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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.

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.

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.

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.

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.

Recently, independent studies have shown that the expression of two integrin chains,β4 andα2, plus the epithelial cadherin are related to tumour progression in human bladder carcinomas. For the first time, we compare the expression of these three cell adhesion molecules using immunohistochemical analysis of consecutive cryosections from a series of 50 bladder tumours. E‐cadherin,β4, andα2 were strongly expressed in normal urothelium. A majority of non‐invasive bladder cancers stained positively for E‐cadherin (62%), whereas only 29% expressed normal positivity forα2, and only 35% forβ4. However, most invasive tumours presented an aberrant expression ofα2 (81%),β4 (100%), and E‐cadherin (75%). We studied the correlation of immunoreactivity with histological grade and stage. Theα2 pattern was not correlated with stage and grade. In contrast, loss of normalβ4 expression was significantly related to increasing tumour grade and deep invasion with a higher correlation for grade. Finally, E‐cadherin expression was highly correlated with stage, but not with grade. Thus our results indicate that, although many invasive bladder tumours presented a disorder in expression of the two integrinsα2 andβ4, E‐cadherin appeared to be a better marker of invasiveness in bladder carcinomas.

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