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PLoS ONE
2013

Reduced Placental Telomere Length during Pregnancies Complicated by Intrauterine Growth Restriction

J{\'e}r{\^o}me Toutain, Martina Prochazkova-Carlotti, David Cappellen, Ana Jarne, Edith Chevret, Jacky Ferrer, Yamina Idrissi, Fanny Pelluard, Dominique Carles, Brigitte Maugey-Laulon, Didier Lacombe, Jacques Horovitz, Jean-Philippe Merlio, Robert Saura

Recent studies have shown that telomere length was significantly reduced in placentas collected at delivery from pregnancies complicated by intrauterine growth restriction secondary to placental insufficiency. Placental telomere length measurement during ongoing pregnancies complicated by intrauterine growth restriction has never been reported. This was the main objective of our study.

J Hered
October, 2012

Development and Application of Camelid Molecular Cytogenetic Tools.

Felipe Avila, Pranab J. Das, Michelle Kutzler, Elaine Owens, Polina Perelman, Jiri Rubes, Miroslav Hornak, Warren E. Johnson, Terje Raudsepp

Cytogenetic chromosome maps offer molecular tools for genome analysis and clinical cytogenetics and are of particular importance for species with difficult karyotypes, such as camelids (2n = 74). Building on the available human-camel zoo-fluorescence in situ hybridization (FISH) data, we developed the first cytogenetic map for the alpaca (Lama pacos, LPA) genome by isolating and identifying 151 alpaca bacterial artificial chromosome (BAC) clones corresponding to 44 specific genes. The genes were mapped by FISH to 31 alpaca autosomes and the sex chromosomes; 11 chromosomes had 2 markers, which were ordered by dual-color FISH. The STS gene mapped to Xpter/Ypter, demarcating the pseudoautosomal region, whereas no markers were assigned to chromosomes 14, 21, 22, 28, and 36. The chromosome-specific markers were applied in clinical cytogenetics to identify LPA20, the major histocompatibility complex (MHC)-carrying chromosome, as a part of an autosomal translocation in a sterile male llama (Lama glama, LGL; 2n = 73,XY). FISH with LPAX BACs and LPA36 paints, as well as comparative genomic hybridization, were also used to investigate the origin of the minute chromosome, an abnormally small LPA36 in infertile female alpacas. This collection of cytogenetically mapped markers represents a new tool for camelid clinical cytogenetics and has applications for the improvement of the alpaca genome map and sequence assembly.

Digital object identifier (DOI): 10.1093/jhered/ess067

Prenat Diagn, 32(8), 742--751
August, 2012

Identification of circulating fetal cell markers by microarray analysis.

Marie Brinch, Lotte Hatt, Ripudaman Singh, Kristine M{\o}ller, Steffen Sommer, Niels Uldbjerg, Britta Christensen, Steen K{\o}lvraa

Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester.Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis.Twenty-eight genes were identified as fetal cell marker candidates.Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment.

Leukemia, 26(7), 1695--1697
July, 2012

Molecular characterization of deletions of the long arm of chromosome5 (del(5q)) in 94 MDS/AML patients.

N. Douet-Guilbert, E. {De Braekeleer}, A. Basinko, A. Herry, N. Gueganic, C. Bovo, K. Trillet, A. {Dos Santos}, M. J. {Le Bris}, F. Morel, J. R. Eveillard, C. Berthou, M. {De Braekeleer}

Deletion of the long arm of chromosome 5 (del(5q)) is a common finding in myelodysplastic syndrome (MDS) and in acute myeloid leukemia (AML). First described in 1974 by Van den Berghe et al.,1 the 5q- syndrome, more frequently found in old-aged females, is characterized by erythroid hypoplasia, macrocytic anemia, normal to elevated platelets count, preponderance of monolobulated megakaryocytes, isolated 5q deletion and low rate of progression to AML.

Am J Med Genet B Neuropsychiatr Genet, 159B(5), 598--604
July, 2012

Mild cognitive impairment identified in older individuals with Downsyndrome by reduced telomere signal numbers and shorter telomeresmeasured in microns.

Edmund C. Jenkins, Lingling Ye, Milen Velinov, Sharon J. Krinsky-McHale, Warren B. Zigman, Nicole Schupf, Wayne P. Silverman

Previously, we established that short-term T lymphocyte cultures from people with Down syndrome (DS) and dementia (Alzheimer's disease) had shorter telomeres than did those from age- and sex-matched people with DS only, quantified as significantly reduced numbers of signals of peptide nucleic acid (PNA) telomere probes in whole metaphases [Jenkins et al. (2008); Neurosci Lett 440:340-343] as well as reduced telomere probe light intensity values in interphases [Jenkins et al. (2010); Neurobiol Aging 31:765-771]. We now describe shorter telomere length in adults with DS and mild cognitive impairment (MCI) compared to age- and sex-matched individuals with DS without MCI. Telomere length is quantified by reduced telomere signal numbers and shorter chromosome 1 telomeres measured in micrometers (microns). These findings were in agreement with quantitative light intensity measurements of chromosome 1 and chromosome 21 PNA telomere probes with and without the use of a #normalizing##ratio# involving the fluorescence exhibited by a PNA probe for centromere 2, and with the use of light intensity measurements of interphase preparations. Most importantly, the distributions of chromosome 1 telomere lengths (in microns) were completely non-overlapping for adults with and without MCI, indicating that this measure has great promise as a biomarker for MCI as well as dementia in this population.

J Clin Invest, 122(2), 569--574
February, 2012

Recurrent genomic instability of chromosome 1q in neural derivativesof human embryonic stem cells.

Christine Varela, J{\'e}r{\^o}me Alexandre Denis, J{\'e}r{\^o}me Polentes, Maxime Feyeux, Sophie Aubert, Benoite Champon, Genevi{\`e}ve Pi{\'e}tu, Marc Peschanski, Nathalie Lefort

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.

PLoS One, 7(10), e47185
2012

Evaluation of different biomarkers to predict individual radiosensitivityin an inter-laboratory comparison--lessons for future studies.

Burkhard Greve, Tobias B{\"o}lling, Susanne Amler, Ute R{\"o}ssler, Maria Gomolka, Claudia Mayer, Odilia Popanda, Kristin Dreffke, Astrid Rickinger, Eberhard Fritz, Friederike Eckardt-Schupp, Christina Sauerland, Herbert Braselmann, Wiebke Sauter, Thomas Illig, Dorothea Riesenbeck, Stefan K{\"o}nemann, Normann Willich, Simone M{\"o}rtl, Hans Theodor Eich, Peter Schmezer

Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity.

Blood, 118(26), 6760--6768
December, 2011

Impact of additional cytogenetic aberrations at diagnosis on prognosisof CML: long-term observation of 1151 patients from the randomizedCML Study IV.

Alice Fabarius, Armin Leitner, Andreas Hochhaus, Martin C M{\"u}ller, Benjamin Hanfstein, Claudia Haferlach, Gudrun G{\"o}hring, Brigitte Schlegelberger, Martine Jotterand, Andreas Reiter, Susanne Jung-Munkwitz, Ulrike Proetel, Juliana Schwaab, Wolf-Karsten Hofmann, J{\"o}rg Schubert, Hermann Einsele, Anthony D Ho, Christiane Falge, Lothar Kanz, Andreas Neubauer, Michael Kneba, Frank Stegelmann, Michael Pfreundschuh, Cornelius F Waller, Karsten Spiekermann, Gabriela M Baerlocher, Michael Lauseker, Markus Pfirrmann, Joerg Hasford, Susanne Saussele, R{\"u}diger Hehlmann, f{\"u}r Klinische Krebsforschung (SAKK), Schweizerische Arbeitsg, the German CML Study Group,

The prognostic relevance of additional cytogenetic findings at diagnosis of chronic myeloid leukemia (CML) is unclear. The impact of additional cytogenetic findings at diagnosis on time to complete cytogenetic (CCR) and major molecular remission (MMR) and progression-free (PFS) and overall survival (OS) was analyzed using data from 1151 Philadelphia chromosome-positive (Ph(+)) CML patients randomized to the German CML Study IV. At diagnosis, 1003 of 1151 patients (87\%) had standard t(9;22)(q34;q11) only, 69 patients (6.0\%) had variant t(v;22), and 79 (6.9\%) additional cytogenetic aberrations (ACAs). Of these, 38 patients (3.3\%) lacked the Y chromosome (-Y) and 41 patients (3.6\%) had ACAs except -Y; 16 of these (1.4\%) were major route (second Philadelphia [Ph] chromosome, trisomy 8, isochromosome 17q, or trisomy 19) and 25 minor route (all other) ACAs. After a median observation time of 5.3 years for patients with t(9;22), t(v;22), -Y, minor- and major-route ACAs, the 5-year PFS was 90\%, 81\%, 88\%, 96\%, and 50\%, and the 5-year OS was 92\%, 87\%, 91\%, 96\%, and 53\%, respectively. In patients with major-route ACAs, the times to CCR and MMR were longer and PFS and OS were shorter (P

Am J Clin Pathol, 136(5), 712--720
November, 2011

Detection of genomic abnormalities in multiple myeloma: the applicationof FISH analysis in combination with various plasma cell enrichmenttechniques.

Luise Hartmann, Julie Sanford Biggerstaff, Douglas B. Chapman, Janice M. Scott, Krystal R. Johnson, Keely M. Ghirardelli, Wayne K. Fritschle, Dolores L. Martinez, Richard K. Bennington, Monica E. {de Baca}, Denise A. Wells, Michael R. Loken, Barbara K. Zehentner

Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells (PCs) that accumulate in the bone marrow. A number of different genomic abnormalities are associated with MM; however, detection of these by fluorescence in situ hybridization (FISH) can be limited by the percentage of PCs in the specimen. In this study, we tested 20 bone marrow specimens with known MM and a low concentration of monoclonal PCs for the presence of genomic abnormalities using FISH in combination with various PC enrichment techniques: magnetic cell sorting, targeted manual scoring, and automated image analysis. In addition, flow cytometric cell sorting of PCs in combination with FISH analysis was also tested for minimal residual disease applications. Different parameters were evaluated when assessing the detection efficiency of each approach. FISH results are highly dependent on the chosen enrichment method. We describe the evaluation of different techniques applicable for various laboratory settings and specimen parameters.

Leuk Res, 35(8), 1114--1116
August, 2011

Evolutionary sequence of cytogenetic aberrations during the oncogenesisof plasma cell disorders. Direct evidence at single cell level.

Zs{\'o}fia Nagy, B{\'e}la Kajt{\'a}r, P{\'a}l J{\'a}ks{\'o}, Mariann D{\'a}vid, Szabolcs Kosztol{\'a}nyi, Judit Hermesz, L{\'a}szl{\'o} Kereskai, L{\'a}szl{\'o} Pajor, Don{\'a}t Alp{\'a}r

Bone marrow specimens from 185 patients with plasma cell disorders (PCD) were investigated by fluorescence in situ hybridization (FISH) in order to determine the temporal sequence of cytogenetic aberrations. In 25 cases combined FISH analysis has also been performed at single cell level. Clonal evolution was observed in 16\% of cases. The ?13 was preceded by t(4;14)(p16;q32) and t(14;16)(q32;q23) translocations. Deletion of p53 gene was a secondary aberration compared to ?13 and t(11;14)(q13;q32) translocation. In 22\% of all cases with recurrent IGH translocation, this aberration was presented only in a subset of purified plasma cells questioning its initiating role.

Stem Cell Rev, 7(2), 471--477
June, 2011

An improved technique for chromosomal analysis of human ES and iPScells.

Daniela Moralli, Mohammed Yusuf, Mohammad A Mandegar, Suhail Khoja, Zoia L Monaco, Emanuela V Volpi

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.

Cytometry A, 79(5), 375--382
May, 2011

Urovysion: Considerations on modifying current evaluation scheme,including immunophenotypic targeting and locally set, statisticallyderived diagnostic criteria.

Gabor Pajor, Laszlo Somogyi, Bela Melegh, Donat Alpar, Bela Kajtar, Laszlo Farkas, Maria Kneif, Daniel Bollmann, Laszlo Pajor, Norbert Sule

Urovysion multitarget fluorescence in situ hybridization (FISH) assay is a promising tool for detection of bladder cancer, however, there is still no consensus regarding abnormal signal pattern and cut-off level, and the recommended targeting carries limitations similar to urine cytology. Aim of this study was to explore diagnostic benefits of a recently introduced method featuring target specific genotyping, as well as to investigate the feasibility of locally and statistically determined cut-off, compared with conventional evaluation scheme. Histology, cytology, and comparative FISH approaches were performed on 42 patients with high clinical suspicion for urothelial carcinoma (UC). FISH parallels were (1) Urovysion-alone (according to manufacturer's instruction); (2) Targeted-Urovysion (cytokeratin7 immunophenotyping followed by Urovysion), both of which evaluated by both conventional and statistical evaluation scheme. For statistical evaluation cut-offs and sufficient sample size were determined on controls and ratio of positive cells was recorded, whereas conventional evaluation relied on manufacturer's recommendations. The specificity of cytology, Urovysion-alone in general and targeted-Urovysion in general appeared 86\%, 86\%, and 100\%, respectively. In the same comparison, overall sensitivity was 60\%, 80\%, and 93\%, respectively. In superficial cases sensitivity was 48\% for cytology, 72\% for Urovysion-alone and 91\% for targeted-Urovysion, while no prominent differences were seen in muscle invasive cases. The ratio of FISH positive cells was proportionate with both stage and grade, however, targeted genotyping could separate high grade/high stage cases more effectively. In conclusion, CK7 targeting raises diagnostic efficiency of Urovysion, and could be an ideal tool for identifying tumor cells in ambiguous cases or when other tumors are present. Statistical evaluation produces accuracy comparable with results of conventional evaluation, and with laboratories setting cut-offs individually but according harmonized protocol, it could aid method standardization. Furthermore, by providing additional quantitative information about tumor characteristics, is likely to have therapy relevant value in the future.

PLoS Genet, 7(4), e1002042
April, 2011

DNA damage, somatic aneuploidy, and malignant sarcoma susceptibilityin muscular dystrophies.

Wolfgang M Schmidt, Mohammed H Uddin, Sandra Dysek, Karin Moser-Thier, Christine Pirker, Harald H{\"o}ger, Inge M Ambros, Peter F Ambros, Walter Berger, Reginald E Bittner

Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD-pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD-gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases.

Blood, 117(15), e161--e170
April, 2011

Myelodysplasia and leukemia of Fanconi anemia are associated witha specific pattern of genomic abnormalities that includes crypticRUNX1/AML1 lesions.

Samuel Quentin, Wendy Cuccuini, Raphael Ceccaldi, Olivier Nibourel, Corinne Pondarre, Marie-Pierre Pag?s, Nadia Vasquez, Catherine {Dubois d'Enghien}, J{\'e}r{\^o}me Larghero, {Peffault de Latour}, R{\'e}gis, Vanderson Rocha, Jean-Hugues Dalle, Pascale Schneider, Mauricette Michallet, G{\'e}rard Michel, Andr{\'e} Baruchel, Fran{\c{c}}ois Sigaux, Eliane Gluckman, Thierry Leblanc, Dominique Stoppa-Lyonnet, Claude Preudhomme, G{\'e}rard Soci{\'e}, Jean Soulier

Fanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8\%), 3q+ (41.4\%), -7/7q (17.2\%), and 11q- (13.8\%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7\%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBP? mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.

DNA Repair (Amst), 10(3), 322--337
March, 2011

Validation of a fully automated COMET assay: 1.75 million singlecells measured over a 5 year period.

Albert Rosenberger, Ute R{\"o}ssler, Sabine Hornhardt, Wiebke Sauter, Heike Bickeb{\"o}ller, H-Erich Wichmann, Maria Gomolka

The COMET assay is recognized as a rapid and sensitive method in quantifying radiation induced DNA damage. We investigated the distorting influence of endogenous, assay-inherent factors onto base (single cell level) and primary outcome measures (experimental/slide level), such as olive tail moment (OTM) and percentage DNA in the tail (\%tail-DNA). From 2003 to 2008, we performed the assay on lymphocytes isolated from the blood samples of 355 lung cancer patients, 170 controls, and 610 relatives, as well as one single reference individual, repeated 170 times. In total, the data from 10,016 single experiments containing around 1,750,000 cells have been included in this study. This is the first time that the endogenous variability of the COMET assay has been validated systematically on such a huge data set over a 5 year period. Assuming that the reference sample reflects assay specific white noise, we estimated a proportion of 7-95\% of the variability of the outcome measures due to assay variation (white noise) depending on parameter, exposure level, and study group. The proportion of white noise was largest for the initial radiation damage. The specific endogenous factors considered attribute to 14.8\% of the total variability in the primary outcome measurements of the OTM and 6.9\% of the \%tail-DNA. OTM turns out to be a sensitive parameter to detect variation, but is also more susceptible to disturbance caused by endogenous factors than \%tail-DNA. To reduce the experimental variability in COMET assays, we recommend a highly standardized operation protocol as well as inspecting and/or adjusting the primary outcome measures according to endogenous factors before calculating secondary outcome measures, e.g. DNA repair capacity (DRC) or testing statistical inference. A human reference (HR) sample is also useful to inspect homogeneity in the temporal progression of long lasting investigations, but not for calibrating primary outcome measurements.

J Biomed Biotechnol, 2011, 693691
2011

Chromosomal rearrangements in post-Chernobyl papillary thyroid carcinomas:evaluation by spectral karyotyping and automated interphase FISH.

Ludwig Hieber, Reinhard Huber, Verena Bauer, Quirin Sch{\"a}ffner, Herbert Braselmann, Geraldine Thomas, Tatjana Bogdanova, Horst Zitzelsberger

Structural genomic rearrangements are frequent findings in human cancers. Therefore, papillary thyroid carcinomas (PTCs) were investigated for chromosomal aberrations and rearrangements of the RET proto-oncogene. For this purpose, primary cultures from 23 PTC have been established and metaphase preparations were analysed by spectral karyotyping (SKY). In addition, interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation (FISH) for the presence of RET/PTC rearrangements using RET-specific DNA probes. SKY analysis of PTC revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20, 21, and 22. FISH analysis for RET/PTC rearrangements showed prevalence of this rearrangement in 72\% (16 out of 22) of cases. However, only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity. The comparison of visual and automated scoring of FISH signals revealed concordant results in 19 out of 22 cases (87\%) indicating reliable scoring results using the optimised scoring parameter for RET/PTC with the automated Metafer4 system. It can be concluded from this study that genomic rearrangements are frequent in PTC and therefore important events in thyroid carcinogenesis.

Mol Cytogenet, 4(1), 8
2011

A rare case of t(11;22) in a mantle cell lymphoma like B-cell neoplasiaresulting in a fusion of IGL and CCND1: case report.

Cristiano Krings Rocha, Inka Praulich, Iris Gehrke, Michael Hallek, Karl-Anton Kreuzer

ABSTRACT: The chromosomal translocation (11;14)(q13;q32) rearranging the locus for cyclin D1 (CCND1) to that of the immunoglobulin heavy chain (IGH) can be found in virtually all cases of mantle cell lymphoma (MCL), while other CCND1 translocations are extremely rare. As CCND1 overexpression and activation is a hallmark of MCL it is regarded as a central biological mechanism in the development and maintenance of this disease.Here we present a patient initially diagnosed with chronic lymphocytic leukemia (CLL) where chromosome banding analysis revealed, among other aberrations, a translocation (11;22)(q13;q11.2). We show by fluorescence in situ hybridization (FISH) analysis that on chromosome 22 the immunoglobulin light chain lambda (IGL) is involved in this cytogenetic aberration. Additionally, we demonstrate the resulting overexpression of CCND1 on the RNA and protein level, thereby consolidating the new diagnosis of a MCL-like B-cell neoplasia. Summing up, we described a rare case of t(11;22)(q13;q11.2) in a MCL-like neoplasia and showed that this aberration leads to an overexpression of CCND1 which is regarded as a key biological feature in MCL. This case underlines the importance of cytogenetic analyses especially in atypical cases of B cell lymphomas.

PLoS One, 6(12), e28368
2011

Characterization of Abcc4 gene amplification in stepwise-selectedmouse J774 macrophages resistant to the topoisomerase II inhibitorciprofloxacin.

B{\'e}atrice Marquez, Genevi{\`e}ve Ameye, Coralie M Vallet, Paul M Tulkens, H{\'e}l{\`e}ne A Poirel, Fran{\c{c}}oise Van Bambeke

Exposure of J774 mouse macrophages to stepwise increasing concentrations of ciprofloxacin, an antibiotic inhibiting bacterial topoisomerases, selects for resistant cells that overexpress the efflux transporter Abcc4 (Marquez et al. [2009] Antimicrob. Agents Chemother. 53: 2410-2416), encoded by the Abcc4 gene located on Chromosome 14qE4. In this study, we report the genomic alterations occurring along the selection process. Abcc4 expression progressively increased upon selection rounds, with exponential changes observed between cells exposed to 150 and 200 µM of ciprofloxacin, accompanied by a commensurate decrease in ciprofloxacin accumulation. Molecular cytogenetics experiments showed that this overexpression is linked to Abcc4 gene overrepresentation, grading from a partial trisomy of Chr 14 at the first step of selection (cells exposed to 100 µM ciprofloxacin), to low-level amplifications (around three copies) of Abcc4 locus on 1 or 2 Chr 14 (cells exposed to 150 µM ciprofloxacin), followed by high-level amplification of Abcc4 as homogeneous staining region (hsr), inserted on 3 different derivative Chromosomes (cells exposed to 200 µM ciprofloxacin). In revertant cells obtained after more than 60 passages of culture without drug, the Abcc4 hsr amplification was lost in approx. 70\% of the population. These data suggest that exposing cells to sufficient concentrations of an antibiotic with low affinity for eukaryotic topoisomerases can cause major genomic alterations that may lead to the overexpression of the transporter responsible for its efflux. Gene amplification appears therefore as a mechanism of resistance that can be triggered by non-anticancer agents but contribute to cross-resistance, and is partially and slowly reversible.

Diagn Pathol, 6, 76
2011

FISH as an effective diagnostic tool for the management of challengingmelanocytic lesions.

Mathew W. Moore, Robert Gasparini

The accuracy of melanoma diagnosis continues to challenge the pathology community, even today with sophisticated histopathologic techniques. Melanocytic lesions exhibit significant morphological heterogeneity. While the majority of biopsies can be classified as benign (nevus) or malignant (melanoma) using well-established histopathologic criteria, there exists a cohort for which the prediction of clinical behaviour and invasive or metastatic potential is difficult if not impossible to ascertain on the basis of morphological features alone. Multiple studies have shown that there is significant disagreement between pathologists and even expert dermatopathologists in the diagnosis of this subgroup of difficult melanocytic lesions.A four probe FISH assay was utilized to analyse a cohort of 500 samples including 157 nevus, 176 dysplastic nevus and 167 melanoma specimens.Review of the lesions determined the assay identified genetic abnormalities in a total of 83.8\% of melanomas, and 1.9\% of nevus without atypia, while genetic abnormalities were identified in 6.3\%, 6.7\%, and 10.3\% of nevus identified with mild, moderate and severe atypia, respectively.Based on this study, inheritable genetic damage/instability identified by FISH testing is a hallmark of a progressive malignant process, and a valuable diagnostic tool for the identification of high risk lesions.