Daniel Seigneurin

Significance of low levels of thyroglobulin in fine needle aspirates from cervical lymph nodes of patients with a history of differentiated thyroid cancer

OBJECTIVE Measurement 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. METHOD Prospective 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 I(131)-iodine treatment (1 patient) and the outcome measure was pathological or scintigraphic evidence of DTC LN metastasis. RESULTS All 26 negative controls showed FNATg <1 ng/FNA and all 21 positive controls showed high levels of FNATg (127-210,000 ng/FNA, median 38,000). Among DTC patients in 25 LNs with a benign FNA cytology, FNATg was undetectable in 24 and low in 1 (6 ng/FNA); in 19 LNs with a malignant FNA cytology, FNATg was high in 17 (80-140,000 ng/FNA, median 7174 ng/FNA) and low in 2 (6.6 and 7.1 ng/FNA), which proved to be low Tg immunostaining oncocytic DTC metastasis; in 9 LNs with a non-informative cytology, FNATg was undetectable in 8 but 11,825 ng/FNA in 1, which proved a DTC metastasis. Measurement of FNA albumin demonstrated that contamination of FNA by serum proteins was negligible. CONCLUSION Low FNATg levels can indicate a DTC metastasis. It cannot be related to clinically relevant levels of serum Tg.

EXPRESSION OF E-, P-, N-CADHERINS AND CATENINS IN HUMAN BLADDER CARCINOMA CELL LINES

PURPOSE 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSION These results indicate that N-cadherin may participate in intercellular adhesion, while facilitating bladder tumorigenesis.

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: Simultaneous assessment of proliferation, apoptosis, and necrosis

BACKGROUND The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

Expression of Three Cell Adhesion Molecules in Bladder Carcinomas: Correlation with Pathological Features

Recently, independent studies have shown that the expression of two integrin chains, β4 and α2, 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, and α2 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 for α2, and only 35% for β4. However, most invasive tumours presented an aberrant expression of α2 (81%), β4 (100%), and E‐cadherin (75%). We studied the correlation of immunoreactivity with histological grade and stage. The α2 pattern was not correlated with stage and grade. In contrast, loss of normal β4 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 integrins α2 and β4, E‐cadherin appeared to be a better marker of invasiveness in bladder carcinomas.

Progesterone Receptor Heterogeneity in MCF-7 Cell Subclones Is Related to Clonal Origin and Kinetics Data

Heterogeneity of progesterone receptor (PR) expression in MCF-7 cells is generally attributed to the coexistence of several sublines, each possessing different stages of differentiation. One hypothesis is that the variation of PR distribution relates to the genotype cell heritage and cell cycle phases. The aim of this study was to demonstrate the implication of cell subclones in PR heterogeneity. MCF-7 cell line subclones were obtained initially by the limit dilution method on microscopic slides. On these slides PR was assessed by immunofluorescence. 20 of the subclones were PR-negative, 10 were positive with varying degrees of PR expression. As these cell populations arose from a single cell, they can be considered as monoclonal. These results show that PR heterogeneity (positive vs. negative clones) is based on a clonal origin and could be genotypically explained. In a second experiment four PR-positive MCF-7 cell subclones were maintained in continuous culture and studied. On each one a triple fluorescent staining (PR, Ki-67 antigen and DNA) was performed and the reactions were quantified by videofluoro microscopy. These results demonstrated that a relation between cell PR content and cell cycle stages exists in these four subclones. Cells in G0 express only little PR; PR level increases during the S phase to reach a maximum in the G2 phase; after mitosis PR level decreases with cell division and degradation may occur in G1: PR level reaches a minimum in late G1 and in the early S phase. The doubling times of the different MCF-7 subclones shows that those that are rapidly cycling were preferentially PR-positive, whereas slowly cycling MCF-7 subclones were PR-negative. We conclude that in MCF-7 cells some subclones are able or not able to synthesize PR; PR content is directly dependent on cell cycle phase and population doubling time.

Quantitative analysis of three-dimensional distribution of AgNOR proteins during interphase in leukemic cells

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.

Adénocarcinome mammaire avec composante neuroendocrine majoritaire

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.

Limits of flow-cytometry histogram analysis methods to assess bladder tumour antigen expression

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 include threshold setting (Histogram Shape Dependent (HSD) threshold developped in this study, 2% preset or 5% preset background), subtraction of the blank from the test histograms, and Kolmogorov–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.

Videomicrofluorometry of Progesterone Receptors and Their Genes in Breast Cancer Cells

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.

PROGNOSIS SIGNIFICANCE OF IMAGE CYTOMETRY IN NON INVASIVE BLADDER CANCER

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