The Extramedullar Dissemination of Acute Myeloid Leukemic Cells Depends on CD31 (PECAM-1) and CD38 Coexpression Level.
Authors: Herault Olivier , Gallay Nathalie , Domenech Jorge , Colombat Philippe , Binet Christian 
Published: October 2019

Abstract
The number of peripheral white blood cells (WBC) shows a wide range in acute myeloblastic leukemia (AML) patients. A high WBC count constitutes an adverse prognosis factor when at presentation and (myelo)monocytic subtypes were more frequently associated with extramedullar tumor sites. Marrow endothelial cells express CD31 and CD31/CD31 interaction is known to promote the migration in physiological mechanisms, as for example transendothelial migration of neutrophiles and monocytes in diapedesis. CD31 is a specific receptor of CD38 which is associated with numerous molecules on the surface of blood cells. Moreover, CD38 could interact with hyaluronate, a component of the extracellular matrix, 2 hyaluronate-binding sites having been reported in its extracellular domain. These elements lead us to postulate that CD31 and CD38 are colocalized on AML cells and that an excess of CD31 promotes the egress of the cells from the marrow compartment, while an excess of CD38 favors their anchorage to marrow microenvironment.
The CD38/CD31 colocalization was demonstrated using FRET and cocapping strategies. FRET experiments were performed with a 488 nm laser flow cytometer, Cy3-conjugated anti-CD38 mAb and FITC-conjugated mAb against CD31. For cocapping experiments, we induced capping of CD31 with anti-CD31 mAb at 37°C; the cells were then fixed and labeled with anti-CD38 mAb. Transendothelial migration has been studied using anti-CD31 mAb and anti-CD38 blocking mAbs in Transwell® experiments performed with TrHBMEC cell line. To study the CD38/hyaluronate interaction, cells were treated or not with all-trans retinoic acid to induce CD38 overexpression just before plating the cells onto hyaluronate-coated dish. The in vivo influence of CD31 and CD38 coexpression level was evaluated by studying the phenotype of marrow leukemic cells (S/N ratio of the MFI) and the WBC count of 78 consecutive patients with newly diagnosed de novo AML. In all experiments, P value <0.05 has been considered as statistically significant.
FITC-conjugated anti-CD31 induced increase in MFI of Cy-3 by 108.4% and we observed a CD31/CD38 cocapping. The inhibition of migration by anti-CD31 blocking mAb ranged from 2% to 66% of control and was correlated to the CD31 expression level (R=0.9236; P<0.0001), while migration in presence of anti-CD38 blocking mAb was about 80% of the control without correlation with CD38 expression level, suggesting a lesser role in the transendothelial migration process. The blasts exposed to ATRA exhibited a 1.3±0.1-fold increase in the CD38 expression (S/N of MFI) and a 40±10 % increase in adhesion to hyaluronate. The expression of CD31 and CD38 on the membrane of marrow leukemic cells of the AML patients were correlated (R=0.6621; P<0.0001). Moreover, peripheral WBC count was correlated with the CD31/CD38 ratio (R=0.5286; P=0.0129) and an excess of CD31 antigen was associated with an increased peripheral WBC count when comparing patients with ratio >1 to those with ratio <1 (P<0.0001). Interestingly, CD31/CD38 ratio was higher in AML (myelo)monocytic subtypes when comparing M4/M5 to all other FAB subtypes (1.2±0.1 and 0.7±0.1, respectively; P<0.0001).
These findings suggest that extramedullar dissemination of AML cells depends on CD31 and CD38 coexpression level and give new insights into novel strategies to reduce the number of extramedullar tumor sites, notably in AML (myelo)monocytic subtypes.