Transcriptomic analysis of peripheral blood mononuclear cells in head and neck squamous cell carcinoma patients.

OBJECTIVES: To investigate the gene expression profile of peripheral blood mononuclear cells (PBMCs) from head and neck squamous cell carcinoma (HNSCC), including oral cancer (OC) and oropharyngeal cancer (OPC) patients, and compare them with healthy controls (HC). MATERIALS AND METHODS: Transcriptomic analysis of PBMCs was performed by RNA-sequencing. The upregulated candidate genes were selected for validation by quantitative real-time polymerase chain reaction (qPCR). In addition, related plasma protein levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Three significantly upregulated genes, including high mobility group nucleosomal binding domain 2 (HMGN2), folate receptor gamma (FOLR3), and amphiregulin (AREG), were selected. In the first cohort, the results showed that only HMGN2 expression was significantly increased in OC patients. In the larger sample size, the overall results demonstrated that HMGN2 expression had a tendency to increase in both OC and OPC patients compared with HC. Interestingly, the plasma HMGN2 (HMG-17) protein level exhibited the same trend as that observed at the transcriptional level. CONCLUSION: HMGN2 expression and plasma HMG-17 (HMGN2 protein) were increased in both cancer patients compared with HC. This gene may be important for further functional studies in the PBMCs of HNSCC patients.

High 4-1BB Expression in PBMCs and Tumor Infiltrating Lymphocytes (TILs) in Patients with Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: 4-1BB is a costimulatory immune-activating molecule. Increased amounts of this protein have previously been found in the plasma of patients with oropharyngeal and oral cancer. Here, we focused on this molecule that functions as part of the immune system. We investigated 4-1BB in the peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) of patients with head and neck squamous cell cancer (HNSCC). MATERIALS AND METHODS: The expression level of 4-1BB in the PBMCs was determined using real-time polymerase chain reaction (PCR). The TIMER (Tumor Immune Estimation Resource) web server was utilized to approximate the 4-1BB level in HNSCC TILs. Moreover, 4-1BB immunohistochemistry (IHC) was used to validate TILs in four organs of HNSCC, including oral cancer (OC), oropharyngeal cancer (OPC), sinonasal cancer (SNC), and laryngeal cancer (LC), in both the tumor area and adjacent normal epithelium. The difference in 4-1BB expression levels in various groups was assessed using a Kruskal-Wallis test and an independent sample t-test. RESULTS: The level of 4-1BB expression in PBMCs was highest in OPC, followed by OC and healthy controls (HC). Significant differences were discovered between HC and OPC and between OC and OPC. Bioinformatics revealed a substantial correlation between 4-1BB expression level and lymphocyte infiltration in HNSCC, including B cells, CD8+ T cells, and CD4+ T cells. IHC validation in HNSCC tissue revealed that the average number of 4-1BB positive TILs in all four HNSCC subtypes was considerably greater than the number of lymphocytes seen in adjacent normal tissue. Interestingly, the number of lymphocytes that were 4-1BB positive increased in relation to the TIL level. CONCLUSION: A higher number of 4-1BB expression levels were found in the PBMCs and TILs of HNSCC patients, implying that 4-1BB may be a promising approach for HNSCC patients to improve their immune function. It is important to study and create a treatment that uses 4-1BB medicine as well as existing drugs.

Immune-associated plasma proteins in oral and oropharyngeal cancer patients.

Background: Plasma protein patterns differ between cancer patients and healthy donors. This study aimed to examine the plasma levels of several cytokines and immunological checkpoint proteins between patients with oral and oropharyngeal cancer and healthy donors. Materials and methods: Plasma samples from healthy donors, oral cancer patients, and oropharyngeal cancer patients were analyzed using the Human Th Cytokine Panel 13-plex (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, and TNF-α) and Human Immune Checkpoint Panel1 12-plex [sCD25 (IL-2Ra), 4-1BB, sCD27, B7.2 (CD86), Free Active TGF-ß1, CTLA-4, PD-L1, PD-L2, PD-1, Tim-3, LAG-3, and Galectin-9]. The plasma 4-1BB levels were verified by Western blot method. In addition, the study of the receive operating curve (ROC) yielded the calculation of a number of diagnostically significant indicators. Results: Significantly increased levels of IL-6, 4-1BB, PDL-1, PD-1, and CTLA-4 and decreased levels of IL-13 and sCD27 were observed in cancer patients compared with healthy donors. These levels were highly significant, particularly for cancer patients in stage IV. Validation by Western blot revealed that cancer patients had higher plasma levels of 4-1BB than healthy donors (p < 0.05), and ROC curve analysis revealed that plasma 4-1BB had the highest cancer detection capability. Intriguingly, plasma levels of 4-1BB were significantly positively correlated with PDL-1 and PD-1 levels (p < 0.0001). Conclusion: This data provided descriptive knowledge of oral and oropharyngeal cancer immunity at a fundamental level. Additional research should concentrate on the significantly different factors, especially 4-1BB, PDL-1, and PD-1, which may contribute to the development of novel alternative diagnostic tools or therapies for patients with oral and oropharyngeal cancer.

Evaluation of lymphocyte apoptosis in patients with oral cancer.

OBJECTIVES: To evaluate apoptotic levels of peripheral blood mononuclear cells (PBMCs) and apoptotic regulatory proteins (Bax and Bcl-2) in lymphocyte subsets of oral cancer (OC) patients and healthy controls (HC). METHODOLOGY: The percentage of apoptotic cells and lymphocyte counts were measured in the first cohort using PBMCs obtained from 23 OC patients and 6 HC. In the second cohort, (OC, 33; HC, 13), the mean fluorescence intensity (MFI) of Bax and Bcl-2 in CD19+ B, CD4+ T, CD8+ T, and CD16+56+ natural killer (NK) cells was determined via flow cytometry. RESULTS: The percentage of apoptotic cells was higher in the PBMCs of OC patients than in HC patients, particularly in patients with stage IV cancer (p<0.05). However, lymphocyte counts were significantly lower in stage IV patients (p<0.05). NK CD19+ B and CD16+56+ cell counts were significantly lower in OC patients compared with HC patients (p<0.001 and p<0.01, respectively), but CD4+ T cells were interestingly significantly higher in OC patients (p<0.001). While Bax MFI was slightly higher, Bcl-2 MFI was significantly lower for all four lymphocyte subsets in OC samples, particularly in stage IV patients, when compared with HC. Consequently, Bax/Bcl-2 ratios showed an upward trend from HC to OC patients, particularly those in stage IV. We found similar trends in Bax and Bcl-2 MFI for tumor stage, tumor size, and lymph node involvement. CONCLUSIONS: The increased lymphocyte apoptosis in stage IV OC patients may be related to higher Bax levels and lower Bcl-2 levels. The Bax/Bcl-2 ratio in lymphocytes may be useful to determine the prognosis of OC patients, and could be considered a mean for supportive treatment in the future.

Induction of ZCCHC6 expression in peripheral blood mononuclear cells by HNSCC secretions.

Cancer secretion can change the properties of adjacent cells, including peripheral blood mononuclear cells (PBMCs). We investigated whether such secretion influences messenger RNA expression in PBMCs of patients with head and neck squamous cell carcinoma (HNSCC). In the present study, co-culture model of normal PBMCs and HNSCC cell lines were established. The PBMCs were subsequently subjected to RNA sequencing for transcriptome analysis. Furthermore, expression data from the Gene Expression Omnibus repository, platform GPL4133, series GSE39400, were gathered to analyze, afterward identify zinc finger CysCysHisCys (CCHC)-type domain-containing protein 6 (ZCCHC6) as the main gene involved in HNSCC. This gene was then validated by a quantitative real-time polymerase chain reaction. The results showed that ZCCHC6 was expressed at significantly higher levels in the patients with HNSCC than in the healthy controls, and the sensitivity and specificity of these findings for diagnostic purposes were 100.00% and 70.83%, respectively. In summary, our findings demonstrated that the secretion of HNSCC cells could cause the alterations in messenger RNA expression by PBMCs. The ZCCHC6 expression level may apply in HNSCC screening.

Evaluation of lymphocyte apoptosis in patients with oral cancer

Abstract Objectives To evaluate apoptotic levels of peripheral blood mononuclear cells (PBMCs) and apoptotic regulatory proteins (Bax and Bcl-2) in lymphocyte subsets of oral cancer (OC) patients and healthy controls (HC). Methodology The percentage of apoptotic cells and lymphocyte counts were measured in the first cohort using PBMCs obtained from 23 OC patients and 6 HC. In the second cohort, (OC, 33; HC, 13), the mean fluorescence intensity (MFI) of Bax and Bcl-2 in CD19+ B, CD4+ T, CD8+ T, and CD16+56+ natural killer (NK) cells was determined via flow cytometry. Results The percentage of apoptotic cells was higher in the PBMCs of OC patients than in HC patients, particularly in patients with stage IV cancer (p<0.05). However, lymphocyte counts were significantly lower in stage IV patients (p<0.05). NK CD19+ B and CD16+56+ cell counts were significantly lower in OC patients compared with HC patients (p<0.001 and p<0.01, respectively), but CD4+ T cells were interestingly significantly higher in OC patients (p<0.001). While Bax MFI was slightly higher, Bcl-2 MFI was significantly lower for all four lymphocyte subsets in OC samples, particularly in stage IV patients, when compared with HC. Consequently, Bax/Bcl-2 ratios showed an upward trend from HC to OC patients, particularly those in stage IV. We found similar trends in Bax and Bcl-2 MFI for tumor stage, tumor size, and lymph node involvement. Conclusions The increased lymphocyte apoptosis in stage IV OC patients may be related to higher Bax levels and lower Bcl-2 levels. The Bax/Bcl-2 ratio in lymphocytes may be useful to determine the prognosis of OC patients, and could be considered a mean for supportive treatment in the future.