Longitudinal Analyses of CXCR4^dimCD5^brCD19⁺ Fractions of Chronic Lymphocytic Leukemia Clones Reveal Features Consistent with a Source of Clonal Heterogeneity

Abstract 804

Significant inter-and intra-clonal heterogeneity, manifested by phenotypic and molecular differences among cell subsets, is a key feature of chronic lymphocytic leukemia (CLL). However, the definition of the subset of cells within a clone more likely to diversify and understanding the likelihood that these cells would have a survival advantage once altered have not been addressed. Most CLL cells express CXCR4, and interaction with its ligand, CXCL12, promotes survival as well as directing migration, two key factors for perpetuation of genetically-modified subclones. We have shown that clonal fractions with lower CXCR4 density contain cells with increased expression of activation markers and represent recently divided/born cells (“proliferative compartment”) as evidenced by enrichment of Ki-67⁺ cells and in vivo²H-labeled DNA. The current study was undertaken to better characterize the proliferative compartment and track the evolution of this more actively cycling fraction to determine if it may be the wellspring of clonal heterogeneity in CLL.

To follow the natural history of the cells comprising the proliferative compartment, we analyzed changes in 10 CLL (5 U-CLL and 5 M-CLL) cases for whom cryopreserved PBMCs were available over a 3–11yr period during the course of the disease. PBMCs from 3 or 4 successive time points for every case were subjected to immunofluorescent staining and the CXCR4^brCD5^dim, CXCR4^intCD5^int, and CXCR4^dimCD5^br fractions analyzed at every time point for expression of a series of surface and intracellular molecules of functional relevance: CD11a, CD23, CD27, CD38, Ki67, Mcm6, MDR (Pgp120), Survivin, and hTERT. The CXCR4^dimCD5^br subset exhibited the highest percentages and levels of CD11a, CD23, CD27, and CD38 on all CLL clones at all time points tested. Although Ki-67, Mcm6, MDR (Pgp120), Survivin, and hTERT also followed the same trend, with disease course there was an overall increase in Survivin, MDR, and Mcm6 levels specifically in the CXCR4^dimCD5^br subset, which were paralleled by a decrease in the Bim:Mcl-1 ratio. Levels of TERT protein and telomerase enzyme activity in B cells at each time point from 8 of 10 cases were consistently higher in the CXCR4^dimCD5^br B-cell subclones than the corresponding CXCR4^brCD5^dim and CXCR4^intCD5^int fractions. Therefore, CXCR4/CD5 B-cell subsets from 8 B-CLL cases frozen over a period of time were flow-sorted and differences in telomere lengths over time analyzed by flow-FISH. Post-sort, in ∼95% of the measurements the CXCR4^dimCD5^br fraction had significantly longer telomeres than the total B-cell fraction and the CXCR4^intCD5^int fractions, implying elevated telomerase activity counteracted telomere erosion.

Finally, since activation of CLL cells can be achieved through BCR stimulation, we used phosphoflow to analyze differences in the response of CXCR4^brCD5^dim, CXCR4^intCD5^int, and CXCR4^dimCD5^br fractions to anti-IgM stimulation, measured by changes in phospho- Akt, Erk, p38MAPK, NFkB, Syk, Btk and STAT5 levels after a 5 minute exposure to the agonist. CXCR4^brCD5^dim cells had the highest constitutive phosphorylation levels for all molecules except NFkB. However after anti-IgM stimulation, the greatest increase in phospho- Akt, Btk, Erk and Syk levels (>25%) occurred in the CXCR4^dimCD5^br fraction. Notably, CXCL12 elicited the highest (>40%) phosphorylation of NFkB and Erk among CXCR4^brCD5^dim cells.

Collectively, these findings indicate that the CXCR4^dimCD5^br fraction of CLL clones contains cells with the highest levels of molecules involved in cell proliferation and longevity, whereas the CXCR4^brCD5^dim fraction responds best to the pro-survival and trafficking influences of CXCL12. This is consistent with new DNA abnormalities more likely originating and surviving among the proliferating cells of the CXCR4^dimCD5^br fraction and when these mature to a CXCR4^brCD5^dim phenotype having the best chance of trafficking back to solid tissues and entering a long-lived pool. This sequence would lead to a survival advantage to altered clonal members and permit ongoing clonal heterogeneity and evolution.