[CANCER RESEARCH 43, 2876-2879, June 1983] 0008-5472/83/0043-0000$02.00 Neodymium-Yttrium Aluminium Garnet Laser Destruction of Nonsensitized and Hematoporphyrin Derivative-sensitized Tumors1

Thierry Patrice, Marie-Françoise Le Bodic,2 Louis Le Bodic, Thierry Spreux, GérardDabouis, and Luc Hervouet DéparlementLaser CHU Nantes [T. P., L L B., T. S., G. D., L H.] and Laboratoires d'Anatomie Pathologique A et B [M-F. L. B.], Facultéde Médecine,1 rue Gaston Veil, 44035 Nantes, France

ABSTRACT in darkness. The solution for injection was prepared by mixture of one part of HPD and 50 parts of 0.1 N soda solution left at room temperature The injection of hematoporphyrin derivative (5 mg/kg ¡.v.) for 1 hr. The pH was adjusted at 7.2 to 7.4 with the addition of followed 24 hr later by a neodymium-yttrium aluminium garnet hydrochloric acid solution and made isotonic by the addition of sodium laser irradiation shows the destruction of CX1 tumors grafted on chloride. The final 5-mg/ml solution was sterilized by filtration through a Millipore filter and kept in darkness at -20° until used. nude mice. This acidophilic necrosis occurred with a significantly The energy was produced by a Nd-YAG laser (Cilas-Marcoussis increased frequency in tumors treated by hematoporphyrin de rivative injection and irradiated with the neodymium-yttrium alu Laboratory, 91460, France) emitting in the near IR (1.06 //m). This laser is a solid-state laser commonly used in our medical applications of laser minium garnet laser as compared with noninjected but irradiated therapy. A mechanical device held the tip of the fiber constantly at 5 mm tumors or with injected tumors irradiated with sunlight. from the target. A system combining a TRG 101 thermopile and a TRG On the basis of our data, it seems difficult to maintain the 102 energy meter (Control Data Corp., Yellow Springs Institute, Colo.) hypothesis of singlet oxygen production as the only mechanism was used to measure the power at the end of the fiber. of the phenomenon. Further studies will be necessary to explain The visualization beam of the helium:neon laser was cut off throughout the necrosis that we observed. the experiment.

Methods INTRODUCTION All of the mice received a tumor graft on the first day of the experiment. Envisaged in the 1900s by Tappeiner et al. (13), the affinity of Groups were constituted as follows. hematoporphyrin for neoplastic tissues has been confirmed by Experimental Series (Group I; 14 Tumors). On the ninth day, the Figge ef al. (5) and Lipson et al. (9) and was demonstrated by mice received an injection of HPD (5 mg/kg). On the 10th day, following Rasmussen-Taxdall ef al. (12), Gregorie ef al. (6), and Kelly and skin incision (to prevent secondary skin necrosis caused by laser heating) Snell (7) using HPD.3 Later, the oxidizing properties of HPD which did not interfere with the deep vascular supply of the tumor, the under different non-ionizing rays were demonstrated regardless tumors received a series of Nd-YAG laser exposures. The incision was of the wavelength used (mercury vapor lamps or xenon arc then stitched up to avoid septic contaminations. On the 12th day, the lamps) or whether or not it was filtered (1, 2). These studies led mice were sacrificed, and the tumors were removed for histopathological Dougherty ef al. (3) to use heliunrneon or dye lasers as sources examination. of excitation of HPD. This led us to study the effects of photo- Comparative Control Series (Group II) with the Following coagulation by the Nd-YAG laser on human colonie adenocarci- Subgroups. In Group II-A, 4 tumors were removed on the 12th day, the mice having received one tumor graft only; in Group II-B (8 tumors), on noma-type tumors grafted in the hip of the nude mouse after the ninth day, the mice received an injection of HPD (5 mg/kg). They sensitization of the tumor tissue by HPD. were sacrificed on the 12th day for examination; in Group II-C (8 tumors), The aim of the study was to evaluate under these conditions the mice received a similar injection of HPD, but on the 10th day, after the efficacy of the Nd-YAG laser and to study the lesions created skin incision, the tumors were exposed to sunlight through the window in order to determine whether they were specific or similar to glass for 5 min. This group was formed to show that the observed effect those seen in control series. of Group I was not linked to the surrounding light. These mice were sacrificed on the 12th day; in Group II-D (8 tumors), the mice did not receive an injection of HPD, but the tumors were treated with the laser MATERIALS AND METHODS on the 10th day and removed for examination on the 12th day. Materials The energy level of the laser has been controlled for each tumor of Groups I and II-D. For every shot, the power was 45 watts, and time Male albino nude mice were used. A human colonie adenocarcinoma- exposure was 1.5 sec (Table 1). type tumor (C x 1) was grafted by bilateral injection of 0.2 ml of crushed The same experimental conditions used to obtain sample material, tumor in solution in culture medium. anesthetic techniques, and darkness were applied to all of the different PD was prepared using the method of Gregorie ef al. (6) and Lipson groups. All of the mice were sacrificed on the 12th day for histopatho ef al. (9). Hematoporphyrin hydrochloride (Roussel) was dissolved in logical examination of the tumor. The latter was fixed in 12% formol for acetic acid:sulfuric acid (9:1). The mixture was left at room temperature 24 hr, cut along its long axis, and mounted in its entirety in several for 12 hr and was then filtered and neutralized by addition of a 3% paraplast blocks before being studied by numerous serial sections sodium acetate solution until a pH of 6 was obtained. The precipitate stained with hematoxylin:eosin:safran. collected by filtration was washed until a neutral washing water was obtained, and it was then dried and stored at -20° in a solid state and RESULTS 1This work has been supported by C. N. E. H. (French Ministry of Health). 1 To whom requests for reprints should be addressed. 3 The abbreviations used are: HPD, hematoporphyrin derivative; Nd-YAG, neo Group I (cf. Fig. 1). The 14 tumors which photocoagulated dymium-yttrium aluminium garnet. after sensitization with HPD showed necrosis affecting approxi Received May 18,1982; accepted February 8,1983. mately 90% of the total tumor mass. In 10 of them, there was

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1983 American Association for Cancer Research. Laser Destruction of Non- and HPD-sensitized Tumors total acidophilic necrosis leaving no detectable tissue or cellular structure. In 3 tumors, there was necrosis although it was not acidophilic, with loss of glandular arrangement, disappearance of cell borders, and retraction of the nuclei when they could still be identified. At the extreme periphery of the tumor, and in particular at depth, in 12 of 14 cases, rare neoplastic glandular tubes per sisted, representing approximately 10% of the total tumor mass. On the surface and laterally, at the level of the cutaneous tissue, edema, congestion, and moderate inflammatory infiltrate were observed. In depth and beyond the tumor, the muscular layer was affected in more than one-half of all cases. Group II-A (cf. Fig. 2). Upon histological examination, these 4 control tumors, varying from 7 to 10 mm in diameter, were found to be weakly mucosecreting adenocarcinomas. Tumor spread developed in the s.c. tissue. The arrangement was frankly glan dular and was sometimes polyadenoid with neoplastic tubules lined by an epithelium rich in cytological and nuclear abnormalities

Table 1 Tumor size and dose exposures in Groups I and II-D tumor diame ter (mm)86.8Av. no. ofshots5.4 energy(J)364.5 Group I Group II-DAv. 4.8Av. 324 ^¿aäKS5Ä%afl:Wm$&^£w an^'WkSi»*Vf SS w^'ViF.

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Fig. 2. Group II-A, control tumor:human colonie adenocarcinoma type tumor (Cx1). Hematoxylin, eosin, and safran; A, x 3.7; B, x 250.

and with a high mitotic index. There were few fibroblastic stroma. There was neither localized abscess formation nor necrosis apart from one case where there was a very limited area of necrosis. Group II-B (cf. Fig. 3). In these mice, which had received HPD, the nonphotocoagulated tumors were the site of rare areas of scanty punctiform necrosis with persistence of >90% of the total tumor mass. However, in 2 cases, there was an area of complete acidophilic necrosis. Group II-C (cf. Fig. 4). These mice received an injection of HPD. These tumors were exposed to sunlight 24 hr later. Five tumors were the site of areas of punctiform necrosis slightly larger than those in Group II. Approximately 85% of the tumor tissue persisted. By contrast, 3 tumors showed areas of acidophilic necrosis. There were no notable congestive phenomena, but the tumor was surrounded by an inflammatory infiltrate consisting of histio- cytes, plasmocytes, and a few rare polynuclear cells. Group II-D (cf. Fig. 5). These 8 tumors received no HPD but were photocoagulated. They never showed areas of total aci dophilic necrosis. The tumor tissue was nevertheless destroyed, 95% necrotic, with isolated cellular elements with retracted nuclei in the course of lysis. In one half of the cases, no neoplastic Fig. 1. Group l, acidophilic necrosis of tumor photocoagulated after sensitization glandular tubules remained in the periphery. In the other half, with HPD. Hematoxylin, eosin, and safran; A, x 3.5; B, x 250. there were a few glandular islets which persisted laterally.

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In 7 of 8 cases, the necrotic tumor was limited at its extremities (c) Similar findings were reported by Dougherty ef al. (4) who by a congested zone. The deep muscular layer was affected in used a helium:neon or dye laser pumped by an argon laser to all cases. destroy breast tumors. However, they did not make any study of the histopathological lesions. Similarly, Thomson (14) obtained DISCUSSION tumor destruction (using an argon laser) of tumors sensitized Analysis of the results of this experimental study leads to the with acridine orange instead of HPD. following remarks. In summary, in answer to the question raised at the beginning (a) The comparison by the x2 test of Group I with Groups II-B, of this report, the following conclusions may be drawn: (a) The II-C, and II-D combined shows a significant difference between Nd-YAG laser destroyed colonie adenocarcinoma-type tumors these 2 groups (x2 = 22.8; 1 d.f.). grafted in the mouse when these tumors were previously sensi The comparison of Group I with Groups II-B, II-C, and II-D tized with hematoporphyrin derivative; (b) The coagulation necro individually also has been done with the x2 test. After the sis observed after the use of the laser alone was replaced here correcting Yates test, a significant difference between Group I by acidophilic necrosis; (c) This lesion was in no way specific. and each Group II is shown (x2 = 9.62; 1 d.f.). The same type of lesion was seen in nontreated lesions. Never (b) In the nontreated tumors, virtually no necrosis was seen theless, the laser increased necrosis considerably, while this on the 12th day. In the mice receiving HPD only, very few increase did not occur when tumors sensitized by hematopor changes were seen within the tumors even when exposed to phyrin were irradiated with ordinary light. light on the 10th day. By contrast, in the 2 series subjected to A doubt remains as far as the mechanism of action is con either the action of the laser alone or the action of the laser after cerned. From this point of view, this paper raises more questions injection of HPD, there was virtually total destruction of tumor than it gives answers. Singlet oxygen production as mentioned tissue. by Weishaupt ef al. (15) could be one of the mechanisms; The type of necrosis seen was not the same in Group I (laser however, that would require an absorption of HPD at 1.06 ^m. plus administration of HPD) and in Group II-D (laser only). When Although we believe it could exist, technical problems prevent the tumor had been previously sensitized by the administration us from asserting it clearly and drawing a comparison with the of HPD, there was total massive acidophilic necrosis with dis 0.632-MiTi absorption. A direct alteration of HPD into a toxic appearance of all tissue or cell structures. This acidophilic necro compound under the heating effect could be another mechanism. sis appeared to be different from the coagulation obtained by Further studies are being conducted by our department to try to the laser alone. Furthermore, the distribution of the few residual provide an answer. glandular tubules is different in the 2 cases. After use of the laser REFERENCES alone, the glandular tubules which were found only in one-half of 1. Diamond, I., Granelli, S., Mac Donagh, A. F., Nielsen, S., Wilson, C. B., and the cases studied were invariably distributed on either side of Jeanicke, R. Photodynamic therapy of malignant tumors. Lancet, 2: 1175- the coagulation lesion and never at depth. The underlying mus 1177, 1972. cular layer was affected. By contrast, when the tumor had been 2. Dougherty, T. J., Grindtey, G. B., Fiel, R., Weishaupt, K. R., and Boyte, D. G. Photoradiation therapy II: cure of animal tumors with hematoporphyrin and previously sensitized with HPD (Group I), residual glands were light. J. Nati. Cancer Inst., 55: 115-119, 1975. found in 3 of 4 cases and were distributed in a wreath-like 3. Dougherty, T. J., Kaufman, J. E., Goldfarb, A., Weishaupt, K. R., Boyle, D. G., and Mittleman, A. Photoradiation therapy for the treatment of malignant arrangement laterally as well as in depth. The underlying mus tumors. Cancer Res., 38: 2628-2635, 1978. cular layer was affected less frequently than that in Group II-D. 4. Dougherty, T. J., Lawrence, G., Kaufman, J. H., Boyle, D., Weishaupt, K. R., It appeared as if the destructive effect of the laser was poten- and Goldfarb, A. Photoradiation in the treatment of recurrent breast carcinoma. J. Nati. Cancer Inst., 62: 231-237, 1979. tialized by HPD with regard to the tumor itself but limited with 5. Figge, F. H., Weiland, G. S., and Manganello, L. O. J. Cancer detection and regard to the depth of the effects. In the absence of the admin therapy. Affinity of neoplastia embryonic and traumatized regenerating tissues for porphyrins and metalloporphyrins. Proc. See. Exp. Bid. Med., 68: 640- istration of HPD, congestive phenomena seen at the periphery 641,1948. of the tumor tissue were more frequent and more marked. 6. Gregorie, H. B., Horger, E. 0., Ward, J. L., Green, J. F., Richards, T., It should be noted that HPD alone, in the absence of photo- Robertson, H. C., and Stevenson, T. B. Hematoporphyrin derivative fluores cence in malignant neoplasms. Ann. Surg., 767: 820-828,1968. coagulation, had an effect on the tumor cells. Islets of punctiform 7. Kelly, J. F., and Snell, M. E. Hematoporphyrin derivative: a possible aid in the necrosis were seen in Group II-B. This was already reported in diagnosis and therapy of carcinoma of the bladder. J. Urol., 775: 150-151, vitro by Malik and Djaldetti (10) in 1980 when following the 1976. 8. Kinsey, J. H., Cortese, D. A., Moses, H. L., Ryan, R. J., and Branum, E. L. administration of HPD scanning electron microscopy revealed Photodynamic effects of hematoporphyrin derivative as a function of optical destruction of plasmic membranes and of the coat of Burkitt- spectrum and incident energy density. Cancer Res., 47: 5020-5026,1981. 9. Lipson, R. L., Baldes, E. J., and Olsen, A. M. The use of a derivative of type tumor cells with inhibition of RNA and DNA synthesis. hematoporphyrin in tumor detection. J. Nati. Cancer Inst., 26:1-12,1961. This effect was increased with exposure to light (Group II-C), 10. Malik, Z., and Djaldetti, M. Destruction of erythroteukemia, myelocytic leukemia and necrotic areas then were more extensive. Moan ef al. (11) and Burkitt lymphoma cells by photoactivated protoporphyrin. Int. J. Cancer, 26:495-500,1980. have emphasized the fact that the action of rays appears to be 11. Moan, J., Petersen, E. D., and Christensen, T. The mechanism of photody- more marked when HPD is intracellular. namic inactivation of human cells in vitro in the presence of hematoporphyrin. Using tumor cell cultures, Kinsey ef al. (8) estimated that the Br. J. Cancer., 39: 398-407,1979. 12. Rassmussen-Taxdall, D. S., Ward, G. E., and Figge, F. H. Fluorescence of mass of cells destroyed was greater when the wavelength of the human lymphatic and cancer tissues following high doses of intravenous light source was shorter. All of this did not seem to be corrobo hematoporphyrin. Cancer (Phila.), 8: 78-81, 1955. 13. Tappeiner, H. N., and Jesionek, A. Therapeutische Versuche mit Fluoreszieren rated by our own study. den Stoffe. Muench. Med. Wochenschr., 7: 2042-2044,1903. While there was no evidence to show that the laser lesions 14. Thomson, S. H. Photodestruction of mouse epithelial tumors after oral acridine were specific, there was nevertheless no similarity whatsoever orange and argon laser. Cancer Res., 34: 3124-3127,1974. 15. Weishaupt, K. R., Gomer, C. J., and Dougherty, T. J. Identification of singlet between the few areas of necrosis seen in the control series and oxygen as the cytotoxic agent in photo-inactivation of a murine tumor. Cancer the total necrosis of tumors subject to photocoagulation. Res., 36: 2326-2329,1976.

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Fig. 3. Group II-B, rare areas of scanty punctiform necrosis after administration of HPD. Hematoxylin, eosin, and safran; A, x 2.2; B, x 250. Fig. 4. Group II-C, areas of punctiform necrosis after administration of HPD followed by local exposures to light. Hematoxylin, eosin, and safran; A, x 2.7; B, x 250. Fig. 5. Group II-D, necrosis of tumor photocoagulated by Nd-YAG laser. Hematoxylin, eosin, and safran; A, x 2.8; B, x 250.

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Thierry Patrice, Marie-Françoise Le Bodic, Louis Le Bodic, et al.

Cancer Res 1983;43:2876-2879.

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