Epigenetic sampling effects : nephrectomy modifies the clear cell renal cell cancer methylome

Abstract

Background: Sample collection for clinical epigenetics research often occurs at the time of surgical excision of a diseased organ. However, this approach may compromise the epigenetic profile under study. The use of different sampling procedures during a study can often not be avoided, but may in theory lead to biased results. The effect of tissue sampling approach on DNA methylation is studied here using clear cell renal cell cancer (ccRCC) as a model. A comparison of the DNA methylation profiles between vascularised tumour biopsy samples and subsequent devascularised nephrectomy samples obtained from the same two individuals (total of 6 samples per individual) was undertaken. Validation of the results was performed using biopsy and nephrectomy samples obtained from 14 patients included in a ccRCC clinical trial (SuMR; ClinicalTrials.gov identifier: NCT01024205). Findings: Using MBD2 sequencing, the methylome was analysed for all samples and differential methylome regions were retrieved. The results, from the test set, show six differentially methylated genes, of which four were clearly linked to ischaemia or hypoxia (REXO1L1, TLR4, hsa-mir-1299, and ANKRD2). To validate these findings, it was evaluated whether these loci were also featured by differential methylation in the clinical trial cohort with a similar experimental design to the test set. Three of the six genes are again significantly differentially methylated, showing an overall clear impact of renal artery clamping on DNA methylation. Conclusions: Renal artery ligation modulates the ccRCC methylome, impacting methylation of ischaemia and hypoxia associated genes. Results from devascularised surgical resection specimens do not accurately reflect findings from tumour biopsies.