Presence of XMRV RNA in urine of prostate cancer patients
Gupta et al
Cleveland Clinic and Glickman Urological and Kidney Institute
Introduction: Urine specimens from cancer patients are often used as a source of RNA for detectin expression of genes of interest involved in cancer development and metastasis. A noninvasive urine based test for XMRV detection would be useful for screening and monitoring men with prostate cancer.
Methods: We have examined urine samples from 120 unselected prostate cancer patients after prostate massage during digital rectal examination. Also urine samples from 22 normal, healthy control individuals were tested. RNA was extracted from the samples using magnetic bead-based Magmax viral RNA isolation kit from Ambion. Quantitative RT-PCR was used to measure low copies of XMRV RNA in samples. Standard curves were performed using in vitro transcribed XMRV env RNA. Confirmation of the presence or absence of XMRV nucleic acids was obtained by nested RT-PCR followed by sequencing of the amplicon. These methods specifically amplify env sequences encoding a region of XMRV gp79 envelope protein (variable regions A and C)
Results: We have identified XMRV specific nucleic acids from prostate cancer patient urine samples. The sensitivity of the qRT-PCR assay was determined to be about 10 copies of XMRV RNA per reaction. About 26% of prostate cancer patients (31/120) showed the presence of XMRV env RNA by qRT-PCR analysis, whereas only 10 (8.3%) were scored as positive by nested nRT-PCR. However, all 10 of the samples that were positive for XMRV RNA by nRT-PCR were also positive by qRT-PCR. In contrast, 1 out of 22 or (4.5%) control urine samples from normal healthy individuals were scored as positive for XMRV env RNA by qRT-PCR. A subset of positive samples was retested by qRT-PCR for another region of env and confirmed as positive. The range of XMRV RNA copy numbers detected was 15 to 60 copies per ml of urine. RT-PCR products of selected samples containing XMRV RNA were confirmed by DNA sequencing.
Conclusions: The presence of XMRV RNA in urine is significant because such data can provide the basis for a urine-based XMRV detection assay that is noninvasive, rapid and easy to perform, avoiding the difficulty of obtaining blood or tissue biopsies. Results showed that qRT-PCR was more sensitive than nRT-PCR. Because men with XMRV infections, especially those that fail to clear the virus, may be at increased risk of prostate cancer these studies could result in a new diagnostic tool for evaluating risk of prostate cancer initiation and or progression.