Daiki Mochizuki

Tumor suppressor activity and inactivation of galanin receptor type 2 by aberrant promoter methylation in head and neck cancer.

There is accumulating evidence that galanin receptors (GALRs) may be tumor suppressors in head and neck squamous cell carcinoma (HNSCC). Promoter methylation status and gene expression were assessed in a large panel of head and neck primary tumors, based on the hypothesis that cytosine‐guanine dinucleotide (CpG) hypermethylation might silence the galanin receptor 2 (GALR2) gene.

Aberrant Methylation Inactivates Somatostatin and Somatostatin Receptor Type 1 in Head and Neck Squamous Cell Carcinoma

Purpose The aim of this study was to define somatostatin (SST) and somatostatin receptor type 1 (SSTR1) methylation profiles for head and neck squamous cell carcinoma (HNSCC) tumors at diagnosis and follow up and to evaluate their prognostic significance and value as a biomarker. Methods Gene expression was measured by quantitative RT-PCR. Promoter methylation status was determined by quantitative methylation-specific PCR (Q-MSP) in HNSCC. Results Methylation was associated with transcription inhibition. SST methylation in 81% of HNSCC tumor specimens significantly correlated with tumor size (P = 0.043), stage (P = 0.008), galanin receptor type 2 (GALR2) methylation (P = 0.041), and tachykinin-1 (TAC1) (P = 0.040). SSTR1 hypermethylation in 64% of cases was correlated with tumor size (P = 0.037), stage (P = 0.037), SST methylation (P < 0.001), and expression of galanin (P = 0.03), GALR2 (P = 0.014), TAC1 (P = 0.023), and tachykinin receptor type 1 (TACR1) (P = 0.003). SST and SSTR1 promoter hypermethylation showed highly discriminating receiver operator characteristic curve profiles, which clearly distinguished HNSCC from adjacent normal mucosal tissues. Concurrent hypermethylation of galanin and SSTR1 promoters correlated with reduced disease-free survival (log-rank test, P = 0.0001). Among patients with oral cavity and oropharynx cancer, methylation of both SST and SSTR1 promoters correlated with reduced disease-free survival (log-rank test, P = 0.028). In multivariate logistic-regression analysis, concomitant methylation of galanin and SSTR1 was associated with an odds ratio for recurrence of 12.53 (95% CI, 2.62 to 59.8; P = 0.002). Conclusions CpG hypermethylation is a likely mechanism of SST and SSTR1 gene inactivation, supporting the hypothesis that SST and SSTR1 play a role in the tumorigenesis of HNSCC and that this hypermethylation may serve as an important biomarker.

ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

Prognostic value of aberrant promoter hypermethylation of tumor-related genes in early-stage head and neck cancer.

Staging and pathological grading are useful, but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Accordingly, molecular biomarkers that predict the risk of recurrence are necessary to improve clinical outcomes. The methylation statuses of the promoters of 11 tumor-related genes (p16, RASSF1A, E-cadherin, H-cadherin, MGMT, DAPK, DCC, COL1A2, TAC1, SST, and GALR1) were analyzed in 133 HNSCC cases using quantitative methylation-specific PCR. We detected frequent methylation of p16 (44%), RASSF1A (18%), E-cadherin (53%), H-cadherin (35%), MGMT (35%), DAPK (53%), DCC (42%), COL1A2 (44%), TAC1 (61%), SST (64%), and GALR1 (44%) in HNSCC. Disease-free survival was lower in patients with 6–11 methylated genes than in those with 0–5 methylated genes (log-rank test, P = 0.001). In a multivariate Cox proportional hazards analysis, the methylation of E-cadherin, COL1A2, TAC1, and GALR1 was associated with poor survival, with hazard ratios of 4.474 (95% CI, 1.241–16.124). In a joint analysis of these four genes, patients with 2–4 methylated genes had a significantly lower survival rate than those with 0–1 methylated genes in early-stage HNSCC. Importantly, the methylation of some genes was closely related to poor prognosis in early-stage HNSCC, providing strong evidence that these hypermethylated genes are valuable biomarkers for prognostic evaluation.

Site‐specific methylation patterns of the GAL and GALR1/2 genes in head and neck cancer: Potential utility as biomarkers for prognosis

The aim of this study was to evaluate the prognostic value of the promoter methylation status of galanin (GAL) and galanin receptor 1/2 (GALR1/2) by assessing their association with disease‐free survival and known prognostic factors in head and neck cancer. We generated methylation profiles of GAL and GALR1/2 in tumor samples obtained from 202 patients with head and neck squamous cell carcinoma (HNSCC); these included 43 hypopharynx, 42 larynx, 59 oral cavity, and 58 oropharynx tumor samples. CpG island hypermethylation status of the three genes was analyzed using quantitative methylation‐specific PCR (Q‐MSP). In order to determine the prognostic value of the methylation status of these genes, the associations between methylation index and various clinical characteristics, especially tumor site, were assessed for tumors from patients with HNSCC. The methylation index was positively correlated with female gender (P = 0.008) and disease recurrence (P = 0.01) in oral cancer and human papillomavirus (HPV)‐positive (P = 0.004) status and disease recurrence (P = 0.005) in oropharyngeal cancer. Among patients with oral and oropharyngeal cancer, promoter hypermethylation of GAL, GALR1, or GALR2 was statistically correlated with a decrease in disease‐free survival (log‐rank test, P = 0.036 and P = 0.042, respectively). Furthermore, methylation of GAL, GALR1, or GALR2 exhibited the highest association with poor survival (log‐rank test, P = 0.018) in patients with HPV‐negative oropharyngeal cancers. As such, GAL and GALR1/2 methylation status may serve as an important site‐specific biomarker for prediction of clinical outcome in patients with HNSCC.

Epigenetic silencing of SALL3 is an independent predictor of poor survival in head and neck cancer

BackgroundThis study examined Sal-like protein (SALL)3 methylation profiles of head and neck cancer (HNSCC) patients at diagnosis and follow-up and evaluated their prognostic significance and value as a biomarker. SALL3 expression was examined in a panel of cell lines by quantitative reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the SALL3 promoter was examined by quantitative methylation-specific PCR.ResultsSALL3 promoter methylation was associated with transcriptional inhibition and was correlated with disease recurrence in 64.8% of cases, with an odds ratio of 1.914 (95% confidence interval: 1.157–3.164; P = 0.011) by multivariate Cox proportional hazard regression analysis. SALL3 promoter hypermethylation showed highly discriminatory receiver operator characteristic curve profiles that clearly distinguished HNSCC from adjacent normal mucosal tissue, and was correlated with reduced disease-free survival (DFS) (log-rank test, P = 0.01). Hypermethylation of tumor-related genes was higher among patients with SALL3 methylation than among those without methylation (P < 0.001). Furthermore, SALL3 hypermethylation was associated with expression of TET1, TET2, and DNMT3A genes.ConclusionsThis study suggests that CpG hypermethylation is a likely mechanism of SALL3 gene inactivation, supporting the hypothesis that the SALL3 gene may play a role in the tumorigenesis of HNSCC and may serve as an important biomarker.

Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma

The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51–6.75 (p = 0.002); 2.24, 1.06–4.76 (p = 0.035); and 2.56, 1.09–6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

Genes encoding neuropeptide receptors are epigenetic markers in patients with head and neck cancer: a site-specific analysis

Staging and pathological grading systems are useful but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). To identify potential prognostic markers, we examined the methylation status of eight neuropeptide receptor gene promoters in 231 head and neck squamous cell carcinomas. The NPFFR1, NPFFR2, HCRTR1, HCRTR2, NPY1R, NPY2R, NPY4R, and NPY5R promoters were methylated in 80.5%, 79.2%, 67.1%, 73.2%, 35.1%, 36.4%, 38.5%, and 35.9% of the samples, respectively. In a multivariate Cox proportional hazards analysis, the odds ratio for recurrence was 2.044 (95% confidence interval [CI], 1.323-3.156; P = 0.001) when the NPY2R promoter was methylated. In patients without lymph node metastasis (n = 100), methylation of NPY2R (compared with methylation of the other seven genes) best correlated with poor disease-free survival (DFS) (odds ratio, 2.492; 95% CI, 1.190-5.215; P = 0.015). In patients with oral cancer (n = 69), methylated NPY1R and NPY2R were independent prognostic factors for poor DFS, both individually and, even more so, in combination (odds ratio, 3.90; 95% CI, 1.523-9.991; P = 0.005). Similar findings were observed for NPY2R and NPY4R in patients with oropharyngeal cancer (n = 162) (odds ratio, 5.663; 95% CI, 1.507-21.28; P = 0.010).Staging and pathological grading systems are useful but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). To identify potential prognostic markers, we examined the methylation status of eight neuropeptide receptor gene promoters in 231 head and neck squamous cell carcinomas. The NPFFR1, NPFFR2, HCRTR1, HCRTR2, NPY1R, NPY2R, NPY4R, and NPY5R promoters were methylated in 80.5%, 79.2%, 67.1%, 73.2%, 35.1%, 36.4%, 38.5%, and 35.9% of the samples, respectively. In a multivariate Cox proportional hazards analysis, the odds ratio for recurrence was 2.044 (95% confidence interval [CI], 1.323–3.156; P = 0.001) when the NPY2R promoter was methylated. In patients without lymph node metastasis (n = 100), methylation of NPY2R (compared with methylation of the other seven genes) best correlated with poor disease-free survival (DFS) (odds ratio, 2.492; 95% CI, 1.190–5.215; P = 0.015). In patients with oral cancer (n = 69), methylated NPY1R and NPY2R were independent prognostic factors for poor DFS, both individually and, even more so, in combination (odds ratio, 3.90; 95% CI, 1.523–9.991; P = 0.005). Similar findings were observed for NPY2R and NPY4R in patients with oropharyngeal cancer (n = 162) (odds ratio, 5.663; 95% CI, 1.507–21.28; P = 0.010).

Human papillomavirus-associated small cell carcinoma/neuroendocrine carcinoma of the oropharynx: a report of two cases

IntroductionSmall 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.Case descriptionWe 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.Discussion and EvaluationSCNEC of the oropharynx is a rare and novel HPV-associated disease with neuroendocrine granules and aggressive clinical behavior.ConclusionsHerein, 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.

Primary combined small and squamous cell carcinoma of the hypopharynx: A case report.

We herein report a very rare case of combined small-cell carcinoma (SmCC) of the hypopharynx, with a squamous cell carcinoma (SqCC) element. A 74-year-old man presented with a 3-month history of throat pain and hoarseness. On hypopharyngoscopy, a tumor was identified in the right anterior wall of the piriform sinus and, following examination of a biopsy sample, the lesion was diagnosed as SqCC. Total laryngectomy with bilateral neck dissection was performed and the malignancy was diagnosed as combined SmCC. One month after surgery, concomitant chemoradiotherapy with cisplatin and etoposide was administered. Immunohistochemically, the SmCC element was positive for CD56 and Ki-67 (50.2%), whereas the SqCC element was positive for cytokeratin 34βE12 and Ki-67 (47.5%). Furthermore, the SmCC element was positive for KIT and platelet-derived growth factor α (PDGFRα), while the SqCC element was positive for epidermal growth factor receptor (EGFR) and PDGFRα. By genetic analysis, a silent mutation in the PDGFRα gene was recognized. The expression of KIT, PDGFRα and EGFR in this case provided evidence that combined SmCC may be a candidate for molecular-targeted therapy, although further investigations are required.