p16, MGMT, RARβ2, CLDN3, CRBP and MT1G gene methylation in esophageal squamous cell carcinoma and its precursor lesions

Abstract

Abstract. Esophageal squamous cell carcinoma (ESCC) is a common cancer with a very poor prognosis. New methods are needed to screen high-risk populations and identify curable tumors and precursor lesions early. Molecular markers may be useful in such screening efforts. This study was designed to determine the prevalence of p16, MGMT, RARß2, CLDN3, CRBP and MT1G gene methylation in patients with ESCC to evaluate the variation of gene methylation across a spectrum of preneoplastic lesions, and assess the feasibility of using gene methylation in a primary screening test utilizing frozen esophageal cells collected by balloon cytology samplers. Samples were obtained from high-risk subjects from north central China. These samples included 11 foci of histologically normal mucosa, 8 foci of low-grade squamous dysplasia, 7 foci of high-grade squamous dysplasia, and 13 foci of ESCC from 6 fully embedded resection specimens; endoscopic biopsies from 6 individuals with no histological evidence of disease; and frozen esophageal balloon samples from 12 asymptomatic subjects. Promoter CpG site-specific hypermethylation status was determined for each gene using realtime methylation-specific PCR (qMS-PCR) based on Taqman chemistry. Of the 6 ESCC patients, 5 showed methylation of at least one gene. For most genes, methylation occurred with increasing frequency during neoplastic progression, with the largest increase found between low-and high-grade dysplasia. There was considerable variation in methylation patterns among different foci of the same histological grade, even within individual patients, but 16/20 (80%) of high-grade dysplastic and cancer foci had ≥ 2 methylated genes, while 17/19 (89%) of normal and low-grade dysplastic foci had <2 methylated genes. These genes were rarely methylated in histologically normal mucosa from patients with or without ESCC. Gene methylation was common and easily detectable in the frozen esophageal cells collected by balloon cytology samplers. Our data suggest that methylation of p16, MGMT, RARß2, CLDN3, CRBP, and MT1G is common in the esophageal mucosa of patients with ESCC in this high-risk population, and tends to increase in prevalence in foci with increasing histological severity of disease. Methylation data from panels of genes may be able to identify patients with high-grade lesions. Balloon cytology may be able to screen the length of the esophagus effectively for a subset of cells with abnormal methylation, and may be useful in a primary screening test for ESCC and its precursor lesions.

Methylation in oral cancer and pre-cancerous lesions (Review)

“…Interestingly, one study detected 72.73% methylation (21) in 22 samples of healthy peritumoural tissue, suggesting that p16 methylation may be an early event in carcinogenesis and might therefore serve as a prognostic and diagnostic marker. In studies on pre-cancerous lesions, it was reported that p16 methylation was not related to the malignant transformation of lichen planus (23) but was significantly associated with the malignization of leukoplakia, especially in relation to tobacco (but not alcohol) use (24). A correlation was also found between p16 methylation and higher-grade dysplasia (18,23,25,26).…”

“…In studies on pre-cancerous lesions, it was reported that p16 methylation was not related to the malignant transformation of lichen planus (23) but was significantly associated with the malignization of leukoplakia, especially in relation to tobacco (but not alcohol) use (24). A correlation was also found between p16 methylation and higher-grade dysplasia (18,23,25,26). However, another study of patients in whom p16 methylation was detected reported that the time interval between methylation and oscc onset varied widely (0-70 months, mean of 18 months), suggesting that p16 methylation may not be correlated with the pathological status of the lesion (27).…”

“…the data for salivary gland cancer are less clear: in the tumour, MgMt methylation of <20% was observed in 5 cell lines and 69 human salivary gland carcinoma samples, while methylation of up to 15% was found in healthy mucosa (29). However, reports of MgMt methylation of 68-73% in healthy perilesional tissue (21,28) and of 56 and 80% in leukoplakia samples (23,25,28) suggest that, as with p16, MgMt methylation may be related to an early stage of the lesion and might be useful as a prognostic or diagnostic factor. In fact, it was observed in lesions with a low-grade dysplasia and even in clinically normal oesophageal mucosa with no detectable histological lesion (23).…”

“…However, reports of MgMt methylation of 68-73% in healthy perilesional tissue (21,28) and of 56 and 80% in leukoplakia samples (23,25,28) suggest that, as with p16, MgMt methylation may be related to an early stage of the lesion and might be useful as a prognostic or diagnostic factor. In fact, it was observed in lesions with a low-grade dysplasia and even in clinically normal oesophageal mucosa with no detectable histological lesion (23). studies of saliva samples (16,18,25,19) obtained methylation percentages of 48-62%, indicating that saliva analysis is an effective method to detect methylation of this gene.…”

“…As in the case of p16, MgMt has been widely studied in different types of sample from oscc and from head and neck cancer in general. studies have detected MgMt methylation percentages of 23 to 56.4% in tumour tissue (16,18,19,21,23,28) versus the 9% found in oral mucosa of healthy individuals (23). the data for salivary gland cancer are less clear: in the tumour, MgMt methylation of <20% was observed in 5 cell lines and 69 human salivary gland carcinoma samples, while methylation of up to 15% was found in healthy mucosa (29).…”