Biomedical subjects

D Seigneurin

Publications and source records attributed to D Seigneurin.

At least 37 records · Page 2Linked to original sources

PubMed1996

Prognostic value of quantitative cytometry in a series of 415 T1T2/N0N1/M0 breast cancer patients--preliminary results.

Identifying prognostic markers in local regional breast carcinomas remains an important challenge today. DNA content obtained by flow cytometry, has been found to be of prognostic value; results with other methods remain less clear. This report describes DNA image cytometry patterns which are assessed with respect to disease-free survival. From June 1982 to December 1992, 415 patients under 75 years of age, without any previous or synchronous carcinoma, suffering from an invasive breast cancer classified as T1 (52.8%), T2 (47.2%), N0 (65.1%) N1 (34.9%), MO according to clinical TNM staging, were enrolled in this study. The median age was 53 (28-75) and 58.8% of the patients were premenopausal; 85.3% underwent a breast conservative procedure and 14.7% a modified radical mastectomy followed by postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade and, oestrogen receptor content were used in decision-making for adjuvant treatment: hormonotherapy (48%) or chemotherapy (18.8%). Imprints were taken from the macroscopically visible lesion at the time of surgery, and a Feulgen staining was carried out on air dried smears to be analysed using the Samba 200 cell image processor (Alcatel TITN, France). Five parameters were systematically assessed: proliferation index; DNA histogram, integrated optical density, DNA malignancy grade, ploidy balance. With a median follow-up of 36 months (0-105), proliferation index (P = 0.0008), DNA histogram (P = 0.0017), integrated optical density (IOD) (P = 0.018) and DNA malignancy grade (P = 0.017) had a significant prognostic value on disease-free survival estimated by the Kaplan-Meier method. When these parameters were included in a Cox proportional regression hazards model, PR (P = 0.01), Scarff-Bloom histological grading (P = 0.02), axillary clearance (P = 0.04) were significant; however, in the same model, taking into account the axillary lymph node histological status, IOD was significant for pN- patients (P = 0.03), and proliferation index (P = 0.03) was significant for pN+. Such results need to be updated with a longer median follow-up, but they suggest that the mean DNA content, as measured by the integrated optical density (IOD), should be considered when deciding on medical adjuvant treatment with respect to patients with a negative axillary clearance.

Adult↗

PubMed1996

Methods for simultaneous interphase in situ hybridization and nuclear antigen immunocytochemistry in T47-D cells.

Procedures that combine immunocytochemistry (ICC) and in situ hybridization (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapid procedures for simultaneous detection of a nuclear antigen, progesterone receptors (PR), and the centromeric region of chromosome 11 (to which the human PR gene has been assigned) in T47-D cells. Proteins were stained by precipitates of horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (APase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable contrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X-phosphate-immunostained cells with interphasic fluorescent in situ hybridization (FISH). By contrast, we combined the APase-Fast Red ICC with an immunocytochemical ISH using alkaline phosphatase-NBT-X-phosphate detection. Only the procedure combining APase-NBT-X-phosphate ICC and FISH ensures optimal visualization of both the PR content and the number of chromosome 11. This method easily provides simultaneous localization of DNA and protein targets in the same cells and should be applicable to many other situations.

Antigens, Nuclear↗

PubMed1996

[Prognostic value of quantitative DNA analysis, expressed in integrated optical density, in a series of 415 T1-T2, N0N1, M0 UICC breast cancers].

From June 1982 to December 1992, 415 patients less than 75 years of age, without any previous or synchronous carcinoma, suffering from an invasive breast cancer classified as T1 (52.8%), T2 (47.2%), NO (65.1%) N1(34.9%), MO according to clinical TNM staging, were enrolled in this study. The median age was 53 (28-75), and 58.8% of the patients were menopaused; 85.3% underwent a breast conservative procedure and 14.7% a modified radical mastectomy followed by postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade, estradiol receptor content, were used to set up medical adjuvant treatment: hormonotherapy (52%) or chemotherapy (18.8%). Imprints were taken from the macroscopically visible lesion at the time of surgery, and a Feulgen staining was done on air dried smears to be analyzed using the Samba 200 cell image processor (Alcatel TITN, France). Five parameters were systematically assessed: proliferation index, DNA histogram, integrated optical density, DNA malignancy grade, and policy balance. With a median follow-up of 36 months (0-105), proliferation index (P = 0.0008), DNA histogram (P = 0.0017), integrated optical density (P = 0.018), DNA malignancy grade (P = 0.017) have a significant prognostic value on disease free survival estimated by the Kaplan-Meir method. When these parameters were included in a Cox proportional regression hazards model, Scarff-Bloom histological grading (P = 0.002), positives nodes (P = 0.02), optical integrated density (P = 0.045) were significant. Such results need to be updated with a longer follow-up, but they suggest that the mean DNA content, as measured by the integrated optical density (IOD), has to be considered when deciding on medical adjuvant treatment with respect to patients with a negative axillary clearance.

Adult↗

PubMed1995

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.

Breast Neoplasms↗

PubMed1995

Interest of argyrophilic proteins nucleolar organizer regions (AgNOR) to estimate the reactivity of T cell clones against autologous malignant B-NHL cells.

The silver-stained acidic proteins of interphase nucleolar organizer regions (AgNOR) were studied to assess the reactivity of 11 T cell clones (Tcc) against autologous B-NHL cells. Tcc, derived from tumour-infiltrating T lymphocytes of seven patients, were cultured in the presence of irradiated autologous B-NHL cells with recombinant IL-2. Then the percentage of activated T cells expressing the CD25 antigen and their proliferating rate (measured by [3H]thymidine incorporation) were estimated. Simultaneously, at the end of this culture period, B-NHL cells were eliminated by using a cell-sorter, and the resulting purified T cells were studied for AgNOR expression. Tcc cultured without B-NHL cells served as controls. In eight out of the 11 Tcc, the increase in [3H]thymidine incorporation and CD25 expression paralleled the increase of AgNOR area and number. By contrast, in the three remaining Tcc, we observed a decrease (one Tcc) or an increase (two Tcc) of AgNOR parameters, whereas CD25 expression and/or [3H]thymidine incorporation remained unchanged in comparison to control cultures. We concluded that quantification of AgNOR should be a more sensitive technique than thymidine incorporation and CD25 expression for detecting the activation in vitro of T cells induced by autologous B-NHL cells.

Clone Cells↗

PubMed1995

Flow and image cytometric DNA measurements in fine needle aspiration samples of prostatic neoplasms.

OBJECTIVE: To compare the DNA content measured by flow cytometry (FCM) and image analysis (IA) from prostatic fine needle aspiration (FNA) samples. STUDY DESIGN: A total of 48 samples were studied. FCM was performed on propidium iodide-stained nuclei according to the Vindelov method, and image analysis was performed on Feulgen-stained slides. Positive FNA results were grade (1-3) and compared with Gleason grades. RESULTS: Aneuploidy was closely related to positive FNA results (P < .01). DNA histograms were found to be aneuploid in all grade 3 carcinomas (n = 13) by IA and in 11 cases (84.6%) by FCM. Grade 2 carcinomas (n = 9) were found to be aneuploid with both methods. In grade 1 carcinomas (n = 10), 2 cases exhibited IA aneuploid profiles, whereas all FCM cases were diploid. Aneuploid profiles were more often associated with high Gleason scores than were diploid ones (P < .01). Among the 16 patients with negative FNA results, two cases had tetraploid DNA profiles related to contaminating seminal vesicle cells. The difference in DNA measurements reached 10.4% but was not statistically significant. CONCLUSION: These findings show that the two methods, as applied to prostatic FNA samples, give comparable results and that seminal vesicle cells may be responsible for false tetraploid profiles.

Biopsy, Needle↗

PubMed1995

Developmentally regulated chromatin acetylation and histone H1(0) accumulation.

There exists a close relationship between core histone acetylation and the induced expression of the histone H1(0) gene. We took advantage of this fact to evaluate the influence of chromatin hyperacetylation on the developmentally regulated expression of this specific gene. In this study, the in situ immunodetection approach has been used to analyze both the acetylated histone H4 isoforms and histone H1(0) accumulation during early Xenopus laevis development. We have chosen two stages of development, gastrula stage, when H1(0) is not expressed and not inducible by butyrate treatment, and stage 27 when H1(0) is not expressed but is inducible by butyrate. At stage 27 of development, the early induced accumulation of histone H1(0) under butyrate treatment, occurs mainly in tissues that express the protein normally during later development. These experiments suggest that histone acetylation may be part of a pathway which, in a specific set of cells, keeps H1(0) and probably a series of specific genes, competent for transcription, but cell-specific factors are involved in the induced expression of these genes.

Acetylation↗

PubMed1995

[Value of image cytometry to oncologic diagnosis and prognosis].

For almost 100 years, malignant diseases diagnoses have been made by subjective evaluation of morphologic criteria in stained cellular preparations or tissue sections. This diagnosis remains the basis and the first step of any oncological work. Cell quantification by image analysis has only gained scientific but not yet routine application. However it is useful in screening, diagnosis and prognosis of malignant tumors. SCREENING: DNA quantification allows the distinction between non specific abnormalities and atypias considered as pre-cancerous lesions in cervical neoplasia. DIAGNOSIS: althought not often used, image cytometry may be useful for the diagnosis between benign and malignant lesions as we demonstrated it in mammary tumors, studied after Feulgen staining of imprints. PROGNOSIS: grading of malignancy can be done using image cytometry. Numerous parameters have been found useful for the prognosis evaluation of various tumors. For breast tumors, these are: nuclear or nucleolar area, DNA content, proliferating rate, hormone receptors positive cell percentage.... Quantification of oncogene amplification or chromosomal aberrations are under evaluation. Thus, computerised microscopy is now a necessary tool for the pathologist.

Biomarkers, Tumor↗

PubMed1994

[Modulation of indications of adjuvant hormone therapy with tamoxifen in T1T2/N0N1 breast cancers. Preliminary results of a multicenter study with 695 cases].

From 1982 to 1990, patients less than 75 years, without any previous or synchonous carcinoma, suffering from an invasive breast cancer classified as T1T2/N0N1/MO according to clinical TNM staging, were enrolled in this study; 82.4% underwent a breast conservative procedure and 17.2% a modified radical mastectomy followed by a postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade, estradiol receptor content, were used to define three groups of patients. The breakdown of patients is not well balanced: 416 women were included in group I (N-, grade I II, ER+) when there was no adjuvant medical treatment, 110 in group II (N-, grade III, ER+), 169 in group III (N+ < or = 3, grade I II, ER+). Patients from the latter two groups were receiving tamoxifene, 20 mg per day for 2 years; Those women not menopaused received first a pelvic irradiation. With a median follow-up of 35 months (1-138) the overall survival is respectively for the three groups 95%, 96%, 96% (P = 0.5) and the disease free survival 86%, 93%, 90% (P = 0.1). The actuarial local regional remission rate is 94%, 97%, 99% (P = 0.07). Such results need to be updated with a longer follow-up, but they show the ability of adjuvant hormonotherapy to tailor the short term survival thanks to prognostic factors.

Adult↗

PubMed1994

Image analysis as a tool for quantitative enzyme determination at the cellular level: application for monocytic differentiation of the UM-384 cell line.

Image analysis has been used to determined enzyme activity at the cellular level in individual smeared cells. The counterstains used to visualize smeared cells were chosen to avoid overlap with the chromogene. The amount of the reaction product was quantified by computerised scanning cytophotometry when the conditions of incubation, time and temperature of the reaction, and substrate concentration varied. Under optimal conditions for time, temperature and substrate concentration, a linear relationship was found between enzyme activity determined on smeared cells and in cell lysate. Using these defined conditions, differentiation of UM-384 cells was studied by measuring enzyme activity. After a monocytic differentiation process, induced by sodium butyrate, non-specific esterase cell activity was compared either with differentiation markers (HLA-DR, plasminogen activator inhibitor type 2 and lysozyme) or with markers of proliferation (DNA content) or functional properties (nitroblue tetrazolium reduction and phagocytosis). The results show that, using image analysis, non-specific esterase seems to be a useful means for the assessment of monocytic differentiation whereas myeloperoxidase is not. More generally, quantification of enzyme activity at the cellular level using image analysis can be applied to the study of the differentiation process and may help in the classification of leukemic cells.

Acid Phosphatase↗

PubMed1994

The value of DNA image cytometry for the cytological diagnosis of well-differentiated breast carcinomas and benign lesions.

Cytological evaluation of breast diseases can lead to false diagnosis if benign lesions show epithelial atypia or if carcinomas are orthoplasic and well differentiated. In order to determine the potential usefulness of image cytometry for discrimination between benign and malignant breast lesions, we studied 120 Feulgen-stained imprints, half from benign lesions with epithelial atypia and half from well-differentiated carcinomas and determined values of parameters expressing DNA content, morphometry and chromatin texture. Twenty-five out of the 60 carcinoma imprints were found to be DNA-aneuploid and could thus be easily distinguished from benign imprints, as, in our and other authors' experiences, aneuploidy very rarely occurs in benign breast lesions. When the remaining 35 carcinoma imprints were compared with 38 benign lesion imprints, randomly selected from the 60 cases studied, only four cases were misclassified after a linear discriminant analysis; the most discriminant parameters were those expressing the heterogeneity of DNA content and the chromatin texture. Thus, in cases where cytological diagnosis is difficult, image analysis of Feulgen-stained imprints may be a very useful tool for correctly discriminating benign from malignant cases.

Aneuploidy↗

PubMed1993

Flow and image cytometry for DNA analysis in bladder washings: improved concordance by using internal reference for flow.

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 detection. In this flow cytometry study, cells were doubly stained for panurothelial antigens 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 assessment, and we conclude that, on these conditions, flow or image analysis can be considered as equivalent methods for DNA content studies of tumors.

Adult↗

PubMed1993

Simultaneous quantitation of DNA and nucleolar organizer regions by image cytometry.

This paper describes a DNA/nucleolar organizer region-associated protein (NOR) double staining technic and quantitation method. On smeared cell slides, the acidic proteins associated with nucleolar organizer regions were revealed by the silver colloid technic, and DNA was stained with the fluorescent dye Hoechst 33342. The simultaneous quantitation of silver stained proteins (AgNORs) and DNA was performed with image cytometry. Two applications of this method are presented: a study of AgNOR expression during the MCF-7 cell line cycle and the simultaneous quantitation of DNA and AgNORs in non-Hodgkin's lymphoma node imprints. This method offers the advantage of evaluating, on the same cell at the same time, the DNA content (ploidy) and AgNOR expression, which constitute a good approximation of the cell's proliferation state. Since ploidy and proliferation index are being used more and more in tumor assessment, it is useful to obtain them in minimum time and with a minimum of biologic material.

Animals↗

PubMed1993

[Usefulness of cytoprognostic classification combined with histoprognostic classification in breast cancer].

Histopathological grading according to Scarff Bloom Richardson can provide powerful prognostic information. However one criticism of this system is that 60% of patients cluster in grade 2 with no clear separation of good versus bad prognosis. In an effort to solve this problem we propose to add cytological grading performed on fine needle aspirates or imprints. Indeed histological grade 2 (H2) group can be subdivided into 2 groups according to cytological grading: cytological grade 2 (C2) and grade 3 (C3). Analysis of cytometrical and proliferative features shows that H2 C3 tumors are closer from H3 C3 than from H2 C2 tumors. So, using morphological evaluation, we propose to classify breast carcinoma into 2 groups: 1 group associating histological grade 1 tumors and histological grade 2, cytological grade 2 tumors; group 2, associating histological grade 3 tumors and histological grade 2, cytological grade 3 tumors.

Breast Neoplasms↗

PubMed1993

Image cytometry of hepatocellular carcinoma: comparison with alcoholic cirrhosis and normal liver.

Image cytometry can provide valuable information for the prognosis of patients with malignant tumours. This study was performed on three cell populations: hepatocellular carcinoma (n = 30), alcoholic cirrhosis (n = 12) and normal liver (n = 2). The results showed that hepatocellular carcinoma cell populations contained more hyperploid and aneuploid cells than cirrhosis and normal liver cell populations; two groups of hepatocellular carcinoma were separated, called A and B. The values of group A cytometric parameters were close to those of cirrhosis whereas group B nuclei had a much higher DNA content and were more often hyperploid or aneuploid. These two groups were also different according to biological (alpha-foetoprotein) or clinical (survival time) variables. Since group A seemed to be made up of patients with better prognosis than group B, cytometric analysis of hepatocellular carcinoma could be a tool for prognosis and clinical management of patients with hepatocellular carcinoma.

Adult↗

PubMed1993

Absence of both subunits of cytochrome b558 in the UM384 cell line relative to the inability to generate superoxide anions.

Phagocytic cells are characterized by their ability to generate superoxide anions upon activation by appropriate stimuli. UM384, a myelomonocytic cell line, was shown to be defective in this oxidase activity as measured by nitroblue tetrazolium or cytochrome c reduction. Cytochrome b558, a unique pigment present in phagocytes and implicated in electron transfer from NADPH to O2, was absent in the differentiated UM384 cells. Both subunits of the cytochrome b558 appeared to be absent or present in strongly reduced amounts compared to the mother cell line U937, as indicated by immunocytochemistry or Western blot analysis using monoclonal antibodies (MABs). On the other hand, cytosolic factors also involved in NADPH oxidase activity were shown to be present, either immunologically or by using the capacity of the cytosol to activate the oxidase in a membrane fraction from bovine neutrophils. At the molecular level, the mRNA that encodes the gp91-phox was shown to be absent in the differentiated UM384 cells, whereas the mRNA that encodes the p22-phox was normally expressed. These results suggest that the defect in superoxide production by the UM384 cells is related to the absence of cytochrome b558, a situation mimicking that observed in phagocytes from patients with X-linked chronic granulomatous disease (X-CGD).

Antibodies, Monoclonal↗

PubMed1993

DNA image cytometry of bladder tumours: comparison of washings and tumour imprints from 61 patients.

Cellular DNA cytometry is commonly used to assess the prognosis of bladder tumours. Measurements are made mainly on voided urine or irrigation fluids by flow or image cytometry. In order to determine whether this material is really representative of the bladder tumour, we compared DNA assessments of bladder washings and tumour imprints from the same patients using image cytometry on Feulgen-stained preparations. DNA aneuploidy was found in 41.0% of the washings, 42.6% of the imprints and 49.2% of the patients if both samples were considered. If DNA histograms are classified into three groups (diploid, diploid+aneuploid, aneuploid), a 70% concordance rate is obtained between washings and imprints. In 15% of the cases, an aneuploid population was found in both samples, but in different proportions. In the latter 15%, an aneuploid population was found in one sample but not in the other; this discrepancy, which is probably due to poor tumour exfoliation or to tumour heterogeneity, is of clinical significance since it indicates the importance of making DNA assessments on both washings and cells directly obtained from the tumour.

Adult↗

PubMed1991

Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment.

Simultaneous quantification of DNA and Ki-67 proliferation-associated antigen was performed using fluorescence image cytometry. In the MCF-7 cell line, the Ki-67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki-67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki-67 antigen remains constant during G1 (Ki-67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki-67 (Ki-67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate-sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki-67 content (Ki-67 increase pathway) was observed from intermediate-sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki-67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki-67 decrease pathway and Ki-67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.

Animals↗