Prognostic role of targeted methylation analysis in paraffin-embedded samples of adrenocortical carcinoma

Context: Adrenocortical carcinoma (ACC) is a rare aggressive disease with heterogeneous prognosis. Previous studies identified hypermethylation in the promoter region of specific genes to be associated with poor clinical outcome.

Objective: Comparative analysis of promising hypermethylated genes as prognostic markers and evaluation of their added value to established clinical prognostic tools.

Design: We included 237 patients with ACCs. Tumor DNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples. Targeted pyrosequencing was used to detect promoter region methylation in 5 preselected genes (PAX5, GSTP1, PYCARD, PAX6, G0S2). The prognostic role of hypermethylation pattern was compared to S-GRAS score. Primary endpoints were progression-free (PFS) and overall survival (OS), with disease-free (DFS) as secondary endpoint.

Results: 27.9%, 13.9%, 49%, 49% and 25.3% of cases showed hypermethylation in PAX5, GSTP1, PYCARD, PAX6, and G0S2, respectively. Hypermethylation in all individual genes – except GSTP1 – was significantly associated with both PFS and OS - with Hazard Ratios (HR) between 1.4 and 2.3. However, only hypermethylation of PAX5 remained significantly associated with OS (p=0.013; HR=1.95, 95%CI 1.2-3.3) in multivariable analysis. A model for risk stratification was developed, combining PAX5 methylation status and S-GRAS groups, showing improved prognostic performance compared to S-GRAS alone (Harrell’s C index: OS=0.751, PFS=0.711, DFS=0.688).

Conclusions: This study demonstrated that hypermethylation in PAX5 is associated with worst clinical outcome in ACC, even after accounting for S-GRAS score. Assessing methylation in FFPE material is straightforward in the clinical setting and could be used to improve accuracy of prognostic classification, enabling the direction of personalized management.