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.

Evaluation of NID2 promoter methylation for screening of Oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is an aggressive human malignancy. Because of late diagnosis and recurrence of OSCC, the treatment of patients with OSCC is often ineffective. Thus, finding novel biomarkers of OSCC are essential. Here we derived a methylation marker by utilizing methylation microarray data and testing its capacity in cross-sectional study designed for OSCC detection and screening. METHODS: According to bioinformatics analysis of total of 27,578 cg sites, cg22881914 of Nidogen 2 (NID2) methylation was selected for evaluation. Next, we confirmed the methylation status by bisulfite sequencing from the microdissected OSCC cells in comparison with the microdissected oral epithelia. Subsequently, we developed a simple technique using real-time PCR with the specific probe to examine the ability for the detection of OSCC in the oral epithelial samples, which included 103 oral rinse and 82 oral swab samples. RESULTS: Based on the comparison of microdissected tissue, cg22881914 of NID2 was proved to be methylated in most OSCC cells but unmethylated in the normal oral epithelia. Furthermore, the methylated NID2-relied quantitative PCR approach has demonstrated that this marker assists in distinguishing among patients with OSCC from normal oral epithelia, smokers, and patients with oral lichen planus using the non-invasive oral rinse and swab samples. CONCLUSIONS: Specific methylation at cg22881914 of NID2 of OSCC could be used as an important potential marker for detecting OSCC. Thus, to certify the utility of this marker, further studies with a larger sample size are needed.

Head and neck squamous cell carcinoma drives long interspersed element-1 hypomethylation in the peripheral blood mononuclear cells.

OBJECTIVE: Alteration of long interspersed element-1 (LINE-1) methylation in peripheral blood mononuclear cells (PBMCs) has been simultaneously activated to breast carcinogenesis due to its secretion. To evaluate the effect in head and neck squamous cell carcinoma (HNSCC), LINE-1 methylation levels and patterns have been measured both in vitro and in vivo. METHODS: Analysis of LINE-1 methylation in cocultured models between HNSCC cell lines and normal PBMCs was performed. The observation of PBMCs of HNSCC patients compared to PBMCs of normal controls was performed using the semiquantitative combined bisulfite restriction analysis technique. RESULTS: Downregulation of LINE-1 methylation was significantly found in the PBMCs cocultured with all HNSCC cell lines compared to normal PBMCs. Likewise, a reduction in LINE-1 methylation levels was observed in PBMCs of HNSCC compared to normal PBMCs (p < 0.0001). Receiver operating characteristic analysis demonstrated the potential of the unmethylated alleles (u Cu C) of LINE-1 for distinguishing the PBMCs of HNSCC patients from normal controls with 100% sensitivity and specificity. CONCLUSION: Our data supported that the alteration of LINE-1 methylation levels in PBMCs was influenced by HNSCC secretions. Moreover, the unmethylated LINE-1 allele of PBMCs was proved to be an effective tumor marker and possesses a potential as HNSCC diagnostic tool.

Concordance between whole exome sequencing of circulating tumor DNA and tumor tissue.

Next generation sequencing of circulating tumor DNA (ctDNA) has been used as a noninvasive alternative for cancer diagnosis and characterization of tumor mutational landscape. However, low ctDNA fraction and other factors can limit the ability of ctDNA analysis to capture tumor-specific and actionable variants. In this study, whole-exome sequencings (WES) were performed on paired ctDNA and tumor biopsy in 15 cancer patients to assess the extent of concordance between mutational profiles derived from the two source materials. We found that up to 16.4% ctDNA fraction can still be insufficient for detecting tumor-specific variants and that good concordance with tumor biopsy is consistently achieved at higher ctDNA fractions. Most importantly, ctDNA analysis can consistently capture tumor heterogeneity and detect key cancer-related genes even in a patient with both primary and metastatic tumors.

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.