Noninvasive Diagnosis of Bladder Carcinoma by Enzyme-Linked Immunosorbent Assay Detection of CD44 Isoforms in Exfoliated Urothelia

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.

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 describing 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).

Preparation of Protein Lysates FluroTag FITC-labeling kit (Sigma) according to the manufac- Cell Lines. RT112 (human bladder carcinoma cell line) turer’s instruction. The protein concentration of the labeled and HT29 (human colon carcinoma cell line) were grown to antibody was determined by absorbance at 280 nm, and the 75% confluence in RPMI 1640 medium (Sigma, Poole, United FITC labeling ratio was determined by measuring the absorb- Kingdom) containing 10% fetal bovine serum (Sigma) and 20 ance at 490 nm. Stock aliquots of 1 mg/ml were kept at 4°C. mM HEPES at 37°C. The cells were harvested using a flexible ϫ cell scraper, pelleted by centrifugation (600 g, 4°C, 5 min) Western Blotting and washed with ice-cold wash buffer [PBS containing a pro- Samples of exfoliated urothelial cell lysates and tumor cell tease inhibitor cocktail of aminoethylbenzenesulfonyl fluoride, lines, prepared as described above, were subjected to 6% SDS- ␮ 0.4 mg/ml EDTA-Na (0.5 mg/ml), leupeptin (0.5 g/ml), and PAGE under nonreducing conditions. The separated proteins pepstatin (0.5 ␮g/ml) supplied by ICN/Flow (Thame, United 2 were electroblotted (0.8 mA/cm ; 1 h) onto an Immobilon-P Kingdom)]. The cell pellet obtained by centrifugation (600 ϫ g, membrane (Millipore) using Tris-Glycine transfer buffer [48 4°C, 5 min) was resuspended 1:1 (w/v) in lysis buffer [20 mM mM Tris, 39 mM glycine, 0.1% SDS, 20% methanol (pH 9.2)]. tris (pH 8.0), 150 mM NaCl, 20 mM CHAPS, and protease Nonspecific reactions were blocked with TBS containing 5% inhibitor cocktail], snap-frozen in liquid nitrogen, and stored at skimmed milk before the membrane was incubated with mAb Ϫ 80°C until required. Hermes-3 at 4°C overnight and then with peroxidase-conjugated Exfoliated Urothelial Cells. Naturally voided urine antimouse IgG (Sigma; 1/1000 dilution) for 1 h at room tem- specimens were collected from urology out-patient clinics at the perature. All antibodies were diluted in 5% skimmed milk in Oxford Churchill Hospital and processed as described previ- TBS, and after each incubation, the membrane was washed with ϳ ously (19). Briefly, specimens of 50 ml were collected from TBS containing 0.1% Tween 20. Signals were detected by 53 patients with newly diagnosed bladder cancer, 54 patients enhanced chemiluminescence using an enhanced chemilumines- who had previously received treatment for bladder cancer but cence detection kit (Amersham). were presently disease free as assessed by cystoscopy, and from 65 persons with no evidence of disease. (Details of age, sex and clinical status for each of these three groups is summarized Assay Optimization Using Cultured Human Tumor within the results presented in Table 2). Additional specimens Cell Lysates were also investigated from eight patients with benign prostatic To optimize the assay conditions, CD44 protein isoforms hyperplasia and from six with confirmed carcinoma of the were analyzed in cell lysates from two human tumor cell lines. prostate. To assess the effect of hematuria on the assay, 10 We have previously described the presence of CD44 standard patients with macroscopic bleeding and 7 patients with cystic and variant protein isoforms in human bladder (RT112) and stents in situ, prone to microscopic hematuria, were also as- colon carcinoma (HT-29) cell lines (19–21) using Western sessed. The urine was collected into vessels containing protease blotting and immunohistochemistry. Analysis of these cell lines inhibitor cocktail at a final concentration as described above and was used to define the conditions for sample preparation, sample kept on ice during transport to the laboratory. By assaying the concentration, and composition of sample diluent buffer. Neg- inability of urine cell lysate preparations to cleave resofurin- ative controls used for all ELISA analyses were: (a) no capture labeled casein, we have previously documented that complete antibody; (b) no detection antibody; (c) no antigen; and (d) BSA inhibition of protease activity in the urine is obtained using this as nonspecific protein in place of antigen at appropriate con- protocol (28). The exfoliated urothelial cells were subsequently centrations. All data were corrected by subtraction of negative pelleted, washed, and lysed as described above for cell lines. control values. Before the assay, all lysates were thawed and kept on ice Sample Preparation. Incomplete solubilization of mem- for 1 h to ensure complete lysis, and insoluble debris was brane proteins limits the availability of CD44 protein isoforms removed by centrifugation (15,000 ϫ g, 4°C, 30 min). The in the resulting lysate, so incorporation of detergents in sample concentration of total protein was determined (Bio-Rad protein buffers is advisable to maximize solubilization. However, the assay kit) and adjusted to 500 ␮g/ml by dilution with wash choice of detergent is dependent on several factors, including buffer for analysis by ELISA. For Western blot analysis, ali- the intended assay system and the interference of detergents on quots were adjusted to 1 mg/ml and mixed 1:1 (v/v) with antibody epitope binding. To optimize the incorporation of SDS-PAGE gel loading buffer and boiled for 5 min. detergents in the sample lysis buffer, a comparative assay was undertaken on lysates prepared by cell disruption by passage Labeling of Capture and Detection Antibodies through a 21-gauge needle (physical shearing) or with hypotonic mAbs F-10–44-2 [recognizing protein domain encoded by buffer, containing either CHAPS, Tween 20 (1% v/v), or NP40 exon 1 (s1)] and VFF-18 [recognizing protein domain encoded (1% v/v). HT29 cells were harvested as described and split into by exon 11 (v6); both at 1 mg/ml PBS] were biotinylated with four aliquots containing an equal number of cells. The differing Biotin-NHS (Boehringer Mannheim) according to the manufac- efficiency of protein solubilization was clearly observed on turer’s protocol. The labeled antibodies were column-purified measuring total protein within each of the clarified lysates. Lysis (Sephadex G50), and concentration was determined by absorb- buffers containing either CHAPS or NP40 released almost 25 ance at 280 nm. Stock aliquots were diluted to 20 ␮g/ml PBS mg/ml total protein, whereas with Tween 20, this was reduced to and stored at Ϫ20°C. 15 mg/ml. Extraction using detergent buffers but without shear- FITC-labeling of mAb Hermes-3 (1 mg/ml; recognizing ing resulted in protein yields of ϳ3 mg/ml. protein domain encoded by exon 5) was performed using a Analysis of HT29 (100 ␮g/ml total protein) cell lysates by

Fig. 2 Optimization of ELISA. Determination of optimal sample concentration for the evaluation of CD44 protein isoforms. Samples were extracted using CHAPS lysis buffer as described in “Materials and Methods.” Results are mean Ϯ SE of quadruplicate wells from three separate assays.

ELISA demonstrated that similar results could be obtained after lated VFF-18 (epitope encoded by exon 11; Bender Medsystem, extraction of protein using physical disruption or the incorpo- Vienna, Austria). A fourth ELISA was performed using all three ration of CHAPS in the lysis buffer. However, if NP40 or antibodies at 2 ␮g/ml; hence, in each ELISA, the total concen- Tween 20 were used in the lysis buffer, no CD44 could be tration of capture antibodies was 6 ␮g/ml. detected by the ELISA (data not shown). Thus, because CHAPS lysis buffer appeared to have no detrimental effects upon the Optimum ELISA Protocol for Detection of ELISA, yet was superior to physical disruption in releasing total Urothelial Neoplasia protein, this buffer was used in all further studies. One hundred ␮l of ELISA blocking buffer (0.5% protein) Concentration of Sample. Before the assessment of containing biotinylated F-10–44-2 (2 ␮g/ml) and biotinylated CD44 in exfoliated urothelial cells, the optimum sample con- VFF-18 (2 ␮g/ml) were bound to streptavidin-coated microtiter centration was determined by constructing titration curves for plates (Pierce and Warriner, Chester, United Kingdom) for 30 the detection of CD44 in the RT112 and HT29 cell lines. These min at room temperature, with shaking at 250 rpm. All exfoli- studies demonstrated that the optimal sample concentration ated cell lysates (extracted with CHAPS lysis buffer) were should be between 100 and 1000 ␮g/ml total protein (Fig. 2). diluted to a total protein concentration of 500 ␮g/ml, and 10-␮l Concentration of Blocking Protein in ELISA Diluent samples were assayed in triplicate by incubation with shaking Buffer. Initial studies were conducted in ELISA blocking (250rpm) for1hatroom temperature. A single preparation of buffer (Boehringer Mannheim) reconstituted according to the cell line RT112 cell lysate (100 ␮g/ml) was used as a positive manufacturer’s instruction, producing a buffer containing 1% control for all assays, allowing interassay comparison. Detection (w/v) protein. When the data obtained for both RT112 and HT29 of the captured proteins was achieved by incubation with FITC- was compared with the intensity of signal observed in Western labeled Hermes-3 (1 ␮g/ml in blocking buffer) for 1 h followed blotting (data not shown), the absorbance values in the ELISA by peroxidase-labeled mouse anti-FITC antibody (0.15 units in were lower than may have been anticipated, particularly with ELISA blocking buffer) for a further 30 min at room tempera- HT29. A reduction in blocking protein to 0.5% (w/v) resulted in ture with shaking (250 rpm). Plates were washed between each an increase in assay sensitivity without an increase in back- incubation step with four changes of wash buffer (PBS contain- ground signal. Specifically, when diluted with buffer (1% pro- ing 0.05% Tween 20). tein) a maximum absorbance (Ϯ SE) of 0.68 (Ϯ 0.03) was obtained; however, with a reduction to 0.5% protein, the max- RESULTS imum absorbance was 1.53 (Ϯ0.12). The absorbance values for ELISA Design. Previous studies on bladder cancer (19, the negative controls were 0.187 (Ϯ 0.01) and 0.199 (Ϯ 0.02), 21, 22) have indicated which epitopes are most useful for the respectively. detection of CD44 protein isoforms in neoplastic urothelia. On this basis, combinations of mAbs recognizing epitopes encoded Optimization of Multiple-Capture Antibody ELISA by exons 1 and 5 of the standard region and exons 7 and 11 of Triplicate (10 ␮l) RT112 protein extracts (0–1000 ␮g/ml) the variant region were tested in a sandwich-ELISA design for were assessed in an ELISA using either biotinylated F-10–44-2 accurate CD44 detection by comparison with Western blot (6 ␮g/ml) as the sole capture antibody, or at a concentration of profiles from the same samples. 3 ␮g/ml in combination with 3 ␮g/ml biotinylated mAb 23.6.1 Comparison of ELISA with Western Blot Profiles. To (epitope encoded by exon 7; Ref. 19), or with 3 ␮g/ml biotiny- validate the reliability of ELISA results, data obtained by this

Fig. 3 Optimization of ELISA. Evaluation of the efficacy of various combinations of capture antibodies in detecting CD44 protein isoforms extracted from the RT112 bladder tumor cell line. Samples were extracted using CHAPS lysis buffer as described in “Materials and Methods.” Re- sults are mean Ϯ SE of quadruplicate wells from three separate assays.

Table 1 Inter- and Intra-assay variability for the multiple capture ELISAa Well No. Plate 12345678910Mean CVb % 1 1.26 1.31 1.31 1.28 1.29 1.30 1.29 1.28 1.27 1.29 1.288 1.62 2 1.39 1.32 1.34 1.35 1.34 1.37 1.35 1.33 1.35 1.32 1.346 2.17 3 1.45 1.43 1.38 1.39 1.41 1.42 1.37 1.37 1.44 1.41 1.407 2.87 4 1.28 1.29 1.34 1.33 1.35 1.32 1.31 1.33 1.29 1.30 1.314 2.37 5 1.41 1.40 1.40 1.38 1.37 1.45 1.39 1.35 1.38 1.37 1.390 2.75 Mean intra-assay 2.36 Mean interassay 1.349 4.97 a Samples, RT112 bladder tumor cell line Ϫ total protein concentration ϭ 100 ␮g/ml. b CV, coefficient of variation.

technique were compared with levels of CD44 overexpression several samples exhibiting a characteristic range of high visualized by Western blotting. Naturally voided exfoliated molecular weight CD44 isoforms (19) presented low absorb- urothelial cells were obtained from 16 patients with no malig- ance values in the ELISA. It appeared that this ELISA design nant disease and from 17 patients with histologically confirmed was unable to detect a significant proportion of the samples, bladder cancer, and cellular protein was extracted as described which by clinical examination/histology and Western blot above. Western blots were performed with the Hermes-3 (exon analysis, displayed malignant features. A possible explana- 5 epitope) antibody as described previously (19). Sandwich tion for this discrepancy was the presence of large variant ELISA was conducted using the F10.44.2 (exon 1 epitope) as isoforms in tumor samples not being effectively captured capture antibody and Hermes-3 as the detection antibody. The onto the surface of the microtiter plate. To examine this protein concentration in each assay was 500 ␮g/ml. possibility, we investigated the efficacy of using a multiple- The Western blot profile of these samples was as described capture antibody modification of the ELISA. previously (19). Both nonmalignant and malignant samples ex- Multiple-Capture Antibody Protocol. We reasoned pressed standard form CD44, as demonstrated by the presence of a that the binding of large CD44 isoforms by multiple-capture ϳ Mr 85,000 isoform. Malignant samples also exhibited multiple antibodies should reduce the “shearing” of the molecule from ϳ protein isoforms in the range of Mr 150,000–200,000, a pattern the plate and provide a more reliable measure of the analyte. indicating the overproduction of variant CD44 isoforms. Samples Accordingly, additional capture antibodies were selected for from normal counterparts did not exhibit this pattern. testing on the basis of our previous studies showing that ele- Statistical analysis of the data obtained using this sand- vated expression of CD44 exon 7 (v2) and exon 11 (v6) occurs wich-ELISA protocol did reveal significant difference (P Ͻ in many bladder tumors (19, 21, 22). The epitope recognized by

0.005) between the patient groups (data not shown). How- F-10–44-2 is at the NH2 terminal end of CD44: therefore, the ever, comparison with the Western blot profiles revealed that incorporation of capture antibodies recognizing these variant

Table 2 Results of CD44 ELISA analysis of urine samples from bladder cancer patients either currently presenting with a tumor or in remission compared with control volunteers Control group (no malignancy) Mean age, 55.4 yr, 14 F (21.5%) Mean, 0.270 Total, 65 cases Age range, 23–86 yr 51 M (78.5%) SE, 0.010 Tumor group Patient No. Age Sex Clinical status ELISA absorbance T558 72 M pTaG1L0 0.492 T561 82 M pT1G1L0 0.440 T563 63 F pT2G3L0 0.860 T567 77 M pT1G2L0 0.499 T568 86 M pTaG1L0 0.271 T571 62 M pT1G2L0 0.664 T573 75 M pT1G1L0 0.508 T574 36 M pT1G2L0 0.597 T577 28 M pT2G1L0 0.596 T580 80 M pT1G2L0 0.919 T581 75 M pT2G3L0 0.769 T583 69 M pT1G1L0 0.353 T585 76 M pT1G1L0 0.332 T586 78 F pT1G2L0 0.547 T592 85 F pTaG1L0 0.502 T604 86 M pT1G1L0 0.532 T611 58 M pTaG1L0 0.437 T613 83 M PT1G1L0 0.499 T614 37 M pT2G2L0 0.474 T618 65 M pT1G2L0 1.079 T624 75 M Severe dysplasia 0.451 T633 54 F pT1G1L0 0.406 T655 36 F pT2G2L0 1.497 T684 77 M pTaG1L0 0.267 T700 63 M TCCa (no stage/grade) 0.805 T703 75 M pTaG1L0 0.561 T705 72 F Severe dysplasia 0.267 T713 67 F pT1G2L0 0.692 T714 64 M pT1G1L0 0.362 T716 52 M pT1G2L0 0.676 T717 58 F pTaG1L0 0.208 T725 52 M pT1G2L0 0.431 T729 68 M pT2G2L0 1.112 T730 69 M pTaG2L0 0.265 T731 57 M pTaG1L0 0.246 T733 48 M pTaG2L0 0.548 T735 56 M pT1G2L0 0.524 T736 73 M pT2G2L0 0.647 T741 62 M pT1G1L0 0.527 T743 74 M pT2G3L0 0.770 T747 77 M pT1G2L0 0.613 T750 72 M pT1G2L0 0.438 T754 79 M pT2G3L1 0.456 T761 71 M pT1G2L0 0.888 T762 73 M pTaG1L0 0.306 T764 83 M pT1G2L0 0.345 T766 80 F pT1G1L0 0.857 T769 59 M pT1G1L0 0.459 T771 68 F pT1G2L0 0.880 T772 73 M pT2G3L0 0.761 T780 51 M Severe dysplasia 0.424 T792 82 F pT1G1L0 0.498 T797 57 M pTaG1L0 0.445 Total 53 cases Mean age, 70 yr, 11 F (20.8%) Mean, 0.566 Age range, 28–86 yr 42 M (79.2%) SE, 0.034

Table 2. Continued. Remission group (currently tumor-free) No. Age Sex Clinical status ELISA absorbance P566 56 F Remission (pT2G2) 0.449 P589 58 F Remission (pT1G1) 0.382 P590 57 M Remission 0.297 P615 58 M Remission 0.302 P632 57 M Remission 0.359 P636 73 M Remission (pTaG2) 0.304 P649 75 F Remission (pT1G1) 0.226 P650 65 M Remission 0.484 P651 61 M Remission 0.219 P653 52 M Remission (pTaG1) 0.202 P663 54 M Remission (pT2G2) 0.209 P664 43 M Remission (pT1G1) 0.259 P666 58 F Remission (pT1G1) 0.233 P667 56 M Remission 0.596 P671 58 F Remission 0.390 P672 69 M Remission (pT1G1) 0.202 P674 72 M Remission (pT1G2) 0.292 P675 60 M Remission (pTaG1) 0.363 P677 72 M Remission (pT1G1) 0.396 P681 78 M Remission (pT1G2) 0.306 P683 58 M Remission (pT2G3) 0.429 P684 67 M Remission 0.613 P685 83 M Remission 0.250 P686 65 M Remission (pT1G2) 0.379 P687 80 F Remission (pT1G1) 0.184 P701 74 M Remission (pT1G1) 0.388 P702 52 M Remission 0.227 P708 73 M Remission (pT1G2) 0.248 P709 59 M Remission 0.654 P711 55 M Remission (pT2G2) 0.262 P712 52 F Remission (pT1G2) 0.409 P718 65 M Remission 0.298 P721 59 F Remission (pT1G1) 0.398 P722 53 M Remission 0.340 P723 83 M Remission 0.271 P724 69 M Remission (pT1G1) 0.217 P726 52 F Remission (pT1G1) 0.401 P728 63 M Remission (pT1G2) 0.319 P737 55 M Remission 0.239 P739 84 M Remission 0.266 P744 57 F Remission 0.288 P745 52 M Remission 0.256 P746 73 M Remission (pT1G2) 0.299 P748 77 M Remission (pT1G2) 0.359 P749 55 M Remission (pT1G1) 0.279 P751 64 M Remission 0.375 P752 70 M Remission 0.396 P753 86 M Remission (pT2G2) 0.514 P773 68 M Remission (pT2G3) 0.323 P774 51 F Remission 0.650 P775 57 M Remission (pT1G2) 0.302 P777 69 M Remission (pT1G2) 0.396 P778 63 M Remission 0.859 P779 66 M Remission 0.368 Total, 54 cases Mean age, 63.7 yr, 11 F (20.4%) Mean, 0.380, Range, 43–86 yr 43 M (79.6%) SE, 0.018 a TCC, transitional cell carcinoma.

epitopes would give additional anchorage by binding at a car- were performed, as described in “Materials and Methods.” All boxy-proximal position (Fig. 1). other components of the ELISA were unchanged. To investigate whether the use of a multiple antibody Fig. 3 presents the titration curve for the detection of CD44 capture protocol could increase reliability of identification of isoforms under various assay conditions using total protein tumor cells, ELISAs containing one to three capture antibodies extracted from the RT112 cell line. The sensitivity of the assay

Table 3 Summary data and statistical assessment of the evaluation of CD44 protein expression in exfoliated urothelial cells using the multiple- capture ELISA protocol Clinical statusa No. of cases Mean absorbance SE Significance Normal 65 0.270 0.010 Not applicable Tumor 53 0.566 0.035 P Ͻ 0.005 Remission 54 0.390 0.018 P Ͻ 0.005 BPH 8 0.418 0.055 Not determined Prostate tumor 7 0.389 0.047 Not determined Frank hematuria 19 0.545 0.066 Not determined Stent in 9 0.641 0.090 Not determined a Normal, no urological malignancy; tumor, histologically confirmed transitional cell carcinoma of the bladder; remission, currently tumor-free having had a transitional cell carcinoma of bladder treated previously; BPH, benign prostatic hyperplasia; prostate tumor, histologically confirmed prostate tumor; frank hematuria; macroscopic hematuria; stent in, patient has urinary stent in place when sample was obtained.

was modulated by altering the combination of capture antibod- Further analysis of these data using a one-tailed Student’s ies used. Maximum sensitivity was achieved using F-10–44-2 t test (data normalized by Box-Cox root transformation) showed (3 ␮g/ml) ϩ VFF-18 (2 ␮g/ml) as capture antibodies, manifest that the ELISA values for the samples obtained from patients by a shift of the titration curve to the right compared with that with confirmed bladder cancer were significantly greater than obtained using F-10–44-2 alone. The combination of using the values obtained with either nonmalignant samples (P Ͻ F-10–44-2 with mAb 23.6.1 was less sensitive than with VFF- 0.005). Although the mean absorbance value obtained from 18. When a sample total protein concentration of 200 ␮g/ml was patients who were presently in remission was significantly assayed, an absorbance of ϳ1.00 was measured, whereas the lower than those with a present tumor (P Ͻ 0.005), it was, F10–44-2/VFF-18 capture antibody combination gave an ab- however, significantly higher than that of the normal control ϳ sorbance of 2.4 (Fig. 3). group (P Ͻ 0.005). When the malignant and nonmalignant exfoliated urothe- To investigate the diagnostic potential of these data, a lial cell samples were reassessed using the F-10–44-2/VFF-18 “gate” was imposed at N (max), i.e., the highest absorbance capture-Hermes-3 detection ELISA, the malignant samples gave value obtained from a sample known to be tumor-free. All higher ELISA values in the new assay and compared more values above this value were then assumed to be indicative of a closely with the Western blot profile of the same sample. There tumor, whereas all those below were taken to be tumor-free. was no corresponding increase in the ELISA values of the Using this parameter, the assay is 100% specific for first-time nonmalignant samples with the improved multiple capture an- tumor detection, with a sensitivity of 81.1%. Presented in a more tibody assay, and background values remained comparably low. conventional form, the positive predictive value for this assay Intra- and Interassay Variation of the Multiple- was 100%, and the negative predictive value was 81.1%. Capture ELISA. Before a detailed assessment of the ability The values for specificity and sensitivity obtained by of the multiple-capture ELISA to discriminate between malig- ELISA are similar to those we obtained previously using West- nant and nonmalignant bladder disease, the intra- and interassay ern blot analysis alone (19). To assess the correlation of diag- variability was determined using the RT112 cell line. nosis with the two assays, all samples were analyzed by Western Ten (10 ␮l) RT112 protein extracts (100 ␮g/ml) were blotting and the F-10–44-2/VFF-18/Hermes-3 ELISA. Fig. 5 is assessed in five separate assays. The results are summarized in a representative sample of the comparative data for six normal Table 1. These results show that the assay is reliable and reproducible with a mean intra-assay variability of 2.36% and a and six malignant samples. The diagnostic “end point” for the mean interassay variability of 4.97%. Western blot analysis was the presence or absence of isoforms Ͼ Detection of CD44 Standard and Variant Isoforms in with an apparent molecular weight of Mr 150,000 (intensity of Exfoliated Urothelial Cells. Using the F-10–44-2/VFF-18/ bands was not determined), whereas for the ELISA, the “end Hermes-3 multiple-capture ELISA, the presence of CD44s point” was a quantitative measure as described above. When the and CD44v isoforms was determined in 53 malignant and 65 same urothelial cell samples were analyzed by both Western nonmalignant samples of exfoliated urothelial cells. Addi- blotting and the F-10–44-2/VFF-18/Hermes-3 ELISA, the tionally, samples were assayed from 54 patients who had larger the number, size, and range of CD44 isoforms detected by previously had a bladder tumor, but were presently clinically Western blotting, the higher the absorbance values obtained believed to be clear of any malignant disease. Tables 2 and 3 with the ELISA. Significantly, all samples deemed to be posi- along with Fig. 4 summarize the ELISA values obtained for tive by Western blot were also positive by ELISA, whereas each sample (mean of triplicates after subtraction of negative those that were negative in the one system were also negative in control values). The mean absorbance (ϮSE) value for sam- the other. ples from patients with confirmed tumors was 0.566 Ϯ 0.035 During the course of this study, a limited number of sam- compared with 0.270 Ϯ 0.01 in the group with no malignant ples from patients with benign prostatic hyperplasia or carci- disease. In the group that had previously presented with noma of the prostate were also assessed, but no striking evi- bladder tumors but was presently disease-free, the mean dence of elevated CD44 was observed in the urine from patients absorbance was 0.390 Ϯ 0.018. with either condition (Table 3).

Fig. 4 Evaluation of CD44 protein expression in exfoliated urothelial cells using a sandwich ELISA. Detection of CD44 protein isoforms in exfoliated urothelial cells from patients with histologically confirmed bladder tumors or currently in remission and clinically adjudged to have no urological malignancy and compared with samples from healthy volunteers with no known malignancy. A limited number of samples were also evaluated from patients with benign prostatic hyperplasia, prostatic cancer, frank hematuria, and with a urinary stent in situ. Capture antibodies, F-10–44-2 (3 ␮g/ml) ϩ VFF-18 (3 ␮g/ml); detection antibody: Hermes-3 (1 ␮g/ml); sample, 100 ␮g/ml total protein. Results are mean Ϯ SE of significantly elevated mean compared with normal group (one-tailed t test on normalized ,ء .triplicate samples measured on two separate occasions data; P Ͻ 0.005).

Effect of Hematuria on the CD44 ELISA. Frank he- ence of bladder cancer. The presence of hematuria can interfere maturia interferes with the detection of CD44 isoforms (13, 19) with the assay, but this does not limit the potential clinical value and Fig. 4. This does not reduce the clinical value of assays of the test because direct cystoscopic investigation of patients based upon this molecule because patients presenting with he- presenting with this symptom is imperative. However, the fea- maturia would routinely be investigated by cystoscopy. How- sibility of noninvasive diagnosis of bladder cancer by examina- ever, we considered it prudent to examine whether microscopic tion of naturally micturated urine is attractive because it elimi- hematuria could interfere with the assay. Samples of urine (50 nates investigative discomfort, thereby increasing the likelihood ml) from a healthy volunteer with no urological disease were of patient compliance with diagnostic procedures. The develop- “spiked” with 0–2000 ␮l of whole peripheral blood, and the ment of this ELISA was driven by such needs, and now that a presence of CD44s and CD44v was determined using the proof of principle has been demonstrated, more comprehensive ELISA described above. It was found that the addition of up to patient-based studies will be undertaken. Urinary CD44 levels in ␮ 750 l of whole blood to 50 ml of urine had no significant effect samples collected from larger groups of urology patients will be on the absorbance values obtained (Table 4). After the addition correlated with further clinical parameters now that this ELISA ␮ of 750 l of blood, the resultant lysate has a light pink color enables high throughput analyses to be handled. Repeat sam- indicative of macro- as opposed to microscopic hematuria. With pling regimes and sampling before and after surgical or thera- ␮ up to 1250 l of blood, an increase in absorbance was observed. peutic intervention will also be focused on. ␮ Above 1250 l, the absorbance values declined to below control Several new markers for analysis of voided urine have values obtained with normal exfoliated urothelial cells alone. recently been approved for clinical use, including bladder tumor This phenomena may be related to total cell numbers in the antigen (29, 30), nuclear matrix protein 22 (NMP22; Refs. 31 assayed sample. Spiking the 50-ml sample with 1–1.25 ml of and 32), and fibrin/fibrinogen degradation products (33). Such whole blood may increase the absorbance of the sample because markers can help to detect clinically occult bladder cancer and of CD44 release and/or absorbent molecules such as haem. can increase the interval of cystoscopic evaluation. However, Beyond that, the increased cell number in the centrifuged pellet although these assays are more accurate than urine cytology, may either interfere with efficient protein extraction, or high they do have problems of low sensitivity and specificity, espe- concentrations of released factors may directly inhibit the bind- cially in detection of low-grade tumors (30, 32, 34). In view of ing of soluble ligand in the assay. the continuing need for a reliable, noninvasive test for bladder cancer, a number of alternative molecular markers are under DISCUSSION evaluation. These include the Lewis-X antigen (35), cytokeratin The data obtained in this investigation demonstrate that the 20 (36, 37), microsatellite analysis of urine DNA (38), and measurement of CD44 isoforms in protein lysates of naturally measurement of telomerase activity (28, 39, 40). The suitability exfoliated cells in urine by ELISA can reliably detect the pres- of these assays for routine diagnostic practice awaits further

Fig. 5 Comparative analysis of normal and malignant exfoliated urothelial cells by West- ern blot and multiple-capture ELISA. All samples were analyzed by Western blot using the Hermes-3 antibody and by sandwich ELISA in triplicate on two separate occasions. The blot and mean ELISA values obtained from six tumor and six normal samples are presented. For both assays, the sample concentration ϭ 100 ␮g/ml total protein. Arrows, the position of protein size markers (kDa).

investigation. Widespread use of molecular assays could lead to Table 4 Effect of whole blood contamination on performance of the reduction in the burden of cystoscopic investigations for pri- multiple capture ELISA mary and recurrent disease. The introduction of cheap reliable Equal numbers of RT112 bladder tumor cells were “spiked” with assays in routine practice would also lead to improvement of volume of whole blood as indicated before protein extraction. Samples early detection of the disease even in asymptomatic individuals. were then handled and assayed as indicated in the text. Sample, 100 ␮g/ml total protein. Results were analyzed using a one-sided t test to Previously, using immunohistochemistry, Western blot- determine whether “spiking” significantly elevated the absorbance val- ting, and RT-PCR (13, 19), we reported that inappropriate CD44 ues as compared with nonspiked controls. protein is detectable in exfoliated urothelial cells. These studies “Spiked” clearly demonstrated that the detection of abnormal CD44 gene blood volume Mean expression in exfoliated cells in naturally voided urine could be (␮L) Absorbance SE Significance used to identify patients with bladder cancer with a high level of 0 1.921 0.080 Not applicable accuracy (13, 19). RT-PCR/Southern blot hybridization analy- 250 2.011 0.091 NSE ses had a specificity of 91% and a sensitivity of 83%. Western 500 2.043 0.102 NSE blot assays recorded values of 100% specificity and 75% sen- 750 2.003 0.095 NSE 1000 2.458 0.102 P Ͼ 0.005 sitivity for the detection of bladder tumors. However, both are 1250 2.851 0.110 P Ͼ 0.005 technically demanding and time-consuming with many steps 1500 1.521 0.045 Not determined that are inappropriate for use in routine laboratories, including 2000 1.070 0.062 Not determined electrophoresis, hybridization protocols, and autoradiography. NSE, not significantly elevated. In contrast, the ELISA method produces an unequivocal digital result that can define normal ranges and diagnostic thresholds. There are a number of parameters that must be optimized posed to normal exfoliated urothelial cells (19). Such refinement for the deployment of an ELISA. Our initial studies using mAb of the assay resulted in a 66% increase in sensitivity as deter- F-10–44-2 for capture and mAb Hermes-3 for detection clearly mined by the absorbance value obtained with 200 ␮g/ml protein showed that the diversity of CD44 isoforms generated by alter- extracted from the RT112 bladder tumor cell line, while retain- native splicing complicates the design of a consistently reliable ing a low intra- and interassay variation. assay. For this reason, we devised the ELISA described above The F-10–44-2/VFF-18/Hermes-3 ELISA was found to be using Western blot analysis as a visual index of the overall level an accurate yet simple tool for the noninvasive detection of of CD44 expression in a given sample for purposes of stand- bladder tumors. On a sample of 53 patients with newly diag- ardization. This led to the recognition that introduction of a nosed bladder cancer and 65 persons with no evidence of second capture antibody would prove useful in tailoring the malignant disease, this ELISA resulted in a specificity of 100%, assay to detect a large and complex molecule. This improvement a sensitivity of 80.2%, and a positive predictive value of 100% was interpreted to be attributable to the availability of additional for the presence of bladder cancer. These values surpass those “anchorage” sites and to the overexpression of CD44 isoforms reported for almost all molecular markers in bladder tumor containing epitopes encoded by exon 11 in malignant as op- detection.

Although CD44 expression was found to be significantly gous to the W3/13 antigen of the rat. Eur. J. Immunol., 10: 745–750, lower in the “remission” group compared with those with con- 1980. firmed tumors, the mean ELISA values for those patients who 4. Jalkanen, S., Jalkanen, M., Bargatz, R., Tammi, M., and Butcher, were at that time tumor-free are significantly greater than in E. C. Biochemical properties of glycoproteins involved in lymphocyte recognition of high venules in man. J. Immunol., 141: 1615–1623, 1987. “normal” subjects. This raised “basal” level of CD44 expression in such patients may be attributable to “field-change” effects, 5. Lesley, J., Hyman, R., and Kincade, P. W. CD44, and its interaction with extracellular matrix. Adv. Immunol., 54: 271–335, 1993. which are known to occur throughout the transitional urothelium 6. Rudzki, Z., and Jothy, S. CD44 and the adhesion of neoplastic cells. in cancer patients. Although a significant difference was ob- Mol. Pathol., 50: 57–71, 1997. served between the mean ELISA values for the “remission” and 7. Lesley, J., and Hyman, R. CD44 structure, and Function. Front. tumor-bearing groups, the test still accurately discriminated Biosci., 3: D616–D630, 1998. between individuals with and without bladder cancer. 8. Gunthert, U., Hofmann, M., Rudy, W., Reber S., Zoller, U., Hauss- In this study, we observed that macroscopic hematuria, in mann, I., Matzku, S., Wenzel, A., Ponta, H., and Herrlich, P. A new the absence of a bladder tumor, elevates the mean ELISA variant of glycoprotein CD44 confers metastatic potential to rat carci- absorbance values. We therefore investigated the influence of noma cells. Cell, 65: 13–14, 1991. trace amounts of blood in urine samples by “spiking” the urine 9. Rudy, W., Hofmann, M., Schwartz-Albiez, R., Zoller, M., Heider, K. H., Ponta, H., and Herrlich, P. The two major CD44 proteins with increasing amounts of whole blood. It was found that expressed on a metastatic rat tumor cell line are derived from different hematuria had no effect on the assay until the blood became splice variants: each one individually suffices to confer metastatic visible and by definition macroscopic. In such cases, immediate behavior. Cancer Res., 53: 1262–1268, 1993. cystoscopy would be indicated regardless of the status of the 10. Heider K-H., Hofmann, M., Hors, E., van der Berg, F., Ponta, H., molecular assay. The use of stents to aid urinary flow is com- Herrlich, P., and Pals, S. T. A human homologue of the rat metastasis- mon in cases of urinary obstruction, and assessment of seven associated variant of CD44 is expressed in colorectal carcinomas and patients with stents in situ revealed that this procedure can lead adenomatous polyps. J. Cell Biol., 120: 227–233, 1993. to an elevation in ELISA values in patients without tumor. We 11. Matsumura, Y., and Tarin, D. Significance of CD44 gene products for cancer diagnosis and disease evaluation. Lancet, 340: 1053–1058, hypothesize that the presence of indwelling stents causes in- 1993. creased desquamation of bladder epithelium, resulting in ele- 12. Tanabe, K. K., Ellis, L. M., and Saya, H. Expression of Cd44R1 vated CD44 levels caused by a raised turnover of basal cells. It adhesion molecule in colon carcinomas and metastasis. Lancet, 341: should be noted that irritation and/or inflammation per se, such 725–726, 1993. as is common after bacillus Calmette-Gue´rin therapy, did not 13. Matsumura, Y., Hanbury, D., Smith, J., and Tarin, D. Non-invasive significantly interfere with the assay. detection of malignancy by identification of unusual CD44 gene activity One of the primary target groups for a noninvasive test in exfoliated cancer cells. Br. Med. J., 308: 619–624, 1994. such as this ELISA would be those with early recurrence. 14. 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Anthony C. Woodman, Steve Goodison, Marcus Drake, et al.

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