Identification of leukaemic stemcells in B-cells CLL

As described for other leukaemic haematological disorders we hypothesize that also in B-CLL a small compartment of leukaemic stem cells exists, the identification and characterization of this cell compartment being crucial to gain insight into the pathogenesis of the disease and to develop new effective therapeutic strategies. Both, normal and neoplastic stem cells exhibit three key features (criteria): 1) the ability to differentiate and mature sufficiently for long-term survival of functional elements and for the reconstitution of the mature cell compartment; 2) the capacity of self-renewal (without loss of their proliferative abilities in the absence of cell differentiation/maturation), thus allowing maintenance of the undifferentiated stem cell pool over the lifetime of the host, and; 3) self-regulation of stem cell numbers through balancing self-renewal against differentiation of dividing daughter cells. Recently the terms cancer stem cell, neoplastic stem cell, and leukaemic stem cell (LSC) have emerged and been proposed to identify a typical small number of tumour cells with stem cell properties. In fact, the general definition is the same as that used for normal stem cells: these cells have the capacity to repopulate the host (patient) with the corresponding neoplasm (including all elements and all lineages and subclones of the primary tumour) for an unlimited period of time. Accordingly, to test this hypothesis, we will combine the evaluation of subpopulations of normal B-cells and B- CLL cells in 10 untreated patients and normal healthy adults, per month. In addition, we will also investigate primary B-CLL cells from treated patients with a minimal residual disease status, since the remaining treatment- resistant B-CLL-cells have been shown to be responsible for recurrence of the disease and to reproduce the tumour indicating the persistence of minimal residual disease and thus, they could be the cause for the emergence of new B-CLL clones resistant to therapy. These B-CLL subpopulations will be examined by using multiparameter flow cytometry-based immunophenotyping and cell sorting (FACS). After FACS-sorting the different B-cell subsets will be investigated for the presence of chromosomal and/or genetic aberrations. In addition, to identify the self-renewal capacity in vitro culture assays (colony-forming assay or long-term suspension culture of primary purified cells) as well as animal models (xenotransplant mouse model based on the use of nonobese, diabetic, severe combined immunodeficiency (NOD-SCID) mice) will be used for the long-term growth of true stem cells. B-CLL-subpopulations will repopulate the mouse body in the context of B-CLL stromal cells; after target structures will be identified, a specific treatment or newly developed drugs could be assayed. In addition an increasing number of reports suggests that this could be an universal concept for almost every tumour subtype although it still remains to be proven in many tumours including B-CLL.