KEYWORDS: Tumors, Photodynamic therapy, Bladder, Luminescence, Bladder cancer, Control systems, Animal model studies, Fluorescence correlation spectroscopy, Tissues, Green fluorescent protein
The prevalence of bladder cancer is very high, due to its high recurrence rate in superficial bladder
cancer (30 to 85%), which is the staging of approximately 80% of the patients at first diagnosis. Risk of
recurrence and progression is associated with grade, stage, presence of concomitant carcinoma in situ, size and
number of lesions, as well as time to first recurrence. Recurrences can be partly attributed to new occurrences
but also to residual tumors after resection. Incomplete tumor removal has been observed in 30 to 50% of TUR's,
especially when dealing with T1 or poorly visible malignant or pre-malignant disease1. Fluorescence guided
resection with 5 amino levulinic acid (ALA) or its hexyl ester derivative (Hexvix, has now unequivocally
been demonstrated to increase detection rate and a growing number of studies indicate this has a positive impact
on recurrence and progression ratesImplantation of viable tumor cells, dispersed during resection, is a third factor influencing bladder cancer recurrence. The aim of early intravesical therapy is to interfere with cell
viability and thus reduce implantation risks.
Carcinoma in situ (CIS) of the bladder is a confounding disease that is difficult to recognize endoscopically since it is a flat cancer. Many studies have suggested its relationship with subsequent invasive disease. Early recognition of CIS therefore is essential in order to offer the patients the most appropriate treatment and the highest cure rate. Since white light cystoscopic examination is not sufficient to reveal areas of dysplasia or carcinoma in situ random biopsies are recommended. We wanted to evaluate whether amino levulinic acid (ALA) fluorescence detection could be helpful in diagnosing carcinoma in situ and if the specificity could be enhanced by reducing the ALA dose. Sixteen patients with papillary bladder cancer and carcinoma in situ and dysplasia were instilled with low dose ALA. Fluorescence detection of the metabolized ALA was performed three hours later, with the naked eye, after blue light illumination. CIS or dysplasia was found in 50 biopsies. The sensitivity for detecting CIS was 94% with a specificity of 89%. Carcinoma in situ can be diagnosed with a very high accuracy through fluorescence detection after ALA instillation. Fluorescence detection can be achieved with the naked eye and does not necessitate complex equipment neither specifically trained personnel.
Carcinoma in situ (CIS) of the bladder is a treacherous entity, that will develop into invasive cancer. Early treatment is mandatory in order to prevent progression. When conservative measures, such as Bacillus Calmette Querin (BCG) instillations have failed, radical cystectomy and urinary diversion is recommended. Whole bladder wall photodynamic therapy (PDT) with Photofrin II has been shown to be effective in eradicating carcinoma in situ, but often resulted in bladder shrinking. We wanted to evaluate the effects of PDT after aminolevulinic acid (ALA) sensitization. Six patients with refractory carcinoma in situ of the bladder were treated with whole bladder wall photodynamic therapy, after intravesical sensitization with aminolevulinic acid. The total light dose (scattered plus non scattered) was 75 J/cm2. No skin sensitization occurred, nor loss of bladder capacity. One patient did not respond and was successfully treated with BCG. Another patient developed distant metastases. Carcinoma in situ was completely absent after 3 months in four patients (66%).
Laser-induced fluorescence spectra were recorded in patients undergoing urinary bladder cystoscopy. The measurements were performed in vivo and the spectra were collected from normal and diseased tissue. The patients were divided into two groups. An instillation of a 1% delta-amino-levulinic acid (ALA) solution was performed 2 - 4 hours prior to the investigation of one group of patients. A second group of patients was investigated without any tumor marking substance. The fluorescence was detected following laser excitation at 405 and 337 nm. Fluorescence emission related to ALA-induced protoporphyrin IX (PpIX) was detected in the ALA group for 405 nm excitation. The data were evaluated at the PpIX emission peak at 635 nm and at 490 nm, which approximately corresponds to the peak of the tissue autofluorescence. The data obtained with 337 nm excitation were evaluated at 400 and 460 nm as well as at 390 and 431 nm. The ratios of the respective wavelength pairs were formed in order to investigate the demarcation between tumor and normal tissue. The tumor demarcation results were better and more consistent utilizing the autofluorescence signal following excitation at 337 nm than the PpIX-related signal excited at 405 nm.
Bladder tissue autofluorescence spectra are obtained in vivo at two excitation wavelengths (334 nm and 365 nm) with a cystoscopic fiber- optic device based on a small mercury arc lamp. Upon 365 nm excitation, both normal and cancerous bladder tissue have nearly identical fluorescence spectra, characterized by a broad peak at 455 nm. However, the fluorescence yield from malignant tissue is approximately a factor 3 lower compared to normal tissue. A similar decrease in fluorescence yield is observed upon 334 nm excitation. More importantly, at this excitation wavelength, the spectra from normal and malignant tissue have different lineshape. Normal tissue shows two distinct fluorescence peaks (at 385 nm and 455 nm), while malignant tissue only shows the 455 nm peak. Based on these insights, we have developed a simple spectroscopic algorithm, to differentiate normal from malignant bladder tissue with our device. The main underlying biophysics will be addressed. The integration of the diagnostic method with a reliable therapeutic technique for tumor cell destruction, may open the way for cost- effective preventive care of high-risk patients.
Katarina Svanberg, Stefan Andersson-Engels, Luc Baert, Elisabeth Bak-Jensen, Roger Berg, Arne Brun, Stig Colleen, Ingrid Idvall, Marie-Ange D'Hallewin, Christian Ingvar, Jonas Johansson, Sven-Erik Karlsson, Rolf Lundgren, Leif Salford, Unne Stenram, Lars-Goran Stromblad, Sune Svanberg, Ingrid Wang-Nordman
Laser-induced fluorescence (LIF) can be used for noninvasive spectroscopic identification of biological tissue and is of special interest in early tumor detection. The basis for this optical biopsy method is the interaction of the laser light with tissue chromophores, such as tryptophan, collagen, elastin, NADH, beta-carotene and hemoglobin. The UV-excited fluorescence that arises from the native chromophores, the autofluorescence, has a broad distribution, peaking at about 490 nm with a lower intensity in tumor compared to normal tissue. The tumor detection potential is enhanced with exogenously administrated tumor- marking agents, such as hematoporphyrin (HPD, commercial name Photofrin), with two fluorescence peaks at about 630 and 690 nm. We have developed clinical instrumentation both for tissue point monitoring and for full real-time image processing. Seventy-one patients were investigated in vivo and surgical samples from additional 20 patients. In 46 patients the autofluorescence only was monitored. In 45 patients low-dose Photofrin injection was used. The in vivo investigations included different kinds of lung tumors, urinary bladder tumors, and malignant gliomas. The in vitro measurements were performed in breast tumors and prostatic tumors. Invasive and early tumors and also precancerous lesions can be revealed utilizing LIF in low-dose Photofrin injected patients.
Transitional bladder cell carcinoma (TCC) is easily recognizable, but for the diagnosis of severe dysplasia and carcinoma in situ (CIS) one can only rely on randomly taken biopsies. Fluorescence tagging of tumors by sensitizing agents such as hematoporphyrin derivatives (HpD) is possible but presents, even at low doses, a number of serious drawbacks for the patient. We demonstrate a cystoscopic fiber optic instrument, based on a small mercury arc lamp, for in vivo demarcation of human bladder carcinoma. The instrument detects the tissue autofluorescence upon UV excitation (365 nm), thus eliminating the need for sensitizing agents. The average demarcation contrast obtained for CIS and TCC is respectively 2.6 and 3.2, which is about 60% higher than what can be expected from photodynamic imaging with low-dose HpD. The main underlying biophysics are derived from a spectral analysis of the observed autofluorescence signals. The integration of the diagnostic method with a reliable therapeutic technique for tumor cell destruction, opens the way for cost-effective preventive care of high-risk patients.
Neodymium-Yag (Nd:YAG) laser irradiation for superficial bladder cancer is a valuable and safe treatment modality. The local recurrence rate is significantly reduced as compared to transurethral resection (TUR) alone (2.6 % versus 14.3 %). Remote recurrences can also significantly be reduced in solitary and multiple tumors if biopsies of the tumor are avoided. We state that the macroscopic appearance of a tumor is sufficiently reliable since only 0.67 % of the pathology proven Ti tumors were understaged macroscopically. The recurrence free interval for solitary tumors after Nd:YAG laser irradiation alone without biopsy was 19.i months as compared to i6 months after TUR with adjuvant intravesical instilations and in multiple tumors i3.6 months for Nd YAG laser irradiation alone without biopsies as compared to iO.4 months after TUR.
We report on 15 patients with multifocal carcinoma in situ of the bladder, treated with whole bladder wall photodynamic therapy (PDT). The total light dose, measured in situ (scattered plus nonscattered light) was 100 J/cm2 in the first six patients and 75 J/cm2 in the remaining nine patients. Follow-up ranges were from 6 to 27 months (average 15 months). Two cystectomies had to be performed in the first treatment group because of permanent shrunk bladders. Pathology of the resection specimens showed extensive granulation and fibrosis throughout the whole bladder wall. In the second treatment group, the maximal bladder capacity measured three months after PDT had increased on the average of 63% compared to the initial pretreatment values. No increased fibrosis could be detected on microscopical examination of random biopsies. Four recurrences necessitated cystectomy after 5 to 9 months, two in each treatment group. Three out of these originated in patients with a previous history of invasive bladder cancer. The preliminary data demonstrate the importance of in-situ light dosimetry for minimizing local side effects of PDT as well as the importance of strict inclusion criteria to optimize the therapeutic ratio.
A point-monitoring fluorescence diagnostic system based on a low-energy pulsed laser, fiber transmission optics, and an optical multichannel analyzer was used for diagnosis of patients with bladder malignancies. Twenty-four patients with bladder carcinoma, carcinoma in situ, and/or dysplasia were injected with Photofrin (0.35 or 0.5 mg/kg body weight) 48 hours prior to the investigation. The ratio between the red sensitizer emission and the bluish tissue autofluorescence provided excellent demarcation between papillary tumors and normal bladder wall. Certain cases of dysplasia could be also be differentiated from normal mucosa. Benign exofytic lesion such as malakoplakia appeared different from malignant tumors in fluorescence. Flat suspicious bladder mucosa such as that seen in infectious diseases or after radiation therapy appeared normal in terms of fluorescence.
Two series of investigations utilizing laser-induced fluorescence (LIF) in characterizing diseased tissue are presented. In one in vitro investigation the fluorescence from normal and atherosclerotically diseased arteries are studied. In another clinical study the fluorescence in vivo from superficial urinary bladder malignancies in patients who had received a low-dose injection of Hematoporphyrin Derivative (HpD) is investigated. Additionally, the fluorescence properties of L-tryptophan, collagen-I powder, elastin powder, nicotinamide adenine dinucleotide and (beta) -carothene were investigated and compared with the spectra from the tissue samples. A nitrogen laser (337 nm) alone or in connection with a dye laser (405 nm) was used together with an optical multichannel analyzer (OMA) to study the fluorescence spectra. The fluorescence decay characteristics of atherosclerotic plaque were examined utilizing a mode locked argon ion laser, synchronously pumping a picosecond dye laser. A fast detection system based on photon counting was employed. The fluorescence decay curves were evaluated on a PC computer allowing up to three lifetime components to be determined. A fluorescence peak at 390 nm in fibrotic plaque was identified as due to collagen fibers, while a fluorescence peak at 520 nm was connected to (beta) -carotene. The in vivo measurements of urinary bladder malignancies were performed with the optical fiber of the OMA system inserted through the biopsy channel of a cystoscope during the diagnostical procedure. The spectral recordings from urinary bladders, obtained at 337 nm and 405 nm excitation, revealed fluorescence features which can be used to demarcate tumor areas from normal mucosa. The fluorescence emission might also be useful to characterize different degrees of dysplasia.
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