DETECTION OF HTR, HTERT AND CKS2 MRNA INBLADDER WASHINGS: POTENTIAL MOLECULARMARKERS OF BLADDER CANCER

Background: Although the field of tumour markers in bladder cancer (BC) is rapidly evolving, no ideal marker currently exists. Combined analysis of molecular biomarkers could enhance the diagnostic power of assay. In this exploratory study, we quantified the transcript levels of three different genes associated with carcinogenesis: human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT) and CDC28 protein kinase regulatory subunit 2 (CKS2). Patients and Methods: The study included 99 consecutive patients undergoing flexible cystoscopy for needs related and unrelated to bladder cancer (BC). The patients were classified into two age- and sex-matched groups (P=0.693 and P=0.720, respectively). The first one included 36 patients (32 male, 4 female) with a histopathological diagnosis of BC. The mean age ± SD of the BC group was 68.8 ± 10.8 (range 48 to 87) years. The second group (controls) included 63 patients (59 male, 11 female) with a mean age ± SD of 69.9 ± 10.6 (range 41 to 86) years. Tumour stage was determined using TNM (tumour lymph nodes and metastasis) and grading according to the World Health Organization (WHO 1973) guidelines. Tumours were classified as: 72.2% (26/36) superficial [pTaG1 (n=24), pT1G1 (n=2)], 27.8% (10/36) invasive [pT2G3,. At the time of sampling, all controls were BC-free. Messenger RNA amounts were measured by quantitative real-time reverse transcriptase chain reaction in bladder washings of patients with and without BC. Non parametric receiver operating characteristics analysis was performed to assess the accuracy of study variables to discriminate between BC and controls. The diagnostic value of concomitant examination of these markers was evaluated by logistic regression analysis. Results: Washing fluids transcripts detection of hTR, hTERT and CKS2 genes revealed highly significant differences between BC patients and controls. In particular, hTR, hTERT and CKS2 showed a significant 2.1-fold decrement, 9.3-fold and 8.45-fold increment, respectively, in the expression levels of relative genes compared to controls. The area under the curve (AUC) was 0.67 (95% CI: 0.53- 0.80) for hTR, 0.68 (95% CI: 0.54-0.83) for hTERT and 0.72 (95% CI: 0.58-0.86) for CKS2, indicating an average discrimination power between BC and controls, for all these tests when singularly considered. A model including hTR and CKS2 showed a higher clinical performance in comparison to each marker singularly considered. The analysis was also useful when stratifying BC in superficial or invasive forms. Detection of transcripts hTR, hTERT and CKS2 in washing fluid and, most importantly, their combination in different models, represent molecular markers of urothelial malignancy (AUChTR/CKS2=0.90, 95% CI: 0.82 - 0.98). Conclusion: Taken together these findings suggest that hTR, hTERT and CKS2 gene expression and, most importantly, their combination in different models, represent molecular markers of urothelial malignancy. Confirmation of our results in urine might provide a useful non-invasive tool in early detection and clinical evaluation of BC.