Ana Paula Ambrósio

Translocation (8;17;15;21)(q22;q23;q15;q22) in acute myeloid leukemia (M2) : A four-way variant of t(8;21)

We report the results of cytogenetic, fluorescence in situ hybridization (FISH) and molecular analyses in a 15-year-old boy diagnosed with acute myeloid leukemia subtype M2 (AML-M2). Cytogenetic and FISH analyses, the latter with whole chromosome painting probes, revealed a complex translocation involving four chromosomes: t(8;17;15;21)(q22;q23;q15;q22). The observation of breakpoints at 8q22 and 21q22 suggested a rearrangement of the ETO and AML1 genes, respectively. Using a dual-color FISH test with ETO and AML1 probes, we demonstrated an AML1/ETO fusion signal on the derivative chromosome 8. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of AML1/ETO fusion transcripts identical to those found in classical t(8;21). The present case highlights the relevant role of the rearranged chromosome 8, which encodes the AML1/ETO fusion product in the pathogenesis of AML-M2.

Three-way translocation (X;20;16)(p11;q13;q23) in essential thrombocythemia implicates NFATC2 in dysregulation of CSF2 expression and megakaryocyte proliferation.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm essentially characterized by excessive production of platelets. Molecular pathogenesis of ET is linked in approximately half of the patients to intracellular cytokine signaling dysregulation as a result of thrombopoietin receptor or Janus kinase 2 (JAK2) mutations. However, genetic defects underlying cytokine transcription have not been associated with ET. Using molecular cytogenetics and whole‐genome array analyses, we uncovered a submicroscopic deletion at 20q13.2 in a JAK2V617F‐positive ET patient with an acquired complex chromosome translocation. The deletion encompassed the nuclear factor of activated T‐cells, cytoplasmic, calcineurin‐dependent 2 (NFATC2) gene that encodes a transcription factor involved in the regulation of hematopoietic cytokines. RNA interference‐mediated suppression of NFATC2 mRNA or pharmacological inhibition of NFATC2 protein with 11R‐VIVIT in cultured JAK2V617F‐positive SET‐2 megakaryocytes increased colony stimulating factor 2 (granulocyte‐macrophage) (CSF2) mRNA and promoted cell proliferation. Moreover, impairment of NFATC2–calcineurin interaction with 11R‐VIVIT further reduced the transcription of the NFATC2 gene. Antibody‐mediated neutralization of CSF2 cytokine in inhibitor‐treated cells prevented 11R‐VIVIT‐induced cell proliferation, indicating that impairment of NFATC2–calcineurin interaction promotes megakaryocyte proliferation through up‐regulation of CSF2 transcription. Our results suggest a model in which haplo‐insufficiency of NFATC2 cooperates with activation of the JAK–STAT signaling pathway in the pathogenesis of JAK2V617F‐positive ET with del(20q). These results further indicate that pathogenesis of ET may be linked to genetic defects of other transcription factor genes involved in the regulation of cytokine expression.

Combined molecular diagnosis of B-cell lymphomas with t(11;14)(q13;q32) or t(14;18)(q32;q21) using multiplex- and long distance inverse-polymerase chain reaction.

Translocations t(14;18)(q32;q21) and t(11;14)(q13;q32) are recurrent findings in follicular lymphoma (FL) and mantle cell lymphoma (MCL), respectively. However, the molecular counterparts of these translocations can only be detected in up to 75% of FL and 50% of MCL cases using routine techniques. To improve the efficiency of detection, we first devised a single-tube multiplex-polymerase chain reaction (PCR) assay with primers located within a conserved immunoglobulin heavy chain (IGH) sequence and 5′ to the main breakpoint cluster regions of BCL2 and CCND1. Using this assay in 17 FL and 11 MCL diagnostic DNA samples, we readily identified a BCL2-IGH fusion in 65% of FL patients and a CCND1-IGH fusion in 55% of MCL patients. In the remaining cases, we used long distance inverse-PCR to detect BCL2-IGH and CCND1-IGH fusion genes with different BCL2 and CCND1 breakpoint locations. We found additional translocations in 3 patients (17%) with FL and in 4 patients (36%) with MCL. Taken together, we show that multiplex-PCR combined with long distance inverse-PCR detected a t(14;18) in 82% of FL patients and a t(11;14) in 91% of MCL patients, demonstrating that this 2-step protocol is an effective approach for molecular detection of t(11;14) and t(14;18) in B-cell lymphomas.