NEW YORK (GenomeWeb News) – Scientists have developed a method for evaluating DNA methylation in archival tissue samples that have been formalin-fixed and paraffin-embedded, according to a new technical advance paper appearing in the current issue of the Journal of Molecular Diagnostics.
Using a technique called high-resolution melting, or HRM, analysis, Austrian and American researchers have demonstrated that they can assess promoter methylation patterns in archived colorectal cancer samples. Their results suggest that HRM can provide high quality information about DNA methylation in formalin-fixed paraffin-embedded tissue samples.
"The methylation status in archival tumor specimens from patients with colorectal cancer can ... be determined with high quality by HRM," lead author Marija Balic, an oncology researcher at Austria's University of Graz, and her colleagues wrote. "The ability to analyze archival tissues greatly facilitates further research and its clinical implementation."
DNA modifications, such as methylation, can alter gene expression patterns and, in some cases, have been linked to cancer development and progression. For instance, promoter hyper-methylation can silence the transcription of important cellular pathways and has been implicated in carcinogenesis. Consequently, assessing methylation can be useful for some early cancer diagnoses or for predicting risk or treatment response in some cases.
But the quality of DNA in archived samples is often poor and detecting methylation patterns in these samples can be difficult. In addition, the researchers noted, techniques for assessing fresh tissue, such as fluorescence-based, real-time PCR, and bisulfite sequencing can be expensive and time-consuming.
In an effort to come up with new methods for assessing methylation in fixed samples, Balic and her colleagues tested HRM, a relatively new technique that is also known as high-resolution melt curve analysis, which is based on melting analyses of bisulfite-treated DNA.
"Archival tissues represent an enormous source for testing of clinically important tissues ... The aim of this study was to validate HRM for methylation status detection on archival [formalin-fixed paraffin-embedded] tissues from colorectal cancer patients," the authors wrote. "To our knowledge this is the first report on the use of HRM for detection of the promoter methylation status on [formalin-fixed paraffin-embedded] tissues."
After standardizing their protocol and testing the sensitivity of HRM using control tissues that were known to be either unmethylated or fully methylated, the researchers validated their HRM approach in eight different cancer cell lines — five human breast cancer lines and three human prostate cancer lines — comparing cultured and formalin-fixed paraffin-embedded samples.
Their results suggest that HRM can detect promoter hyper-methylation in fixed cells, with results consistent with those obtained using the fluorescence-based, real-time PCR method MethyLight.
Next, the team tested the approach in surgically resected, formalin-fixed paraffin-embedded samples taken from 48 male and 18 female colorectal cancer patients, checking for promoter methylation at two genes: MGMT and APC. They also looked at nine normal tissue samples.
About 42 percent of the cancer samples tested displayed MGMT promoter methylation and about a third displayed APC promoter methylation, though the levels of methylation varied. As expected, normal tissues showed no promoter methylation for these genes. The results were concordant to those obtained using MethyLight assays between 91 and nearly 99 percent of the time.
In the future, the researchers reportedly plan to apply a similar protocol to look at a well-defined prostate cancer cohort in an effort to determine whether HRM-based methylation analyses can predict lymph node metastases and other events in cancer progression.
"Identification of the methylation profiles in primary tumors that are associated with metastasis would not only elucidate those epigenetic events involved in disease progression but may aid in the development of a genomic prediction marker panel for patient outcome that can readily be assessed from paraffin-embedded tissue specimens," the authors concluded. "[O]ne application may be the risk stratification of patients based on methylation status of specific markers."