Absence of Bcl-xL down-regulation in response to cisplatin is associated with chemoresistance in ovarian carcinoma cells.

OBJECTIVE: Recurrence and subsequent acquired chemoresistance to platinum-based treatments constitute major hurdles to ovarian carcinoma therapy. Our objective was to examine the involvement of Bcl-xL anti-apoptotic protein in resistance to cisplatin. METHODS: We described the effect of cisplatin on cell cycle and apoptosis induction in sensitive (IGROV1 and OAW42) and resistant (IGROV1-R10 and SKOV3) ovarian carcinoma cell lines. We correlated it with Bcl-xL mRNA and protein expression after exposure to cisplatin. We then used bcl-xS gene transfer to impede Bcl-xL activity. RESULTS: Our study showed that Bcl-xL basal expression was high in both sensitive and resistant cell lines, as well as in all the studied ovarian tumor samples. Thus, Bcl-xL basal expression could not allow to predict sensitivity. Wondering whether variation of Bcl-xL level in response to cisplatin could be a better determinant of sensitivity, we investigated the expression of this protein in the cell lines after treatment. Cisplatin-induced down-regulation of Bcl-xL was strictly associated with apoptosis and absence of recurrence in vitro. Conversely, the maintenance of Bcl-xL expression in response to cisplatin appeared as a sine qua non condition to escape to treatment. To try to sensitize SKOV3 cells by impeding anti-apoptotic activity of Bcl-xL, we transfected bcl-xS gene in these cells. Bcl-xS exogenous expression was only slightly cytotoxic on its own, but highly sensitized SKOV3 resistant cells to cisplatin-induced apoptosis, and delayed recurrence. CONCLUSION: This work thus provides one more argument to put Bcl-xL forward as a pertinent target of inhibition to overcome chemoresistance of epithelial ovarian carcinoma.

The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells.

The seriousness of ovarian cancer, which is related to the observed link between recurrency and cell cycle control defect, prompted us to explore the effect of ectopic expression of the cdk inhibitor p21(cip1/waf1) on ovarian carcinoma chemosensitivity. The transfection of p21(cip1/waf1) cDNA into SKOV3 and OVCAR3 cells led to reduction of tumor cell growth, enhanced susceptibility to cisplatin-induced apoptosis, and abolition of recurrency after cisplatin exposure. p21(cip1/waf1) gene transfer allowed a marked reduction of the cisplatin concentration needed to erradicate the tumor cell population. These results suggest exploring the possible use of p21(cip1/waf1) as an adjunctive to conventional chemotherapy.

Prognostic value of DNA cytometry in 281 premenopausal patients with lymph node negative breast carcinoma randomized in a control trial: multivariate analysis with Ki-67 index, mitotic count, and microvessel density.

BACKGROUND: The clinical relevance of DNA image cytometry (ICM) and flow cytometry (FCM) remains under investigation in breast carcinoma. The objective of the current work was to study the prognostic value of DNA ICM and FCM in a series of patients randomized in a control trial. A multivariate analysis has been performed including other factors still under investigation such as Ki-67 index, mitotic count, microvessel density, and P53 and Bcl-2 expression. METHODS: Two hundred and eighty-one patients were randomized in the European Organization for Research and Treatment of Cancer 10854 trial comparing surgery followed by one course of perioperative chemotherapy versus surgery alone. Tumor parameters studied were pT, multicentricity, tumor grading according to modified Scarff-Bloom-Richardson, estrogen receptors, mitotic count per 1.7 mm(2), MIB-1, and BCL-2 scores, microvessel density, and p53 expression. ICM DNA parameters studied from paraffin embedded specimens, were DNA ploidy, proliferative index, 2c deviation index, malignancy grade, and Auer-Baldetorp typing. FCM DNA parameters analyzed on the same samples were ploidy and S-phase fraction statistics. The influence of tumor parameters, and DNA parameters on overall survival (OS), disease free survival (DFS), and metastasis-free survival (MFS) was evaluated using the Cox model. Median follow-up was 82 months. RESULTS: For OS, the prognostic parameters retained were pathologic tumor size (pT) and mitotic index (MI). Overall survival was 94% and 68% for tumors pT1/MI less than 10 and pT2-3 MI greater than or equal to 10, respectively. For DFS, age, multicentricity, and grading according to modified Scarff and Bloom were predicting factors with the same relative risk. Disease free survival was 96%, 78% and 68% respectively, when 1, 2, or 3 of those factors were present. For MFS, the only retained predicting factor was MI. MFS was 97% and 73% when MI was less than 10 and MI was greater than or equal to 10, respectively. CONCLUSIONS: Evaluation of proliferative compartment was the most important predicting factor for OS and MFS in the current series of premenopausal lymph node negative patients with breast invasive carcinoma. When working on paraffin embedded tissue, the best way of assessing it was MI count. ICM DNA analysis results were not selected in multivariate analysis. DNA analysis by FCM should be considered as an unsuitable technique when working on paraffin embedded tissue.

Flow cytometry in primary breast carcinomas. Prognostic impact of proliferative activity.

From 1990 to 1996, 607 previously untreated, node-negative, invasive breast carcinomas were sampled by a pathologist for flow-cytometric DNA analysis. The aim of the present work was to study the correlations between flow cytometric results obtained thanks to the American Consensus (AC) guidelines of 1993 and the established clinico-pathological prognostic factors (T, grade, receptors), and despite a short global follow-up (mean of 4 years), to correlate flow cytometry with the outcome of the patients. In this study S-phase fraction (SPF) correlated strongly with tumor size, histological grade, lack of steroid receptors, histological type and was together with the mitotic activity a paramount prognostic factor even after multivariate analysis. This study compared also the technical criteria proposed by the AC with our own more stringent ones and concluded that the criteria of the AC are relevant and allow, thanks to the use of tertiles in the reporting of SPF values, a comparison of values obtained by different teams. Our review of the literature, focused on series using fresh material, enabled us to show that there is a rather wide agreement concerning the relationship between SPF and prognosis most often after multivariate analysis. This despite the lack of standardization in the design of the studies (implementation of the technical steps or reporting of results). When estimated from fresh or frozen material following AC's guidelines. SPF along with mitotic activity should become a prognostic factor used in the daily practice by oncologists in the management of breast carcinomas.

Variation of flow cytometric DNA measurement in 1,485 primary breast carcinomas according to guidelines for DNA histogram interpretation.

From 1990-1996, 1,485 previously untreated invasive breast carcinomas were sampled by a pathologist for flow cytometric DNA analysis. The aim of the present work was to study the variations of flow cytometric DNA ploidy and S-phase evaluation according to the conditions of DNA histogram interpretation. Results obtained with the American Consensus guidelines of 1993 and the François Baclesse Department of Pathology's own guidelines are presented. According to the percentage of events taken into account to identify a DNA aneuploid peak, the proportion of DNA diploid cases can change from 35-39%. For S-phase evaluation, although the two guidelines were quite different, the results of S-phase cutoff were identical. Whichever guidelines were used, there was a strong relationship between DNA ploidy and/or S-phase and classical clinicopathological factors (T, N, histological type, grade, receptor status, or lymphatic invasion), with the exception of age, whose correlation was discrepant with S phase according to the set of guidelines. Whichever guidelines were used, ploidy and S phase correlated strongly with survival (overall, metastasis-free, or recurrence-free). Hence we recommend the use of the American consensus guidelines, despite minor imperfections, because they are now well-known, allow a high yield in the ratio of assessable S phases, and permit standardization in the technical processing and reporting of S phases, thanks to the use of terciles.

Image analysis software for automatic DNA ploidy assessment of archival solid tumours.

BACKGROUND: Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS: An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS: DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS: Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.

Acquisition of chemoresistance in a human ovarian carcinoma cell is linked to a defect in cell cycle control.

Chemoresistance is a major concern in cancer erradication; it involves various mechanisms, including defects in the apoptosis program induced by anticancer drugs. In order to further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin treatment, we established an in vitro model, mimicking a clinical protocol of administration of cisplatin. Therefore, IGROV1 ovarian carcinoma cells were exposed for 2 hr to the drug and allowed to recover for several weeks; this way of exposure was reiterated with escalating doses. We followed changes in cytotoxicity of the drug, cell cycle kinetics and long-term survival of cells after cisplatin treatment, and found that resistance to cisplatin was not associated with altered apoptosis pathway, since both cisplatin sensitive and resistant cells underwent apoptosis in a similar way. Acquisition of resistance to cisplatin was associated with the ability of the treated cells to progress through the cell cycle beyond the G1/S checkpoint; although most cells died by apoptosis, a few surviving cells proliferated and recolonized the cultures. Compared to sensitive cells, the chemoresistant variants were able to override the G1/S checkpoint whatever the dose, and the recurrent cells recolonized the cultures much faster. Analysis of alterations in gene expression suggests that the defect in cell cycle regulation could take place at the level of the cdk inhibitor p21(CIP1/WAF1).

[Automation of the cytometric analysis of the DNA content of solid tumors]. / Automatisation de l'analyse cytodensitométrique du contenu en ADN des tumeurs solides.

An automatic machine, dedicated to solid tumor DNA ploidy quantitation has been built in order to provide pathologists with a tool usable in clinical practice. Main efforts were focused on an automation of each step of the analysis and on an elimination of any subjective choice, while preserving the quality of measurement. As the software is independent of the machine architecture, it offers performances which increase in parallel with the rapid evolution of the computers. An illustration of the various functionalities of the automaton is proposed through the study of deparaffined breast cancer samples.

[Flow cytometry in ORL cancers]. / Cytométrie en flux dans les cancers ORL.

The possibility to perform flow cytometry was examined in a series of 167 patients with primary untreated head and neck carcinoma referred to our Institution from February 1989 to January 1992. In all cases, flow cytometry was carried out on frozen tumour samples. The Cox model was used including age, tumour size, nodal status on clinical assessment, topography, treatment, malignancy grade, S phase fraction and ploidy as independent variables and overall survival as dependent variable. In this study, ploidy could be assessed in only 73% of cases and S phase fraction and G2M in 65% of the population studied. No correlation could be evidenced between ploidy or SPF with other clinical, pathologic characteristics or clinical outcome. We conclude that flow cytometry should remain a research tool until the method has proved to be relevant in clinical routine, and until the yield of the technique can be improved.

Improvements in slide preparation from archival material for automated DNA measurement by image analysis.

OBJECTIVE: To optimize slide preparation for DNA content measurement by automated image analysis and to obtain a rapid and easy method for routine use. STUDY DESIGN: Some improvements in previously described methods were achieved: automatic dewaxing, reduction of washing times, accurate adjustment of final concentration of nuclei, sedimentation and temperate drying of nuclei. RESULTS: The complete preparation of 12 slides required four intermittent working hours. Accurate adjustment of the final concentration and sedimentation of nuclei resulted in a constant and homogeneous distribution of nuclei. Cell loss was prevented and the most fragile structures preserved. CONCLUSION: High-quality preparations of nuclei lead to high-resolution histograms obtained from a large collection of events. This promotes automated image analysis method instead of flow cytometry when only archival material is available for DNA content measurement and proliferating fraction evaluation.

Glucose up-regulates expression of the differentiation-associated brush border binding site for enterotoxigenic Escherichia coli colonization factor antigen I in cultured human enterocyte-like cells.

The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding of CFA/I to a brush border-associated component. Binding of the purified radiolabeled [125I]CFA/I- and 14C-labeled CFA/I-positive bacteria could be displaced by an increasing concentration of unlabeled CFA/I. Moreover, we showed that expression of the specific CFA/I binding developed as a function of cell differentiation in Caco-2 cells, whereas expression of the nonspecific binding did not. Expression of the brush border differentiation-associated component acting as a binding site for CFA/I was up-regulated by glucose. Indeed, the enterocyte-like HT-29 glc- cell subpopulation not expressing the CFA/I binding site when cultured in dialyzed serum and hexose-free medium regained the ability to bind CFA/I when the cells were returned to culture medium containing glucose. Furthermore, expression of the brush border-associated CFA/I binding site in the enterocyte-like Caco-2 cells was repressed when the cells were cultured in hexose-free conditions.

In vivo activity and pharmacodynamics of cefotaxime or ceftriaxone in combination with fosfomycin in fibrin clots infected with highly penicillin-resistant Streptococcus pneumoniae.

Using a clinical pneumococcal strain for which MICs were 2, 0.5, 0.5, and 16 mg/liter for penicillin, cefotaxime, ceftriaxone, and fosfomycin, respectively, we studied the efficacies of these antibiotics alone and in combination in one or two doses or in continuous infusion over 6 h in the treatment of the prolonged (48-h) experimental fibrin clot infections in rabbits. Doses were chosen to obtain low antibiotic concentrations. We observed the highest bacterial reductions (change in log10 CFU per gram) with the following five regimens: combination of cefotaxime plus fosfomycin given in two divided doses 6 h apart (each at 50 mg/kg of body weight given intravenously (4.2 +/- 0.7 CFU/g), ceftriaxone (8 mg/kg given once intravenously) along with one or two doses of fosfomycin (3.79 +/- 0.6 and 3.95 +/- 0.5 CFU/g), cefotaxime alone administered in two divided doses (3.6 +/- 0.4 CFU/g), and a 6-h continuous infusion of cefotaxime (100 mg/kg) with fosfomycin (100 mg/kg) (3.5 +/- 0.4 CFU/g). The bacterial reductions obtained with these five regimens were all higher than those obtained with the other regimens tested (P < 0.05). The time of bacterial regrowth was significantly delayed with the two doses of the cefotaxime-fosfomycin regimens (23.2 +/- 11 h) compared with those with the other combinations (P < 0.05). The rate of bacterial regrowth with this regimen was even lower than that observed with cefotaxime alone given in two doses (P < 0.05). By a multivariate analysis, the most important independent parameters for efficacy were the maximal concentrations of beta-lactam antibiotics and the residual concentration of fosfomycin and, for the combinations, the log of the area under the concentration-time curve/MIC ratio for beta-lactam antibiotics. From these findings, the combinations cefotaxime or ceftriaxone plus fosfomycin could be proposed for the treatment of infections caused by highly penicillin-resistant pneumococci.

DNA cytometric abnormalities in human esophageal squamous intraepithelial and invasive carcinomas.

DNA ploidy abnormalities of 21 archival human esophageal intraepithelial neoplasia samples were assessed, using image cytometry of deparaffinized samples, with reference to invasive squamous cell carcinoma and corresponding uninvolved squamous epithelium. Cytometric parameters investigated were proportion of G0G1 aneuploid cell population, histogram typing, proportion of G0G1 diploid nuclei, coefficient of variation, mean DNA content, crude 5c exceeding proportion, 2c deviation index, malignancy index and grade, and entropy. The distributions of the above parameters were compared using the paired t test and Fisher's exact test. Among 10 parameters used, Auer typing of DNA histograms, crude 5c exceeding rate, 2c deviation index and malignancy grade according to Böcking allowed discrimination between uninvolved epithelium and invasive squamous cell carcinoma as well as intraepithelial neoplasia. In particular, the distribution of 2c deviation index in the uninvolved epithelium did not overlap that of intraepithelial and invasive carcinomas. The above four parameters, however, were unable to discriminate intraepithelial neoplasia from invasive carcinoma.

Development of an automatic program for the stereological estimation of the volume weighted mean volume of cell nuclei.

A stereological method was recently developed for the estimation of the volume weighted mean volume of particles from 2D sections. Several authors successfully applied this manual method to paraffin sections of bladder cancers and malignant melanomas and found a good correlation between the mean nuclear volume and the histological grading and prognosis of these tumors. The implementation of this measuring method on an automatic image analyser, devoted to routine work in tumor pathology, is presented. The accuracy of the automatic measurement has been evaluated together with the first results obtained from human esophageal cancers.

Fast liquid chromatography for the determination of drugs in plasma and combination with liquid-solid extraction in a fully automated system.

Fast liquid chromatography was applied to the assay of several drugs in plasma. Short columns, 3.3-4 cm long, packed with C18 material, 3 microns particle size, were used. The peaks were little subject to extra-column band-broadening because the investigated drugs were eluted with high capacity factors in order to obtain an adequate separation from plasma components. The main influences on efficiency were the response time of the detector and the solvent composition of the injected sample. Conventional apparatus was used. A fully automated analytical system combining liquid-solid extraction via disposable extraction columns and fast liquid chromatography on a small-dimensioned 3 microns particle size column is described for the assay of drugs in plasma. Automation was accomplished by using the Automatic Sample Preparation with Extraction Columns system.

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks.

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 microns deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-500 (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.

Determination of oxiracetam in plasma and urine by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic method was developed for the determination of oxiracetam in plasma and urine. A sample of plasma (250 microliters) or urine (10 microliters) is mixed with the internal standard solution, 4.2 ml of acetonitrile-water (1000:4, v/v) and 0.8 ml of dichloromethane, and 1 ml of the clear solution is injected onto a first column filled with Li-Chrosorb NH2. The sample is eluted with acetonitrile-water (95:5, v/v). The portion of the eluate (heart-cutting) from this column containing the compounds of interest is selected and loaded on a Nucleosil NH2 column and eluted with acetonitrile-water (90:10, v/v). During this chromatography the first column (LiChrosorb NH2) is rinsed with acetonitrile-water (50:50, v/v). Ultraviolet detection at 200 nm is used for quantitation. The limit of quantitation of oxiracetam is ca. 1.5 microM (240 ng/ml) in plasma and 76 microM (12 micrograms/ml) in urine. Oxiracetam was stable in plasma and urine samples kept frozen at -20 degrees C for nine months and one year, respectively.

Analysis of the results of the micronucleus test in patients presenting upper digestive tract cancers and in non-cancerous subjects.

A micronucleus test was performed on 75 subjects of whom 38 presented with cancer of the upper digestive tract and 37 were free of disease; the absence of cancerous or pre-cancerous lesions in this latter group was confirmed by endoscopy and vital staining. The daily levels of alcohol and tobacco consumption of the 75 subjects were determined by precise questioning: 78% of the non-cancerous subjects smoked less than 10 g of tobacco per day whereas 79% of the cancer patients smoked 10 g or more daily. The alcohol intake of 78% of the non-cancerous subjects and 63% of the cancer patients was less than 101 ml per day. Only 10% of the cancer patients had combined daily intake levels corresponding to the threshold of sensitivity of the micronucleus test as defined by previous studies. The mean frequency of micronucleated buccal cells was 0.26% in the cancer patients and 0.13% in the non-cancerous subjects. All non-cancerous patients presented a negative test. Only 5% of the cancer patients presented a micronucleated cell frequency above 1% and could thus be considered as positive. It thus appears that the micronucleus test was not significantly positive in our population of 38 cancer patients.