Poster Board II-634
Adhesion of AML blasts within the bone marrow microenvironment confers chemotherapy resistance. CXCR4 plays a primary role in the retention of stem cells within the marrow, and is associated with poor prognosis in AML (Rombouts et al Blood 104:550, 2004, Konoplev et al Cancer 109:1152, 2007; Spoo et al Blood 109:786, 2007). A CXCR4 inhibitor enhanced chemotherapy cytotoxicity for human AML cells on stroma (Zeng et al Mol Cancer Ther 5:3113, 2006). VLA4 expression is also correlated with prognosis in AML, and our group and others have shown that disruption of VLA4 enhances chemotherapy cytotoxicity (Matsunaga et al Nat Med 9:1158, 2003; Becker et al Blood 113:866, 2009). However, given the number of potential ligands and receptors involved in the interaction of leukemia cells within the bone marrow stroma, there are likely other important binding sites. Fundamental to determining mechanisms to overcome drug resistance will be identification of all critical interactions responsible for retention and/or adhesion to the bone marrow stroma. We therefore sought to fully characterize all potential adhesive interactions utilized by AML blasts. We also examined the leukemia stem cell population, the small number of leukemia cells that are able to sustain and perpetuate the leukemia. We have now defined the adhesion receptor expression pattern (18 receptors) for populations of AML blasts, including circulating vs. marrow, and putative stem cell populations as defined by surface markers. Bone marrow and peripheral blood samples have been obtained at presentation for a total of 38 patients with AML. We performed flow cytometry analysis, gating on sequential populations of cells by forward scatter/side scatter, CD45 vs. side scatter, CD34+, or CD34+/CD38-/CD123+ populations. Expression levels were measured by percent of positive cells (%EXPR) and mean fluorescence intensity (MFI). Our analysis of adhesion receptor expression by AML blasts revealed that some receptors were expressed by 85%–92% of blasts, including CD11a, CD29, CD44, CD49d, CD49e, CD31; some were expressed by <5%, including CD49a, CD49c, CD106; and others exhibited moderate expression 18–62%, such as L-selectin, ICAM-1, CXCR4, CD49f. For a given patient, there was no significant correlation between CXCR4 % EXPR or MFI and expression of other receptors, such as CD49d, CD49f, or CD29. However, there was significant positive correlation (by Spearman rank order correlation) between %EXPR on marrow vs. circulating blasts from the same patient for CD29 [p=.01], CD11a (LFA-1) [p=.004], CXCR4 [p=.03], CD31 (PECAM-1) [p=.03], L-selectin [p=.007], CD44 [p=.04], while others were different, including CD49d [p=.41], CD49e [p=.08], and CD29f [p=.17] (all two-sided p values). When comparing the whole CD34+ population to the CD34+CD38-CD123+ population, there was no significant difference in MFI for the receptors CD29, CD44, or CD49f, but the MFI of CD49d and CD49e was significantly lower on the more primitive (stem) cells [p=0.003 for each ]. When we examined the CD34+/CD38-/CD123+ population isolated by sequential CD38 depletion then CD34 selection, and gated on CD123+ cells, we found uniform high percent expression >90% for CD49d, CD49f, CD44, and the percent expression of CD49f was also lower for the primitive cells in some patients. Thus, there are several adhesion receptors expressed at high level that may contribute to biological behavior and chemotherapy resistance. Overcoming chemoresistance in leukemia stem cells may require targeting of multiple receptors in order to achieve a clinical response.
Becker:Leukemia and Lymphoma Society: Research Funding.
Asterisk with author names denotes non-ASH members.