Vol. 6, 2381–2392, June 2000 Clinical Cancer Research 2381 Noninvasive Diagnosis of Bladder Carcinoma by Enzyme-linked Immunosorbent Assay Detection of CD44 Isoforms in Exfoliated Urothelia Anthony C. Woodman, Steve Goodison, positive predictive value of 87% and a negative predictive Marcus Drake, Jeremy Noble, and David Tarin1 value of 100%. The data presented demonstrate that the rapid and accurate detection of elevated levels of CD44 Cranfield Biomedical Centre, Institute of Bioscience and Technology, Cranfield University, MK43 0AL Bedfordshire, United Kingdom protein isoforms in exfoliated urothelial cells is applicable to [A. C. W.]; University of California, San Diego Cancer Center and the identification and monitoring of primary and recurrent Department of Pathology, La Jolla, California 92093-0658 [S. G., bladder cancer. D. T.]; and Department of Urology, Oxford Churchill Hospital, OX3 7LJ Oxford, United Kingdom [M. D., J. N.] INTRODUCTION Bladder carcinoma has a high prevalence in many indus- ABSTRACT trialized countries, which is in part a consequence of the disease The expression of variant isoforms of the adhesion having a strong association with cigarette smoking and a num- molecule CD44 is correlated with the onset of neoplasia in ber of occupations, e.g., the rubber industry and the use of many carcinomas. We have previously shown that noninva- organic solvents. Bladder cancer rates are stable at around 17 sive detection of bladder carcinoma is possible by analysis of per 100,000, and it is estimated that there will be Ͼ50,000 new anomalous CD44 expression in exfoliated urothelia. Al- cases in 1998 in the United States.2 Approximately 60% of though the sensitivity and specificity values obtained for the superficial bladder tumors recur after initial resection, and 20% detection of bladder tumors using RT-PCR and Western of these will progress to invasive malignancy (1). Treatments blotting methods were superior to those obtained using using chemotherapy and/or surgery are generally successful, urine cytology, the application of such techniques is incon- particularly when the tumors are detected before invasion venient for routine diagnostic use. We now report the design through the basement membrane of the bladder wall. However, and development of a sandwich-ELISA system for the reli- because of the inaccessibility of the bladder to unaided visual able detection of CD44 protein extracted from sedimented examination, internal investigation is only considered when urothelial cells in voided urine. Naturally micturated urine macroscopic hematuria or other symptoms occur. Although samples were obtained from 53 patients with newly diag- cystoscopy, contrast urography, and ultrasound are to date the nosed bladder cancer and from 65 subjects with no evidence most powerful methods for the diagnosis and monitoring of of disease; patients with gross hematuria were excluded bladder tumors, they are uncomfortable and labor-intensive pro- because of interference with the assay. To demonstrate the cedures. Even with such invasive procedures, it is sometimes diagnostic potential of the system, a “gate” was imposed at difficult to reach a definitive diagnosis, especially after the N (max), i.e., the highest absorbance value obtained from a resection of an earlier neoplasm, either because the lesion is too sample known to be tumor free. All values above this value small to find or is inaccessible. Thus the development of sim- were assumed to be indicative of the presence of a tumor. pler, preferably noninvasive methods for the detection of urothe- Using this parameter, 42 of 53 (81.1%) patients with histo- lial malignancy are urgently required. Furthermore, because logically confirmed bladder tumors were correctly diag- bladder lesions have a strong tendency to recur, the monitoring nosed. Correspondingly, under these conditions, the assay is of asymptomatic patients for recurrence after treatment is par- 100% specific for tumor detection, with a sensitivity of ticularly important. Urine cytology, although simple and highly 81.1%, which equates to a positive predictive value of 100% specific, has insufficient sensitivity to be routinely useful, par- and a negative predictive value of 81.1%. A further 54 ticularly in the diagnosis of well-differentiated, early-stage neo- patients who had previously received treatment for bladder plasms, which are the most amenable to successful treatment cancer but were currently clinically disease-free were also (2). Recently identified molecular abnormalities that occur in investigated. Of these, 47 of 54 (87%) were correctly diag- neoplasia offer new opportunities for the early diagnosis of nosed to be tumor-free, which in this group equates to a 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 Received 10/27/99; revised 3/6/00; accepted 3/6/00. constantly expressed in almost all tissues (CD44s), with the The costs of publication of this article were defrayed in part by the inclusion of the remaining exons (CD44v) being subject to 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 2 Vital Statistics Of the United States. American Cancer Society Facts Jolla, CA 92093-0658. Phone: (619) 822-1222; Fax: (619) 822-0207. and Figures, http://www.cancer.org/statistics/cff98, 1997. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2000 American Association for Cancer Research. 2382 Noninvasive Diagnosis of Bladder Carcinoma Fig. 1 Schematic map of CD44 gene structure. Arrows, gene sequences encoding for antibody binding domains. alternative splicing. This processing of CD44 transcripts can those obtained using microscopic urine cytology, the application theoretically lead to the production of several hundred protein of the techniques used is presently not feasible for routine isoforms, all of which are subject to extensive posttranslational diagnostic use. modifications, particularly glycosylation, yielding isoforms with Immunohistochemistry and ELISA are techniques more apparent molecular weights of between Mr 85,000 and Mr suitable for the high-throughput, routine assessment of diagnos- 250,000. Having been initially characterized as the lymphocyte tic molecular markers. The immunohistochemical assessment of homing receptor (3, 4), the CD44 family of proteins is now CD44 protein isoforms in solid bladder tissue is informative believed to play a role in many cellular activities, including (19–21) and has revealed useful information concerning the T-lymphocyte activation, cellular adhesion, embryonic develop- expression of the CD44 gene during tumor progression in the ment, and hyaluron metabolism (5–7). However, much interest bladder (22). Furthermore, immunocytochemical analysis of has recently been directed to the role of CD44 in malignancy. CD44 proteins on exfoliated urothelial cells has been shown to Animal studies with transfected cell lines have implicated a role be a useful adjunct to cytology (19). However, the application of for one of the splice variants in experimental tumor metastasis immunohistochemistry to the rapid, large volume analysis of (8–10), whereas RT-PCR3 based studies of human tissues have exfoliated cells is inconvenient and labor-intensive in interpre- demonstrated aberrant CD44 gene expression in many human tation. The assay of choice for such purposes is an ELISA-based tumors (11–15), indicating that it could be a useful diagnostic system, where high throughput is coupled with a nonsubjective marker. Subsequently there have been many publications de- digital result. ELISA systems have been described for the de- scribing abnormalities of CD44 transcription and translation in tection of circulating soluble CD44 in the serum of patients with a variety of types of malignant disease (for recent reviews, see colon (23), breast (24, 25), and ovarian cancers (26, 27), but the Refs. 6, 16, 17, and 18). technology has not been applied to the measurement of CD44 Previously, we have presented data describing the nonin- proteins in exfoliated cells. We report the design and develop- vasive detection of bladder carcinoma by detection of anoma- ment of a sandwich-ELISA system for the detection of CD44 lous CD44 mRNA and proteins by RT-PCR/Southern hybrid- protein isoforms in exfoliated urothelial cells and discuss its ization and Western blotting, respectively (13, 19), in exfoliated application for the noninvasive detection of bladder cancer. urothelial cells collected from naturally micturated urine. Al- though the sensitivity and specificity values obtained for the MATERIALS AND METHODS detection of bladder tumors were superior in those studies to CD44 Gene Nomenclature The following nomenclature, as described previously (6, 17, 21), is used for description of CD44 gene exon arrangement. 3 The abbreviations used are: RT-PCR, reverse transcription-PCR; mAb, Alternatively spliced variant exons 6 to 15 (v1–10) can be monoclonal antibody; TBS, Tris-buffered saline; CHAPS, 3-[(3-chol- included in the transcript by alternative splicing between stand- amidopropyl)dimethylammonio]-1-propanesulfonate. ard exons 5 (s5) and 16 (s6). Downloaded