Prevalence of somatic mutl homolog 1 promoter hypermethylation in Lynch syndrome colorectal cancer

Abstract

Background: Colorectal cancers (CRCs) that have microsatellite instability (MSI) and mutL homolog 1 (MLH1) immunoloss are observed in 3 clinical scenarios: Lynch syndrome (LS), sporadic MSI CRC, and Lynch-like syndrome (LLS). v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational analysis is used to differentiate LS from sporadic MSI CRC. The role of MLH1 promoter methylation status for the differential diagnosis of these clinical forms is not well established. The objectives of this study were: 1) to analyze MLH1 promoter methylation in MLH1-deficient CRCs by pyrosequencing, and 2) to assess its role in the differential diagnosis of MLH1-deficient CRCs.

Methods: In total, 165 CRCs were analyzed, including LS (n = 19), MSI BRAF-mutated CRC (n = 37), MSI BRAF wild-type CRC (n = 60), and a control group of CRCs without MSI (microsatellite stable [MSS] CRC; n = 49). MLH1 promoter methylation status was analyzed by pyrosequencing, and the ability of different strategies to identify LS was assessed.

Results: The average ± standard deviation methylation in LS (9% ± 7%) was significantly lower than that in MSI BRAF-mutated CRC (42% ± 17%; P < .001) and in MSI BRAF wild-type CRC (25% ± 19%; P = .002). Somatic MLH1 hypermethylation was detected in 3 patients (15.8%) with LS, in 34 patients (91.9%) with MSI BRAF-mutated CRC, and in 37 patients (61.7%) with MSI BRAF wild-type tumors. Patients with MSI BRAF wild-type, unmethylated tumors (ie, LLS) had a stronger family history of CRC than those who had tumors with MLH1 methylation (P < .05). The sensitivity for ruling out LS was 100% for BRAF analysis, 84.2% for MLH1 methylation analysis, and 84.2% for the combination of both analyses.

Conclusions: Somatic MLH1 promoter methylation occurs in up to 15% of LS CRCs. Somatic BRAF analysis is the most sensitive strategy for ruling out LS. Patients who have CRCs with loss of MLH1 protein expression and neither BRAF mutation nor MLH1 methylation resemble patients with LS.

Keywords: CpG island methylator phenotype; Lynch syndrome; colorectal cancer; methylation; microsatellite instability; mutL homolog 1; v-Raf murine sarcoma viral oncogene homolog B1.

Figure 1.

This flow chart is a schematic representation of the classification of the mutL homolog 1 (MLH1)-deficient colorectal carcinomas (CRCs) that were included in the current study based on an analysis of both somatic v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational status and MLH1 methylation. Gray boxes indicate the final diagnosis based on molecular analysis. MSS indicates microsatellite stability; BRAF, v-Raf murine sarcoma viral oncogene homolog B1; wt, wild type; MSI, microsatellite instability.

Figure 2.

MutL homolog 1 (MLH1) methylation results are illustrated for the 4 study groups. Box-and-whisker plots show the median methylation level expressed as a percentage (horizontal lines), the 25th and 75th percentiles (boxes), and the maximum and minimum levels (whiskers). Means and standard deviations also are indicated, and statistical comparisons between groups are depicted. MSS indicates microsatellite stability; LS, Lynch syndrome; MSI, microsatellite instability; BRAF, v-Raf murine sarcoma viral oncogene homolog B1.

Figure 3.

The distribution of mutL homolog 1 (MLH1) somatic hypermethylation is illustrated for the 4 study groups. For each group, the dark gray bars indicate methylated tumors. The percentages of MLH1-methylated tumors are indicated, and statistical comparisons between the groups are depicted. MSS indicates microsatellite stability; LS, Lynch syndrome; MSI, microsatellite instability; BRAF, v-Raf murine sarcoma viral oncogene homolog B1.