Photodynamic therapy of ovarian cancer peritoneal metastasis with hexaminolevulinate: a toxicity study.

CONTEXT: While photodynamic therapy (PDT) is a promising treatment for peritoneal carcinomatosis, its use is often limited because of the toxicity of photosensitizers. In this study, safety of PDT with hexaminoevulinate (HAL), a second generation photosensitizer, is assessed. METHODS: PDT of the peritoneal cavity was performed in a rat model of peritoneal carcinomatosis. Rats were treated according to different protocols: with full or half HAL dose, after intraperitoneal or oral administration of HAL, 4 or 8h after its injection, using red or green light, after protection of the liver or cooling of the abdominal wall. Toxicity was assessed by blood tests quantifying hematocrit, liver and muscular enzymes and by pathological examination of abdominal and intrathoracic organs after treatment. The results were analyzed in the light of quantification of fluorescence and protoporphyrin IX (PPIX) content of the same organs. RESULTS: PDT with HAL induced rhabdomyolysis, intestinal necrosis and liver function test anomalies, leading to death in 2 out of 34 rats. The liver and the intestine contained high levels of PPIX (3-5 times more than tumor nodules). CONCLUSION: HAL PDT lacked specificity. However, the strategy associating diagnosis, treatment and evaluation of the results in one single procedure was effective and should be tested with other photosensitizers.

Adenoid basal hyperplasia of the uterine cervix: a lesion of reserve cell type, distinct from adenoid basal carcinoma.

Adenoid basal hyperplasia is an underrecognized cervical lesion, resembling adenoid basal carcinoma, except the absence of deep invasion into the stroma. We report a series of 10 cases, all extending less than 1 mm from the basement membrane. Our results support the hypothesis that adenoid basal hyperplasia arises from reserve cells of the cervix. Lesions were found close to the squamocolumnar junction, in continuity with the nearby subcolumnar reserve cells. They shared the same morphology and immunoprofile using a panel of 4 antibodies (keratin 5/6, keratin 14, keratin 7 and p63) designed to differentiate reserve cells from mature squamous cells and endocervical columnar cells. We detected no human papillomavirus infection by in situ hybridization targeting high-risk human papillomavirus, which was concordant with the absence of immunohistochemical p16 expression. We demonstrated human papillomavirus infection in 4 (80%) of 5 adenoid basal carcinoma, which is in the same range as previous studies (88%). Thus, adenoid basal hyperplasia should be distinguished from adenoid basal carcinoma because they imply different risk of human papillomavirus infection and of subsequent association with high-grade invasive carcinoma. In our series, the most reliable morphological parameters to differentiate adenoid basal hyperplasia from adenoid basal carcinoma were the depth of the lesion and the size of the lesion nests. Furthermore, squamous differentiation was rare in adenoid basal hyperplasia and constant in adenoid basal carcinoma. Finally, any mitotic activity and/or an increase of Ki67 labeling index should raise the hypothesis of adenoid basal carcinoma.

Continuous or fractionated photodynamic therapy? Comparison of three PDT schemes for ovarian peritoneal micrometastasis treatment in a rat model.

OBJECTIVE: This experimental study aimed to compare three illumination schemes to optimize hexaminolaevulinate (HAL)-PDT in a rat tumor model with advanced ovarian cancer. MATERIALS AND METHODS: Peritoneal carcinomatosis was induced by intraperitoneal 5×10(6)NuTu-19 cells injection in 60 female rats Fisher 344. Carcinomatosis was obtained 50 days post-tumor induction. Four hours post-intraperitoneal HAL (Photocure ASA, Oslo, Norway) injection, three different schemes of PDT were performed during 25 min on a 1cm(2) area. (A) Fractionated illumination (n=20) with an on-off cycle ("on": 2 min and "off": 1 min) at 30mW cm(-2) until a fluence of 30J cm(-2), (B) continuous illumination (n=20) at 30mW cm(-2) with a fluence of (45J cm(-2)C) continuous illumination (n=20) at 20mW cm(-2) with a fluence of 30J cm(-2). Laser light was generated using a 532nm KTP laser (Laser Quantum, Stockport, UK). Biopsies were taken 24h after treatment. Quantitative histology was performed. Necrosis value was determined: 0-no necrosis to 4-full necrosis. Depth of necrosis was then measured for each sample and correlated to Necrosis value. RESULTS: HAL-PDT was efficient in producing necrosis irrespective of the scheme. Tumor destruction was superior with fractionated illumination compared to both continuous illumination schemes regarding to the depth of necrosis (213±113µm vs 154±133µm vs 171±155µm) (p<0.05) or to the full necrosis rate (50% vs 30% vs 10%) (p<0.0001). CONCLUSION: Fractionated illumination during photodynamic therapy (PDT) was shown to improve tumor response. Fractionated illumination with short intervals should be considered for an effective PDT of advanced ovarian cancer.

Comparison of continuous and fractionated illumination during hexaminolaevulinate-photodynamic therapy.

OBJECTIVE: This experimental study aimed to compare continuous and fractionated illumination to optimize hexaminolaevulinate (HAL)-photodynamic therapy (PDT) in a rat tumour model with advanced ovarian cancer. MATERIALS AND METHODS: Intraperitoneal 10(6) Nu Tu-19 cells were injected in 36 female rats Fisher 344. Peritoneal carcinomatosis was obtained 26 days post-tumour induction. Four hours post-intraperitoneal HAL (Photocure ASA, Oslo, Norway) injection, two schemes of PDT were performed at 30 mW cm(-2) on a 1cm(2) area: fractionated illumination (n=16) with a on-off cycle ("on": 2 min and "off": 1 min) until a fluence of 30 J cm(-2) was delivered, and continuous illumination (n=20) with a fluence of 45 J cm(-2). Laser light was generated using a 532 nm KTP laser (Laser Quantum, Stockport, UK). Biopsies were taken 24h after treatment. Semi-quantitative histology was performed. Necrosis value was determined-0: no necrosis to 4: full necrosis. RESULTS: HAL-PDT was efficient in producing necrosis irrespective of the scheme (NV=3.34+/-0.91). Tumour destruction was superior with fractionated illumination compared to continuous illumination (3.67+/-0.70 vs. 3.10+/-0.94) (p<0.05). CONCLUSION: Fractionated illumination during photodynamic therapy was shown to improve tumour response. Fractionated illumination with short intervals should be considered for an effective PDT of advanced ovarian cancer.

Specific MALDI imaging and profiling for biomarker hunting and validation: fragment of the 11S proteasome activator complex, Reg alpha fragment, is a new potential ovary cancer biomarker.

MALDI imaging mass spectrometry represents a new analytical tool to directly provide the spatial distribution and relative abundance of proteins in tissue. Twenty-five ovary carcinomas (stages III and IV) and 23 benign ovaries were directly analyzed using MALDI-TOF MS. The biomarker with the major prevalence (80%) has been fully identified using MALDI MS and nanoESI MS and MS/MS after separation by RP-HPLC and trypsin enzymatic digestion. This marker with an m/z of 9744 corresponds to 84 amino acid residues from the 11S proteasome activator complex, named PA28 or Reg-alpha. Validation of this marker has been performed using MALDI imaging, classical immunocytochemistry with an antibody raised against the C-terminal part of the protein, specific MALDI imaging, and Western blot analysis. The validation, using immunocytochemistry, confirmed the epithelial localization of this fragment with nucleus localization in benign epithelial cells and a cytoplasmic localization in carcinoma cells. This indicates that this antibody could be used to discriminate the borderline tumor cases. At this point, a multicentric study needs to be conducted in order to clearly establish the potential of this biomarker. Taken together these studies reflect that direct tissue analysis and specific MALDI imaging strategies facilitate biomarker hunting and validation which can be named pathological proteomics.

Comparison of aminolevulinic acid- and hexylester aminolevulinate-induced protoporphyrin IX fluorescence for the detection of ovarian cancer in a rat model.

OBJECTIVES: This study set out to compare the photodetection of peritoneal micrometastases in an ovarian cancer model following administration of two precursors of protoporphyrin IX (PpIX): 5-aminolevulinic acid (ALA) and hexylester aminolevulinate (He-ALA). METHODS: ALA or He-ALA (100 mg/kg) was injected into the peritoneal cavity of 16 rats with induced peritoneal metastases of ovarian cancer. Two hours later, the tumors were visualized laparoscopically using both white light for standard exploration and blue light for fluorescence. Peritoneal micrometastases were counted. Fluorescence intensities of tumoral and normal surrounding tissues were compared. The distribution of PpIX throughout the peritoneum was studied on frozen biopsies using fluorescence microscopy and correlated with pathological findings. RESULTS: The number of micrometastases detected by the fluorescence blue mode was significantly higher (p < 0.05) than with standard white light for both ALA (235 versus 198) and He-ALA application (248 versus 199). The mean fluorescence intensity ratio between tumor and normal surrounding tissue was significantly (p < 0.05) higher for He-ALA (1.55 +/- 0.1) compared to ALA (1.45 +/- 0.1). Fluorescence microscopy confirmed that the PpIX fluorescence remained limited to cancer cells. Macroscopically fluorescing nodules were histopathologically confirmed as malignant. CONCLUSION: He-ALA is an excellent precursor for PpIX synthesis, giving the highest PpIX fluorescence contrast between normal and tumoral peritoneal tissues. Imaging with He-ALA improves the detection of peritoneal metastases compared to ALA.

Laparoscopic photodynamic diagnosis of ovarian cancer peritoneal micro metastasis: an experimental study.

The goal of this study was to assess the interest of photodynamic diagnosis (PDD) for laparoscopic detection of peritoneal micro metastasis in ovarian carcinoma. Using an experimental animal model, intraperitoneal injection of aminolevulinic acid (ALA) and hexylester of aminolevulinic acid (He-ALA) were compared in order to improve laparoscopic detection of ovarian peritoneal carcinomatosis. Twenty-one 344 Fischer female rats received an intra peritoneal injection of 106 NuTu-19 cells. At day 22, carcinomatosis with micro peritoneal metastasis was obtained. Rats were randomized in three groups concerning intra peritoneal injection before laparoscopic staging: 5-ALA hydrochloride, HE-ALA and sterile water. Using D Light system, laparoscopic peritoneal exploration was performed with white light (WL) first and then with blue light (BL). The main objective was to assess feasibility and sensibility of laparoscopic PDD for nonvisible peritoneal micro metastasis of ovarian cancer. The main parameter was the confirmation of neoplasic status of fluorescent foci by histology. Concerning PDD after intraperitoneal injection of 5-ALA, mean values of lesions seen is higher than without fluorescence (32 vs 20.7; P = 0.01). Using He-ALA, mean values of detected lesions is higher than without fluorescence (42.9 vs 33.6; P < 0.001). Neoplasic status of fluorescent foci was confirmed in 92.8% of cases (39/42). Using 5-ALA, fluorescence of cancerous tissue is significantly higher than that of normal tissue in all the rats (ratio 1.17) (P = 0.01). With He-ALA, intensity of fluorescence is significantly higher in cancerous tissue compared to normal tissue, irrespective of the rat studied (ratio 1.22; P < 0.001).

Fluorescence diagnosis of cervical squamous intraepithelial lesions: A clinical feasability study.

UNLABELLED: The potential of fluorescence diagnosis (FD) is still undeveloped in gynaecology. In order to diagnose and localize squamous intraepithelial lesion (SIL) of the cervix, a new method improving the low specificity of colposcopy, would be useful. OBJECTIVE: The goal of this study was to assess the feasability and safety of colposcopic FD of SIL after topicaly application of methyl aminolevulinate (MAL). MATERIALS AND METHODS: Patients with histologic proved cervical intraepithelial neoplasia (CIN) and planned for loop electrosurgical excision procedure (LEEP) under general anesthesia, were included in a prospective study. Before general anesthesia, a thick layer of MAL (Metvix(®)-160mg/mL cream) was applied on the cervix for 35-150min. Fluorescent colposcopic inspection of the cervix was performed using a rigid 10-mm laparoscope inserted in the vaginal cavity and connected to D-light AF system (Karl Storz Endoskope, Tuttlingen Germany). In order to confirm neoplasic status, fluorescent foci underwent directed punch biopsy(ies). RESULTS: Fourteen patients were included in the study. Colposcopic fluorescence imaging revealed red fluorescent foci in 71.4% of cases (10/14) (neoplasic status of fluorescent foci was confirmed in 80%). Concerning ME-ALA, the mean of application time was 73min (35-150). Two cases presented a false-positive fluorescence and four cases of false-negative fluorescence. For all cases of false-negative fluorescence, application time of MAL was less than 60min. We observed no systemic or local toxicity of MAL application in any of the groups. CONCLUSION: Using topical application of MAL to the cervix, we demonstrated that FD of SIL is feasible. This study justifies the further development of fluorescence imaging that combines classical white light colposcopy with the use of a photosensitizer.