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In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients., (© 2014 by The American Society of Hematology.)

We report here the case of a sixty-eight-year old woman with chronic myeloid leukemia. Molecular techniques identified the presence of the rare e19a2 BCR-ABL1 transcript. The patient was treated by 1(st) generation tyrosine-kinase inhibitor (TKI) (imatinib). Disease monitoring was performed by cytogenetic analyses and quantification of the BCR-ABL1 transcript. After 3 months, the treatment was modified due to an absence of biological response and poor tolerance. After 21 months with 2nd generation TKIs (nilotinib), the patient was responding optimally to treatment, with a complete cytogenetic response and a major molecular response. This observation emphasizes the importance of determining the chromosomal breakpoints at diagnosis to enable adequate molecular monitoring of residual disease. Careful monitoring of minimal residual disease is important to thoroughly assess the response to treatment, detect resistance and adapt the therapeutic strategy. The kinetics and the depth of the response to TKI also represent major prognostic factors. Molecular monitoring is performed using real-time quantitative PCR, which has to be adapted to each type of transcript. For rare BCR-ABL1 transcripts, an international standardization, as it is developed for conventional transcripts, is lacking. Yet, such a harmonization would be useful to assess in an optimal and large scale way the response to TKI in these patients, and to determine what the best management is.

The significance of chromosomal translocations (CTRAs) and karyotype complexity (KC) in chronic lymphocytic leukemia (CLL) remains uncertain. To gain insight into these issues, we evaluated a series of 1001 CLL cases with reliable classic cytogenetic data obtained within 6 months from diagnosis before any treatment. Overall, 320 cases were found to carry ≥ 1 CTRAs. The most frequent chromosome breakpoints were 13q, followed by 14q, 18q, 17q, and 17p; notably, CTRAs involving chromosome 13q showed a wide spectrum of translocation partners. KC (≥ 3 aberrations) was detected in 157 cases and significantly (P < 0.005) associated with unmutated IGHV genes and aberrations of chromosome 17p. Furthermore, it was identified as an independent prognostic factor for shorter time-to-first-treatment. CTRAs were assigned to two categories (i) CTRAs present in the context of KC, often with involvement of chromosome 17p aberrations, occurring mostly in CLL with unmutated IGHV genes; in such cases, we found that KC rather than the presence of CTRAs per se negatively impacts on survival; (ii) CTRAs in cases without KC, having limited if any impact on survival. On this evidence, we propose that all CTRAs in CLL are not equivalent but rather develop by different processes and are associated with distinct clonal behavior., (Copyright © 2013 Wiley Periodicals, Inc.)

Whether sex chromosome loss (SCL) is an age-related phenomenon or a cytogenetic marker of hematological disease is unclear. To address this issue in chronic lymphocytic leukemia (CLL), we investigated 20 cases with X or Y chromosome loss detected by conventional cytogenetics (CC). The frequency of SCL was low in CLL (2.3%). It was the sole abnormality, as detected by CC, in 10/20 (50%) patients. Fluorescence in situ hybridization (FISH) analyses confirmed SCL in all patients tested, present in 5-88% of cells (median: 68%). Deletions of 13q were observed by FISH in 16/20 (80%) patients. Compared with CLL without SCL, SCL was significantly associated with 13q deletion, especially when bi-allelic (P = 0.04). Co-hybridization analyses showed that SCL could be a concomitant, primary or secondary change, or be present in an independent clone. FISH analyses were performed on blood sub-populations isolated by Ficoll or flow cytometry. Comparing mononuclear cells (including CLL cells) and polynuclear cells separated by Ficoll, a maximum of 2% of polynuclear cells were found with SCL, whereas mononuclear cells exhibited a significantly higher loss frequency (range: 6-87%) (P = 0.03). Comparing B-cells (including CLL cells) and T-cells sorted by flow cytometry, the proportion of B-CD19+ cells with SCL was significantly higher (range: 88-96%) than that observed in T-CD3+ cells (range: 2-6%) (P = 0.008). We conclude that SCL has to be considered as a clonal aberration in CLL that may participate in the oncogenic process., (Copyright © 2013 Wiley Periodicals, Inc.)

Chromosomal translocations involving the immunoglobulin heavy chain locus (IGH@) are recurrent but rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and various partner genes have been described. Here, we report a new series of 29 cases of BCP-ALL with IGH@ translocations. The partner gene was identified by fluorescence in situ hybridization and/or molecular cloning in 20 patients. Members of the CEBP gene family (n = 11), BCL2 (n = 3), ID4 (n = 3), EPOR (n = 2), and TRA/D@ (n = 1) were identified and demonstrated by quantitative real-time reverse transcriptase-polymerase chain reaction to be markedly up-regulated. The present cases, added to those already reported, confirm the diversity of the partner genes, which, apart from BCL2, are specific to BCP-ALL. Collectively, patients with IGH@ translocations may represent a novel sub-group of BCP-ALL occurring in adolescents and young adults., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Waldenström's macroglobulinemia is a disease of mature B cells, the genetic basis of which is poorly understood. Few recurrent chromosomal abnormalities have been reported, and their prognostic value is not known. We conducted a prospective cytogenetic study of Waldenström's macroglobulinemia and examined the prognostic value of chromosomal aberrations in an international randomized trial. The main aberrations were 6q deletions (30%), trisomy 18 (15%), 13q deletions (13%), 17p (TP53) deletions (8%), trisomy 4 (8%), and 11q (ATM) deletions (7%). There was a significant association between trisomy of chromosome 4 and trisomy of chromosome 18. Translocations involving the IGH genes were rare (<5%). Deletion of 6q and 11q, and trisomy 4, were significantly associated with adverse clinical and biological parameters. Patients with TP53 deletion had short progression-free survival and short disease-free survival. Although rare (<5%), trisomy 12 was associated with short progression-free survival. In conclusion, the cytogenetic profile of Waldenström's macroglobulinemia appears to differ from that of other B-cell lymphomas. Chromosomal abnormalities may help with diagnosis and prognostication, in conjunction with other clinical and biological characteristics.

Translocations involving MYC are rare in chronic lymphocytic leukemia (CLL), and up to now, their prognostic significance remains unclear. We report the characteristics of 21 patients with CLL and nine patients with prolymphocytic leukemia (PLL), diagnosed in multiple centers (n = 13), which showed an MYC translocation demonstrated by fluorescence in situ hybridization. The prevalence was estimated to be <1%. Advanced age and male predominance were observed. Morphological analysis frequently revealed the presence of prolymphocytes. A typical "CLL-immunophenotype" was found in four of nine cases with PLL. Moreover, CD5 and CD23 were frequently expressed in PLL. The latter findings are atypical for PLL and may suggest transformation or progression of an underlying CLL. MYC translocations were frequently observed with concomitant adverse cytogenetic markers, such as del(11q) (n = 8/30) and/or del(17p)/monosomy 17 (n = 7/30). In addition, the presence of unbalanced translocations (n = 24 in 13/30 cases) and complex karyotype (n = 16/30) were frequent in cases with MYC translocations. Altogether, del(17p)/monosomy 17, del(11q), and/or complex karyotype were observed in 22 of 30 patients. Survival outcome was poor: the median time to treatment was only 5 months, and overall survival (OS) from clinical diagnosis and from genetic detection was 71 and 19 months, respectively. In conclusion, CLL/PLL with MYC translocations is a rare entity, which seems to be associated with adverse prognostic features and unfavorable outcome.

Loss-of-function mutations affecting one or both copies of the Ten-Eleven-translocation (TET)2 gene have been described in various human myeloid malignancies. We report that inactivation of Tet2 in mouse perturbs both early and late steps of hematopoiesis including myeloid and lymphoid differentiation in a cell-autonomous manner, endows the cells with competitive advantage, and eventually leads to the development of malignancies. We subsequently observed TET2 mutations in human lymphoid disorders. TET2 mutations could be detected in immature progenitors endowed with myeloid colony-forming potential. Our results show that the mutations present in lymphoid tumor cells may occur at both early and later steps of lymphoid development and indicate that impairment of TET2 function or/and expression predisposes to the development of hematological malignancies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Long-term responses have been reported after autologous stem cell transplantation (ASCT) for chronic lymphocytic leukemia (CLL). We conducted a prospective, randomized trial of ASCT in previously untreated CLL patients. We enrolled 241 patients < 66 years of age with Binet stage B or C CLL. They received 3 courses of mini-CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone/prednisolone) and then 3 courses of fludarabine. Patients in complete response (CR) were then randomized to ASCT or observation, whereas the other patients were randomized to dexamethasone, high-dose aracytin, cisplatin (DHAP) salvage followed by either ASCT or 3 courses of fludarabine plus cyclophosphamide (FC). The primary end point was event-free survival (EFS). After up-front treatment, 105 patients entered CR and were randomized between ASCT (n = 52) and observation (n = 53); their respective 3-year EFS rates were 79.8% and 35.5%; the adjusted hazard ratio was 0.3 (95% CI: 0.1-0.7; P = .003). Ninety-four patients who did not enter CR were randomized between ASCT (n = 46) and FC (n = 48); their respective 3-year EFS rates were 48.9% and 44.4%, respectively; the adjusted hazard ratio was 1.7 (95% CI: 0.9-3.2; P = .13). No difference in overall survival was found between the 2 response subgroups. In young CLL patients in CR, ASCT consolidation markedly delayed disease progression. No difference was observed between ASCT and FC in patients requiring DHAP salvage.

Fluorescence in situ hybridization was performed to characterize 81 cases of myeloid and lymphoid malignancies with cytogenetic 1p36 alterations not affecting the PRDM16 locus. In total, three subgroups were identified: balanced translocations (N = 27) and telomeric rearrangements (N = 15), both mainly observed in myeloid disorders; and unbalanced non-telomeric rearrangements (N = 39), mainly observed in lymphoid proliferations and frequently associated with a highly complex karyotype. The 1p36 rearrangement was isolated in 12 cases, mainly myeloid disorders. The breakpoints on 1p36 were more widely distributed than previously reported, but with identifiable rare breakpoint cluster regions, such as the TP73 locus. We also found novel partner loci on 1p36 for the known multi-partner genes HMGA2 and RUNX1. We precised the common terminal 1p36 deletion, which has been suggested to have an adverse prognosis, in B-cell lymphomas [follicular lymphomas and diffuse large B-cell lymphomas with t(14;18)(q32;q21) as well as follicular lymphomas without t(14;18)]. Intrachromosomal telomeric repetitive sequences were detected in at least half the cases of telomeric rearrangements. It is unclear how the latter rearrangements occurred and whether they represent oncogenic events or result from chromosomal instability during oncogenesis.

Background: Chromosomal translocations are usually analyzed as a single entity, and are associated with a poor outcome in chronic lymphocytic leukemia. Translocations involving immunoglobulin genes are recurrent, but uncommon (<5%), and their individual prognosis is not clear. The two most frequent partners are BCL2 (18q21) and BCL3 (19q13)., Designs and Methods: Herein, 75 cases are reported of chronic lymphocytic leukemia and t(14;18) (BCL2-CLLs). Our series benefits from morphological, immunological and cytogenetical reviews. The IGHV status analyses were performed by referring laboratories. Comparison was made with our previously published series of chronic lymphocytic leukemia patients with t(14;19) (BCL3-CLLs, n=29)., Results: Compared with BCL3-CLLs, lymphocytosis was lower in BCL2-CLLs (p<0.008), and splenomegaly was less frequent (p<0.0001). There were more "typical" morphologies (p<0.005) and Matutes scores >4 (p<0.001) in the BCL2-CLLs group, and less CD38 expression (p<0.04). More variant BCL2-translocations were observed (t(18;22), n=11; 2t(2;18), n=2; p<0.02), and BCL2-translocation was frequently single (p<0.002). Complex karyotypes (p<0.02), trisomy 12 (p<0.03), 6q deletion (p<0.002) and TP53 deletion (p<0.02) were less frequent in BCL2-CLLs, whereas 13q deletion was more frequent (p<0.005). The IGHV gene was frequently mutated in BCL2-CLLs (p<0.0001). Treatment-free survival was longer in BCL2-CLLs (p<0.0001)., Conclusions: BCL2-CLL.S express CD5 and lack expression of CD38, and have a Matutes score ≥4, frequent trisomy 12, no ATM and 6q deletions, and a mutated IGHV status. Compared to BCL3-CLLs, BCL2-CLLs are much less aggressive; indicating that identifying individual translocations and cytogenetic partners would allow improved patient stratification.

Recent developments in the management of chronic lymphocytic leukemia (CLL) patients have made necessary the availability of dependable prognostic factors. We have developed a prognostic index derived from the multivariate analysis of 339 stage A patients at diagnosis, exhaustively studied for classical and recent predictive markers. Only 4 biologic parameters were found to be independent predictors of progression-free survival (PFS): serum thymidine kinase (sTK), lymphocytosis, β2-microglobulin, and CD38 expression. Two groups were distinguishable: cases with no or 1 risk factor (among whom 85% did not progress after 7 years), and cases with 2 or more factors showing a median PFS of 20 months. Finally, we propose an easy, fast, cost-effective strategy for a trustworthy prognostication in stage A patients, who currently represent more than 80% of the CLL population, allowing physicians to adapt follow-up individually.

Background: The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both in vitro and in vivo, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes., Methods: Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent in situ labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test., Results: Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, RELB was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells., Conclusion: The molecular defects in the silencing of RELB involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic RELB silencing as a new marker of the progressive disease in males.

Evolution to myelofibrosis (MF), acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) may occur over time in myeloproliferative neoplasms (MPN) patients most likely due to the acquisition of additional mutations. The Groupe Francophone de cytogenetique hematologique (GFCH) has collected and reviewed 82 patients with transformation of MPN (66 AML/MDS and 16 MF). JAK2V617F and TET2 mutations were searched for in 40 and 32 patients, respectively. Significantly more -7/del(7q) (P = 0.004) and -5/del(5q) (P = 0.03) were found in AML/MDS with a higher incidence of dup1q (P = 0.01) in MF. Some specific chromosomal abnormalities occurred together, for example -5/del(5q) and -17/del(17p) (P = 0.0007). In multivariate analysis, two factors were independently associated with an inferior overall survival (OS); AML/MDS transformation (P < 0.0001) and -5/del(5q) abnormality (P = 0.02). Although both giving rise to loss of 7q, der(1;7) differed from other 7q deletions in terms of distribution (lower frequency of AML/MDS, P = 0.02), association with chromosomal abnormalities (absence of -5/del(5q), P = 0.003; increased del(20q), P = 0.05), and longer OS (P = 0.0007). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in samples following transformation, ranging from wild-type to mutated forms of both genes. The mutated and wild-type forms of the genes were not found to be associated with a specific chromosomal abnormality. There was no evidence that JAK2 or TET2 mutations were associated with the type of MPN transformation, whereas the type of cytogenetic abnormalities were strongly linked, perhaps indicating that they play a specific role in the transformation process.

In somatic cells, eroded telomeres can induce DNA double-strand break signaling, leading to a form of replicative senescence or apoptosis, both of which are barriers to tumorigenesis. However, cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis, enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction, and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs, 2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably, these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions, or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together, our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction.

Recurrent cytogenetic abnormalities observed in chronic lymphocytic leukaemia (CLL) are frequent. They involve the 13q, 11q, 12q, 17p and 6q chromosomal regions, by decreasing order of frequency. These abnormalities can be isolated or associated. They can also appear during the course of the disease. Some of them, as 11q and 17p deletions, give prognostic information for the management of CLL patients.

Using array-based CGH, we identified 2p gain in 22/78 (28%) untreated Binet stages B/C CLL, which was the second most frequent copy number change after 13q deletion. It never occurred as a sole abnormality and was associated with other changes (6q deletion; 1p gain). The region of 2p gain frequently included two oncogenes, REL and MYCN. All patients with gain of REL were unmutated for IGHV (p=0.03). Gain of MYCN was associated with increased mRNA expression (p=0.005), suggesting a pathogenic role for MYCN. Gain of 2p appears to be a marker of progression and may contribute to the poor prognosis., (2009 Elsevier Ltd. All rights reserved.)

Several prognostic factors can predict the rapid progression in chronic lymphocytic leukaemia (CLL), including IGHV mutational status, cytogenetic abnormalities and, more recently, LPL/ADAM29 expression. In contrast, few studies have been devoted to the influence of these factors on clinical outcome in responding patients after therapy. We here propose to analyse the impact of IGHV gene status, LPL and ADAM29 gene expression on disease-free survival (DFS) and overall survival (OS) in 41 stage B or C CLL patients in remission after oral fludarabine plus cyclophosphamide. The median follow-up was of 64 (16-74) months. Sequencing of IGHV showed mutated (M) VH genes in 16 of 41 cases and unmutated (UM) in 25 cases. Analysis of LPL and ADAM29 expression in 35 of 41 cases showed overexpression of ADAM29 in 17 cases (14 M and three UM) and LPL in 18 cases (all UM). Patients expressing UM IGHV and LPL had shorter DFS and OS when compared to patients expressing M IGHV and/or ADAM29. Furthermore, blood minimal residual disease (MRD) evaluation using four-colour flow cytometry was performed in 33 out the 41 patients. We showed that patients who achieved phenotypic remission displayed longer DFS than those with MRD(+). Our results support the use of LPL and ADAM29 gene expression associated to IGHV mutational status for predicting the clinical outcome of patients treated by oral fludarabine + cyclophosphamide and could be considered for treatment strategies.

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

Waldenström macroglobulinemia (WM) is now defined as an uncommon lymphoplasmocytic proliferation associated with an immunoglobulin M peak. The associated chromosomal abnormalities are not specific to the disease, and changes in the diagnostic criteria and techniques used as well as low-level abnormal cell proliferation made their analysis difficult. A literature review however, shows that if specific abnormalities were not recognized until now, it is the frequency of some chromosomal abnormalities (for instance partial deletion of the long arm of chromosome 6 and trisomy 4) that distinguishes WM from other chronic malignant B-cell proliferations. The data collected in the present review show directions for future research which will benefit from use of more recent techniques such as fluorescent in situ hybridization, comparative genomic hybridization and expression microarrays.

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse-polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5' or 3' of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)-PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.

Subtle variation in the expression or function of a small group of transcription factors can drive leukemogenesis. The CEBPA protein is known to regulate the balance between cell proliferation and differentiation during early hematopoietic development and myeloid differentiation. In human myeloid leukemia, CEBPA is frequently inactivated by mutation and indirect and posttranslational mechanisms, in keeping with tumor suppressor properties. We report that CEBPA is activated by juxtaposition to the immunoglobulin gene enhancer upon its rearrangement with the immunoglobulin heavy-chain locus in precursor B-cell acute lymphoblastic leukemia harboring t(14;19)(q32;q13). Overexpression of apparently normal CEBPA RNA or protein was observed in 6 patients. These data indicate that CEBPA may exhibit oncogenic as well as tumor suppressor properties in human leukemogenesis.

Objectives: The t(12;14)(p13;q11)--a recurrent translocation in childhood T-cell acute lymphoblastic leukemia (T-ALL)--has very recently been molecularly characterized in one case, which displayed overexpression of the cyclin D2 gene (CCND2)., Patients and Methods: We have characterized two pediatric t(12;14)-positive T-ALLs using fluorescence in situ hybridization (FISH), cDNA microarray, and real-time polymerase chain reaction (PCR)., Results: FISH revealed breakpoints (BPs) in the T-cell receptor alpha/delta locus (14q11) and in the vicinity of the CCND2 gene at 12p13. To investigate the expression of genes in 12p13, cDNA microarray analysis was performed. Expression data for eight genes, including CCND2, surrounding the 12p BP were compared with those in other T-ALLs. The t(12;14)-positive T-ALL displayed an increased expression of CCND2 compared to the controls, whereas the expression of the other genes was similar in all T-ALLs. Expression of CCND2 and two additional genes (PARP11 and FGF23), close to the 12p BP, was investigated with real-time PCR of the two t(12;14)-positive cases and four controls. Neither PARP11 nor FGF23 displayed expression differences among the T-ALLs, whereas CCND2 was clearly overexpressed in both t(12;14)-positive cases as compared to the mean expression level in the controls., Conclusion: We have confirmed, in two additional cases, that the recurrent T-ALL-associated t(12;14) results in overexpression of cyclin D2. The t(12;14) is the first neoplasia-associated translocation shown to result in overexpression of cyclin D2. Furthermore, it is the first example of a T-cell neoplasm with a targeted deregulation of a member of a cyclin-encoding gene family.

Double minutes (dmin)-circular, extra-chromosomal amplifications of specific acentric DNA fragments-are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in approximately 1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of approximately 500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the 'episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplifications.

Burkitt-type acute leukemia cells were present in the bone marrow of a patient with B-prolymphocytic leukemia diagnosed from peripheral blood cell morphology. Immunophenotype analysis confirmed morphological patterns. Cytogenetic and fluorescence in situ hybridization (FISH) analysis showed an identical t(8;22)(q24;q21) with MYC locus rearrangement in blood and bone marrow cells, with additional chromosome abnormalities in the bone marrow. In addition, the loss of one copy of the TP53 gene and identical IGH DNA clonal rearrangements were shown with FISH and polymerase chain reaction analysis respectively in the two types of leukemic cells. These data indicated the common origin of the two coexisting leukemias and are the first example of such occurrence in a leukemic patient.

A systematic cytogenetic study was performed in 49 patients with polycythemia vera (PV) in order to investigate the occurrence of subtelomeric rearrangements of chromosome 20, the most frequently rearranged chromosome in this myeloproliferative disorder. Partial deletion of the long arm of chromosome 20 was observed in two patients and two cryptic translocations, t(1;20)(p36;q13) and t(18;20)(p11;q13) in two others, all previously treated. The localization of the breakpoints of the translocated 20 chromosomes was different in the two translocations, as shown by fluorescence in situ hybridization (FISH) to metaphase chromosomes using BAC clones. Although infrequent (2/49), cryptic translocations of chromosome 20 deserve to be detected as preliminary to identification of molecular defects in PV.

A case of de novo acute myeloblastic leukemia (AML) M2, with trisomy 4 and double minute (dmin) chromosomes is reported. Amplification of the MYC gene ascertained by FISH was associated with dmin. A review of the literature of trisomy 4-dmin-associated AML shows that this entity preferentially occurs in elderly women and is not always associated with previously identified exposition to mutagens.

The improvement of molecular biology techniques and human genome mapping and sequencing boosted the molecular analysis of chromosomal abnormalities observed in human hematological malignancies. The characterization of structural abnormalities (translocation, deletion) has proven particularly seminal. A better understanding of the pathology itself and of its generation arose from the identification of the genes involved in the chromosomal translocations of human leukemia. This work summaries some of the present knowledge regarding human leukemogenesis.

Two patients with Ph-positive chronic myelocytic leukemia in erythroblastic transformation and rearrangement of the short arm of chromosome 18 are reported. Fluorescence in situ hybridization studies showed that the 18p rearrangement resulted from translocation of the main part of chromosome 22 long arm to 18p, including BCR-ABL1 fusion. The 18p abnormality resulted, thus, in loss of 18p and duplication of BCR-ABL1 in both patients. The possible relation to the erythroblastic type of blastic phase is briefly discussed. In addition an apparently intact germline ABL1 gene was duplicated and inserted into chromosome 6 at band p21 in one of these patients.

Translocation t(1;22)(p13;q13) is associated with a peculiar subtype of acute megakaryocytic leukemia (M7) occurring in infants. We have recently characterized a fusion gene, OTT-MAL, resulting from this translocation. We now report three additional cases and show that this gene fusion is present in all five t(1;22) cases studied to date. Nucleotide sequence analysis of two translocation breakpoints suggests a nonhomologous end joining mechanism in the genesis of this translocation and reveals a noncanonical topoisomerase II-like consensus sequence within the OTT gene. FISH and PCR techniques described in this work are useful for identifying t(1;22) associated with M7.

Fluorescence in situ hybridization (FISH) studies were performed in three cases of acute lymphoblastic leukemia (ALL) with marker chromosomes to analyze the contribution of chromosome 21 in these markers. FISH with a chromosome 21 painting probe confirmed that chromosome 21 was involved in all three cases. FISH with YAC probes showed that the number of extra copies varied according to their location on chromosome 21. Attention was focused on the AML1 gene, which was present as five copies in most of the cells exhibiting the marker chromosomes. As controls, 11 cases of childhood ALL were studied with PAC probes covering AML1. The results agreed with the banded karyotypes in 10 patients. FISH uncovered a clone with four copies of AML1 which were only observed by FISH analysis of interphase nuclei in one patient. No point mutation was detected in exons 3-5, encoding the runt domain of AML1, in the three cases, suggesting an oncogenic role of wild-type AML1 amplification., (Copyright 2001 Wiley-Liss, Inc.)

The recurrent t(1;22)(p13;q13) translocation is exclusively associated with infant acute megakaryoblastic leukemia. We have identified the two genes involved in this translocation. Both genes possess related sequences in the Drosophila genome. The chromosome 22 gene (megakaryocytic acute leukemia, MAL) product is predicted to be involved in chromatin organization, and the chromosome 1 gene (one twenty-two, OTT) product is related to the Drosophila split-end (spen) family of proteins. Drosophila genetic experiments identified spen as involved in connecting the Raf and Hox pathways. Because almost all of the sequences and all of the identified domains of both OTT and MAL proteins are included in the predicted fusion protein, the OTT-MAL fusion could aberrantly modulate chromatin organization, Hox differentiation pathways, or extracellular signaling.