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Different lineage involvement in myelodysplastic/myeloproliferative disease with combined MPL W515L and JAK2 V617F mutation

2009; Wiley; Volume: 145; Issue: 5 Linguagem: Inglês

10.1111/j.1365-2141.2009.07671.x

1365-2141

Kais Hussein, Katharina Theophile, T. Buhr, Alexandra Beller, Hans Kreipe, Oliver Bock,

Kruppel-like factors research

According to the World Health Organization classification, the myelodysplastic/myeloproliferative diseases comprise heterogeneous clonal myeloid neoplasms that, at the time of initial presentation, have clinical, laboratory or morphological attributes of a myelodysplastic syndrome as well as a chronic myeloproliferative neoplasm/disorder (CMPD) (Tefferi & Vardiman, 2007). Refractory anaemia with ringed sideroblasts associated with chronic thrombocytosis (RARS-T) provides a provisional entity within this group that has no specific cytogenetic or molecular findings known as yet. Recently, JAK2V617F or MPLW515L mutations have been described in RARS-T (Steensma et al, 2006; Malcovati & Cazzola, 2008). There is accumulating evidence that JAK2V617F can combine with other stem cell defects, as could be demonstrated for JAK2V617F-mutated CMPD with concurrent MPLW515K/L mutation (Pardanani et al, 2006; Hussein et al, 2009) or BCR-ABL1 junction (Hussein et al, 2007a). The former combination has, to date, been found exclusively in primary myelofibrosis (PMF), essential thrombocythaemia (ET) and non-fibrotic CMPD unclassifiable (Pardanani et al, 2006; Hussein et al, 2009). Here, we present a case that demonstrated that the JAK2V617F/MPLW515L double mutation also occurs in RARS-T and thus, as suggested by Malcovati and Cazzola (2008), provides further evidence for the overlapping relationship of PMF, ET, RARS-T and refractory anaemia with ringed sideroblasts. A 57-year-old male presented with thrombocytosis and anaemia. Clinical examination was inconspicuous and sonography revealed no organomegaly. Bone marrow (BM) examination in an external clinic revealed a CMPD, which was most likely an ET with an additional conspicuous partial macroblastic immaturity of the erythropoiesis. Several BM follow-up biopsies were taken after 41, 60, 69, 88 and 109 months, and the initial diagnosis of an ET was confirmed, but enlarged islands of macrocytic erythropoiesis and an elevated number of ringed sideroblasts (frequently >20%) were found in addition to an increase of enlarged and clustered megakaryocytes (Fig 1A). Chronic thrombocytosis was present (platelet count >500 × 109/l). Thus the diagnosis was amended to RARS-T. Cytogenetic analysis of BM cells showed a normal karyotype. RARS-T with MPLW515L and JAK2V617F mutations. (A) Bone marrow histology showed macrocytic erythropoiesis (arrows) and an increased number of enlarged and clustered megakaryocytes (Giemsa stain, 400×). More than 20% ringed sideroblasts were demonstrable in bone marrow smears (inset; Prussian blue, 1000×). The 60-month biopsy and smear are shown as representative of other samples. Images were produced with a microscope (BX51), digital camera (DP50) and software (DP 3.1) all produced by Olympus (Hamburg, Germany). (B) Dynamics of MPLW515L and JAK2V617F allele burden and clinical data during the course of disease. Abbreviations: total bone marrow cell-derived MPLW515L and JAK2V617F mutant allele frequencies (BM MPLW515L, BM JAK2V617F), haemoglobin (HB), not determined (n.d.), white blood cell counts (WBC) (all left-hand axis on graph), platelet counts (PLT; right-hand axis on graph), no splenomegaly (−), splenomegaly (+). Despite splenomegaly, which developed during a follow-up period of 9 years, no significant changes over time, in particular any progression to fibrosis and no signs of blastic transformation, had been observed at 140 months after initial presentation. Thrombocytosis and anaemia persisted. Drug treatment had been based on hydroxycarbamide and anagrelide. Retrospective molecular analysis of the BM sample taken 60 months after initial diagnosis revealed 41%MPLW515L alleles coexisting with 4%JAK2V617F alleles. DNA extraction, primer sets/polymerase chain reaction amplification and quantification of mutant alleles by pyrosequencer® assay (Biotage, Uppsala, Sweden) were performed as described (Hussein et al, 2007a). After 109 months, BM cells revealed an increase of up to 13%JAK2V617F alleles, not significantly affecting chronic anaemia. At the same time, MPLW515L alleles remained at higher levels (between 39%–51%) and were associated with constant thrombocytosis (Fig 1B). Thus, similar to previous reported RARS-T cases (Malcovati & Cazzola, 2008), the JAK2V617F mutation, presumed to induce erythrocytosis, was not sufficient to counteract the sideroachrestic defect although the increase of mutant allele burden was roughly paralleled by an increase in haemoglobin. Fresh peripheral blood or bone marrow cells were not available. Therefore, we selected the 109-month sample with the highest JAK2V617F frequency for parallel JAK2V617F and MPLW515L analysis of laser-microdissected BM-derived CD15+ granulocytic cells, haemoglobin-positive erythropoietic islansds, and megakaryocytes (anti-CD15 monoclonal mouse antibody, 1:50; Becton-Dickinson, Franklin Lakes, NJ, USA; anti-haemoglobin polyclonal rabbit antibody, 1:20; Quartett, Berlin, Germany; SmartCutPlus-System; MMI, Eching, Germany). MPLW515L was detectable in all myeloid lineages investigated (erythropoietic, megakaryocytic and granulocytic cells) whereas JAK2V617F was exclusively detectable in erythropoietic and megakaryocytic cells but, remarkably, not in granulocytes (Fig 2). Due to insufficient amounts of DNA analysis on the single cell level could not be performed. MPLW515L and JAK2V617F mutations in different myeloid lineages. CD15+ granulocytic cells (left-hand column), haemoglobin+ (HB+) erythroid progenitors (middle column) and megakaryocytes (right-hand column), were separated by laser microdissection from 3 μm sections of the formalin-fixed and paraffin-embedded BM biopsy, which was taken 109 months after initial disease presentation. Immunohistochemistry was visualised by FAST-red (Sigma-Aldrich, St Louis, MO, USA) and counterstained with haematoxylin. Sequence begins from the top with accurate isolation and melting of cells into an adhesive lid of a polymerase chain reaction tube for further processing (original magnification 400×, SmartCutPlus-System based on a CKX41 inverse microscope; Olympus, Hamburg, Germany). For each of the three laser-microdissected samples as well as all BM biopsies under investigation, MPLW515L and JAK2V617F frequencies (percentage mutant alleles) were measured in duplicate. Note that, except for JAK2wild-type granulocytes, lineage-specific MPLW515L and JAK2V617F allele burden were similar to the corresponding unsorted BM cells. In some cases of sporadic and familial JAK2V617F-positive polycythaemia vera, JAK2V617F was limited to erythropoietic cells and megakaryocytes (Zehentner et al, 2006; Hussein et al, 2007b). In a MPLW515L/JAK2V617F double mutated CMPD an even more complex lineage involvement could be demonstrated, including MPLwild-type/JAK2V617F status in erythropoietic, megakaryocytic and eosinophilic cells (Hussein et al, 2009). By comparing colony formation by CD34+ precursor cells from MPLW515K/L or JAK2V617F-mutated patients, Pardanani et al (2007) suggested that, within the haematopoietic hierarchy of maturation, MPLW515K/L might affect more primitive cells than JAK2V617F. The first case of MPLW515L/JAK2V617F-mutated RARS-T, presented here and analysed in situ, lends support to this view because MPLW515L involved all lineages whereas JAK2V617F did not affect the granulocytic lineage. In conclusion, we report the first case of RARS-T with combined MPLW515L/JAK2V617F mutation. The increased number of JAK2V617F alleles had no effect on anaemia. In situ analysis of lineage involvement revealed that MPLW515L and JAK2V617F affected progenitor cells at different levels of the maturation hierarchy. The authors wish to thank Ms. Sabine Schröter for her skilful work in the laboratory. Furthermore, we thank Prof. Dr. med. Christian Wittekind, Institute of Pathology, Universitätsklinik Leipzig, Prof. Dr. med. Peter Jehle from the Hospital of the Paul-Gerhardt-Stiftung, Wittenberg, Germany, and Dipl.-med. Alvith Brandl, Wittenberg, Germany, for their kind support. Research grants: Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung 10-2191 (O.B., H.K.), Deutsche Forschungsgemeinschaft –DFG BO 1954/1-1 (O.B., H.K.), Hochschul-interne Leistungsförderung – HiLF 11/07, Medizinische Hochschule Hannover (K.H.).