[Tools of molecular biology. Diagnostic use of DNA arrays].
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Breast Neoplasms↗
Biomedical subjects
F Bertucci
Publications and source records attributed to F Bertucci.
Expression scanning of an array of growth control genes in human tumor cell lines.
Analysis of gene expression on a medium- or large-scale is an increasingly recognized method for functional and clinical investigations based on the now extensive catalog of known or partially sequenced genes. The accessibility of this approach can be enhanced by using readily available technology (macroarrays on Nylon, radioactive detection) and the IMAGE resource to assemble sets of targets. We have set up such a medium-scale, flexible system and validated it by the study of quantitative expression levels for 120 genes in six cell lines, including three mammary carcinoma cell lines. A number of important parameters are identified as necessary for the assembly of a valid set and the obtention of good-quality quantitative data. The extensive data assembled in this survey identified potential targets of carcinogenesis, for example the CRABP2 and GATA3 transcription factor genes. We also demonstrate the feasibility of this procedure for relatively small tumor samples, without recourse to probe amplification methods.
Breast Neoplasms↗
Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line.
NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.
Antigens, CD↗
Expression profiling: DNA arrays in many guises.
DNA arrays have become the preferred method for large-scale expression measurement. Such data are needed in view of the large amounts of sequence data available: expression levels in a number of different tissues or situations provide a first step toward functional characterisation of new entities revealed by DNA sequencing. Although the basic principle of measurement is in all cases based on hybridisation of a mixed probe derived from tissue RNA to large sets of DNA fragments representing many genes, a number of different forms of implementation of this principle are at hand. They are briefly described and compared, emphasizing the important issue of sensitivity and sample requirements and the accessibility of the methods to academic scientists. When these factors are taken into account, it appears that, contrary to a largely prevalent impression, the "best" approach is not necessarily always provided by the widely advertised glass microarrays or oligonucleotide chips.
DNA↗
Association of angiogenesis and poor prognosis in node-positive patients receiving anthracycline-based adjuvant chemotherapy.
UNLABELLED: Tumoral angiogenesis has been described as associated with poor prognosis in breast cancer, particularly for node negative breast cancer. The purpose of this study was to evaluate the influence of angiogenesis in node-positive breast cancer and particularly its potential impact on adjuvant chemotherapy. PATIENTS AND METHODS: One hundred and thirty-five node-positive breast cancer patients who received anthracycline or derivative based adjuvant chemotherapy were selected from the data base of the Institut Paoli Calmettes. Angiogenesis was evaluated using CD31 antibody. Other prognosis variables studied were: hormonal status, tumor size, hormonal receptors, Elston and Ellis grade, and number of involved lymph nodes. RESULTS: In multivariate analysis, a high level of angiogenesis was independently associated with a diminution of survival (p = 0.007), and of metastasis-free survival (p = 0.003). Other variables associated with poor survival were progesterone receptor status (p = 0.003) and Elston's grade (p = 0.003), and with metastasis-free survival, progesterone receptor status (p = 0.018). CONCLUSION: Tumoral angiogenesis appears to be an independent prognostic factor for node-positive breast cancer, when treated with adjuvant chemotherapy. Adjuvant strategies for patients with a high level of angiogenesis should be discussed.
Adult↗
Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples.
DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon micro-arrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 microl) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for all these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with(33)P-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.
Biosensing Techniques↗
Angiogenesis as a prognostic marker in breast carcinoma with conventional adjuvant chemotherapy: a multiparametric and immunohistochemical analysis.
It has now been clearly established that quantitative immunohistochemical methods applied to tumour angiogenesis under suitable quality control conditions are a powerful prognostic tool for use in the initial assessment of breast carcinomas. Appropriate parameters for predicting the aggressiveness of tumours and their sensitivity to treatment are, however, still required. To determine whether the microvessel count (MVC) may serve to predict the chemotherapeutic response, a retrospective study was carried out on a series of 162 patients with breast carcinoma, who were all treated with the same standard adjuvant chemotherapy. Angiogenesis was assessed by performing CD31 immunostaining and MVC per mm2. Several other factors such as P53, ERBB2, BCL2, and Ki67 were also measured, and their prognostic value was compared with that of the MVC. The MVC was not found to be correlated with any of the other prognostic parameters, but turned out to be of great prognostic value whatever the threshold value chosen, which suggests that it is continuously valid at all levels. The median value of the MVC (43.5 per mm2) divided this series into two significantly different prognostic categories, in terms of both disease-free survival (P = 0.0002) and overall survival (P = 0.037). Univariate analysis showed that most of the parameters analysed were of prognostic value regarding the disease-free survival, namely grade (P = 0.029), mitotic index (P = 0.049), size (P = 0.015), oestrogen receptors (P = 0.022), progesterone receptors (P = 0.018), P53 (P = 0.0045), ERBB2 (P = 0.046), and Ki67 (P = 0.0008). As regards overall survival, grade and ERBB2 showed a loss of prognostic value. In multivariate analysis on disease-free survival, the MVC was the most accurate prognostic factor (RR = 2.64), followed by Ki67 (RR = 2.06) and P53 (RR = 1.69). With respect to overall survival, the MVC ranked third among the prognostic parameters analysed. Standard chemotherapy did not reduce the high prognostic value of the MVC performed on tumour angiogenesis. This suggests that the MVC may predict the degree of resistance to chemotherapy. Patients with high levels of angiogenesis, particularly node-negative patients, might therefore be able to benefit from adjuvant therapy of another kind.
Antigens, Neoplasm↗
High-dose chemotherapy and haematopoietic stem cell transplantation for inflammatory breast cancer: pathologic response and outcome.
Inflammatory breast cancer still has a poor prognosis despite improvements related to the introduction of neoadjuvant chemotherapy. The purpose of this study was to evaluate pathologic response rate and outcome of patients receiving high-dose chemotherapy with haematopoietic stem cell support for IBC. Seventeen consecutive patients with IBC received an association of mitoxantrone (36 mg/m2), cyclophosphamide (120 mg/kg), melphalan (140 mg/m2), with stem cell transplantation (SCT) following four to five cycles of cyclophosphamide (1000 mg/m2), doxorubicin (75 mg/m2) and 5FU (500 mg/m2). Mastectomy was performed a median of 2 months (range 1.5-45) after high-dose chemotherapy and was followed by radiotherapy. Macroscopic and microscopic pathologic complete response rates were respectively 56 and 39%. With a median follow-up of 36 months (range 17-52) 10 patients remain alive free of disease and seven patients have relapsed. Two relapses occurred in the group of patients with pathologic CR and five in the group with residual tumour. These results show that high-dose chemotherapy (HDC) with alkylating agents followed by SCT allows a very high tumour eradication in inflammatory breast cancer, suggesting a possible global benefit in progression-free survival and survival which remains to be demonstrated prospectively.
Adult↗
Benefits of granulocyte-colony-stimulating factor after stem cell transfusion in intensive sequential chemotherapy for breast cancer.
The aim of this study was to evaluate the clinical and economic benefit of filgrastim given with intensive sequential chemotherapy. Women with poor-prognosis breast cancer received four cycles of high-dose cyclophosphamide (3 g/m2) and doxorubicin (75 mg/m2), followed by filgrastim 5 microg/kg/dy, stem cell collection after the cycle 1, and stem cell infusion after cycle 3 and cycle 4. The first cohort received filgrastim after the fourth cycle but the second cohort did not.Thirty three patients were included in the first cohort and 13 in the second. The results indicate that the duration of grade IV neutropenia was shorter in the group given filgrastim as was the median time to recover an absolute neutrophil count (ANC) > 1.0 x 10(9)/L. The rate and duration of the rehospitalizations were higher in the group not receiving filgrastim. We found that costs such as drugs and hospitalizations were significantly higher (p = 0.032 and p = 0.049) in the non-filgrastim-treated group. Using ANC > 1.0 x 10(9)/L as an intermediary efficiency criterion it was more cost effective to give filgrastim. It can be concluded from this study that filgrastim can decrease the duration of grade IV neutropenia in patients receiving intensive sequential chemotherapy. This, in turn, reduces the cost of hospitalization. However, in our study, this reduction of neutropenia did not have any impact on further therapy.
Adult↗
High-dose sequential chemotherapy with stem cell support for non-metastatic breast cancer.
The importance of dose-intensity has been suggested in breast cancer. The aim of this study was to evaluate the feasibility of a high-dose intensity doxorubicin-cyclophosphamide regimen with supporting G-CSF and blood stem cells. Twenty-five patients with non-metastatic breast cancer received four cycles of doxorubicin (75 mg/m2) and cyclophosphamide (3000 mg/m2) at 3 week intervals. Apheresis was performed after the first cycle and if necessary after the second cycle. Stem cells were reinfused after the third and fourth cycles. G-CSF was started on day 3 of each cycle (5 microg/kg/day) and was stopped the day before the last apheresis or when absolute neutrophil count was above 0.5 x 10(9)/l. Median received dose-intensity was respectively 25 mg/m2/week (range 22-26) and 1000 mg/m2/week (range 904-1065) for doxorubicin and cyclophosphamide. Grade IV thrombocytopenia occurred in 8% of cycles. Two patients needed platelets and 12 red cell transfusion. Fifteen patients were readmitted for a median duration of 4 days (range 1-7). We have established a safe, outpatient, high-dose intensity doxorubicin-cyclophosphamide regimen with supporting G-CSF and blood stem cells which can be submitted for comparison with the current standards.
Adult↗
[Intensive chemotherapy with autologous stem cell transplantation in ovarian cancers: analysis of 67 patients treated at the Paoli-Calmettes Institute and a review of the literature].
Despite important initial chemosensitivity, advanced ovarian cancer has a bad prognosis with a median survival of 20 to 30 months. These results might be better with intensive chemotherapy. We analysed 67 patients treated by intensive chemotherapy with autologous stem cell transplantation for advanced ovarian cancer at Institute Paoli-Calmettes between 1980 and 1994. Population was divided in two groups: salvage group (n = 30) for initial chemotherapy-refractory patients and consolidation group (n = 37) for sensitive patients. Several successive conditioning regimens were used, all based on alkylating agents. Principal toxicities were severe aplasia and mucositis. Four patients died from toxicity related to infection during strong immunosuppression. In salvage group, 9 out of 21 evaluable patients responded (43%), but duration of responses was short (median range of 5 months) and 2-year overall survival rate was 8% after transplantation. In consolidation group, 19 patients are alive and 15 are without disease progression with a median follow-up of 42 months (17, 161) after diagnosis. Five-year disease-free survival rate is 28% (median range of 35 months) and 5-year overall survival rate is 48% (median range of 41 months). Intensification does not seem to be long term beneficial for initial chemotherapy refractory patients, despite objective responses rate better than classical treatment. On the other hand, results seem better than conventional treatments in case of chemosensitive disease and should be confirmed prospectively in larger cohort of patients. Moreover, other research directions are open like intensification supported by hematopoietic growth-factors and peripheral stem cells, definition of best conditioning regimen, use of taxanes, and intensification in first line chemotherapy after initial surgery.
Adult↗
Expression of FGF and FGF receptor genes in human breast cancer.
The family of FGF growth factors is involved in several biological processes and might play an important role in tumorigenesis. We have studied the respective expression of 8 of the 9 characterized FGF genes, and of the 4 known FGF receptor genes, in a panel of 10 tumor-cell lines and 103 breast-tumor samples, using RT-PCR and Northern-blot analyses. FGF1 and FGF2 were expressed in almost all samples, while expression of FGF5, FGF6, FGF7, and FGF9 was more restricted. FGFR1, FGFR2 and FGFR4 were expressed at high levels in respectively 22%, 4% and 32% of tumors. FGFR3 expression was not detected. The transcript encoding an FGFR1 isoform with 2 immunoglobulin-like domains was the most prevalent.
Base Sequence↗
[Sarcoma of the aortic intima: a rare cause of arterial embolism].
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Aged↗
Prognostic value of simultaneous expression of p21 and mdm2 in breast carcinomas treated by adjuvant chemotherapy with antracyclin.
One hundred and sixty-two breast carcinomas treated by adjuvant chemotherapy were investigated in immunohistochemistry for expression of p53 and two wild-type p53-regulated induced proteins, mdm2 and p21/waf1. p21 and mdm2 were expression stongly correlated with Ki67 but not with survival. The p53+/p21+, p53+/p21- and p53+/mdm2- phenotypes were associated with the worst prognosis. The p53+/p21+/ mdm2+ tumors were associated with a better outcome than the other phenotypes, they may be tumors expressing wild-type p53 and p21, and a form of mdm2 that might lead to the stabilization of p53. It is suggested that p21/mdm2 expression should be investigated in all cases of p53 positive breast cancer.
Antibiotics, Antineoplastic↗
High-dose chemotherapy with hematopoietic stem cell support in patients with advanced epithelial ovarian cancer: analysis of 67 patients treated in a single institution.
BACKGROUND: Advanced ovarian carcinoma is a chemosensitive tumor, but its prognosis is poor with 20 to 30% 5-year survival using conventional therapy. Increasing doses of chemotherapy might improve the prognosis because of the dose-effect. MATERIALS AND METHODS: Between 1980 and 1994 at Institut Paoli-Calmettes, 67 patients with advanced ovarian cancer were treated with different alkylating agents-based regimens of high dose chemotherapy (HDC) and hematopoietic stem cell support (HSCS). The population was divided in 2 groups: a salvage group (n = 30) including initial conventional chemotherapy-refractory patients, and a consolidation group (n = 37) including patients whose disease was sensitive to classical first-line chemotherapy after debulking surgery. RESULTS: Toxicity was essentially hematological (severe aplasia) and digestive (mucositis). Four toxic deaths occurred related to infection during immunosuppression. In the salvage group, 9 out of 22 evaluable patients responded (41%), but the duration of responses was short (median range of 6 months) and the 2-year overall survival rate was 13%. In the consolidation group, 17 patients are still alive, 12 with progression with a median follow-up of 63 months. The 5-year disease-free survival rate was 32% while the 5-year overall survival rate was 46%. CONCLUSIONS: Toxicity of HDC with HSCS is acceptable for poor-prognosis patients. In the long term, this therapy is not beneficial for chemotherapy refractory patients, despite objective response rates better than the conventional treatment, confirming the dose-effect for alkylating agents in ovarian cancer. On the other hand, the results seem better than classical therapy in case of chemosensitive disease and should be confirmed prospectively in a larger cohort of patients.
Antineoplastic Agents, Alkylating↗
Outpatient sequential high dose alkylation with stem cell support for patients with advanced breast cancer: a phase I-II study.
We evaluated the feasibility of administering, in an out-patient setting, a sequential high dose alkylating regimen with hematopoietic growth factor (HGF) and stem cell support to patients with advanced breast cancer. Peripheral blood stem cells (PBSC) were previously collected after chemotherapy and HGF. Two consecutive cycles of alkylating agents were planned: Thiotepa (T) then, 15 days later, BCNU (B). Three dose levels of each agent were administered in cohorts of consecutive patients: 400, 500 and 600 mg/m2 respectively. HGF and reinfusion of PBSC followed both cycles. Toxicity and response were evaluated according to the WHO recommendations. From April 1996 to August 1988, 30 women were enrolled: 8 in the first, 12 in the second and 10 in the third dose level. In all cases, B was administered after T with a median delay of 25 days because of grade 3/4 hematological toxicity. 4 patients did not receive B because of previous lung radiotherapy, persistent tricytopenia or insufficient PBSC collection. 19 patients with measurable lesions were considered for response. The objective response rate was 48% (11% CR, 37% PR). We recommended T and B at a dose of 600 mg/m2 to conduct a phase II study in metastatic breast cancer and even to administer B before T.
Antineoplastic Agents, Alkylating↗
Phase II study of a combination of cisplatin, all-trans-retinoic acid and interferon-alpha in squamous cell carcinoma: clinical results and pharmacokinetics.
UNLABELLED: Preclinical and clinical data suggest a certain antitumor efficacy for combination of retinoids, cytokines and cytotoxic compounds. PATIENTS AND METHODS: Between November 1994 and October 1996, 38 patients with advanced squamous cell carcinoma were enrolled in a phase II study to investigate an association of low-dose all-trans-retinoic acid (tRA) (40 mg/m2/day, 84 days), interferon-alpha (IFN-alpha) (6.10(6) UI/day, 84 days s.c.) and cisplatin (40 mg/m2/day, day 1, 28 and 56, i.v.). A Pharmacokinetic evaluation was performed on 12 patients. RESULTS: The incidence of grade 3/4 hematologic toxicities was moderate (< 20%). Extra hematological toxicities were frequent but easily manageable and not life threatening. However, treatment delivery was poor since only 6 patients (16%) received full therapy. Seven objective responses were observed (21%), suggesting some degree of synergism between tRA, IFN-alpha and cisplatin. Interestingly, continuous tRA treatment in combination with IFN-alpha and cisplatin did not induce a significant decrease in plasma levels, as had been previously described. CONCLUSIONS: Regarding the short median response duration and the frequency of toxic events, this regimen should no longer be recommended in pretreated patients with advanced disease. However, the consistent response rate reported here may warrant further investigation in an early setting.
Adult↗