title: Dynamic epigenetic changes to VHL occur with sunitinib in metastatic clear cell renal cancer.
abstract: 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 methylome/genetic ITH and to evaluate specific epigenetic and genetic changes associated with sunitinib therapy.
  
  
  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 the statistical computing environment R.
  
  
  RESULTS
  Unsupervised hierarchical clustering revealed complete methylome clustering 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. The 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 was found with sunitinib treatment.
  
  
  CONCLUSIONS
  Demonstration of relative methylome homogeneity and consistent VHL hypermethylation, after sunitinib, may overcome the hurdle of ITH present at other molecular levels for biomarker research.
authors.affiliation: Edinburgh Urological Cancer Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.|Scottish Collaboration On Translational Research into Renal Cell Cancer (SCOTRRCC), Scotland, UK.|Academic Urology Group, University of Cambridge, Addenbrooke's Hospital, Cambridge Biomedical Campus, Cambridge, UK.
authors.forename: Grant D
authors.initials: GD
authors.lastname: Stewart
authors.affiliation: Renal Cancer Unit, The Royal Free Hospital, London, UK.|Centre for Experimental Cancer Medicine, Bart's Cancer Institute, Queen Mary University of London, London, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Powles
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Christophe
authors.initials: C
authors.lastname: Van Neste
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Meynert
authors.affiliation: Edinburgh Urological Cancer Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.|Scottish Collaboration On Translational Research into Renal Cell Cancer (SCOTRRCC), Scotland, UK.
authors.forename: Fiach
authors.initials: F
authors.lastname: O'Mahony
authors.affiliation: Edinburgh Urological Cancer Group, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.|Scottish Collaboration On Translational Research into Renal Cell Cancer (SCOTRRCC), Scotland, UK.|MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Laird
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Filip
authors.initials: F
authors.lastname: Van Nieuwerburgh
authors.affiliation: Biobix: Laboratory of Bioinformatics and Computational Genomics, Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Trooskens
authors.affiliation: Biobix: Laboratory of Bioinformatics and Computational Genomics, Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Van Criekinge
authors.affiliation: Biobix: Laboratory of Bioinformatics and Computational Genomics, Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Ghent, Belgium.
authors.forename: Tim
authors.initials: T
authors.lastname: De Meyer
authors.affiliation: Scottish Collaboration On Translational Research into Renal Cell Cancer (SCOTRRCC), Scotland, UK.|School of Medicine, University of St Andrews, Fife, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Harrison
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000235:genetics*; D016000:Cluster Analysis; D019175:DNA Methylation / Q000187:drug effects; D004252:DNA Mutational Analysis; D044127:Epigenesis, Genetic / Q000187:drug effects; D005260:Female; D006801:Humans; D007211:Indoles / Q000627:therapeutic use*; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D010865:Pilot Projects; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011758:Pyrroles / Q000627:therapeutic use*; D000077210:Sunitinib; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics*
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; D011758:Pyrroles; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human; D000077210:Sunitinib
keywords: VHL; heterogeneity; methylation; mutations; renal cancer