title: Diagnostic utility of MS-MLPA in DNA methylation profiling of adenocarcinomas and neuroendocrine carcinomas of the colon-rectum.
abstract: Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) is a fast, new, inexpensive method that has rarely been exploited in DNA methylation profiling of colorectal cancers (CRCs). The aim of this study was to test the diagnostic utility of MS-MLPA to evaluate the methylation status of 34 genes in normal colonic mucosa samples and in a well-characterized series of 83 adenocarcinomas and 21 neuroendocrine carcinomas of colon-rectum. Two commercial MS-MLPA kits (SALSA MS-MLPA ME001-C1 Tumor suppressor-1 Kit and SALSA MS-MLPA ME002-B1 Tumor suppressor-2 Kit) were used to perform promoter methylation analysis on formalin-fixed and paraffin-embedded tissues. MS-MLPA analysis was validated by bisulfite pyrosequencing, bisulfite cycle sequencing, and methylation-specific PCR. MS-MLPA analysis identified a subset of 27 CRCs (26 % of cases) showing high levels of gene methylation involving a mean percentage of 34 % of the promoters examined. These tumors exhibited all the main clinicopathological and genetic features described for CRCs with CpG island Methylator Phenotype-High. High levels of methylation were observed with similar frequency in adenocarcinomas and in neuroendocrine carcinomas (25 % versus 29 %, respectively), but different methylation profiles were observed in the two tumor types. In both groups, tumors with microsatellite instability and widespread methylation represented a homogeneous clinicopathological entity. MS-MLPA assay is an easy and reliable system for epigenetic characterization of tumor tissues and leads to a rapid identification of CRCs with the highest levels of gene methylation. Aberrant gene methylation is a common abnormality in CRC initiation and may be observed in tumors with very different genetic and clinicopathological profiles.
authors.affiliation: Department of Surgical and Morphological Sciences, Anatomic Pathology Unit, University of Insubria and Ospedale di Circolo, Via O. Rossi, 9, 21100, Varese, Italy. daniela.furlan@uninsubria.it
authors.forename: Daniela
authors.initials: D
authors.lastname: Furlan
authors.forename: Nora
authors.initials: N
authors.lastname: Sahnane
authors.forename: Mara
authors.initials: M
authors.lastname: Mazzoni
authors.forename: Roberta
authors.initials: R
authors.lastname: Pastorino
authors.forename: Ileana
authors.initials: I
authors.lastname: Carnevali
authors.forename: Michele
authors.initials: M
authors.lastname: Stefanoli
authors.forename: Andrea
authors.initials: A
authors.lastname: Ferretti
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Chiaravalli
authors.forename: Stefano
authors.initials: S
authors.lastname: La Rosa
authors.forename: Carlo
authors.initials: C
authors.lastname: Capella
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018278:Carcinoma, Neuroendocrine / Q000175:diagnosis / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D004273:DNA, Neoplasm; D025521:Tumor Suppressor Proteins