You have accessJournal of UrologyKidney Cancer: Basic Research (IV)1 Apr 2013610 DEVELOPMENT OF A TEST FOR RCC BY DETECTION OF VHL MUTATIONS IN TISSUE AND FLUIDS OF PATIENTS WITH RENAL CELL CARCINOMA Shilpa Sreedharan, Rebecca S Arnold, Kimberly Kerley, Kristina B Mercer, Kerry Ressler, Viraj A Master, Kenneth Ogan, David L Roberts, John G Pattaras, and John A Petros Shilpa SreedharanShilpa Sreedharan Atlanta, GA More articles by this author , Rebecca S ArnoldRebecca S Arnold Atlanta, GA More articles by this author , Kimberly KerleyKimberly Kerley Atlanta, GA More articles by this author , Kristina B MercerKristina B Mercer Atlanta, GA More articles by this author , Kerry ResslerKerry Ressler Atlanta, GA More articles by this author , Viraj A MasterViraj A Master Atlanta, GA More articles by this author , Kenneth OganKenneth Ogan Atlanta, GA More articles by this author , David L RobertsDavid L Roberts Atlanta, GA More articles by this author , John G PattarasJohn G Pattaras Atlanta, GA More articles by this author , and John A PetrosJohn A Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.161AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Surgery is the only highly effective therapy for renal cell carcinoma (RCC) and there is no effective screening or blood test for RCC. Somatic changes in the von Hippel-Lindau gene (VHL) are found in approximately 80% of clear cell RCC. Cancer cells and/or DNA may be recovered from body fluids of patients with RCC in nearly every case. The objective of this research is to develop a blood and/or urine test for RCC based upon the detection of small amounts of circulating tumor DNA characterized by the somatic mutations in VHL. METHODS DNA from archived patient tissue and fluids was used to compile a panel of acquired VHL polymorphisms for use in a diagnostic urine or serum test identifying circulating RCC cells. The sensitivities of methods capable of such identification were contrasted to more easily detect mutations present in early stages of RCC at a time when they can be surgically cured. DNA from 50 archived renal cancer patient tissue, serum, and urine was purified, amplified, and sequenced using an ABI3100 DNA Sequencer. The mutations detected were confirmed with restriction fragment length polymorphism (RFLP), when possible. The sensitivity of RFLP was contrasted with Sequenom's© ability to detect minute amounts of mutated DNA in urine and serum via a single base extension reaction and mass spectrometry. RESULTS Sequencing of VHL revealed the presence of mutations which subsequently created alterations in restriction enzyme digestion. After analysis of the commonly mutated exon regions of VHL for 50 patients, 78% were found to have mutations. 17% of those mutations occurred in multiple patients. DNA from serum and/or urine from these patients was then examined for the identified mutations. 27% of patients had mutations detected in the DNA purified from their serum and/or urine. Contrasting the sensitivity of mutation detection via RFLP of body fluids versus detection via Sequenom indicated that Sequenom was able to identify mutations present in DNA as dilute as 0.05ng, compared to 12.5ng DNA required for RFLP detection. CONCLUSIONS A panel of the most highly observed VHL polymorphism DNA biomarkers is a potential diagnostic tool for the detection of early stage RCC. High-throughput identification methods of such mutations, such as mass spectrometry, have an increased sensitivity and specificity compared to PCR/RFLP analysis and would therefore better translate into clinical application. VHL mutations identified in DNA purified from patient urine or serum via RFLP and mass spectrometry support the feasibility of developing such a test. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e249-e250 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shilpa Sreedharan Atlanta, GA More articles by this author Rebecca S Arnold Atlanta, GA More articles by this author Kimberly Kerley Atlanta, GA More articles by this author Kristina B Mercer Atlanta, GA More articles by this author Kerry Ressler Atlanta, GA More articles by this author Viraj A Master Atlanta, GA More articles by this author Kenneth Ogan Atlanta, GA More articles by this author David L Roberts Atlanta, GA More articles by this author John G Pattaras Atlanta, GA More articles by this author John A Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...