Abstract
Despite many years of research efforts and continued progress in the identification of urine markers for detection of bladder cancer, none of the markers described to date has been able to replace cystoscopy and urine cytology, the current gold standards for diagnosis. Here, we present a comprehensive gel-based proteomic study in which we have analyzed the presence and origin of fibrinogen (FG) and its degradation products (FDPs) in the urine of patients with and without urothelial carcinoma (UCs), with the aim of evaluating the potential of two-dimensional (2D) gel FDP profiling for detecting bladder cancer. A total of 151 urine samples collected from patients with Ucs of varying degrees of atypia and stages of invasion were compared with a control group consisting of 34 healthy volunteers with no clinical history of bladder cancer. The level and degree of degradation of FG in the urine were determined by 2D gel Western blotting in combination with enhanced chemilumenscence detection. Elevated levels of urine FG/FDPs were found in 99% of patients bearing superficial tumors, in 97% of the cases with early invasive disease, and in 96% of patients with highly invasive tumors. 2D gel profiling of urine FG/FDPs showed that the FG chains exhibited differential susceptibility to in situ proteolysis, with the α-chain being the most susceptible and the γ-chain the most resistant. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified peptide sequence regions in several of the most representative and common FDPs, which can be of value for producing novel specific antibodies to detect FG/FDPs in the urine. In addition, we present evidence indicating that FG is not produced by normal or malignant urothelium, although it is present both in the stroma of malignant tissue and tumor lesions. Taken together, the data indicate that increased levels of FG/FDPs amounts in the urine are a characteristic feature of bladder cancer, and emphasize the value of 2D gel profiling of urine FG/FDPs for detecting low-grade, noninvasive UCs.
Highlights
Bladder cancer is the fifth most common malignancy in the world, and represents the second most common type of urological cancer
A total of 151 urine samples collected from patients diagnosed with urothelial carcinomas (UCs) of various degrees of atypia and stages of invasion, and 34 control urine samples collected from healthy volunteers with no known clinical record of bladder malignancy were analyzed by 2D gel immunoblotting and enhanced chemiluminescent (ECL) detection using the A0080 fibrinogen antibody (DAKO)
The intact form of the fibrinogen γ-chain was detected in all the urines from patients diagnosed with UCs, including highly invasive stages of the disease (Fig. 2B–E), whereas the α-chain was more susceptible to degradation and as a result could not be detected in most of the superficial lesions (Fig. 2B,C)
Summary
Bladder cancer is the fifth most common malignancy in the world, and represents the second most common type of urological cancer. It includes a wide spectrum of histologically heterogeneous tumor types that arise predominantly in the transitional epithelium (urothelium) lining of the urinary bladder and the ureters. UCs comprise more than 90% of the diagnosed bladder tumors in the Western hemisphere [4,5] and are broadly divided in two groups—low-grade superficial papillary lesions and high-grade lesions (carcinoma in situ [CIS]) [1,2]—based on histological examination and clinical parameters [6,7]. About 40% are pTa and 30% are pT1 lesions
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