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International audience; Malignant mesothelioma (MM) is one of the worst cancers in terms of clinical outcome, urging the need to establish and characterize new preclinical tools for investigation of the tumorigenic process, improvement of early diagnosis and evaluation of new therapeutic strategies. For these purposes, we characterized a collection of 27 cell lines established from F344 rats, after 136 to 415 days of induction with crocidolite asbestos administered intraperitoneally. Four mesotheliomas were distinguished from 23 preneoplastic mesothelial cell lines (PN) according to their propensity to generate tumors after orthotopic transplantation into syngeneic rats, their growth pattern, and the expression profile of three genes. PN cell lines were further discriminated into groups / subgroups according to morphology in culture and the expression profiles of 14 additional genes. This approach was completed by analysis of positive and negative immunohistochemical MM markers in the four tumors, of karyotype alterations in the most aggressive MM cell line in comparison with a PN epithelioid cell line, and of human normal mesothelial and mesothelioma cells and a tissue array. Our results showed that both the rat and human MM cell lines shared in common a dramatic decrease in the relative expression of Cdkn2a and of epigenetic regulators, in comparison with PN and normal human mesothelial cells, respectively. In particular, we identified the involvement of the relative expression of the Ten-Eleven Translocation (TET) family of dioxygenases and Dnmt3a in relation to the 5-hydroxymethylcytosine level in malignant transformation and the acquisition of metastatic potential. INTRODUCTION Malignant mesothelioma (MM) is a rare, aggressive cancer mainly related to asbestos exposure [1], the long latency time between exposure and occurrence of clinical symptoms limiting the efficacy of therapeutic interventions. Given its chemoresistance, current therapies have a negligible impact on overall survival due to a lack of understanding of the complex biology of MM [2]. Thus, a better understanding of the different steps in the development of this disease should be of great interest for the identification of early markers of MM AU 1

Clonal heterogeneity is a major difficulty in the analysis of chromosome rearrangements within tumor tissue. Using in situ hybridization, a cell-to-cell analysis can be performed and should allow a better understanding of the genetic process. In addition, detection of pre-neoplastic lesions with only a few cells involved may improve the diagnosis of such lesions and their precocious treatment.Automated analysis was performed on tissue sections with our previously described two-color fluorescence in situ hybridization-based method for quantitative determination of chromosome arm imbalance. The imbalance between the long and short arms of chromosome 3 was determined in 24 cases of non-small-cell and small-cell lung cancers in which only small snap-frozen sections were used, allowing other simultaneous molecular analyses, such as TP53 gene mutation detection.Specifically developed software allowed localization of each nucleus within the section with regard to its chromosome imbalance and to reconstitute a multi-clonal panel within an apparently homogeneous sample. In some cases, discrepancies in the imbalance values were observed between the biopsy and the tumor obtained after surgery from the same patient.The discrepancies observed between biopsies and tumors, likely linked to the samples' heterogeneity, demonstrate the necessity to analyze tissue sections collected at various locations. The fully automated approach developed in this study rendered such investigations possible.

Objectives Presently, conventional cytogenetic analysis of metaphase chromosomes remains the reference approach in prenatal diagnosis. However, this method is labor-intensive and time-consuming. The first step toward the rapid identification of aneuploidies is achieved by interphase fluorescence in situ hybridization (FISH) with centromeric or locus-specific probes. Spot counting using this type of probes is a reliable approach, but is very time-consuming with some technical and biological limitations. In this study, we present a new FISH method using image cytometry for the detection of trisomy 21 within interphase nuclei. Methods The method is based on a comparative quantitation of the fluorescence signals emitted by whole chromosome 21 and 22 painting probes cohybridized on interphase nuclei. The chromosomal imbalance was determined with an automated image cytometer by detecting an abnormal ratio of both fluorescence emissions when compared with the ratio obtained in normal cells. Results Ten blood samples and twenty amniotic fluids were analyzed. Results from FISH and standard cytogenetics were compared and 100% correlation was achieved. Conclusions This method, which enables an easy detection of chromosomal imbalances without a need for metaphase preparations, can be applied to the diagnosis of trisomy 21 and extended to other disorders with chromosomal imbalances. Compared to other interphase FISH techniques, it avoids spot-scoring difficulties. Copyright © 2003 John Wiley & Sons, Ltd.

The detection of DNA aneuploid cells using flow cytometry is an indication for the presence of tumor cells, but when DNA diploid cells are found in 25-33% of the cases, the diagnostic and prognostic significance of DNA ploidy is more limited. We analyzed interphase nuclei after in situ hybridization and using image cytometry on 50 breast tumors with diploid DNA content to investigate whether early chromosome rearrangements were detectable and if their occurrence was clinically significant. Imbalances between the two arms of chromosome 1 were found in 55% of the cases and values ranged from 1.5-3.0. Comparison with histological data showed that Grade I tumors mainly have imbalances (67%) and that Grade III tumors were mainly without the imbalance (67%), whereas Grade II tumors were intermediate (50% imbalance). These data suggest that the diagnosis of DNA diploid cases may be improved by using interphase FISH. In addition, the data also indicates that early breast tumors may have different genetic origins, which is important in the comprehension of tumor malignancy in early stages, especially for preinvasive lesions.

The diagnosis of lung cancer is quite often hampered by the existence of various cell types within samples such as biopsies or pleural effusions. We have established a new marker for image cytometry of interphase tumor cells of the lung by using the most recurrent and early cytogenetic event in lung cancer, the loss of the short arm of chromosome 3. The method is based on the detection of the imbalance between the long and the short arms of chromosome 3 by performing two-color fluorescence in situ hybridization on both arms. Fourteen tumors were analyzed after short-term culture and compared with the corresponding cytogenetic data obtained from metaphase analysis. Results on interphase nuclei and control experiments on metaphases were the same, with imbalance ratios ranging from 1.0 to 2.0 (mean value 1.6, median 1.5). To assess the clinical significance of this approach, three pleural effusions were analyzed. Data showed that normal cells within the sample could have been distinguished from the tumor cells based on different imbalance values between the long and the short arms. Thus, our method allows refined detection of lung tumor cells within samples containing heterogeneous cell populations.

A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody-labeled human and hamster chromosomes were 4-10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.

Little information exists on how various nucleolar proteins function in ribosome biogenesis. Of special interest is that group of nucleolar proteins which are not incorporated into mature ribosomes because they are candidates for a role in the regulation of ribosome construction. Non-ribosomal nucleolar proteins can be analyzed using autoimmune sera from scleroderma patients which often contain antinucleolar antibodies. One such serum, designated ScBr, is shown by indirect immunofluorescence to react specifically with nucleoli in cells of 3 different mammalian species, indicating that the antigen is at least partly conserved evolutionarily. It is not RNase-sensitive, but is completely eliminated after incubation with pronase and 2 M NaCl. Immuno-electron microscopy was carried out on Lowicryl ultrathin sections to localize the antigen. The labeling was observed over both the granular and the dense fibrillar component but not the fibrillar centers, indicating that the antigen is associated with ribosomal RNA transcription sites and ribosome assembly into precursor particles. In addition, the antibody was localized to small nucleoplasmic entities, termed dense nuclear bodies. This could indicate a relationship between nucleoli and dense nuclear bodies. By immunoprecipitation, only a single protein of 94 kDa molecular weight was revealed. By immunoblotting, the band at 94 kDa was found to be the only positive band for high ScBr dilutions. Observation of the behavior of the antigen during mitosis revealed that it became dispersed into the cytoplasm after breakdown of the nuclear envelope, lining most of the chromosomes rather than remaining associated with the NOR-chromosomes. The antigen appeared to be restored to nucleoli only in late telophase; phase-dense prenucleolar bodies of early telophase cells did not show positive staining for the antigen. During actinomycin-D RNA synthesis inhibition as well as in non-stimulated lymphocytes the positive staining is greatly decreased. These results were consistent with a role for the 94 kDa nucleolar protein in the process of preribosome assembly.