Abstract

Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment is known to protect the cells from chemotherapy-induced apoptosis. Understanding the mechanisms of blast cell adhesion and adhesion-mediated chemotherapy resistance may be critical to improving response to chemotherapy. One study in AML patients (pts) proposed that expression of the adhesion receptor, VLA-4, is associated with chemotherapy resistance, leading to reduced survival (Matsunaga et al. 2003). VLA-4, composed of α4 and β1, binds to both the CS-1 domain of fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1), and has a major role in retaining stem/progenitor cells within the BM. We examined frozen BM samples from 175 AML pts (age 18–80, median 48) who underwent induction chemotherapy with anthracycline and cytarabine on Southwest Oncology Group clinical trials. Thawed samples were incubated in serum containing media with IL-3 and SCF for 16h prior to analysis. To assess the effect of freezing and thawing, we examined VLA-4 expression by cell lines and fresh versus frozen AML BM samples treated similarly, and found no difference in % expression. Flow cytometry was performed with gating on blasts by both forward vs. side scatter and CD45 vs. side scatter, and on viable cells by exclusion of 7-AAD. Both % positive cells and mean fluorescence intensity were measured. VLA-4 expression varied widely, with mean expression 60.6% (range 6.2–99.6%) for α4. VLA-4 functional adhesion was also measured. An in vitro cell adhesion assay to Fn peptide CH-296, performed on 20 randomly selected samples, revealed that pts with low α4 expression (<40% by flow cytometry) had virtually no adherent cells. The mean % of bound soluble VCAM-1 examined in 102 patient samples was 28.2%. Interestingly, overall survival (OS; p=0.028) and perhaps complete response (p=0.065) increased with increasing level of bound soluble VCAM-1. Estimated 5-yr OS was 29% (95% CI 12–46%) in 29 pts with VCAM-1 binding ≥40%, compared to 11% (CI 4–19%) in 79 pts with lower binding. Expression of the β1 activation dependent epitope 9EG7 was examined in 24 randomly selected samples before and after treatment with activating antibody 8A2, revealed pts expressed, on average, 57.5% of their maximum potential level of functional activation (range 6.0–100%). VLA-4 expression was also measured by real-time quantitative RT-PCR in 23 randomly selected pts. A significant correlation (Spearman’s R=0.44, p=0.037) was found between molecules VLA-4/μg RNA and α4 expression by flow cytometry. Fourteen pts with high levels of α4 (≥40% by flow cytometry) had a mean of 2.34x104 molecules VLA-4/μg RNA, compared to 1.68x104 in pts with lower α4. In these 175 pts, VLA-4 expression was not significantly associated with response to chemotherapy, or with disease free or OS. However, we found a significant correlation between functional status of α4, as indicated by soluble VCAM-1 binding, and longer OS. These data may suggest an adverse effect of functionally inactive α4 v. physiological response of α4+ cells to the BM microenvironment. Disruption of this and other binding interactions may enhance susceptibility to chemotherapy. These approaches need to be prospectively evaluated.

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