DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM)

Abstract

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples.

Resistance to cis- and carboplatin initiated by epigenetic changes in ovarian cancer patients

“…Once the selected material has been removed from the sections, a relevant method for analysis of methylation levels needs to be selected. Some methods utilized for the detection of DNA methylation levels [42][43][44][45] : Digestion-based assay (PCR, qPCR, RT-PCR, cold PCR); High resolution melting luminometric methylation assay; HPLC-UV; Mass spectrometry; ELISA-based methods; Amplified fragment length polymorphism; Restriction fragment length polymorphism; Luminometric methylation assay; Pyrosequencing; Bisulfite sequencing; ELISA; Methylation ligation-dependent macro-array; High-throughput measurement technologies.…”

A DNA Methylation-based Test for Esophageal Cancer Detection

“…COL14A1 aberrant methylation has been reported in ESCC (16), as well as in renal cell carcinoma, sarcomas, and endometrial carcinoma (28,31,32), whereas hypomethylation has been shown in coronary artery disease (33). GPX3 promoter methylation has been shown in ESCC and esophageal glandular lesions, including Barrett's esophagus and EA (30,34,35). In the same vein, ZNF569 promoter hypermethylation has been associated with glandular lesions, like Barrett's esophagus (17) in comparison with the normal esophagus.…”

A DNA Methylation-based Test for Esophageal Cancer Detection

“…COL14A1 aberrant hypermethylation has been reported in ESCC (16), as well as renal cell carcinoma, sarcomas and endometrial carcinoma (35)(36)(37), whereas hypomethylation has been shown in coronary artery disease (38). GPX3 promoter hypermethylation has been reported in ESCC and esophageal glandular lesions, including Barrett's esophagus and EA (17,18,39). In the same vein, ZNF569 promoter hypermethylation has been associated with glandular lesions, like Barrett's esophagus (19) in comparison with normal esophagus.…”

“…Conversely, GPX3 promoter methylation levels did not differ between EA from normal tissues, although a few cases disclosed higher methylation levels (data not shown). Notwithstanding, different sample processing (fresh frozen tissues vs. FFPE) (17,39), different methodologies to assess GPX3 methylation levels among studies (qMSP vs. MSP vs. methylation ligation-dependent macroarray vs. pyrosequencing) and the smaller size of some cohorts (17,18) may explain some disparate results.…”

“…Based on the literature evidence, we aimed to identify and validate methylome alterations that might constitute biomarkers for early detection, as well as identi cation of minimal residual disease after neoadjuvant treatment, eventually precluding the need for esophagectomy. Hence, promoter methylation levels of three genes selected after extensive literature review (16)(17)(18)(19)) -GPX3 (glutathione peroxidase 3), COL14A1 (collagen type XIV alpha 1 chain) and ZNF569 (zinc nger protein 569) -were tested in a series of ECa and normal esophageal tissues. Table 1).…”