Splenic pooling and loss of VCAM-1 causes an engraftment defect in patients with myelofibrosis after allogeneic hematopoietic stem cell transplantation

Abstract

Myelofibrosis is a myeloproliferative neoplasm that results in cytopenia, bone marrow fibrosis and extramedullary hematopoiesis. Allogeneic hematopoietic stem cell transplantation is the only curative treatment but is associated with a risk of delayed engraftment and graft failure. In this study, patients with myelofibrosis (n=31) and acute myeloid leukemia (n=31) were analyzed for time to engraftment, graft failure and engraftment-related factors. Early and late neutrophil engraftment and late thrombocyte engraftment were significantly delayed in patients with myelofibrosis as compared to acute myeloid leukemia, and graft failure only occurred in myelofibrosis (6%). Only spleen size had a significant influence on engraftment efficiency in myelofibrosis patients. To analyze the cause for the engraftment defect, clearance of hematopoietic stem cells from peripheral blood was measured and immunohistological staining of bone marrow sections was performed. Numbers of circulating CD34⁺ were significantly reduced at early time points in myelofibrosis patients, whereas CD34⁺CD38^- and colony-forming cells showed no significant difference in clearance. Staining of bone marrow sections for homing proteins revealed a loss of VCAM-1 in myelofibrosis with a corresponding significant increase in the level of soluble VCAM-1 within the peripheral blood. In conclusion, our data suggest that reduced engraftment and graft failure in myelofibrosis patients is caused by an early pooling of CD34⁺ hematopoietic stem cells in the spleen and a bone marrow homing defect caused by the loss of VCAM-1. Improved engraftment in myelofibrosis might be achieved by approaches that reduce spleen size and cleavage of VCAM-1 in these patients prior to hematopoietic stem cell transplantation.

Figure 1.

Myelofibrosis (MF) patients show significant delayed early and late engraftment as compared to acute myeloid leukemia (AML) patients. Cumulative incidence of early (A) and late (B) neutrophil and early (C) and late (D) platelet engraftment in MF and AML patients.

Figure 2.

Spleen size and splenectomy but not donor type are associated with improved engraftment in myelofibrosis (MF) patients. Cumulative incidence of engraftment according to spleen and donor characteristics. Early (A) and late (B) neutrophil and early (C) and late (D) platelet engraftment in MF patients with regard to spleen size. Early and late neutrophil engraftment in MF (E and F) and acute myeloid leukemia (G and H) patients with regard to donor type. MRD: matched related donor; MUD: matched unrelated donor; MMUD: mismatched unrelated donor.

Figure 3.

Overall survival is equal in myelofibrosis (MF) and acute myeloid leukemia (AML) patients but transfusion needs significantly differ. (A) Kaplan-Meier estimate of survival in MF and AML patients with a follow up of 24 months after allogeneic stem cell transplantation. Average number of erythrocyte (B) and platelet (C) transfusions in MF and AML patients on day 28 and 100 after allogeneic stem cell transplantation.

Figure 4.

Clearance of CD34⁺ cells in myelofibrosis (MF) patients is significantly different to acute myeloid leukemia (AML) patients. Clearance of CD34⁺ cells after allogeneic stem cell transplantation in MF and AML patients at defined time points. (A) Representative dotplot of CD34 and CD38 stained HSC within the peripheral blood of one recipient. Clearance of CD34⁺ cells (B) and proportion of CD38⁻ cells within the CD34⁺ cell fraction (C) in MF (n=5) and AML (n=5) patients. (D) Representative white (CFU-M) and red (BFU-E) colonies after hematopoietic stem cell transplantation. (E) Clearance of colony-forming cells (CFC) in MF and AML patients at defined time points after transplantation.

Figure 5.

Peripheral blood (PB) hematopoietic stem cells (HSC) express common homing receptors but loss of VCAM-1 is detected in the bone marrow of myelofibrosis patients prior to hematopoietic stem cell transplantation (HSCT). (A) Flow cytometric analysis of the homing receptors CD44, CD184, CD49d, CD49e and α9β1 integrin on CD34⁺CD38⁺ and CD34⁺CD38⁻ cells. (B) Representative immunohistology staining of VCAM-1 on acute myeloid leukemia (AML) and MF patients derived BM sections shortly before conditioning chemotherapy as compared to isotype control and one representative MF BM section eight months after HSCT. (C) Immunohistochemical rating score (IRS) for VCAM-1 expression in BM of MF patients (before HSCT n=11, after HSCT n=5), AML patients (n=4) and healthy controls (n=6). Each data point represents the individual VCAM-1 IRS and bar represents the median of sample groups. (D) Expression of soluble VCAM-1 in the serum of MF patients (n=8) as compared to AML patients (n=3) and healthy controls (n=8). Each data point represents the mean individual sVCAM-1 concentration assayed in duplicate and bar represents the mean of sample groups. *P<0.05