S1965 Novel Evidence for Soma-Wide MLH1 Hypermethylation and a Global Methylation Defect in Non-Familial Early-Onset Colorectal Cancer

Introduction: Recently soma-wide monoallelic germline epigenetic inactivation of MLH1 has been proposed as a potential mechanism for DNA mismatch repair (MMR) gene inactivation in some individuals with colorectal cancer (CRC). The present study aimed to investigate whether this methylation defect would be limited to the MLH1 promoter, or represents a broader epigenetic defect involving multiple genes. Methods: A 20 year old woman presented with a sigmoid colon cancer, and a negative family history for any cancer. Her tumor was evaluated for DNA mismatch repair (MMR) gene defects, and she was tested for germline mutations in MMR genes. Quantitative pyrosequencing and bisulfite sequencing of cloned PCR products was used to ascertain MLH1 methylation status in all three germ-layers (blood, hair and buccal tissues), as well as in her Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Genome wide/global methylation status for ~1500 genes/methylation loci was determined in the patient's blood and controls (which included both parents, her sibling, and 3 unrelated controls) using Illumina GoldenGate methylation microarrays. Results: The patient's tumor showed MSI and loss of expression of MLH1, PMS2 and MSH6 proteins. However, no germline mutations were detected in MLH1, MSH2 or MSH6. A single nucleotide polymorphism (SNP) permitted allele identification; 14%monoallelicMLH1 methylation was found in the tumor, but there was no allelic imbalance at MLH1 in the tumor. MLH1 methylation was observed in the patient's blood (10%), EBV transformed lymphocytes (10%), buccal mucosa (22%) and hair follicles (48%). No evidence for MLH1 methylation was present in patient's family members or any healthy control (all <1%). Interestingly, MLH1 methylation increased to 24% in the patient's blood after 5-FU based chemotherapy treatment. Genome wide methylation analyses showed that aberrant methylation in the blood was not limited to MLH1, but included 43 additional hypermethylated genes (including RUNX3, IGF1, PPARγ and NOTCH4) and another 34 genes which were hypomethylated (including CDK2, STAT5 and IL10). Conclusions: This study reports the youngest patient with soma-wide MLH1 hypermethylation and CRC. The increase in MLH1 methylation following chemotherapy suggests that cells with methylated MLH1 promoters are relatively resistant to the toxic effects of this drug. More importantly, our observation that the epigenetic defects were not restricted to the MLH1 promoter suggests the possibility of a broader epigenetic defect in those individuals with soma-wide methylation of MLH1.