A refined molecular taxonomy of breast cancer.

The current histoclinical breast cancer classification is simple but imprecise. Several molecular classifications of breast cancers based on expression profiling have been proposed as alternatives. However, their reliability and clinical utility have been repeatedly questioned, notably because most of them were derived from relatively small initial patient populations. We analyzed the transcriptomes of 537 breast tumors using three unsupervised classification methods. A core subset of 355 tumors was assigned to six clusters by all three methods. These six subgroups overlapped with previously defined molecular classes of breast cancer, but also showed important differences, notably the absence of an ERBB2 subgroup and the division of the large luminal ER+ group into four subgroups, two of them being highly proliferative. Of the six subgroups, four were ER+/PR+/AR+, one was ER-/PR-/AR+ and one was triple negative (AR-/ER-/PR-). ERBB2-amplified tumors were split between the ER-/PR-/AR+ subgroup and the highly proliferative ER+ LumC subgroup. Importantly, each of these six molecular subgroups showed specific copy-number alterations. Gene expression changes were correlated to specific signaling pathways. Each of these six subgroups showed very significant differences in tumor grade, metastatic sites, relapse-free survival or response to chemotherapy. All these findings were validated on large external datasets including more than 3000 tumors. Our data thus indicate that these six molecular subgroups represent well-defined clinico-biological entities of breast cancer. Their identification should facilitate the detection of novel prognostic factors or therapeutical targets in breast cancer.

NFAT3 transcription factor inhibits breast cancer cell motility by targeting the Lipocalin 2 gene.

NFAT1 and NFAT5 act as pro-invasive and pro-migratory transcription factors in breast carcinoma, contributing to the formation of metastases. We report that NFAT3 is specifically expressed in estrogen receptor alpha positive (ERA+) breast cancer cells. We show that NFAT3 inhibits by itself the invasion capacity of ERA+ breast cancer cells and needs to cooperate with ERA to inhibit their migration. Conversely, NFAT3 downregulation results in actin reorganization associated with increased migration and invasion capabilities. NFAT3 signaling reduces migration through inhibition of Lipocalin 2 (LCN2) gene expression. Collectively, our study unravels an earlier unknown NFAT3/LCN2 axis that critically controls motility in breast cancer.

Changes in allelic imbalances in locally advanced breast cancers after chemotherapy.

In advanced breast cancers, TP53 mutation is highly predictive of complete response to high-dose epirubicin/cyclophosphamide chemotherapy. In these tumours with an altered control of genomic stability, accumulation of chemotherapy-induced genetic alterations may contribute to cell death and account for complete response. To explore the effects of chemotherapy on stability of the tumour genome, allelic profiles were obtained from microdissected tumour samples before and after chemotherapy in 29 unresponsive breast cancers (9 with TP53 mutation). Ninety-four per cent allelic profiles remained unchanged after treatment. Interestingly, 11 profiles (6%) showed important changes after treatment; allelic imbalances significantly increased (four cases) or decreased (seven cases) after chemotherapy in three distinct experiments, two of which using laser microdissected tumour cells. These genetic changes were not linked to the TP53 status, but one tumour showed complete disappearance of TP53-mutated cells in the residual tumour after treatment. Altogether, these observations carry important implications for the clonal evolution of breast cancers treated with DNA-damaging agents, as they point both to the importance of tumour heterogeneity and chemotherapy-driven selection of subclones.

Targeted gene correction with 5' acridine-oligonucleotide conjugates.

Single-stranded oligonucleotides (SSOs) mediate gene repair of punctual chromosomal mutations at a low frequency. We hypothesized that enhancement of DNA binding affinity of SSOs by intercalating agents may increase the number of corrected cells. Several biochemical modifications of SSOs were tested for their capability to correct a chromosomally integrated and mutated GFP reporter gene in human 293 cells. SSOs of 25 nucleotide length conjugated with acridine at their 5' end increased the efficiency of gene correction up to 10-fold compared to nonmodified SSOs. Acridine and psoralen conjugates were both evaluated, and acridine-modified SSOs were the most effective. Conjugation with acridine at the 3' end of the SSO inhibited gene correction, whereas flanking the SSO by acridine on both sides provided an intermediate level of correction. These results suggest that increasing the stability of hybridization between SSO and its target without hampering a 3' extension improves gene targeting, in agreement with the "annealing-integration" model of DNA repair.

[Acute dyspnea in the emergency room: the utility of troponin, natriuretic, procalcitonin and D-dimers]. / Dyspnée aiguë aux urgences: utilité des troponines, des peptides natriurétiques, de la procalcitonine et des D-dimères.

In emergency, some "low cost" biological tests are too often systematically performed while others are not prescribed because they are considered "too expensive". Good practices for biological testing are in fact the real means for saving money. Here, in order to help for defining those good practices, we review emergency medical approach and, as an example, specify findings concerning the clinical utility of key blood analyses in patients with acute dyspnea. Emergency laboratory testing is usefull when it contributes to establish the diagnosis or to evaluate comorbidity or to stratify disease severity. In a given emergency context, clinical utility must be anticipated according to a bayesian approach with an estimation of the post-test probabilities using the likelihood ratios (estimated from literature) and the pretest probabilities (established by examination at the bedside). The likelihood ratio is the best criterion for diagnostic accuracy of a biological test. According to this criterion, troponin, natriuretic peptides, procalcitonin and D-dimers are four "costly" markers but which can significantly contribute to the etiologic diagnosis of an acute dyspnea. Troponin, natriuretic peptides and procalcitonin are also prognostic markers and are valuable parameters for stratifying disease severity according to their initial value and their plasmatic kinetic during the clinical course of the disease. In conclusion, it is not only the cost of the test but overall the potential impact of its result on the management of the patient's care which makes the decision of performing the test or not.

Improving gene replacement by intracellular formation of linear homologous DNA.

BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.

[Biochemical and pharmacological check-up in emergency orientation]. / Bilans biochimiques and pharmacologiques d'orientation en urgence.

Biochemical and pharmacological tests usually prescribed in casualty department were reviewed taking into account their physiological significance and predictive value : ions, total proteins, carbohydrate and nitrogenous metabolites, enzymes, tissue markers, pharmacological drugs. Few blood components were kept with the first intention, ideally with a turn around time below one hour: sodium, potassium, chloride, bicarbonate, total proteins, pCO2 and pO2, creatinine, glucose, ketone compounds, calcium, bilirubin, transaminases, lipase, C-reactive protein, myoglobin, troponin, chorionic gonadotropin hormone. Those tests do not have to be systematically performed but prescribed only after the evaluation of pre-test probabilities by the clinician.

Prevalence, risk factors and outcome of hyperlactataemia in HIV-infected patients.

OBJECTIVE: We describe the prevalence, risk factors and outcome of hyperlactataemia (HL) in a cohort of 140 HIV-infected patients. PATIENTS AND METHODS: Patients were enrolled consecutively within a 3-month period (July to September 1999) and followed until 31 October 2000. One hundred and forty HIV-infected patients had venous plasma lactate levels measured. HL was defined at baseline by two consecutive lactate levels > 2.1 mmol/L (upper limit of normal). We compared baseline demographic characteristics, immuno-virological parameters, antiretroviral therapy and outcome between patients with HL (cases) or without HL (controls). We described the clinical features of patients with HL. RESULTS: Among 129 patients included in the analysis, HL was found in 11 patients (8.5%), all of whom were receiving nucleoside reverse transcriptase inhibitors (NRTIs). Cases were more likely than controls to receive didanosine or stavudine (82% vs. 19%, P= 2.7 x 10(-6) and 82% vs. 48%, P= 0.03, respectively). Only 4/11 cases (36%) had symptoms consistent with HL. After a median follow-up of 15 months, lactate level returned to normal in all three patients who discontinued NRTIs, but in only 2/8 patients who did not (P = 0.06). Only one case experienced lactic acidosis and died during follow-up. Mortality rate was similar in cases and controls. CONCLUSION: HL is associated with NRTI use, in particular didanosine and stavudine, and discontinuation of NRTIs seems to be associated with rapid resolution of HL. Lactic acidosis remains rare and the long-term outcome of patients with HL does not seem to be poorer than controls.

A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination.

Directed mutagenesis in mammalian cells has been the focus of intense research because of its promising application for gene correction and engineering. Both natural and modified oligonucleotides (ODN), RNA-DNA chimeric oligonucleotide (RDO) and small fragment DNA (SFHR), as well as vector DNA were used for promoting homologous replacement with varying success. It was recently shown that a triple helix-forming oligonucleotide (TFO) tethered to an oligonucleotide (donor DNA) can enhance mutagenesis by homologous recombination in cells. The basic idea is to accelerate homology search by oligonucleotide-directed triple helix formation in the vicinity of the target site for donor DNA. Here we describe a new method named GOREC (guided homologous recombination) which shares similar gene targeting, but has notable difference in the concept with the previous method. It is made of a homing device (TFO) and a donor DNA for effecting distinct functions. They are linked together by non-covalent or covalent interaction. This modular concept allows guidance of either an oligonucleotide (ODN, RDO) or a small DNA fragment to the target site for homologous replacement. Therefore, the triple helix site can be hundreds of base pairs away from the target site. An episomal assay for proof-of-principle study will be presented and discussed.

Naturally processed peptides from HLA-DQ7 (alpha1*0501-beta1*0301): influence of both alpha and beta chain polymorphism in the HLA-DQ peptide binding specificity.

Self peptides bound to HLA-DQ7 (alpha1*0501-beta1*0301), one of the HLA molecules associated with protection against insulin-dependent diabetes mellitus, were characterized after their acid elution from immunoaffinity-purified HLA-DQ7 (alpha1*0501-beta1*0301) molecules. The majority of these self peptides derived from membrane-associated proteins including HLA class I, class II, class II-associated invariant chain peptide and the transferrin-receptor (TfR). By in vitro binding assays, the specificity of these endogenous peptides for HLA-DQ7 (alpha1*0501-beta1*0301) molecules was confirmed. Among these peptides, the binding specificity of the TfR 215-230 self peptide was further examined on a variety of HLA-DQ and DR dimers. Several findings emerged from this analysis: (1) this peptide displayed HLA-DQ allelic specificity, binding only to HLA-DQ7 (alpha1*0501-beta1*0301); (2) when either the DQalpha or DQbeta chain was exchanged, little or no binding was observed, indicating that specificity of HLA-DQ peptide binding was determined by polymorphic residues of both the alpha and beta chains. (3) Unexpectedly, the TfR 215-230 self peptide, eluted from DQ, was promiscuous with regard to HLA-DR binding. This distinct DR and DQ binding pattern could reflect the structure of these two molecules as recently evidenced by crystallography.

Influence of metabolic and genetic factors on tumour necrosis factor-alpha and lymphotoxin-alpha production in insulin-dependent diabetes mellitus.

The potential role of tumour necrosis factors (TNFs) in autoimmunity and insulin-dependent diabetes mellitus (IDDM) led us to determine in vitro TNF-alpha and lymphotoxin-alpha (LT-alpha, TNF-beta) production in IDDM patients according to TNF polymorphism. LT-alpha production of peripheral blood mononuclear cells (PBMC) was lower in diabetic subjects (m = 0.30 +/- 0.2 ng.10(-6) cells) than controls (m = 0.68 +/- 0.3 ng.10(-6) cells, p < 0.05), and early age-at-onset was correlated with low LT-alpha production (rs = 0.8, p = 0.0006). TNF-alpha production was the same in patients and controls, but patients with HbA1c > or = 8% had a higher TNF-alpha production (m = 3.05 +/- 1.2 ng.10(-6) cells) than those with HbA1c < 8% (m = 1.31 +/- 0.33 ng.10(-6) cells, p < 0.05). A study of the microsatellite TNFa region close to the LTA gene showed that the presence of the TNFa1 allele in HLA-(DR3) subjects was associated with increased risk of IDDM. TNFa1-positive subjects (both patients and controls) also had lower LT-alpha production than other subjects. These results indicate that low LT-alpha production is an additional risk factor for IDDM and that poor glycaemic control in patients is associated with enhanced PBMC TNF-alpha production which causes an imbalance between TNF-alpha and LT-alpha production in IDDM patient.

Analysis of HLA haplotypes in families with type 1 diabetes mellitus in La Réunion island.

To analyse HLA and insulin-dependent diabetes mellitus (IDDM) association in the ethnically mixed population of La Réunion island, we carried out a family study on 70 diabetic subjects. HLA-DQA1, -DQB1 and -DRB1 typing was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), completed by PCR-sequence-specific oligonucleotide (SSO) and PCR-sequence-specific priming (SSP). Haplotype-relative risks (HRR) were determined with the non-transmitted parental haplotypes as controls, and relative risks (RR) were calculated with a classical case-control study. The most significant risks were found for the cis and trans combinations between DQA1*03 or *0501 (Arg52+) and DQB1*02 or *0302 (Asp57-) alleles, suggesting a direct role for the HLA-DQ heterodimer in IDDM susceptibility. Interestingly, due to the mixed origin of the population, the trans-encoded DQ molecules in the (DR3)-DQA1*0501-DQB1*02/(DR4)-DQA1*03-DQB1*0302 subjects were also found cis-encoded in patients with the (DR7 or 9)-DQA1*03-DQB1*02 haplotype and in a patient with the rare (DR11)-DQA1*0501-DQB1*0302 haplotype. A relative predispositional effect (RPE) analysis gave significant haplotype-IDDM+ associations in the following order: (DR3)-DQA1*0501-DQB1*02 > (DR4)-DQA1*03-DQB1*0302 > (DR9)-DQA1*03- DQB*02 > (DR7)-DQA1*03-DQB1*02 > (DR2)-DQA1*01-DQB1*0502. No protective effect remained significant once the susceptible haplotypes were removed. A stratification study showed a stronger influence of the DQ genes than DRB1 alleles within the DR7 haplotypes. On the other hand, IDDM subjects with only one susceptible haplotype had inherited this haplotype more often from their father than from their mother. This paternal effect could be related to the greater risk of IDDM in offspring of diabetic fathers than the risk in offspring of diabetic mothers.

Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying beta (1-6)N-acetylglucosamine-branched N-linked oligosaccharides.

The circulating forms of malignant cells from patients with Sezary syndrome exhibit on their glycoproteins a high level of beta (1-6)GlcNAc-branched N-linked oligosaccharides, a particular species of glycans related to the metastatic potential of several tumors and T lymphocytes activation. An increased activity of the N-acetylglucosaminyltransferase V and of the beta (1-4)galactosyltransferase, two enzymes implicated in beta (1-6)GlcNAc-branching is also found. Nevertheless, contrary to activated normal T lymphocytes, Sezary lymphocytes in agreement with their non-proliferating state, do not exhibit increased thymidine uptake. This result suggests that expression of the beta (1-6)GlcNAc-branched N-linked carbohydrates could be related to some of the malignant properties of Sezary lymphocytes.

HL 60 leukaemia cells chemically induced to differentiate retain some surface glycan features of undifferentiated cells not found in normal leukocytes.

Human HL 60 myeloid leukaemia cells have the potential to differentiate into either macrophage-like cells or granulocyte-like cells under the stimulus of chemical treatments. Using glycotechnology procedures, the glycosylation patterns of differentiated and undifferentiated HL 60 cells were analysed and compared with those of normal human peripheral monocytes. Both in vitro differentiations result in significant morphologic and functional changes, but we observed that the glycosylation patterns of undifferentiated and differentiated HL 60 cells exhibit several common glycosidic features that are absent in normal peripheral monocytes: the presence of (i) bisecting beta-N-acetylglucosamine attached at the C-4 position of the beta-mannose of polyantennary complex-type carbohydrate chains and (ii) complex-type carbohydrate chains enriched with non-reducing terminal beta-N-acetylglucosamine residues. Moreover, the three populations of HL 60 cells express small amounts of biantennary complex-type structures (< 6%), whereas normal peripheral monocytes expressed > 20% of such structures. Thus, the cell glycosylation pattern could reflect the pathological state of the HL 60 cells.

[Dysregulation of in vitro TNF-beta production in insulin-dependent diabetes mellitus]. / Dysrégulation de la production in vitro de TNF-beta au cours du diabète insulinodépendant.

The possible role of tumor necrosis factors (TNF) in autoimmunity led us to explore the relationship between TNF production, polymorphism of the TNF-beta gene and type one diabetes. In the diabetic group we found a low production of TNF-beta. This abnormality appeared not to be exclusively related to the TNF-beta gene and could be also due to polymorphic regulatory sequences in the major histocompatibility complex region.

Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity.

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the Vmax. In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.

[Variability of biological glucose transport activity in human erythrocytes]. / Variabilité de l'activité biologique du transporteur du glucose dans les érythrocytes humains.

Glucose transport activity was determined on erythrocytes from healthy adult and children. A large variability of values was found between each donor, but transport rate was significantly higher in erythrocytes from adults than those from children. In opposite, the same number of glucose transport sites was found on erythrocytes from adult and children, suggesting that transporters from adult erythrocytes exhibited an higher own activity than transporters from child erythrocytes. Involvement of various factors in glucose transport activity was discussed.