Raman Molecular Imaging: A Novel Spectroscopic Technique for Diagnosis of Bladder Cancer in Urine Specimens

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1 EUROPEAN UROLOGY 59 (2011) available at journal homepage: Bladder Cancer Raman Molecular Imaging: A Novel Spectroscopic Technique for Diagnosis of Bladder Cancer in Urine Specimens Amos Shapiro a, *, Ofer. N. Gofrit a, Galina Pizov b, Jeffrey Kirk Cohen c, John Maier d a Department of Urology, Hadassah Hebrew University Medical Center, Jerusalem, Israel b Department of Pathology, Hadassah Hebrew University Medical Center, Jerusalem, Israel c Department of Urology, Western Pennsylvania Prostate Cancer Foundation, Pittsburgh, PA, USA d Application Science Department, ChemImage Corporation, Pittsburgh, PA, USA Article info Article history: Accepted October 13, 2010 Published online ahead of print on October 28, 2010 Keywords: Raman spectroscopy Bladder cells Cancer Abstract Background: Raman molecular imaging (RMI) is an optical technology that combines the molecular chemical analysis of Raman spectroscopy with high-definition digital microscopic visualization. This approach permits visualization of the physical architecture and molecular environment of cells in the urine. The Raman spectrum of a cell is a complex product of its chemical bonds. Objective: In this work, we studied the possibility of using the Raman spectrum of epithelial cells in voided urine for diagnosing urothelial carcinoma (UC). Design, setting, and participants: Raman signals were obtained from UC tissue, then from UC touch preps obtained from surgical specimens and studied using the FALCON microscope (ChemImage, Pittsburgh, PA, USA), with a 100 collection objective and green laser illumination (532 nm). Then, urine samples were obtained from 340 patients, including 116 patients without UC, 92 patients with low-grade tumors, and 132 patients with high-grade tumors. Spectra were obtained from an average of five cells per slide. Measurements: Raman spectroscopy of cells from bladder cancer (BCa) tissues and patients. Results and limitations: The Raman spectra from UC tissue demonstrate a distinct peak at a 1584 cm 1 wave shift not present in benign tissues. The height of this peak correlated with the tumor s grade. The signal obtained from epithelial cells correctly diagnosed BCa with sensitivity of 92% (100% of the high-grade tumors), specificity of 91%, and a positive predictive value of 94% and a negative predictive value of 88%. The signal correctly assigned a tumor s grade in 73.9% of the lowgrade tumors and 98.5% of the high-grade tumors. RMI for diagnosis of BCa is limited by the need for specialized equipment and training of laboratory personnel. Conclusions: RMI has the potential to become a powerful diagnostic tool that allows noninvasive, accurate diagnosis of UC. # 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Department of Urology, Hadassah Hebrew University Medical Center, PO Box 12000, Jerusalem 91120, Israel. Tel ; Fax: address: amossh@cc.huji.ac.il (A. Shapiro) /$ see back matter # 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 EUROPEAN UROLOGY 59 (2011) Introduction Noninvasive diagnosis of bladder cancer (BCa) is a continual challenge. Various characteristics of malignant cells found in the urine have been used for this purpose. The only clinical method used today urinary cytology is characterized by high specificity but low sensitivity, especially in low-grade tumors [1]. During the past two decades, multiple urine-based tests have been studied [2,3], but none is recommended in the current guidelines for diagnosis and treatment of BCa [4]. Raman spectroscopy, technique historically applied in analytical chemistry for the evaluation of chemical compounds, is based on excitation of vibrational modes in the chemical bonds that hold molecules together, illuminating a sample with laser light. Because Raman signals originate from the vibration of bonds within molecules, Raman spectroscopy provides a measure of biologically important molecular groups. In a molecularly complex system such as a cell, Raman spectroscopic measurements carry information about the mixture of chemicals present in the system. Variations in the relative compositions of different molecular species lead to changes in the measured Raman spectrum of such a complex system. Near-infrared absorption spectroscopy is an alternative spectroscopy technique that derives molecular specificity from molecular vibrations and has been applied to tissue samples [5]. Previous authors have shown that it is possible to discriminate between tumor and nontumor bladder tissue using Raman spectroscopy [6,7]. In this work, we studied the possibility of using the Raman spectrum of epithelial cells in voided urine for diagnosing BCa with the FALCON Raman microscope (ChemImage, Pittsburgh, PA, USA) [8]. We demonstrate a correlation between the Raman spectrum measured from individual epithelial cells and diagnoses of BCa made independently. 2. Methods The study was performed under institutional review board approval from Allegheny General Hospital in Pittsburgh, Pennsylvania, USA, and Hadassah University Hospitals, Jerusalem, Israel. Tissues for the study were obtained from cystectomy specimens (containing malignant tissue only) and from open prostatectomy surgery for benign disease (urothelium of the bladder neck removed routinely during this type of surgery). Pathologic staging was performed according to the TNM system, and grading was done according to the 1998 World Health Organization/International Society of Urological Pathology system. Initially, Raman signals were obtained from urothelial carcinoma (UC) tissue, then from UC touch preps obtained from surgical specimens, and finally from voided urinary cells. Paraffin blocks of these tissues were cut into 5-mm sections and placed on aluminum slides. The preparations were deparaffinized according to the following protocol: The sections were warmed to just above the melting point of paraffin, and then immersed in xylene to dissolve the paraffin. The slides were then immersed in a second change of xylene followed by two changes in 100% alcohol and 95% and 80% alcohol, respectively. Raman spectra were obtained from epithelial cells using the FALCON microscope, with a 100 collection objective using green laser illumination (532 nm) [8]. Fig. 1 provides a schematic diagram of the instrumentation used in this investigation. To compare the Raman signal obtained from tissue to cells, a touch prep of UC was obtained from surgical specimens. The rest of the sample was frozen, sectioned (5-mm thickness), and placed on aluminum slides, with analysis as described above. Routine hematoxylin and eosin stained tissue was obtained from the rest of the sample. Cellular analysis was performed on 50 ml of urine collected in the operating room in a sterile fashion from patients with and without UC of the bladder. The urine was spun at 3000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in 50 ml of distilled water, and then centrifuged again at 3000 rpm for 5 min, after which the supernatant was discarded. Filter preparation to remove excess white blood cells and residue of lysed red blood cells was made in cases of high turbidity. Filtration was performed using a filter with 20-micron pores to capture the epithelial cells and allow other constituents to be washed off. The filter with the epithelial cells was then washed to resuspend the Fig. 1 Schematic diagram of the ChemImage FALCON microscope instrumentation used for Raman molecular imaging in this investigation. LC TF = liquid crystal tunable filter; CCD = charge-coupled device.

3 108 EUROPEAN UROLOGY 59 (2011) Fig. 2 Raman spectrum obtained from urothelial carcinoma (dashed) and from three cases of normal bladder tissue (solid). The high 2950-cm S1 wave signals the C H bond and is present in any biologic material. The 1600-cm S1 wave is typical to human tissues. Notice that a 1584-cm S1 wave is present in malignant tissue only. urothelial cells of interest. Two drops of the remaining material was placed into a cytospin chamber and spun at 1100 rpm for 5 min, which resulted in cells being deposited on an aluminum-coated slide. Each slide was then analyzed with the FALCON Raman molecular imaging (RMI) microscope. The epithelial cells were centered and photographed using 50 objective magnification. Raman spectra were obtained using 100 objective so as to minimize the empty space around the cell. Spectra were obtained from an average of five cells per slide, and every spectrum was baseline corrected and normalized. A positive 1584-cm 1 wave shift in a single cell was considered a positive reading. 3. Results To understand the signal differences observed from normal bladder and UC using Raman spectroscopy, initial experiments were performed on thin sections of tissues known to be normal or cancerous. Four different samples of stage T2, high-grade UC; six samples of stage Ta, low-grade; three samples from normal bladder; and two samples from Tis were studied to obtain baseline spectral features. Fig. 2 shows the Raman spectrum obtained from three specimens of normal bladder mucosa and a single case of high-grade UC. The Raman spectra of both low-grade and high-grade UC demonstrate a distinct peak at a 1584-cm 1 wave shift not present in benign tissue (Fig. 3). The height of this peak correlates with UC grade Raman spectroscopy of cells from bladder cancer tissues To compare the distinctive Raman signals of tissue and cells, touch preps of benign and malignant bladder tissues were created. Spectra were obtained from the cells and compared with the tissues of origin. Fig. 4 shows a spectrum from tissue with high-grade UC and a cellular touch prep of the same sample. The spectra of the cell and tissue were virtually identical Raman spectroscopy of cells from the urine of bladder cancer patients To show the unique Raman signals of benign and malignant epithelial cells, urine was collected from patients. Fig. 5 shows the unique Raman spectroscopic signature observed from voided cells: The spectrum appears to be distinct. Specifically, malignant urinary cells demonstrate a peak at 1584 cm 1. Raman spectroscopic study of a series of patients was used to construct an objective model for classification based on spectra. The sample included 172 specimens, including 50 patients without UC, 50 patients with low-grade tumor, and 72 patients with high-grade tumor (including 22 patients with Tis). Table 1 shows the distribution of disease Table 1 Disease distribution and Raman molecular imaging results of the cases used to develop the model and the accuracy of prediction No. of cases Positive Raman signal for bladder tumor, n (%) Grade-appropriate Raman signal tumor, n (%) No tumor 50 5 (5) All classified as low grade Low grade (90) 44 (88) High grade (100) 71 (98.6)

4 EUROPEAN UROLOGY 59 (2011) Fig. 3 Raman spectrum obtained from high-grade urothelial carcinoma (UC; dashed) tissue, low-grade UC tissue (dotted), and normal tissue (solid). The 1584-cm S1 wave is higher in high-grade tumors but is also present in low-grade tissue. and model performance on this set of samples. The signal obtained from epithelial cells correctly diagnosed BCa with a sensitivity of 96% (100% of the high-grade tumors) and a specificity of 90%. The positive predictive value (PPV) and negative predictive value (NPV) were 96% and 90%, respectively. The model developed for classification of spectra takes a normalized spectrum and evaluates the height of the 1584-cm 1 band above the baseline at 1500 cm 1 and uses a set of thresholds to classify the spectrum. The threshold for low grade was found to be 6 units, and the threshold for high grade 15 units (Fig. 6). The signal correctly assigned the tumor s grade in 88% of the lowgrade tumors and 98.6% of the high-grade tumors. Fig. 7,for example, depicts the thresholds for no tumor, low-grade tumors, and high-grade tumors. To test the model developed in the first step, the algorithm was applied to additional 166 urine samples. Table 2 shows the distribution of disease and the performance of the model on the validation set of data. As further statistical analysis of the model set of cases, principal component analysis (PCA) indicates that the Fig. 4 The Raman spectrum of high-grade urothelial carcinoma tissue (dashed) and cells from a touch prep of the tissue (solid). The spectra of the cell and tissue were virtually identical.

5 110 EUROPEAN UROLOGY 59 (2011) Fig. 5 Raman spectra of cumulative single, exemplary cells from cases of high-grade (solid bold), low-grade (dotted), and normal cells (solid). The figures at the right show the digital microscopy images of the cells from which the spectra were acquired as they appear on the FALCON RMI microscope. Fig. 6 Segmental view (fingerprint) of the cm S1. The thresholds used to determine cancer grade classification are indicated. ROI = region of interest. Table 2 Disease distribution and Raman molecular imaging results of the cases used to validate the model and the accuracy of prediction No. of cases Positive Raman signal for bladder tumor, n (%) Grade-appropriate Raman signal tumor, n (%) No tumor 66 6 (9.1) All classified as low grade Low grade (80.9) 24 (57) High grade (100) 59 (98.3)

6 EUROPEAN UROLOGY 59 (2011) Table 3 Disease distribution and Raman molecular imaging results of all patients participating in the study No. of cases Positive Raman signal for bladder tumor, n (%) Grade-appropriate Raman signal tumor, n (%) No tumor (9.5) All classified as low grade Low grade (95.8) 68 (73.9) High grade (100) 130 (98.5) Fig. 7 A scatter plot of the spectra used to develop the model projected onto a plane in principal component analysis. The color-coded figure at the right indicates the classification of the spectra based on the location in the scatter plot, as indicated by the colored ovals. PC = principal component. spectra are distinct among different grades. Fig. 7 shows a cluster plot of the model set in a projection of PCA space, indicating the distinctiveness of spectra from cells of different grades of cancer. Biologic variability is evident in the scatter plot but is less than the variability related to cancer grade. When analyzing the combined results of the spectrum used to develop and validate the model (Table 3), a sensitivity of 95% and a specificity of 90.5% were obtained. 4. Discussion RMI combines digital imaging and Raman spectroscopy in a modality that is reproducible and quantifiable [7]. The ability of Raman spectroscopy to discriminate between malignant and benign bladder tissue was studied by Crow et al. [7].Our investigation demonstrates that the sensitivity of RMI for BCa diagnosis on cells obtained from urine is remarkably high (92% in the validation set). In addition, the Raman spectrum of 105 of 116 urine samples of patients without UC did not contain a 1584-cm 1 wave shift. Furthermore, RMI was able to accurately discriminate low-grade from high-grade tumors (Table 3). Low-grade tumors were correctly assigned in 74% of the cases and high-grade tumors in 98.5%. Besides noninvasiveness and accuracy, RMI using the FALCON microscope has several advantages: It is fast (15 50 min, depending upon the cleanliness of the urine), does not require expert spectroscopists or pathologists, and does not produce any toxic waste. These advantages could make RMI an appropriate tool for UC diagnosis and possibly for screening of UC in high-risk populations (ie, heavy smokers >40 yr of age). Currently available methods for noninvasive diagnosis of UC, including urinary cytology [1], Lewis X [9], the NMP22 test (Matritech, Köln, Germany), the BTA stat test [10,11], microsatellite assays [12], cytokeratin stains [13], and UroVysion (Abbott Molecular, Abbott Park, IL, USA) [14]. Cytology in low-grade disease has a sensitivity and PPV of 20% and 100%, respectively [1]. In addition, the cost factors make it prohibitive for screening large numbers of high-risk patients. Microsatellite assay, Lewis X, and cytokeratin stains are operator dependent. The NMP22 and BTA stat tests are US Food and Drug Administration approved but have a sensitivity of 80% with high-grade disease and a 15% falsepositive result in the control group. UroVysion, although highly accurate, is an expensive test. Currently, these methods are not suitable for early detection in large numbers of patients with UC. Raman spectroscopy of bladder tissue has been investigated by Crow et al for correlation between spectra measured on tissue and independent diagnosis [7]. These authors showed that Raman spectra can discriminate between malignant and benign tissues and even predict cancer grade. A notable difference between this study and the previous work, besides the use of voided urine, is using the 532-nm excitation light. This wavelength is not often used for examination of biologic samples because of its notorious autofluorescence. In our experience, however, this wavelength provided a useful Raman spectrum, with higher Raman scattering relative to excitation wavelengths in more red spectral regions. Our study demonstrates high sensitivity for cancer diagnosis (95% for all cancer patients and 100% for high-grade cancer). In 9.5% of the patients without UC, a 1584-cm 1 wave shift was found. All of these patients were classified as suffering from low-grade cancer. The use of RMI for diagnosis of UC, however, requires the purchase of expensive equipment and some training. 5. Conclusions This is the first study using RMI to diagnose and classify UC in urine samples and in a broader sense to diagnose cancer in isolated cell samples. A remarkable sensitivity and specificity were found. Further investigations are needed to verify the performance of this technology in urinary samples and in other cellular systems to determine the applicability to other malignancies. Author contributions: Amos Shapiro had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Shapiro, Maier, Cohen. Acquisition of data: Shapiro, Gofrit, Pizov. Analysis and interpretation of data: Shapiro, Gofrit, Pizov, Cohen, Maier.

7 112 EUROPEAN UROLOGY 59 (2011) Drafting of the manuscript: Shapiro, Gofrit, Pizov, Cohen, Maier. Critical revision of the manuscript for important intellectual content: Shapiro, Gofrit, Pizov, Cohen, Maier. Statistical analysis: Shapiro, Gofrit, Cohen, Maier. Obtaining funding: None. Administrative, technical, or material support: Shapiro, Gofrit, Pizov, Cohen, Maier. Supervision: Shapiro, Cohen. Other (specify): None. Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/ affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: Dr. Maier is an employee of and Dr. Cohen is a board member of ChemImage. Funding/Support and role of the sponsor: This study was sponsored in part by the Western Pennsylvania Prostate Cancer Foundation, which was involved with preparation of this manuscript. References [1] Koss GL, Dietch D, Ramanathan R, Sherman AB. Diagnostic value of cytology of voided urine. Acta Cytol 1985;29: [2] Van Tilborg AA, Bangma CH, Zwarthoff EC. Bladder cancer biomarkers and their role in surveillance and screening. Int J Urol 2009;16: [3] Vrooman OPJ, Witjes JA. Urinary markers in bladder cancer. Eur Urol 2008;53: [4] Babjuk MW, Oosterlink W, Sylvester R, Kaasinen E, Böhle A, Palou J. Guidelines on TaT1 (non-muscle invasive) bladder cancer. Arnhem, The Netherlands: European Association of Urology; [5] Kidder LH, Kalazinsky VF, Luke J, Levin IW, Lewis EN. Visualization of silicone gel in human breast tissue using a new infrared imaging spectroscopy. Nat Med 1997;3: [6] de Jong BW, Bakker Schut TC, Wolffenbuttel KP, Nijman JM, Kok DJ, Puppels GJ. Identification of bladder wall layers by Raman spectroscopy. J Urol 2002;168: [7] Crow P, Barrass B, Kendall C, et al. The use of Raman spectroscopy to differentiate between different prostatic adenocarcinoma cell lines. Br J Cancer 2005;92: [8] Morris H, Hoyt CC, Miller P, Treado PJ. Liquid crystal tunable filter Raman chemical imaging. Appl Spect 1996;50: [9] Pode D, Golijanin D, Sherman Y, Lebensart P, Shapiro A. Immunostaining of Lewis X in cells from voided urine, cytopathology and ultrasound for noninvasive detection of bladder tumors. J Urol 1998;159: [10] Theodoros GAM, Constantinos GAC, Constantinos CPHD. Comparative evaluation of the diagnostic performance of BTA stat test, NMP22 and urinary bladder cancer antigen for primary and recurrent bladder tumors. J Urol 2001;166: [11] Friderich MG, Hellstern A, Hautmann SH, et al. Clinical use of urinary markers for the detection and prognosis of bladder carcinoma: a comparison of immunocytology with monoclonal antibodies against Lewis X and 486p3/12 with BTA STAT and NMP22 tests. J Urol 2002;168: [12] Sourvinos G, Kazanis I, Delakas D, Cranidis A, Spandidos DA. Genetic detection of bladder cancer by microsatellite analysis of p16, RB1 and p53 tumor suppressor genes. J Urol 2001;165: [13] Golijanin D, Shapiro A, Pode D. Immunostaining of cytokeratin-20 in cells from voided urine for detection of bladder cancer. J Urol 2000;164: [14] Gofrit ON, Zorn KC, Silvestre J, et al. The predictive value of multitargeted fluorescent in-situ hybridization in patients with history of bladder cancer. Urol Oncol 2008;26:246 9.