Rabbit Anti-Human RARB (a.a. 155-250) Polyclonal antibody

Objective: To examine epigenetic events of aberrant promoter methylation as diagnostic markers in primary head and neck squamous cell carcinoma using a novel multigene approach. Promoter methylation-mediated silencing is a hallmark of several established tumor suppressor genes. Changes in DNA methylation have been reported to occur early in carcinogenesis and therefore are potentially important early indicators of existing disease. Design: A multicandidate gene probe panel interrogated DNA for aberrant methylation status in 22 cancer genes using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Aberrant promoter hypermethylation was confirmed using methylation-specific polymerase chain reaction after bisulfite treatment. Setting: Primary care medical center. Subjects: We examined fresh-frozen primary head and neck tumor specimens from 28 patients, including 21 late-stage (19 stage IV and 2 stage III) and 7 early-stage (6 stage II and 1 stage I) tumors. Results: Promoter hypermethylation was observed in 14 of the 28 patients (50%). Genes for RARB, APC, and CHFR were most frequently hypermethylated, occurring in 11 (39%) for RARB, 7 (25%) for CHFR, and 6 (21%) for APC. Aberrant methylation of CHFR was solely a stage IV event. Methylation-specific polymerase chain reaction after bisulfite treatment with conventional and real-time polymerase chain reaction confirmed aberrant methylation for RARB and CHFR. Conclusions: Promoter methylation profiling of primary head and neck squamous cell carcinoma using multiple target genes identified RARB, APC, and CHFR as frequent epigenetic events. The clinical implications of these genes as diagnostic and treatment biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing.

Objective: To evaluate promoter methylatioln quantitation using recently described pyrosequencing techniques by correlation with messenger RNA (mRNA) expression. Design: DNA was extracted from tissue samples and was subjected to bisulphite conversion. Quantitative methylation data for multiple CpG sites in each of 9 gene promoters were obtained for. tumors using pyrosequencing. RNA was extracted and converted to complementary DNA, and this formed the template for relative quantitation assays of the expression of each gene by real-time reverse transcription-polymerase chain reaction. Setting: Academic research. Patients: Thirty-seven patients with head and neck squamous cell carcinoma. Main Outcome Measures: The genes studied were PI 6 (OMIM 600160), cyclin A1 (OMIM 604036), RARB (OMIM 180220), E-cadherin (OMIM 192090), MGMT (OMIM 156569), STAT1 (OMIM 600555), ATM (OMIM 607585), hMLH1 (OMIM 120436), and TIMP3 (OMIM 188826). Immunohistochemistry was also performed for p16. Results: STAT1, TIMP3, ATM, and hMLH1 promoters were essentially unmethylated in all cases. The data for cyclin Al (Spearman rank correlation, p=-0.53; P<.001), MGMT (p=-0.53, P <.001), and RARB (p= -0.34, P=.02) showed the expected negative correlation between levels of methylation and mRNA expression. The data relating to E-cadherin were inconclusive. Surprisingly, PI 6 expression was statistically significantly greater in those cases with higher levels of methylation (p= 0.57, P <.001), a finding at odds with assumptions usually made in the literature relating gene promoter methylation to reduced gene expression. The results from p16 immunohistochemistry were in keeping with the mRNA data, but the number of positive staining samples proved too few for statistical analysis. Conclusions: These data present a novel perspective on head and neck cancer epigenetics and reveal new and some unexpected associations and findings. The advantages of pyrosequencing over nonquantitative techniques are discussed in analyses of this nature.