A NOVEL URINE CYTOLOGY STAIN FOR THE DETECTION AND MONITORING OF UROTHELIAL CARCINOMA. Running head: A Novel Stain For The Detection Of Bladder Cancer

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1 A NOVEL URINE CYTOLOGY STAIN FOR THE DETECTION AND MONITORING OF UROTHELIAL CARCINOMA Authors Noa Davis 1, Yoram Mor 4, Pavel Idelevich 1, Dov Terkieltaub 1, Vivi Ziv 1, Adi Elkeles 1, Sylvia Lew 2, Elimelech Okon 3, Menachem Laufer 4,Jacob Ramon 4, Daniel Kedar 5, Jack Baniel 5, Ofer Yossepowitch 5 1 Zetiq Technologies Ltd. 2 Patho-Lab Diagnostics Ltd, Ness Ziona, Israel 3 LEM Pathology Laboratory, Ness Ziona, Israel 4 Department of Urology, The Chaim Sheba Medical Center, Ramat-Gan, Israel 5 Institute of Urology, Rabin Medical Center, Petach-Tikva, Israel Running head: A Novel Stain For The Detection Of Bladder Cancer Correspondence: Noa Davis Micromedic Technologies Ltd. Kiryat Atidim, Building 3, 5th Floor Tel Aviv Mobile: Fax: noa@m-medic.com Key Words: Cancer, Urine cytology, Urothelial carcinoma, Urinary bladder, CellDetect

2 Abstract Purpose: CellDetect is a unique platform technology which enables color discrimination between malignant (red) and benign (green) cells based on specific metabolic alterations exclusive to the former. Pre-clinical studies and clinical trials have demonstrated applicability of CellDetect in a plethora of cell culture lines and in various cancers. The purpose of the current study was to explore the performance characteristics of CellDetect in bladder cancer. Materials and Methods: An open-label, two-step study conducted in tertiary medical centers enrolling patients with newly diagnosed or history of urothelial carcinoma (UC). The first step of the study involved staining of archived biopsy. Slides were evaluated by 2 independent pathologists and the concordance between CellDetect and H&Ebased diagnosis was determined. The second step included staining of urine specimens by CellDetect and comparing the findings to the patients final diagnosis and the results of standard urine cytology. Results: A total of 58 archived biopsies were collected. The concordance between the CellDetect staining and H&E-based diagnosis was 100%. CellDetect was applied to 44 urine smears demonstrating 94% sensitivity and 89% specificity in detecting UC. Compared to standard urine cytology, CellDetect demonstrated overall superior sensitivity (94% versus 46%, respectively, P<0.005), particularly in low grade tumors (88% versus 17%, P<0.005). There was no significant difference in specificity between the 2 staining techniques Conclusion: The findings demonstrate the capability of CellDetect in accurately identifying UC, indicating that this technology can be further developed to provide an alternative urine cytology test with diagnostic values that may have significant clinical benefits. 1

3 Introduction Bladder cancer is the fourth most common cancer in males, and the eighth most common cause of cancer death [1], with an estimated incidence of 72,000 new cases and mortality rate of 15,000 in 2013 in USA [1]. For those diagnosed with nonmuscle invasive tumors, there is up to 80% chance of tumor recurrence [2], rendering bladder cancer as one of the most prevalent malignancies. Clinical guidelines recommend that patients with stages Ta, Tis, or T1 bladder cancer should be followed with cystoscopy every 3 months for the first 2 years after tumor resection, semiannually during the subsequent 2 years, and annually thereafter [3]. However, the actual surveillance of these patients often deviates from the standard protocols [4] mostly due to the heavy work burden imposed on physicians as well as the associated pain and discomfort discouraging patients. Seeking for alternative non-invasive diagnostic tools, numerous urinary biomarkers for detection of bladder cancer have been developed and commercialized during the last two decades [5], [6]. Thus far, none of these tools has been implemented in routine clinical practice to supplant cystoscopy as a standard of care. The CellDetect technology is a novel cell staining method based on a proprietary plant extract that enables color discrimination between benign and malignant cells, while preserving critical features of cell morphology [7-10]. The discriminative capacity of the stain is related to specific metabolic alterations and increased metabolic activity observed in neoplastic cells. Pre-clinical studies and clinical trials have demonstrated applicability of the CellDetect technology in a plethora of cell culture lines and in various cancers. [8, 10]. The purpose of the current study was to explore the performance characteristics of the CellDetect technology in bladder cancer. 3

4 Methods An open label, two-step study was conducted in accordance with the Good Clinical Practice, Declaration of Helsinki 2008 and Ministry of Health requirements and regulations in Israel. The first step involved CellDetect staining of archived bladder tumor specimens. The second step included CellDetect staining of voided urine specimens obtained from: (a) Normal controls: patients with intact bladders undergoing routine cystoscopic follow-up after resection of non-muscle invasive bladder cancer, all deemed free of disease, 12 months or longer prior to participating in the study. (b) Patients diagnosed with bladder cancer by cystoscopy and scheduled for TURBT; urine samples were obtained prior to cystoscopy or tumor/bladder removal. The minimal volume of urine required for analysis was 50 ml, and all samples were analyzed by microscopy using a Neubauer hemocytometer. Samples that contained high levels of obscuring elements (e.g., erythrocytes, leukocytes), or were oligocellular, were considered technically inadequate and were excluded from analysis. Serial sections from archived biopsies were deparaffinized and rehydrated. One section from each biopsy was stained by Hematoxylin-Eosin (H&E) according to standard protocol. Adjacent sections from the tumor were stained by CellDetect according to manufacturer s instructions (Zetiq Technologies LTD). Briefly, the CellDetect kit contains four principal components, a proprietary plant extract and three dyes. The staining protocol includes fixation with 10% Trichloroacetic acid, followed by nuclear staining with Hematoxylin and serial incubations in the kit s proprietary components with intermittent washes. All CellDetect stained sections were analyzed by 2 independent pathologists using white light microscopy and the diagnosis was established based upon cell color and morphology. The CellDetect readings were compared to the diagnosis based on H&E-staining and the concordance between the 2 methods was assessed. Urine samples were collected in clinic during morning hours, but not as first morning urine, and were processed to cytospin smears and fixed with 96% Ethanol. Smears (one slide per patient) were stained by CellDetect and analyzed by an expert /

5 cytopathologist using 20X magnification. The cells morphology was determined according to standard cytological criteria (namely, enlarged nucleus to cytoplasm [NC] ratio, nuclear irregularity, nuclear polymorphism and presence of nucleoli), and the color of the cytoplasm and nucleus was documented. The CellDetect cytology readings were compared to the patients final diagnosis and the standard urine cytology readings based on hospital records when available. Results A total of 58 eligible archived biopsy specimens were retrieved, including 22 (38%) with normal mucosa, 17 (29%) with stages Ta, T1 and Tis bladder cancer, and 19 (33%) with muscle invasive tumor (stage T2 and above). Figure 1 (A-F) illustrates representative images of the CellDetect staining technology applied to tissue specimens. As noted in the figure, cells comprising the normal transitional epithelium (Figs. 1A, C) had a greenish-blue cytoplasm, whereas morphologically recognizable neoplastic cells exhibited red/magenta tinged cytoplasm (Figs. 1B, D). In fact, based exclusively on the tinctorial status of the epithelium, it was possible to recognize neoplasia from normal epithelium clearly, even at low magnification. The concordance between the diagnoses made by CellDetect and that established by H&E staining was 100%. One hundred and forty-eight urine samples were collected: 69 were utilized for staining calibration (i.e., adaptation of the staining protocol from tissue samples to cell smears), 30 were considered technically inadequate owing to severe inflammation, low cellularity or poor cell preservation and 5 were excluded from analysis due to lack of a subsequent confirmatory biopsy, leaving 44 samples available for analysis of the performance characteristics of the CellDetect technology in urine. Multiple slides were prepared from the urine collected from each individual patient, each stained independently by the same technician or by 2 different operators. We found minimal variation in terms of cell color and nonspecific background, none of which altered the final reading or diagnosis. Case distribution, as detailed in Table 1, included 27 healthy individuals and 17 cancer cases. In general, the CellDetect technology was capable of discriminating 1

6 neoplastic cells, which stained red, from the surrounding normal cells, stained green (Figure 2). The red dye highlighted the nucleus, and often the cytoplasm, of neoplastic/dysplastic cells, whereas the target of the green dye was the cytoplasm of normal cells. (Figure 2). Cytomorphological features consistent with neoplasia/dysplasia were not altered by the staining technique and assisted in confirming the diagnosis. The nucleus of cells with reactive changes stained green to purple (Figure 2B) and their NC ratio was significantly smaller, enabling their discrimination from dysplastic/neoplastic cells. Other cells detected on the slides were erythrocytes and leukocyte, both easily identified by morphology and size. To evaluate the association between the CellDetect color readings and cellular morphology, we studied the morphological features of 1031 cells obtained from 14 cancer patients and 8 healthy controls. A positive CellDetect staining was defined as red/purple nucleus on the background of pink or green cytoplasm. A negative CellDetect staining was defined as green/blue nucleus and a greenish cytoplasm. We found that 93% of the cells exhibiting morphologic features consistent with malignancy (362 out of 389 cells) were categorized as "positive staining", whereas 97% of the normal cells (669 out of 690 cells) were categorized as "negative staining" (Table 3). Next, we compared between the CellDetect urine staining results and the final pathological diagnosis established by either cystoscopy (in healthy individuals) or H&E staining of biopsy specimens in patients undergoing TURBT. A total of 44 eligible urine samples were analyzed, including 27 normal subjects and 17 with either recurrent (15 cases) or primary UC. All normal subjects were deemed tumor-free 1 year or longer prior to study enrollment. 19 of the 27 normal subjects received instillation: 7 received intravesical BCG, 8 intravesical mitomycin C, and 4 a combination of both. The CellDetect technology identified accurately 16 of the 17 cancer cases and 24 of the 27 healthy controls, translating into 94% sensitivity, 89% specificity, 84% negative predictive value and 96% positive predictive value (Table 2). Noteworthy, the CellDetect staining identified accurately patients with low grade non-invasive tumors (7 of the 8 patients) as well as patients with high grade urothelial carcinoma (9 out of 9 patients). 6

7 We further compared the performance of the CellDetect technology with that of urine cytology, based on hospital records of the participating subjects. A total of 34 cytology records were available for this analysis. Compared to standard urine cytology, the CellDetect technology demonstrated overall superior sensitivity (94% versus 46%, respectively, P<0.005, table 2), and particularly in low grade tumors (88% versus 17%, P<0.005, data not shown). There was no significant difference in specificity between the 2 staining techniques (89% for the CellDetect versus 95% for urine cytology, P=0.2, not shown). /

8 Discussion The high prevalence of bladder cancer and the requirement for frequent long-term bladder surveillance renders bladder cancer as the most expensive malignancy [11], with an estimated lifetime cost-per patient as high as 200,000 US$ [12]. Hence, the development of an effective, low cost, non-invasive urine-based assay to detect bladder cancer would be highly valuable for both patients and the healthcare system. This study represents the first application of the CellDetect technology in urologic oncology. We found this novel staining technology to discriminate fairly accurately between benign and malignant transitional epithelium. The nuclear staining of neoplastic cells was found consistently red-to purple, thus affording clear distinction from the green shade of non-neoplastic (normal and reactive) cells. We also found the application of the staining procedure in urine smears and histological specimens to be straightforward and reproducible. Nonetheless, as there was no apparent advantage to CellDetect over the traditional H&E staining in tissue biopsy specimens, the added benefit and future application of this technology is likely to be in voided urine specimens. CellDetect identified 16 of the 17 cancer cases correctly in voided urine specimens, specifically 7 of the 8 patients with low grade disease. One acknowledged limitation of urine cytology, particularly in the low grade setting, is its low sensitivity. The latter has been a major hurdle in routine bladder cancer surveillance [13], and thus the high sensitivity of the CellDetect staining observed in this study may allow patients to forgo some of the required invasive cystoscopies. Notably, this high sensitivity did not come at the expense of compromised specificity: 24 of the 27 normal subjects were diagnosed accurately by CellDetect. Whether the 3 patients classified as "false positive" may develop bladder tumors in the near future remains to be determined. Taken together, we believe that CellDetect represents a promising novel non-invasive staining technology, serving as an adjunct to 8

9 cystoscopy. These findings, however, are based on a limited number of cases and need further validation in a large, prospective study. What is the biological ground for CellDetect staining? Neoplastic cells have been shown repeatedly to exhibit striking alternations in metabolism, manifesting as a shift in glucose metabolism from oxidative phosphorylation to glycolysis, culminating in intracellular alkalosis. The CellDetect technology is assumed to rely on this phenomenon, capturing the unique metabolic signature resulting from the shift in energy metabolism. Based on differential ph affinity, the combination of dyes enables color discrimination accentuated and stabilized by the proprietary plant extract. This differential metabolic activity of malignant cells leads to prominent changes in intracellular proteins, which are preserved in fixated cells and in turn react differently with the dyes upon exposure to the plant extract. Further research is now conducted to explore the exact molecular mechanism underlying this phenomenon. A major limitation of this study remains its small sample size, primarily due to the exclusion of a large number of technically inadequate samples resulting from either hypocellularity or presence of inflammatory cells. Urine is generally considered to be oligocellular, and slides containing little-to-no cells are very common in assessment of urine cytology smears [14]. However, unlike in other cytology tests (e.g., cervical cytology), there are no consensus guidelines regarding the minimal number of cells defining a sample as adequate. Further work is warranted to determine the minimal number of cells needed for a reliable CellDetect analysis. Inflammation is a known cause of false positive findings in bladder cancer diagnostics [15] and many studies in the field consider it as an exclusion criteria, in order to improve the specificity of the assays [16]. As this was our initial proof of concept study aimed to calibrate the staining protocol we refrained from including suboptimal samples such as oligocellular or those with a large amount of inflammatory cells, acknowledging the bias it might have introduced. Current studies are conducted to overcome these hurdles including usage of liquid based cytology to filter the inflammatory cells and increasing the volume of urine volume to collect more cells. 9

10 Conclusions The presented findings demonstrate the ability of the CellDetect technology in identifying accurately UC in biopsy and voided urine specimens. This technology may provide an alternative to standard urine cytology with significant clinical benefit. 50

11 Table 1: Cases categorized according to clinical diagnosis groups Abbreviations: NED: no evidence of disease; LG: low grade; HG: high grade. Diagnosis No. of Subjects NED 27 Cancer 17 Total 44 Cancer grade No. of Subjects LG 8 HG 9 Total 17 Cancer stage No. of Subjects Ta 10 Tis 1 T1 5 T3 1 Total 17 Table 2: CellDetect and urine cytology performances (in all cancer stages) Cutoff: suspicious for malignancy. Abbreviations: NPV: negative predictive value; PPV: positive predictive value. Test No. of cases Sensitivity Specificity NPV PPV CellDetect 44 94% 89% 84% 96% Urine cytology 34 42% 95% 83% 75% Table 3: Color/morphology correlation of the CellDetect stain Positive staining = Red/purple color of the nucleus with either pink or green cytoplasm Negative staining = green/blue color of the nucleus and a green cytoplasm. Category Positively stained cancer cells Negatively stained cancer cells Negatively stained normal cells Positively stained normal cells No. of cells % 93% 7% 97% 3% 55

Figure 1: Photomicrographs of biopsies of bladder transitional epithelium. A-B: Images of histological sections stained with CellDetect C-D Images of histological sections stained with H&E.

12 Figure 1: Photomicrographs of biopsies of bladder transitional epithelium. A-B: Images of histological sections stained with CellDetect C-D Images of histological sections stained with H&E. A, C: Normal epithelium, B, D: UC. Cytoplasmic green/blue staining is characteristic of non-neoplastic states, whereas neoplasia consists of cells with pink/magentastained cytoplasm. Magnification: 40x. 51

Figure 2: Photomicrographs of urine smears stained with CellDetect. A-F are images of urine cytospin smears of a normal subject (A,B) or UC patients (C-F).

13 Figure 2: Photomicrographs of urine smears stained with CellDetect. A-F are images of urine cytospin smears of a normal subject (A,B) or UC patients (C-F). The majority of the cells are normal, epithelial cells that are stained in green. Image B shows reactive cells (enlarged in a box), stained in green as well. In images C-F a few foci of reddish cancer cells are seen (enlarged in boxes). Cancer cells preserve their distinctive morphology with enlarged, polymorphic nucleus and little cytoplasm. Magnification: 40x. 53

14 References 1. The American Cancer Society's publication, Cancer Facts & Figures 2013 Available from: 2. Grossman HB, Soloway MS, Messing E, et al., Surveillance for recurrent bladder cancer using a point-of-care proteomic assay. JAMA, (3): p Scher H, Bahnson R, Cohen S, et al., NCCN urothelial cancer practice guidelines. National Comprehensive Cancer Network. Oncology (Williston Park), (7A): p Schrag D, Hsieh LJ, Rabbani F, et al., Adherence to surveillance among patients with superficial bladder cancer. J Natl Cancer Inst (8): Liou LS, Urothelial cancer biomarkers for detection and surveillance. Urology, (3 Suppl 1): p ; discussion van Rhijn BW, van der Poel HG and van der Kwast TH, Urine markers for bladder cancer surveillance: a systematic review. Eur Urol, (6): p Idelevich P, Elkeles A, Okon E, et al., Novel dual-function CellDetect staining technology: wedding morphology and tinctorial discrimination to detect cervical neoplasia. Diagn Pathol, : p Idelevich, P, Kristt D, Okon E, et al., Novel histochemical stain for tinctorial detection of cancer and neoplastic cells. J Histotechnol, : p Idelevich P, Kristt D, Schechter E, et al., Screening for cervical neoplasia: a community-based trial comparing Pap staining, human papilloma virus testing, and the new bi-functional Celldetect stain. Diagn Cytopathol, (12): p Sagiv I, Idelevich P, Rivkin I, et al., A color discriminating broad range cell staining technology for early detection of cell transformation. J Carcinog, : p van Rhijn BW, Burger M, Lotan Y, et al., Recurrence and progression of disease in non-muscle-invasive bladder cancer: from epidemiology to treatment strategy. Eur Urol, (3): p Tiu A, Jenkins LC and Soloway MS, Active surveillance for low-risk bladder cancer. Urol Oncol, (1):33.e Sheaff MT and Singh N, Cytopathology. 2013, The Urinary Tract and Retroperitoneal Cytology. p London: Springer-Verlag 14. The College of American Pathologists homepage. Available from: Sharma S, Zippe CD, Pandrangi L, et al., Exclusion criteria enhance the specificity and positive predictive value of NMP22 and BTA stat. J Urol, (1): p Lotan Y and Roehrborn CG, Sensitivity and specificity of commonly available bladder tumor markers versus cytology: results of a comprehensive literature review and meta-analyses. Urology, (1): p ; discussion /

Combining Color and Morphology Improves Identification of Low-Grade Urothelial Cancer Cells

Combining Color and Morphology Improves Identification of Low-Grade Urothelial Cancer Cells Alexander Shtabsky 1, Sylvia Lew 2, Ronny Rona 3, Noa Davis 4, Yoram Mor 5, Uri Lindner 6, Ofer Nativ 7, Ofer