Background: New methods for detection of bladder cancer are needed because cystoscopy is both invasive and expensive and urine cytology has low sensitivity. We screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and healthy tissues and investigated the diagnostic accuracy of one such marker in urine. Methods: Three specimens of bladder cancer and healthy urothelium, respectively, were used for proteome differential display using narrow-pH-range twodimensional electrophoresis. To evaluate the presence of calreticulin (CRT) as detected by Western blotting, we obtained 22 cancerous and 10 noncancerous surgical specimens from transurethral resection or radical cystectomy. To evaluate urinary CRT, we collected 70 and 181 urine samples from patients with and without bladder cancer, respectively. Anti-CRT COOH-terminus antibody was used to detect CRT in tissue and urine. Results: Proteomic analysis revealed increased CRT (55 kDa; pI 4.3) in cancer tissue. Quantitative Western blot analysis showed that CRT was increased in cancer tissue (P ؍ 0.0003). Urinary CRT had a sensitivity of 73% (95% confidence interval, 62-83%) at a specificity of 86% (80 -91%) for bladder cancer in the samples tested. Conclusions: Proteomic analysis is useful in searching for candidate proteins as biomarkers and led to the
Using proteomic analysis, we previously identified calreticulin (CRT) as a potentially useful urinary marker for bladder cancer. Now, we have also identified γ γ γ γ-synuclein (SNCG) and a soluble isoform of catechol-o-methyltransferase (s-COMT) as novel candidates for tumor markers in bladder cancer, by means of proteomic analysis. In the process of establishing a superior tumor marker system, we investigated the diagnostic value of a combination assay of these three proteins. Voided urine samples were obtained from 112 bladder cancer and 230 control patients. Urinary CRT, SNCG, and s-COMT were measured as a combined marker by quantitative western blot analysis. Relative concentration of each protein was calculated and the diagnostic value of a concomitant examination of these markers was evaluated by receiver operator characteristic analysis. With the best diagnostic cutoff, the overall sensitivity of the combined markers was 76.8% (95% confidence interval, 69-81%) with a specificity of 77.4% (72-80%), while those of a single use of CRT were 71.4% and 77.8%, respectively. When evaluated in relation to tumor characteristics, such as grade, stage, size, and outcome of urinary cytology, the diagnostic capacity of the combined markers was equal to or better than that of CRT in all categories. Concomitant use of CRT, SNCG, and s-COMT had higher sensitivity for detection of bladder cancer than did single use of CRT. Our study suggests that use of this panel of markers will improve the diagnosis of bladder cancer and may allow the development of a protein microarray assay or multi-channel enzyme-linked immunosorbent assay. (Cancer Sci 2004; 95: 955-961) ladder cancer is a common urothelial cancer with an estimated 57,400 and 13,000 diagnosed cases per year in the United States and Japan, respectively. 1, 2) Approximately 70% of bladder cancers are superficial 3) and respond well to endoscopic transurethral resection. However, 50% to 70% of these patients experience tumor recurrence, and 10% to 15% of recurrent tumors progress to muscle invasive disease. 4) Because the propensity for local recurrence extends over the lifetime, patients with superficial bladder cancer must undergo life-long surveillance.Cystoscopy is the mainstay in diagnosing bladder cancer. However, this procedure is unsuitable for screening of large groups because of its invasiveness and expense. In addition, follow-up cystoscopy for bladder cancer patients treated endoscopically represents a considerable part of the workload of any urological unit. Therefore, new tests with high specificity and sensitivity that are easy to perform are needed for both screening and monitoring the response to treatment of bladder cancer. Voided urine cytology (VUC) has been used in both diagnosis and follow-up of superficial bladder cancer since its first description by Papanicolaou and Marshall in 1945, but it has poor sensitivity although high specificity, particularly in well-differentiated cancer.5) To date, several urine-based markers for bladder cancer have ...
The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.
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