Int Urol Nephrol (2012) 44:761–767 Table 2 Percent of positive qRT-PCR signal for EGFR, COL1A1 and COL3A1 mRNAs and relative expression levels of KIT and CD47 in blood samples collected from controls and patients treated for bladder tumors Controls Days after TURBT (P values) Before Positive signal of expression (percent) EGFR 0% 27% (0.0006) COL1A1 3% 27% (0.007) COL3A1 28% 40% N.S. Relative expression levels KIT 0.061 CD47 0.020 0.231 (8.66E-07) 0.279 (2.71E-08) 1 20% (0.005) 45% (0.0004) 38% N.S. 0.265 (0.003) 0.233 (1.09E-08) Control group consisted of 17 healthy volunteers selected from laboratory staff 3 14% N.S. 25% (0.007) 30% N.S. 0.224 (1.43E-06) 0.264 (1.30E-07) 7 10% N.S. 36% (0.0004) 36% N.S. 0.258 (1.70E-06) 0.219 (1.10E-08) 765 30 4% N.S. 23% N.S. 22% N.S. 0.291 (1.23E-06) 0.200 (8.46E-08) selection of mRNA markers strongly determines the ﬁnal results of the study. Therefore, the study started with the identiﬁcation of urothelium-speciﬁc genes. As a result, circulating EGFR mRNA-positive cells were found in 27% of blood samples collected from bladder cancer patients and in none of the control blood samples. Also, positive RT-PCR signals for COL1A1 and CD47 mRNAs were found signiﬁcantly more frequent and at higher levels, respectively, in blood samples collected from cancer patients compared to controls. EGFR and COL1A1 are not expressed in hemopoietic cells [20], while expression of CD47 seems to be signiﬁcantly lower in those cells compared to expression in epithelial cells. Thus, from potential molecular markers selected for our analyses, expression of only few genes exhibited the desired speciﬁcity. It should however be noted that the results of our statistical analysis may be underestimated. Compari- sons utilizing only qualitative information in datasets of moderate size, such as employed in this study, require large relative frequency differences to con- clude signiﬁcant group bias. Moreover, results are susceptible even to single sample readout alteration. For example, when one group features 20/31 pos/neg ratio, there is no possibility of proving signiﬁcantly lower pos/neg proportions in other groups using 17 samples (assuming P value \ 0.01). Only a 14/3 (or higher) pos/neg ratio is signiﬁcant in that group (Fisher test, signiﬁcance level = 0.01, Online Resource 2). Previous studies have reported EGFR expression in 30–50% of bladder tumors [29], and the presence of EGFR transcript in peripheral blood was proposed as a marker for circulating BCa cell detection [13]. A study by De Luca et al. [30], which detected EGFR- expressing cells in the blood of lung, breast and colon cancer patients, suggests that this assay rather may be used in detecting cancer cells of epithelial origin than different carcinoma types. Nevertheless, the combi- nation of several markers could increase speciﬁc cancer type detection, and such molecular indicators were also proposed for BCa. Expression of UPK2 was found to be highly speciﬁc to the uroepithelia [31, 32] and was suggested as a highly speciﬁc but not sensitive blood marker for CTCs in bladder cancer patients [3]. However, in our study, a standard bowel preparation by polyethylene glycol (Fortrans) followed by a cleansing phosphate enema 30 min before the colonoscopy signiﬁcantly affected expression of UPK2 and KIT in blood samples from healthy subjects. Therefore, bowel preparation before colonoscopy may release epithelial cells into circulation or activate transcription of epithelia-speciﬁc genes in hemopoietic cells or both. An optimal method for detecting CTCs should be not only sufﬁciently speciﬁc but also sensitive enough to deﬁne the relatively low number of tumor cells in blood samples. Recently, the most popular methods employed for analyzing CTCs have used PCR 123