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.

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.

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.