Dasatinib is a second-generation tyrosine kinase inhibitor (TKI), which is used successfully in the treatment of chronic myeloid leukemia (CML). Dasatinib has two unique features when compared to other TKIs (imatinib, nilotinib); first, dasatinib has a significantly shorter half-life in the plasma and second, dasatinib inhibits a wider spectrum of kinases, including several kinases known to be important in the function of the immune system (src, tec, and syk families), which are not affected by the other TKIs. Interestingly, it has been recently shown that both short-term exposure to dasatinib in vivo and long-term treatment with dasatinib improves NK-cell cytotoxicity, however, the mechanisms are not known.
To study the improved cytotoxicity observed in dasatinib-treated patients, we aimed to perform a complete NK-cell phenotyping in these patients. Finally, our goal is to correlate NK-cell phenotype with NK-cell cytotoxicity, and to study the possible correlation between phenotypical changes, NK-cell function and clinical outcome.
This study included 19 dasatinib-treated (DA) CML patients, both first-line (n=7) and second-line (n=12). To investigate the specificity of the immunomodulatory effects of dasatinib, a control group of 9 CML patients treated with imatinib (IM) and another group of 12 healthy donors (HD) were included. Peripheral blood samples obtained before the patients took their daily drug dose were phenotyped with a comprehensive 8-color flow cytometry panel (total 32 antibodies, table 1).
To study the correlation between phenotypical changes and NK-cell cytotoxicity, we performed a standard CD107 degranulation assay. Mononuclear cells were incubated for 6 hours in the presence of the target cell line K562 and a CD107 antibody. CD107 positive NK-cells were then phenotyped with the same panel of 32 antibodies.
All results are summarized in table 1. In brief, DA- and IM-treated CML patients and HD had equal proportions of NK-cells (CD3negCD56+) of total lymphocytes. Regarding trafficking molecules, NK-cells in both DA- and IM-patients had a lower frequency of the chemokine receptor CCR7 when compared to HD. This suggests a reduction in the NK-cell population that is able to migrate to lymph nodes, and is likely caused by the disease or TKIs in general. Moreover, DA-treatment specifically decreases the expression of the homing molecule CD62L in NK-cells.
In addition, NK-cells in DA-patients, when compared to IM and HD, expressed less CD11b and significantly more often CD11c and HLA-DR, which reproduce the immunophenotypic changes that typically occurs in recently activated NK-cells and has been shown to associate with improved clinical benefits. Conversely, increased expression of CD57 together with a lower frequency of CD27 and CD28 were observed in both groups of patients and were similar to those typically observed in conditions of chronic NK-cell stimulation.
In contrast, DA-patients had a lower frequency of most of the studied NK-receptors (Nkp30, Nkp46, NKG2D, CD94, CD161, KIR2DL1/S1) when compared to IM and HD. This suggests that NK-cells in DA-treated patients have a more mature phenotype, which is caused by the treatment.
NK-cells in TKI-treated CML patients display a mature phenotype, which is often observed after chronic stimulation suggesting that TKIs have immunomodulatory effects on NK-cells or the disease itself causes the changes. Interestingly, NK-cells in DA-treated patients express a highly differentiated phenotype characterized by high expression of CD57, and decreased expression of Nkp30, Nkp46 and CD161. Similar changes were not seen in IM-patients or HD. It is possible that NK-cells expressing this phenotype might also represent those NK-cells that have previously been driven into clonal expansion by encounters with pathogens because of the specific immunomodulatory effects of dasatinib. This phenotype of highly mature NK-cells, which is associated with high cytolytic potential, could be responsible for the previously described enhanced NK-cytotoxicity caused by dasatinib. In accordance, our preliminary results suggest that these unique phenotypic changes observed in DA-treated patients correlates with the cytotoxic potential. Studies to correlate these results with therapy outcome are ongoing.
Garcia:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Research Funding. Steegmann:Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau.
Asterisk with author names denotes non-ASH members.