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In Vivo Bladder Cancer Diagnosis by High-Volume Raman Spectroscopy Ronald O. P. Draga,*,† Matthijs C. M. Grimbergen,‡ Peter L. M. Vijverberg,§ Christiaan F. P. van Swol,| Trudy G. N. Jonges,⊥ J. Alain Kummer,# and J. L. H. Ruud Bosch† Department of Urology, C.04.236, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands, Department of Medical Technology and Clinical Physics, Room F.01.126, University Medical Center Utrecht, The Netherlands, Department of Urology, St Antonius Hospital, P.O. Box 2500, 3430 EM Nieuwegein, The Netherlands, Department of Medical Physics, St Antonius Hospital, Nieuwegein, The Netherlands, Department of Pathology, Room H.04.312, University Medical Center Utrecht, The Netherlands, Department of Pathology, St Antonius Hospital, Nieuwegein, The Netherlands, Department of Urology, University Medical Center Utrecht, The Netherlands We studied the feasibility of Raman spectroscopy for the diagnosis of bladder cancer in vivo. Since the invasion stage is crucial for the treatment choice, a high-volume based Raman probe was used to investigate the potential of determining the invasiveness of bladder cancer. High quality spectra were obtained from suspicious and nonsuspicious bladder locations during the procedure of transurethral resection of bladder tumors (TURBT) with collection times of 1-5 s. Multivariate analysis was used to generate the classification models. The algorithm was able to distinguish bladder cancer from normal bladder locations with a sensitivity of 85% and a specificity of 79%. The Raman spectra of bladder cancer stages showed a gradual increase in the intensity of specific amino acid peaks and, most likely, an increase in the intensity of DNA peaks. Bladder cancer is the ninth most frequently diagnosed cancer1 and accounts for 5-10% of all malignancies in males worldwide.2 Approximately 75-80% of the patients with bladder cancer present with nonmuscle-invasive bladder cancer (NMIBC). Disease recurs in 50-70% of the patients with NMIBC after transurethral resection alone,3 of which approximately 13% have the tendency to progress to muscle-invasive bladder cancer or worse.4 Therefore, early detection, complete resection and surveillance of bladder cancer are essential for preventing early recurrence and progression. * To whom correspondence should be addressed. Phone: +31 88 75 539 50. Fax: +31 30 2540532. E-mail:
[email protected]. † Department of Urology, University Medical Center Utrecht. ‡ Department of Medical Technology and Clinical Physics, University Medical Center Utrecht. § Department of Urology, St Antonius Hospital, St Antonius Hospital. | Department of Medical Physics, St Antonius Hospital. ⊥ Department of Pathology, University Medical Center Utrecht. # Department of Pathology, St Antonius Hospital. (1) Parkin, D. M.; Bray, F.; Ferlay, J.; Pisani, P. Ca-Cancer J. Clin. 2005, 55, 74. (2) Kirkali, Z.; Chan, T.; Manoharan, M.; Algaba, F.; Busch, C.; Cheng, L.; et al. Urology 2005, 66, 4. (3) Brausi, M.; Collette, L.; Kurth, K.; van der Meijden, A. P.; Oosterlinck, W.; Witjes, J. A.; et al. Eur. Urol. 2002, 41, 523. (4) Kurth, K. H.; Denis, L.; Bouffioux, C.; Sylvester, R.; Debruyne, F. M.; Pavone-Macaluso, M.; et al. Eur. J. Cancer 1995, 31A, 1840. 10.1021/ac100448p 2010 American Chemical Society Published on Web 06/04/2010
Transurethral resection of bladder tumors (TURBT) followed by histopathology and urine cytology are the gold standard for the diagnosis of bladder cancer. However, flat neoplastic lesions such as carcinoma in situ (CIS) and small papillary lesions are easily overlooked during TURBT which contribute to increased residual rates after 6 weeks varying between 25 and 78%5,6 and a recurrence rate of 50% within 18 months.7 The use of photodynamic diagnosis or fluorescence cystoscopy (PDD) improves the detection of bladder cancer during cystoscopy and increases the recurrence free survival by 67% compared to white light cystoscopy.8,9 Fluorescence cystoscopy enhances the visual contrast between benign and malignant tissue using violet-blue light after instilling the bladder with a photosensitive dye such as 5-aminolevulinic acid (5-ALA) or hexaminolevulinic acid (HAL). Aminolevulinic acid is a precursor in the heme biosynthesis pathway and leads to the accumulation of protoporphyrin IX (PpIX) in malignant cells. After illumination with violetblue light, PpIX will show red fluorescence in contrast to the blue reflection of normal bladder tissue. Although PDD has a much higher sensitivity, 97% compared to 76% of white light endoscopy (WLE), higher false detection rates of approximately 38% have been reported.10,11 Patients that might benefit most from highly specific optical diagnosis, such as Raman spectroscopy, to improve the overall diagnostic accuracy in combination with fluorescence cystoscopy, are female patients and patients with a TURBT within 3 months before the PDD procedure.12 Raman spectroscopy has the capability of characterizing tissues at a molecular level and could be used to ascertain the pathological nature of suspicious fluorescent positive lesions in photodynamic diagnosis. The Raman effect is an inelastic scattering process, (5) (6) (7) (8) (9) (10) (11) (12)
Jocham, D.; Stepp, H.; Waidelich, R. Eur. Urol. 2008, 53, 1138. Soloway, M. S.; Sofer, M.; Vaidya, A. J. Urol. 2002, 167, 1573. Allard, P.; Bernard, P.; Fradet, Y.; Tetu, B. Br. J. Urol. 1998, 81, 692. Denzinger, S.; Burger, M.; Walter, B.; Knuechel, R.; Roessler, W.; Wieland, W. F.; et al. Urology 2007, 69, 675. Daniltchenko, D. I.; Riedl, C. R.; Sachs, M. D.; Koenig, F.; Daha, K. L.; Pflueger, H.; et al. J. Urol. 2005, 174, 2129. Hungerhuber, E.; Stepp, H.; Kriegmair, M.; Stief, C.; Hofstetter, A.; Hartmann, A.; et al. Urology 2007, 69, 260. Grossman, H. B.; Gomella, L.; Fradet, Y.; Morales, A.; Presti, J.; Ritenour, C.; et al. J. Urol. 2007, 178, 62. Draga, R. O.; Grimbergen, M. C.; Kok, E. T.; Jonges, T. N.; Bosch, J. L. Urology 2009, 74, 851.
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Figure 1. Left: picture taken of the Raman probe (front). Right: diagram of the clinical Raman system.
where scattered photons donate energy to the sample’s molecular bonds which results the Raman scattered light to be shifted to longer wavelengths (red-shifted inelastic scattering or “Stokes” Raman scattering). Detection of the scattered photons results in a spectrum of peaks, each of which is characteristic for a specific molecular bond and, thus, provides an intrinsic “molecular fingerprint” of the sample. The advantages of in vivo Raman spectroscopic diagnosis is that it is nondestructive and does not require any pretreatment. The microfiber optic probes can be applied endoscopically with clinically acceptable measurement times (
