Gene expression of MLH1 related to microsatellite instability, immuno-histochemistry, and promoter hypermethylation in colorectal cancer

Abstract

3601 Background: The subgroup of highly microsatellite instable (MSI-H) colorectal cancers (CRC), is well described with DNA microsatellite and immuno-histochemistry (IHC) analyses. MSI-H CRCs are most often caused by hypermethylation inactivation of MLH1 or to a lesser extent by hereditary mutations in this or other mismatch repair genes. Microsatellite analysis and IHC are qualitative rather than quantitative methods. Reports showing that MMR deficient CRC has a favorable prognosis and that these cancers may respond less to chemotherapy may be more informative if based on a quantitative method instead. The aim of the present study was to correlate the expression of MLH1 to microsatellite analysis, promoter hypermethylation, and IHC. Methods: Sections of paraffin-embedded tumors from 206 unselected consecutive patients with CRC were microdissected and RNA was isolated and reverse-transcribed to cDNA. The relative gene expression of MLH1 was measured with real-time PCR. DNA from tumor and blood was examined with five standard microsatellite markers to find length mutations indicating instability. Tumor DNA was further analyzed with a methylation specific PCR in the C region of the MLH1 promoter. Protein expression of MLH1 was analyzed with IHC. Results: For all quantifiable tumors the median (and lower and upper quartiles) of the relative MLH1 gene expression was 0.90 (0.68, 1.42). For MSI-H tumors, defined as presense of instability in at least two markers or lack of protein expression with IHC, the median was 0.36 (0.26, 0.56). For microsatellite stable cancers the median was 0.96 (0.73, 1.44), p<0.0001 (Wilcoxon test). Median and quartiles of MLH1 expression in hypermethylated and non-methylated tumors was 0.31 (0.26, 0.45) and 0.96 (0.73, 1.43) respectively (p<0.0001). Relative MLH1 expression in MSI-H tumors range up to 0.64. Fourteen % of MSS tumors are in this range, too. Conclusions: In this large unselcted group of CRC patients we found a significant relation between low gene expression, MSI-H, MLH1-hypermethylation and negative IHC. We further identify a subgroup of MSS patients with low gene expression of MLH1 and suggest this quantitative marker in studies of prognosis and treatment effect. [Table: see text]