br Acknowledgements br Introduction The chronic myeloprolife

Acknowledgements
Introduction The chronic myeloproliferative neoplasms (MPN) comprise a group of clonal stem cell disorders with a high prevalence of the JAK2 V617F mutation (JAK2+) causing excessive production of mature blood cells. Transformation to acute myeloid leukemia (AML) is seen in 2–5% of patients with polycythemia vera and essential thrombocythemia (ET), and in 15–30% with primary myelofibrosis [1]. Overt AML in these cases is usually preceded by an accelerated phase of disease characterized by deranged hematopoiesis [2]. The JAK2+ aberration by itself is not sufficient to cause blastic transformation but induces genomic instability, which may predispose to leukemic transformation. Additionally, other mutations have been described in this setting although it remains uncertain to what extent they contribute to disease progression [3]. TET2, a member of the ten-eleven-translocation family of genes, is mutated in 7–13% of all MPN cases, but less frequently so in ET [4]. Mutated TET2 has been suggested to found a pre-MPN methylergometrine while other data suggest it to be a late event in the progression of MPN [5,6]. As in AML, specific cytogenetic aberrations in MPN hold prognostic potential. Such aberrations tend to accumulate and up to 95% of the patients have an abnormal karyotype in the transformation phase [7]. Despite the obvious clinical importance of AML development in MPN patients, the causative genetic mechanisms and clinical development are poorly understood. We here report a case of JAK2+ ET with multiple TET2 mutations that progressed to del(7q) positive AML after an almost three years lag phase prominent by normalized blood cell counts. In February 2009 a 64-yr-old woman presented with peripheral thrombosis. The platelet count was 1263×109/L, total leukocytes 14.4×109/L, hemoglobin 9.4mmol/L and hematocrit 0.45. Abdominal ultrasound did not reveal splenomegaly. The JAK2 allele burden was 14%. Morphology was consistent with ET with trilineage hyperplasia dominated by excess of large, hyperlobulated and loosely clustered mekaryocytes. The iron content was normal and there was no reticulin fibrosis. Standard G-Banding revealed a normal karyotype. She was diagnosed with ET and was subsequently treated with hydroxyurea and low dose aspirin (Fig. 1). Therapy was discontinued due to cytopenias in June 2009. Despite withdrawal of cytoreductive treatment, peripheral blood cell counts stayed normal (Fig. 1). A routine check in February 2012 revealed leukocytosis of 129×109/L with neutrocytosis of 98×109/L, hemoglobin 5.7mmol/L, and platelets 214×109/L. The JAK2+ allele burden had declined to 0.2% (Fig. 1). Morphology and flow cytometry did not disclose blastic transformation (Fig. 2A and B). Now standard G-Band karyotyping revealed a del(7q) in 21/25 metaphases (Fig. 2D). The patient was deemed in accelerated phase of MPN with high risk of transformation and was offered cytoreduction with 2 days of anthracycline and 5 days of cytarabine, “2+5”, and allocated for hematopoietic stem cell transplantation. Unfortunately, she progressed during treatment and was diagnosed with AML in May 2012 and succumbed to this in January 2013. The almost three year long lag phase with stable normalized peripheral blood cell counts preceding the acceleration phase, led us to further delineate the pathogenetic mechanisms by analyzing bone marrow (BM) cells from critical time points. At the time of initial diagnosis as well as during progression and in the AML phase, the patient was negative for mutations in CEBPa, FLT3-ITD, FLT3-D835, cKIT D816V, IDH1 R132, NPM1, and WT1 exon 7 indels as analyzed by a sensitive multiplex fluorescent-labeled capillary gel electrophoresis. In addition, she was negative for balanced reciprocal AML- and CML-related translocations by standard qPCR and FISH and negative for gene rearrangements of PDGFRA and PDGFRB. TET2 mutation screening, by a combination of denaturing gradient gel electrophoresis and Sanger sequencing of the entire coding sequence and splice sites, disclosed two loss-of-function mutations (c.2601 del T and c.3810-3811 INS C) as well as a previously unreported TET2 missense mutation (c.A1505T) in all samples from ET diagnosis, accelerated phase, and AML (Fig. 2C).