Molecular and Functional Analysis of Large Granular Lymphocyte Expansions in Chronic Myelogenous Leukemia Patients Undergoing Tyrosine Kinase Inhibitor Therapy.

Abstract 2204

Poster Board II-181

Tyrosine kinase inhibitor (TKI) therapy has become the standard treatment of chronic myelogenous leukemia (CML). Although all of these drugs directly inhibit bcr-abl, off target effects are potentially responsible for their side effects. Of these TKIs, dasatinib is effective for the treatment of imatinib-resistant Philadelphia chromosome-positive CML. It has been shown that some dasatinib treated patients' exhibit adverse side effects resulting in colitis, pleural effusions and fevers. In conjunction with these adverse side effects, patients have been shown to present with oligoclonal expansions of large granular lymphocytes (LGL). We have now further characterized this phenomenon in fifty-six patients treated with five different TKIs. Age matched healthy controls as well as untreated newly diagnosed CML patients were used for comparisons. TCR clonality was determined using total cellular DNA subjected to amplification with a series of oligonucleotide primers for regions of the T-cell receptor genes and a flow cytometry based V-beta repertoire panel. Levels of activated nuclear T-bet and NFκB were analyzed by utilizing the respective Active Motif's Trans-AM T-bet and NFκB family kit in conjunction with the nuclear extraction kit according to established protocol. An analysis of membrane-bound IL-15 in LGL cells was determined by flow cytometry with commercially available mouse anti-human IL-15 in conjunction with CD3, CD8, CD16, CD56 and CD57. PDGF BB plasma levels were determined with ELISA techniques using plates generated from commercially available antibodies. Regulatory T cell percentages were investigated also by flow cytometry using mouse anti-human CD4, CD25 and CD127. Patients on dasatinib therapy with and without side effects show an elevated membrane bound IL-15, as well as modest elevated PDGF-BB plasma levels, both of which have been shown to be important in LGL survival. Patients with LGL expansions had constitutively active NFκB and variable T-bet expression in the CD8+ compartment in the setting of alpha/beta TCR clonality. Imatinib treated patients without significant side effects show broader plasma levels of PDGF-BB as well as an elevated membrane bound IL-15 expression with variable T-bet and NFκB expression. All patients treated with nilotinib showed low expression of IL-15, T-bet and NFκB expression as well as low levels of PDGF-BB. TCR clonality with or without LGL expansions is a relatively common phenomenon in CML patients under therapy with TKI's. In patients treated with dasatinib, some adverse side effects typically related with this drug are more frequently associated with this event. However, the ultimate mechanism of these clonal expansions is still unclear. A decreased percentage of regulatory T-cells or enhancement of antigen presentation capability that overcomes tolerance mechanisms has been described by our group and could play an important role. Finally, the inhibition of tyrosine kinases involved in the regulation of memory lymphocytes development is under investigation in our laboratory.