762 documented that diagnostic and therapeutic proce- dures [6, 9] can additionally release dysplastic cells from the tumor and may affect the level of CTCs in the peripheral blood. In turn, dissemination of tumor cells from locally advanced tumors may signiﬁcantly increase risk for metastatic disease, although only limited number of CTCs may colonize distant sites propagating the metastases. An optimal method for detecting CTCs should be sufﬁciently speciﬁc and sensitive with the most popular methods utilizing polymerase chain reaction (PCR)-based analyses. They allow detection of expres- sion of so-called epithelium-speciﬁc genes, which are expressed in cancer cells but not in nucleated cells of the blood. While the detection of mRNA markers speciﬁc for CTCs by reverse transcription (RT)-PCR is sensitive, its speciﬁcity may signiﬁcantly differ depending on the marker. Therefore, marker selection is considered vital for a proper analysis of CTCs. Here, we asked the question whether the electrore- section of a bladder tumor, which uses high pressure of washing ﬂuid, may induce a spread of cells from resected tissues into blood stream leading to the increase level of circulating urothelial cells in the peripheral blood. To answer it, we used quantitative RT-PCR to amplify the transcripts of selected genes, which might be speciﬁc and sensitive indicators of circulating urothelial cells. Methods Patients A total number of 51 patients (43 men and 8 women; mean age 67.5 years, range 34–89) with primary or recurrent urothelial BCa submitted to TURBT between April 2009 and May 2010 were prospectively selected for the study. Pathological staging was as follows: pTa—2 pts (2 patients; 4%), pT1—26 pts (26 patients; 51%), pT2—11 pts (11 patients; 21%), while in 12 cases (24%) primary tumor stage could not have been deﬁnitely assessed (pT C1; lack of muscularis propria in the specimen). The grade of the tumor was deﬁned as G1, G2 and G3 according to 1973 WHO classiﬁcation in 8 (16%), 25 (49%) and 18 (35%) cases, respectively. Details on patient’s age, sex and pathological diagnosis are given in the Online Resource 1. The control group consisted of 32 healthy 123 Int Urol Nephrol (2012) 44:761–767 volunteers, 17 among them were recruited from the laboratory staff and 15 were selected among individ- uals who underwent a screening colonoscopy with no known history of any malignancy. The study protocol was approved by the Ethics Committee, and all patients signed informed consent before inclusion. Urothelial-speciﬁc genes Literature searching [10–19] allowed selection of the following genes: uroplakin 2 (UPK2), EGFR, a-type platelet-derived growth factor receptor (PDGFRA), Insulin-like growth factor-binding protein 7 (IG- FBP7), Sorting nexin-16 (SNX16), TP53, KIT, Vas- cular endothelial growth factor A (VEGFA), CD47 and telomerase reverse transcriptase (TERT), which expression was reported previously to be highly speciﬁc and sensitive markers for the detection of CTCs, including circulating urothelial cells. In addi- tion, computation of the fold change of transcript levels between nucleated blood cells and urothelium was performed in searching for new candidate biomarkers of CTCs. Appropriate data were imported from the GSE4251 [20] dataset, which contained gene expression measurements for blood cells analyzed in three healthy subjects, and from the GSE3167 [21] and GSE5287 [22] datasets, which contained measure- ments of normal bladder and various dysplastic lesions. Datasets were deposited in the Gene Expres- sion Omnibus [23] database and represented measure- ments performed on Affymetrix Human Genome U133A and U133A 2.0 platforms, respectively. All imported measurements were processed with the GCRMA algorithm [24] and then averaged and sorted according to minimal fold change between any given blood cell population (white blood cells, peripheral blood monocytes and T lymphocytes) and urothelia. Calculation basing on the highest minima transcript levels has selected several urothelial-speciﬁc genes (not shown). Of them, four genes, COL1A1, COL3A1, KRT7 and AGR2, were considered as potential markers of circulating urothelial cells. RNA studies Blood samples were collected a day before, immedi- ately after (1 day) and 3, 7 and 30 days after surgery by venipuncture into PAXgene Blood RNA Tubes (QIAGEN) and were kept at –80 C until further use.