Pascale Cornillet-Lefebvre

Genotype-phenotype correlations in Down syndrome identified by array CGH in 30 cases of partial trisomy and partial monosomy chromosome 21

Down syndrome (DS) is one of the most frequent congenital birth defects, and the most common genetic cause of mental retardation. In most cases, DS results from the presence of an extra copy of chromosome 21. DS has a complex phenotype, and a major goal of DS research is to identify genotype–phenotype correlations. Cases of partial trisomy 21 and other HSA21 rearrangements associated with DS features could identify genomic regions associated with specific phenotypes. We have developed a BAC array spanning HSA21q and used array comparative genome hybridization (aCGH) to enable high-resolution mapping of pathogenic partial aneuploidies and unbalanced translocations involving HSA21. We report the identification and mapping of 30 pathogenic chromosomal aberrations of HSA21 consisting of 19 partial trisomies and 11 partial monosomies for different segments of HSA21. The breakpoints have been mapped to within ∼85u2009kb. The majority of the breakpoints (26 of 30) for the partial aneuploidies map within a 10-Mb region. Our data argue against a single DS critical region. We identify susceptibility regions for 25 phenotypes for DS and 27 regions for monosomy 21. However, most of these regions are still broad, and more cases are needed to narrow down the phenotypic maps to a reasonable number of candidate genomic elements per phenotype.

Compilation of published comparative genomic hybridization studies

The power of comparative genomic hybridization (CGH) has been clearly proven since the first paper appeared in 1992 as a tool to characterize chromosomal imbalances in neoplasias. This review summarizes the chromosomal imbalances detected by CGH in solid tumors and in hemopathies. In May of 2001, we took a census of 430 articles providing information on 11,984 cases of human solid tumors or hematologic malignancies. Comparative generic hybridization has detected a number of recurrent regions of amplification or deletion that allows for identification of new chromosomal loci (oncogenes, tumor suppressor genes, or other genes) involved in the development, progression, and clonal evolution of tumors. When CGH data from different studies are combined, a pattern of nonrandom genetic aberrations appears. As expected, some of these gains and losses are common to different types of pathologies, while others are more tumor-specific.

Identification of chromosomal loci associated with non‐P‐glycoprotein–mediated multidrug resistance to topoisomerase II inhibitor in lung adenocarcinoma cell line by comparative genomic hybridization

In order to identify genomic changes associated with an etoposide resistance acquisition, we used comparative genomic hybridization (CGH) to compare a human lung adenocarcinoma cell line, A549 wild type, and three sublines, A549‐VP1‐3, exposed to increasing concentrations of the topoisomerase II inhibitor, VP16. R‐banding karyotype, fluorescence in situ hybridization (FISH), and Southern blot for the MLL gene were also performed. The CGH analysis showed that the A549‐VP3 cell line shared chemoresistance‐specific abnormalities (amplification of 11q23–qter, loss of chromosome 17, and deletions of 2p14–pter and 2q23–q24). FISH analysis confirmed the loss of one chromosome 17 in the three resistant sublines and revealed an increased fragmentation of chromosome 2 in more than two segments, depending on the etoposide concentration. FISH with an MLL gene probe showed additional signals of MLL (from three in the A549‐WT to seven in the A549‐VP3 cell line) translocated onto several other chromosomes. Southern blot indicated an amplification of the MLL gene, dependent on the etoposide concentration, without gene rearrangement. The CGH results are suggestive of loci that could be associated with the acquisition of an etoposide‐chemoresistant phenotype. Deletion of the 2p region has already been reported, without any candidate gene being identified. The role of MLL in leukemogenesis has previously been demonstrated, but its role in the development of other tumors or its significance in the chemoresistance process remains to be elucidated.

Cytogenetic characterization of chromosomal rearrangement in a human vinblastine-resistant CEM cell line: use of comparative genomic hybridization and fluorescence in situ hybridization.

In order to identify genomic changes associated with drug-resistance acquisition, we performed R-banding karyotyping, fluorescence in situ hybridization, and comparative genomic hybridization to compare a human T-cell lymphoblastic leukemia cell line, CEM-wild type, and a subline with resistance to vinblastine (CEM-VLB) and overexpressing P-glycoprotein. Comparative genomic hybridization analysis showed that the CEM-VLB cell line carried chemoresistance-associated chromosomal abnormalities (amplification of 7q11 approximately q22, losses of chromosomes 2, 3, 5, 9, 10, and 16, and deletion of 4q13 approximately qter). Fluorescence in situ hybridization identified an amplified 7q21 region translocated on the short arm of a chromosome 2. This region contained the MDR1 gene locus and probably neighboring genes, such as SRI or MDR3/ABCB4. According to previous reports, this chromosomal rearrangement occurred during drug selection and attested a resistance acquisition.

A subpopulation of human B lymphocytes can express a functional Tissue Factor in response to phorbol myristate acetate

Tissue Factor (TF,CD142) is a transmembrane glycoprotein that belongs to the cytokine receptor superfamily. This multifunctional protein is constitutively present in most tissues. Among circulating blood cells, TF expression is known to be restricted to monocytes which do not constitutively produce TF but express TF in response to various stimuli. Here, we report that highly purified B lymphocytes can support a procoagulant activity (PCA) in response to phorbol myristate acetate (PMA). Using flow cytometry (FCM), we observed that a subpopulation of B lymphocytes (CD19+) can express TF and anionic phospholipids in response to PMA. TF protein was identified and characterized by immunofluorescence, immunocytochemistry and electronic microscopy. Using RT-PCR, we identified the presence of TF mRNA in response to PMA. In conclusion, B lymphocytes are a potential source of functional TF in human.

Long-term incubation with IL-4 and IL-10 oppositely modifies procoagulant activity of monocytes and modulates the surface expression of tissue factor and tissue factor pathway inhibitor

Monocytes can be induced to express both tissue factor (TF) and its inhibitor, TF pathway inhibitor‐1 (TFPI‐1). A short incubation (<6u2003h) with interleukin (IL)‐4 and IL‐10, two potent deactivators of monocyte functions, has been shown to modulate the synthesis and expression of TF by monocytes activated by lipopolysaccharide, but the consequences of longer incubations (up to 96u2003h) on both TF and TFPI‐1 are unknown. The results of this study showed that adherent monocytes in culture spontaneously expressed TF and TFPI and that prolonged incubation with IL‐10 induced a time‐ and dose‐dependent decrease of monocyte TF synthesis, and an accumulation of TF/TFPI‐1 complexes at the moncyte surface, suggesting a decreased clearance of these complexes. In contrast, IL‐4 induced a time‐ and dose‐dependent increase in TF synthesis, which remained intracytoplasmic, as shown by confocal microscopy. Surprisingly, TF:antigen (Ag) was decreased at the monocyte surface, but the procoagulant activity (PCA) of IL‐4‐treated monocytes was increased, as a result of more pronounced decrease of TFPI‐1:Ag expression than that of TF. In conclusion, prolonged incubation with IL‐4 and IL‐10 oppositely modified PCA of cultured monocytes, and altered TF and TFPI trafficking and clearance. These data explain the respective deleterious or benefit effects of IL‐4 or IL‐10 in atherothrombosis.

Cytogenetic evolution of human ovarian cell lines associated with chemoresistance and loss of tumorigenicity

In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH) to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties. Multicolor FISH (Multiplex‐FISH and Spectral Karyotype) and conventional FISH are also used to elucidate the karyotype of parental cell line. The drug‐resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype. However, these cell lines shared two common rearrangements: an unbalanced translocation der(8)t(8;13)(p22;q?) and a deletion of the 11p. These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8)) or the loss of tumorigenicity properties (del(11p)). Colour figure can be viewed on http://www.esacp.org/acp/2003/25‐3/struski.htm.

Label free technologies: Raman micro-spectroscopy and multi-spectral imaging for lymphocyte classification

Background Current diagnostic and prognostic approaches in oncology use morphological and molecular techniques which lead to patient personalized therapies. However, they are still complex and hard to standardize. This is also true for Chronic Lymphocytic Leukemia (CLL) that has been chosen for the IHMO project [1,2]. Simplifying diagnostic processes and making easier the standardization would be highly suitable. In order to develop such a simpler automated method, the IHMO project, funded by the French National Research Agency, proposed to develop a multimodal microscopy scanning platform that includes in a single machine a Raman micro-spectrometer (RMS) combined with a multispectral imager (MSI) [3,4]. RMS is a quick non-invasive and non-destructive technique for tissues and cells analysis [5]. It is very sensible to molecular changes and it could be used as a powerful diagnostic and prognostic tool when used in association with multivariate statistical methods. It is particularly useful for characterizing pathological tumors especially at the cellular level [6,7]. Multispectral imaging in the visible spectrum could confirm RMS classifications and provide new morphometric findings [8]. CLL disease is characterized by the proliferation of lymphocytes (lymphocytosis). It is the most common leukemia, preferentially affecting people aged over 50 years old. It is incurable and in most cases shows no clinical signs. Thus, it is often discovered by chance during a blood test. If necessary, morphological and immunological studies are led by analyzing blood smears colored with May-Grunwald Giemsa, by making a complete blood count and by computing a Matutes score. Such studies are necessary because it is impossible to distinguish a healthy cell from a cancerous one only using a conventional microscope.