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Photodynamic Therapy for the Treatment of Squamous Cell Carcinoma Using Benzoporphyrin Derivative JEFFREY MARCUS, MD, MPH EDWARD GLASSBERG, MD LYNN DIMINO-EMME, MD RICHARD YAMAMOTO, MD RONALD L. MOY, MD SANDOR G. VARI, MD THANASSIS PAPAIOANNOU, MS VAN1 R. PERGADIA, MS WENDY J. SNYDER, MS WARREN S. GRUNDFEST, MD GARY P. LASK, MD

BACKGROUND.Photodynamic

therapy (PDT) involves laser light excitation of a tumor-localizing photosensitizer to destroy neoplasms. Benzoporphyrin derivative BPD) is a new photosensitizer with several favorable characteristics. OBJECTIVE.Studies were designed to: I) assess the efficacy of BPD-mediated PDT in treating in vivo squamous cell carcinomas (SCC); 2) obtain dosimetry data for BPD and laser parameters; and 3) establish clinical and histologic correlates of BPD-induced tumor regression. METHODS. Human SCC was implanted into nude mice. One group received BPD followed by laser light of 250 J/cmZ

M

ultiple treatment modalities currently exist for nonmelanoma skin cancer. The more frequently utilized approaches include excision with or without Mohs micrographic surgery, cryosurgery, and electrodesiccation and curettage.' Radiotherapy has been used either by itself or as a postoperative adjuvant. Newer approaches include immunotherapy and photodynamic therapy (PDT). PDT relies on the excitation of a pharmacologically inactive photosensitizing dye in vivo by laser light of an appropriate wavelength to produce a local toxic reaction. Essential to this technique is the selective retention of the photosensitizer within malignant tissue, after intravenous administration, at higher levels than in the normal surrounding tissue. Subsequent localized laser activation From the Division of Dermatology (JM, EG, RLM, GPU, UCLA Medical Center, Los Angeles, California; the Division of Dermatology (EG, LDE, RY, GPU, UCLA-Harbor Medical Center, Los Angeles, California; and the Laser Research and Technology Development Department (SGV, TP, VRP, WJS, WSG),Cedars-Sinai Medical Center, Los Angeles, California. Address correspondence and reprint requests to: Jeffrey Marcus, MD, MPH, Division of Dermatology, UCLA Medical Center, 200 UCLA Medical Plaza, Suite 450, Los Angeles, CA 90024.

0 1994 by Elsevier Science Inc. 0148-0812/94/$7.00

from an argon-pumped dye laser at 690 nm. Three control groups included laser energy alone, BPD alone, and no treatment. RESULTS.A t day 21 posttreatment only PDT-treated tumors showed a statistically significant decrease in tumor volume and complete cure rate. Clinical resolution (scar) correlated perfectly with histologic resolution (scar). CONCLUSION. Human SCC in a nude mouse model responds to BPD-mediated PDT. J Dermatol Surg Oncol 1994; 20:375-382.

of the photosensitizer, in the presence of oxygen, generates free radicals, which then destroy malignant tissue.2 The photodynamic effect was first investigated by Raab3in 1900 when he observed that neither low doses of acridine dye nor light had an effect on paramecia, but together they were lethal. The last two decades have witnessed an intensified effort to clarify and maximize the mechanisms of the photodynamic effect through basic science research. Clinical experience has been gained for head and neck malignancie~,~,~ central nervous system cancer^,^,^ esophageal and gastrointestinal tumors,*-1° ocular malignancies," gynecologic cancers,12 bladder turn or^,'^-'^ and cutaneous neoplasm^.'^-^^ Cutaneous lesions are ideal for investigation of the photodynamic effect because their superficial location allows direct access for both photosensitizer and laser light. The cutaneous neoplasms that have been treated with photodynamic therapy include squamous cell carcinoma (SCC), Bowen's disease, basal cell carcinoma, melanoma, Kaposi's sarcoma, cutaneous T cell carcinoma, and cutaneous rnetastase~.'~-~~ Most clinical studies have been done using hematoporphyrin derivative (HPD) and Photofrin I1 (Quadra

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Logic Technologies, Inc., Vancouver, BC, Canada), a purified form of HPD.33These photosensitizers have an absorption peak at approximately 630 nm. Recently these photosensitizers have been successfully used to treat a variety of cutaneous tumors in humans. However, photosensitivity is a major limiting side effect of HPD.24 While many normal tissues clear HPD faster than tumors, the skin clears it very slowly. The skin can retain HPD for 30 days after injection, making it necessary to shield patients from sunlight for a significant period of time.34 Furthermore, the HPD-activating wavelength of 630 nm does not allow for as deep penetration into tissue as do other photosensitizers with longer absorbance wavelengths. Therefore, there is interest in findmg new photosensitizers that absorb greater at longer wavelengths and localize better in tumors. Benzoporphyrin derivative (BPD), a type of chlorin, is a promising new photosensitizer synthesized from protop ~ r p h y r i n BPD . ~ ~ has certain properties that make it a superior photosensitizer to HPD. It has an absorbance peak in the red spectrum of approximately 690 nm that is nearly four times greater than Photofrin 11’s red peak absorbance of approximately 630 nm, resulting in more efficient light a b ~ o r p t i o n . ~ Furthermore, ~ , ~ ~ , ~ ~ at a wavelength of approximately 690 nm, light penetrates deeper into tissues than at 630 nm.35In addition, photosensitivity in animal models is reduced to only 24 hours compared with 30 days as for HPD.34,37-39 Because of the potential improvement in cure rate and reduction in the consequent side effects that a new photosensitizer like BPD offers, our study was designed as the first attempt to assess the efficacy of BPD-mediated PDT in treating SCC in a nude mouse model. In the process, we hoped to establish some preliminary parameters for photosensitizer and laser light dosimetry for future studies of cutaneous neoplasms in vivo. Finally, we sought to establish a correlation between histologcal and clinical resolution of these treated tumors so as to support the concept that clinical resolution of a tumor by PDT is not just a temporary reduction in tumor volume with consequent regrowth of residual tumor cells, but rather that BPD-mediated PDT can provide a complete cure for certain cutaneous neoplasms.

Materials and Methods Tumor Growth and lmplantation Human SCC line SCC-25 (American Type Culture Collection, Rockville, MD) was obtained in lo8 cell lots. Thirty-six male, athymic nude mice, species nu-nu, 6 - 7 weeks old (Harlan Sprague Dawley, Indianapolis, IN), were obtained with an average weight of 20 g. The mice were housed five per cage in a sterile microenvironmental

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Figure 1. Subcutaneous injection of lo6 human SCC cells into the hindquarter of an athymic nude mouse.

habitat in the vivarium at Cedars-Sinai Medical Center (Los Angeles, CA). The tumor cells were shipped in proliferating lots in Dulbecco’s Modified Eagles Medmm-Hams F12 nutrient media (Sigma Chemical Co., St. Louis, MO). Ten million SCC cells in 0.15 ml of phosphate-buffered saline (Signla Chemical Co.) were injected subcutaneously into the hindquarter of each mouse (Figure 1). These cells were allowed to proliferate until they obtained 0.4 - 0.6-cm visible nodules at 2 weeks postimplantation (Figure 2). At 2 weeks postimplantation, all tumors were measured in three dimensions with digital calipers accurate to 0.1 mm. Tumor volumes were calculated using the formula: 4/ 3n(D, X D2 X D 3 / 8 ) .

Figure 2. Clinically apparent tumor 2 weeks after implantation of the cells into athymic nude mouse,

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Treatment and Control Groups Thirty-six mice were implanted, and of these, 33 developed a visible tumor. A total of 24 mice survived to complete the study and one control mouse had its tumor excised prior to any treatment, at day 0, in order to ascertain the presence of a true SCC tumor, rather than artifact. The mice were grouped according to the following scheme: five mice were in the absolute control, no treatment (group A); five mice were in the BPD only (group B); six mice were in the laser light only (group C); and eight mice were in the BPD and laser light (photodynamic therapy) treatment (group D).

Photodynamic Therapy The photosensitizing compound used in this study was a porphyrin-related compound, benzoporphyrin derivative (a generous gift from Quadra Logic Technologies, Inc., Vancouver, BC, Canada). The BPD was shipped as a soluble powder and was reconstituted before the experimental trial as a 1-mg/ml stock solution in dimethylsulfoxide (Sigma Chemical Co.) and stored in sterile covered plastic 15-ml conicals. The stock solution was diluted in colorless balanced salt solution (Sigma Chemical Co.) and injected intravenously into the tail vein of group B and D mice at a concentration of 2 mg/kg. The dye was allowed to incubate for 4 hours. All dye preparation and in vivo work was done under very dim light to avoid photobleaching and phototoxic effects. The laser light only group C and the PDT group D tumors were exposed to 150 J/cmZof power at a density of 200 mW/cmz from an argon-pumped dye laser (model MD-90; Meditic of America, Clearwater, FL) at a wavelength of 690 nm. The spot size at the target tissue measured l .O cm in diameter, and laser output was measured before and after each treatment. Energies varied less than 10% over time. During the approximately 15-minute laser sessions, the mice were under light general anesthesia using 50 mg/kg Ketamine and 50 mg/kg Xylazine intraperitoneally and supervised by the Cedars-Sinai Veterinary Department (Figure 3).

Data Collection Pretreatment measurements and photographs of each tumor were obtained. A biopsy was taken pretreatment to determine that the tumor had indeed proliferated. Biopsies, photographs, and measurements were again taken at 2 and 5 weeks postimplantation in all groups and volumetric measurements were calculated at the above time parameters (3 weeks posttreatment). Final excisional biopsies were taken under general anesthesia with an approximately 1.O-cm margin and serial sections were obtained to assess the presence or absence of tumor.

Figure 3. Laser treatment of the tumor in an athymic nude mouse 4 hours after intravenous injection of BPD (ie, PDT).

Statistical Methods Analysis of variance was used to compare the four groups on baseline values and difference from baseline after 21 days, with Bonferroni adjustments to P values for multiple comparisons. Tumor resolution rates were compared using Fisher’s exact test.

Results Tumor Growth Out of 36 animals injected with SCC tumor cells, 33 developed visible and palpable cutaneous tumors at the right flank injection site by 2 weeks postinjection. Tumors measured from 0.4 to 0.6 cm in diameter, with volumes ranging from 40 to 64 mm3. There were no statistical differences between tumor volumes of any of the experimental groups (A- D) at day 0 (2 weeks postimplantation, first day of treatment).

Clinical Resolution (Table 1) Out of eight tumors in the PDT group, all developed thick eschars by 1 week after treatment (Figure 4); by day 21, three of these animals had no palpable tumor, only residual scar tissue (Figure 5). Animals in the laser control group developed some transient tan pigmentation at the treatment site, but all tumors continued to enlarge with no signs of involution (Figure 6). All of the PDT treatment tumors that did not resolve completely showed partial

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Table 1. Clinical Resolution Group

Complete Clinical Resolution*

PDT Laser BPD Control

3/8t (scar) 0/6 0/5 0/5

Cfinicaf resolution correlated perfectly with histologic resolution.

t P = ,032.

regression, or at least temporary cessation in growth during the first 7- 14 days posttreatment. All other tumors in the pure control, and BPD-only groups continued to slowly enlarge by clinical observation during the first 2 weeks posttreatment. By 7- 14 days most tumors seemed to plateau somewhat in their growth, though none spontaneously decreased in size.

Figure 5 . Complete clinical resolution of the tumor with residual scar in an athymic nude mouse 3 weeks after PDT.

Tumor Volume By day 7 after treatment, average tumor volume of the PDT group decreased by 78%, while all other groups increased by 190-288% over the same period (Table 2). Although the average tumor volume of the PDT group slowly increased after day 7 (due to recovery of partial response tumors), the average tumor size of all other groups was significantly higher (two- to three-fold) than PDT-treated tumors at day 21 (end of experimental period). No other tumors in any of the other three experimental groups showed either complete or partial responses with regard to tumor volume.

Histologic Response All tumors evaluated for histology were completely excised. The control (untreated) tumor excised at day 0 for Figure 4 . Thick eschar formation in an athymic nude mouse 3 days after PDT.

tumor growth confirmation showed the typical changes of SCC with some cystic components. There was invasive anaplastic proliferation into the dermis, keratin pearl formation, mitotic activity, and marked pleomorphism of tumor nuclei (Figure 7A and B). Representative samples from each control group (AC) were biopsied at day 21 as well as all eight tumors in the PDT group. The three clinically resolved tumors were also histologically free of tumor showing only residua 1 cicatrix (Figure 8A and B); a fourth PDT-treated tumor showed only focal epithelial proliferation and residual in situ changes, and was counted as a partial response. Th'e remaining four PDT tumors showed frank SCC histologically. Most tumors in all groups showed some cystic comFigure 6 . N o change in the tumor except for tan pigmentation at the site of treatment in an athymic nude mouse 3 days after receiving only laser treatment.

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Table 2. Tumor Volume Group

Baseline (mm3)

I Week(mm3)

PDT Laser BPD Control

64

42 40

14 68 115 116

60

ponents, probably reflecting the mode of tumor implantation and possibly the xenograft origin. The complete cure rate of three out of eight tumors was statistically significant at P = .032 (Table 1).

Discussion In this study, we have shown that human SCC in a nude mouse model are responsive to PDT using a BPD photosensitizer. As Figure 9 shows, there was a clear and significant difference in mean change in tumor volume from baseline between the PDT group and various control groups. At 1 week posttreatment there was a 78% decrease in tumor volume in the PDT group. In contrast, all the other experimental groups increased by 190- 288% over the same period (Table 2). After day 7, the average tumor volume of the PDT groups slowly increased due to the recovery and growth of partial response tumors. However, at day 21 posttreatment, only PDT-treated tumors showed a mean decrease in tumor volume. Neither laser alone nor BPD had any negative effect on tumor growth, therefore, at 21 days posttreatment both groups showed a significant overall increase in tumor volume. We demonstrated a 38% total cure in the PDT group with three out of eight tumors showing clinical and histologic resolution of tumor at day 21. A fourth PDT tumor showed a partial response with only focal epithelial proliferation and in situ changes. In contrast, in the other three experimental groups, none of the tumors showed either complete or partial responses with regard to tumor volume or clinical and histolop resolution. It was also demonstrated in this study that clinical resolution correlated perfectly with histologic cure. The three clinically resolved PDT tumors were also free of tumor on histopathologic examination, showing only residual cicatrix. The remaining tumors showed frank SCC histologically. Furthermore, although we observed minimal localized tissue necrosis of the tumor and overlying skin, generalized photosensitivity was not observed under ambient low light conditions. In addition, we have obtained preliminary data on effective dosimetry using fixed BPD and laser parameters. Although the optimal dose of BPD is yet to be determined, we have demonstrated that PDT with 2 mg/kg of BPD and 150 J/cm2 is adequate for cure of SCC in a nude mouse model.

A

B Figure 7. Untreated tumor excised from an athymic nude mouse revealing the typical changes of SCC with cystic components. There is invasive anaplastic proliferation info the dermis, keratin pearl formation, mitotic activity, and marked pleomorphism of tumor nuclei. A) H 6 E, original magnification XlOO; B) H 6 €, original magnification X400.

PDT is a promising new therapeutic modality for the treatment of various malignancies. It involves the destruction of a neoplasm by the administration of a tumorlocalizing photodynamic sensitizer followed by direct exposure to visible i r r a d i a t i ~ n . An ~ , ~empirically ~,~~ determined lag period is necessary to allow some clearance of the photosensitizer from normal tissues. The laser energy used will depend on the individual photosensitizer used. A photosensitizer is best activated by light at its most effectively absorbed ~ a v e l e n g t h In .~~ theory, the best photosensitizer for PDT, in addition to good tumor-localizing ability, should absorb strongly at the wavelength where tissue attenuation is the least and light penetration of tissue the best, which is at approximately 700 - 850 nm or 1,000-1,100 nm.35


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A

B Figure 8. Scar excised from an athymic nude mouse 3 weeks after PDT. The specimen is histologically free of tumor with only residual cicatrix. A) H 6 E, original magnification XlOO; B) H 6 E, original magnification X400.

As stated previously, BPD has several characteristics that make it theoretically a more ideal photosensitizer than the previously studied HPD. In addition to less phototoxicity, BPD allows for more efficient light absorption because of an absorption peak at 690 nm. Furthermore, all of BPD's components are potent photosensitizers for photodynamic destruction of many types of normal and cancer cells in ~ i t r oIn. ~a ~study by Richter et al,36BPD was found to be 10-70 times more cytotoxic than HPD on cultured human lung carcinoma and leukemia cells. Furthermore, in a tumor localization study by Richter et al,35the monoacid derivative of BPD (BPD-MA) selectively localized in mastocytomas (P815) and rhabdomyosarcomas (Ml) better than normal tissue, except kidney, liver, and spleen. They showed that BPD-MA had a favorable tumor-to-skin distribution. The majority of the intravenously injected BPD cleared from the body during

the first 24 hours.35In addition, in a mouse skin photlosensitization study comparing BPD and Photofrin, animals receiving BPD analogues, exposed to light 24 hours or more after injection, showed only minimal photosensitivity, whereas Photofrin photosensitivity persisted throughout the testing period (up to 72 hours postinject i ~ n )In. ~ fact, ~ one of the strong advantages of BPD over HPD is its short-lasting photosensitivity of approximately 24 hours compared with 30 days.34*37-39 Other animal studies have shown BPD to be a promising new photosensitizer with a greater tumoricidal effect than HPD. Morgan et a141 conducted a study showing BPD's cytotoxic effect on rat bladder tumors (A-Y27) transplanted into rats. At a dose of 5.0 mg/kg of BPD, there was no clinical or histologic evidence of tumor at 112 days posttreatment, whereas the control tumors continued to At a dose of 1 mg/kg of BPD, the tumors had decreased to 73% of control tumor size at 12 days posttreatment, but no animals were tumor free." In contrast, HPD at a dose of 1 mg/kg showed 99% of control tumor size with no animals cured of tumor at 12 days.41 Clearly BPD showed an increased tumoricidal action compared with HPD. Additional studies have shown an enhanced therapeutic effect upon irradiation of tumors

Figure 9. Mean change in tumor volume at the end of the 3week follow-up period after each respective treatment. Only the group that received PDT experienced a reduction in tumor volume, while the three control groups (pure control, BPD alonrp, and laser alone) all experienced an increase in tumor volume.

N m (Control)

BPD

Treatment

Laser

Both

(PDT)

Groups

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when associated with 1ip0protein.s.~~ An experiment by Allison et a138showed that precomplexing BPD with low density lipoprotein or high density lipoprotein led to significantly greater tumor localization than in aqueous solution. PDT is not intended to replace current treatment modalities but rather be developed for its own ideal indications. Some of the indications for PDT include: patients with tumors in functionally or cosmetically significant locations for which surgery might be disfiguring; medically unfit or elderly patients; patients for whom other treatment approaches have failed; patients for whom surgery might be impractical secondary to widespread malignancies; and patients for whom radiotherapy might be contraindicated, such as patients with SCC due to prior radiation therapy or in patients with xeroderma pigmentosum, patients with the nevoid basal cell carcinoma syndrome, patients with multiple tumors due to previous arsenical exposure, or immunocompromised patient^.^," The major advantages of PDT include: tumor fluorescence that enhances visualization; selective tumor destruction that spares surrounding normal tissue; ability to use PDT repeatedly without the cumulative dose risk of radiation therapy; and excellent cosmetic results. The disadvantages of PDT include: inability to check for clearance of tumors as with excisions; prolonged photosensitivity with some photosensitizers; moderate pain in some patients; inadequate penetration of pigmented lesions; inability to effectively treat lesions greater than 1.0 cm deep without the help of intralesional fiberoptic cylinders; and unknown potential systemic toxicities due to internal organ retention of photo sensitizer^.^,^^,^^ In conclusion, we have shown that human SCC in a nude mouse model is responsive to PDT using a BPD photosensitizer. Since our study is the first in vivo PDT experiment using BPD on SCC, we hope our research can lay some framework for future endeavors in this area. Future studies changing laser energy or BPD dose parameters would certainly increase the patient cure rate. Further animal studies to ascertain optimal parameters and as a prerequisite to human trials are necessary. In addition, clinical experience with HPD-mediated PDT has revealed significantly better cure rates for basal cell carcinomas than for SCC, so that once BPD reaches the point where it can be safely studied in humans, it is likely that even higher cure rates would be expected for basal cell carcinomas. Our experiment, as well as previous BPD studies, suggest a potentially high efficacy of PDT with BPD in the treatment of human malignancies. Coupled with its short-lasting photosensitivity and high tumor-localizing ability, BPD-mediated PDT holds future promise for the treatment of certain human malignancies, and therefore warrants further in vitro and in vivo studies.

Acknowledgments This study was supported by grants from the Dermatology Foundation and the Research and Educational Institution, UCLA-Harbor Medical Center. We are also indebted to Leo lndianer, MD, for his histopathologic analysis of the tumor and scar excisions.

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