We developed a model of orthotopic transplantation of bladder tumor cells in female Fischer rats using a new reproducible technique. After first performing the mechanical abrasion of a portion of the bladder urothelium with an Abrader© inserted transurethrally via a catheter, we administered a suspension of 5–40 × 10 6 viable AY‐27 tumor cells in sterile phosphate‐buffered saline to the bladder cavity. This rapidly led to a tumor growth incidence of approximately 100%. The induced bladder tumors grew expansively into the bladder cavity from the surface (mucosa) and gradually invaded the submucosa, muscles, serosa and surrounding tissue (high‐stage invasive transitional cell carcinoma). Size and staging were related to the quantity of tumor cells instilled into the bladder cavity. This model matches the characteristics of human bladder tumor more closely than other bladder cancer models induced with tumor cells. Moreover, it presents many advantages: the method is reproducible, tumors grow rapidly, they are directly attached to the bladder surface and they are always located on the bladder wall, in line with the urethra. This proves especially helpful for evaluating chemotherapeutic agents by different means such as in vivo fluorescence spectroscopy, a noninvasive method used in photodynamic therapy, or other methods designed to detect and treat transitional cell carcinoma. © 2002 Wiley‐Liss, Inc.

Abstract Coutier, S., Bezdetnaya, L., Foster, T. H., Parache, R-M. and Guillemin, F. Effect of Irradiation Fluence Rate on the Efficacy of Photodynamic Therapy and Tumor Oxygenation in Meta-Tetra (Hydroxyphenyl) Chlorin (mTHPC)-Sensitized HT29 Xenografts in Nude Mice. Radiat. Res. 158, 339–345 (2002). We present direct experimental evidence of the fluence-rate-dependent, radiation-induced variations in intratumor oxygen partial pressure (pO2) in HT29 human colon adenocarcinoma xenografts subjected to meta-tetra(hydroxyphenyl)chlorin (mTHPC)-based photodynamic therapy (PDT). The data establish a correlation between tumor oxygenation and treatment outcome. Tumor-bearing mice were injected with 0.3 mg/kg photosensitizer and subjected 72 h later to a 12 J/cm2 red light dose administered at fluence rates of 5, 30, 90 and 160 mW/cm2. A significant decrease in mean and median pO2 was registered at approximately half of the total radiation fluence was delivered in tumors treated at rates of 160 and 90 mW/cm2. Conversely, with the two lower fluence rates, intratumor pO2 was maintained at levels comparable to those measured before illumination. Tumor oxygenation values registered shortly after every treatment protocol were at least equal to baseline levels, thus excluding the possibility of significant acute vessel damage during illumination. The tumor regrowth profile correlated with the pO2 values monitored during irradiation. Tumors treated with fluence rates of 5 and 30 mW/cm2 exhibited significantly longer tumor quadrupling times than those treated at 160 and 90 mW/cm2. Improved tumor destruction could be expected by reducing the rate and the extent of oxygen depletion during meta-tetra(hydroxyphenyl)chlorin photodynamic therapy using low fluence rates.

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm−2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen-mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 ± 2.2 mM in this system.

Primary non-Hodgkin's lymphoma of the testicle is rare. We analysed cases treated in French anticancer centres from 1969 to 1995. All cases were reviewed and classified according to the R.E.A.L. Classification. Eighty-four cases were included in this study. The median age was 67 years (17-85). Disease was classified as stages I in 42 cases, stages II in 19 and stages III-IV in 23. Diffuse large B-cell lymphoma was diagnosed in 75% of cases. Treatment included orchidectomy and radiotherapy and/or chemotherapy. A complete response was obtained in 72.6% of the patient population and in 100%, 68% and 33% of stage I, II and III-IV disease respectively. Recurrence occurred in 32 cases and the most frequent site was the central nervous system: six of these patients presented stage I disease. Median overall survival was 32 months for the entire population, 52 months for stage I, 32 months for stage II, and 12 months for stage III-IV cases (P < 0.0001). Among patients presenting stage I disease, no difference was found between those treated with combined surgery and chemotherapy or surgery followed or not followed by radiotherapy. This study confirms that non-Hodgkin's lymphoma of the testicle carries a poor prognosis. Systemic adjuvant chemotherapy should be discussed because of the high recurrence rate. Inclusion of these cases in large co-operative prospective studies is recommended.

The presence of the c-erbB2 oncoprotein was demonstrated by immunohistochemistry in a study involving 173 mammary lesions. The lesions included infiltrating cancers, non-invasive neoplasia, as well as atypical and benign lesions. Our aim was to investigate the correlation between the c-erbB2 oncoprotein overexpression and the morphological features of the different mammary tissues analyzed to obtain a better characterization for the growth potential of certain lesions, with emphasis placed on the non-invasive neoplasia and the atypical lesions. Nearly 30% of infiltrating ductal carcinomas (27/89 cases) and 2 out of 24 infiltrating lobular carcinomas were positive. The comedocarcinomas were mostly stained (83%). In contrast, the intraductal carcinomas of cribriform or papillar patterns were consistently negative. No staining was observed in the atypical epithelial hyperplasia located in the vicinity of positive cancers for anti-oncoprotein c-erbB2 antibody. Furthermore, the only 5 positive cases for c-erbB2 out of 32 cancer-free cases were three fibroadenomas and two fibrocystic diseases with atypical ductal hyperplasia. A close correlation was thus observed between c-erbB2 oncoprotein overexpression and cancerous cell morphology, characterized by a marked nuclear hypertrophy often associated with cellular pleomorphism. However, predictive abnormalities of malignant transformation in nonneoplastic epithelial proliferation was difficult to identify, considering only the c-erbB2 expression. A group of tumors with little nuclear abnormalities were found positive for c-erbB2 immunostaining. These probably corresponded to a particular cellular phenotype. Further studies involving other oncogenes should lead to a better characterization of the different tumor phenotypes and help to clarify breast carcinogenesis.

The treatment of skin tumors is an application of photochemotherapy (PCT) which involves an initial administration of a photosensitizer (PS) followed by irradiation with a light beam that causes the PS to produce cytotoxic oxygen species within the tumors. As the PS is also present in normal skin, it is necessary to know how it is distributed between the two tissues. In this study, we have used SKH-1 hairless mice bearing papillomas or carcinomas chemically induced. The biodistribution of hematoporphyrin derivative (HpD) and the tissue autofluorescence measurements were studied by light induced fluorescence spectroscopy. The tumor and normal autofluorescence spectra measured on control mice with papillomas or carcinomas had a very similar shape. However, the principal endogenous porphyrin peak at about 630 nm showed a fluorescence signal amplitude 2 (for papilloma) and 1.5 (for carcinoma)-fold higher than the one found for the normal skin. Moreover, the fluorescence intensity of carcinoma spectrum is 1.4-fold lower than the one of papilloma spectrum at 630 nm. The tissue autofluorescence can be used to distinguish tumor from normal skin and benign from malignant tumor. This difference in fluorescence intensity at 630 nm was directly related to the concentration of endogenous porphyrins in the tumor. Fluorescence intensity ratios between tumor and normal skin were measured 4, 8, 24, 48, 72 and 96 hours after intraperitoneal injection of HpD (5 mg/kg body weight). The best tumor/normal skin ratio was 6.2 for HpD and the time required to reach this ratio was 48 h. HpD showed a moderate selectivity since the ratio was higher than 1 during the four first days. Photodynamic therapy with the same dose of HpD used in this biodistribution study must also be carried out to verify that the maximal tumor/skin ratio corresponds to the maximal efficiency of HpD.

This study aimed to determine whether haptocorrin (HC), a vitamin B12 binder, is stored in hepatic cells and whether this storage is modified by hepatic carcinogenesis. It was carried out using immunohistochemistry on different liver tissues (normal liver and steatosis, N = 22; cirrhosis, N = 13; and hepatocellular carcinoma, N = 31). No significant immunostaining of HC was detected in noncancerous biopsies with the exception of in one case of cirrhosis. Hepatocellular carcinoma (HCC) sections showed a weak to moderate cytoplasmic staining of cancerous cells (93% of cases) and of noncancerous hepatocytes surrounding the tumor (95%) of cases. Sections with pseudoglandular structures showed a moderate to strong staining of their secretion products. These results and previous studies would seem to confirm the hypothesis that the raised HC serum level observed in HCC is due both to the increased hepatic synthesis of HC and to a decreased uptake by the liver of the particular isoform of this glycoprotein present in the serum of HCC patients.

We report a new case of glial tissue in uterine cervix found in a 63 year-old woman with an abortion 30 years ago. The lesion, measuring 0.5 cm in diameter, showed a typical feature of mature glial tissue immunoreactive for the glial fibrillary acid protein. Hypothesis concerning its histogenesis (fetal graft, tumor, metaplasia) are discussed. Fetal implantation is likely as in the majority of published cases.

A retrospective histological and immunohistochemical study was performed on 66 basal cell carcinomas (BCC). To determine the differentiation stages of epithelial cells in these BCC, three monoclonal antibodies directed against cytokeratins K1, K2, K9 and K10-11 (EE21-06), to cytokeratins K1 to K19 (F12-19), and to corneodesmosin (G36-19) were used in indirect immunofluorescence on paraffin-embedded sections. Three histological groups of BCC with specific cytokeratin immunohistochemical features were distinguished: (1) superficial BCC were unlabelled, (2) nodular and variant (keratotic, adenoid) BCC showed an homogeneous labelling, and (3) infiltrative aggressive-type BCC showed a heterogeneous cell to cell labelling. Some nodular BCC cells presented characteristics of granular keratinocytes, i.e. they were labelled by the anticorneodesmosin antibody. All the clinically recurrent tumors were found to be of the infiltrative aggressive-type. If these aggressive forms of BCC were not identified by specific marker, their topographic patterns of labeling with antibodies directed to cytokeratins allowed them to be distinguished. We suggest that an immunohistochemical analysis with antibodies specific for different stages of keratinocyte differentiation is an efficient complement to histological diagnosis of BCC.

A tamoxifen-resistant cell line (MCF7TAM) was established from tamoxifen-sensitive MCF-7 human breast cancer cells expressing estrogen receptors. Though the resistant cell line grows in the presence of tamoxifen, estrogen receptors continue to be expressed at similar levels as in the parental cell line. However, estrogen receptors appeared to be altered in the resistant cell line since important discrepancies are observed between results obtained with ligand binding assays and immunoenzymatic assays, tending to show modifications of estrogen receptor ligand binding capacity. The intracellular distribution of tamoxifen in sensitive and resistant cell lines was investigated using fluorescence of eosin–tamoxifen ionic association. Fluorescence emission spectra of eosin, tamoxifen and eosin–tamoxifen complex (λex = 480 nm) were analyzed and the maximal fluorescence intensity found for the complex (λem = 540 nm) was four times higher than that of eosin alone, while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be mainly located surrounding the nucleus, although nuclear fluorescence intensity was significantly lower. No highly fluorescent granules were observed in the resistant cell lines as opposed to sensitive cells. Improvement of this fluorescence microscopy methodology could appear of interest, taking into account the complexity of tamoxifen resistance molecular pathways.

In a case-control study, including 135 cases of endometrial carcinoma diagnosed after breast cancer and 467 controls, the relative risks for endometrial carcinoma were higher for women treated with tamoxifen (4.9; P = 0.0001) and those who undergone pelvic radiotherapy (7; 8; P = 0.0001). After adjusting for confounding factors, a multivariate analysis has shown an increased risk of endometrial carcinoma for tamoxifen users, especially for treatment longer than 3 years and for pelvic radiotherapy. Patients treated with tamoxifen had more advanced tumours and lower survival rates than those who had not received tamoxifen.

As the biochemical assay, the measurement of hormone receptors by immunocytochemistry in invasive breast cancers may predict the ability of patients to respond to hormone therapy. The objective of this work is to determine the reliability of hormone receptors analysis on cytologic spreadings. Estrogen and progesterone receptors analysis was carried out in 109 invasive breast carcinomas: (1) with a SAMBA 2005 image analysis system on frozen cytologic spreadings (ER/PR-ICA, Abbott); (2) by visual evaluation on paraffin sections (ER-1D5, Dako; PR-ICA, Abbott); (3) by biochemistry (EIA, Abbott). There is a significant correlation between the three methods of hormone receptors analysis (0.716 to 0.842). The sensitivity of immunocytochemical methods ranges from 88.0 to 94.3%, and the specificity from 70.0 to 94.7%. The minimum concordance is 87.2%. This study demonstrates that immunocytochemistry of hormone receptors is a good alternative to biochemical measurement, especially when applied to cytologic spreadings. Therefore, immunocytochemistry can be used, when conventional biochemical assay cannot be performed for hormone receptors evaluation, particularly on cytoponctions.

Thirty-eight previously untreated patients with a histologically proved diagnosis of nasopharyngeal carcinoma were evaluated for in vivo cell kinetics before treatment by conventional radiation therapy. Thirty-seven tumors were analyzed by flow cytometry. Values of median potential doubling time (Tpot), labelling index (LI), and duration of S phase (Ts) were, respectively, 10.9 days, 3.8%, and 10.8 hours. In 35 cases, the results obtained from two biopsies of the same tumor were compared. A good reproducibility was obtained for LI and Tpot (P < 0.01). Thirty-one tumors were analysed by immunohistochemistry and labelling index (HLI) was determined in 24 tumors with a percentage of labelled cells varying from 6.1% to 39.2% (median value = 18.5%). No correlation was found between LI and HLI, but when observations were focused on the restricted group of DNA aneuploid samples, mean values of LI and HLI were closer (respectively, 12.8 +/- 4.5% and 18.3 +/- 7.7%) and a good correlation was obtained (P = 0.01). Moreover, no difference in proliferation was found between diploid and aneuploid tumors. Considering these results, a combined Tpot was calculated that allowed classification of tumors as highly or slowly proliferative.

A tamoxifen resistant cell line (MCF7(TAM)) was established from tamoxifen sensitive MCF-7 human adenocarcinoma cells expressing estrogen receptors. The resistant cell line was found to express estrogen receptors to similar level as the parent cell line but the receptors were found to be altered, having lost their ability to bind estradiol or tamoxifen. The fluorescence of eosin-tamoxifen ionic association was used to investigate intracellular location of tamoxifen in both sensitive and resistant cell lines. Fluorescence emission spectra of eosin, tamoxifen and eosin-tamoxifen complex (lambda exc=480mn) were analysed and showed that maximal fluorescence intensity of the complex (lambda em=540 nm) was four times higher than that of eosin alone while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be diffusively located in the cytoplasm and nuclear fluorescence intensity was significantly lower. No difference was observed in fluorescence intensity or location in tamoxifen resistant cells, although it has been previously correlated with clinical responsiveness. Improvement of this fluorescence microscopy methodology appears necessary to provide accurate results taking into account the complexicity of tamoxifen resistance molecular pathways.

As the biochemical assay, the measurement of hormone receptors by immunocytochemistry in invasive breast cancers may predict the ability of patients to respond to hormone therapy. The objective of this work is to determine the reliability of hormone receptors analysis on cytologic spreadings. Estrogen and progesterone receptors analysis was carried out in 109 invasive breast carcinomas: (1) with a SAMBA 2005 image analysis system on frozen cytologic spreadings (ER/PR-ICA, Abbott); (2) by visual evaluation on paraffin sections (ER-1D5, Dako; PR-ICA, Abbott); (3) by biochemistry (EIA, Abbott). There is a significant correlation between the three methods of hormone receptors analysis (0.716 to 0.842). The sensitivity of immunocytochemical methods ranges from 88.0 to 94.3%, and the specificity from 70.0 to 94.7%. The minimum concordance is 87.2%. This study demonstrates that immunocytochemistry of hormone receptors is a good alternative to biochemical measurement especially when applied to cytologic spreadings. Therefore, immunocytochemistry can be used, when conventional biochemical assay cannot be performed for hormone receptors evaluation, particularly on cytoponctions.

An endometrioid ovarian adenocarcinoma cell line CAVEOC-2 was characterized. Maintained in monolayered culture, CAVEOC-2 cells exhibited a 33-hr doubling time. When xenografted into nude mice, these cells produced fast growing tumors. Colony-forming efficiency in agar was 50%. DNA index was 1.5 and cytogenetic analysis showed a triploid karyotype. CAVEOC-2 cells did not express mdr-1 gene and were chemosensitive to doxorubicin (IC50 = 1.82 +/- 0.76 mumol/l), paclitaxel (IC50 = 3.33 +/- 0.26 nmol/l) and docetaxel (IC50 = 0.68 +/- 0.28 nmol/l), while they showed an intermediate sensitivity to cisplatin (IC50 = 9.40 +/- 1.02 mumol/l). CAVEOC-2 cells seemed highly radioresistant (SF2 = 0.81, alpha = 0.02 Gy-1, beta = 0.025 Gy2, and MID = 4.31 Gy). Activities of glutathione S transferase and gamma-glutamyl transpeptidase were respectively 23.5- and 3.4- fold higher than those of sensitive A2780 cell line. These characteristics make the CAVEOC-2 cells a suitable model for the study of human endometrioid ovarian adenocarcinoma.

The effects of a classic two-stage carcinogenesis protocol on the formation of skin tumors in hairless female SKH-1 mice were studied in order to carry out photochemotherapy on the mice bearing tumors later. Mice were initiated with a single application of 100 nmol of 7,12- dimethylbenz[a]anthracene in 0.1 ml acetone and promoted one week later, twice weekly with topical applications of 1.8 nmol (first protocol) or 5 nmol (second protocol) 12-o- tetradecanoylphorbol-13-acetate in 0.1 ml acetone. The first tumors occurred between 4 and 6 weeks after the beginning of the promotion process depending on the protocol and the percentage of mice bearing tumors increased and reached 41% and 100% at the end of the treatment respectively for the first and the second protocol. Depending on the protocol, the tumor yield was 0.8 for the first one and approximately 10 for the second one whereas we expected 3 tumors per mouse. Histology of some skin tumors revealed that all were papillomas, hence benign tumors. These papillomatous lesions seem characteristic of a viral attack as seen in other strains of mammals including humans.

Les récepteurs stéroïdiens dans les cancers du sein sont traditionnellement dosés par les biochimistes. Actuellement, les anatomopathologistes peuvent fournir rapidement et à moindre coût une évaluation chiffrée de ces récepteurs. La méthode immunohistochimique permet d'obtenir un marquage nucléaire quantifiable des récepteurs hormonaux. Cette quantification est utile au clinicien pour le choix d'un protocole de traitement.