Hypermethylation of the p16<sup>INK4a</sup> Promoter as an Early Epigenetic Event in Nodular Hyperplasia Associated with Secondary Hyperparathyroidism

Secondary hyperparathyroidism (SHPT) associated with chronic kidney disease (CKD) is characterized by parathyroid hyperplasia, which progresses from diffuse-type to nodular-type lesions. Nodular hyperplasia in SHPT is often considered to exhibit monoclonal proliferation, suggesting a shift toward neoplastic behavior, but the molecular mechanisms underlying this transformation remain poorly defined. In this study, we analyzed 340 surgically resected parathyroid glands from long-term dialysis patients who met clinical indications for parathyroidectomy. Based on histological architecture, lesions were classified into diffuse, nodular, or diffuse-nodular (mixed) hyperplasia. We conducted immunohistochemical analysis of Ki-67 and p16^INK4a (CDKN2A), and further assessed region-specific DNA methylation of the p16^INK4a promoter using a bisulfite padlock probe method combined with rolling circle amplification. Nodular-type lesions exhibited significantly higher Ki-67 indices and lower p16^INK4a expression compared to diffuse-type lesions. <i>In situ</i> methylation analysis revealed increased methylation of the p16^INK4a promoter specifically in nodular regions, suggesting epigenetic silencing. Our findings suggest that p16^INK4a silencing through promoter methylation may play a critical role in the clonal expansion and histopathological transformation of parathyroid tissue in SHPT. These results underscore the importance of epigenetic regulation in SHPT progression and suggest that p16^INK4a methylation could represent a potential biomarker for nodular transformation. The padlock probe-based detection system enabled high-resolution spatial analysis of methylation patterns and may serve as a valuable tool for dissecting epigenetic events in early phase of cellular alterations.