MLH1 Hypermethylation and BRAF Mutation Testing in Colorectal Cancer Patients with Mismatch Repair Defect
Ming Jin, Mark Clendenning, Heather Hampel, Wendy L Frankel. Ohio State University Wexner Medical Center, Columbus, OH; Queensland Institute of Medical Research, Herston, Queensland, Australia
Background: Lynch syndrome (LS) is associated with germline mutations in DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2), and accounts for 2-5% of all colorectal cancers (CRCs). Patients with LS almost never have a BRAF mutation or MLH1 hypermethylation in their tumor. We evaluated MLH1 hypermethylation and BRAF mutation testing following microsatellite instability (MSI) testing or immunohistochemistry (IHC) for MMR proteins to determine their utility in testing algorithms for identifying LS.
Design: 93 newly diagnosed CRC patients with either MSI-H (82) or at least one absent MMR protein (89) were included from a local LS study. BRAF mutation and MLH1 hypermethylation analyses were compared for cases with MSI-H and/or absence of MLH1 and PMS2 proteins. BRAF mutation (V600E) analysis was carried out by PCR amplification of exon 15 followed by direct sequencing. MLH1 hypermethylation status was studied by methylation specific PCR. Genetic germline mutation testing was performed on CRCs with MSI-H and/or any absent MMR protein.
Results: Of 82 MSI-H cases, 53/79 were BRAF mutation negative and 50/82 were not MLH1 hypermethylated. BRAF mutation was found in 25/32 (78%) MLH1 hypermethylated cases, and MLH1 hypermethylation was present in 25/26 (96%) with BRAF mutation. 3 fewer patients (50 vs. 53) would have needed further germline testing if MLH1 hypermethylation analysis were used rather than BRAF testing. Genetic testing confirmed 43/82 MSI-H had LS. Of 89 with at least one absent MMR protein, 46 had MLH1 and PMS2 absent and 43 had other stain(s) absent. Among 46 with MLH1 and PMS2 absent, 19/43 were not BRAF mutated and 14/46 were not MLH1 hypermethylated. 25/32 (78%) MLH1 hypermethylated cases had BRAF mutation, and 24/24 (100%) with BRAF mutation had MLH1 hypermethylation. 5 fewer patients (14 vs. 19) would have needed further germline testing if MLH1 methylation analysis were used rather than BRAF testing. 9/46 with absent MLH1 and PMS2 and 34/43 with absence of other MMR protein(s) were found to have LS by confirmatory genetic testing. None of 43 LS cases were found to have BRAF mutation or MLH1 hypermethylation.
Conclusions: Both BRAF mutation and MLH1 hypermethylation testing following MSI or MMR IHC are useful in identifying LS. MLH1 hypermethylation testing excludes more patients from further germline analysis, and is therefore potentially more cost effective.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 100, Monday Morning