In this study, we used CIBERSORT to assess TME, TMB and characterized 22 subsets of TIICs based on GSE6631 and HNSCC-TCGA database in individuals with HNSCC, to explore molecular subpopulation, survival, function and expression difference of these cells in tumor conditions. We found that the composition of TIIC subtypes differs substantially in different HNSCC. Tumor mutations generates novel epitopes. Understanding the immunogenicity of these epitopes may deepen our knowledge on the clinical immunology and prognosis of HNSCC tumors. Based on TCGA data TME was associated with TMB, supporting the hypothesis that TMB is a strong proxy for TME. This is an interesting revelation, particularly in the face of immunomodulatory therapies, and may uncover potential biomarkers for immunotherapy or prognosis of HNSCC tumors.
Infiltration of immune cells has been postulated to be a significant factor influencing the prognosis of tumors. In this study, we first explored the distribution of TIICs in HNSCC patients based on GSE6631 and HNSCC-TCGA datasets. Macrophages which mediate phagocytosis and inflammation aid in controlling tumor progression, by facilitating invasion of tumor tissues and angiogenesis25-27. Macrophages are mainly classified into M0/M1/M2 Under specific conditions, M0 macrophages can be polarized to M1/M2 types, Liu et al28 found that RhoA pathway could obstruct elongation (hummingbird phenotype) of M0 macrophages, suggesting that M0 may be important in bone-marrow-derived macrophages. As subpopulations of T cells, resting memory CD4+ T cells can differentiate to perform other functions (separate to those mediated by memory CD8 + T cells, but aiding in suppressing growth of tumors)29-30. Resting memory CD4 + cells must be activated by IL-7 and IL-15, yet not MHC class II, for their survival and intermittent homeostatic proliferation31. A recent study suggested that the homologous interaction between tumor antigen-specific CD4 + Th1 cells and tumor-associated macrophages may shift the M1/M2 ratio within the tumor to M132. So, infiltration of immune cells plays a critical role in HNSCC and therefore more research was needed. Meanwhile, we also analyzed the interaction between the 22 different TIICs and elucidated on co-expression of immune cells in HNSCC, to open up future research on the same. This analysis though, may be subject to biasness, because the interactions may be influence by the other factors in the TME.
The role of varying TMB in HNSCC-TCGA was also investigated. Missense mutation we found to be the most common somatic alterations, while SNP was the most common DNA sequence polymorphism with C>T single nucleotide substitution being the most frequent. Changes that modify amino acid sequences are known as Missense mutations. These changes may influence the structure and stability of proteins, which may interfere their interactions with other biomolecules, translation of functional proteins and enhance progression of tumours33. For example, BRAF mutation in melanoma and KRAS G12D mutations in colorectal cancer34-35. Establishing impacts of cancer missense mutations may help in identifying drivers of mutations as well as illustrating molecular mechanisms for developing HNSCC. SNP have been identified in nearly all types of cancers, such as colorectal36, breast37, prostate38, and HNSCC39. SNP is a third-generation genetic marker for detecting numerous phenotypic differences in human, and susceptibility to diseases and drugs may be associated with SNP40. Top 10 mutated genes that related to TMB were TP53, TTN, FAT1, MUC16, CDKN2A, CSMD3, SYNE1, LRP1B, NOTCH1 and PIK3CA. Kubesova et al41 reported that TP53 mutations with low variant allele frequency irrespective of disease subtype, driver gene status and cytoreduction during myeloproliferative neoplasm. Mukhopadhyay et al42 suggested that ESR2-mutant TP53 combination prognosticates survival in triple negative breast cancer. But in this study, there was no statistical difference between TMB and clinical survival time which may have been influenced by too small samples size in HNSCC-TCGA database. We finally divided TMB into high and low groups to analyze the functions of DEGs. Analysis of biological processes performed by DEGs suggested that these genes may play a significant role in tissue development and response to acidic chemicals. Molecular function was enhanced in structural constituents in the extracellular matrix, and affected cellular structure as well. KEGG analysis revealed that for most part, metabolism of glutathione is enhanced in DEGs. Glutamine is a significant metabolite utilized in the development of malignant cells. Reducing the level of GLN through chemotherapy and radiotherapy has been found to restores diminished glutathione, enhancing the recovery of intestine epithelium and immunological system43. Our results also suggested that NK cells resting and Eosinophils was differently expressed between high and low TMB group indicating these genes are essential in tumor immune response.
Meanwhile, our study had several limitations. First, our results were not validated in clinical settings and may not provide precise data. We also analyzed samples from relatively few patients. Second, due to multiple types of histology and anatomical sites for HNSCC, tumor-infiltrating immune cells may vary widely. Finally, the interacting of different immune cells is affected by environmental conditions, thus ours corHeatmap analysis may be subject to bias. Meanwhile, these particular tumors may lack substantial tumor infiltrating immune cells (immune deserts) and needs more related studies conduct. We will do follow-up studies to develop new, accurate interventions for cancer medicine. Here, we described TME in relation to immune system, revealing variable infiltration of various HNSCC subtypes. The intricate connection between TIIC, TMB and genomic alterations was also elucidated the mechanism of immune response revealed in this study provides a strong foundation for future research that can enhance tumor immunotherapy.