Abstract
The cure of chronic myeloid leukemia (CML) following allogeneic stem cell transplantation (SCT) is attributed to a graft-versus-leukemia (GvL) effect which eliminates residual leukemia cells that might otherwise have the capacity to regenerate the leukemia. Whether the GvL effect is directed against the most primitive leukemia stem cell or only against a more mature leukemia progenitor cell is unknown. In contrast, although tyrosine kinase inhibitors (TKI), notably imatinib, effectively suppresses at least 2 logs of CML hematopoiesis in most patients, they appear not to affect the leukemia stem cell or the most primitive progenitor and so fail to eradicate the leukemia. To characterize further the GvL reaction and to identify targets that might be exploited for vaccine therapy outside the context of a stem cell transplant, we studied expression of selected proteins in CML progenitors at various levels of commitment. CD34+ progenitor subpopulations were purified by fluorescence activated cell sorting from 10 CML patients (5 chronic phase, 3 accelerated phase and 2 blastic phase) and from 8 healthy individuals. We used real-time quantitative polymerase chain reaction (RQ-PCR) for BCR-ABL, WT1, proteinase 3 (PR3), β-catenin and BMI-1. Hematopoietic stem cells, CD34+CD38-Lin-CD90+ and primitive progenitor subpopulations CD34+CD38- Lin- CD7+, CD34+CD38+ Lin-CD7+, and more differentiated CD34+CD38+ Lin + CD7- and CD34+CD38+ Lin+ CD7+ populations were collected. In addition, CD34+CD38+Lin- cells were sorted into common myeloid progenitor (CMP) CD34+CD38+Lin- IL3Rα+CD45RA- and granulocyte-macrophage progenitor (GMP) CD34+CD38+Lin- IL3Rα+CD45RA+ populations and their CD7+ and CD7- subpopulations collected. Mature myeloid progenitor subpopulations CD34+CD38+Lin-CD33+ were also separated into CD7+ and CD7- groups. All primitive leukemic CD34+ subpopulations expressed similar levels of BCR-ABL mRNA and contained greater than 80% BCR-ABL positive cells by fluorescence in situ hybridization. By RQ-PCR we detected higher expression of the leukemia-associated antigen WT1 in the most primitive CD34+CD38-CD90+Lin- subset of hematopoietic progenitors in CML compared to normal. In more mature CMP and GMP populations, the expression of both WT1 and PR3 was higher in CML than normal. The CD7+ subpopulation of GMPs in CML, which are implicated in disease progression, also have higher expression of leukemia-associated antigens than normal. The expression of β-catenin and BMI-1 was lower in CML CD34+ progenitors in comparison to those from healthy individuals in all subpopulations except in CD7+ GMPs, indicating low self-renewal activity. These results suggest that the most primitive leukemic hematopoietic progenitors, as well as their more differentiated progeny, express WT1 and PR3 at relatively high levels and may thus be targets for peptide-based vaccines. The mechanism that protects primitive leukemic progenitors from the effects of TKI remains undefined but may be related to their quiescent status or to their high levels of BCR-ABL expression.
The Elusive Chronic Myeloid Leukemia Stem Cell: Does It Matter and How Do We Eliminate It?
