Noninvasive Diagnosis of Bladder Carcinoma by Enzyme-linked Immunosorbent Assay Detection of CD44 Isoforms in Exfoliated Urothelia Anthony C. Woodman, Steve Goodison, Marcus Drake, Jeremy Noble, and David Tarin 1 Cranfield Biomedical Centre, Institute of Bioscience and Technology, Cranfield University, MK43 0AL Bedfordshire, United Kingdom [A. C. W.]; University of California, San Diego Cancer Center and Department of Pathology, La Jolla, California 92093-0658 [S. G., D. T.]; and Department of Urology, Oxford Churchill Hospital, OX3 7LJ Oxford, United Kingdom [M. D., J. N.] ABSTRACT The expression of variant isoforms of the adhesion molecule CD44 is correlated with the onset of neoplasia in many carcinomas. We have previously shown that noninva- sive detection of bladder carcinoma is possible by analysis of anomalous CD44 expression in exfoliated urothelia. Al- though the sensitivity and specificity values obtained for the detection of bladder tumors using RT-PCR and Western blotting methods were superior to those obtained using urine cytology, the application of such techniques is incon- venient for routine diagnostic use. We now report the design and development of a sandwich-ELISA system for the reli- able detection of CD44 protein extracted from sedimented urothelial cells in voided urine. Naturally micturated urine samples were obtained from 53 patients with newly diag- nosed bladder cancer and from 65 subjects with no evidence of disease; patients with gross hematuria were excluded because of interference with the assay. To demonstrate the diagnostic potential of the system, a “gate” was imposed at N (max), i.e., the highest absorbance value obtained from a sample known to be tumor free. All values above this value were assumed to be indicative of the presence of a tumor. Using this parameter, 42 of 53 (81.1%) patients with histo- logically confirmed bladder tumors were correctly diag- nosed. Correspondingly, under these conditions, the assay is 100% specific for tumor detection, with a sensitivity of 81.1%, which equates to a positive predictive value of 100% and a negative predictive value of 81.1%. A further 54 patients who had previously received treatment for bladder cancer but were currently clinically disease-free were also investigated. Of these, 47 of 54 (87%) were correctly diag- nosed to be tumor-free, which in this group equates to a positive predictive value of 87% and a negative predictive value of 100%. The data presented demonstrate that the rapid and accurate detection of elevated levels of CD44 protein isoforms in exfoliated urothelial cells is applicable to the identification and monitoring of primary and recurrent bladder cancer. INTRODUCTION Bladder carcinoma has a high prevalence in many indus- trialized countries, which is in part a consequence of the disease having a strong association with cigarette smoking and a num- ber of occupations, e.g., the rubber industry and the use of organic solvents. Bladder cancer rates are stable at around 17 per 100,000, and it is estimated that there will be 50,000 new cases in 1998 in the United States. 2 Approximately 60% of superficial bladder tumors recur after initial resection, and 20% of these will progress to invasive malignancy (1). Treatments using chemotherapy and/or surgery are generally successful, particularly when the tumors are detected before invasion through the basement membrane of the bladder wall. However, because of the inaccessibility of the bladder to unaided visual examination, internal investigation is only considered when macroscopic hematuria or other symptoms occur. Although cystoscopy, contrast urography, and ultrasound are to date the most powerful methods for the diagnosis and monitoring of bladder tumors, they are uncomfortable and labor-intensive pro- cedures. Even with such invasive procedures, it is sometimes difficult to reach a definitive diagnosis, especially after the resection of an earlier neoplasm, either because the lesion is too small to find or is inaccessible. Thus the development of sim- pler, preferably noninvasive methods for the detection of urothe- lial malignancy are urgently required. Furthermore, because bladder lesions have a strong tendency to recur, the monitoring of asymptomatic patients for recurrence after treatment is par- ticularly important. Urine cytology, although simple and highly specific, has insufficient sensitivity to be routinely useful, par- ticularly in the diagnosis of well-differentiated, early-stage neo- plasms, which are the most amenable to successful treatment (2). Recently identified molecular abnormalities that occur in neoplasia offer new opportunities for the early diagnosis of bladder cancer, and the analysis of the expression of the trans- membrane glycoprotein CD44 may be useful in this context. Occupying 60-kb of the short-arm of chromosome 11, the CD44 gene (Fig. 1) contains at least 20 exons, of which 10 are constantly expressed in almost all tissues (CD44s), with the inclusion of the remaining exons (CD44v) being subject to Received 10/27/99; revised 3/6/00; accepted 3/6/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at University of California, San Diego Cancer Center, 9500 Gilman Drive, 0658, La Jolla, CA 92093-0658. Phone: (619) 822-1222; Fax: (619) 822-0207. 2 Vital Statistics Of the United States. American Cancer Society Facts and Figures, http://www.cancer.org/statistics/cff98, 1997. 2381 Vol. 6, 2381–2392, June 2000 Clinical Cancer Research Research. on June 26, 2017. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from