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The vast majority of chronic myeloid leukemia patients express a BCR-ABL1 fusion gene mRNA encoding a 210 kDa tyrosine kinase which promotes leukemic transformation. A possible differential impact of the corresponding BCR-ABL1 transcript variants e13a2 (“b2a2”) and e14a2 (“b3a2”) on disease phenotype and outcome is still a subject of debate. A total of 1105 newly diagnosed imatinib-treated patients were analyzed according to transcript type at diagnosis (e13a2, n=451; e14a2, n=496; e13a2+e14a2, n=158). No differences regarding age, sex, or Euro risk score were observed. A significant difference was found between e13a2 and e14a2 when comparing white blood cells (88 vs. 65 × 109/L, respectively; P

Background Rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints.Design and Methods We established a generic quantitative reverse transcriptase polymerase chain reaction to detect overexpression of the 3′-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion.Results At diagnosis, all patients with known fusion genes involving PDGFRA (n=5; 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 versus 0.0066 versus 0.0064, P

Background The formation of fusion genes plays roles in both oncogenesis and evolution by facilitating the acquisition of novel functions. Here we describe the first example of a human polymorphic in-frame fusion of two unrelated genes associated with a copy number variant.Design and Methods Array comparative genomic hybridization was used to identify cryptic oncogenic fusion genes. Fusion gene structure and origin was examined using molecular biological and computational methods. Phenotype associations were examined using PopGen cohorts.Results Targeted array comparative genomic hybridization to identify cryptic oncogenic fusion genes in patients with atypical myeloproliferative neoplasms identified a 111 kb amplification with breakpoints within the TRK-fused gene (TFG, a target of translocations in lymphoma and thyroid tumors) and G-protein-coupled receptor 128 (GPR128) resulting in an expressed in-frame TFG-GPR128 fusion transcript. The fusion gene was also identified in healthy individuals at a frequency of 0.02 (3/120). Normally both genes are in identical orientations with TFG immediately downstream of GPR128. In individuals with a copy number variant amplification, one or two copies of the TFG-GPR128 fusion are found between the two parental genes. The breakpoints share a region of microhomology, and haplotype and microsatellite analysis indicate a single ancestral origin. Analysis of PopGen cohorts showed no obvious phenotype association. An in silico search of EST databases found no other copy number variant amplification-associated fusion transcripts, suggesting that this is an uncommon event.Conclusions The finding of a polymorphic gene fusion in healthy individuals adds another layer to the complexity of human genome variation and emphasizes the importance of careful discrimination of oncogenic changes found in tumor samples from non-pathogenic normal variation.

The BCR-ABL K247R change is based on a rare single nucleotide polymorphism occurring likewise in healthy controls and non-hematologic cell types. Despite its juxtaposition to the P-loop, functional analysis showed no alteration compared to non-mutated BCR-ABL. We sought to investigate if other changes in the BCR-ABL kinase domain should be considered as single nucleotide polymorphisms rather than acquired mutations. A total of 911 chronic myeloid leukemia patients after failure or suboptimal response to imatinib were screened for BCR-ABL kinase domain mutations. Single nucleotide polymorphism analysis was based on the search for nucleotide changes in corresponding normal, non-translocated ABL alleles by ABL allele-specific PCR following mutation analysis. In addition to the K247R polymorphism we uncovered five new single nucleotide polymorphisms within the BCR-ABL kinase domain; two of them led to amino acid changes. Single nucleotide polymorphisms could theoretically contribute to primary but not to secondary resistance to tyrosine kinase inhibitors and must therefore be distinguished from acquired mutations. Novel point mutations should be confirmed by analyzing the normal ABL alleles to exclude polymorphisms.

Actual BCR-ABL kinase inhibition in vivo as determined by phospho-CRKL (pCRKL) monitoring has been recognized as a prognostic parameter in patients with chronic myelogenous leukemia treated with imatinib. We report a biomarker sub-study of the international phase I clinical trial of nilotinib (AMN107) using the established pCRKL assay in imatinib-resistant chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia. A minimum dose (200 mg) required for effective BCR-ABL inhibition in imatinib resistant/intolerant leukemia was determined. The pre-clinical activity profile of nilotinib against mutant BCR-ABL was largely confirmed. Substantial differences between peripheral blood baseline pCRKL/CRKL ratios were observed when comparing chronic myeloid leukemia with Ph+ acute lymphoblastic leukemia. Finally, rapid BCR-ABL-reactivation shortly after starting nilotinib treatment was seen in acute lymphoblastic leukemia patients with progressive disease carrying the P-loop mutations Y253H, E255K, or mutation T315I. Monitoring the actual BCR-ABL inhibition in nilotinib treated patients using pCRKL as a surrogate is a means to establish effective dosing and to characterize resistance mechanisms against nilotinib.

Background Mutations of the BCR-ABL tyrosine kinase domain constitute a major cause of resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia. We sought to improve the diagnostic armamentarium by screening and to analyze the dynamics of mutated clones in chronic myeloid leukemia patients who experienced hematologic or cytogenetic relapse.Design and Methods Ninety-five patients who relapsed during imatinib therapy were screened for BCR-ABL kinase domain mutations using sensitive denaturing high-performance liquid chromatography (D-HPLC) and direct sequencing. To investigate the dynamics of mutated clones D-HPLC was applied to 453 cDNA samples tracking back from relapse towards the start of imatinib therapy.Results Twenty-two different point mutations affecting 18 amino acids were detectable in 46/79 (58%) and in 7/16 patients (44%) with hematologic or cytogenetic relapse, respectively. A deletion of 81 nucleotides (del248-274) of ABL exon 4 was observed in two patients. Three patients had exclusively single nucleotide polymorphisms (K247R, T315T, E499E, n=1 each) within the BCR-ABL kinase domain. In patients harboring mutations, hematologic relapse occurred after a median of 12.9 months (range, 0.9–44.2), and BCR-ABL mutations first became detectable at a median of 5.8 months (range, 0–30.5) after starting imatinib therapy (p

Clonal cytogenetic aberrations of the Philadelphia chromosome (Ph) positive hematopoiesis have been associated with the natural evolution of chronic myeloid leukemia (CML) to advanced disease. Clonal aberrations of Ph negative metaphases have been described after treatment with interferon or imatinib. This study evaluates the effect of dasatinib on Ph positive clones with additional cytogenetic aberrations and the frequency of novel aberrations in Ph positive and negative metaphases. Seventy-one patients treated with dasatinib after imatinib failure for a median of nine months were evaluated. Novel aberrations within Ph positive and negative clones appeared in six and three patients, respectively.

The vast majority of chronic myeloid leukemia patients express a BCR-ABL1 fusion gene mRNA encoding a 210 kDa tyrosine kinase which promotes leukemic transformation. A possible differential impact of the corresponding BCR-ABL1 transcript variants e13a2 ("b2a2") and e14a2 ("b3a2") on disease phenotype and outcome is still a subject of debate. A total of 1105 newly diagnosed imatinib-treated patients were analyzed according to transcript type at diagnosis (e13a2, n=451; e14a2, n=496; e13a2+e14a2, n=158). No differences regarding age, sex, or Euro risk score were observed. A significant difference was found between e13a2 and e14a2 when comparing white blood cells (88 vs. 65 × 10(9)/L, respectively; P<0.001) and platelets (296 vs. 430 × 10(9)/L, respectively; P<0.001) at diagnosis, indicating a distinct disease phenotype. No significant difference was observed regarding other hematologic features, including spleen size and hematologic adverse events, during imatinib-based therapies. Cumulative molecular response was inferior in e13a2 patients (P=0.002 for major molecular response; P<0.001 for MR4). No difference was observed with regard to cytogenetic response and overall survival. In conclusion, e13a2 and e14a2 chronic myeloid leukemia seem to represent distinct biological entities. However, clinical outcome under imatinib treatment was comparable and no risk prediction can be made according to e13a2 versus e14a2 BCR-ABL1 transcript type at diagnosis. (clinicaltrials.gov identifier:00055874)., (Copyright© Ferrata Storti Foundation.)

Background: The formation of fusion genes plays roles in both oncogenesis and evolution by facilitating the acquisition of novel functions. Here we describe the first example of a human polymorphic in-frame fusion of two unrelated genes associated with a copy number variant., Design and Methods: Array comparative genomic hybridization was used to identify cryptic oncogenic fusion genes. Fusion gene structure and origin was examined using molecular biological and computational methods. Phenotype associations were examined using PopGen cohorts., Results: Targeted array comparative genomic hybridization to identify cryptic oncogenic fusion genes in patients with atypical myeloproliferative neoplasms identified a 111 kb amplification with breakpoints within the TRK-fused gene (TFG, a target of translocations in lymphoma and thyroid tumors) and G-protein-coupled receptor 128 (GPR128) resulting in an expressed in-frame TFG-GPR128 fusion transcript. The fusion gene was also identified in healthy individuals at a frequency of 0.02 (3/120). Normally both genes are in identical orientations with TFG immediately downstream of GPR128. In individuals with a copy number variant amplification, one or two copies of the TFG-GPR128 fusion are found between the two parental genes. The breakpoints share a region of microhomology, and haplotype and microsatellite analysis indicate a single ancestral origin. Analysis of PopGen cohorts showed no obvious phenotype association. An in silico search of EST databases found no other copy number variant amplification-associated fusion transcripts, suggesting that this is an uncommon event. Conclusions The finding of a polymorphic gene fusion in healthy individuals adds another layer to the complexity of human genome variation and emphasizes the importance of careful discrimination of oncogenic changes found in tumor samples from non-pathogenic normal variation.