinct between HIM and LIM. As presented in Fig. 3A and B, APC regulator of WNT signaling pathway (APC), tumor protein p53 (TP53), titin (TTN), KRAS proto-oncogene (KRAS) and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) were among the top 10 mutated genes in HIM and LIM samples. It was further identified that most of the mutations were single base substitutions and the substitution of C->T was the most common type in all samples (Fig. 3C and D).
Neoantigen burden in association with immune scores
It was also investigated whether the mutational/neoantigen patterns were associated with the immune scores. The neoantigen counts and neoantigen origin protein counts for each CRC sample were obtained from the TCIA database. The LIM samples had a significantly lower neoantigen burden (Wilcoxon rank-sum test P=0.0068; Fig. 3E) and neoantigen origin protein burden (P=0.0051; Fig. 3F) than the samples from the HIM group, which suggested that the HIM samples had a higher number of mutations accumulated in the tumor cell genome than the LIM samples, resulting in a corresponding increase in neoantigen burden and thereby activating more T cells and producing a stronger immune response.
Comparison of gene expression profiles with immune scores
To reveal the correlation between gene expression profiles and immune scores, a differential analysis of the count data of the genes in the samples from the HIM and LIM groups was performed. As indicated in the volcano plots in Fig. 4A, 80 genes were upregulated and 1,630 genes were downregulated in the high score group compared with the low score group (|log2fold change|>1.5 and FDR<0.05). A total of 283 differentially expressed genes were identified from 1,378 immune-associated genes. To outline the potential function of the differentially expressed genes, functional enrichment (GO and KEGG) analysis of the 283 genes was performed (Figs. 4B and S2). The aforementioned genes were enriched in some immune-related pathways, such as B cell receptor signaling pathway, Natural killer cell mediated cytotoxicity and Fc gamma R-mediated phagocytosis. Besides, they were also enriched in disease-related pathways, such as transcriptional misregulation in cancer and inflammatory bowel disease (Figs. 4B and S2).
Potential function of differentially expressed genes
Kaplan-Meier curve analysis with the log-rank test for each of the 283 genes provided 13 genes whose expression was significantly correlated with the survival of the patients with CRC. To investigate the functional association of the 13 genes, the genes were analyzed using the GeneMANIA plugin of Cytoscape to generate an interaction network (Fig. 4C). Most of the network interactions were coexpression. The Kaplan-Meier survival curves that were significantly different for the 13 genes (high vs. low expression) are presented in Figs. 4D and S3. Further clinical analysis of these genes demonstrated that the SERPINE1 and UCHL1 were significantly associated with multiple types of clinical stage (Fig. 4E). The association between the expression profiles of these two genes and TIICs was also analyzed and it was revealed that their copy number variation was significantly associated with changes in the proportion of multiple types of TIIC, such as B cells, CD8+ T cells, neutrophils and dendritic cells (Fig. S4).
Comprehensive validation in GEO datasets
To examine the universality of the results from the TCGA cohort, the GSE17536 and GSE21510 datasets were analyzed for validation. After batch correction (Fig. S5A), the same cutoff as that for the TCGA sample analysis was used to stratify all GEO samples into HIM and LIM groups. Due to the particularity of HIM samples, the CIBERSORT algorithm was used to assess the difference in the proportion of 22 immune cell subsets in these samples (Fig. S5B). It was revealed that the proportions of the immune cell subsets in the TCGA and GEO datasets were similar. In addition, differential analysis of gene count data in samples with HIM and LIM revealed that SERPINE1 was still significantly differentially expressed (|log2fold change|>1.5, P=0.00142). However, the difference in UCHL1 expression was not significant.
Discussion
The present study sought to identify tumor microenvironment-associated factors that contribute to the OS of patients with CRC with HIM or LIM. The results indicated that MSIH tumors rather than MSS tumors were significantly associated with high immune scores. A previous study indicated that in CRC, MSIH tumors have 10s of times more somatic mutations than MSS tumors (26). Under these conditions, lymphocyte infiltration is prominent due to increased neoantigen burdens and more stroma cells are present in tumor tissues (27). In addition, the cytolytic activity of tumor cells is positively correlated with the neoantigen burden (15). In line with this, the present results suggested that the HIM samples were characterized by higher cytolytic activity and stromal scores than LIM samples. This means that the samples in the HIM group had a good cytolytic immune response and relatively abundant stromal cells due to the higher levels of TIICs. In addition, a previous study indicated that cytolytic activity was associated with immune responses and improved prognoses (28). The present study also indicated significantly improved OS in patients with HIM vs. LIM.
Using CIBERSORT, the proportional changes of the 22 immune cell subsets in the HIM and LIM samples were analyzed. The TIIC proportions of the samples in LIM did not meet the criterion of P<0.05, which was due to their comparatively lower immune score. It is worth noting that the P-value obtained with CIBERSORT only reflects one part of a sample that contains immune cells and non-immune cells. Therefore, P>0.05 does not mean that TIICs do not exist in LIM samples. However, the present results indicated that the proportion of TIICs was not significant in samples with lower immune scores. Tumor-associated microenvironments, which include immune cells, are able to inhibit malignant cells. Numerous studies have indicated that the degree of infiltration of immune cells, tissue localization and cell type are significantly associated with CRC progression and survival. For instance, the 5-year OS values for stage III CRC patients with lower levels of TIICs were determined to be significantly lower than those for stage III CRC patients with high levels of TIICs (29). The present study also indicated that HIM samples have significant changes in the proportion of TIIC and the survival time of patients with CRC with high immune scores was significantly improved. In addition, the present results provided details on the infiltration of the 22 TIIC subsets in CRC. The proportions of resting memory CD4+ T cells and macrophages were the highest, while gamma delta T cells were not present in any of the samples. The present study also indicated that resting CD4+ memory T cells were significantly associated with the survival of patients with CRC. Resting CD4+ memory T cells may help CD8+ T cells inhibit tumors and block CD8+ T-cell activation and NK cell activity (30). The present study also confirmed that they have a key role in the development of CRC.
The neoantigen burden is an effective biomarker in cancer immunotherapy and neoantigens may be the focus of the development of novel therapeutic approaches to modify T-cell reactivity against this class of antigens (31). The probability of the presence of CD8+ T cells in cancer lesions is higher in tumors with high mutation burdens than in those with low mutation burdens (32). T-cell reactivity against neoantigens is common in melanoma (33). In CRC, the present study indicated that the neoantigen peptide burden of HIM samples was significantly higher than that of LIM samples. More than one neoantigen peptide may be derived from a protein. In the HIM sample, the proportions of infiltration of activated memory CD4+ T cells were significantly increased. One reason is that the HIM samples have a high neoantigen burden and studies have also indicated that most of the new antigen-specific T-cell responses in melanoma are against neoantigens (34).
The present study indicated that certain genes were highly mutated in both HIM and LIM samples. The tumor suppressor gene TP53 exhibited a discontinuous mutation distribution in the two groups. TP53 has a regulatory role in cell proliferation and apoptosis (35). The loss of TP53 may lead to CRC development and progression through a multistep process (36). An association between TP53 mutations and worse outcome in stage III CRC has been reported (37). The present study indicated that 68 (64%) LIM samples had TP53 gene mutations. The number of correspondingly mutated HIM samples was 169 (52%). Of note, the APC gene had the highest mutation frequency in HIM and LIM samples, at 84 and 70%, respectively. In addition, the CRC proto-oncogene KRAS was mutated in 39 and 53% of HIM and LIM samples, respectively, and its mutations cause the RAS/RAF/MAPK pathways to remain active with loss of normal regulation of cell growth (38).
By comparing the gene expression profiles in HIM and LIM samples and screening immune-associated genes, 283 tumor microenvironment-related genes were identified in the present study. Functional enrichment analyses indicated that these genes mainly participated in the immune response and cell adhesion. The study further focused on SERPINE1 and UCHL1, which are immune-associated genes that were significantly differentially expressed between HIM and LIM samples, and their expression was significantly associated with OS and multiple types of clinical stage in patients with CRC. In addition, the present analysis revealed that their copy number variation led to significant changes in infiltration of the multiple immune cell subsets. Previous studies have indicated that SERPINE1 overexpression occurs in primary tumors caused by KRAS mutations in CRC (39) and UCHL1 is associated with lymph node metastasis in CRC (40). Therefore, it was concluded that SERPINE1 and UCHL1 may be additional biomarkers for CRC. The difference in expression of SERPINE1 was also verified in the GEO dataset, however, the difference in UCHL1 expression was not significant. How they affect tumor progression in tumor microenvironments requires further investigation. However, the present study had certain limitations, as all of the results were obtained in silico. Further in vivo or clinical studies will contribute to the further elucidation of the relationship between immune scores and microenvironmental changes in CRC.
In conclusion, in CRC samples that had high immune scores, a good cytolytic immune response and relatively abundant stromal cells, significant infiltration of 22 immune cell subsets and a high non-synonymous mutation burden and neoantigen burden was identified, and a set of genes associated with the tumor microenvironment was extracted. The present study revealed that a tumor microenvironment of CRC with a high immune score is associated with favorable survival.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This work was partly supported by the Shanghai Natural Science Foundation (grant no. 15ZR1420800 to SP) and the Quality Engineering Project of Colleges and Universities in Anhui Province (grant nos. 2015zytz070 and 2019rcsfjd088 to XH).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
WY and SP conceived the study, collected the data and performed the bioinformatics analyses. WC and XH performed quality control of the raw data and participated in part of the data analyses. WY and SP wrote the manuscript and supervised the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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