Stephen P. Mulligan, M.B., B.S., Ph.D. University of Sydney (Australia)

IMMUNOLOGY

Global phenotype profiling of chronic lymphocytic leukemia

Grant awarded in 2006

Abstract:

We have developed a new method called 'DotScan' that enables 82 markers to be identified on leukemia cells rather than the usual 15-20 using standard techniques. This method is simple and uses a glass slide coated with a specialized membrane (nitrocellulose) on which tiny amounts - about 10 nanolitres (10-8 litres) - of 82 different antibodies are applied. Cells from leukemia patients are then incubated on the slide, and particular markers on the cells are recognized by their corresponding antibodies; the cell is 'captured' on the array and does not wash off. The results are read by a simple scanner and displayed on a computer screen. By comparison, flow cytometry is the standard method for leukemia typing with relatively high capital cost, requiring a dedicated operator and relatively high volumes of expensive monoclonal antibodies - typically 5-20 µl (10-5 litres) per test. This technique becomes cost-limiting when more than 20 antigens are studied.

The DotScan microarray enhances our capacity to diagnose and classify different types of leukemia. In a study involving 796 patients, DotScan provided an unequivocal diagnosis when a clear leukemia clone or population is present. We have also obtained preliminary data using DotScan showing different antigen expression between patients with Chronic Lymphocytic Leukemia (B-CLL) with adverse prognostic factors (unmutated immunoglobulin variable genes and the intracellular signaling protein, ZAP-70) compared to patients with favorable prognostic factors.

We therefore propose to expand the array capacity to study CLL specifically by examining all surface antigens that are important in the biology of CLL cells. These include receptors involved in homing of the leukemia cell (adhesion receptors), receptors for survival and proliferation signaling, and receptors that induce the cell death pathway (apoptotic receptors). We believe this "Global CLL Phenotype Profiling" will lead to new insights on the biology of CLL and will be very valuable to both researchers and patients.