EUF-224; No. of Pages 7 E U R O P E A N U R O L O G Y F O C U S X X X ( 2 0 1 6 ) X X X – X X X ava ilable at www.sciencedirect.com journa l homepage: www.europeanurology.com/eufocus Bladder Cancer A Novel Urine-Based Assay for Bladder Cancer Diagnosis: Multi-Institutional Validation Study a, b c d e f Noa Davis *, Alexander Shtabsky , Sylvia Lew , Ronny Rona , Ilan Leibovitch , Ofer Nativ , g h i a j Michael Cohen , Yoram Mor , Uri Lindner , Yael Glickman , Haim Matzkin , k l m Alexander Tsivian , Ofer Gofrit , Ofer Yossepovitch a b Micromedic Technologies Ltd., Tel Aviv, Israel; Institute of Pathology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel c d e f Aviv, Israel; Patho-Lab Diagnostics Ltd., Ness Ziona, Israel; Kfar Saba, Israel; Department of Urology, Meir Medical Center, Kfar Saba, Israel; Department g h of Urology, Bnai Zion Medical Center, Haifa, Israel; Department of Urology, HaEmek Medical Center, Afula, Israel; Department of Urology, Sheba Medical i j Center, Tel Aviv, Israel; Department of Urology, Kaplan Medical Center, Rehovot, Israel; Department of Urology, Sourasky Medical Center, Tel Aviv, Israel; k l m Department of Urology, Wolfson Medical Center, Holon, Israel; Department of Urology, Hadassah Medical Center, Jerusalem, Israel; Department of Urology, Rabin Medical Center, Petach Tikva, Israel Article info Abstract Article history: Background: CellDetect is a unique histochemical stain enabling color and morphological discrimination between malignant and benign cells based on differences in metabolic signature. Accepted October 11, 2016 Objective: The objective of the present study was to validate the performance of this assay in a controlled, blinded, multicenter study. Associate Editor: Design, setting, and participants: The study, conducted in nine hospitals, included patients James Catto with documented history of bladder cancer, monitored for urothelial carcinoma (UCC) or scheduled for bladder cancer surgery. Outcome measurements and statistical analysis: Cystoscopy and/or biopsy were used as a Keywords: reference standard to determine sensitivity and specificity. Smears were stained by CellDetect Cancer and interpreted by two cytologists blinded to the patient’s final diagnosis. The findings were Urine cytology compared with those of standard urine cytology and BTA stat. Results and limitations: Two hundred and seventeen voided urine specimens were included. Urothelial cell carcinoma Ninety-six (44%) were positive by histology and 121 (56%) were negative by either cystoscopy or UCC histology. The overall sensitivity of CellDetect was 84%. Notably, the sensitivity for detecting low- Urinary bladder grade nonmuscle-invasive bladder cancer tumors was greater than this of BTA stat (78% vs 54%) CellDetect and more than two-fold higher compared with standard cytology (33%, p 0.05). The specificity was 84% in patients undergoing routine surveillance by cystoscopy. At a median follow-up of 9 mo, 21% of the patients with positive CellDetect and negative reference standard developed UCC, which was significantly higher compared with the 5% of the true negative cases. Limitations include the lack of instrumental urine samples and the lack of patients with nongenitourinary cancers in the study population. Conclusions: This study validates the performance of CellDetect as a urine-based assay to identify UCC in patients with history of bladder cancer. The high sensitivity was maintained across all cancer grades and stages without compromising the assay specificity. Further studies are required to test whether this novel stain can be incorporated in routine bladder cancer surveillance as a noninvasive alternative to cystoscopy. Patient summary: Surveillance of bladder cancer requires frequent invasive procedures. In the present study, we validate the ability of a novel biomarker to accurately identify early-stage tumors in urine specimens for the noninvasive monitoring of patients with history of bladder cancer. # 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Micromedic Technologies Ltd., Kiryat Atidim, Tel Aviv 6158101, Israel. Tel. +972-732753427; Fax: +972-73-2753451. E-mail address: [email protected] (N. Davis). http://dx.doi.org/10.1016/j.euf.2016.10.004 2405-4569/# 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved. Please cite this article in press as: Davis N, et al. A Novel Urine-Based Assay for Bladder Cancer Diagnosis: Multi-Institutional Validation Study. Eur Urol Focus (2016), http://dx.doi.org/10.1016/j.euf.2016.10.004 EUF-224; No. of Pages 7 2 E U R O P E A N U R O L O G Y F O C U S X X X ( 2 0 1 6 ) X X X – X X X 1. Introduction Adult patients monitored for UCC were consecutively enrolled if they had a documented history of UCC, if at least 4 wk had passed since any treatment or procedure for UCC was performed, and if they were able to Urothelial cell carcinoma (UCC) is the most common provide a spontaneous urine sample. Patients with catheters, neoblad- malignancy in the urinary system [1], with a worldwide der, ileal conduit, or kidney stones, patients under any other cancer prevalence of 2.7 million patients [2] and an annual treatment, or patients with suspicious/positive cystoscopy without a incidence of 429 800 new cases [3]. With up to an 80% subsequent biopsy were excluded. No further selection was performed recurrence rate, UCC often requires a lifelong routine in these patients. surveillance depending on disease severity [4], and is thus Voided urine samples were collected from a first cohort of patients considered one of the most costly cancers in terms of undergoing routine cystoscopic surveillance. To enrich the study with lifetime expenditure per patient [5,6]. Cystoscopy remains positive cases, a second cohort of patients, scheduled for transurethral the reference standard for UCC diagnosis and management, resection (TURBT) or radical cystectomy, was enrolled as well. The notwithstanding its invasive nature and relatively high cost patients from both cohorts had a documented history of bladder cancer. A minimum of 50-ml voided urine was collected from each [7]. The need for noninvasive, accurate, cost-effective participant. For urinalysis, Multistix 10SG (Siemens Healthcare GmbH, markers for UCC surveillance is evident. Erlangen, Germany) was used. Fresh specimens were tested with the Urine cytology is the most frequently used, noninvasive predicate Food and Drug Administration-approved BTA stat (Polymedco assay for UCC detection [8]. However, its low sensitivity, Inc., Cortlandt Manor, NY, USA) according to manufacturer’s instructions particularly in low-grade tumors, remains a major hurdle and an aliquot was separated subsequently for standard urine cytology. [9]. Over the past 2 decades, additional urinary markers The remaining urine volume was treated with a designated fixative and have been developed [10–12]; however, none of them have preserved at 4 8C. Samples were then processed to cytocentrifuge smears been proven sufficiently accurate and cost-effective to be in a central lab and fixed in 96% ethanol. integrated in routine patient management [13]. CellDetect is a novel histochemical staining platform 2.2. Staining allowing color and morphological discrimination between normal and neoplastic cells [14,15]. The discriminative Smears were stained by CellDetect according to manufacturer’s instruc- capacity of the stain is assumingly related to the increased tions (Zetiq Technologies Ltd., Tel Aviv, Israel). Briefly, the CellDetect kit contains a proprietary plant extract and two histological dyes. The metabolic activity inherent in cancer cells. This so called staining procedure includes fixation with 10% trichloroacetic acid, nuclear Warburg Effect, characterized by the aerobic glycolysis of staining with hematoxylin followed by differentiation in HCl/ethanol, malignant tumors, is commonly used in cancer diagnostics conditioning with the plant extract, staining with the red dye, such as tumor imaging using labeled glucose analogues (ie, differentiation in acetic acid/ethanol, and staining with the green dye. positron emission tomography-computed tomography). With CellDetect, the nucleus of the malignant cell is stained in red. CellDetect is composed of a unique plant extract (Ficus The cytoplasm of cancer cells is often stained in pink, especially when elastica) and generic dyes. The active component of the plant cells are arranged in clusters. Normal urothelial or squamous epithelial extract was found to be a member of the proantocyanidin cells typically have a dark purple or green nucleus and greenish family, a class of polyphenols present in a variety of plants. cytoplasm. Inflammatory cells are stained in purple/red, and are While the molecular mechanism is not yet fully elucidated, a distinguishable based on their morphology. series of experiments performed on multiple cell lines All CellDetect stained smears were assessed under a light microscope (Olympus Life Science Solutions, Center Valley, PA, USA) by two showed that the purified active component interacts independent cytologists. Five optional results were assigned: negative, specifically with proteins found in malignant cells, thus reactive/inflammatory, suspicious, highly suspicious, and positive for leading to their specific labeling with the red dye. Benign UCC. These options were implemented based on a practice routinely epithelial cells are counter-stained in green (Fig. 1). In