New Approach to the Mechanism of Hyperleukocytosis in Patients with APL After Treatment with ATRA.

To detect the new approach to the mechanism of hyperleukocytosis during induction therapy with ATRA in patients with acute promyelocytic leukemia.

Diagnosis of acute promyelocytic leukemia (APL) was performed according to the FAB cytological classification criteria and cytogenetic criteria. The collected leukemia cells were re-suspended in ATRA at 37°C for 96 h. Differentiation of leukemia cells was assessed by NBT reduction assay and indicated as percent of CD11b positive cells. Constant MTT assay and cell number count were used to detect the proliferation of leukemia cells after treatment with ATRA. For FACS analysis, cells were incubated with CD11b, CD54, CD106 and Ki67 mAb or isotypic control IgG1 antibody. L-CFU assay was taken to measure the colony formation. GM-CFU assay was used to detect the granulocyte-macrophage colony-forming unit. The mRNA expression of MMP-9, TIMP-1 was detected by RT-PCR.

(1) The results showed that the total number of WBC was up-regulated after treatment with ATRA in patients with APL, which was chiefly due to the up-regulation of myelocytes and more matured granulocytes. (2) The primary APL cells, unlike normal promyelocyte, have little requirement for survival factors, Its plating efficiency was also lower and could be up-regulated by exposure to ATRA. MTT assay showed that the OD value of primary APL cells was lower than that of NB4 clone cells or normal hematopoitic cells. Otherwise, the proliferation of primary APL cells, post exposure to ATRA, was up-regulated and more cytokine-dependent or more sensitive to G-CSF stimulation. (3) Most importantly, the division hypothesis should be taken into consideration, because the leukocytosis chiefly contributed to the up-regulation of differentiated myelocytes and more matured neutrophils, which should be the key proliferation pool as the normal myelocytes. The primary APL cells had a low proliferation potential, the mitotic cells are often scarcely detected even for 4 d in culture. Most of primary APL cells had a growth arrest in G0/G1 or G0 phase. The APL cells occur to switch from G0 into cell cycle that was indicated by the up-regulation of Ki67 antigens. In our opinion, most of APL cells enter into cell cycles after exposure to ATRA or arsenic trioxide in vivo, with more mitotic cells. (4) The rheological hypothesis was taken into consideration. The up-regulation of adhessive ability of APL cells, which resulted in more release from bone marrow into peripheral blood. The results indicated that ATRA can induce differentiation of the malignant cells, most remarkable feature was the progressive change of malignant cells with signs of their terminal differentiation and with Auer rods being sometimes observed in mature cells. Usually, the percent of promyelocyte was down-regulated and the percent of myelocyte, metamyelocyte and more matured myeloid cells was obviously up-regulated, especially for the percent of myelocyte or myelocyte-like cells. These results suggest that an asynchronism between rheological and morphological maturation in each APL cell might explain the occurrence of hyperleukocytosis in some patients during ATRA therapy. For example, the serum sICAM1 and sVCAM1level was up-regulated, the secretion up-regulation of adhesive molecules ICAM1 and VCAM1 in primary APL cells was also observed, as inagreement with the up-regulation of CD11b, CD54 and CD106. The adhessive coefficiet was also up-regulated.

The leukocytosis or hyperleukocytosis chiefly contributed to the up-regulation of myelocyte-like cells, which chiefly contributed to the more myelocytes divisions and more sensitive to cytokine stimulation. Partly due to the up-regulation of adhesive index and adhesion molecules of differentiationed leukemia cells, which could easily resulted in the release of APL cells from bone marrow to peripheral blood.

No relevant conflicts of interest to declare.