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

PTEN Is Associated With Worse Local Control in Early Stage Supraglottic Laryngeal Cancer Treated With Radiotherapy

Objectives: The aim of this study was to establish the prognostic value of the epidermal growth factor receptor (EGFR) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on local control in patients with early stage supraglottic laryngeal squamous cell carcinoma (LSCC) treated with radiotherapy only. Study design: Retrospective cohort study. Methods: Immunohistochemical staining for EGFR and PTEN was performed on pretreatment biopsies of a selected well-defined homogeneous group of 52 patients with T1-T2 supraglottic LSCC treated with radiotherapy between 1990 and 2008. Kaplan-Meier analysis and univariate and multivariate Cox Regression analyses were performed to correlate clinical data and expression levels of EGFR and PTEN with local control. Results: Kaplan-Meier survival analysis and Cox Regression analysis showed a significant association between PTEN expression and local control (hazard ratio [HR] = 3.26, 95% confidence interval [CI] = 1.14-9.33, P = .027) and between lymph node status and local control (HR = 3.60, 95% CI = 1.26-10.31, P = .017). Both were independent prognostic factors in a multivariate analysis (HR = 3.28, 95% CI = 1.14-9.39, P = .027 and HR = 3.62, 95% CI = 1.26-10.37, P = .017, respectively). There was no significant association between EGFR expression and local control (HR = 1.32, 95% CI = 1.17-10.14, P = .79). Conclusion: This study showed an association between both high PTEN expression and the presence of lymph node metastasis and deteriorated local control in early stage supraglottic LSCC treated with radiotherapy. Level of Evidence: NA

Cortactin expression assessment improves patient selection for a watchful waiting strategy in pT1cN0-staged oral squamous cell carcinomas with a tumor infiltration depth below 4 mm

BACKGROUND: In this feasibility study we aimed to evaluate the value of previously reported molecular tumor biomarkers associated with lymph node metastasis in oral squamous cell carcinoma (OSCC) to optimize neck strategy selection criteria. METHODS: The association between expression of cortactin, cyclin D1, FADD, RAB25, and S100A9 and sentinel lymph node status was evaluated in a series of 87 (cT1‐2N0) patients with OSCC treated with primary resection and SLNB procedure. RESULTS: Tumor infiltration depth and tumor pattern of invasion were independent prognostic markers for SLN status, while none of the tumor makers showed a better prognostic value to replace SLNB as neck staging technique in the total cohort. However, in the subgroup of patients with pT1N0 OSCC, cortactin expression (OR 16.0, 95%CI 2.0–127.9) was associated with SLN classification. CONCLUSIONS: Expression of cortactin is a promising immunohistochemical tumor marker to identify patients at low risk that may not benefit from SLNB or END

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteom

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 similar to 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. British Journal of Cancer (2010) 102, 1778-1785. doi: 10.1038/sj.bjc.6605707 www.bjcancer.com Published online 25 May 2010 (C) 2010 Cancer Research U

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

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

<p>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 (<i>OCLN, CDKN2A, MGMT,</i> <i>MLH1</i> and <i>DAPK1</i>) were frequently differentially methylated in OOSCC. Of these, <i>MGMT</i> methylation was associated with pN0 status (<i>P</i> = 0.02) and with lower immunoexpression (<i>P</i> = 0.02). <i>DAPK1</i> methylation was associated with pN+ status (<i>P</i> = 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, <i>DAPK1</i> and <i>MGMT</i>, 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.</p

FADD Expression as a Prognosticator in Early-Stage Glottic Squamous Cell Carcinoma of the Larynx Treated Primarily With Radiotherapy

PurposeWe recently reported on the identification of the Fas-associated death domain (FADD) as a possible driver of the chromosome 11q13 amplicon and the association between increased FADD expression and disease-specific survival in advanced-stage laryngeal carcinoma. The aim of this study was to examine whether expression of FADD and its Ser194-phosphorylated isoform (pFADD) predicts local control in patients with early-stage glottic carcinoma primarily treated with radiotherapy only.Methods and MaterialsImmunohistochemical staining for FADD and pFADD was performed on pretreatment biopsy specimens of 92 patients with T1–T2 glottic squamous cell carcinoma primarily treated with radiotherapy between 1996 and 2005. Cox regression analysis was used to correlate expression levels with local control.ResultsHigh levels of pFADD were associated with significantly better local control (hazard ratio, 2.40; 95% confidence interval, 1.04–5.55; p = 0.040). FADD overexpression showed a trend toward better local control (hazard ratio, 3.656; 95% confidence interval, 0.853–15.663; p = 0.081). Multivariate Cox regression analysis showed that high pFADD expression was the best predictor of local control after radiotherapy.ConclusionsThis study showed that expression of phosphorylated FADD is a new prognostic biomarker for better local control after radiotherapy in patients with early-stage glottic carcinomas

Prognostic significance of HIF-1a, CA-IX, and OPN in T1-T2 laryngeal carcinoma treated with radiotherapy

<p>Objectives/Hypothesis: To examine the prognostic value of hypoxia inducible factor HIF-1a, CA-IX, and OPN on clinical outcome in patients with T1-T2 supraglottic laryngeal squamous cell carcinoma (LSCC) treated with primarily radiotherapy (RT).</p><p>Study Design: Retrospective cohort study.</p><p>Methods: Tumor tissue sections of 60 patients with T1-T2 supraglottic LSCC treated with primarily radiotherapy were assessed immunohistochemically for expression of HIF-1a, CA-IX, and OPN. The relationship of protein expression and classical clinical parameters with clinical outcome was studied, using Cox regression and Kaplan-Meier survival analyses.</p><p>Results: Neither HIF-1a nor CA-IX was of prognostic significance toward local control or overall survival in T1-T2 supraglottic LSCC. Cox regression survival analysis showed no relation between HIF-1a or CA-IX expression and local control (HR [hazard ratio] 1.07, CI [95% confidence interval] 0.29-3.87; HR 0.34, CI 0.04-2.58). Furthermore, OPN expression was not associated with local control (HR 1.37, CI 0.45-4.17) and overall survival (HR 0.99, CI 0.44-2.21). Our earlier findings in T1-T2 glottic LSCC (Schrijvers et al., 2008) could not be confirmed.</p><p>Conclusion: The absence of prognostic significance for HIF-1a and CA-IX toward local control in supraglottic LSCC, unlike glottic LSCC, suggests that supraglottic LSCC might represent another biological entity.</p>

Comparing the Cervista HPV HR Test and Hybrid Capture 2 Assay in a Dutch Screening Population: Improved Specificity of the Cervista HPV HR Test by Changing the Cut-Off

The diagnostic performance of the widely-used Cervista HPV HR test was compared to the Hybrid Capture 2 (HC2) test in a Dutch population-based cervical cancer screening program. In 900 scrapings of women with normal cytomorphology, specificity was 90% (95%CI: 87.84-91.87) for the Cervista HPV HR test and 96% (95%CI: 94.76-97.37) for the HC2 test with 93% agreement between both tests (κ = 0.5, p<0.001). The sensitivity for CIN2+ using 65 scrapings of women with histological-confirmed CIN2+ was 91% (95%CI: 80.97-96.51) for the Cervista HPV HR test and 92% (95%CI: 82.94-97.43) for the HC2 test with 95% agreement between both tests (κ = 0.7, p<0.001). Fifty-seven of 60 HC2 negative/Cervista positive cases tested HPV-negative with PCR-based HPV assays; of these cases 56% were defined as Cervista triple-positive with FOZ values in all 3 mixes higher than the second cut-off of 1.93 (as set by manufacturer). By setting this cut-off at 5.0, specificity improved significantly without affecting sensitivity. External validation of this new cut-off at 5.0 in triple-positive scrapings of women selected from the SHENCCASTII database revealed that 22/24 histological normal cases now tested HPV-negative in the Cervista HPV HR test, while CIN2+ lesions remained HPV-positive. The intra-laboratory reproducibility of the Cervista HPV HR test (n = 510) showed a concordance of 92% and 93% for cut-off 1.93 and 5.0 (κ = 0.83 and κ = 0.84, p<0.001) and inter-laboratory agreement of the Cervista HPV HR test was 90% and 93% for cut-off 1.93 and 5.0 (κ = 0.80 and κ = 0.85, p<0.001). In conclusion, the specificity of the Cervista HPV HR test could be improved significantly by increasing the second cut-off from 1.93 to 5.0, without affecting the sensitivity of the test in a population-based screening setting