Sune Svanberg

Photodynamic Therapy in Interplay with Fluorescence Diagnostics in the Treatment of Human Superficial Malignancies

In the present paper we address the question if fluorescence diagnostics can be used to monitor and possibly predict the outcome of photodynamic therapy (PDT) using the tumor seeking agents Photofrin and (delta) -aminolevulinic acid (ALA). The degree of selective uptake may vary from patient to patient and it would be interesting to use the drug-related fluorescence signal as a tool to tailor the treatment strategy. Clearly, the fluorescence intensity cannot be directly related to the tissue drug contents because of varying absorption and scattering properties of the tissue. However, because of the real-time capability of fluorescence it is interesting to see how far the fluorescence information can be utilized for optimizing the light delivery. Patients with basal cell carcinoma and spread metastatic breast cancer in the skin were treated. Two different doses, 1 and 2 mg/kg b.w. of Photofrin (Quadra Logic, Vancouver, Canada) were used. The treatment laser was a Nd:YAG pumped dye laser (Multilase Dye 600, Technomed International, Paris/Bron, France). The system provides 1064 nm IR and 532 nm green light from the Nd:YAG laser as well as red light in the region 620 - 670 nm from a dye laser. The treatment procedure was preceded by fluorescence measurements for allowing comparisons between the diagnostic signals and the treatment outcome. At the end of the treatment, fluorescence was again monitored to assess the degree of bleaching manifested by the appearance of an additional red peak. Our data on the connection between fluorescence signals, delivered dose and observed treatment outcome are presented and the potential of imaging fluorescence monitoring in PDT dosimetry is discussed.

Fluorescence Diagnostics of Head and Neck Cancer Utilizing Oral Administration of Delta-amino Levulinic Acid

Tissue fluorescence measurements utilizing a point monitoring fluorosensor and a multi-color fluorescence imaging system were performed in patients with various malignant tumors in the head and neck region. Also precancerous lesions were investigated and characterized by means of laser-induced fluorescence. Oral administration of low-dose (delta) -amino levulinic acid was used for the induction of protoporphyrin IX, which exhibits a strong fluorescence in the red wavelength region. The blue-green fluorescence emitted from the tissue natural chromophores was also utilized for tumor demarcation. By incorporating both Protoporphyrin IX and natural fluorescence in a tumor demarcation criterion a tumor demarcation with a tumor-to-normal-surrounding-tissue of about 5 - 10:1 contrast was achieved. Hypertrophic tissue did not exhibit tumor specific signals.

Fluorescence characteristics of atherosclerotic plaque and malignant tumors

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.

Diffusely scattered femtosecond white-light examination of breast tissue in vitro and in vivo

Multispectral studies of light propagation in female breast tissue have been performed. Short pulses of white light were generated by using self-phase modulation of a high-power laser pulse focused into a cuvette filled with water. The white light pulses illuminated the tissue and the scattered light was recorded with time- and wavelength dispersion by a streak camera. Measurements were performed on breast mastectomies in vitro and measurements on healthy breast tissue in vivo. The reduced scattering coefficient and the absorption coefficient of breast tissue were obtained in different wavelength regions by fitting solutions of the diffusion equation to the experimental data. Significant variations in the magnitude of the optical properties could be seen between the different individuals. No characteristic spectral discrepancy for tumor tissue was found.

Multicolor fluorescence imaging system for tissue diagnostics

A multicolor fluorescence imaging system for tissue diagnostics has been constructed. Examples given to illustrate the system performance are atherosclerotic plaque lesions from human artery samples and a malignant rat brain tumor model. The system simultaneously monitors fluorescence images at four different wavelengths, enabling spatial as well as spectral information to be extracted. By selection of band pass filters an artificial image of the lesion under study can be formed, based on an optimal contrast function of four fluorescence intensities. The image is constructed in a computer and displayed in false colors on a monitor. Pulsed excitation light from an N2 laser provides the possibility of using a gated detector, and in this way suppress room light to an undetectable level. Images can thus be recorded under white light illumination during visual inspection, increasing the usefulness of the system.

Optical detection of human urinary bladder carcinoma utilising tissue autofluorescence and protoporphyrin IX-induced fluorescence following low-dose ALA instillation

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.

Tumor detection using time-resolved light transillumination

A time-gated technique to reduce the effect of light scattering when transilluminating turbid media such as tissue is demonstrated. The concept is based on transillumination with picosecond laser pulses and time-resolved detection. By detecting only the photons with the shortest travelling time, and thus the least scattered photons, the contrast can be enhanced. Measurements on a tissue phantom as well as breast tissue in vitro are presented. It is demonstrated that the spatial resolution can be enhanced by using the time-gated technique. It is also shown that differences in scattering properties may be more pronounced than differences in absorption properties when demarcating tumor from normal tissue.

Laser-induced fluorescence diagnostics of basal cell carcinomas of the skin following topical ALA application

Fourteen patients with superficial basal cell carcinomas (BCCs) and fifteen patients with nodular BCCs were investigated by means of laser-induced fluorescence (LIF) in connection with photodynamic therapy (PDT). Topical application of (delta) -amino levulinic acid (ALA) was performed six hours prior to the treatment session. Fluorescence spectra were recorded, using a point-monitoring system with an excitation wavelength of 405 nm. The measurements were performed in scans over the lesion and the surrounding normal skin before application of ALA, and immediately before and after the laser treatment. The selective uptake of the photosensitive resulted in a fluorescence intensity ratio of 2.4:1 for superficial BCCs and 2.5:1 for nodular BCCs. If the fluorescence intensity was divided by the autofluorescence, this resulted in a contrast enhancement of about a factor 6 for tumor tissue. In seven patients (five with nodular BCC and two with superficial BCC), additional fluorescence measurements were performed two and four hours following the ALA application, and two hours after the PDT procedure. Thus, the kinetics of the transformation of ALA to protoporphyrin IX (PpIX) could be followed, which indicated that the synthesis of PpIX was more rapid in the tumor than in the normal tissue. After four hours, the PpIX level inside the tumour was saturated, while there still was an accumulation in the surrounding skin. The highest contrast between tumor and normal skin was reached within two hours after the ALA application.

Clinical fluorescence diagnosis of human bladder carcinoma following low-dose photofrin injection

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.

Laser-induced fluorescence in medical diagnostics

We have performed extensive investigations using laser-induced fluorescence in animal as well as human tissue in order to localize diseased tissue and thus discriminate such tissue from normal surrounding areas. In characterizing different tissue types the endogenous fluorescence (autofluorescence) as well as specific fluorescence from different photosensitising substances was utilized. We have investigated different experimental and human malignant tumors in vivo and in vitro as well as atherosclerotic lesions in vitro. A fiber-optic fluorosensor was constructed and used in the experiments and in the clinical examination of patients. Dimensionless spectroscopic functions were formed to ensure that the signals were independent of clinically uncontrollable variables such as distance variations, tissue topography, light source fluctuations and variations in detection efficiency. A multi-color two-dimensional imaging system was constructed for real-time imaging. The system was tested peroperatively and during standard examination patient procedures. Besides utilizing the time-integrated fluorescence signal we have also investigated the possibility of incorporating time-resolved fluorescence characterization.