In Vitro Chemosensitivity of Leukaemic Stem and Progenitor Cells to Gemtuzumab Ozogamicin (Mylotarg) in AML.

Preliminary data from 1115 patients entered into the MRC AML 15 trial indicated that the addition of Gemtuzumab Ozogamicin (GO) to induction chemotherapy improved disease free survival (Abstract #13, ASH 2006). We hypothesised that this improved survival may be underpinned by the specific therapeutic targeting of leukaemic stem and progenitor cells (LSPC). The LSPC subset of AML cells contains those cells capable of self-renewal in culture and of recapitulating leukaemia in animal models. Successful chemotherapeutic targeting of this subset is essential for complete eradication of leukaemia. We have devised a flow cytometric assay which allows us to measure the in vitro chemosensitivity of the LSPC (CD34+CD38-CD123+) subset in as few as 100 cells and we have used the assay to screen the effectiveness of GO against LSPC. CD123 expression is a determining cell surface marker for leukaemic versus normal stem cells and we were able to demonstrate a significant difference in CD123 MFI values between CD34+CD38- of leukaemic (n= 16) versus normal CD34+ CD38- cells (n= 5; p=0.03), demonstrating the sensitivity of our flow cytometric assay in detecting this leukaemic subset. Blast cells from 14 AML samples were treated with GO (10ng/ml) for 48 hours in an in vitro culture system that maintains LSPC viability. A significant reduction in the number of LSPC (n=14; median 46% cell kill; p= 0.002) as well as AML bulk cells (n=14; median 16% cell kill; p= 0.005) was achieved. This data demonstrates the chemosensitivity of AML cells to GO, particularly to the LSPC subset (p=0.001). Also, the total percentage of LSPC at the start of the assay was found to be positively correlated with GO chemosensitivity (p<0.0001) at 48 hours in in vitro culture (n=14). We have extended culture time for up to 96 hours and preliminary data suggest a further achievable LSPC kill (median 51% cell kill; n= 8). CD33 expression in bulk and CD34+ CD38- populations was explored in the same AML patients. Although CD33 MFI values were highly variable (n= 16; Median = 34.82 and range= 3.7 – 116.54 in bulk fraction and median = 13.69 and range= 0.47 – 436.73 in CD34+ CD38- fraction), we found a significant correlation in CD33 MFI values between bulk and CD34+ CD38- cells (p< 0.0001). Also, the total percentage of CD34+CD38-CD33+ cells was found to be positively correlated with LSPC GO chemosensitivity (n= 14; p= 0.04) after 48 hours of in vitro culture. The GO chemosensitivity of mononuclear cells from mobilised healthy donors was investigated and these were found to be insensitive to this agent both at the bulk cell level and in the CD34+ CD38- subset (mean % cell kill of 10% and 5%, respectively; n=3) after 48 hour in vitro culture. This data establishes the specific targeting of GO to CD123+ CD34+ CD38- and CD33+CD34+ CD38- LSPC, while sparing normal stem and progenitor cells. In conclusion, with many novel agents and drug combinations available for research, we have developed an assay for screening drug effectiveness against LSPC and have demonstrated that GO targets this subset effectively. Combination drugs with GO now need to be further investigated for the complete eradication of LSPC.