Evaluation Of CLL Adhesion Molecule Expression As a Diagnostic Tool For Disease Progression

Chronic lymphocytic leukemia (CLL) is a chronic B cell lymphoproliferative disorder characterized by small mature neoplastic lymphocytes, which primarily involve blood, bone marrow, and/or lymph nodes. Complex cellular and molecular interactions between CLL cells and the bone marrow or lymph node have been shown to affect CLL proliferation, survival and confer drug resistance that may be responsible for residual disease after conventional therapy. The standard methods used to assess CLL patient survival and treatment requirements involve the Rai and Binet staging systems. Pathologic markers such as CD38 expression by flow cytometric analysis, immunoglobulin heavy chain variable region (IgVH) mutation status, and conventional cytogenetics also demonstrate loose association with overall patient survival. However, these systems cannot identify stable or progressive forms of the disease in individual patients, especially in the early stages of CLL.

The objective of this study is to evaluate the expression profile of specific adhesion molecules that may regulate critical interactions between CLL cells and the bone marrow as potential dynamic biomarkers that correlate with CLL disease progression and response to chemotherapy. We evaluate the co-surface-expression of the molecular scaffold protein, CD82, with its interacting integrin partner, alpha 4 on CLL cells. We correlate the expression of these adhesion molecules with the patient’s clinical stage, lymphocyte doubling time (LDT), IgVH mutation status, conventional cytogenetics and the expression level of CD38 antigen on CLL cells.

A single-institution study prospectively enrolled CLL patients diagnosed and followed up at a university-based cancer center. Peripheral blood samples were obtained from consented patients at the time of their follow up appointments. Peripheral blood mononuclear cells were isolated from the whole blood using a ficoll density gradient and CLL cells were isolated by magnetic bead negative selection. Using flow cytometry, CLL cells were analyzed for alpha 4 and CD82 surface expression and the median fluorescence intensity values were correlated with the patient’s clinical staging parameters (Ann Arbor system, Rai stage classification) and LDT. A non-parametric measure (Spearman’s rho) was used to assess the correlation between variables. The correlation analyses were stratified by indolent cases versus aggressive disease cases and, also by the IgVH mutation status and the presence of CD38 overexpression and cytogenetics at the time of diagnosis. An unpaired t-test was used to compare differences in the CD82 and alpha 4 expression among groups.

At the time of abstract submission, a total of 18 patients were enrolled in the pilot study. Nine patients had indolent disease and had not received treatment for CLL, whereas nine patients had received treatment for CLL at the time of enrollment. CD82 and alpha 4 expression data were not obtained for three patients on active treatment due to severe lymphopenia. CD82 and alpha 4 data were obtained for all of the indolent cases. IgVH mutation status and CD38 expression data at diagnosis were evaluated when available. A statistically significant correlation (P< 0.05) was observed for the co-expression of CD82 and alpha 4 in all of the cases. When analyzed by subgroups, the co-expression of CD82 and alpha 4 remained statistically significant in the indolent disease group and there was a similar trend in the aggressive disease group that did not reach statistical significance. In the aggressive disease group, CD82 expression negatively correlated (P<0.05) with lower absolute lymphocyte counts and a lower platelet counts at the time of enrollment in the study. In addition, we observed a trend of negative correlation between the CD82 expression and LDT in the indolent disease group. For those patients with CD38 overexpression a trend to a higher expression of CD82 and alpha 4 was found.

In this initial data set, we observed trends of correlation of CD82 and alpha 4 integrin expression with platelet count, absolute lymphocyte count, LDT and CD38 overexpression. In future work, we will continue to recruit more patients to enhance the statistic power of the study. Furthermore, we will evaluate the expression level changes of CD82 and alpha 4 during treatment and their role in response to chemotherapy and CLL metabolic activity.

No relevant conflicts of interest to declare.