Efficacy studies are required for regulatory approval of new medical treatments in the United States and elsewhere. Although efficacy studies may demonstrate safety and efficacy, they are not always sufficient for characterizing the effects of a treatment in actual clinical practice. Ongoing outcomes research is necessary to identify outcomes of treatment and treatment patterns in actual clinical practice. Criteria for evaluating palliative treatments in outcomes research must reflect the treatment's capacity to relieve symptoms while entailing minimal risks and adverse effects. However, the measurement of symptom relief as a result of treatment is prone to error because symptoms are inherently subjective and may be influenced by a variety ofnon-treatment factors, such as individual perception, physical exertion, and concurrent symptom management strategies. PDT patients treated with Photofrin® and 630-nm light at our center have had reduced dysphagia grade and stable performance status for approximately one month after PDT (N= 7-26), but these effects did not necessarily persist at the three-month followup interval. Preliminary data on five patients collected in a pilot study of a new symptom burden measurement tool suggest that the perceived burden ofphotosensitivity may increase with time. Fatigue, poor appetite and decreased overall quality of life appear to be the most troubling symptoms for our late-stage esophageal cancer PDT patients. The least burdensome symptoms were anxiety, pain and depression.

In this presentation, the utility of PDT will be described in a variety of clinical situations. Attention will not only be paid to areas of recognized utility in skin and mucosal membrane lesions, but also areas of clinical investigation which are ongoing. Special attention will be given to lesions of pleural and peritoneal cavities and some discussion will be aimed at other targets as well, including treatments of selected solid tumors by interstitial techniques.

We have performed 800 photodynamic therapy (PDT) treatments in over 300 patients at the University of Pittsburgh since 1996. Over 150 patients have undergone PDT for palliation of dysphagia for esophageal cancer. Of the first 77 dysphagia improved in 90.8% with a mean dysphagia-free interval of 80 days. An expandable metal stent was required for extrinsic compression in 19 patients. We have treated 14 high-risk patients with early esophageal cancer or Barrett's high-grade dysplasia for curative intent. At a median follow-up of 12.8 months eight remain free of cancer. Over 100 patients have undergone PDT for lung cancer. Sixty-two patients received 77 courses for palliation. Thirty-five patients were treated for non-massive hemoptysis with resolution in 90%. Forty-four patients were treated for dyspnea with improvement in 59%. A subset of seven high-risk patients with early lung cancer were treated with curative intent. A complete response was seen in 7/10 lesions at a mean follow-up of 30 months. PDT offers good palliation for both advanced esophageal and lung cancer. The role of PDT for curative intent needs further investigation in protocol settings. In our preliminary experience we have treated a small number of non-surgical, high-risk patients with a reasonable success rate.

In a phase II trial we treated more than 100 patients with malignant brain tumors with 2-mg/kg Photofrin iv. and intraoperative cavitary PDT. We concluded that PDT was safe in patients with newly diagnosed or recurrent supratentorial malignant gliomas. Regression analysis showed that pathology, performance grade and light dose were significantly related to survival time. We identified a prolongation of survival in selected patients when an adequate light dose was used. The surgical mortality rate was less than 3%. We have initiated two randomized prospective trials - the first, to determine if the addition of PDT to standard therapy [surgery, radiation and/or chemotherapy] prolongs the survival of patients with newly diagnosed malignant astrocytic tumors; and the second, to determine whether high light dose PDT [120 J/cm2] is superior to low light dose PDT [40 J/cm2] in patients with recurrent malignant astrocytic tumors. In the first 20 months of these clinical studies, 90 patients have been recruited. There were 52 in the recurrent study and 37 in the newly diagnosed study. 64% of the tumors were glioblastoma and 23% malignant astrocytoma or malignant mixed glioma. In the trial of newly diagnosed tumors 17 were randomized to surgery with a mean age of 58 ! 2.9 [sem] and 20 to surgery plus PDT with a mean age of 54 ! 2.5. In recurrent glioma trial 26 were randomized to low light dose PDT [mean age 48.1 ! 2.7] and 26 to high light dose [age 52 ! 2.7]. An update of our phase 2 data and a description of brain tumor PDT techniques is presented below. The clinical studies are supported in part by grant CA 43892 awarded by DHHS/NIH/NCI.

Photodynamic Therapy (PDT) using second-generation photosensitizer Aluminium Phtalocyanine (NIOPIC, Russia) (PS) in dose 0.5-0.8 mg per kg of body weight have been provided in 15 patients with breast cancer. In 5 patients with T2-T3NOMO primary tumor was treated as the preoperarive treatment, radical mastectomy has been fulfilled after PDT with subsequent histological examination. 10 patients had local recurrencies of breast cancer after combined treatment, chemotherapy and radiotherapy. Fluorescent diagnostics of tumor, accumulation of PS in tumor, adjacent tissue before and during PDT was fulfilled. For PDT semiconductive laser -X=672+2nm, P=1,5 W was used. Treating primary tumor interstitial irradiation has been done in light dose l5OJIcm3. In patients with skin metastases multiple surface irradiations were provided with interval 24-72 hours and total light dose 400-900 J/cm2. In cases of preoperative PDT pathomorphosis of different degree has been found in all cases. In patients with advanced skin recurrencies of breast cancer partial regression of tumor has been got in 2 monthes after PDT or progression with lung or bone metastasis. In 5patients with early skin metastases in 80% complete responce've been got 2 monthes after treatment with recurrencies in 6 monthes in 60% patients. Our experience show pronounced efficacy of PDT for treating breast cancer.

Objective: The goal ofthis study was to determine a relationship between light dose and the delay time between administration of Verteporfin and light to ablate canine esophageal mucosa. Materials and Methods: Verteporfin was administered IV (0.75 mg/lg). 630-nm light from KTP/Dye laser was delivered using a 9-cm diffuser inside a specially designed reflective 7-cm windowed balloon. Initially, animals were treated at doses of 160 J/cm, 180 J/cm, 200 J/cm, and 220 J/cm 2-3 hours after Verteporfin injection. Based on the acute response in these animals and the plasma Verteporfin clearance, other treatments were devised and tested at 60 J/cm (at 1 5 minutes), 80 J/cm (22 minutes), 100 J/cm (30 minutes), and 145 J/cm (60 minutes). Results: In initial animals, 200 J/cm at 2 hours induced acceptable mucosal ablation. Using this light dose and the plasma drug clearance, subsequent treatments were tested resulting in similar injuries using 60 3/cm (15 minutes), 80 3/cm (22 minutes), 100 3/cm (30 minutes), and 145 J/cm (60 minutes). Conclusions: A relationship was established relating the light dose and the delay time between administration oflight and Verteporfin for ablation ofnormal canine esophageal mucosa.

Tumor hypoxia, either pre-existing or as a result of oxygen bleaching during PDT light irradiation, can significantly reduce the effectiveness of a PDT treatment. To overcome the effect oftumor hypoxia, we propose using supplemental hyperoxygenation during a PDT treatment. The mechanism for the tumor cure enhancement ofthe combined therapy is investigated in the study by using an in vivo/in vitro assay. Tumors treated with PDT/hyperoxygenation are harvested at various times after the light irradiation and cultured in vitro for colony formation, thus separating the direct cell killing by PDT and secondary vascular effect. The results show that, when combined with hyper-oxygenation, the cell survival (colony formation) rate immediately after a PDT treatment is significantly improved over that treated without hyper-oxygenation, suggesting an enhanced direct cell killing. Cell survival rates were unchanged for the first 4 hours after a PDT treatment, but decreased significantly at 1 8 hours, suggesting that secondary vascular damage did take effect, but not until a period of at least 4 hours after light irradiation. These results further confirm our earlier observation that when a PDT treatment is combined with hyper-oxygenation, it can be more effective in controlling hypoxic tumors.

The P3-Approximation was used to optically characterize ex-vivo human prostate and to investigate the role that optical inhomogeneity, at the boundary between the prostate and its surrounding tissues, will play in tissue light dosimetry. Each prostate was found to be optically homogeneous, observing only slight variations in the scattering coefficient (o = 29 mm' to 34 mni'). Every prostate had the same absorption coefficients (o =0.08 mm') and anisotropy factors (g = 0.94). The variation in scattering coefficient had nearly inconsequential effects on theoretical fluence calculations, fluence being the key method of quantifying light for dosimetry purposes. To explore the boundary effects, a prostate was suspending above the bottom of a black painted bowl. Radiance measurements were taken and then intralipid was added until the prostate was completely submersed. The radiance measurements after the addition of intralipid varied greatly from the initial measurements without intralipid. An increase in light intensity was observed, as well as alteration in the shape of the radiance profile implying that surrounding tissues will have an effect on light transport. These must be characterized and integrated into a comprehensive light dosimetry model for PDT ofprostatic carcinoma.

We present a simple fluorescence imaging system for use in the demarcation and monitoring of skin cancers. The imaging system simultaneously acquires four spectrally specific images at 635, 600, 540 and 400nm. The incorporation of an image intensifier, coupled with high speed image processing gives video update rates. The integrated light source is based on low cost fluorescent bulbs, shaped to fit around the lens and phosphor coated to give a peak emission near 400nm, matched to the absorption maximum of PpIX. We apply this system to monitoring the fluorescence of the PpIX photosensitiser before during and after ALA based PDT.

Recently there is some interest in using photobleaching rates in vivo to correctly interpret dose deposition, and even to predict the dose deposited within tissue. There are many factors causing photobleaching rate changes related to localization, transport and oxygen availability which can confound the interpretation of a bleaching rate measured in vivo, and perhaps the most problematic issue is that the fluorescence signal can come from multiple depths within the tissue and this depth can change during the course of treatment. In this study we examine the use of a micro-fiber bundle designed to sample fluorescence from superficial depths, and use this device to illustrate that spatially localized measurements can be used to extract the intrinsic photobleaching rate of a photosensitizer within a scattering medium. At the same time, while the depth from which the tissue signal originates can be confined, in vivo measurements illustrate that multiple bleaching rates exist in vivo in addition to the spatial problem. This latter observation, suggests that bleaching in tissue is complicated enough, that spatially localized measures of intrinsic fluorescence photobleaching are needed to allow mechanistic interpretation from in vivo measurements.

High-dose chemotherapy, followed by autologous hematopoietic stem cell (HSC) transplantation, is widely used for the treatment of cancer. However, contaminating tumor cells within HSC harvests continue to be of major concern since re-infused tumor cells have proven to contribute to disease relapse. Many tumor purging methods have been evaluated, but all leave detectable tumor cells in the transplant and result in significant loss of HSCs. These shortcomings cause engraftment delays and compromise the therapeutic value of purging. A novel approach integrating automated scanning cytometry, image analysis, and selective laser-induced killing of labeled cells within a cell mixture is described here. Non-Hodgkin's lymphoma (NHL) cells were spiked into cell mixtures, and fluorochrome-conjugated antibodies were used to label tumor cells within the mixture. Cells were then allowed to settle on a surface, and as the surface was scanned with a fluorescence excitation source, a laser pulse was fired at every detected tumor cell using high-speed beam steering mirrors. Tumor cells were selectively killed with little effect on adjacent non-target cells, demonstrating the feasibility of this automated cell processing approach. This technology has many potential research and clinical applications, one example of which is tumor cell purging for autologous HSC transplantation.

We describe a compact endoscopic imaging system for the detection of gastro-intestinal cancers. This system is designed to image ALA-induced PpIX fluorescence and allows the clinician to perform fluorescence endoscopy and white light endoscopy simultaneously. The system comprises a filtered mercury arclamp for illumination and fluorescence excitation, a dual camera system coupled to an endoscope for detection and a desktop PC for processing and display of images. The result is a real-time colour image onto which fluorescence information is superimposed. Preliminary in vivo results indicate an increased fluorescence level within cancers in comparison with normal tissue. In addition, the system allows point spectroscopy to be carried out by the insertion of an optical fibre probe down the biopsy channel of the endoscope.

The investigations of tumour damage in vivo due to photodynamic therapy (PDT) using aluminium sulphophthalocyanine were performed. Obtained results showed that antiturnour action of PDT is connected with different mechanisms of tumour damage: necrosis, apoptosis, and xocliroinato1is of tumour cells as well as vascular damages (thrombosis, destruction ofvascular walls).

Photodynamic therapy (PDT) elicits a strong acute inflammatory response that has both local and systemic (acute phase response) attributes. The insult mediated by PDT-induced oxidative stress at the targeted site triggers a complex multifactorial response engaging host defence mechanisms associated with the inflammatory process to participate in the eradication of the treated tumor. Inflammatory cytokines are important mediators of critical events in this process as they regulate the activity of inflammatory, endothelial and other cells. The initial stimulus for enhanced production and release of cytokines likely originates from several types of events, such as activated transcription factors and complement deposition. The PDT-induced complement activation appears to be directly linked to the enhanced expression of various cytokines, including chemokines such as KC (in mouse models), and classic inflammatory cytokines such as IL-1β, TNF-α , IL-6 and IL-10. A variety of interventions that modulate the activity of particular cytokines performed in conjunction with PDT were shown to influence the therapy outcome. The treatments such as using blocking antibodies and local or systemic cytokine delivery may either reduce or dramatically improve the curative effect of PDT. The inflammatory and related cytokines that at present appear particularly interesting and merit further investigation for use as adjuvants to PDT are IL-3, IL-8, IL-15, TNF-α, IFN-γ, G-CSF and GM-CSF.

A new photosensitizer, presently designated QLT0074, may be useful for the treatment of skin conditions, particularly those mediated by T lymphocytes, with photodynamic therapy (PDT). QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. Low concentrations of QLT0074 and blue light were sufficient to induce apoptosis in peripheral blood T cells or Jurkat T lymphoma cells as indicated by expression of the apoptosis-associated mitochondrial 7A6 marker, annexin-V labeling or activation of capsase-3 and cleavage of the capsase substrate poly(ADP-ribose)polymerase (PARP). Flow cytometry studies performed following PDT showed that peripheral blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater degree than T cells with low CD25 levels. This effect of PDT was also shown by the reduction in the percentage of T cells that expressed other activation-associated markers such as very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). In the case of T cells that remained viable following PDT, CD25 expression was lower while CD54, CD95 and HLA-DR levels were unchanged. Thus, PDT with QLT0074 has selective, dose-dependent effects on T cells in vitro.

Early molecular damages have been studied in a series ofhuman tumor and rodent cell lines following photodynamic therapy (PDT) sensitized by the silicon phthalocyanine Pc 4. Pc 4 preferentially localizes in mitochondria, and upon photoirradiation, immediate photodamage to the anti-apoptotic oncoprotein Bcl-2 is observed. The loss ofthe native 26-kDa protein, as evidenced by western blot analysis, is accompanied by the generation of a 23 -kDa fragment from a small portion of the molecules as well as a variety ofhigher molecular weight protein bands indicative ofphotochemical crosslinking ofBcl-2 to itself, to (pro-apoptotic) homologs, or to other nearby proteins. The changes in Bcl-2 are apparent immediately upon exposure ofPc 4-loaded cells to activating red light, occur in the cold, and are not dependent upon caspase-3 or other proteases. Crosslinking is also observed for the intermediate filament protein vimentin. The results implicate Bcl-2 (and perhaps vimentin) as important molecular targets that lead to apoptosis in Pc 4-PDT-treated cells.

In order to determine the optimum light irradiation condition to treat deep lesions, we studied influence oflight intensity and repetition rate of nanosecond light pulses on photodynamic therapy(PDT) with PAD-S3 1(1 3,1 7-bis[lcarboxypropionyl]carbamoylethyl-3-ethenyl-8-ethoxyiminoethylidene-7-hydroxy-2,7,12,18-tetramethyl porphyrin sodium) to mouse renal carcinoma cell line(Renca) in vitro. The oxygen consumption and photobleaching were measured to explain this influence. We used the short light pulses(2: 670 nm, FWHM: 5 ns) at the peak intensity of 06, 1 .8 and 3.6 MW/cm2, repetition rate of 30 and 5 Hz, and used the total fluence of 40 J/cm2. We obtained over 80% cell growth inhibition rate at 0.6 MW/cm2 and 5 Hz. This irradiation condition was the lowest peak intensity and lowest repetition rate in our study. From the measurement for oxygen consumption in the well(culture medium) by a microelectrode and oxygen diffusion calculation by the finite element model, we predicted that the low repetition rate may supply sufficient oxygen for PDT by diffusion process. The photobleaching detection by fluorescence measurement showed that bleaching occurred more intensively at lower peak intensity i.e., the PDT process advanced in this intensity. With high peak intensity irradiation such that we used, it is supposed that the waste energy which was not absorbed by photosensitizer to suppress effective light dose for the PDT, since the number of photon per pulse was extremely larger than the number of the photosensitizer molecule in the cell. The PDT effect at our optimum irradiation condition (Over 80% cell growth inhibition rate) was higher than that by continuous wave irradiation at the same average power density and energy dose(40%). Therefore, we conclude that the irradiation condition of low peak intensity(O.6MW/cm2) and low repetition rate (5Hz) using the nanosecond pulsed irradiation was suitable for the PDT in cultured cell in vitro with PAD-S3 I.

Measurements of tissue oxygen partial pressure (pO2), blood flow and blood pressure were recorded in rat mammary R323OAc tumors during treatment with verteporfin-based photodynamic therapy. Microelectrodes of 12 micron tip diameter were used in a fixed position throughout the measurements to provide data on the temporal changes at a single location within the tissue. The microelectrode signal changes in P°2 indicated an overall trend to acute loss of P°2 after treatment, but with some notable exceptions where the P02 did not decrease either during the treatment or after treatment. These regions which showed little change in P02 were correlated with locations in which the P°2 was high to begin with before the initiation of treatment. Blood flow decreased during treatment, with the dominant effect immediately upon the initiation of treatment.

The effects of fluence rate are investigated in human glioma spheroids incubated in 5-aminolevulinic acid (ALA). It is shown that the response of glioma spheroids to ALA-mediated PDT depends strongly on the rate at which the light dose is delivered. At low doses, lower fluence rates are more effective. For example, at a dose of 50 JIcm2, near total spheroid kill is observed at fluence rates of as low as 1 0 mW/cm2. Below 1 0 mW/cm2, however, treatments are not as effective. The fluence rate effect is not as pronounced at higher doses where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications ofthese findings are discussed.

In cell culture, photodamage induced by clinically-effective agents (e.g., Photofrin, SnET2, mTHPC, LuTex, HPPH, NPe6, ALA, Pc-4 or BPD) appears to involve mitochondria and/or lysosomes. When mitochondria are the major PDT target, a rapid apoptotic response is often observed, associated with release of cytochrome c, a trigger for caspase-9 activation. Fluorescence localization studies revealed that many of the 'mitochondrial-targeting' agents do not actually localize in mitochondria, but bind to a variety of intracellular membranes. The initial photodynamic effect involved photodamage to the anti-apoptotic protein Bcl-2, leaving the pro-apoptotic protein Bax unaffected. Subsequent apoptotic interactions between Bax and mitochondria may therefore represent the first step in the mitochondrial apoptotic pathway. We recently reported that certain bile acids can lower the threshold of photodynamic mitochondrial interactions leading to apoptosis. We suggest that this effect derives from the promotion of an interaction between Bax and the mitochondrial membrane interaction leading to cytochrome c release.

Photodynamic treatment of cancer cells is known to eventually cause cell death in most cases. The precise pathways and the time course seem to vary among different cell types and modes of photodynamic treatment. In this contribution, the focus was put on the responses of human colon carcinoma cells HCT-116 within the first 15 minutes after laser irradiation in the presence of Photofrin® II (PII). To monitor the cell response in this early time period laser phase microscopic imaging was used, a method sensitive to changes in overall cell shape and intracellular structures, mediated by changes in the local refractive index. Laser irradiation of cells loaded with PII induced a significant reduction of the phase shifts, which probably reflects the induced damage to the different cellular membrane structures. The data suggest that even within the first 30 s after the onset of laser illumination, a significant reduction of the phase shifts can be detected. These results underline that laser phase microscopy is a suitable diagnostic tool for cellular research, also in the early time domain.

It has been suggested by us earlier that interactions of excited triplet sensitizer (3PS) and native free radicals compete with Type I (sensitizer radical mediated) and Type II (singlet oxygen mediated) mechanisms during PDT. Evidence such as fall in the overall radical concentration in vivo ( in mice tumors) during PDT and in the life time of 3PS caused by free radicals supported this assumption In addition, following results have been obtained recently. 1.) Excited Photofrin II and m-THPC affected luminol dependent chemiluminescence (CL) generated by respiratory burst of macrophages like free radical inhibitors. 2.) Quantification of spin trapping for chemical and in vitro systems by kinetic ESR spectrometry yielded detailed knowledge of triplet-doublet interactions 3.)Measurements in open systems (tank reactor) yielded data for the interactions between 3PS and peroxy type radicals 4.)Simulation of experimental data based on mechanisms suggested gave fair agreement. Based on experimental results new PS-s called Antioxidant Carrier Sensiters (ACS-s) have been devised, synthesized and tested one of them showing enhanced activity for PDT.

The exogenously stimulated formation of intracellularly generated Protoporphyrin IX, a precursor of heme, is becoming one of the fastest developing areas in the field of Photodynamic Therapy (PDT). We have examined the degree of protection of several scavengers, aminoacids and compounds related to glutathione metabolism, to the photodamage induced by 5-aminolevulinic acid (ALA)-mediated PDT, employing the LM2 cell line, derived from a mammary murine adenocarcinoma. We have exposed the cells to different concentrations of the scavengers, 24 before PDT, during PDT, and 19 hr after treatment. We defined the protection grade (PG) as the ratio between cell survival after ALA-PDT treatment in the presence of the protector and cell survival after ALA-PDT treatment. We found that L-tryptophan (PG=8.3 at 2mM), N-acetyl-L-cysteine (PG= 7.9 at 30 mM), L-cysteine (PG=7.81 at 8mM), S-adenosyl-L-methionine (PG= 7.86 at 8mM), melatonin (PG=6.81 at 8mM) and glycine (PG=6.8 at 40 mM) are the best protectors to PDT damage, followed by L-methionine (PG=4.38 at 0.8 mM), mannitol (PG=2.32 at 2 mM) and reduced glutathione (PG=3.41 at 0.8 mM), whereas oxidized glutathione does not exert any protection. The implications of these results in the photodamage induced by ALA-PDT is discussed.