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S Chevillard

Publications and source records attributed to S Chevillard.

At least 37 records · Page 2Linked to original sources
PubMed1997

Positive prostate-specific antigen circulating cells detected by reverse transcriptase-polymerase chain reaction does not imply the presence of prostatic micrometastases.

OBJECTIVES: Detection of circulating tumor cells may improve the preoperative local staging of prostate cancers. The aim of this study was to perform enhanced reverse transcriptase-polymerase chain reaction (RT-PCR) of prostate-specific antigen (PSA) mRNA to define the predictive value of PSA-positive circulating cells in a large series of patients. METHODS: The study included 46 patients with Stage T1 to T2 prostate cancer, 94 with benign prostatic hyperplasia (BPH), and 51 (including 9 women) with nonprostatic disease. PSA-positive cells from peripheral blood samples were detected by Southern blot analysis of the RT-PCR products. Original oligonucleotide primers were defined to exclusively detect the three PSA mRNA splices. RESULTS: Circulating PSA-positive cells were observed in 8 (8.5%) of 94 patients with BPH, 10 (22%) of 46 with Stage T1 to T2 prostate cancer, and 9 (17.6%) of 51 with nonprostatic disease. The detection rate of PSA-positive circulating cells was significantly increased in patients with prostate cancer versus patients with BPH (P = 0.03). Among clinically localized prostate cancers with a Gleason score less than 8, a correlation was observed between PSA-positive circulating cells and Stage pT3 cancer (P = 0.038), capsular penetration (P = 0.04), and a positive margin (P = 0.038). The specificity of the assay for Stage pT3 cancer detection was 84.6%, with a positive predictive value of 60%. CONCLUSIONS: Although RT-PCR assay may have a role in preoperative local staging, this study demonstrated the absence of tissue and tumor specificity of PSA-positive circulating cells, accounting for the weak positive predictive value of this technique.

Aged↗
PubMed1997

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis.

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte-derived growth factors in its pathogenesis has been documented. We previously reported increased expression of TGF-beta in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients' platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium. consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.

Aged↗
PubMed1997

Value of the preoperative detection of prostate-specific-antigen-positive circulating cells by nested RT-PCR in patients submitted to radical prostatectomy.

OBJECTIVE: To assess the value of the detection of circulating prostate cells [prostate-specific antigen (PSA) positive] by reverse-transcriptase nested polymerase chain reaction (nested RT-PCR) to improve the staging of clinically localized prostate cancer. METHODS: Nested PCR was performed on blood samples of 29 patients submitted to radical prostatectomy for clinically localized (T1-T2) prostate cancer. Nine patients with various benign urologic diseases comprised the negative control group. Incubation was for 25 cycles for each PCR, using beta 2-microglobulin to test the integrity of RNA samples. Each sample was tested in quadruplicate and analyzed by agarose gel electrophoresis, blotted and hybridized with specific internal primers. Nested PCR results were compared with the pT stage of the prostate specimen, processed according to the Stanford method. RESULTS: In 6 out of 29 patients (20.7%) with clinically localized prostate cancer, circulating prostate cells were detected by nested PCR. There was no relationship between pathologic stage and RT-PCR results. Eleven out of 14 pT2 patients (78.6%) were PCR negative and only 3 out of 15 pT3 patients (20%) were PCR positive. All control samples were PCR negative. CONCLUSIONS: In selected patients with T1-T2 prostate cancer, there was no relationship between pathologic stage and the presence of circulating PSA-positive cells detected by nested PCR. However, in 20.7% of patients with clinically localized prostate cancer, circulating prostate cancer cells were detected. A further follow-up based on PSA is necessary to clarify the clinical relevance of this biologic anomaly.

Aged↗
PubMed1997

Measuring multidrug resistance expression in human malignancies: elaboration of consensus recommendations.

Before the prognostic significance of P-glycoprotein (P-gp) expression can be properly evaluated in prospective clinical trials of P-gp modulators, standard techniques for the measurement of P-gp must be widely accepted. Several multicenter trials have demonstrated large discrepancies in the observed levels of P-gp expression in the same clinical samples evaluated at different centers. The greatest discrepancies occurred with samples that expressed low levels of P-gp. Although standardized procedures have dramatically increased the interlaboratory reproducibility of flow cytometry and polymerase chain reaction assays, data from immunocytochemistry remain difficult to interpret. Consensus recommendations are presented for improving data reproducibility. These recommendations emphasize the importance of using calibrated batches of antibodies and two different antibodies for immunocytochemistry, the need for an internal standard for reverse transcriptase-polymerase chain reaction (RT-PCR) assays, the need for the presentation of data as a continuous variable, and the need for setting standard parameters for flow cytometry. It is also extremely important for the success of clinical trials that multiple techniques be employed to insure accurate measurement of P-gp expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Biological and clinical significance of concurrent p53 gene alterations, MDR1 gene expression, and S-phase fraction analyses in breast cancer patients treated with primary chemotherapy or radiotherapy.

We investigated the interrelationship between p53 gene alterations, MDR1 gene expression, and S-phase fraction (SPF) in breast carcinomas treated primarily with chemotherapy or radiotherapy and correlated the results with patient outcome to determine the potential clinical significance of these factors. In a consecutive series of 64 fine-needle samplings of breast cancer patients who underwent either neoadjuvant chemotherapy (n = 53) or radiotherapy (n = 11), p53 (exons 5-9) gene alterations by denaturating gradient gel electrophoresis and subsequent direct sequencing, MDR1 gene expression by semiquantitative reverse transcription-PCR, and SPF by DNA flow cytometry were determined. Our results show that p53 mutations (n = 20) were significantly associated (P = 0.01) with high SPF but not with de novo MDR1 gene expression. Most patients with wild-type p53 tumors were found to be resistant to neoadjuvant chemotherapy. No correlation was observed between p53 mutations and the induction of MDR1 gene expression during treatment. Although a significant correlation between shorter distant disease-free survival and high (>/=5%) SPF (P = 0.016) was found, no correlation between distant disease-free survival and p53 status or intrinsic MDR1 gene expression was found. Poor overall survival was observed in patients with tumors with high SPF (P < 0.0004) or lacking MDR1 gene expression (P = 0.03) before treatment, but not with p53 alterations. These data suggest that SPF remains the most relevant biological factor for breast cancer patients treated by primary chemotherapy or radiotherapy and that p53 and MDR1 status may identify a small subset of patients that may resist therapy or pursue an aggressive course.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Differential expression of transforming growth factor-beta, basic fibroblast growth factor, and their receptors in CD34+ hematopoietic progenitor cells from patients with myelofibrosis and myeloid metaplasia.

Myelofibrosis with myeloid metaplasia (MMM) is a myeloproliferative disorder characterized by clonal expansion of hematopoiesis and marrow fibrosis. Previous results from our group have shown an increased production of two potent fibrogenic factors also involved in the regulation of primitive hematopoietic cells, namely transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF), in patients with MMM. It is likely to assume that the myeloproliferation characteristic of this disease may result from an abnormal proliferation of CD34+ hematopoietic progenitors. Thus, we were particularly concerned in studying the gene and protein expression of these cytokines and their receptors in CD34+ progenitors purified from the peripheral blood of MMM patients by using semiquantitative reverse transcriptase-polymerase chain reaction and immunolabeling methods. Our data showed that the expression of TGF-beta1 is not altered in patients CD34+ cells; in contrast, the expression of TGF-beta type II receptor is significantly decreased in such cells, as compared with CD34+ cells from healthy subjects. Regarding bFGF, the very low expression of the cytokine and its type I and II receptors detected in normal CD34+ cells contrasts with that observed in patients' CD34+ cells, which is significantly higher. Our results might be a clue for a better understanding of the mechanism(s) involved in the dysregulation of hematopoiesis in MMM. Actually, the increased expression of bFGF and its receptors associated with the reduction of the TGF-beta binding receptor in CD34+ progenitors from MMM patients might facilitate the expansion of hematopoietic progenitors, not only by stimulating their growth and/or survival, but also by overcoming negative regulatory signals.

ADP-ribosyl Cyclase↗
PubMed1996

Different p53 mutations produce distinct effects on the ability of colon carcinoma cells to become blocked at the G1/S boundary after irradiation.

The LoVo colon carcinoma cell line that presents two wild type p53 alleles was used as the recipient for a series of transfections with p53 expression vectors coding for wild-type or three different mutants (143ala, 175his or 273his). The parental cell line as well as all clones that had rearranged the plasmid with consequent loss of p53 c-DNA were readily blocked at the G1/S boundary following 10 Gy of irradiation. For each mutation two clones with different levels of mutant protein expression were selected. Confirmation of the integration of the exogenous sequence was obtained by the expression of the mutant m-RNA, established by reverse transcription and DGGE or Southern blot. Flow cytometric measurements of 5-bromodeoxyuridine incorporation revealed a total G1/S block of the 143ala transfectants, similarly to the parental and control transfectant cells, but little or no cell cycle block for the 175his and 273his clones. Although it has been shown in vitro that all three mutations interfere with transcriptional activation by the wild-type protein, not only did we observe p53 protein induction and nuclear accumulation following irradiation, but WAF-1/CIP-1 m-RNA was increased in some of the clones for which the G1/S block was abolished. Our results show that mutant p53 proteins are to some extent submitted to the control of the cellular environment in cancer cells with wild type p53 alleles, but with an efficacy that depends on the mutation.

Alanine↗
PubMed1996

Sequential assessment of multidrug resistance phenotype and measurement of S-phase fraction as predictive markers of breast cancer response to neoadjuvant chemotherapy.

BACKGROUND: The authors examined the relevance of S-phase fraction (SPF) and multidrug resistance (MDR) phenotype as predictive tests of breast cancer response in a series of patients treated by conventional doses of neoadjuvant chemotherapy with (FAC) or without (FTC) doxorubicin. METHODS: Fine needle samplings of tumors were used to measure SPF by flow cytometry before treatment (Day 0), and to assess the MDR phenotype using semiquantified reverse transcriptase polymerase chain reaction and immunocytochemistry, before and after (Days 8 and 28) the first cycle of chemotherapy. RESULTS: Measurement of SPF before treatment was significantly associated with clinical response, but sequential assessment of MDR phenotype identified three groups of tumors with distinct outcomes: (1) tumors with a positive and constant expression of MDR1, in which prediction of resistance was restricted to patients treated by FAC; (2) tumors without any detectable expression, in which resistance to FAC or FTC treatments was rarely observed; and (3) tumors with an early (Day 8) acquired or increased MDR1 gene expression, which were always resistant to therapy to both treatment regimens. These results were confirmed at the protein level. CONCLUSIONS: Sequential assessment of MDR phenotype is a relevant tool for monitoring breast cancer response in neoadjuvant chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Long term prognostic value of growth fraction determination by Ki-67 immunostaining in primary operable breast cancer.

An immunohistochemical determination of the growth fraction (GF) with the Ki-67 monoclonal antibody has been performed in a prospective series of 140 patients with primary operable breast carcinoma. GF ranged from 0% to 43% Ki-67 stained cells with a median value of 8%. High GF (> 8%) was significantly associated with axillary node involvement (p = 0.006), aneuploidy (p = 0.008), histologic grade (p = 0.03), and S-phase fraction > 5% determined by flow cytometry (p = 0.01). After a median follow-up of 6 years, the univariate analysis did not show significant correlation between high GF and worse relapse-free survival (p = 0.10) or shorter overall survival. However, a multivariate analysis on relapse-free survival, performed in 127 comparable patients, showed that GF was an independent predictive factor (p = 0.03) together with nodal status (p = 0.00001), age under 45 years (p = 0.0008), and chemotherapy (0.006). In node negative patients, GF was still an independent prognostic indicator (p = 0.002) together with age under 45 years (p = 0.0003). Tumor proliferative activity evaluated by the monoclonal antibody Ki-67 appears to be an effective indicator of prognosis in breast cancer and could be of assistance in the decision making of adjuvant therapy in node negative patients.

Adult↗
PubMed1996

Reverse transcription-polymerase chain reaction (RT-PCR) assays of estrogen and progesterone receptors in breast cancer.

The biochemical assay for estrogen (ER) and progesterone receptors (PR) as a routine procedure in the clinical evaluation of human breast cancer is well established. Since there are various and complex phenotypic alterations in breast cancer, there is a need for a multiparametric assessment of the biological profile of breast tumours. However, multiparametric analysis requires a large amount of tissue and various methods of quantitative analysis involving expensive reagents. Thus, an evaluation of the diagnostic and prognostic applications of the measurement of mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) has been initiated. A series of 105 surgical samples of breast cancer was assayed for ER and PR expression in parallel by semi-quantitative RT-PCR and standardized enzymoimmunoassays (EIA). 79 (75%) tumour samples were positive for ER expression by EIA, and 86 (82%) by RT-PCR. This shows a good concordance of the two methods (90%). In the case of PR expression 65 (62%) tumour samples were positive by EIA and only 53 (51%) samples by RT-PCR. In conclusion ER-RT-PCR appears to provide information concerning ER expression similar to ER-EIA, and may be an alternative to this assay. The information derived by PR-RT-PCR appears somewhat different from PR-EIA. We are currently evaluating the biological and clinical significance of this discrepancy.

Adult↗
PubMed1996

[A study of the expression of MDR1 gene in solid tumors. Initial results of a multicenter evaluation].

The results obtained from 12 laboratories, dealing with six identical malignant solid tumors, assessing MDR1 phenotype using molecular techniques and immunohistochemistry have been compared. Moreover, comparisons between results of MDR1 gene expression, quantified by RT-PCR or Northern blot analysis from 10 RNA and 10 cDNA samples, were also compared between eight laboratories. Results concerning solid tumors show frequent discrepancies between the results obtained by immunohistochemistry and molecular biology techniques. Moreover, inter-laboratory discrepancies concerning immunohistochemistry techniques are observed, suggesting that the interpretation of staining is critical. Results of RT-PCR and Northern blot using RNA and cDNA show that discrepancies are less frequent than those observed using immunohistochemistry. However, Northern blot is not sensitive enough to be used in routine. The problems encountered using RT-PCR are the following: positivity threshold level, reproducibility and risks of cross-contamination.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Cellular pharmacology of azatoxins (topoisomerase-II and tubulin inhibitors) in P-glycoprotein-positive and -negative cell lines.

Azatoxin (NSC 640737), a synthetic molecule, was rationally designed as a topoisomerase-II inhibitor and was shown to be a potent cytotoxic agent that inhibits both tubulin and topoisomerase II. A structure-activity relationship study allowed to select 3 derivatives that inhibit either tubulin (methylazatoxin) only or topoisomerase II (fluoroanilinoazatoxin and nitroanilino-azatoxin) in MTT assays performed on K562 and K562/ADM cells; the latter, expressing P-glycoprotein, indicated cross-resistance of K562/ADM cells to all 4 compounds. DNA double-strand breaks induced by the 3 azatoxins that inhibit topoisomerase II in vitro were decreased in K562/ADM as compared with K562 cells. Nitroanilino-azatoxin was the only compound for which resistance and reduced DNA damage observed in K562/ADM cells was partially reversed by verapamil, suggesting that nitroanilinoazatoxin was a substrate for P-glycoprotein. These results were confirmed by testing the cytotoxic activity of azatoxins on P-glycoprotein-expressing rat colon-carcinoma DHDK12/TRb cells in the absence and the presence of verapamil. Cell-cycle and mitotic-index studies indicated that azatoxin- and methyl-azatoxin-induced M-phase arrest was less in K562/ADM than in K562 cells. The G2 block induced by fluoro- and nitroanilinoazatoxins was delayed in K562/ADM cells. Verapamil increased cell-cycle inhibition induced by nitroanilinoazatoxin in K562/ADM cells without modifying cell-cycle effects of fluoroanilinoazatoxin. These results (i) are consistent with the specific inhibition of topoisomerase II or tubulin by azatoxin derivatives in cells; (ii) indicate that the nitro group of nitroanilinoazatoxin allows recognition and efflux by the P-glycoprotein; and (iii) suggest that cross-resistance of K562/ADM cells to other azatoxin derivatives is not mediated by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Transforming growth factor-beta and megakaryocytes in the pathogenesis of idiopathic myelofibrosis.

Although the disease is well described, the pathogenesis of bone marrow fibrosis in idiopathic myelofibrosis still remains unclear. We previously reported elevated intraplatelet transforming growth factor-beta (TGF-beta) levels in patients with this myeloproliferative disorder, compared with healthy subjects. Here, in a series of 16 patients, we show that TGF-beta expression is also increased in patients' peripheral blood mononuclear cells (PBMC): (i) at the mRNA level analysed by Northern blot hybridization and/or reverse transcription-polymerase chain reaction (RT-PCR); (ii) and/or at the secreted peptide level as evaluated in conditioned media from patients' mononuclear cells by a growth inhibition assay on CC164 cells. By immunostaining with a polyclonal anti-TGF-beta 1 antibody, TGF-beta was localized in morphologically heterogenous cells; these cells were characterized as megakaryocytes by labelling with a gpIIbIIIa monoclonal antibody. Thus we provide evidence that both TGF-beta and megakaryocytes are linked in the pathogenesis of idiopathic myelofibrosis.

Aged↗
PubMed1993

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis.

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg83b27/vgBG heterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.

Aminopterin↗