Atypical mRNA fusions in PML-RARA positive, RARA-PML negative acute promyelocytic leukemia.
ABSTRACT Reciprocal RARA-PML transcripts are not detected in approximately 25% of patients with PML-RARA positive acute promyelocytic leukemia (APL), but the reasons for this are poorly understood. We studied 21 PML-RARA positive/RARA-PML negative cases by bubble PCR and multiplex long template PCR to identify the genomic breakpoints. Additional RT-PCR analysis was performed based on the DNA findings. Three cases were found to have complex rearrangements involving a third locus: the first had a PML-CDC6-RARA forward DNA fusion and expressed a chimeric PML-CDC6-RARA mRNA in addition to a PML-RARA. The other two had HERC1-PML and NT_009714.17-PML genomic fusion sequences at their respective reciprocal breakpoints. Six patients were falsely classified as RARA-PML negative due to deletions on chromosome 15 and/or 17, or alternative splicing leading to atypical RARA-PML fusion transcripts, which were not identified by conventional RT-PCR assays. This study demonstrates that the frequency of RARA-PML expression has been underestimated and highlights remarkable complexity at chromosomal breakpoint regions in APL even in cases with an apparently simple balanced t(15;17)(q24;q12).
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ABSTRACT: The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2012; 26(9):1976-85. · 10.16 Impact Factor
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ABSTRACT: Acute myeloid leukemia (AML) is not a single pathologic entity but represents a heterogeneous group of malignancies. This heterogeneity is exemplified by the variable clinical outcomes that are observed in patients with AML, and it is largely the result of diverse mutations within the leukemic cells. These mutations range from relatively large genetic alterations, such as gains, losses, and translocations of chromosomes, to single nucleotide changes. Detection of many of these mutations is required for accurate diagnosis, prognosis, and treatment of patients with AML. As such, many testing modalities have been developed and are currently employed in clinical laboratories to ascertain mutational status at prognostically and therapeutically critical loci. The assays include those that specifically identify large chromosomal alterations, such as conventional metaphase analysis and fluorescence in situ hybridization, and methods that are geared more toward analysis of small mutations, such as PCR with allele-specific oligonucleotide primers. Furthermore, newer tests, including array analysis and next-generation sequencing, which can simultaneously probe numerous molecular aberrancies within tumor cells, are likely to become commonplace in AML diagnostics. Each testing method clearly has advantages and disadvantages, an understanding of which should influence the choice of test in various clinical circumstances. To aid such understanding, this review discusses both genetic mutations in AML and the clinical tests-including their pros and cons-that may be used to probe these abnormalities. Additionally, we highlight the significance of genetic testing by describing cases in which results of genetic testing significantly influence clinical management of patients with AML.Molecular Diagnosis & Therapy 11/2012; · 2.59 Impact Factor
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ABSTRACT: The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the nucleophosmin (NPM) gene at 5q35 with the retinoic acid receptor alpha (RARA) at 17q12-22. We have previously shown that leukemic cells express both NPM–RAR and RAR– NPM reciprocal translocation products. In this study we investigated the potential role of both proteins in modulating myeloid differentiation. Expression of NPM–RAR inhibited vitamin D3/transforming growth factor β (TGFβ)-mediated differentiation of U937 cells by more than 50%. In contrast, RAR–NPM expression did not alter vitamin D3/TGFβ-induced differentiation of U937 clones. These results indicate that NPM–RAR, not RAR–NPM, is the prime mediator of myeloid differentiation arrest in t(5;17) APL.Leukemia and Lymphoma 06/2014; 55(6). · 2.61 Impact Factor