RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis.

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma.

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.

Lack of claudin-7 is a strong predictor of regional recurrence in oral and oropharyngeal squamous cell carcinoma.

OBJECTIVES: Adequate treatment of oral and oropharyngeal squamous cell carcinoma (OSCC) is dependent on correctly predicting the presence of lymph node metastases. Current methods to diagnose nodal metastases partly result in overtreatment with associated morbidity and undertreatment with decreased disease-free survival. E-cadherin has been studied extensively as potential marker for lymph node metastases. EpCAM and claudin-7 have a functional relationship with E-cadherin, forming a complex that promotes tumourigenicity in vitro. We hypothesize that the co-expression patterns of these related molecules is a better prognostic marker for nodal status and regional recurrences. MATERIALS AND METHODS: We constructed separate tissue microarrays of tumour centre and tumour invasive front of 227 OSCC with complete clinicopathological and follow-up data, including HPV status, and performed immunohistochemistry for these molecules. RESULTS: Lack of E-cadherin and presence of cytoplasmic EpCAM expression in the tumour front were predictive for nodal metastasis, but no co-expression pattern was found clinically relevant. Lack of claudin-7 in the tumour centre was highly and independently predictive for shorter regional disease-free survival (HR=0.19; 95%CI: 0.06-0.62) and disease-specific survival (HR=0.43; 95%CI: 0.21-0.87). High-risk HPV was not associated with any marker. CONCLUSIONS: The expression of E-cadherin and EpCAM, depending on the specific tumour sublocalization, is predictive for nodal status. However, co-expression did not improve the prediction of nodal status, indicating that the proposed in vitro complex is not functional in clinical samples. Additionally, lack of claudin-7 expression in the tumour centre may be used to identify patients with increased risk for regional recurrence.

FADD expression is associated with regional and distant metastasis in squamous cell carcinoma of the head and neck.

AIMS: The Fas-associated death domain gene (FADD) is often overexpressed in squamous cell carcinoma of the head and neck (HNSCC), and is considered to be a driver gene in amplification of the chromosomal 11q13.3 region. Amplification of 11q13.3 is associated with increased metastasis in HNSCC and breast cancer. The aim of this study was to investigate the association between FADD protein expression in advanced-stage HNSCC and clinicopathological features and outcome. METHODS AND RESULTS: Tumour tissues of 177 HNSCC patients uniformly treated with primary surgery and postoperative radiotherapy were collected. FADD expression was assessed on pretreatment tumour biopsies using immunohistochemistry. High FADD expression was detected in 44% of the HNSCC patients. High expression was associated with an increased rate of lymph node metastasis (P = 0.001) and with a shorter distant metastasis-free interval (DMFI) (HR 2.6, 95% CI 1.0-6.7, P = 0.046) when lymph node metastases were present. CONCLUSIONS: Our data show that an increase in FADD expression is associated with a higher incidence of lymph node metastasis at presentation, and with shorter DMFI when lymph node metastases are present. High FADD expression in the primary tumour could be a useful marker to select patients for systemic treatment strategies that reduce the risk of distant metastases.

The phosphatase and tensin homologue deleted on chromosome 10 mediates radiosensitivity in head and neck cancer.

BACKGROUND: For locally advanced squamous cell carcinoma of the head and neck (HNSCC), the recurrence rate after surgery and postoperative radiotherapy is between 20 and 40%, and the 5-year overall survival rate is approximately 50%. Presently, no markers exist to accurately predict treatment outcome. Expression of proteins in the human epidermal growth factor receptor (EGFR) pathway has been reported as a prognostic marker in several types of cancer. METHODS: The aim of this study was to investigate the prognostic value of proteins in the EGFR pathway in HNSCC. For this purpose, we collected surgically resected tissue of 140 locally advanced head and neck cancer patients, all treated with surgery and postoperative radiotherapy. RESULTS: In a multivariate analysis, expression of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was significantly related to worse locoregional control (LRC; HR: 2.2, 95% CI: 1.1-4.6; P=0.03), independent of lymph node metastases (HR: 5.6, 95% CI: 1.2-27.4; P=0.03) and extranodal spread (HR: 2.7; 95% CI: 1.2-6.5; P=0.02). In vitro clonogenic radiosensitivity assays confirmed that overexpression of PTEN resulted in increased radioresistance. CONCLUSION: Our study is the first report showing that expression of PTEN mediates radiosensitivity in vitro and that increased expression in advanced HNSCC predicts worse LRC.

Overexpression of intrinsic hypoxia markers HIF1alpha and CA-IX predict for local recurrence in stage T1-T2 glottic laryngeal carcinoma treated with radiotherapy.

PURPOSE: To examine the prognostic value of three endogenous hypoxia markers (hypoxia inducible factor 1 alpha subunit [HIF1 alpha], carbonic anhydrase IX [CA-IX], and glucose transporter type 1 [GLUT-1]) on the clinical outcome in patients with early-stage glottic carcinoma primarily treated with radiotherapy (RT) and to determine the predictive hypoxic profile to choose the optimal treatment of early-stage laryngeal carcinoma. METHODS AND MATERIALS: Immunohistochemistry for HIF1 alpha, CA-IX, and GLUT-1 was performed on formalin-fixed, paraffin-embedded, pretreatment tissue samples of 91 glottic squamous cell carcinoma specimens. The patient group consisted only of those with early-stage (T1-T2) glottic carcinoma, and all patients were treated with RT only. Relative tumor staining was scored on the tissue samples. Receiver operating curve analysis was performed to determine the optimal cutoff value for each tumor marker. Cox regression analyses for the variables HIF1 alpha, CA-IX, GLUT-1, gender, age, hemoglobin level, T category, N category, tobacco use, and alcohol use were performed with local control and overall survival as endpoints. RESULTS: HIF1 alpha overexpression in early-stage glottic carcinoma correlated significantly with worse local control (hazard ratio [HR], 3.05; p = 0.021) and overall survival (HR, 2.92; p = 0.016). CA-IX overexpression correlated significantly with worse local control (HR, 2.93; p = 0.020). GLUT-1 overexpression did not show any correlation with the clinical outcome parameters. Tumors with a nonhypoxic profile (defined as low HIF1 alpha and low CA-IX expression) had significantly better local control (HR, 6.32; p = 0.013). CONCLUSION: The results of our study have shown that early-stage glottic laryngeal carcinomas with low HIF1 alpha and CA-IX expression are highly curable with RT. For this group, RT is a good treatment option. For tumors with HIF1 alpha or CA-IX overexpression, hypoxic modification before RT or primary surgical treatment should be considered.

Cortactin expression predicts poor survival in laryngeal carcinoma.

Amplification of the 11q13 region is one of the most frequent aberrations in squamous cell carcinomas of the head and neck region (HNSCC). Amplification of 11q13 has been shown to correlate with the presence of lymph node metastases and decreased survival. The 11q13.3 amplicon carries numerous genes including cyclin D1 and cortactin. Recently, we reported that FADD becomes overexpressed upon amplification and that FADD protein expression predicts for lymph node positivity and disease-specific mortality. However, the gene within the 11q13.3 amplicon responsible for this correlation is yet to be identified. In this paper, we compared, using immunohistochemical analysis for cyclin D1, FADD and cortactin in a series of 106 laryngeal carcinomas which gene correlates best with lymph node metastases and increased disease-specific mortality. Univariate Cox regression analysis revealed that high expression of cyclin D1 (P=0.016), FADD (P=0.003) and cortactin (P=0.0006) predict for increased risk to disease-specific mortality. Multivariate Cox analysis revealed that only high cortactin expression correlates with disease-specific mortality independent of cyclin D1 and/or FADD. Of genes located in the 11q13 amplicon, cortactin expression is the best predictor for shorter disease-specific survival in late stage laryngeal carcinomas.