Evaluation of Mutation Risk Using Patient-Derived Organoids in Patients With Lynch Syndrome

Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by germline mutation of DNA mismatch repair (MMR) genes, most notably associated with colorectal cancer. Although LS patients face high risk of CRC, risk can vary even among those with the same pathogenic MMR germline mutations. We suggest a functional assay platform for assessing mutation risk using patient-derived organoid (PDOs). We measured organoid response to the cytotoxic effects of a methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) based on DNA damage-induced apoptosis. When normal (non-LS) colon organoids were used, treatment with MNNG and four passages of organoid culture led to decreased total growth (organoid area), but there was no significant change in number. To increase the effect of MNNG and induce apoptosis in normal colon organoids, we added O⁶BG (an MGMT inhibitor) and an ATR inhibitor. The cytotoxic effects of combined treatment with MNNG, O⁶BG and ATR inhibitor on non-LS-PDOs were significantly higher than in PDOs of LS patients. To detect the difference at an earlier phase, we examined DNA damage response by analyzing γH2AX expression after treatment with MNNG and O6BG for 24 h. Then, we found higher expression of γH2AX in non-LS-PDOs than in PDOs of LS patients, suggesting continued cell survival with mutation accumulation in LS-PDOs. In conclusion, treatment with MNNG and O6BG using PDOs and measurement of resulting DNA damage response could be used as a preclinical platform for risk stratification in LS patients.