Identifying CD1c as a potential biomarker by the comprehensive exploration of tumor mutational burden and immune infiltration in diffuse large B cell lymphoma
Authors: Xiang Xiaoyu , Gao Li-Min , Zhang Yuehua , Zhu Qiqi , Zhao Sha , Liu Weiping , Ye Yunxia , Tang Yuan , Zhang Wenyan 
Published: December 2023

Background
Tumor mutational burden (TMB) is a valuable prognostic biomarker. This study explored the predictive value of TMB and the potential association between TMB and immune infiltration in diffuse large B-cell lymphoma (DLBCL).

Methods
We downloaded the gene expression profile, somatic mutation, and clinical data of DLBCL patients from The Cancer Genome Atlas (TCGA) database. We classified the samples into high-and low-TMB groups to identify differentially expressed genes (DEGs). Functional enrichment analyses were performed to determine the biological functions of the DEGs. We utilized the cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm to estimate the abundance of 22 immune cells, and the significant difference was determined by the Wilcoxon rank-sum test between the high- and low-TMB group. Hub gene had been screened as the prognostic TMB-related immune biomarker by the combination of the Immunology Database and Analysis Portal (ImmPort) database and the univariate Cox analysis from the Gene Expression Omnibus (GEO) database including six DLBCL datasets. Various database applications such as Tumor Immune Estimation Resource (TIMER), CellMiner, konckTF, and Genotype-Tissue Expression (GTEx) verified the functions of the target gene. Wet assay confirmed the target gene expression at RNA and protein levels in DLBCL tissue and cell samples.

Results
Single nucleotide polymorphism (SNP) occurred more frequently than insertion and deletion, and C > T was the most common single nucleotide variant (SNV) in DLBCL. Survival analysis showed that the high-TMB group conferred poor survival outcomes. A total of 62 DEGs were obtained, and 13 TMB-related immune genes were identified. Univariate Cox analysis results illustrated that CD1c mutation was associated with lower TMB and manifested a satisfactory clinical prognosis by analysis of large samples from the GEO database. In addition, infiltration levels of immune cells in the high-TMB group were lower. Using the TIMER database, we systematically analyzed that the expression of CD1c was positively correlated with B cells, neutrophils, and dendritic cells and negatively correlated with CD8+ T cells, CD4+ T cells, and macrophages. Drug sensitivity showed a significant positive correlation between CD1c expression level and clinical drug sensitivity from the CellMiner database. CREB1, AHR, and TOX were used to comprehensively explore the regulation of CD1c-related transcription factors and signaling pathways by the KnockTF database. We searched the GETx database to compare the mRNA expression levels of CD1c between DLBCL and normal tissues, and the results suggested a significant difference between them. Moreover, wet experiments were conducted to verify the high expression of CD1c in DLBCL at the RNA and protein levels.

Conclusions
Higher TMB correlated with poor survival outcomes and inhibited the immune infiltrates in DLBCL. Our results suggest that CD1c is a TMB-related prognostic biomarker.