Photodynamic therapy of rabbit endometrial transplants: a model for treatment of endometriosis

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1 FERTILITY AND STERILITY Copyright" 1989 The American Fertility Society Printed in U.S.A. Photodynamic therapy of rabbit endometrial transplants: a model for treatment of endometriosis MichaelJ. Manyak, M.D.*t Lawrence M. Nelson, M.D.:!: Diane Solomon, M.D. Angelo Russo, M.D., Ph.D.* Gunter F. Thomas, B.S.* Robert J. Stillman, M.D.:!: National Cancer Institute, Bethesda, Maryland, and The George Washington University Medical Center, Washington, D.C. The potential of photodynamic therapy for endometriosis was evaluated by autotransplantation of endometrial tissue in 15 female virgin New Zealand white rabbits. After maturation, 14 animals were injected intravenously with 10 mg/kg of dihematoporphyrin ether (DHE) and, 24 hours later, transplants were exposed to 60 nm light at 100 to 210 mw/cm 2 Sixteen transplants received 100 J/cm 2, 10 transplants received 50 J/cm 2 ; 1 untreated transplants served as controls. In the remaining animal that did not receive DHE, transplants were irradiated with 100 J/cm 2 Six days after treatment, transplants were harvested with the underlying musculature, and multiple sections were examined histopathologically. endometrial epithelial destruction was seen in 1 of 16 transplants (81%) in the 100 J/cm 2 group and in 6 of 10 transplants (60%) in the 50-J/ cm 2 group. Nearly complete endometrial epithelial destruction was seen in 2 other transplants in each group. No damage occurred in either the transplants that received 100 J/cm 2 without prior DHE or in the 1 transplants with DHE and no irradiation. The sensitivity of ectopic rabbit endometrial tissue encourages further evaluation of photodynamic therapy for the treatment of human endometrial disorders. Fertil Steril 52: 140,1989 Photodynamic therapy (PDT) is an experimental treatment modality that destroys tissue through an interaction between absorbed light and retained photosensitizer. Although there is worldwide interest in several photosensitizers for potential clinical use, only dihematoporphyrin ether (DHE), a purified product of hematoporphyrin derivative (HPD), is currently approved by the Food and Drug Administration (FDA) for human experimental trials. 1,2 The potential biologic advantage of Received November 15, 1988; revised and accepted February 2,1989. * Radiation Oncology Brandl, Experimental Phototherapy Section, National Cancer Institute. t Present address and requests for reprints: Michael J. Manyak, M.D., Department of Urology, George Washington University Medical Center, 2150 Pennsylvania Avenue, N.W., Washington, D.C :j: Department of Obstetrics and Gynecology, The George Washington University Medical Center. Laboratory of Pathology, National Cancer Institute. PDT arises from the observations that DHE, like HPD, is retained in neoplastic tissue after intravenous administration and that cell death occurs after sufficient light energy is absorbed. 2 Retention of porphyrin photo sensitizers also has been reported in inflammatory, traumatized, and embryonic tissue.,4 Photodynamic therapy has been used to treat a wide variety of tumors in over,000 people throughout the world since ,6 The use of photodynamic therapy for gynecologic lesions, although not extensive, has been encouraging. Hematoporphyrin derivative fluorescence has been reported in both dysplasia and cervical carcinomas. 7 Primary and recurrent vaginal cancers, cervical, endometrial and, more recently, ovarian tumors have been treated by PDT Endometriosis is a common disorder characterized by an invasive but nonneoplastic growth pattern of hormonally responsive endometrial tissue outside the endometrial cavity. It is confined, with 140 Manyak et al. Endometrial photodynamic therapy Fertility and Sterility

2 few exceptions, to women during the reproductive age and can pose both diagnostic and therapeutic dilemmas. 15 Patients with endometriosis have increased rates of infertility, pelvic pain, dysmenorrhea, and dyspareunia. 15 In this study, photodynamic therapy was evaluated to determine its effect on an endometriosis model. MATERIALS AND METHODS Endometrial transplants were prepared according to the method established by Schenken and Asch 16 and modified by Hahn et al. 17 Under sterile conditions, 15 female virgin New Zealand white rabbits (2.5 to.5 kg) under general anesthesia via endotracheal intubation had laparotomies performed through lower midline incisions. A - to 4- cm portion of one uterine horn was excised from each animal and hemostasis was maintained. Mesovarium was dissected from the specimen, which then was incised longitudinally and divided into three or four approximately 1-cm sections. Fifty full-thickness uterine sections were transplanted unilaterally to the peritoneal surface and anchored with 5-0 chromic sutures in an interrupted (n = 47) or continuous (n = ) fashion. In 46 sections, the serosal surface was placed adjacent to the peritoneum, and 4 other sections were placed with the endometrial surface next to the peritoneum to determine the influence of position on cyst formation. After hemostasis with electrocautery, closure was completed in layers. All animals received 5 mg/ kg of gentamicin at surgery and 24 hours postoperatively. The transplants were allowed to mature for 10 to 14 weeks, and 14 animals were injected intravenously with 10 mg/kg of dihematoporphyrin ether (Photofrin II, Photomedica, Inc., Raritan, NJ). One animal (no. 6) received one-half of the dose intraperitoneally because of difficult intravenous access. Twenty-four hours later, a laparotomy was performed through a midline incision and adhesions were lysed as necessary to expose the transplants. After hemostasis, the peritoneum was everted, the lesions were measured, and a 600-Jl,m, fused silica, flat end optical fiber (QPC, Plainfield, NJ) attached to an argon-pumped dye laser (Coherent PRT 100, Palo Alto, CA) was positioned perpendicularly above the treatment lesion. The laser delivered 60 nm light at 100 to 210 m W /cm 2, for a total of 50 or 100 J/cm 2 Light energy from the optical fiber was verified by a Model 210 power meter (Coherent, Palo Alto, CA). Lesions not treated were shielded by a reflective surface. Fourteen rabbits had laser irradiation in this fashion. In 1 rabbits, there were 24 transplants treated, 14 with 100 J/cm 2 and 10 with 50 J/cm 2 Each of these animals had at least 1 transplant left untreated as a control. An additional animal had both transplant lesions partially shielded so that one half of each lesion received 100 J/cm 2 A control animal that did not receive intravenous photosensitizer had all transplants irradiated with 100J/cm 2 Animals were killed 6 days after PDT by intravenous injection of 1.5 ml of embutramide and mebezonium (T-61, Taylor Pharmaceuticals, Decatur, IL). Transplants were harvested with the underlying peritoneum and rectus muscle and placed in 10% formalin solution. After hematoxylin and eosin staining (Baker Labs, Great Falls, VA), multiple sections of each specimen were mounted and examined histopathologically by an investigator not present at treatment (D.S.). Each transplant was measured from the furthest point of the exterior cyst wall to its base. Without knowledge of treatment, each slide was scored according to approximate percentage of normal-appearing endometrial epithelium and its relationship to surrounding abnormal tissue (i.e., whether it was lateral to or within destroyed tissue). In addition, the depth of apparent destruction was estimated. RESULTS All animals tolerated both laparotomies and laser treatment. During the interim between implantation and treatment, 10 cystic lesions had coalesced to form inseparable cystic masses and were treated as single entities, and 1 transplant did not survive. Therefore, of the 50 transplanted uterine sections, 42 separate, viable entities at the time of the second laparotomy were included in the study, 7 in the treatment animals and 5 in control animals. Fifteen transplants were grossly cystic in appearance, while 27 appeared flat. Thickness of the transplants varied from 4.5 to 1 mm, with an average thickness of 7.1 mm. Most of the flat-appearing lesions had some degree of cystic structure on microscopic examination. There was no difference in cyst formation in transplants sutured with serosal or endometrial surface adjacent to the peritoneum. Cysts were lined with endometrial epithelium and contained serous fluid free of blood except Manyak et al. Endometrial photodynamic therapy 141

3 Table 1 Histopathologic Destruction in 42 Rabbit Endometrial Transplants After Photodynamic Therapy or 60 nm Laser Light Alone" Therapy Degree of destruction DHE J/cm 2 No. 100% >75% <75% 0% " Although shielded from irradiation, four controls had mild destruction at the lateral border directly adjacent to a treated transplant. for cysts (all in no. 6), which contained dense papillary endometrial tissue. Cyst formation occurred despite the use of continuous suture to prevent involution of the transplant edge. Cyst fluid was clear in all instances and samples aspirated from control transplants readily transmitted 60 nm light. Adhesions were found before PDT on of 16 transplants treated at 100 J/cm 2, 1 of 10 transplants treated at 50 J/cm 2, and on 5 of 1 control transplants. Following PDT, adhesions were found on 10 of 16 transplants treated at 100 J/cm 2, 4 of 10 transplants treated at 50 J/cm 2, and on 6 of 1 control transplants. Only one animal had adhesions before treatment, but no adhesions after PDT. Conversely, five animals had adhesions both before and after treatment, while five had adhesions only after PDT. Two flat lesions, treated with 50 and 100 J/cm 2, became hemorrhagic immediately after treatment (no. 11). Four cystic lesions, two treated with 50 J/ cm 2 and two with 100 J/cm 2 (one each in no. 7 and no. 12), were hemorrhagic when harvested. Temperature did not rise more than C at any time on the monitored lesions. The only significant temperature rise was noted in the control animal, in which half of each lesion received 100 J/cm 2 at a dose rate of 126 mw/cm 2 An initial rapid temperature rise decreased and stabilized when the dose rate was decreased to 100 m W / cm 2 The effects of PDT on the endometrial transplants at 50 J/cm 2 and 100 J/cm 2 compared with controls are demonstrated in Tables 1 and 2. In the group treated with 100 J/cm 2, 1 of 16 lesions (81 %) had complete destruction of the endometrial epithelium (Fig. 1A). This group included one animal that received 100 J/cm 2 to each of two partly shielded transplants after photosensitizer injection and showed a correspondingly sharp demarcation of damage between treated and nontreated areas (Fig. 1B). Two additional transplants (nos. and 12) had between 75% and 90% epithelial destruction. Although parts of the transplant with dense papillary growth (no. 6) showed destruction, 50% to 80% of the epithelium of this large, macroscopically cystic structure remained intact (Fig. 1C). This animal received only one-half of the photosensitizer dose intravenously because of difficult intravenous access. Endometrial epithelium in six often transplants (60%) treated with 50 J/cm 2 showed complete destruction. Two other transplants in this group (nos. 2 and 1) had >75% of the epithelium obliterated. Histopathology of one transplant (no. ) showed anterior cyst wall destruction with an intact posterior cyst wall (Fig. 1D). Similar to the 100 J/cm 2 group, the densely papillary variant in the animal that received half the photosensitizer dose intravenously (no. 6) showed less destruction (0%) than any other treated transplant. In the study animals, eight untreated control transplants were completely intact (Fig. IE) and four others had a very small amount of damage at the extreme lateral border of the tissue adjacent to a treated field, most likely from reflected light. One control had significant damage in continuity with complete destruction of the adjacent treatment transplant, while the portion furthest from the laser source remained intact, suggesting inadequate shielding from the laser. A separate control animal received 100 J/cm 2 to all three transplants without sensitizer and sustained no damage to any lesion (Fig. IF). DISCUSSION Endometriosis is a common disorder responsible for a significant portion of gynecologic operations. Pathologically, endometriosis is defined as the presence of endometrial epithelium, glands, and stroma, with or without evidence of menstrual cyclicity, but clinical decisions are frequently based on diagnosis by visual inspection of the pelvis at endoscopy without confirmatory tissue diagnosis. 15 Endometriotic tissue is stimulated by estrogens and inhibited by androgens, and this polarity of response is the basis for current medical management. 1S The most effective FDA-approved agent for creation of an acyclic, low-estrogen, high-androgen environment is the synthetic steroid danazol.19 Although danazol relieves pain in 90% ofpatients, its use may be associated with major side effects, some of which persist after treatment Manyak et al. Endometrial photodynamic therapy Fertility and Sterility

4 Table 2 Histopathologic Degree of Endometrial Destruction in Transplants Treated with 100 J/cm2 and 50 J /cm2 Compared with Controls No. lesions Animal 100J/cm2 50J/cm2 1 (2) 2 >75% (papillary) >50% 0% (partial shielding) 90% (2) >75% 1/16 complete (81%) 6/10 complete (60%) 9 10 Totals 9 >85% <50% damage, cyst wall intact posteriorly Control Mild damage adjacent to Mild damage adjacent to Mild damage adjacent to Damage contiguous with Damage to control transplant endometrium was only seen contiguous to a destroyed treated transplant. Surgical removal of both ovaries and uterus remain the only definitive therapy of endometriosis. Therefore, young women with endometriosis frequently are faced with difficult decisions regarding their reproductive futures. Figure 1 The effects of photodynamic therapy on rabbit endometrial transplants: (A), after 100 J/cm2, complete destruction; (B), after 100 J/cm2, sharp demarcation between treated and shielded areas showing complete destruction in the treated area; (C), after 100 J/cm2, thick papillary transplant with little destruction; (D), after 50 J/cm2, anterior cyst wall destruction only; (E), intact untreated control; and (F), after 100 J/cm2, no photosensitizer, no damage. Photodynamic therapy is an experimental modality used to destroy malignant tumors through activation by visible light absorption of a relatively selectively retained photosensitizer. Fluorescence has been used to detect retained porphyrin photosensitizers.5,6 In the gynecologic field, HPD fluorescence has been reported in both dysplasia and cervical carcinomas. 7,21 One series reported HPD fluorescence in 18 of 2 patients (78%) with cervical carcinoma and 9 of 11 patients (82%) with dysplasia, but in only 2 of 10 patients (20%) with chronic cervicitis. 7 Another investigator reported HPD fluorescence in all 14 patients with carcinoma in situ, dysplasia, dr atypical squamous metaplasia, but in only 2 of 5 patients with chronic cervicitis or squamous metaplasia; one patient showed fluorescence without any abnormality.21 Based on our laboratory observations of significant DHE fluorescence in rabbit endometrial tissue, photodynamic therapy was considered as a possible means to destroy ectopic endometrial tissue. Although endometriosis is not malignant and the risk of carcinoma in endometriosis is less than 1%,22 this disease entity does share some characteristics with neoplastic behavior, notably, its propensity to invade tissue and its ability to spread by lymphatic or hematogenous dissemination. 2,24 Few quantitative animal models of endometriosis exist for laboratory experiments. About 60 years Manyak et ai. Endometrial photodynamic therapy 14

5 ago, Sampson surgically produced primate models for endometriosis, but the ease with which endometrial tissue proliferates within the abdominal cavity of research animals does not, by itself, constitute sufficient evidence of a good endometriosis model.16,17 A true model of this disease must produce symptoms of the disease; the most common symptoms are pain and infertility. While the former is difficult to assess in an animal, the influence of endometriosis on infertility has been quantified in the monkey, rat, and, most commonly, the rabbit.16 In rabbits, previous investigators have produced endometriomas through "seeding" or suturing of endometrial squares to the peritoneal cavity.16,17 Following the surgical transplantation of rabbit endometrium, Schenken and Asch as well as Hahn et al. reported around 50% reduction in fertility rates unrelated to intraperitoneal adhesion formation.16,17 Both these groups reported separation of endometrium from myometrium with ease. In our model, this separation was not accomplished without damage to the friable endometrial tissue, and, therefore, we deviated from their model by transplanting full thickness uterine tissue. In our study, all but 1 uterine section became established following transplantation. Fifteen transplants were grossly cystic, 7 in the 100 J/cm 2 group and 5 in the 50 J /cm 2 group, but nearly all had some cystic formation, which occurred despite attempts to anchor the transplant circumferentially. The cyst fluid was observed to transmit 60 nm light readily when tested and, therefore, cyst drainage was not performed before treatment. The cyst fluid did not appear to interfere with treatment in the 100 J/cm 2 group, where 6 of 7 cystic transplants had complete endometrial destruction and the remaining 1 was greater than 75% destroyed. However, in the 50 J/cm 2 group, endometrium in only 2 of 5 cystic transplants was destroyed completely, while the remaining transplants had between 50% and 85% destruction. It is possible that, at the lower energy density, slight turbidity not grossly apparent could sufficiently attenuate the light and, thereby, interfere with endometrial destruction. This could account for preservation of the posterior cyst wall seen in Figure 1D. Tissue penetration of red light varies according to inherent scatter characteristics and chromophore absorption and, in most cases, fluence of 60 nm light is reduced by a factor of 10 at a depth of 2 to 8 mm. 25 From the histopathology slides, the thickness of the transplants measured from 4.5 to 1 mm but was probably greater due to tissue shrinkage during preparation. Survival of endometrial tissue, when found, was always at the furthest point from the laser source, suggesting that light penetration was the limiting factor in endometrial tissue destruction. However, endometrial destruction in a 12-mm cyst by 50 J /cm 2 (no. 7), compared with little destruction at either 50 or 100 J/cm 2 in solid papillary lesions (no. 6), demonstrates the variability of light penetration within tissue. endometrial epithelial destruction was evident in 1 of 16 transplants (81 %) treated by 100 J/cm 2 and in 6 of 10 transplants (60%) treated by 50 J/cm 2 Endometrial epithelial destruction was greater than 75% in 4 other transplants, 2 in each treatment group. Therefore, complete or nearly complete destruction of endometrial epithelium was seen in 2 of 26 transplants (88%). The sharp demarcation of damage between shielded and nonshielded areas of the same transplant, together with lack oftissue injury in transplants irradiated without DHE, further demonstrated that this effect resulted from treatment. The poorest response at both energy densities occurred in the dense papillary transplants and was most likely related to lack of light penetration. Although adhesions to the transplants were found more frequently after treatment, they also were found on untreated control transplants, and, therefore, were believed to have no adverse affect on the transplant biology. The potential for clinical use of this modality in the treatment of endometriosis is intriguing. Aggressive intraperitoneal use of PDT would be tempered by injury potential to other organ systems involved by endometriosis. It is encouraging that a recent report of three patients treated for ovarian carcinomatosis with intraperitoneal PDT using a light-diffusing medium had no demonstrable damage to other abdominal organs,14 although the energy density was much less than those used in this study. However, early stage disease with smaller endometriotic lesions may be responsive to lower energy densities. This study demonstrates the sensitivity of ectopic rabbit endometrial tissue to these energy densities and encourages further evaluation of photodynamic therapy for the treatment of human endometriosis and other disorders where ablation of endometrial tissue is desired. REFERENCES 1. Dougherty TJ, Potter WR, Weishaupt KR: The structure of the active component of hematoporphyrin derivative. In 144 Manyak et al. Endometrial photodynamic therapy Fertility and Sterility

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