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Molecular cytogenetics, 9, 90
2016

Inherent variability of cancer-specific aneuploidy generates metastases.

Bloomfield, Mathew, Duesberg, Peter

The genetic basis of metastasis is still unclear because metastases carry individual karyotypes and phenotypes, rather than consistent mutations, and are rare compared to conventional mutation. There is however correlative evidence that metastasis depends on cancer-specific aneuploidy, and that metastases are karyotypically related to parental cancers. Accordingly we propose that metastasis is a speciation event. This theory holds that cancer-specific aneuploidy varies the clonal karyotypes of cancers automatically by unbalancing thousands of genes, and that rare variants form new autonomous subspecies with metastatic or other non-parental phenotypes like drug-resistance - similar to conventional subspeciation. To test this theory, we analyzed the karyotypic and morphological relationships between seven cancers and corresponding metastases. We found (1) that the cellular phenotypes of metastases were closely related to those of parental cancers, (2) that metastases shared 29 to 96% of their clonal karyotypic elements or aneusomies with the clonal karyotypes of parental cancers and (3) that, unexpectedly, the karyotypic complexity of metastases was very similar to that of the parental cancer. This suggests that metastases derive cancer-specific autonomy by conserving the overall complexity of the parental karyotype. We deduced from these results that cancers cause metastases by karyotypic variations and selection for rare metastatic subspecies. Further we asked whether metastases with multiple metastasis-specific aneusomies are assembled in one or multiple, sequential steps. Since (1) no stable karyotypic intermediates of metastases were observed in cancers here and previously by others, and (2) the karyotypic complexities of cancers are conserved in metastases, we concluded that metastases are generated from cancers in one step - like subspecies in conventional speciation. We conclude that the risk of cancers to metastasize is proportional to the degree of cancer-specific aneuploidy, because aneuploidy catalyzes the generation of subspecies, including metastases, at aneuploidy-dependent rates. Since speciation by random chromosomal rearrangements and selection is unpredictable, the theory that metastases are karyotypic subspecies of cancers also explains Foulds' rules, which hold that the origins of metastases are "abrupt" and that their phenotypes are "unpredictable."

Digital object identifier (DOI): 10.1186/s13039-016-0297-x

Atom Indonesia, 42(2), 71-77
2016

Comparison of Radiosensitivity of Human Chromosomes 1, 2 and 4 from One Healthy Donor

Ramadhani, Purnami, Yoshida

In general, it was assumed that the chromosome aberration induced by ionizing radiation is proportional to the chromosome size. From this viewpoint, the higher chromosome size, the more resistant to radiation. However, different opinions, in which chromosomes are particularly sensitive or resistant to radiation, are also still followed until now. Here in this research, we compared the chromosome sensitivity between chromosomes number 1, 2, and 4 using the FISH (fluorescence in situ hybridization) technique. From this research, we expect that the information obtained could show clearly whether a longer chromosome is more frequently involved in translocations and also more resistant to radiation than a shorter one. The type of chromosome aberration considered was limited only to translocation and we used one sample donor in order to avoid donor variability. The whole blood from a healthy female was irradiated with γ-rays with doses of 1, 3 and 5 Gy, respectively. Isolated lymphocytes from the whole blood were then cultured for 48 hours. After the culture process was completed, preparations of harvest and metaphase chromosomes were carried out. Chromosomes 1, 2, and 4 were stained with different fluorochromes. The translocation of each chromosome at each dose point was subsequently evaluated from 50 images obtained from an automated metaphase finder and capturing system. An additional analysis was performed to identify which chromosome arm was more frequently involved in translocation. Further analyses were also conducted with the aim of determining which chromosome band had a higher frequency of radiation-induced breakage. The experimental results showed that chromosome number 4 was more frequently involved in translocations compared to chromosomes 1 and 2 at 5 Gy. In contrast, at doses of 1 and 3 Gy translocations involving chromosomes number 1 and 2 were more numerous compared to the ones involving chromosome 4. However, if the number of translocation was accumulated for all the doses applied, the chromosome number 4 was the chromosome most frequently involved in translocations. Breakpoint analysis revealed that in chromosome 1, chromosome 2, and chromosome 4, the highest chromosome bands as break position were in band q32, p13, and q21, respectively. It can be concluded that chromosome 4 is more sensitive to radiation in all doses point, despite having less DNA content than chromosomes 1 and 2. Thus, it was showed that our research cannot support the general assumption about chromosome aberration induced by radiation being proportional to DNA content.

Gastroenterol Res Pract, 2016, 6089658
2016

The Role of Chromosomal Instability and Epigenetics in Colorectal Cancers Lacking beta-Catenin/TCF Regulated Transcription.

Abdel-Rahman, Wael M., Lotsari-Salomaa, Johanna E., Kaur, Sippy, Niskakoski, Anni, Knuutila, Sakari, Järvinen, Heikki, Mecklin, Jukka-Pekka, Peltomäki, Päivi

All colorectal cancer cell lines except RKO displayed active β-catenin/TCF regulated transcription. This feature of RKO was noted in familial colon cancers; hence our aim was to dissect its carcinogenic mechanism. MFISH and CGH revealed distinct instability of chromosome structure in RKO. Gene expression microarray of RKO versus 7 colon cancer lines (with active Wnt signaling) and 3 normal specimens revealed 611 differentially expressed genes. The majority of the tested gene loci were susceptible to LOH in primary tumors with various β-catenin localizations as a surrogate marker for β-catenin activation. The immunohistochemistry of selected genes (IFI16, RGS4, MCTP1, DGKI, OBCAM/OPCML, and GLIPR1) confirmed that they were differentially expressed in clinical specimens. Since epigenetic mechanisms can contribute to expression changes, selected target genes were evaluated for promoter methylation in patient specimens from sporadic and hereditary colorectal cancers. CMTM3, DGKI, and OPCML were frequently hypermethylated in both groups, whereas KLK10, EPCAM, and DLC1 displayed subgroup specificity. The overall fraction of hypermethylated genes was higher in tumors with membranous β-catenin. We identified novel genes in colorectal carcinogenesis that might be useful in personalized tumor profiling. Tumors with inactive Wnt signaling are a heterogeneous group displaying interaction of chromosomal instability, Wnt signaling, and epigenetics.

Digital object identifier (DOI): 10.1155/2016/6089658

Nat Commun, 7, 10529
2016

RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

Lescale, Chloé, Abramowski, Vincent, Bedora-Faure, Marie, Murigneux, Valentine, Vera, Gabriella, Roth, David B., Revy, Patrick, de Villartay, Jean-Pierre, Deriano, Ludovic

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.

Digital object identifier (DOI): 10.1038/ncomms10529

Trends in Cancer Research
2015

Fluorescence in situ hybridisation assays designed for del(7q) detection uncover more complex rearrangements in myeloid leukaemia cell lines

Yasser Mostafa Kamel, Abdulbasit Naiel, Areej Alshehri, Michael Vetter, Salvatore Saccone, Rhona Anderson, Sabrina Tosi

Fluorescence in situ hybridisation assays designed for del(7q) detection uncover more complex rearrangements in myeloid leukaemia cell lines ABSTRACT Chromosome 7 abnormalities are associated with poor prognosis in myeloid leukaemia. The pathogenetic mechanisms chromosome 7 rearrangements and lead to malignancy are still poorly understood. The use of leukaemia- derived cell lines might be a useful tool to shed some light on these mechanisms. The cytogenetic characterisation of these cell lines is therefore important for the understanding of the genetic alterations leading to the disease. We carried out fluorescence in situ hybridisation (FISH) on three different myeloid leukaemia-derived cell lines (GDM-1, GF-D8 and K562). These were selected on the basis of harbouring rearrangements of chromosome 7. The probes used in these experiments were whole and partial chromosome paints, Multiplex-fluorescence in situ hybridisation (M-FISH) probes as well as locus specific probes for the 7q22, 7q31 and 7q36 regions. Our study confirmed the chromosome 7 abnormalities previously reported in the cell lines GDM-1 and GF-D8. We refined one of the rearrangements of chromosome 7 in the K562 cell line and reported some discrepancies with the data published in earlier reports. With this study, we confirm the importance of using a series of FISH that arise from probes to characterise chromosomal abnormalities in detail, as some rearrangements might go under-detected or mis-interpreted. Moreover, we highlight the importance of monitoring cell lines broadly used in research, as these can lose or acquire characteristics as they evolve in time in different laboratories.

Blood Cancer J, 5, e374
2015

Four genetic lymphoma-specific events (MYC, BCL2, BCL6 and CCND1) identified in a high grade B lymphoma case.

Ittel, A., Hélias, C., Wissler, M. P., Toussaint, E., Miguet, L., Chenard, M. P., Monier, L., Gervais, C., Mauvieux, L.

In the WHO classification, double or triple-hit lymphoma depicts rare and aggressive lymphomas displaying BCL2 and/or MYC and/or BCL6 gene rearrangements that are categorized as B-cell lymphomas unclassified, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma. Bacher et al.2 described an interesting series of 10 cases of such neoplasms. In addition, they reported the two first cases displaying four different lymphoma-specific events (quadruple hit) involving the genes MYC, BCL2, BCL6 and CCND1. We describe here a third case occurring in a 79-year-old male patient suffering from paraesthesias for 4 months who was referred for polyneuritis in a context of poor general condition. Clinical examination showed the presence of numerous axillary, supraclavicular, mediastinal and inguinal lymphadenopathies, neuro-meningeal invasion and skin infiltration. The biopsy of a left arm skin nodule revealed large proliferating cells (Ki-67 80%) stained by anti-CD20, BCL2 and BCL6 antibodies, CD10 and CD23 remaining negative, consistent with the diagnosis of diffuse large B-cell lymphoma (DLBCL), not otherwise specified. Blood cell counts showed 8.1 × 109/l leukocytes, 13.2 g/dl hemoglobin, 166 × 109/l platelets. LDH and β-2 microglobulin were elevated (989 U/I and 9.14 mg/l, respectively). Blood cell film examination showed the presence of 28% abnormal lymphocytes (medium sized, with intense basophilia, irregular nuclear contours, slightly clumped chromatin and frequent prominent nucleoli) suggestive of a high grade lymphoma. Flow cytometry revealed a lambda immunoglobulin light chain restriction. These cells expressed pan-B markers such as CD19, CD20, FMC7, CD22, with weak CD5 and CD43 positivity. CD10 and 23 were negative. Both the morphology and immunophenotype of the blood cells favored a pleomorphic mantle cell lymphoma (MCL) aggressive variant diagnosis. Cytogenetic study performed in the WBCs found a complex hyperdiploid karyotype (47 chromosomes, Figure 1) with a t(3;22) translocation involving the BCL6 and IGL genes, a structural abnormality of chromosome 8 resulting in juxtaposition of 5′ MYC and BCL2 in fluorescence in situ hybridization (with break of the MYC probe), a derivative chromosome 18 from a t(14;18) translocation with fusion of 5′IGH and BCL2, and a t(11;14) complex translocation involving IGH and CCND1 (Figure 2). Other numeral (trisomy 12) and structural abnormalities (involving the 1, 7, 14 and 21 chromosomes) were also detected (Figure 1). Overexpression of cyclin D1 was detected in the WBCs by real-time quantitative PCR, as well as in the skin lesion using immunochemistry. Anti-SOX11 antibody staining was found to be negative. Chemotherapy combining rituximab, ifosfamide, cytosine arabinoside and intrathecal methotrexate was initiated, but the patient died 4 months after the diagnosis. This third case of quadruple-hit lymphoma underlines the complexity of the classification of such aggressive malignancies. Initial rearrangement of the CCND1 gene characterizes MCL that may harbor in very rare cases additional rearrangements of MYC or BCL6, but histological transformation to typical large cell lymphoma is not retained in the WHO classification. In addition, cyclin D1 overexpression is considered to be a rare feature in DLBCL. Recently, Ok et al.3 proposed to reclassify DLBCL with expression of cyclin D1, CCND1 chromosomal rearrangement and CD5 positivity as an aggressive pleomorphic MCL variant. However, no observation of multiple lymphoma-specific gene rearrangements was described in that study. Juskevicius et al.4 suggest the existence of a ‘gray zone’ in which morphologic, clinical and genetic features are insufficient to segregate lymphomas with overexpression of cyclin D1/translocations involving CCND1 between blastoid MCL and cyclin D1-positive DLBCL. Regarding the immunophenotyping and molecular data, our case is possibly a genetically unstable aggressive pleomorphic MCL variant, which acquired three additional genetic hits.

Digital object identifier (DOI): 10.1038/bcj.2015.99

Hematology and Leukemia, 3(4)
2015

Detection of t(7;12)(q36;p13) in paediatric leukaemia using dual colour fluorescence in situ hybridisation.

Temitayo Owoka, Michael Vetter, Concetta Federico, Salvatore Saccone, Sabrina Tosi

The identification of chromosomal rearrangements is of utmost importance for the diagnosis and classification of specific leukaemia subtypes and therefore has an impact on therapy choices in individual cases. The t(7;12)(q36;p13) is a cryptic rearrangement that is difficult to recognise using conventional cytogenetic methods and is often undetected by reverse transcription polymerase chain reaction due to the absence of a fusion transcript in many cases. Here we present a reliable and easy to use dual colour fluorescence in situ hybridisation assay for the detection of the t(7;12)(q36;p13) rearrangement. A comparison with previous similar work is given and advantages and limitations of this novel approach are discussed.

Molecular cytogenetics, 8, 79
2015

Karyotype alteration generates the neoplastic phenotypes of SV40-infected human and rodent cells.

Bloomfield, Mathew, Duesberg, Peter

Despite over 50 years of research, it remains unclear how the DNA tumor viruses SV40 and Polyoma cause cancers. Prevailing theories hold that virus-coded Tumor (T)-antigens cause cancer by inactivating cellular tumor suppressor genes. But these theories don't explain four characteristics of viral carcinogenesis: (1) less than one in 10,000 infected cells become cancer cells, (2) cancers have complex individual phenotypes and transcriptomes, (3) recurrent tumors without viral DNA and proteins, (4) preneoplastic aneuploidies and immortal neoplastic clones with individual karyotypes. As an alternative theory we propose that viral carcinogenesis is a form of speciation, initiated by virus-induced aneuploidy. Since aneuploidy destabilizes the karyotype by unbalancing thousands of genes it catalyzes chain reactions of karyotypic and transcriptomic evolutions. Eventually rare karyotypes evolve that encode cancer-specific autonomy of growth. The low probability of forming new autonomous cancer-species by random karyotypic and transcriptomic variations predicts individual and clonal cancers. Although cancer karyotypes are congenitally aneuploid and thus variable, they are stabilized or immortalized by selections for variants with cancer-specific autonomy. Owing to these inherent variations cancer karyotypes are heterogeneous within clonal margins. To test this theory we analyzed karyotypes and phenotypes of SV40-infected human, rat and mouse cells developing into neoplastic clones. In all three systems we found (1) preneoplastic aneuploidies, (2) neoplastic clones with individual clonal but flexible karyotypes and phenotypes, which arose from less than one in 10,000 infected cells, survived over 200 generations, but were either T-antigen positive or negative, (3) spontaneous and drug-induced variations of neoplastic phenotypes correlating 1-to-1 with karyotypic variations. Since all 14 virus-induced neoplastic clones tested contained individual clonal karyotypes and phenotypes, we conclude that these karyotypes have generated and since maintained these neoplastic clones. Thus SV40 causes cancer indirectly, like carcinogens, by inducing aneuploidy from which new cancer-specific karyotypes evolve automatically at low rates. This theory explains the (1) low probability of carcinogenesis per virus-infected cell, (2) the individuality and clonal flexibility of cancer karyotypes, (3) recurrence of neoplasias without viral T-antigens, and (4) the individual clonal karyotypes, transcriptomes and immortality of virus-induced neoplasias - all unexplained by current viral theories.

Digital object identifier (DOI): 10.1186/s13039-015-0183-y

Molecular cytogenetics, 7, 71
2014

Karyotypic evolutions of cancer species in rats during the long latent periods after injection of nitrosourea.

Bloomfield, Mathew, McCormack, Amanda, Mandrioli, Daniele, Fiala, Christian, Aldaz, C Marcelo, Duesberg, Peter

A century of research has established that cancers arise from tissues exposed to carcinogens only after long latencies of years to decades and have individual clonal karyotypes. Since speciation from known precursors also depends on long latencies and new species also have individual karyotypes, we and others have recently proposed that carcinogenesis is a form of speciation. According to this theory karyotypic evolutions generate new cancer species from normal cells as follows: Carcinogens induce aneuploidy (Figure 1). By unbalancing thousands of genes aneuploidy automatically destabilizes the karyotype and thus catalyzes random karyotypic variations. Selections of variants with proliferative phenotypes form non-clonal hyperplasias with persistently varying karyotypes. Very rare karyotypic variations form new cancer species with individual clonal karyotypes. Despite destabilization by the resulting congenital aneuploidies, cancer karyotypes are stabilized within narrow margins of variation by clonal selections for cancer-specific autonomy. Because all non-cancerous aneuploidies are unstable, all aneusomies of prospective cancers are joined in single-steps, rather than gradually. Since this mechanism is very inefficient, it predicts long latent periods from carcinogens to cancers and individual clonal cancer karyotypes. Here we have tested the predicted roles of karyotypic evolutions during the time course of carcinogenesis in an established experimental system. In this system injection of nitrosourea induces in female rats non-invasive mammary hyperplasias ("tumors") after two or more months, and invasive carcinomas after six or more months. Accordingly four specific predictions were tested: (1) Invasive cancers are late and carry individual clonal karyotypes and phenotypes, (2) Persistent hyperplasias carry non-clonal karyotypes, (3) Non-clonal hyperplasias generate clonal cancers spontaneously but rarely, (4) Cancer-karyotypes arise with all individual clonal aneusomies in single-steps. All four predictions were experimentally confirmed. Our results along with the literature reveal a coherent karyotypic mechanism of carcinogenesis: Carcinogens induce aneuploidy. The inherent instability of aneuploidy automatically catalyzes new karyotypic variations. Aneuploid karyotypes with proliferative phenotypes form varying non-clonal hyperplasias. Rare variations form cancer species with individual clonal karyotypes, which are stabilized by clonal selection for autonomy. The low odds of this mechanism explain the long latencies of carcinogenesis, the individuality and karyotypic clonality of cancers.

Digital object identifier (DOI): 10.1186/s13039-014-0071-x

Neoplasia, 15(11), 1301--1313
November, 2013

Alternative Lengthening of Telomeres: Recurrent Cytogenetic Aberrations and Chromosome Stability under Extreme Telomere Dysfunction.

Despoina Sakellariou, Maria Chiourea, Christina Raftopoulou, Sarantis Gagos

Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.

Br J Haematol
October, 2013

Fusion of the additional sex combs like 1 and teashirt zinc fingerhomeobox 2 genes resulting from ider(20q) aberration in a patientwith myelodysplastic syndrome.

Jana Brezinova, Iveta Sarova, Halka Buryova, Jana Markova, Sarka Ransdorfova, Silvia Izakova, Karla Kostylkova, Jacqueline Soukupova, Zuzana Zemanova, Kyra Michalova

A variant of del(20q), an isochromosome of the long arm with the loss of an interstitial part of 20q, ider(20q), has been reported in patients with myeloid diseases (Li et al, 2004). About 40 cases with this rearrangement have been reported up to 2012 (reviewed by Mullier et al, 2012). Molecular cytogenetic and array techniques have been used for mapping of the deleted region on 20q (Douet-Guilbert et al, 2009). The proximal breakpoints are consistently located in the 20q11.21 band, and the distal breakpoints span from band 20q13.13 to band 20q13.33.

Mutat Res
June, 2013

Persisting ring chromosomes detected by mFISH in lymphocytes of acancer patient-A case report.

Sabine Schmitz, Michael Pinkawa, Michael J. Eble, Ralf Kriehuber

We report the case of an 84 years old prostate cancer patient with severe side effects after radiotherapy in 2006. He was cytogenetically analysed in 2009 and in 2012 in a comparative study for individual radiosensitivity of prostate cancer patients. No other patient had clonal aberrations, but this patient showed ring chromosomes in the range of 21-25\% of lymphocytes. He received 5 cycles of 5-fluorouracil/folic acid for chemotherapy of sigmoid colon carcinoma in 2003, three years before radiotherapy of prostate cancer. Blood samples were irradiated ex vivo with Cs-137 {\~a}-rays (0.7Gy/min) in the G0-phase of the cell cycle. 100 FISH painted metaphases were analysed for the control and the irradiated samples each. Multicolour in situ hybridisation techniques like mFISH and mBand as well as MYC locus, telomere and centromere painting probes were used to characterise ring metaphases. Metaphase search and autocapture was performed with a Zeiss Axioplan 2 imaging microscope followed by scoring and image analysis using Metafer 4/ISIS software (MetaSystems). In 2009 chromosome 8 rings were found in about 25\% of lymphocytes. Rings were stable over time and increased to about 30\% until 2012. The ring chromosome 8 always lacked telomere signals and a small amount of rings displayed up to four centromere signals. In aberrant metaphases 8pter and 8qter were either translocated or deleted. Further analyses revealed that the breakpoint at the p arm is localised at 8p21.2-22. The breakpoint at the q arm turned out to be distal from the MYC locus at 8q23-24. We hypothesise that the ring chromosome 8 has been developed during the 5 FU/folic acid treatments in 2003. The long term persistence might be due to clonal expansion of a damaged but viable hematopoietic stem cell giving rise to cycling progenitor cells that permit cell survival and proliferation.

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.

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.

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

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.

Methods Mol Biol, 730, 203--218
2011

The use of M-FISH and M-BAND to define chromosome abnormalities.

Ruth N. Mackinnon, Ilse Chudoba

Multicolour fluorescence in situ hybridisation (M-FISH) and multicolour banding (M-BAND) are advanced chromosome painting techniques combining multiple chromosome- or region-specific paints in one step. M-FISH identifies all chromosomes or chromosome arms at once, whereas M-BAND identifies the different regions of a single chromosome. The use of either or both can improve the accuracy of karyotyping and help identify cryptic chromosome rearrangements. These probes are prepared by pooling multiple chromosome- or chromosome region-specific DNA libraries, each labelled with a unique combination of fluorochromes. Commercial probes are available, avoiding the need for probe preparation. In the protocol described here, a commercial probe is used. Well-spread metaphases are prepared according to standard techniques, followed by alkaline denaturation and application of the denatured probe. After an incubation period, the slides are washed. A fluorescence microscope with filter sets specific to the fluorescent labels is used for analysis, together with specialised image analysis software. The software interprets the combination of fluorochromes to identify each chromosome and produce a false colour image specific for each chromosome or region. The single colour galleries - which show the hybridisation patterns of the individual fluorochromes - are useful to help interpret and confirm the false colour images produced by the software, including ambiguous signals.

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.

Mol Cytogenet, 4, 16
2011

Biclonal myelodysplastic syndrome involving six chromosomes and monoallelicloss of RB1 - A rare case.

Walid Al-Achkar, Abdulsamad Wafa, Elisabeth Klein, Abdulmunim Aljapawe

ABSTRACT:Myelodysplastic syndrome (MDS) represents a group of clonal hematological disorders characterized by progressive cytopenia, and reflects to defects in erythroid, myeloid and megakaryocytic maturation. MDS is more frequently observed in older aged patients with cytogenetic abnormalities like monosomy of chromosome(s) 5 and/or 7. In 50\% of de novo MDS cases, chromosomal aberrations are found and rearrangements involving the retinoblastoma (RB1) gene in 13q14 are found.Here, we are presenting a case report of a rare biclonal MDS with a karyotype of 45, XY,-4, der(6)t(4;6)(p15.1;p21.3), der(8)t(4;8)(q31.2;q22), t(13;16)(q21.3;p11.2)11/45, XY, der(7)t(7;13)(p22.2~22.3;q21.3),-13 9. The patient was diagnosed according to WHO classification as refractory anemia with excess of blasts (RAEB-II).Immunophenotyping was positive for CD11b, CD11c, CD10, CD13, CD15, CD16 and CD33.We report, a novel and cytogenetically rare case of a biclonal MDS with complex chromosomal aberrations and deletion of RB1-gene in both clones. These findings are associated with a poor prognosis as the patient died 3 months after diagnosis.