Megakaryocytic hyperplasia in myeloproliferative neoplasms is driven by disordered proliferative, apoptotic and epigenetic mechanisms

Abstract

AimsMyeloproliferative neoplasms (MPN) are a heterogeneous group of clonal proliferative bone marrow diseases characterised by extensive megakaryocytic hyperplasia and morphological atypia. Despite knowledge of genomic defects, the pathobiological processes driving these megakaryocytic abnormalities in MPN remain poorly explained. We have explored the proliferative, apoptotic and epigenetic profiles of megakaryocytes in human MPN.MethodsImmunohistochemical staining was performed on bone marrow trephine biopsies of 81 MPN (with and withoutJAK2V617FandCALRmutations) and 15 normal controls to assess the megakaryocytic expression of biomarkers associated with proliferation (Ki67), apoptosis (Bcl-XL, BNIP-3) and epigenetic regulation (EZH2, SUZ12).ResultsMyeloproliferative megakaryocytes showed significantly greater expression of proliferative Ki67 and anti-apoptotic Bcl-XL, reduced pro-apoptotic BNIP-3 and increased SUZ12 compared with controls. In essential thrombocythaemia, large-giant megakaryocytes with hyperlobated nuclei showed a trend towards a proliferative signature. In contrast, myelofibrotic megakaryocytes with condensed nuclear chromatin, and cases withCALRmutations, had significant reductions in pro-apoptotic BNIP-3.ConclusionsUncontrolled megakaryocytic expansion in MPN results from a combination of increased proliferation, attenuated apoptosis and defective epigenetic regulation withCALRmutations favouring apoptotic failure. The higher platelet counts reported to be seen in MPN withCALRmutations may be due to greater dysregulation of megakaryocyte apoptosis.

Megakaryocytes in Myeloproliferative Neoplasms Have Unique Somatic Mutations

“…This results in up-regulated proliferation and impaired apoptosis leading to megakaryocytic hyperplasia with hyperploid nuclei. 21 Here, we demonstrate that these abnormal megakaryocytes in MPNs have the propensity to acquire additional somatic mutations. These are not present in the patient-matched nonmegakaryocytic cells.…”

“…21,46 The predominance of low frequency ( 20%) megakaryocyte-unique variants suggests they were acquired late in the endomitotic process close to full maturation. Alternatively, they could be present in a subpopulation of megakaryocytes, or they may be present in a subset of the chromosomes within these highly polyploid cells.…”

“…These morphologic changes are a consequence of increased megakaryocyte proliferation and impaired apoptosis, resulting in abnormally large cells and hyperploidy (up to 512N) with, presumably, an extended life span. 21 It is postulated that these abnormal megakaryocytes drive the fibrotic process by releasing profibrogenic mediators. However, the precise mechanism by which this occurs and the biological defect that drives this have not been determined.…”

“…However, because they have undergone further DNA replication during endomitosis, they have the capacity to acquire additional genomic aberrations. 21 To date there are no data to substantiate this assertion, in part due to the inherent difficulties in acquiring megakaryocytes to study. Megakaryocytes constitute a relatively small percentage of cells in the marrow, and, because of their size, they are difficult to isolate using conventional methods (eg, flow sorting).…”

Automated enumeration of lymphoid and plasma cells in bone marrow to establish normal reference ranges

“…TMA sections were cut at 4 µm onto charged glass slides (Hurst Scientific, Australia). Immunohistochemical staining was performed with monoclonal antibodies to formalin and mercury-formalin resistant epitopes to identify immature lymphoid precursors (TdT; clone SEN28; Leica Biosystems), T-lymphocytes (CD3; clone LN10; Leica Biosystems), B-lymphocytes (CD20; clone L26; Dako, Australia) and plasma cells (CD138; clone MI15; Leica Biosystems, and MUM1; clone EAU32; Dako) on a Leica BOND-RX immunostainer (Leica Biosystems) as outlined by Malherbe et al 36. Antigen expression was detected with an alkaline phosphatase BOND Polymer Refine Red Detection kit (FASTRED Chromogen, Leica Biosystems) chromogenic substrate.…”