UROTHELIAL EGF-R GENE EXPRESSION IN BLADDER WASHINGS: A FEASIBILITY STUDY

Introduction and Objectives: Epidermal growth factor (EGF) is a strong tumor promoter. Its concentration is reduced in urine of patients affected by bladder cancer supporting the important role of its interaction with the urothelial epidermal growth factor-receptor (EGF-R) in tumor development and progression (1, 2). It would be of great usefulness to evaluate EGF-R gene expression during follow-up of non-muscle invasive bladder cancer (NMI-BC) avoiding tissue biopsy. The objective of our research was to investigate the feasibility of EGF-R evaluation in bladder washings of patients affected by NMI-BC. Materials and Methods: The study included patients undergoing adjuvant intravesical therapy for NMI-BC and age-matched healthy controls. In a preliminary phase of our research, bladder washing was selected for EGF-R analysis due to the high variability and easier contamination of urine. Samples of bladder washings were collected before, during and after adjuvant intravesical therapy and during the follow-up to investigate the gene expression of EGF-R. After collection, the samples were centrifuged twice at 4˚C, 1,200 rpm for 10 minutes, in cold phosphate-buffered saline solution. The cellular pellet was stored at –80˚C and later analyzed by isolation of cellular RNA using a miRNeasy Mini Kit (Qiagen®). Reverse transcription-polymerase chain reaction (RT-PCR) was performed in order to analyze EGF-R gene expression. Changes in the EGF-R content were calculated using the ooCt method after normalization with endogenous reference and calibrating cycle threshold (Ct) value for each RNA obtained for triplicate reactions. The percentage of patients with bladder washings giving a useful pellet for EGF-R determination was considered for the feasibility. EGF-R gene expression was related to tumor characteristics and compared to healthy controls. Descriptive statistical analysis was performed. Results: Thirty-two patients and 13 healthy controls were entered in the study. Fifty-two samples were obtained. A useful pellet to evaluate EGF-R expression was obtained in 26 patients (81.2%), 22 males and 4 females, mean age 71 (range=52-83) and in 10 healthy controls (76.9%). The bladder tumors were Ta, T1 and Tis, respectively in 8, 16 and 2 patients; single in 7 and multiple in 17, primary in 11 and recurrent in 16 patients. In 6 patients, a synchronous Tis was diagnosed. The median EGFR expression resulted 2.4-fold compared to controls (EGFR= 1). Four patients (15%) presented elevated levels of EGFR after transurethral resection (TUR) and before adjuvant therapy with a median value of 4-fold. EGF-R expression increased during follow-up in 9 patients (34%) with a median of 4-fold (range=2.5-8); returning within limits in 3 of them after adjuvant therapy. Discussion: EGF-R is detected in the basal layer of normal urothelium. Its expression in NMI-BC is limited and increases in high grade and invasive tumors. EGF-R activation promotes tumor growth by blocking apoptosis and increasing cell proliferation, motility, adhesion and invasive capacity. The results obtained in NMI-BC on EGF-R as an independent predictor of progression are scarce and conflicting (1). Only few data have been obtained in NMIBC (2). Our study suggests the feasibility of EGF-R evaluation in bladder washings of patients affected by NMIBC avoiding tissue biopsies. It was technically possible to evaluate its expression after TUR in more than 80% of our patients resulting up-regulated in 15% of them. EGF-R upregulation might represent a marker of potential aggressiveness of the tumor, failure of the treatment and progression during follow-up.