Validation of DNA methylation subtype as a prognostic biomarker in pediatric adrenocortical tumors: re-evaluation of results from the COG ARAR0332 clinical trial

Pediatric adrenocortical tumors (pACTs) are rare neoplasms with limited biomarkers for risk stratification. Two DNA methylation-based molecular subtypes (A1 and A2) previously identified using data from the International Pediatric Adrenocortical Tumor Registry cohort were strongly associated with outcome, with A1 linked to poor prognosis and A2 to indolent disease. The objective of the current study was to validate the prognostic utility of pACT molecular subtypes in the clinical trial cohort from the Children’s Oncology Group ARAR0332 protocol (ClinicalTrials.gov: NCT00304070, registration date: 3/15/2006). A DNA-methylation-based neural-network classifier trained on 60 reference samples was used to assign ARAR0332 samples to A1, A2, or normal adrenal classes. The class assignments were correlated with clinical variables, genomic features, and survival outcomes. Among ARAR0332 samples, 11 (22%) were classified as A1 and 38 (78%) as A2. A1 tumors were associated with older age, metastatic disease, Cushing syndrome, and CTNNB1 mutations, while A2 tumors presented in younger patients (mean 4.1 years) and in patients who more often exhibited virilization. A1 tumors had significantly worse 5-year event-free survival and overall survival compared to A2 tumors. In patients with non-metastatic tumors, methylation class remained a significant predictor of risk even after adjusting for clinical stage and histopathologic risk class as defined by Wieneke criteria. Taken together, our findings suggest integration of methylation class with clinical stage and histopathologic features could improve risk stratification on future risk-adapted clinical trials.