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ABSTRACT: Chronic myeloid leukemia in chronic phase (CML-CP) cells that harbor oncogenic BCR-ABL1 and normal ABL1 allele often become resistant to the ABL1 kinase inhibitor imatinib. Here, we report that loss of the remaining normal ABL1 allele in these tumors, which results from cryptic interstitial deletion in 9q34 in patients who did not achieve a complete cytogenetic remission (CCyR) during treatment, engenders a novel unexpected mechanism of imatinib resistance. BCR-ABL1-positive Abl1(-/-) leukemia cells were refractory to imatinib as indicated by persistent BCR-ABL1-mediated tyrosine phosphorylation, lack of BCR-ABL1 protein degradation, increased cell survival, and clonogenic activity. Expression of ABL1 kinase, but not a kinase-dead mutant, restored the antileukemic effects of imatinib in ABL1-negative chronic myelogenous leukemia (CML) cells and in BCR-ABL1-positive Abl1(-/-) murine leukemia cells. The intracellular concentration of imatinib and expression of its transporters were not affected, although proteins involved in BCR-ABL1 degradation were downregulated in Abl1(-/-) cells. Furthermore, 12 genes associated with imatinib resistance were favorably deregulated in Abl1(-/-) leukemia. Taken together, our results indicate that loss of the normal ABL1 kinase may serve as a key prognostic factor that exerts major impact on CML treatment outcomes.
Cancer Research 06/2011; 71(16):5381-6. · 7.86 Impact Factor
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ABSTRACT: Chronic myelogenous leukemia (CML) results from the neoplastic transformation of a haematopoietic stem cell. The hallmark genetic abnormality of CML is a chimeric BCR/ABL1 fusion gene resulting from the Philadelphia chromosome rearrangement t(9;22)(q34;q11). Clinical and laboratory studies indicate that the BCR/ABL1 fusion protein is essential for initiation, maintenance and progression of CML, yet the event(s) driving the transformation from chronic phase to blast phase are poorly understood.
Here we report multiple genome aberrations in a collection of 78 CML and 14 control samples by oligonucleotide array comparative genomic hybridization. We found a unique signature of genome deletions within the immunoglobulin heavy chain (IGH) and T cell receptor regions (TCR), frequently accompanied by concomitant loss of sequences within the short arm regions of chromosomes 7 and 9, including IKZF1, HOXA7, CDKN2A/2B, MLLT3, IFNA/B, RNF38, PAX5, JMJD2C and PDCD1LG2 genes.
None of these genome losses were detected in any of the CML samples with myeloid transformation, chronic phase or controls, indicating that their presence is obligatory for the development of a malignant clone with a lymphoid phenotype. Notably, the coincidental deletions at IGH and TCR regions appear to precede the loss of IKZF1 and/or p16 genes in CML indicating a possible involvement of RAG in these deletions.
BMC Genomics 01/2010; 11:41. · 4.07 Impact Factor
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ABSTRACT: Chronic myeloid leukaemia (CML) is characterized by the expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase that commonly results from the formation of the Philadelphia (Ph) chromosome after a t(9;22)(q34;q11) or variant rearrangement. The duplication of the Ph chromosome is a recurring abnormality acquired during disease progression, whereas intrachromosomal amplification of BCR/ABL1 is a rare phenomenon and has been associated with imatinib therapy resistance. Archival bone marrow chromosome suspensions from 19 CML patients known to carry more than 1 copy of BCR/ABL1 and 10 CML cell lines were analyzed by fluorescent in situ hybridization with a panel of probes from 9q34.1-qter to investigate whether they carried two identical copies of the Ph chromosome or, instead, one or both Ph contained cryptic imbalances of some regions.
A duplication of the entire Ph chromosome with no further events involving the derivative 22 was found in 12 patients. In contrast, a sideline with either 1 or 2 isochromosomes of the Ph chromosome was identified in 6 patients but none of the cell lines. In one of the patients a translocation between the distal end of one arm of the isoderivative chromosome 22 and a third chromosome was revealed. 2 patients were found to carry marker structures harbouring high copy number gains of BCR/ABL1 fusion along with a variable part of 9q34 region downstream of ABL1 breakpoint, similarly to the markers present in the imatinib resistant cell line K562. We identified the following regions of amplification: 9q34.1 → q34.2 and 9q34.1 → qter, with a common minimum amplified region of 682 Kb. One of the patients had 5 BCR/ABL1 positive clones with variable level of 9q34 amplifications on a variety of structures, from an isoderivative 22 to tandem duplications.
These data confirm that the intrachromosomal genomic amplification of BCR/ABL1 that occurs in some CML patients during disease progression also involves amplification of 9q34 gene-rich sequences downstream of ABL1 breakpoint. The variety of rearrangements identified in this relatively small cohort demonstrates that the Ph chromosome is not a stable structure but prone to further rearrangements during disease progression.
Molecular Cytogenetics 01/2010; 3:15.
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Petr Hubacek, Anna Virgili,
Katherine N Ward,
David Pohlreich,
Petra Keslova,
Barbora Goldova,
Marketa Markova,
Miroslav Zajac,
Ondrej Cinek,
Elisabeth P Nacheva,
Petr Sedlacek,
Petr Cetkovsky
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ABSTRACT: Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.
British Journal of Haematology 03/2009; 145(3):394-8. · 4.94 Impact Factor
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Petr Hubacek, Anna Virgili,
Katherine N. Ward,
David Pohlreich,
Petra Keslova,
Barbora Goldova,
Marketa Markova,
Miroslav Zajac,
Ondrej Cinek,
Elisabeth P. Nacheva,
Petr Sedlacek,
Petr Cetkovsky
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ABSTRACT: Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.
British Journal of Haematology 02/2009; 145(3):394 - 398. · 4.94 Impact Factor
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ABSTRACT: Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal integration sites of human herpesvirus 6 (HHV-6) in phytohemagglutinin-stimulated leukocytes and B lymphocytes from Epstein-Barr virus transformed lymphoblastoid cell lines (LCLs). Five different chromosomal integration sites were found in nine individuals. Only one site was identified in each individual, each site was in the vicinity of the telomeric region and was on either the p or q arm of only one of the two chromosome homologues. The sites were 9q34.3, 10q26.3, 11p15.5, 17p13.3, and 19q 13.4, of which three have not been previously identified. For 9q34.3 the site of integration was further mapped using a locus-specific probe for 9q34.3 together with a pan-telomeric probe and both co-localized with the HHV-6 signal. Similarly an arm-specific telomeric probe for 19q co-localized with the HHV-6 signal. It was therefore concluded that the site of integration is actually within the telomere. The number of viral DNA copies/cell was calculated in blood, LCL cells and hair follicles and was one or more in every case for each of the nine individuals. This result was confirmed by FISH where 100% of cells gave an HHV-6 signal. These findings add to previous reports suggesting that integrated HHV-6 DNA is found in every cell in the body and transmitted vertically. Finally, including our data, worldwide seven different chromosomal sites of HHV-6 integration have now been identified. Large epidemiological studies of chromosomal integration are required to identify further telomeric sites, geographical or racial variation and possible clinical consequences.
Journal of Medical Virology 10/2008; 80(11):1952-8. · 2.82 Impact Factor
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Anna Virgili,
Diana Brazma,
Alistair G Reid,
Julie Howard-Reeves,
Mikel Valgañón,
Anastasios Chanalaris,
Valeria As De Melo,
David Marin,
Jane F Apperley,
Colin Grace,
Ellie P Nacheva
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ABSTRACT: Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder, almost always characterized by the presence of the Philadelphia chromosome (Ph), usually due to t(9;22)(q34;q11) or its variants. The Ph results in the formation of the BCR/ABL1 fusion gene, which is a constitutively activated tyrosine kinase. Around 1% of CML patients appear to have a Ph negative karyotype but carry a cryptic BCR/ABL1 fusion that can be located by fluorescence in situ hybridisation (FISH) at chromosome 22q11, 9q34 or a third chromosome. Here we present FISH mapping data of BCR and ABL1 flanking regions and associated chromosomal rearrangements in 9 Ph negative BCR/ABL1 positive CML patients plus the cell line CML-T1.
BCR/ABL1 was located at 9q34 in 3 patients, 22q11 in 5 patients and CML-T1 and 22p11 in 1 patient. In 3 of 6 cases with the fusion at 22q11 a distal breakpoint cluster was found within a 280 Kb region containing the RAPGEF1 gene, while in another patient and the CML-T1 the distal breakpoint fell within a single BAC clone containing the 3' RXRA gene. Two cases had a duplication of the masked Ph while genomic deletions of the flanking regions were identified in 3 cases. Even more complex rearrangements were found in 3 further cases.
BCR/ABL1 formation resulted from a direct insertion (one step mechanism) in 6 patients and CML-T1, while in 3 patients the fusion gene originated from a sequence of rearrangements (multiple steps). The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in Ph negative BCR/ABL1 positive CML.
Molecular Cytogenetics 01/2008; 1:14.
