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International audience; OBJECTIVE: The roles of phosphatidylinositol 3 (PI3K) and mitogen-activated protein kinases (MAPK) have been widely studied in terms of the differentiation process induced by several drugs (phorbol ester, vitamin D-3, retinoic acid, etc.), but their exact functions in leukemic cells' phenotype and their potential therapeutic role remain incompletely clarified. MATERIALS AND METHODS: In order to investigate this query, leukemia cells were cultured in presence of kinase inhibitors (KIs). Proliferation, apoptosis, and differentiation were analyzed at the cellular and molecular levels, using flow cytometry and reverse transcriptase quantitative polymerase chain reaction. RESULTS: SB203580, a P38 MAPK inhibitor, had no effect on cell proliferation, whereas LY294002, a PI3K inhibitor, and PD098059, a selective inhibitor of mitogen-activated extracellular regulated kinase (MEK) phosphorylation, arrested cells in G(0)/G(1). However, LY294002 and PD098059 acted using different mechanisms: LY294002 decreased the expression of phosphorylated S6RP, whereas PD098059 increased P21/waf1 antigen expression. SP600125, an inhibitor of N-terminal c-jun kinases, arrested cells in G(2) and induced an endoreplicative process. SP600125 increased p21 at both the mRNA and protein levels. G(2) blockage is dependent on the PI3K pathway and the endoreplicative process is dependent on the PI3K and extracellular regulated kinase (ERK) pathways and mRNA synthesis. On the other hand, PD098059 potentiated the apoptotic process induced by either SP600125 or LY294002. Modulation of the expression of CD11, CD15, CD18, and CD54 was cell-dependent. CONCLUSION: Our results suggest that KIs modulate proliferation of leukemia cells and that the MEK/ERK inhibitor, PD098059, in combination with either SP600125 or LY294002, could have clinical value.

Objective: Investigating whether extracellular factors are possible actors in tumoral progression in bladder carcinoma. Methods: RT112/G2 bladder tumour cells were grown in presence of TGFβ and analysed by immunological and cDNA microarray techniques. Results: TGFβ inhibited cell proliferation, reduced TNFα- and IFNγ-induced apoptosis by decreasing TNFα-RI and IFNγ-R antigen expression. It also inhibited cleaved caspase 8 and 9 expression, decreased E-cadherin, and increased Bclx L and cyclooxygenase-2 expression. The cDNA microarray approach showed that TGFβ up-regulated the expression of genes with defined roles in tumoral progression sometimes associated with poor outcome in bladder cancer. Conclusion: These results suggest that a part of the bladder tumoral progression process may be related to the action of exogenous TGFβ confirming the possible role for the microenvironment.

Resume L’existence d’une differenciation neuroendocrine est rapportee dans les adenocarcinomes mammaires, le plus souvent sous la forme de cellules dispersees dans la tumeur, plus rarement sous la forme d’un contingent tumoral a part entiere. Nous rapportons un cas d’adenocarcinome mammaire infiltrant avec un contingent neuroendocrine majoritaire. Huit ans apres avoir beneficie d’un traitement conservateur du sein gauche pour un adenocarcinome infiltrant etiquete « canalaire commun » SBRIII pT2N1M0, une femme de 67 ans, a presente une metastase sternale de morphologie purement neuroendocrine. L’existence d’un lien entre la tumeur mammaire et la metastase osseuse est alors discutee. La relecture de la tumeur mammaire, du curage axillaire gauche et des immunomarquages realises a posteriori ont montre que : a) la tumeur du sein comportait un contingent neuroendocrine de haut grade de malignite, a petites et grandes cellules, synaptophysine +, NCAM + et chromogranine — (80 %) et 2 contingents n’exprimant pas les marqueurs neuroendocrines, metaplasique malpighien (10 %) et canalaire de type commun (10 %) ; b) 4 des ganglions du curage etaient envahis par le contingent de type canalaire commun englobant quelques cellules tumorales NCAM+ mais synaptophysine- ; c) les recepteurs hormonaux et HER2 n’etaient pas exprimes ni dans la tumeur mammaire ni dans les ganglions metastatiques. Nous discutons l’histogenese de ces tumeurs mammaires composites avec differenciation neuroendocrine ainsi que les potentiels evolutifs des differents contingents et les implications pronostiques de ce type de proliferation.

The knowledge that cervical neoplasia are caused by human papillomavirus (HPV) infection has led to the evaluation of its role in screening. We evaluated the accuracy of HPV testing by Hybrid capture II (HC II) method in detecting cervical intraepithelial neoplasia grade 2 and 3 (CIN 2 and 3) lesions in 4 cross-sectional studies with common protocol and questionnaire in 3 different locations (Kolkata, Mumbai and Trivandrum) in India. These studies involved 18,085 women aged 25-65 years. The reference standard for final diagnosis was a combination of colposcopy/biopsy. All women were investigated with colposcopy and 3,116 received directed biopsy. The sensitivity of HPV testing for detecting CIN 2-3 lesions varied from 45.7% to 80.9% across the study sites; the specificity varied from 91.7% to 94.6% and the positive predictive value from 6.7% to 13.7%. Retesting of 298 randomly chosen denatured samples in France revealed an agreement rate of 85.9% and a kappa-value of 0.72. Although HPV testing seems to be a promising approach for cervical cancer prevention, a large range in sensitivity was observed in our study, possibly due to variations in the quality of specimen collection and reference standards. A higher sensitivity was associated with the center performing the test well. Further developments in terms of more reproducible, less expensive and less sophisticated testing are essential to make the test feasible and effective in low-resource settings.

The expression of plasminogen- and colony-stimulating factor-1-associated markers was first investigated in seven bladder carcinoma cell lines and in 15 primary bladder tumors using RT-PCR (mRNAs), zymography (protein activity), ELISA and immunocytochemistry analysis (ICC) (protein levels). The mRNAs expression, the activity and the levels of the secreted proteins were not informative. Only urokinase plasminogen activator receptor (uPA-R/CD87) and possibly plasminogen activator inhibitor type-2 (PAI2) antigen expression at the cellular levels seem to be useful markers. uPA-R antigen expression correlated with the secretion of hepatocyte growth factor (HGF) ( P=0.016) and the motility of the bladder tumor cells ( P=0.014), two markers associated with a poor prognosis in bladder carcinoma. To validate our technique and confirm these preliminary results, uPA-R and PAI2 antigen expression was determined in the imprints from 129 resected bladder carcinoma fragments. uPA-R correlated with the grade ( P=0.002), tumor invasion ( P=0.003) and the ploidy ( P=0.05) of the bladder carcinomas and with the low overall survival ( P=0.045) of the patients. PAI2 correlated only with the stage ( P=0.02) and low overall survival ( P=0.038). We conclude that in bladder carcinomas, studying the transcripts of PAs, PAIs, CSF-1 and its receptor, as well as measuring their concentration or activity in culture supernatants was of no clinical interest in terms of diagnostic or prognostic value. Only the ICC of uPA-R, which correlated with the major histopathological parameters of tumors and the low overall survival, proved to be a diagnostic and prognostic marker.

Cell proliferation is a fundamental process involved in growth, development and oncogenesis. Monitoring and quantification of proliferation are essential to analyse the behaviour of cells drug-treated or not. Flow cytometry assessment of cell proliferation requires mathematical models to extract information of interest from fluorescence distributions. Various methods are available for cell cycle analysis, including estimation of cell phase durations and doubling time. In this context, we compare widely used flow cytometric methods based on nuclear labelling (using BrdUrd incorporation in combination with DNA content) to membrane labelling (using intercalating dyes PKH).

We describe a new human bladder carcinoma cell line (DAG-1) established from a resected bladder cancer fragment and maintained in culture for more than 5 years and over 300 passages.Immunological, biochemical and molecular analysis showed that the DAG-1 cells (62 chromosomes) express the cytokeratines 8, 13, 18 and 20 that confirm their epithelial origin as well as numerous cytokine and cytokine receptor mRNAs. They secrete tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitors (PAI-1 and PAI-2), and express u-PA receptors (u-PAR/CD87) at their surface. DAG-1 cells are resistant to TNFalpha- and IFNgamma-induced apoptosis, two cytokines secreted in the urine of Calmette-Guérin bacillus-treated patients and involved in the tumor regression.The DAG-1 cell line is a useful tool, both in vitro and in vivo, to study the progression of bladder tumors and their mechanisms of resistance to immunotherapy in relation with PAI-2 and antioxidant enzymes.

Cadherins are cell surface glycoproteins that mediate Ca2+-dependent, homophilic cell-cell adhesion. The classical cadherins, E-, P- and N-cadherins, are known to self-associate from their extracellular domain, while their cytoplasmic domain interacts with either beta-catenin or plakoglobin (gamma-catenin), which in turn is bound to alpha-catenin that links the complex to the actin cytoskeleton. The aim of the present study was to analyze the expression of E-, P- and N-cadherins and catenins in human bladder carcinoma cells.Five human bladder carcinoma cell lines, representing a variety of differentiation states, were grown in cell culture. We performed a cell aggregation assay, specific for biological cadherin activity. The expression of cadherins and catenins was analyzed by immunocytochemistry, Western blotting and RT-PCR. The interactions between cadherins and catenins were assessed by immunoprecipitation.We observed a reduced E-cadherin expression in the poorly differentiated and invasive-tumor derived cells. Interestingly, immunofluorescence study reveals the persistent localization of catenins at intercellular contacts in two E-cadherin deficient cell lines (T24 and TCCSUP) which yet exhibit an epithelial-like morphology and a calcium-dependent adhesive capacity. This suggests that other cadherin(s) are expressed in these both cell lines. P-cadherin, another epithelial cadherin, is expressed only in E-cadherin positive cells. On the other hand, N-cadherin is present at cell-cell borders in the very anaplastic cell lines, T24 and TCCSUP, and is able to link beta-catenin or plakoglobin.These results indicate that N-cadherin may participate in intercellular adhesion, while facilitating bladder tumorigenesis.

Tumour‐associated antigens detected in cells obtained from bladder washings or tumours are useful markers in bladder cancer. Flow cytometry is commonly used to quantify immuno‐stained cells. A straightforward way to analyze data is to count the fluorescent cells above a threshold empirically determined on a control histogram representation. However, specific antigens expressed at highly variable rates give rise to wide range distributions in flow cytometry as illustrated when a mucin antigen for urinary bladder was titrated by M344 monoclonal antibody in urothelial cancer cells. We have evaluated several methods of background estimation and subtraction in order to determine the proportion of M344 Mab positive cells. These includethreshold setting(Histogram Shape Dependent (HSD) threshold developped in this study, 2% preset or 5% preset background),subtraction of the blankfrom the test histograms, andKolmogorov–Smirnov statistical test. The HSD method appeared to be a more reliable method for background estimation; however, in the case of very low antigen expression, where specific fluorescence histograms could hardly be distinguished from that of the background, fluorescence microscopy remained the only valid method, since it allowed the distinction between specific and non‐specific fluorescence on the basis of structural differences between the two.

Acidic proteins of the nucleolar organizer regions, selectively stained by silver (AgNOR-proteins), were investigated during interphase in leukemia cells with a confocal scanning laser microscope (CSLM). Simultaneous confocal fluorescence (for specific labeling of DNA, using propidium iodide) and transmitted light microscopy combined with digital deconvolution (for the location of the AgNOR proteins in nonconfocal mode) were used. The distribution of the AgNOR proteins measured by 3D microscopy was described by their number, the volume occupation of the nucleus by the AgNOR aggregates, the distance between each AgNOR, the distance of each AgNOR to the nucleolar border, and their anisotropy. The results of the 3D analysis were compared to those obtained by conventional 2D analysis, cytogenetical analysis of metaphase nucleolar organiser regions (NORs), and cell duplication rate. The descriptive power of these 3D parameters were assessed for nine leukemic cell lines. The measurements of the 3D spatial distribution of AgNORs was a better discriminant parameter than the morphological parameters (i.e., number and volume). The 3D expression of AgNORs is also a reliable parameter for assessing proliferative activity of leukemic cells and seems to be in relation with the differentiation stage of these leukemic cells.

Using flow or image cytometry, we compared the DNA distribution of cells from bladder washings from 52 patients with bladder cancer. For image cytometry, urothelial nuclei (recognized visually) were analyzed for DNA content using polymorphonuclear nuclei as internal diploid reference. For flow cytometry, two methods can be used: either all cells can be analyzed, as commonly performed, or urothelial cells can be analyzed alone, after specific derection. In this flow cytometry study, cells were doubly stained for panurothelial antigen T16 and for DNA. All the cells were first analyzed using peripheral lymphocytes as an external reference; 79% of the results were similar with results obtained from image analysis. For discordant results, flow-cytometric data were reprocessed to identify immunologically stained urothelial cells; one additional case became concordant with image cytometry when only urothelial cells were analyzed, with lymphocytes as diploid reference; a better concordance (94%) was found when the nonurothelial cells of the samples served as a diploid reference instead of peripheral lymphocytes. This suggests that we achieved an improvement of the flow-cytometric evaluations for ploidy assessement, and we conclude that, on these conditions, flow or image analysis can be considered as equivalent methods for DNA content studies of tumors. © 1993 Wiley-Liss, Inc.

International audience; BACKGROUND: In a previous investigation, we showed that the janus kinase (JNK) inhibitor SP600125 induced several phenotypic and genomic changes in leukemia cells. However, the molecular mechanisms that sustain these changes remain unknown. The purpose of the present study was to examine gene expression changes in THP-1 leukemia cells treated with SP600125. MATERIALS AND METHODS: Gene expression levels were investigated using Affymetrix hybridization technology and quantitative reverse transcriptase polymerase chain reaction. RESULTS: Affymetrix technology showed that the expression of 1,038 genes with a biological process description well known in gene ontology was modulated. Fifteen genes were related to kinases or phosphatases, 20 genes were involved in the cell cycle regulation, and 23 genes were involved in apoptosis. A network of 15 correlated genes was obtained showing a primordial role for the myelocytomatosis viral oncogene homolog (MYC). CONCLUSION: These findings show that SP600125 exhibits cytostatic and cytolytic activities through MYC gene modulation.

ObjectiveMeasurement of thyroglobulin in the washout of lymph node (LN) fine needle aspirates is recommended in the follow-up of patients with differentiated thyroid cancer (DTC). The significance of low fine needle aspirates thyroglobin (FNATg) levels remains a question, which we addressed.MethodProspective study comparing FNATg with FNA cytology. Exploration of 34 DTC patients (53 cervical LNs), 26 non-thyroidectomized patients with a thyroid-unrelated cervical mass (negative controls) and 13 with 21 thyroid nodules (positive controls). The 12 DTC patients (19 LNs) with a malignant FNA cytology and/or high FNATg level received LN surgery (11 patients) or I131-iodine treatment (1 patient) and the outcome measure was pathological or scintigraphic evidence of DTC LN metastasis.ResultsAll 26 negative controls showed FNATg ConclusionLow FNATg levels can indicate a DTC metastasis. It cannot be related to clinically relevant levels of serum Tg.

International audience; BACKGROUND: Obstructive sleep apnea syndrome (OSA) is associated with systemic and upper airway inflammation. Pharyngeal inflammation has a potential role in upper airway collapse, whereas systemic inflammation relates to cardiovascular morbidity. However, the presence of an inflammatory involvement of lower airway has been poorly investigated. OBJECTIVE: The aim of the study was to demonstrate an inflammatory process at the bronchial level in patients with OSA and to analyze effects of continuous positive airway pressure (CPAP) application and humidification on bronchial mucosa. METHODS: The study was conducted by using sequential induced sputum for cell analysis and IL-8 production, nitric oxide exhalation measurement, and methacholine challenge before and after CPAP. RESULTS: Bronchial neutrophilia and a high IL-8 concentration were observed in untreated OSA compared with controls (75% +/- 20% vs 43% +/- 12%, P < .05; and 25.02 +/- 9.43 ng/mL vs 8.6 +/- 3.7 ng/mL, P < .001, respectively). IL-8 in sputum supernatant was correlated to apnea hypopnea index (P < .01; r = 0.81). After 1 month of CPAP, this inflammatory pattern remained unchanged, and an increase in airway hyperresponsiveness (AHR) was observed (P < .001). CONCLUSION: Obstructive sleep apnea syndrome is associated with bronchial inflammation. Our data demonstrate CPAP effect on the development of AHR, possibly facilitated by the pre-existing inflammation. Both issues should be evaluated during long-term CPAP use. CLINICAL IMPLICATIONS: Results showing a spontaneous bronchial inflammation in OSA and the development of a CPAP-related AHR require a long-term follow-up to evaluate consequences on chronic bronchial obstruction.

International audience; UM384 cells, derived from the human myeloid leukemia U937 cell line, fail to differentiate in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Using cDNA microarray and real-time quantitative PCR (RT-QPCR) approaches, we observed a difference in the response to TPA treatment: all the genes from U937 cells were continuously modulated from 2 to 24h. In UM384 cells, 60% of the genes were transiently modulated at 2h, then returned to control levels at 24h. Moreover, HuR, an AU-rich element-binding protein (ARE-BP), was differentially located in the two cell lines. Therefore, a defect of mRNA stabilization could be responsible for the resistance of UM384 cells to TPA-induced differentiation, suggesting a possible role for the post-transcriptional regulation in the leukemogenesis.

The samples from pleural and pericardial effusion, in which immature haematopoietic cells had been identified cytologically, were re-examined. The results were then analysed along with the clinico-biological context and compared to published data. The aim was to determine the frequency, the type and the context of haematopoietic cell identification in pleural and pericardial fluid effusion. In 10 years, 28 cases were studied. Four sub-groups were described: 1) patients with a severe sepsis, 2) patients with an acute local or regional infection, 3) persistent or recurrent effusion without specific context, 4) patients who underwent a transplantation treated with cyclosporin A. Even when the clinico-pathological context did not suggest a classical extra-medullary haematopoiesis, it was not exceptional to identify immature haematopoietic cells. This hypothesis is supported by published data, which suggests that a local inflammatory state could help mesothelial cells to constitute a favourable environment for division and maturation of circulating haematopoietic progenitor cells.

TNFα and IFNγ, two main cytokines secreted in the urine of bladder cancer patients after Bacillus Calmette Guerin immunotherapy (BCG therapy), exert various responses ranging from growth arrest, apoptosis, phenotypic changes and differentiation.To identify their transcriptional and translational targets, the highly sensitive bladder cancer cell line (RT112) was treated for 24 hours with increasing doses of IFNγ or TNFα and analyzed for cellular and molecular changes using a cDNA microarray technique (Transcriptome) containing 800 genes.High doses (10 ng/ml) induced an apoptotic cell death, whereas low doses (5 ng/ml) induced a survival program. TNFα-inducible genes, IFNγ-inducible genes and genes modulated by TNFα and IFNγ together were identified. All were related to the tumor progression program including cell proliferation, apoptosis/survival, angiogenesis and metastatic processes.These results suggest that the transcriptomic approach could be a good methodology to determine the molecular mechanisms involved in bladder tumor progression processes in relation to a low response to BCG treatment. However, mRNA and protein expression did not always correlate, suggesting that translational regulation is a vital process in bladder tumor progression.

Neuroendocrine differentiation can be identified in a subset of human breast carcinomas, either as scattered cells or as a predominant neuroendocrine component. We report a case of an invasive breast carcinoma largely composed of neuroendocrine cells. Eight years after a left mammary lumpectomy for a pT2N1MO SBR III invasive ductal carcinoma, a 67-years-old woman presented with a metastastic neuroendocrine sternal mass. To establish a relationship between mammary carcinoma and bone metastasis, histological slides of both the breast tumor and axillary lymph nodes were reviewed, and an immunohistochemical study was performed. They showed that: a) the mammary carcinoma was composed of a majority of small and large neuroendocrine cells synaptophysin +, NCAM+, chromogranin - (80%), associated with 2 other differentiated non endocrine components, one of metaplastic squamous carcinoma (10%) and the other of ductal carcinoma (10%); b) 4 axillary lymph nodes were involved by the ductal component which contained few NCAM + but synaptophysin - cells; c) Estrogen and progesterone receptors and HER2 were negative in the breast tumor and the metastatic nodes. We discuss the histogenesis of composite mammary carcinomas with neuroendocrine differentiation, the outcome of each component and the prognostic relevance of such a diagnosis.

IFN-gamma is detected in the urine of bladder cancer patients after intravesical bacillus Calmette Guerin instillation. Because it acts in the anticancer process, we studied its cellular and molecular mechanisms of action on human bladder cancer cell lines.IFN-gamma (5 ng.ml(-1))(400 IU.ml(-1)) inhibited the growth of bladder cancer cell lines and modified the expression of the tumor-associated markers tissue-type plasminogen activators, Plasminogen activator inhibitor-2, urokinase plasminogen activator receptor, colony-stimulating factor 1, intercellular adhesion molecule 1, and class II MHC. Interestingly, IFN-gamma-induced apoptosis of the low-grade bladder cancer cell lines (RT4/G1 and RT112/G2) related to a cleavage of caspases 1, 8, and 9. This process was inhibited by the phosphatidylinositol 3'-kinase inhibitor (LY294002) and the protein synthesis inhibitor (cycloheximide). Moreover, low doses of IFN-gamma (5 ng.ml(-1))(400 IU.ml(-1)) increased the resistance to the cytotoxic effect of tumor necrosis factor alpha in the RT112 cells but not in the RT4 cells. This acquired resistance was associated with morphological changes and with an increase of the cell migration and scattering.We demonstrated that in the low-grade bladder cancer cell lines, the effect of IFN-gamma was dose dependent: high doses (5 ng.ml(-1)) induced apoptosis of RT4 and RT112 cells, whereas low doses (5 ng.ml(-1)) induced a resistance to the cytotoxic effect of tumor necrosis factor alpha and increase the metastatic potential of the RT112 cells. Therefore, we propose that a similar phenomenon could participate to the immunotherapy failure observed during tumor progression of bladder cancer.

The aim of the study was to assess the sensitivity and specificity of fluorescence immunocytochemistry (uCyt+ assay) as combined with urinary cytology for detection of primary and recurrent urothelial carcinomas. We analyzed 694 urinary samples from 236 new symptomatic patients and 458 patients followed after transurethral resection (TUR) for bladder tumor. Lesions suspicious for cancer at cystoscopy were sampled by biopsies or TUR. Sensitivity and specificity of tests were calculated using cystoscopy and histopathology, whether or not combined as gold standards. In new symptomatic patients, sensitivity of uCyt+ was 40%, 88.2%, and 76.7%, whereas that of urinary cytology was 30%, 70.6%, and 83.3%, respectively, in G1, G2, and G3 tumors. In follow-up cases, sensitivity of uCyt+ was 61.9%, 66.7%, and 76.9%, whereas that of urinary cytology was 38.1%, 58.3%, and 64.1%, respectively, in G1, G2, and G3 tumors. The combination of uCyt+ and urinary cytology significantly increased mean sensitivity in newly diagnosed cases (86.4% versus 71.2% with urinary cytology only, p < 0.05), as well as in patients followed after TUR (79.3% versus 55.2%, p < 0.001). Specificity of uCyt+ and urinary cytology was identical in new patients (83.3%) and was 81.9% and 86.2%, respectively, in patients followed after TUR. In patients with negative cystoscopy, positive uCyt+ tests had a strong predictive value for tumor recurrence at 1 year (47.0% versus 11.9% in patients with negative assay, p < 0.01). We conclude that combining uCyt+ with urinary cytology improves the detection of urothelial carcinomas as well in patients with symptoms suggesting bladder cancer as in those followed after treatment.

OBJECTIVE To identify alpha-amylase crystalloid formations in parotid specimens obtained by fine needle aspiration. STUDY DESIGN The study concerned three cases of sialadenitis with crystalloid formation observed between 1993 and 1998. In one of these cases, transmission electron microscopy, mass spectrometry and measurement of amylase activity were used to characterize the nature of the crystalloids. RESULTS Light microscopy revealed the same crystalloid structure in all three cases. In one case, where the material was saved, a biochemical method made it possible to reveal high amylase activity, while protein electrophoresis and mass spectrometry were used to identify salivary alpha-amylase. CONCLUSION Crystalloids of salivary alpha-amylase can be identified by May-Grunwald-Giemsa and Papanicolaou stain and can be rapidly confirmed through determination of amylase activity.

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies, (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.

Expression of progesterone receptors (PR) at the cell level is heterogeneous. Quantification by image analysis of several fluorescent immunostainings (PR, Ki-67 antigen, DNA) on breast cancer cell lines shows that this heterogeneity is partially linked to cell proliferation kinetic. Simultaneous detection of chromosome 11 (on which PR gene is located) by in situ hybridization and PR immunostaining do not demonstrate any relationship between the number of chromosome 11 and PR cell positivity, probably because numbers of chromosome 11 and of PR gene copies are not correlated, as shown by a double fluorescent in situ hybridization on metaphases.

A significant level of estrogen receptors (ER) in breast cancer cells is an indication of tumor differentiation and suggests that a homeostatic control of cell growth may persist in these cancers. In medical practice, the Dextran-coated charcoal assays (DCCA) are still the most frequently used test to characterize patients having ER-positive malignant breast tumors and for whom hormonal therapy is justified. Nevertheless, this routine biochemical technique is not satisfactory because it is a broad method unsuitable for revealing receptor tissue heterogeneity. However, immunocytochemical labeling, such as the ER-ICA method, which involves a monoclonal antibody linked to peroxidase, is a specific reaction for this purpose but which until now was not quantitative. The present study uses an original cell preparation technique combining the PAP reaction with toluidine blue counterstain for image analysis on the SAMBA system. Special software has been developed for the quantitative analysis of immunocytochemistry in cancers. Results obtained showed a high correlation between the DCCA values and the score derived from the mean ER concentration per positive tumor cell and the labeling index. In addition, intracell and intratumor heterogeneity can be displayed according to several parameters and were shown to vary according to tumor and to antiestrogen (Tamoxifen) presurgical therapy.

Auer rods were found in the bone marrow of nine out of 56 patients having all the criteria of refractory anemia with excess of blasts (RAEB). No differences were found in Auer-positive or Auer-negative subgroups according to cytology, kinetics, type of growth in agar, or disease course. Therefore, the presence of Auer rods in the bone marrow of patients with RAEB does not imply overt leukemia.

Les 2 types d'anemie refractaire avec exces de blastes (AREB), l'AREB et l'AREB en remission, definis par le groupe de collaboration FAB, different par l'intensite de l'insuffisance medullaire mais non en matiere de pronostic, evolution de la maladie en transformation leucemique et il n'existe aucune raison d'adopter un programme therapeutique different