Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia

Abstract

A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.

Figure 1

Comparison of clinical parameters between the TN-ET patients and mutated ET patients. (A) Frequencies of driver mutations in the ET patients in our cohort. (B) A diagram presenting the mutation profiles of the TN-ET patients. NC-JAK2/MPL: noncanonical JAK2 and MPL mutation. The WBC count (C), Hb value (D), platelet count (E), and LDH level (F) for the patients classified based on driver mutation status are shown. Gray highlight shows normal range. * < 0.05, ** < 0.01, *** < 0.001, ns: not significant.

Figure 2

Survival data of the ET patients grouped by driver mutation status. The fibrosis-free survival (A), leukemia-free survival (B), and overall survival (C) of the patients stratified by driver mutation status are shown.

Figure 3

Noncanonical mutants of JAK2 and MPL exhibit wild-type-equivalent levels of STAT5 activation. (A,B) STAT5 reporter activity determined by luciferase reporter assay. The y-axis indicates values of STAT5 reporter activity adjusted by the internal control. Plasmids encoding JAK2 (A) or MPL (B) with the indicated mutation were used. Representative data from multiple experiments are presented. * < 0.05, ** < 0.01, ns: not significant.

Figure 4

Polyclonal hematopoiesis in the TN-ET patients harboring comparable serum levels of cytokines for megakaryopoiesis. (A) Typical profiles of capillary electrophoresis of HpaII-digested gDNA from granulocytes (n = 10) or MNCs (n = 5) and CD3-positive cells. Two HpaII-resistant peaks representing maternal and paternal alleles in polyclonal hematopoiesis. One of these alleles becomes HpaII-sensitive in granulocytes, representing clonal hematopoiesis (arrow). (B) A pie chart presenting the frequencies of clonal and polyclonal hematopoiesis judged by the HUMARA assay in TN-ET. Four of 15 patients who exhibited ambiguous patterns in the HUMARA assay (A) were excluded from the analysis. Comparison of TPO (C) and IL-6 (D) concentrations in the serum among the patients with TN-ET, patients with ET harboring driver mutations, and healthy controls.

Figure 5

Cell-autonomous megakaryopoiesis in hematopoietic stem/progenitor cells in TN-ET. (A) Representative images of megakaryocytic colonies (CD42b-positive) from CD34-positive BM cells from the indicated patients and controls. Scale bar indicates 100 µm. (B) Relative ratio of the number of megakaryocytic colonies for the indicated patients and controls formed in the presence and absence of TPO. ** < 0.01, *** < 0.001, ns: not significant.