A new BCR-ABL1 Drosophila model as a powerful tool to elucidate the pathogenesis and progression of chronic myeloid leukemia.

The oncoprotein BCR-ABL1 triggers chronic myeloid leukemia. It is clear that the disease relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. We describe and validate a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. We show that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate our Drosophila model, which promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators in order to better elucidate the mechanism of leukemia onset and progression.

A PNA-mediated clamping PCR for routine detection of KRAS mutations in colorectal carcinoma.

The detection of somatic mutations in a tumor represents a valuable tool for tumor characterization and provides the clinicians with information for setting up the most appropriate therapy. KRAS mutations in codons 12 and 13 are important biomarkers routinely analyzed in the clinic for the management of anti-EGFR treatment in colorectal carcinoma (CRC). Here we report a sensitive and inexpensive assay for KRAS mutations based on a PNA-mediated PCR clamping. The assay displays very high sensitivity (0.7%) and specificity (96.7%) when compared to traditional sequencing (SS) and pyrosequencing (PS), two of the most commonly and routinely used methods employed today by diagnostic laboratories. Furthermore, the PNA assay requires only basic and low-cost laboratory equipment, in contrast with all the most recent PCR-based technologies, which are highly sensitive but also much more expensive. Finally, despite the PNA assay does not allow for the definition of specific mutations, it is the cheapest and easiest screening method to firstly stratify wild-type and mutated patients, information that is strictly necessary to clinicians for the management of CRC and anti-EGFR treatment.

The PAX5 gene is frequently rearranged in BCR-ABL1-positive acute lymphoblastic leukemia but is not associated with outcome. A report on behalf of the GIMEMA Acute Leukemia Working Party.

BACKGROUND: Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric patients with acute lymphoblastic leukemia. So far the occurrence of PAX5 alterations and their clinical correlation have not been investigated in adults with BCR-ABL1-positive acute lymphoblastic leukemia. DESIGN AND METHODS: The aim of this study was to characterize the rearrangements on 9p involving PAX5 and their clinical significance in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Eighty-nine adults with de novo BCR-ABL1-positive acute lymphoblastic leukemia were enrolled into institutional (n=15) or GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) (n=74) clinical trials and, after obtaining informed consent, their genome was analyzed by single nucleotide polymorphism arrays (Affymetrix 250K NspI and SNP 6.0), genomic polymerase chain reaction analysis and re-sequencing. RESULTS: PAX5 genomic deletions were identified in 29 patients (33%) with the extent of deletions ranging from a complete loss of chromosome 9 to the loss of a subset of exons. In contrast to BCR-ABL1-negative acute lymphoblastic leukemia, no point mutations were found, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1-positive acute lymphoblastic leukemia. The deletions were predicted to result in PAX5 haploinsufficiency or expression of PAX5 isoforms with impaired DNA-binding. Deletions of PAX5 were not significantly correlated with overall survival, disease-free survival or cumulative incidence of relapse, suggesting that PAX5 deletions are not associated with outcome. CONCLUSIONS: PAX5 deletions are frequent in adult BCR-ABL1-positive acute lymphoblastic leukemia and are not associated with a poor outcome.

Deferasirox is a powerful NF-kappaB inhibitor in myelodysplastic cells and in leukemia cell lines acting independently from cell iron deprivation by chelation and reactive oxygen species scavenging.

BACKGROUND: Usefulness of iron chelation therapy in myelodysplastic patients is still under debate but many authors suggest its possible role in improving survival of low-risk myelodysplastic patients. Several reports have described an unexpected effect of iron chelators, such as an improvement in hemoglobin levels, in patients affected by myelodysplastic syndromes. Furthermore, the novel chelator deferasirox induces a similar improvement more rapidly. Nuclear factor-kappaB is a key regulator of many cellular processes and its impaired activity has been described in different myeloid malignancies including myelodysplastic syndromes. DESIGN AND METHODS: We evaluated deferasirox activity on nuclear factor-kappaB in myelodysplastic syndromes as a possible mechanism involved in hemoglobin improvement during in vivo treatment. Forty peripheral blood samples collected from myelodysplastic syndrome patients were incubated with 50 muM deferasirox for 18h. RESULTS: Nuclear factor-kappaB activity dramatically decreased in samples showing high basal activity as well as in cell lines, whereas no similar behavior was observed with other iron chelators despite a similar reduction in reactive oxygen species levels. Additionally, ferric hydroxyquinoline incubation did not decrease deferasirox activity in K562 cells suggesting the mechanism of action of the drug is independent from cell iron deprivation by chelation. Finally, incubation with both etoposide and deferasirox induced an increase in K562 apoptotic rate. CONCLUSIONS: Nuclear factor-kappaB inhibition by deferasirox is not seen from other chelators and is iron and reactive oxygen species scavenging independent. This could explain the hemoglobin improvement after in vivo treatment, such that our hypothesis needs to be validated in further prospective studies.

FOXO transcription factor activity is partially retained in quiescent CML stem cells and induced by tyrosine kinase inhibitors in CML progenitor cells.

Chronic Myeloid Leukaemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL. ABL-specific tyrosine kinase inhibitors (TKIs), whilst effective against mature CML cells, induce little apoptosis in stem/progenitor cells. However, in stem/progenitor cells TKIs exert potent anti-proliferative effects through a poorly understood mechanism. We showed that in CD34(+) CML cells FOXO1, 3a and 4 (FOXOs) were phosphorylated, predominantly cytoplasmic and inactive, consequent to BCR-ABL expression. TKIs decreased phosphorylation of FOXOs, leading to their re-localisation from cytoplasm (inactive) to nucleus (active), thus inducing G1 arrest. Of key importance, despite BCR-ABL activity, primitive quiescent CML stem cells showed low levels of FOXO phosphorylation and predominant nuclear localisation, resembling the pattern in normal stem cells. These results demonstrate for the first time that TKI-induced G1 arrest in CML progenitor cells is mediated by re-activation of FOXOs, whilst quiescence of CML stem cells is regulated by sustained FOXO activity. These data contribute to our understanding of CML stem cell quiescence and TKI activity, suggesting new strategies to target CML stem/progenitor cells by preventing or reversing this effect.

Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1-positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working Party (GIMEMA AL WP).

The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Delta4-7) and by removal of exons 2 through 7 (Delta2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Delta4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Delta2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Hypereosinophilic syndrome in childhood: clinical and molecular features of two cases.

Hypereosinophilic syndrome (HES) represents a heterogeneous group of diseases, some of which are being clarified by recent advances in molecular genetics. It is very rare in children. Uncertainties in classification and lack of prospective studies make therapeutic decisions difficult. The authors report two cases of HES in which steroid therapy was effective.

Identification of different Ikaros cDNA transcripts in Philadelphia-positive adult acute lymphoblastic leukemia by a high-throughput capillary electrophoresis sizing method.

BACKGROUND: Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA. DESIGN AND METHODS: In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts. RESULTS: We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus. CONCLUSIONS: Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia.

Expression of spliced oncogenic Ikaros isoforms in Philadelphia-positive acute lymphoblastic leukemia patients treated with tyrosine kinase inhibitors: implications for a new mechanism of resistance.

Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph+ acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non-DNA-binding Ik6 isoform was detected in 49% of Ph+ ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph+ ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph+ ALL patients.

Early prediction of treatment outcome in acute myeloid leukemia by measurement of WT1 transcript levels in peripheral blood samples collected after chemotherapy.

The Wilms' tumor gene WT1 is a reliable marker for minimal residual disease assessment in acute leukemia patients. The study was designed to demonstrate the potential use of WT1 to establish quality of remission in acute leukemia patients for early identification of patients at high risk of relapse. A prospective study based on a quantitative Real-Time PCR (TaqMan) assay in 562 peripheral blood samples collected from 82 acute leukemia patients at diagnosis and during follow-up was established. The evaluation of WT1 in peripheral blood samples after induction chemotherapy can distinguish the continuous complete remission patients from those who obtain only an "apparent" complete remission and who could relapse within a few months. WT1 helps identify patients at high risk of relapse soon after induction chemotherapy allowing post-induction therapy in high risk patients to be intensified.

Nuclear factor kB as a target for new drug development in myeloid malignancies.

The transcription nuclear factor k B (NF-kB) can intervene in oncogenesis through to its capacity to regulate the expression of a large number of genes that regulate apoptosis, cell proliferation and differentiation as well as inflammation, angiogenesis and tumor migration. Impaired NF-kB activity has been demonstrated not only in solid cancers but also in various types of hematologic malignancies including acute myeloid leukemia, chronic myelogenous leukemia and in a subset of myelodysplastic syndromes. The underlying mechanisms, illustrated in the text and although quite diverse in different diseases, provide the rationale for new therapeutic strategies combining different NF-kB or proteasome inhibitors. It has, therefore, been proposed that inhibition of NF-kB could be an adjuvant therapy for cancer and many phase I/II clinical studies are ongoing with different inhibitors. This review highlights the in vitro and in vivo results of NF-kB inhibition in myeloid malignancies.

The efficacy of imatinib mesylate in patients with FIP1L1-PDGFRalpha-positive hypereosinophilic syndrome. Results of a multicenter prospective study.

BACKGROUND AND OBJECTIVES: The hypereosinophilic syndrome (HES) may be associated with the fusion of the platelet derived growth factor receptor a (PDGFRalpha) gene with the FIP1L1 gene in chromosome 4 coding for a constitutively activated PDGFRalpha tyrosine kinase. These cases with FIP1L1-PDGFRalpha rearrangement have been reported to be very sensitive to the tyrosine kinase inhibitor imatinib mesylate. DESIGN AND METHODS: A prospective multicenter study of idiopathic or primary HES was established in 2001 (Study Protocol Registration no. NCT 0027 6929). One hundred and ninety-six patients were screened, of whom 72 where identified as having idiopathic or primary HES and 63 were treated with imatinib 100 to 400 mg daily. RESULTS: Twenty-seven male patients carried the FIP1L1-PDGFRalpha rearrangement. All 27 achieved a complete hematologic remission (CHR) and became negative for the fusion transcripts according to reverse transcriptase polymerase chain reaction (RT-PCR) analysis. With a median follow-up of 25 months (15-60 months) all 27 patients remain in CHR and RT-PCR negative, and continue treatment at a dose of 100 to 400 mg daily. In three patients imatinib treatment was discontinued for few months, the fusion transcript became rapidly detectable, and then again undetectable upon treatment reassumption. Thirty-six patients did not carry the rearrangement; of these, five (14%) achieved a CHR, which was lost in all cases after 1 to 15 months. INTERPRETATION AND CONCLUSIONS: All patients meeting the criteria for idiopathic or primary HES should be screened for the FIP1L1-PDGFRalpha rearrangement. For all patients with this rearrangement, chronic imatinib treatment at doses as low as 100 mg daily ensures complete and durable responses.

Valproate enhances imatinib-induced growth arrest and apoptosis in chronic myeloid leukemia cells.

BACKGROUND: The objective of this study was to evaluate the ability of the clinically available histone deacetylase (HDAC) inhibitor valproate to enhance the cytotoxicity of the Bcr-Abl inhibitor imatinib in imatinib-resistant cell lines. METHODS: Interactions between imatinib, and valproate have been examined in imatinib-sensitive and -resistant chronic myeloid leukemia (CML)cell lines (K562, KCL-22, CML-T1) and in bone marrow mononuclear cells (MNCs) derived from imatinib-resistant CML patients. RESULTS: In imatinib-sensitive cell lines, cotreatment with imatinib 0.5 muM and valproate 5 microM for 48 hours potently enhanced imatinib-induced growth arrest and apoptosis. In resistant cell lines and in primary MNCs derived from imatinib-rsistant patients, valproate restored sensitivity to the cytotoxic effects of imatinib. Coexposure of cells to valproate and imatinib was associated with repression of several genes involved in Bcr-Abl transformation. In particular, the combination valproate-imatinib downregulated the expression of Bcr-Abl and the antiapoptotic protein Bcl-2, which is particularly overexpressed in imatinib-resistant clones. CONCLUSIONS: Data from this study suggested that administration of the clinically available HDAC inhibitor valproate may be a powerful strategy to enhance cytotoxic effects of imatinib in those patient resistant to imatinib or in which complete cytogenetic remission has been not reached.

Genetic abnormalities as targets for molecular therapies in myelodysplastic syndromes.

Recent advances in molecular genetics have increased knowledge regarding the mechanisms leading to myelodysplastic syndrome (MDS), secondary acute myeloid leukemia (AML), and therapy-induced MDS. Many genetic defects underlying MDS and AML have been identified thereby allowing the development of new molecular-targeted therapies. Several new classes of drugs have shown promise in early clinical trials and may probably alter the standard of care of these patients in the near future. Among these new drugs are farnesyltransferase inhibitors and receptor tyrosine kinase inhibitors including FLT3 and VEGF inhibitors. These agents have been tested in patients with solid tumors and hematologic malignancies such as AML and MDS. Most of the studies in MDS are still in early stages of development. The DNA hypomethylating compounds azacytidine and decitabine may reduce hypermethylation and induce re-expression of key tumor suppressor genes in MDS. Biochemical compounds with histone deacetylase inhibitory activity, such as valproic acid (VPA), have been tested as antineoplastic agents. Finally, new vaccination strategies are developing in MDS patients based on the identification of MDS-associated antigens. Future therapies will attempt to resolve cytopenias in MDS, eliminate malignant clones, and allow differentiation by attacking specific mechanisms of the disease.

Sensitivity to imatinib therapy may be predicted by testing Wilms tumor gene expression and colony growth after a short in vitro incubation.

BACKGROUND: The objective of the current study was to verify the ability to predict response to imatinib therapy using in vitro assays to evaluate the inhibition of Wilms tumor gene (WT1) expression and colony growth after samples obtained from patients with chronic myelogenous leukemia (CML) before the start of treatment were subjected to short-term incubation with imatinib. METHODS: WT1 transcript levels and colony growth in bone marrow (BM) samples from 23 patients with CML that was later identified as being responsive to imatinib and from 13 patients with CML that was later identified as not being responsive to imatinib were evaluated after incubation of these samples with imatinib at a concentration of 1 microM for 18 hours. In addition, real-time quantitative polymerase chain reaction (RQ-PCR) analysis of WT1 expression was performed during follow-up, and the results were analyzed for associations with cytogenetic response and with BCR/ABL transcript levels as determined using RQ-PCR analysis. RESULTS: Before treatment, it was found that WT1 expression was elevated in BM samples obtained from all patients with CML. WT1 expression and colony growth were reduced significantly after an 18-hour incubation with imatinib in samples obtained from patients who were later identified as responders to treatment, but not in samples obtained from patients who did not experience responses to treatment. Inhibition of WT1 expression in vitro was associated with inhibition of imatinib-induced BCR-ABL tyrosine kinase activity, a finding that also has been made in studies involving certain Philadelphia chromosome (Ph)-positive and Ph-negative cell lines. CONCLUSIONS: Inhibition of WT1 transcript levels after a short period of in vitro exposure of pretherapy BM samples to imatinib was correlated with inhibition of colony growth and may represent the basis for an easy test that is capable of predicting the sensitivity of CML to treatment with imatinib for individual patients.

Myelodysplastic syndromes.

Efforts made during the last few years have helped unravel the complex pathogenesis of the myelodysplastic syndromes (MDS). A large number of studies, made possible by the introduction of newer technologies, have led to major progress in understanding the heterogeneous genetic and biological abnormalities contributing to the development and progression of myelodysplasia. Better insights into these pathogenetic processes will aid the development of newer and more successful therapies for MDS patients. The identification of specific genes involved in the emergence and progression of the myelodysplastic clone has extended biological findings into the clinic. Recently, several clinical trials have used selective compounds to target and inhibit the disrupted signal transduction pathway in myelodysplastic patients. The demonstration of genetic abnormalities present not only in MDS patients but also in acute myeloid leukemia (AML) patients or in chronic myeloproliferative disorders (CMPD) has prompted extension of a number of clinical trials from AML and CMPD to MDS patients. In spite of this, the more complex and heterogeneous pathogenesis underlying the myelodysplastic process is responsible for the often different and in same cases worse clinical results obtained in MDS patients. Finally, the identification of myelodysplasia-associated antigens that may be targeted by an immunotherapeutic approach represents a future perspective in tailored therapy for MDS patients.