Zhang, Dan

Abstract MGMT is primarily inactivated via promoter methylation, but MGMT homozygous deletion (hmzdel) has been rarely reported as an alternative inactivating mechanism. Although functionally similar to a methylated MGMT promoter, MGMT hmzdel is associated with an unmethylated MGMT promoter test result reflecting the MGMT promoter status of the non-neoplastic cells invariably present in any tumor specimen. Thus, patients whose tumors have MGMT hmzdel (and unmethylated MGMT promoter) would be inaccurately predicted to lack benefit from alkylating chemotherapy based on MGMT promoter methylation testing alone. To evaluate the impact of this clinical scenario, we estimated the frequency and distribution of MGMT hmzdel in routine clinical testing of CNS tumors. Among 12,424 CNS tumors (3,044 with provided diagnosis of glioblastoma) tested using Oncoscan chromosomal microarray (2015-2025), 10 tumors (0.08%) had MGMT hmzdel: 8 with entire gene hmzdel and 2 with partial gene hmzdel favored to be inactivating (RNA-sequencing underway to exclude possibility of activating rearrangements). All tumors were supratentorial and non-midline. Median age at diagnosis was 58.5 years (range, 34-81), with 3:2 male:female. Of the 10 cases, 9 (of 3,044; 0.3%) had integrated diagnosis of glioblastoma, IDH-wildtype; the remaining case was a recurrent astrocytoma, IDH-mutant, CNS WHO grade 4. All 10 cases had loss of chromosome 10q and a highly unmethylated MGMT promoter by droplet digital PCR testing of downstream CpG sites 75-80/84-87, with median fraction abundance of 0.19% (range, 0-0.85). In summary, MGMT hmzdel only occurred in high-grade adult-type diffuse astrocytic gliomas, and is a rare potential pitfall for MGMT promoter methylation testing leading to inaccurate patient stratification. It is critical that laboratories performing routine CNS tumor copy number testing include MGMT in the curated list of reportable genes, and that clinicians be aware of this rare clinical scenario to accurately stratify patients despite misleading MGMT promoter test results.