Flow Cytometry Analysis of Peripheral Blood CD34+ Cells in Patients with Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal chronic myeloproliferative disorder characterized by the accumulation of megakaryocytes in the bone marrow (BM), variable degrees of BM fibrosis, tear-drop erythrocytes, increased numbers of CD34+ hematopoietic progenitors in the peripheral blood (PB), and extramedullary hematopoiesis. Since the antigenic properties of the circulating CD34 cells may yield clues to disease pathogenesis and have not been extensively studied, we used five-color flow cytometry to examine these cells from 20 well characterized patients with PMF and 10 normal controls. Bone marrow biopsies, molecular and cytogenetic studies were also reviewed. As expected, the percentages of peripheral-blood CD34 cells were significantly higher in the PMF patients (mean 1.4%, range, range 0.065–7.15) compared to the controls (mean 0.05%, range 0.01–0.57). The mean fluorescence intensity (MFI) values related to HLA-DR expression were increased (more than 3 fold) on the CD34+ cells in 12/20 (60%) PMF patients relative to normal control levels, while increased levels of CD13 were seen in 5/20 (25%) of PMF patients. CD33 and CD117 expression were similar on the CD34+ cells in both groups. Aberrant expression of lymphoid antigens was observed in 6/20 (30%) with CD7, 6/20 (30%) with CD4, and 3/20 (15%) with CD56 on CD34 positive cells in PMF. In the18 cases also studied with antibodies against CD45RA and CD123, the majority of CD34+ CD38 + cells phenotypically resembled megakaryocyte-erythroid precursors (CD45RA−, CD123−) in 5 cases, common myeloid progenitors (CD45RA−, CD123+) in 12 cases, and granulocyte-macrophage progenitors (CD45RA+, CD123 +) in 1 case. JAK2-V617F mutations were detected in 9 of 20 cases, but were present in only 1 of 5 cases showing predominately megakaryocyte-erythroid precursors. The percentage of CD34+ cells also expressing CXCR4 (CD184) appears to be increased in some patients relative to normal controls in contrast to other reported studies. In conclusion, the peripheral blood CD34+, progenitor cells in PMF patients are heterogeneous phenotypically resembling megakaryocyte-erythroid precursors in approximately 30% of cases, and common myeloid progenitors in approximately 70% of cases. In addition, these cells often show phenotypic abnormalities (increased intensity of HLA-DR and CD13 expression) that can be detected with flow cytometry relative to normal peripheral blood CD34+ cells. Patterns of antigen expression in PMF also appear to differ from those reported for CD34 positive cells in other myeloproliferative disorders which may help in early diagnosis and/or monitoring treatment responses.