Kinetics of CD34+ Cells in the Peripheral Blood of Patients with Primary Myelofibrosis Undergoing Splenectomy
Authors: Campanelli Rita , Massa Margherita , Bonetti Elisa , Viarengo Gianluca , Rosti Vittorio , Gattoni Elisabetta , Moretti Enza , Bergamaschi Gaetano , Villani Laura , Lucioni Marco , Bandiera Laura , Riboni Roberta , Barosi Gianni 
Published: October 2019

Abstract
We have previously shown that primary myelofibrosis (PMF) is characterized by an increased number of circulating CD34+ cells and that the membrane expression of CXCR4 on these cells is significantly lower than in healthy subjects in terms both of percentage and of mean fluorescence intensity (MFI). The advanced phase of PMF is characterized by the accumulation in various organs of myeloid progenitor and precursor cells in a process known as extramedullary hematopoiesis. The accumulation and growth of these cells in the spleen lead to the pathologic enlargement of the organ and, in a limited number of cases, splenectomy is required. We have investigated the kinetic of circulating CD34+ and CD34+CXCR4+ cells in patients with PMF before and after splenectomy. We have also compared the percentages of CD34+ and CD34+CXCR4+ cells found in the peripheral blood (PB) with those found in cells derived from spleen tissue samples, obtained soon after splenectomy. Five milliliter of PB in EDTA was obtained from 8 patients with PMF and from 2 healthy subjects (CTRLs), who underwent splenectomy because of traumatic injury of the organ, before (T0) and at different time points (24 hours, 72 hours, and 14 days) after splenectomy. In 6/8 patients and in the 2 CTRLs, spleen samples were obtained immediately after the removal of the organ. After extensive rinsing, each spleen sample was minced to obtain a single cell suspension. For cytofluorimetric analysis, PB and spleen cells were stained with FITC-conjugated anti-CD34 and PE-conjugated anti-CXCR4 monoclonal antibodies. The membrane expression of CXCR4 was evaluated as percentage of CD34+ cells expressing the antigen and as CXCR4 MFI of CD34+ cells. Results are expressed as median (range) or mean where appropriate. The median percentage of circulating CD34+ cells in patients with PMF gradually decreased after splenectomy [24h: 5.5 (0.6–20.1); 72h: 3.1 (0.9–8.5); 14d: 1.6 (0.5–6.7)] with respect to T0 [3.7 (0.2–17.1)]. Vice versa, the percentage of circulating CD34+ cells expressing the CXCR4 antigen increased after the intervention [(24h: 27.3 (12.0–59.6); 72h: 51.4 (24.7–87.7); 14d: 49.0 (34.0–59.7)] with respect to T0 [23.3 (6.2–27.3)]. CXCR4 MFI similarly increased (not shown). In 3/13 patients, assessed 4 months after splenectomy a further decrease of the percentage of circulating CD34+ cells [0.65 (023–1.95)] and a stable percentage of CD34+ cells expressing CXCR4 [44.0 (29.4– 56.0)] were observed. The mean percentage of circulating CD34+ cells in the 2 CTRLs showed an irregular tendency after splenectomy (24h: 0.39; 72h: 0.6; 14d: 0) with respect to T0 (0.7) whereas the mean percentage of circulating CD34+ cells expressing CXCR4 did not change after the intervention (24h: 72.5; 72h: 76.3; 14d: 88) with respect to T0 (82.5). CXCR4 MFI was stable too (not shown). In the spleen of the 6 PMF patients in whom a spleen tissue sample was obtained, the median percentage of CD34+ cells [6.1(2.4–17.5)] was higher (p<0.05) than in their PB at T0 [1.4 (0.2–7.0)]; on the contrary, the percentage of CD34+ spleen cells expressing CXCR4+ was decreased [10.4 (3.9–20)] with respect to that found at T0 in PB cells [22.6 (21.6–25.1)]. The spleen samples of PMF patients showed a percentage of CD34+ cells higher (p<0.05) than that of CTRLs [0.8 (0.5–1.2)], while both the percentage and the MFI of CD34+CXCR4+ cells were comparable (not shown). Taken together, these data support the hypothesis that most of the circulating CD34+ cells of patients with PMF before splenectomy derives from the spleen. The removal of this organ is associated with a decrease of the percentage of circulating CD34+ cells, with an increased expression of CXCR4 on these cells, which is still evident a few months after the intervention. These observations also suggest that, in addition to the spleen, other organs in which extramedullary hematopoiesis takes place can release into the PB different amounts of CD34+CXCR4+ cells.