Complex Chromosomal Translocations Research Articles

Characterization of complex chromosomal rearrangements by targeted capture and next-generation sequencing

Translocations are a common class of chromosomal aberrations and can cause disease by physically disrupting genes or altering their regulatory environment. Some translocations, apparently balanced at the microscopic level, include deletions, duplications, insertions, or inversions at the molecular level. Traditionally, chromosomal rearrangements have been investigated with a conventional banded karyotype followed by arduous positional cloning projects. More recently, molecular cytogenetic approaches using fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), or whole-genome SNP genotyping together with molecular methods such as inverse PCR and quantitative PCR have allowed more precise evaluation of the breakpoints. These methods suffer, however, from being experimentally intensive and time-consuming and of less than single base pair resolution. Here we describe targeted breakpoint capture followed by next-generation sequencing (TBCS) as a new approach to the general problem of determining the precise structural characterization of translocation breakpoints and related chromosomal aberrations. We tested this approach in three patients with complex chromosomal translocations: The first had craniofacial abnormalities and an apparently balanced t(2;3)(p15;q12) translocation; the second has cleidocranial dysplasia (OMIM 119600) associated with a t(2;6)(q22;p12.3) translocation and a breakpoint in RUNX2 on chromosome 6p; and the third has acampomelic campomelic dysplasia (OMIM 114290) associated with a t(5;17)(q23.2;q24) translocation, with a breakpoint upstream of SOX9 on chromosome 17q. Preliminary studies indicated complex rearrangements in patients 1 and 3 with a total of 10 predicted breakpoints in the three patients. By using TBCS, we quickly and precisely defined eight of the 10 breakpoints.

Establishment of a novel MALT lymphoma cell line, ma‐1, from a patient with t(14;18)(q32;q21)‐positive Helicobacter Pylori‐Independent Gastric MALT Lymphoma

Although t(11;18)(q21;q21), t(1;14)(p22;q32), and a few other genetic mutations are specific markers for the Helicobacter pylori (HP)-independent status of gastric mucosa-associated lymphoid tissue (MALT) lymphoma, the molecular mechanisms responsible for HP-independence of gastric MALT lymphoma without such translocations and mutations remain uncharacterized. In the present study, we describe the establishment and characterization of a novel MALT lymphoma cell line, MA-1, which was derived from a gastric MALT lymphoma which was negative for both t(11;18)(q21;q21) and t(1;14)(p22;q32); the patient had failed HP eradication therapy and chemotherapy. The cell morphology and the immunophenotype of this cell line were similar to that of the original gastric MALT lymphoma. Comparative genomic hybridization analysis showed no significant gene copy number changes. Spectral karyotyping displayed a near-diploid chromosome content (48 < 2n>XY), with at least 13 chromosome structural abnormalities. Furthermore, fluorescence in situ hybridization analyses disclosed the existence of three sub-clones, characterized by t(14;18)(q32;q21)/IGH-BCL2, t(14;18)(q32;q21)/IGH-MALT1, and the presence of both chromosomal translocations in the same cell, respectively; whereas amplification of the genes CRAD9, TRAF2, and BCL10 were not found. In conclusion, we have established the first human gastric MALT lymphoma cell line, which is characterized by unusual and complex chromosome translocations and will be useful to explore further the molecular mechanisms of HP-independence in gastric MALT lymphoma.

Abstract 5346: Dual role of PAX3-NCOA2 in tumorigenesis of rhabdomyosarcoma

Abstract Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults, and can be classified into two major histological subtypes, embryonal RMS and alveolar RMS. Recently, two novel fusion genes, PAX3-NCOA1 and PAX3-NCOA2, were identified in the embryonal and alveolar subtypes. We previously analyzed the complex chromosomal translocation in one case with embryonal RMS by means of spectral karyotyping (SKY) and identified a translocation involving chromosome band 2q35, which is the locus of the PAX3 gene (Hosoi H, Kakazu N, et al. Cancer Genetics and Cytogenetics 189 (2009) 98-104). The patient is alive and in remission at nine years after treatment. We later identified a PAX3 partner gene as NCOA2 using fluorescence in situ hybridization (FISH) and cDNA sequence analysis. Because the role of PAX3-NCOA2 in rhabdomyosarcoma tumorigenesis is unknown, we investigated the biological function of PAX3-NCOA2 in this study. Methods: Mouse myoblast C2C12 cells and human embryonic kidney HEK293 cells were cultured in DMEM containing 10% fetal bovine serum. HEK293 cells were transfected with GFP-PAX3-NCOA2 expression vector and the fusion protein was localized by confocal microscopy. C2C12 cell lines expressing wild type PAX3, PAX3-FOXO1, PAX3-NCOA2 and a C-terminal activation domain deletion mutant of PAX3 were established using a murine stem cell virus (MSCV) retrovirus expression system. Anchorage-independent growth was assessed using soft agar colony formation. Myosin Heavy Chain (MHC), a marker of fully differentiated myocytes, was identified immunohistochemically. Myogenic differentiation was induced by switching the medium to DMEM containing 2% horse serum. Results and Discussion: In transfected HEK293 cells, GFP-PAX3-NCOA2 was observed in the nucleus, consistent with the hypothesis that PAX3-NCOA2 is involved in transcriptional regulation. Expression of PAX3-NCOA2 protein promoted anchorage-independent growth in C2C12 myoblasts. The number of colonies of the PAX3-NCOA2 stable cell line was half the number of colonies of the PAX3-FOXO1 stable cell line. Negligible colony formation was observed in wild-type PAX3 and PAX3 deletion mutant stable cell lines. Expression of PAX3-NCOA2, wild-type PAX3 and PAX3-FOXO1 each blocked the fusion of myoblasts to myotubes in differentiation medium. MHC was not detected in any of the PAX3-NCOA2, wild-type PAX3 or PAX3-FOXO1 stable cell lines. The PAX3-NCOA2 and PAX3-FOXO1 stable cell lines had the comparable phenotype. However, the finding that the PAX3-NCOA2 stable cell line produced fewer colonies than the PAX3-FOXO1 stable cell line may indicate that RMS with PAX3-NCOA2 fusion gene has a less aggressive phenotype and a better prognosis. Conclusions: The PAX3-NCOA2 fusion gene promoted anchorage-independent growth and inhibited myogenic differentiation in mouse myoblasts. These data suggest that PAX3-NCOA2 has a dual role in the tumorigenesis of RMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2011-5346

Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells

AbstractReprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using nonintegrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non–B- or non–T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods.

The RAG2 C terminus suppresses genomic instability and lymphomagenesis

Misrepair of DNA double-strand breaks (DSBs) produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans1 and in mice2–7. Defects in DNA damage response factors such as ATM and combined deficiencies in classical nonhomologous end joining (NHEJ) and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis2–11. Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical NHEJ for proper repair12,13, roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 C-terminus, although dispensable for recombination14,15, is critical for maintaining genomic stability. Thymocytes from “core” Rag2 homozygotes (Rag2c/c mice) show dramatic disruption of Tcrα/δ locus integrity. Furthermore, all Rag2c/c p53−/− mice, unlike Rag1c/c p53−/− and p53−/− animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications, and deletions involving the Tcrα/δ and Igh loci. We also find these features in lymphomas from Atm−/− mice. We show that, like ATM-deficiency3, core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar “end release/end persistence” mechanisms underlie genomic instability and lymphomagenesis in Rag2c/c p53−/− and Atm−/− mice.

Familial Kleefstra syndrome due to maternal somatic mosaicism for interstitial 9q34.3 microdeletions

The Kleefstra syndrome (Online Mendelian Inheritance in Man 607001) is caused by a submicroscopic 9q34.3 deletion or by intragenic euchromatin histone methyl transferase 1 (EHMT1) mutations. So far only de novo occurrence of mutations has been reported, whereas 9q34.3 deletions can be either de novo or caused by complex chromosomal rearrangements or translocations. Here we give the first descriptions of affected parent-to-child transmission of Kleefstra syndrome caused by small interstitial deletions, approximately 200 kb, involving part of the EHMT1 gene. Additional genome-wide array studies in the parents showed the presence of similar deletions in both mothers who only had mild learning difficulties and minor facial characteristics suggesting either variable clinical expression or somatic mosaicism for these deletions. Further studies showed only one of the maternal deletions resulted in significantly quantitative differences in signal intensity on the array between the mother and her child. But by investigating different tissues with additional fluorescent in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) analyses, we confirmed somatic mosaicism in both mothers. Careful clinical and cytogenetic assessments of parents of an affected proband with an (interstitial) 9q34.3 microdeletion are merited for accurate estimation of recurrence risk.

Cytogenetic analysis in a case of multiple deformities with complex chromosome translocations t(3;12;15)

染色体畸变是导致自然流产、死胎和畸形儿的重要病因之一[1].细胞遗传学检查发现,最常见的染色体结构变异包括缺失(deletion)、重复(duplication)、倒位(inversion)和易位(translocation).本研究报道1例多发畸形患儿复杂易位核型分析。

CpG Oligonucleotide Combined with Interleukin-2 Reveals Unexpected Chromosomal Lesions In B-CLL

AbstractAbstract 2411In B-CLL the precise definition of the chromosomal pattern has always been a very difficult goal as leukemic cells poorly respond to the traditionally used set of mitogens. Thus, conventional cytogenetic studies (CC) have always been replaced by interphase FISH (iFISH) analyses. However, recent studies have demonstrated that the CpG oligonucleotide plus interleukin-2 (ODN+IL-2) stimulation can effectively increase the detection rate of chromosomal abnormalities by inducing metaphase spreads from B-CLL leukemic cells. Based on these data, we decided to compare the results obtained by the ODN+IL2 combination, the pokeweed mitogen (PKWM) stimulation and iFISH in ninety-one B-CLL patients diagnosed at our Institution between January 2007 and July 2010. In addition, we evaluated any possible correlation with other prognostic markers and clinical parameters. There were thirty-eight females and fifty-three males with a median age of 64 years (range 42–83). According to Binet, sixty-three patients (69.2%) were considered as stage A, seventeen (18.7%) as stage B and eleven (12.1%) as stage C. PKWM did not provide any mitotic figure in thirteen patients (14.2%) and detected clonal defects in sixteen patients (17.5%). In contrast, the ODN+IL-2 combination was unable to provide metaphase spreads in three patients only (3.3%) and revealed clonal abnormalities in fifty-six patients (61.5%). In eight patients both cultures revealed an equal percentage of cells carrying the same abnormality. In addition, the ODN+IL-2 combination revealed a complex karyotype (≥ three defects) in twelve patients (13.2%), two chromosomal defects in fourteen patients (15.4%)(including five with band 17p13 rearrangement and three with band 11q13 rearrangement) and a single chromosomal defect in thirty patients (32.9%). Fifteen patients harboured various chromosomal rearrangements (16.4%). iFISH was carried out with the B-CLL FISH probe panel (Vysis, Downers Grove, IL, USA) and revealed clonal abnormalities in sixty-two patients (68.1%). The most common defects were: 13q-, +12, 11q- and 17p- having an incidence of 46.1%, 14.2%, 7.7% and 6.5%. Three patients with a normal FISH pattern presented a +12 when analysed with the ODN+IL-2 combination and five, who showed the loss of one p53 signal on iFISH, presented a structural 17p13 defect when investigated with the ODN+IL2 combination. In addition, considering the forty-two patients who harboured a 13q- on iFISH analyses, the ODN+IL2 combination revealed that in five patients this defect was included in a complex karyotype. From a clinical point of view, considering the fifteen patients with various chromosomal rearrangements nine were classified as stage A, four as stage B and two as stage C and considering the twelve patients with a complex karyotype three were considered as stage B and nine as stage C. In conclusion, the ODN+IL2 combination i) allows a precise definition of the chromosomal pattern in B-CLL patients and in our series identified clonal defects in 61% of patients; ii) reveals minor +12 clonal cell populations which may evade iFISH identification; iii) does not always identify a 13q deletion because of the sub-microscopic nature of this chromosomal defect; iv) reveals that the loss of one p53 signal on iFISH analyses is frequently due to an unbalanced rearrangement; iv) is the only technique which allows the identification of complex karyotypes and/or chromosomal translocations both associated with an advanced Binet stage. Thus, the ODN+IL2 combination should be used in conjunction with iFISH to achieve a more accurate karyotype definition in B-CLL patients. Disclosures:No relevant conflicts of interest to declare.

Formation of Complex and Unstable Chromosomal Translocations in Yeast

Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom's-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic genome instability, especially the formation of translocations and the sources of intraclonal heterogeneity, both of which are often associated with human cancers.

Genetic analysis of a complex chromosome rearrangement involving two chromosomes and four breakpoints in an azoospermic man

To perform genetic analysis of a complex chromosome rearrangement (CCR) 46,XY, t(3;11)(q27; q13), ins(11;3)(q13;p26p13) in an azoospermic man. Peripheral blood lymphocytes we re obtained for karyotyping, and metaphases were studied by multicolor fluorescence in situ hybridization procedure, Y chromosomal microdeletions in the azoospermia factor (AZF) region were analyzed with multiplex polymerase chain reaction. The case was a complex chromosomal translocation between chromosomes 3 and 11 with four breakpoints, and accompanied with a band of chromosome 3 inserting into chromosome 11. No Y-chromosome microdeletions were identified at 6 STS sequences of the AZF loci. CCR can have a significant impact on male fertility. Molecular cytogenetic techniques may contribute to improving and personalizing reproductive counseling.

A novel PAX3 rearrangement in embryonal rhabdomyosarcoma

Rhabdomyosarcoma is the most common soft tissue tumor seen in children and young adults, and it can be classified into 2 major histological subtypes, alveolar and embryonal. In the alveolar subtype, 2 recurrent chromosomal translocations, t(2;13)(q35;q14) and its variant t(1;13)(p36;q14), have been identified as the specific cytogenetic abnormalities. These translocations produce the PAX3-FOXO1 and PAX7-FOXO1 fusion genes, respectively. In the embryonal subtype, however, no recurrent chromosomal abnormalities have been identified. In this study, we analyzed the complex chromosomal translocation in one case with embryonal rhabdomyosarcoma by means of spectral karyotyping (SKY) and identified a novel translocation involving chromosome band 2q35, which is the locus of PAX3 gene. Furthermore, we identified the novel PAX3 rearrangement using fluorescence in situ hybridization (FISH) analysis. Additional identification of the partner gene may help disclose the molecular mechanism of the development of this embryonal subtype.

A novel five-way chromosomal translocation observed in chronic myelogenous leukemia

Most patients with chronic myelogenous leukemia (CML) show a Philadelphia (Ph) chromosome with a characteristic translocation between chromosomes 9 and 22. However, there are variant complex translocations involving other chromosomes in addition to the standard translocation. We describe a case of CML showing a complex and novel chromosomal translocation involving five chromosomes, t(4;12;7;9;22). ©2008 Elsevier Inc. All rights reserved.

Dairy breed bull with complex chromosome translocation: fertility and linkage studies.

A chromosome study was performed on the offspring of an American Brown Swiss bull, which was carrier of a complex chromosome translocation. A total of 10 phenotypically normal calves were examined and a similar complex translocation was detected in five calves. The translocation involved identifiable parts of chromosome number one and eight. Furthermore, the terminal band of chromosome one had been translocated to an unidentified chromosome. One of the calves also carried a 1/29 centric fusion chromosome. Pedigree and chromosome analysis showed that the centric fusion originated from another American Brown Swiss bull. A linkage study was performed and an indication of linkage between the Z blood group system and the complex translocation was found. The fertility of the bull was studied, and it was found to have a severely reduced fertility mainly due to an increased rate of early embryonic mortality.

Further delineation of complex chromosomal rearrangements in fertile male using multicolor banding

BackgroundComplex chromosomal rearrangements (CCRs) are defined as structural chromosomal rearrangements with at least three breakpoints and exchange of genetic material between two or more chromosomes. Complex chromosomal translocations are rarely seen in the general population but the frequency of occurrence is anticipated to be much higher due balanced states with no phenotypic presentation. Here, we report a severely mentally retarded fertile male patient in whom further delineation of CCR involving chromosomes 1, 4 and 2 was carried out by using high resolution multicolor banding (MCB) technique. As a FISH based novel chromosome banding approach, high resolution MCB allows for the differentiation of chromosome region specific areas at band and subband levels.ResultsCytogenetic studies using high resolution banding of the proband necessitated further delineation of the breakpoints because of their uncertainty: 46,XY,t(1;4;2)(p21~31;q31.3;q31). After using high resolution MCB based on microdissection derived region-specific libraries, the exact nature of chromosomal rearrangements for chromosomes 1, 2 and 4 were revealed and these breakpoints were located on 1p31.1, 1q24.3 and 4q31.3 giving rise to a balanced situation.ConclusionFurther delineations are certainly required to provide detailed information about the relationship between balanced CCRs and their phenotypes in order to offer proper counseling to the families concerned. Carriers must be investigated with high resolution banding and molecular cytogenetic techniques to determine the exact locations of the breakpoints. High resolution MCB is an alternative and an efficient method to other FISH based chromosome banding techniques and can serve in clarifying the nature of CCR.

Loss or Mutation of TP53 Is Highly Associated with Complex Aberrant Karyotype and Chromosomal Translocations in Chronic Lymphocytic Leukemia.

In chronic lymphocytic leukemia (CLL) cytogenetic aberrations such as del(17p) and del(11q) predict inferior outcome. In addition, complex aberrant karyotypes as well as chromosomal translocations as defined by metaphase cytogenetics were suggested as poor prognostic markers for overall survival. We screened 194 consecutive CLL patients for del(17p)/TP53-deletion by fluorescence in situ hybridization (FISH) and for TP53-mutations by denaturing high performance liquid chromatography (DHPLC) and subsequent direct sequencing of aberrant fragments. In addition 160 of these CLL patients were analyzed by classical metaphase cytogenetics to determine the incidence of TP53-aberration in different cytogenetic subgroups. Interphase FISH on 194 samples detected TP53-deletions in 9.3% (n=18) of cases. In parallel, exons 3–9 of the TP53 gene were screened by DHPLC and an aberrant pattern was detected in 9.8% (n=19) of cases. TP53-mutations were confirmed and further characterized by direct sequencing in 16 of the 19 cases. The residual 3 samples had an aberrant pattern in DHPLC for the amplicon of exons 8–9 which pointed to a small population of TP53-aberrant cells which was beyond the detection limit of sequencing. 16 of 18 (89%) cases with TP53-deletion were accompanied by a TP53-mutation affecting the residual allele. 3 samples with TP53-mutations had no deletion of one TP53 allele. Therefore, the overall incidence of TP53-aberrations was 10.8 % (21/194) with a significant association of TP53-deletion and TP53-mutation (p<0.0001). Metaphase cytogenetics was performed on 160 CLL samples. A complex aberrant karyotype defined by ≥ 3 aberrations was identified in 14% of samples (22/160). The incidence of TP53-aberrations in this cytogenetic subgroup was 50% (11/22) and therefore significantly higher than in other cytogenetic subgroups (p<0.0001). Among 160 samples with cytogenetic analysis 49 (31%) exhibited translocations. We divided these translocations into subgroups with karyotypes carrying balanced translocations only (n=18), carrying unbalanced translocations only (n=20) as well as karyotypes with both balanced and unbalanced translocations (n=11). Within the entire group of translocations the incidence of TP53-aberration was 27% (13/49). The incidence of TP53-aberrations was 5.5% (1/18) in the group with only balanced translocations, 40% (8/20) in the group with only unbalanced translocations and 36% (4/11) where balanced and unbalanced translocation occurred in combination. When the latter two groups with unbalanced translocations were combined TP53-aberration occurred in 39% (12/31) of cases. Altogether the association of TP53-aberration with translocations was strong (p<0.0001) especially with unbalanced translocations (p<0.0001) whereas no coherency with balanced translocations could be demonstrated (p>0.05). Furthermore, translocations were detected in 91% (20/22) and unbalanced translocations in 82% (18/22) of complex karyotypes. The association of translocations, in particular unbalanced translocation with complex aberrant karyotype was significant (for both p<0.0001).In conclusion:Loss of TP53 and TP53 mutations occur with a frequency of 9.3% and 9.8%, respectively and are significantly associated.A highly significant association of TP53-aberrations with complex aberrant karyotypes and unbalanced translocations was observed.We hypothesize that TP53-aberrations might contribute to genetic instability leading to accumulation of cytogenetic aberrations especially unbalanced translocations.

Complex chromosome aberrations persist in individuals many years after occupational exposure to densely ionizing radiation: An mFISH study

Long-lived, sensitive, and specific biomarkers of particular mutagenic agents are much sought after and potentially have broad applications in the fields of cancer biology, epidemiology, and prevention. Many clastogens induce a spectrum of chromosome aberrations, and some of them can be exploited as biomarkers of exposure. Densely ionizing radiation, for example, alpha particle radiation (from radon or plutonium) and neutron radiation, preferentially induces complex chromosome aberrations, which can be detected by the 24-color multifluor fluorescence in situ hybridization (mFISH) technique. We report the detection and quantification of stable complex chromosome aberrations in lymphocytes of healthy former nuclear-weapons workers, who were exposed many years ago to plutonium, gamma rays, or both, at the Mayak weapons complex in Russia. We analyzed peripheral-blood lymphocytes from these individuals for the presence of persistent complex chromosome aberrations. A significantly elevated frequency of complex chromosome translocations was detected in the highly exposed plutonium workers but not in the group exposed only to high doses of gamma radiation. No such differences were found for simple chromosomal aberrations. The results suggest that stable complex chromosomal translocations represent a long-lived, quantitative, low-background biomarker of densely ionizing radiation for human populations exposed many years ago.

Clinical and cytogenetic features of 508 Chinese patients with myelodysplastic syndrome and comparison with those in Western countries

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and leukemia progression. Racial differences may exist on clinical pictures and the molecular events leading to MDS, which are heterogeneous. To better define the clinical and cytogenetic features in Chinese patients, a retrospective multicentric study was performed in 508 MDS cases. Compared with Western countries, Chinese patients showed younger age (median: 49 vs 65-73 years), lower percentages of RARS (2.8 vs 6.6-15.3%), and CMML (5.2 vs 11.7-30.6%). Cytogenetically, among 367 cases with evaluable data, abnormal karyotypes were found in 136 cases, including 56 numerical and 80 structural changes. Incidences of single chromosome 5 and 7 abnormalities were lower than those in Western countries (2.2 vs 17.8-42.5%). However, complex cytogenetic aberrations and chromosome translocations were frequently observed and related to poor prognosis. Both multiple chromosome deletions and translocations were detected in advanced subtypes (RAEB and RAEB-T). Analysis of 200 cases revealed a higher incidence of hepatitis-B-virus infection than that in non-MDS population (21.00 vs 9.75%). This study further confirmed: (1) different genetic/environmental backgrounds between Asian and Western MDS populations; (2) a strong predictive value of cytogenetic abnormalities on disease outcome and involvement of genomic instability in leukemia clone development.

Variant t(14;18) in malignant lymphoma: a report of seven cases

The BCL2 gene was identified through molecular analysis of the breakpoints involved in the t(14;18)(q32;q21) found in the majority of follicular lymphomas (FL). Variant translocations leading to juxtaposing of the BCL2 with either the IGK or IGL gene have been recognized in B-cell malignant lymphoma, although they are rare. We identified seven lymphoma cases that had variant translocations. Three cases had simple translocations involving two chromosomal regions: t(18;22)(q21;q11.2) in two cases and t(2;18)(p11.2;q21) in the third case. Complex translocations affecting more than two chromosomes were seen in the remaining four cases. Fluorescence in situ hybridization using the LSI IGH/BCL2 DNA probes revealed rearrangements of the BCL2 gene locus in all cases. In addition, expression of BCL2 protein was seen in all cases; only five of the seven cases expressed BCL6 protein. Morphologically, the lymphomas were categorized as B-cell follicular lymphoma in six cases and in the seventh case as diffuse large cell lymphoma (Richter syndrome) transformed from preexisting chronic lymphocytic leukemia (CLL). In case 2, the variant t(18;22) was seen as a secondary aberration evolving from a trisomy 12 clone. The findings revealed that BCL2 rearrangements in some malignant lymphomas occur through variant simple or complex chromosomal translocations, but always involving the IGH, IGK, or IGL chromosomal site. In addition, fluorescence in situ hybridization proved to be an important tool in evaluating these cases by showing IGH/BCL2 gene fusion or repositioning of the BCL2 gene.

Cytogenetic Abnormalities Predict Disease Progression in Primary Myelodysplastic Syndrome: A Multicentric Study in 508 Chinese Patients.

Myelodysplastic syndrome (MDS) is a clonal stem cell disorder characterized by ineffective hematopoiesis and frequent leukemia progression. Cytogenetic abnormalities are presented in MDS patients and predict poor prognosis. To better define their relevance to clinical outcome in Chinese patients with primary MDS, cytogenetic analysis was performed in 508 cases and retrospectively compared with clinical data. The median age was 49 years which was 10 years younger than that of western patients. The median overall survival was 34 (range 1–161) months, with the 2-year and 5-year survival rate of 50.0% and 15.0%, respectively. Among 367 cases with evaluable karyotype in bone marrow mononuclear cells, cytogenetic abnormalities were found in 136 of them (37.1%), including 56 cases (15.3%) of numeric abnormality and 80 cases (21.8%) of structural abnormality. Higher incidence of cytogenetic abnormality was found in RAEB-T (43/86, 50.0%) and RAEB (44/106, 41.5%) patients than that of RA patients (38/143, 26.6%). RAEB and RAEB-T had more cases of non-hypodipoidy, double aberrations with translocation, and complex aberrations. In RA, the most frequent aberrations were duplication and numeric changes. This study also showed RAEB appears to be the course of MDS that genome turbulence was under the evolution pressure. In RAEB-T, the genome abnormalities tend to be uniform and behaved as special translocation, which is in accordance with the clinical behavior of AL. Poor prognosis and early leukemia transformation were observed in the patients with cytogenetic abnormalities, especially with complex cytogenetic aberrations and chromosome translocations. In conclusion, cytogenetic abnormalities possess strong predictive value on disease outcome in primary MDS patients.

A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase.

A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.