Dynamic epigenetic changes to VHL occur with sunitinib in metastatic clear cell renal cancer

Background: Genetic intratumoral heterogeneity (ITH) hinders biomarker development in metastatic clear cell renal cancer (mccRCC). Epigenetic relative to genetic ITH or the presence of consistent epigenetic changes following targeted therapy in mccRCC have not been evaluated. The aim of this study was to determine ITH of the methylome relative to genetic ITH and to evaluate specific epigenetic and genetic changes associated with sunitinib. Patients and methods: Multi-region DNA sampling performed on sequential frozen pairs of primary tumor tissue from 14 metastatic ccRCC patients, in the Upfront Sunitinib (SU011248) Therapy Followed by Surgery in Patients with Metastatic Renal Cancer: a Pilot Phase II Study (SuMR; ClinicalTrials.gov identifier: NCT01024205), at presentation (biopsy) and after 3-cycles of 50mg sunitinib (nephrectomy). Untreated biopsy and nephrectomy samples before and after renal artery ligation were controls. Ion Proton sequencing of 48 key ccRCC genes, and MethylCap-seq DNA methylation analysis was performed, data was analysed using R. Results: Unsupervised hierarchical clustering revealed complete methylomeclustering of biopsy and three nephrectomy samples for each patient (14/14 patients). For mutational status, untreated biopsy and all treated nephrectomy samples clustered together in 8/13 (61.5%) patients. Only methylation target significantly altered following sunitinib therapy was VHL promoter region 7896829 which was hypermethylated with treatment (FDR=0.077,P<0.001) and consistent for all patients (pre-treatment 50% patients had VHL mutations, 14% patients VHL hypermethylation). Renal artery ligation did not affect this result. No significant differences in driver or private mutation count found with sunitinib treatment. Conclusions: Demonstration of methylome homogeneity and consistent VHL hypermethylation, after sunitinib, overcome the hurdle of ITH present at other molecular levels.