Expression of Urinary miRNA as a Potential Biomarker for Bladder Cancer Diagnosis

Abstract

Expression of urinarymiRNA-96 as a potential biomarker for bladder cancer diagnosis This study was done at Medical Biochemistry Department, Ain Shams Faculty of Medicine during the period from 2012– 2014 and included 124 patients and 60 normal volunteers. The aim of the present study was to evaluate miRNA-96 usefulness as a urine molecular marker for bladder cancer detection. Urinary miR-96 level was measured using quantitative real-time polymerase chain reaction (qPCR) in cellular pellets from a large number of voided urine samples, including those with bilharziasis in comparison with urine cytologygroups:  Group I: including 94 bladder cancer patients  Group II: including 30 benign bladder lesion patients.  Group III: including 60 healthy volunteers. All patients in our study were subjected to complete detailed history taking, general and local examination, routine laboratory investigations, radio diagnostic investigations as pelvi-abdominal ultrasonography, and IVP. Also all patients were subjected to qualitative detection of bilharzial antibodies in serum, Patients to be enrolled in the study must presented with chronic irritative voiding symptoms or hematuria and cystoscopicevidence confirmed by biopsy consistent with proven bladder carcinoma (in situ, low grade or highgrade),urine cytology, RNA extraction of urine pellets and real-time polymerase chain reaction (qPCR)for measuring urinarymiRNA-96 level.The data were normalized using the endogenous RNU U6 as reference control. The threshold cycle (Ct) value of each sample was calculated with Step One PlusTM software v2.2.2(Applied Biosystems), and the 2-ΔΔCT method was used in the analysis of PCR data for relative quantification of miRNA-96. Patients were examined endoscopically and the tumor was resected by hot loop resection and sent for pathological staging and grading of the tumor according to (TNM) classification. Then the results were calculated and statistically analyzed by the SPSS software. Participants were excluded from the study if they had a past history of occupational exposure to known bladder carcinogens, bladder cancer or another urological malignancies within the past 5 years, received chemotherapy and radiation therapy. GroupI: (Malignant)  miRNA-96qPCR: the percentage of patients who weremiRNA-96positive in this group was 72.3%.  Urine cytology:the percentage of patients who were positive for urine cytology was 34%.  Bilharzial antibody in serum:the percentage of patients who were positive for bilharzial antibody was 37.2%. Group II: (Benign)  miRNA-96qPCR: the percentage of patients who weremiRNA-96positive in this group was 26.7%.  Urine cytology: the percentage of patients who were positive for urine cytology in this group was 6.7%.  Bilharzial antibody in serum: the percentage of patients who were positive for bilharzial antibody was 40%. Group III (Healthy normal)  miRNA-96qPCR:the percentage of patients who weremiRNA-96positive in this group was 3.3%  Urine cytology: all individuals of this group were negative for urine cytology.  Bilharzial antibody in serum: all individuals of this group were negative for bilharzial antibody. The overall urine cytology sensitivity was 34%, specificity was 97.8%, PPV was 94.1%, NPV was 58.7% and accuracy was 65.2%. The positivity rate of urine cytology in different groups of this study showed a high significant increase in malignant group (34%) as compared with benign bladder lesion group (6.7%) and healthy control group (0%), (p≤0.001). No significant difference was observed in positivity rate of urine cytology in relation to sex, smoking, cytology, Bilharziasis, pathological type, stage and grade. Applying (1.63), as a cut off value (calculated by ROC curve), the urinary miRNA-96RQ values show 72.3% sensitivity, 88.9% specificity, 87.2% PPV, 75.5% NPV and 80.4% accuracy. A significant difference was observed in positivity rate of urinemiRNA-96 level(RQ)in relation the malignant group (72.3%) as compared with both benign bladder lesion group (26.7%) and healthy normal group (3.3%) (p≤0.001). There was no significant difference observed in Positivity rate ofurinemiRNA-96 level (RQ) in relation to sex, cytology, Bilharziasis, type,grade , stageand smoking(p> 0.05). A significant difference was found between mean rankofurinarymiRNA-96 level using the RQ valuesin malignantgroup as (121.49) compared with either benign bladder lesion group (76.63) or healthy normal group (55.02) (p≤0.001). When miRNA-96and urine cytology were combined, the overall sensitivity, specificity, PPV, NPV and accuracy were 79.8%, 86.7%, 86.2%, 80.4% and 83.1% and its combination with cytology increases the sensitivity up to 79.8% even in early stage 81.8% and in low grade 81.2% suggesting that miRNA-96 expression level in urine sample is a potentially useful urinary biomarker for early diagnosis of bladder cancer including bilharzial bladder cancer and it improves the sensitivity of urine cytology.