Histopathology of prostate tissue after vascular-targeted photodynamic therapy for localized prostate cancer

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(1)Histopathology of prostate tissue after vascular-targeted photodynamic therapy for localized prostate cancer Caroline Eymerit-Morin, Merzouka Zidane, Souhil Lebdai, Stéphane Triau, Abdel Rahmene Azzouzi & MarieChristine Rousselet Virchows Archiv The European Journal of Pathology ISSN 0945-6317 Volume 463 Number 4 Virchows Arch (2013) 463:547-552 DOI 10.1007/s00428-013-1454-9. 1 23.

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(3) Author's personal copy Virchows Arch (2013) 463:547–552 DOI 10.1007/s00428-013-1454-9. ORIGINAL ARTICLE. Histopathology of prostate tissue after vascular-targeted photodynamic therapy for localized prostate cancer Caroline Eymerit-Morin & Merzouka Zidane & Souhil Lebdai & Stéphane Triau & Abdel Rahmene Azzouzi & Marie-Christine Rousselet. Received: 5 February 2013 / Revised: 23 April 2013 / Accepted: 8 July 2013 / Published online: 16 August 2013 # Springer-Verlag Berlin Heidelberg 2013. Abstract Low-risk prostate adenocarcinoma is classically managed either with active surveillance or radical therapy (such as external radiotherapy or radical prostatectomy), but both have significant side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy proposed as an alternative approach for localized, low-volume, and lowGleason score (≤6) carcinomas. We report histological modifications observed in prostate biopsies of 56 patients, performed 6 months after VTP using the photosensitizer TOOKAD® Soluble (WST11) and low-energy laser administered in the tumor area transperineally by optic fibers. In 53 patients, we observed sharply demarcated hyaline fibrotic scars, with or without rare atrophic glands, sometimes reduced to corpora amylacea surrounded by giant multinuclear macrophages. Mild chronic inflammation, hemosiderin, and coagulative necrosis were also observed. When residual cancer was present in a treated lobe (17 patients), it was always located outside the scar, most often close to the prostate capsule, and it showed no therapy-related modification. Histopathological interpretation of post-WST11 VTP prostate biopsies was straightforward, in contrast with that of prostate biopsies after radio or hormonal therapy, which introduces lesions difficult to interpret. VTP resulted in complete ablation of cancer in the targeted area.. C. Eymerit-Morin : M. Zidane : S. Triau : M.<C. Rousselet Cellular and Tissue Pathology Department, LUNAM University, CHU University Hospital, Angers, France S. Lebdai : A. R. Azzouzi Urology Department, LUNAM University, CHU University Hospital, Angers, France M.<C. Rousselet (*) Département de Pathologie Cellulaire et Tissulaire, CHU Angers, 49933 Angers Cedex 9, France e-mail: mcrousselet@chu-angers.fr. Keywords Photodynamic therapy . Photosensitizer . Targeted therapy . WST11. Introduction Prostate cancer (PCa) stage and Gleason grade are determinants of prognosis. With screening of PCa by PSA measurement having become the mainstream, many men are diagnosed with low-risk, low-volume, and low-grade PCa. In this context, current radical therapies are questioned in terms of benefit for survival and risk of cancer progression, in view of their significant adverse side effects [1]. Another approach is active surveillance (AS) which, however, goes along with a significant risk of progression during the surveillance period. A new approach is the concept of focal therapy which is organ-sparing, as selective tumor ablation is the goal. Of the focal therapy approaches, the most promising are highintensity-focused ultrasound (HIFU), cryotherapy, and vascular-targeted photodynamic therapy (VTP) [2–5]. VTP is intended for patients with localized PCa, as an alternative to AS, as it avoids the risks of morbidity inherent to radical therapy but without compromising life expectancy of the treated patient [6]. In 1990, a letter to the Lancet reported the application of this minimally invasive procedure in PCa for the treatment of two patients [7]. VTP was further assessed for its tolerance and the dose determined in terms of the time of laser illumination with the use of different photosensitizers in phase I or phase II studies [8]. At present, the TOOKAD® Soluble WST11 (padeliporfin), a chlorophyll derivative developed by Steba Biotech, is one of the photosensitizers of choice [9]. Diagnosis, localization, and staging of Pca are determined through preoperative prostate biopsies. The photosensitizer is intravenously infused, absorbs light, and transfers energy to oxygen molecules creating free oxygen species [10]. Light is introduced by transperineal optical fibers at the presumed location of the.

(4) Author's personal copy 548. cancer in the prostate under ultrasound transrectal guidance. Damage to vascular endothelium leads to vascular occlusion by thrombosis followed by ischemia and necrosis of the targeted area. At the cellular level, in vitro studies showed phototherapy-induced apoptosis [11]. Histological effects of WST11 VTP have been mainly reported in animal models using WST09, the precursor photosensitizer of WST11, or WST11; early effects such as hemorrhagic necrosis with inflammation, gland destruction, atrophy, and vascular thrombosis have been observed in the treated area, ultimately followed by dense fibrosis [12–14]. In human PCa, the histological lesions induced by WST11 VTP have been briefly described in review articles focusing on new PCa therapies [15, 16]. They appeared similar to what had been reported for WST09: fibrosis without residual cancer cells but entrapped benign atrophic glands and a very clear demarcation between treated areas and the adjacent normal tissue [17]. In the course of phase II trials using WST11 VTP to treat localized PCa, we had the opportunity to study the prostate biopsies of 56 patients before and 6 months after VTP. The primary aim of our report was to describe the remodeling effects in prostatic tissue induced by the procedure. The secondary aim was to evaluate at 6 months post therapy the presence of residual cancer and its grade and topography, notably in the targeted area and in its relation with the prostate capsule.. Virchows Arch (2013) 463:547–552 Table 1 Inclusion and exclusion criteria for enrollment in the study Inclusion criteria Men over 18 years old Diagnosed PCa histologically proven on biopsies Gleason score ≤3+3 TNM stage up to cT2b-N0/Nx-M0/Mx PSA<10 ng/ml No prior treatment for PCa Eligible for active surveillance Exclusion criteria Contraindication for general anesthesia History of viral or alcoholic hepatitis History of inflammatory bowel disease or other risk factor for fistula formation Previous TURP Medication with photosensitizing effects Anticoagulant drugs History of sun hypersensitivity, porphyria History of renal, hepatic, hematological, or cardiovascular pathology. performed on an automated device (Autostainer, with Envision-plus revelation kit, Dako) using the cocktail antip504S (monoclonal rabbit antihuman P504S, clone 13H4, DaKo, 1:200 dilution) and anti p63 (monoclonal mouse antihuman p63 protein, clone 4A4, Dako, 1:100 dilution) with prior antigen retrieval in EDTA buffer pH 8. Magnetic resonance imaging (MRI) allowed the evaluation of the prostate volume, of any extra-prostatic or nodal extension, and was also necessary to plan the treatment.. Material and methods Treatment procedure Population The University Hospital of Angers included 56 patients between December 2008 and July 2010 in two open multicenter prospective phase II trials, with the primary aim to assess tolerance and feasibility of Tookad® Soluble WST11 VTP. Patient's consent was obtained, as well as the agreement of the Committee of Protection of Persons and the Ethical Committee. Inclusion and exclusion criteria are listed in Table 1. Initial diagnosis and staging Initial diagnosis was based on transrectal ultrasound-guided prostate biopsies of at least 12 cores (six per lobe) performed at the basis, the middle, and the apex of each hemi gland. Formalin-fixed paraffin-embedded tissue block of each biopsy was cut at six levels and analyzed after hematoxylin-phloxinsaffron (HPS) staining. Whenever the diagnosis of cancer was not straightforward on HPS stain, immunohistochemistry was. The procedure was performed under general anesthesia by the same operator. Optical fibers were installed transperineally in the prostate by ultrasound guidance. The location and number of fibers depended on the location of the tumor according to the results of the biopsies and MRI. Then, the patients received a 10-min intravenous infusion of TOOKAD® Soluble WST11. Subsequently, transillumination of the target zone began with a 753 nm wavelength laser at a fixed power of 150 mW/cm and a fixed energy of 200 J/cm during 22.2 min. This induced necrosis of the targeted prostate area. For reasons of security, the minimal distance between the optical fibers and the urethra or the capsule was kept at 5 mm. The patients were protected from light during the entire procedure and for 24 h following treatment. During a 6-month follow-up period, the patients had regular clinical examinations, blood tests (including PSA), and prostate MRI and finally underwent transrectal prostate biopsies, with the same procedure as for the initial biopsies,.

(5) Author's personal copy Virchows Arch (2013) 463:547–552. to avoid sampling bias. Histopathological reported items were biopsy core length, presence of the prostate capsule, presence of cancer and its characteristics (length relative to the total core length, Gleason score, distance between cancer and capsule and between cancer and scar tissue, and presence of perineural invasion and extra capsular extension), and presence of high-grade prostatic intraepithelial neoplasia. Specific reported items for WST11-induced lesions were presence of a fibrotic scar, inflammatory cellular reaction, necrosis, vascular thrombosis, hemosiderin deposit, and morphology of benign glands. Biopsies before and after treatment were systematically reviewed by two pathologists to assure consensus on pathologic diagnosis.. Results Fifty-six patients were included in this study. Mean age was 63 years (range, 55–75 years). All tumors were Gleason VI score (3+3) localized adenocarcinomas, histologically diagnosed on 1 to 5 biopsy cores. The cancer was bilateral in 15 cases. Tumor stage ranged from T1c to T2a. Optical fibers between 6 and 21 were used per patient. In 29 patients, no cancer was found in the 6-month biopsies. Mean initial PSA value was 6.2 ng/ml (range, 1.3–9.8 ng/ml). The mean PSA value 6 months after treatment was 3.7 ng/ml (range, 0.2 to 17.3 ng/ml), which amounts to a 40 % decrease. Post therapy remodeling We observed scarring of variable extent in 53 patients. The scars were mostly in the treated lobe and minimal (ten cases) or inexistent in the nontreated lobe. Three patients did not present any therapy-related remodeling of the prostatic tissue, two had extensive and florid carcinoma, and one had no visible cancer. Scar tissue consisted of rather sharply demarcated areas of fibrosis with dense hyalinized collagen with or without elastosis, sometimes with hemosiderin deposits, and replacing prostatic muscle and glands (Fig. 1). Rare atrophic glands without any cytonuclear atypia were at times observed in the scars, occasionally surrounded by a moderate number of lymphocytes and mononuclear histiocytes. These residual benign glands were sometimes reduced to corpora amylacea directly entrapped in the hyalinized stroma or surrounded by giant multinuclear macrophages (Fig. 2). These lesions were always at least at a 5-mm distance from the prostate capsule, which was either normal or moderately fibrotic. Vascular lesions such as intimal hyaline fibrosis or organized thrombi were rarely observed near the fibrotic tissue. In 30 cases (53 %), foci of necrosis were associated with fibrosis in the treated lobe; it appeared as coagulative. 549. necrosis, usually without containing any benign or malignant gland (Fig. 3). Necrosis was never suppurative. In only one case, rare-altered glands were embedded in necrosis; their epithelium was flat and vacuolated with anisokaryotic nuclei showing altered chromatin without a prominent nucleolus. Immunohistochemistry demonstrated p63-positive basal cells around these dystrophic glands, ruling out malignancy. Persistent cancer at 6 months post therapy Post-VTP adenocarcinoma was observed in 27/56 (48 %) patients, but in ten patients, cancer was only discovered in the nontreated lobe. In nine cases, cancer persisted in the same lobe as prior to therapy or was bilateral. In eight cases with initial bilateral cancer, residual tumor was observed in one lobe only. Successful cancer ablation in treated lobes was therefore observed in 38 cases. The Gleason score was unchanged for 22 patients (3+3) but was upgraded for five patients (3+4 for four patients and 4+3 for one patient). Interestingly, cancer involved only one core biopsy in 52 % (14/27) of the cases, and in 44 % (12/27) of the cases, only a minute adenocarcinoma with a single focus ≤1-mm long (11 patients) or two small foci of 0.5- and 1-mm long (one patient) was found. When cancer was observed in a treated lobe, it was always located outside the scar or an area of necrosis (Fig. 4), either in a biopsy core without significant post therapy lesion (13 patients) or at a variable distance of the remodeled area (range, 0.5 to 6 mm; mean, 2.7 mm). When assessable (in 33 tissue cores), the mean distance between the prostate capsule and the cancer focus was 3.2 mm (range, 0.25–10 mm; median, 1.5 mm). Furthermore, persistent cancer in a treated lobe was located at a distance ≤5 mm from the capsule in most assessable cases (10/12 patients). Persistent carcinomatous glands appeared unaffected by therapy and were easily recognized using morphological criteria, such as the absence of basal cells, prominent nucleoli, and increased nucleocytoplasmic ratio (Fig. 5). A complementary immunohistochemical analysis was necessary in only 11 cases because of small suspicious isolated glands or foci <1 mm, and cancer was confirmed in six cases. Neither extracapsular tumor invasion nor highgrade prostatic intraepithelial neoplasia was observed in any of our cases. Perineural tumor invasion after VTP was observed in eight cases.. Discussion VTP is a minimally invasive ablative therapy currently proposed as an alternative to active surveillance in low-grade.

(6) Author's personal copy 550. Virchows Arch (2013) 463:547–552. Fig. 1 Scar tissue after vasculartargeted phototherapy is sharply demarcated from normal prostatic tissue and consists of hyalinized fibrosis with rare atrophic glands. low-stage prostatic adenocarcinomas. As this therapy is still under investigation, most pathologists are unfamiliar with the histopathology of prostate biopsies performed in the follow-up of treated patients. Our study reports detailed histopathological data obtained 6 months after VTP with TOOKAD® Soluble WST11 as the photosensitizer agent. VTP effects could not be examined in the whole prostate as the primary aim of VTP was to avoid radical prostatectomy. The lesions directly related to VTP were easily recognized in treated areas as well-demarcated hyaline fibrotic scars, with or without coagulative necrosis including mild to moderate. chronic inflammation, a few atrophic benign glands, or only corpora amylacea surrounded by occasional multinuclear macrophages. In only one case, entrapped glands, surrounded by necrosis, showed dystrophic nuclei and vacuolated epithelium but basal cells were easily demonstrated using immunohistochemistry, ruling out residual malignant glands. When persistent cancer was present, tumor foci were always outside the scar tissue and showed no cytonuclear or architectural modification in comparison with the cancer tissue prior to therapy. We encountered no particular interpretation difficulties in these post therapy biopsies, neither in. Fig. 2 Residual corpora amylacea surrounded by giant macrophages in fibrous scar tissue. Fig. 3 Complete coagulative-type necrosis with only ghostly glands and hemosiderin deposits in a treated area.

(7) Author's personal copy Virchows Arch (2013) 463:547–552. Fig. 4 Florid adenocarcinoma (upper right corner) outside the remodeled treated area showing extensive fibrosis. the remodeled area nor in the other parts of the prostate tissue or in persistent cancer tissue when present. This is in contrast with lesions induced by whole prostate irradiation (external radiotherapy or brachytherapy) and by hormonal therapy, which are well-known sources of diagnostic confusion because of severe cytonuclear and architectural atypia in benign as well as in residual cancer glands. After radiation therapy, confusing changes in non-neoplastic prostate tissue include vacuolated cytoplasm, nuclear polymorphism,. Fig. 5 High magnification of the florid adenocarcinoma from Fig. 4. Malignant glands are unaffected by therapy. 551. hyperchromatic smudged nuclei, and even macronucleoli in prominent basal cells. In irradiated cancer, haphazardly scattered glands or single cells with vacuolated cytoplasm and strong variation in nuclear and nucleolar size can be found. Hormonal therapy also drastically alters normal prostate tissue and adenocarcinoma; non-neoplastic glandular atrophy, basal cell hyperplasia, and vacuolization of glandular epithelium are described, while malignant glands tend to be organized in small clusters or cords with inconspicuous lumina and show clear vacuolated cytoplasm and pycknotic nuclei with inconspicuous nucleoli. The histological modifications we observed 6 months after TOOKAD® Soluble VTP are similar to previous descriptions and quite similar to those induced by other focal ablative therapies [15, 16]. HIFU early-on induces areas of coagulation necrosis with secondary fibrosis with hemosiderin deposition and sometimes isolated corpora amylacea as the only evidence of preexisting glands in the treated area [16]. Cryotherapy after several months is followed by stromal fibrosis, basal cell hyperplasia, foreign-body giant cell reactions, coagulative necrosis, and nonspecific inflammation [16]. Interstitial laser ablation induces defined areas of necrosis [16]. Most importantly, if residual adenocarcinoma is still present after focal therapy, it does not present any histological modification that would complicate diagnosis and Gleason grading. Our series confirmed that TOOKAD® Soluble VTP can effectively destroy PCa in the targeted area. Residual cancer, even damaged, was never observed in the scar tissue. However, florid cancer was found in some follow-up biopsies in areas outside the targeted area. In most cases, residual cancer was of low-volume and low-Gleason score and thus eligible either for active surveillance or a second round of focal therapy. In patients with progressing cancer, radical therapy may still be performed. Cancer observed at 6 months post-VTP may be located in the opposite lobe, which might be due to false negative sampling of initial staging biopsies. In a substantial number of patients in this phase II study, of which the primary aim was to assess tolerance and feasibility, florid cancer was located within the area deliberately untreated to avoid prostate capsule necrosis and extra-prostatic tissue damage. This finding may be of interest for improvement of the VTP procedure when tumors are near the capsule or for more appropriate selection of patients after careful evaluation of the distance between cancer and capsule in pre-therapy staging. An international phase III study to assess the efficiency of this promising focal therapy is presently ongoing and will provide further histological data..

(8) Author's personal copy 552 Conflict of interest Abdel Rahmene Azzouzi is a proctor and investigator for Steba Biotech; other authors declare no conflict of interest.. References 1. Eggener SE, Scardino PT, Carroll PR et al (2007) Localized prostate cancer: a critical appraisal of rationale and modalities. J Urol 178:2260–2267 2. Marberger M, Carroll PR, Zelefsky MJ et al (2008) New treatments for localized prostate cancer. Urology 72:36–43 3. Mazzucchelli R, Scarpelli M, Cheng L et al (2009) Pathology of prostate cancer and focal therapy (‘male lumpectomy’). Anticancer Res 29:5155–5161 4. Ahmed HU, Moore C, Emberton M (2009) Minimally-invasive technologies in uro-oncology: the role of cryotherapy, HIFU and photodynamic therapy in whole gland and focal therapy of localised prostate cancer. Surg Oncol 18:219–232 5. Lecornet E, Ahmed HU, Moore CM, Emberton M (2010) Conceptual basis for focal therapy in prostate cancer. J Endourol 24:811– 818 6. Arumainayagam N, Moore CM, Ahmed HU, Emberton M (2010) Photodynamic therapy for focal ablation of the prostate. World J Urol 28:571–576 7. Windahl T, Andersson SO, Lofgren L (1990) Photodynamic therapy of localized prostatic cancer. Lancet 336:1139 8. Lepor H (2008) Vascular-targeted photodynamic therapy for localized prostate cancer. Rev Urol 10:254–261. Virchows Arch (2013) 463:547–552 9. Moore C, Pendse D, Emberton M (2009) Photodynamic therapy for prostate cancer—a review of current status and future promise nature clinical practice. Nat Clin Pract Urol 6:18–30 10. Mazor O, Brandis A, Plaks V et al (2005) WST11, a novel watersoluble bacteriochlorophyll derivative; cellular uptake, pharmacokinetics, biodistribution and vascular-targeted photodynamic activity using melanoma tumors as a model. Photochem Photobiol 81:342–351 11. Liu T, Wu LY, Choi JK, Berkman CE (2010) Targeted photodynamic therapy for prostate cancer: inducing apoptosis via activation of the caspase-8/-3 cascade pathway. Int J Oncol 36:777–784 12. Chen Q, Huang Z, Luck D et al (2002) Preclinical studies in normal canine prostate of a novel palladium-bacteriopheophorbide (WST09) photosensitizer for photodynamic therapy of prostate cancer. Photochem Photobiol 76:438–445 13. Huang Z, Chen Q, Dole KC et al (2007) The effect of Tookadmediated photodynamic ablation of the prostate gland on adjacent tissues–in vivo study in a canine model. Photochem Photobiol Sci 6:1318–1324 14. Chevalier S, Anidjar M, Scarlata E et al (2011) Preclinical study of the novel vascular occluding agent, for photodynamic therapy of the canine prostate. J Urol 186:302–309 15. Evans AJ, Ryan P, Van der Kwast T (2011) Treatment effects in the prostate including those associated with traditional and emerging therapies. Adv Anat Pathol 18:281–293 16. Srigley JR, Delahunt B, Evans AJ (2012) Therapy-associated effects in the prostate gland. Histopathology 60:153–165 17. Trachtenberg J, Weersink RA, Davidson SR et al (2008) Vasculartargeted photodynamic therapy (padoporfin, WST09) for recurrent prostate cancer after failure of external beam radiotherapy: a study of escalating light doses. BJU Int 102:556–562.

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