Enhancer of Zeste homologue 2 (EZH2) overexpression is associated with tumor proliferation, metastasis, and poor prognosis. Targeting and inhibition of EZH2 is a potentially effective therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). We analyzed EZH2 mRNA expression in a well-characterized dataset of 230 (110 original and 120 validation cohorts) human head and neck cancer samples. This study aimed to investigate the effects of inhibiting EZH2, either via RNA interference or via pharmacotherapy, on HNSCC growth. EZH2 upregulation was significantly correlated with recurrence (p < 0.001) and the methylation index of tumor suppressor genes (p < 0.05). DNMT3A was significantly upregulated upon EZH2 upregulation (p = 0.043). Univariate analysis revealed that EZH2 upregulation was associated with poor disease-free survival (log-rank test, p < 0.001). In multivariate analysis, EZH2 upregulation was evaluated as a significant independent prognostic factor of disease-free survival (hazard ratio: 2.085, 95% confidence interval: 1.390–3.127; p < 0.001). Cells treated with RNA interference and DZNep, an EZH2 inhibitor, showed the most dramatic changes in expression, accompanied with a reduction in the growth and survival of FaDu cells. These findings suggest that EZH2 upregulation is correlated with tumor aggressiveness and adverse patient outcomes in HNSCC. Evaluation of EZH2 expression might help predict the prognosis of HNSCC patients.

Differences in the biology of human papillomavirus (HPV)-associated oropharyngeal cancers (OPCs) and HPV-negative OPCs may have implications in patient management. Early detection is imperative to reduce HPV-associated OPC mortality. Circulating tumor DNA (ctDNA) can potentially serve as a biomarker for monitoring clinically relevant cancer-related genetic and epigenetic modifications. We analyzed the methylation status of 24 G protein-coupled receptor (GPCR) genes in verification (85 OPC primary samples) and validation (8 OPC ctDNA samples) studies using quantitative methylation-specific polymerase chain reaction (Q-MSP). The Q-MSP-based verification study with 85 OPC primary samples revealed the GPCR genes that were significantly associated with recurrence in high methylation groups (≥14 methylated genes) with OPC and HPV-associated OPC (p < 0.001). In the Kaplan–Meier estimate and multivariate Cox proportional hazard analyses, 13 GPCR genes were significantly related to increased recurrence in the methylation group. Furthermore, the validation study on ctDNA showed that three of these genes (Prostaglandin D2 receptor 1: PTGDR1, Prostaglandin D2 receptor 2: PTGDR2, and Prostaglandin I2 Receptor: PTGIR) had a prediction performance as emerging biomarkers. We characterized the relationship between the methylation status of GPCR genes and outcomes in HPV-associated OPC. Our results highlight the potential utility of ctDNA methylation-based detection for the clinical management of HPV-associated OPC.

Staging and pathological grading systems are convenient but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Identifying biomarkers for HNSCC that will progress and cause death is a critical research area, particularly if the biomarker can be linked to selection of patients. Therefore, to identify potential alternative prognostic markers, we investigated the methylation status of five neuropeptide gene promoters. The promoter methylation status was determined by quantitative methylation-specific PCR in 230 cases of HNSCC; 58 hypopharynx, 45 larynx, 56 oropharynx, and 71 oral cavity tumor samples were studied. The somatostatin (SST), tachykinin precursor 1 (TAC1), hypocretin neuropeptide precursor (HCRT), neuropeptide Y (NPY), and galanin (GAL) promoters were methylated in 84.3, 63.5, 32.6, 28.3, and 20.0%, respectively, of the samples. The mean number of methylated genes per sample was 2.29 (range, 0–5). Disease-free survival was lower in patients with 3–5 methylated genes than in those with 0–2 methylated genes (log-rank test, P = 0.007). In multivariate Cox proportional hazards analysis, TAC1 and GAL promoter methylation independently predicted recurrence (odds ratios 1.620, 95% confidence interval [CI] 1.018–2.578, P = 0.042, and odds ratios 1.692, 95% CI 1.063–2.694, P = 0.027, respectively). In patients with oral cancer, TAC1 methylation showed the best correlation with poor survival (odds ratio 4.427, 95% CI 1.634–12.00, P = 0.003). Similar findings were observed for HCRT and GAL in patients with laryngeal cancer and oropharyngeal cancer, respectively. In this study, we demonstrated the methylation status of the neuropeptide-encoding genes SST, TAC1, HCRT, NPY, and GAL and its relationship with recurrence and survival in HNSCC. These methylation changes may serve as potential molecular markers for defining the risk and prognosis of HNSCC.

Small cell carcinoma/neuroendocrine carcinoma (SCNEC) of the oropharynx is uncommon. Two cases of SCNEC in an 81-year-old woman and in a 54-year-old man are presented here. We have documented two cases of SCNEC arising in the oropharynx with evidence of high-risk human papillomavirus (HPV) infection. Histologically, both cases were classified as poorly differentiated SCNEC with high nuclear-to-cytoplasmic ratios and nuclear molding. Observations using a transmission electron microscope revealed membrane-bound neuroendocrine granules in some tumor cells. Both tumors expressed high levels of p16, a surrogate marker for high-risk HPV infection. HPV infection was confirmed in both cases using HPV polymerase chain reaction analysis; HPV subtype 16 was identified in one case and HPV subtype 18 in the other. SCNEC of the oropharynx is a rare and novel HPV-associated disease with neuroendocrine granules and aggressive clinical behavior. Herein, we present two cases of SCNEC, focusing on its histologic features and treatment modalities. More studies are required to elucidate the pathophysiology of HPV-associated SCNEC in different organ systems.

The aim of this study was to investigate the efficacy of a trunk stabilization exercise warm-up program in reducing the incidence of lower extremity injuries among male junior soccer players. Two junior soccer teams participated in this study. The intervention (INT) team performed three trunk stabilization exercises before practice sessions and games, while a control (CON) team performed their usual warm-up without trunk exercises. Both teams engaged in regular soccer training and games, and were followed for the incidence of injury. As a result, overall injury incidence rates (IRs) were 2.65 injuries/1,000 h and 4.94 injuries/1,000 h in the INT and CON teams, respectively (incidence rate ratio [IRR]=0.54, 95% confidence interval [CI]=0.32-0.89, p=0.013). The IR of acute injuries was significantly lower in the INT team (1.91 injuries/1,000 h) than in the CON team (4.06 injuries/1,000 h) (IRR=0.47, 95%CI=0.26-0.84, p=0.009). Regarding injury sites, the IRs of ankle injuries in the INT team (0.32 injuries/1,000 h) were significantly lower than that in the CON team (2.28 injuries/1,000 h) (IRR=0.14, 95%CI=0.04-0.47, p<0.001). These results suggest that a warm-up program comprising trunk stabilization exercises alone can prevent acute injuries, especially ankle injuries.