Disease Propagating Blasts in Standard and High Risk Acute Lymphoblastic Leukemia Are Frequent and of Diverse Immunophenotype.

Abstract 1421

Poster Board I-444

Conflicting results in the field of cancer stem cells have reignited debate regarding the frequency and identity of cells with the ability to self renew and to propagate the complete phenotype of the malignancy. Initially it was suggested by different studies that cancer stem cells represent only a small minority of the malignant population and that the immunophenotypes of these cells resemble a rather immature type in the cell hierarchy. More recent data from our own and other groups have challenged these findings by demonstrating that cells at different maturity levels within the leukemic hierarchy have cancer stem cell abilities and that the frequency of the leukemia maintaining cell is higher than previously thought (Cancer Cell 2008, 14(1), p47-58). We use an in vivo NOD/scid IL2Rγnull (NSG) mouse intra-femoral transplant model to determine the clonogenicity of sorted candidate leukemic stem cell populations, characterized by specific immunophenotypes. We selected the surface markers CD10 and CD20, in order to differentiate between rather immature and more mature cells. Furthermore we carried out limiting dilution experiments on sorted (CD20) and unsorted leukemic blasts to investigate the frequency of the proposed leukemic stem cells. Flow sorted ALL blasts of CD19+CD20low and CD19+CD20high as well as of CD19+CD10low and CD19+CD10high immunophenotype were transplanted into NSG mice. Sorts were performed on primary patient material and on leukemic blasts that had been harvested following prior passage in mice. Different subtypes of ALL were included (high risk: BCR/ABL (t9;22) positive (patients L4967, L4951, L49101, L8849, L2510), high hyperdiploid/MRD positive high risk (L754, L835), intermediate risk: high WBC/MRD negative (L736, L784), age >10 years (L803)). CD20 sorts were performed on primary patient material (L4951, L49101, L754, L835 and L776), on secondary samples harvested from engrafted primary mice (L4967, L4951, L2510, L736 and L754) and on tertiary samples harvested from engrafted secondary mice (L4967 and L736). In total 151 mice were transplanted, with 122 showing engraftment in consecutive bone marrow punctures or in bone marrow harvests. CD10 sorts were performed on primary patient material (L784 and L49101) and on secondary samples harvested from engrafted primary mice (L4951, L8849, L2510 and L803) with 31 out of 52 mice transplanted with sorted material showing engraftment as seen with CD20 sorted cells. Blasts of all selected immunophenotypes were able to engraft the leukemia in unconditioned NSG mice as determined by 5 color flow cytometry. In particular, sorted cells of both fractions were able to reconstitute the complete phenotype of the leukemia. Harvested cells from engrafted mice could then be re-sorted into high and low antigen expressing fractions and successfully re-engrafted on secondary and tertiary mice. Cell purities of transplanted cells were usually higher than 90% (range 67-100%). The ability of all populations to serially engraft mice demonstrates long-term self-renewal capacity. Two additional patients were used in the limiting dilution assays (high WBC/t(4;11) high risk (L826); low WBC/MRD negative low risk (L792)) and experiments were performed on primary unsorted and secondary sorted material. Cell numbers necessary for ALL engraftment differed between individual leukemias but as little as 100 cells proved to be sufficient in one unsorted and in both the CD19+CD20low and CD19+CD20high fractions (Table 1). Mice transplanted with 10 cells only are still under observation.

In conclusion we present strong evidence that leukemia-propagating cells are much more prevalent than previously thought and that blasts of diverse immunophenotype are able to serially reconstitute the complete leukemia in immune-deficient mice.