Methylation of p16 and Ras Association Domain Family Protein 1a during Colorectal Malignant Transformation

Abstract

Accurate assessment of gene methylation in formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) by microdissection remains challenging because the tissue volume is small and DNA is damaged. In addition, methods for methylation assessment, such as methylation-specific PCR (MSP), require sodium bisulfite modification (SBM) on purified DNA, which causes major loss of DNA. On-slide SBM, in which DNA is modified in situ before isolation of tumor cells, eliminates DNA purification steps and allows histology-oriented assessment of gene methylation. This study describes a protocol and use of on-slide SBM using 20 FF-PEAT of colorectal cancers with intratumoral adenoma components to detect accumulation of gene methylation during colorectal malignant transformation. Deparaffinized tissue sections were incubated in sodium bisulfite solution for 8 hours at 60°C, stained with hematoxylin, and then microdissected. Proteinase K lysate was directly used as a template in subsequent PCR. Using on-slide SBM, 282-bp-long bisulfite direct sequencing was possible. Yield of modified DNA was 2.6-fold greater than standard SBM on average. The mean conversion rate was 97%, and false-positive or false-negative results were not observed in subsequent MSP. Intratumoral heterogeneity by accumulation of p16 and Ras association domain family protein 1a methylation during malignant transformation were shown by MSP comparing cancer with adenoma parts within a single section. On-slide SBM is applicable in most methylation studies using FF-PEAT. It allows detailed, intratumoral analysis of methylation heterogeneity within solid tumors.On-slide SBM will significantly improve our approach and understanding of epigenetic events in minimal disease and the carcinogenic process. (Mol Cancer Res 2006;4(5):303 -9)

Assessment of Methylation Events during Colorectal Tumor Progression by Absolute Quantitative Analysis of Methylated Alleles

“…Relatively large amounts of DNA are required to compensate for the inevitable loss of DNA during the standard protocol for SBM. We have previously employed a model of comparing, by regular MSP, the methylation status of colorectal cancer cells within the same tissue section showing invasion with cells from the adenomatous precursor lesion (14). In this study, we applied an informative, quantitative technique, providing more detailed information about the methylation status than the dichotomous results of standard MSP.…”

“…DNA from PEAT was modified according to our previously published protocol (14). Briefly, from each tissue block, a single 4-Am section was cut and stained by H&E. Sevenmicrometer PEAT sections were cut consecutively and mounted on adhesive silane-coated slides for DNA studies.…”

“…The adenomatous cells analyzed would represent relevant, high-risk cancer precursors, whereas most studies use randomly selected colorectal adenomas with an unknown likelihood to develop into cancer. We have previously described an approach that enables this direct comparison in colorectal cancer paraffinembedded archival tissue (PEAT) sections by employing in situ sodium bisulfite modification (on-slide SBM) of the DNA (14). Adding a quantitative method to evaluate PEAT sections would allow accurate analysis of epigenetic events related to tumor histopathologic changes.…”

Development of Sporadic Microsatellite Instability in Colorectal Tumors Involves Hypermethylation at Methylated-In-Tumor Loci in Adenoma

“…18 AQAMA of MINT loci 1, 2, 3, 12, 17, 25, and 31 was performed, and data were analyzed in a manner as previously described. 8,28 MLH1 methylation status was analyzed by capillary-array-electrophoresis methylation-specific PCR.…”

“…We recently described an on-slide sodium bisulfite modification (SBM) technique for gene methylation analysis in small (1 to 2 mm 2 ) tissue areas isolated from a single section of paraffin-embedded archival tissue (PEAT). 18 On-slide SBM allows comparison of gene methylation in the primary CRC, the contiguous adenoma lesion, and normal epithelium when these three tissue types are present on the same tissue section. Simultaneous assessment of methylation and MSI changes in CRC, adenoma, and normal tissues from the same patient using the on-slide SBM technique would provide an accurate analysis model to test development of these epigenetic and genetic events during CRC formation.…”