Genomic analyses of chronic lymphocytic leukemia (CLL) identified somatic mutations and associations of clonal diversity with adverse outcomes. Clonal evolution likely has therapeutic implications but its dynamic is less well studied. We studied clonal composition and prognostic value of seven recurrently mutated driver genes using targeted next-generation sequencing in 643 CLL patients and found higher frequencies of mutations in TP53 (35 vs. 12%, p < 0.001) and SF3B1 (20 vs. 11%, p < 0.05) and increased number of (sub)clonal (p < 0.0001) mutations in treated patients. We next performed an in-depth evaluation of clonal evolution on untreated CLL patients (50 "progressors" and 17 matched "non-progressors") using a 404 gene-sequencing panel and identified novel mutated genes such as AXIN1, SDHA, SUZ12, and FOXO3. Progressors carried more mutations at initial presentation (2.5 vs. 1, p < 0.0001). Mutations in specific genes were associated with increased (SF3B1, ATM, and FBXW7) or decreased progression risk (AXIN1 and MYD88). Mutations affecting specific signaling pathways, such as Notch and MAP kinase pathway were enriched in progressive relative to non-progressive patients. These data extend earlier findings that specific genomic alterations and diversity of subclones are associated with disease progression and persistence of disease in CLL and identify novel recurrently mutated genes and associated outcomes.

In order to accomplish their physiological functions leukocytes have the capability to migrate. As a prerequisite they need to adopt a polarized cell shape, forming a leading edge at the front and a uropod at rear pole. In this study we explored the capability of chronic lymphocytic leukaemia (CLL) cells to adopt this leukocyte-specific migration phenotype. Furthermore, we studied the impact of the Toll-like receptor 9 (TLR9) agonists CpGs type A, B and C and the antagonist oligodesoxynucleotide (ODN) INH-18 on the cell polarization and migration process of primary human CLL cells. Upon cultivation, a portion of purified CLL cells adopted polarized cell shapes spontaneously (range 10-38%). Stimulation with CpG ODNs type B (ODN 2006) and CpGs type C (ODN 2395) significantly increased the frequency of morphologically polarized CLL cells, while ODN INH-18 was hardly able to act antagonistically. Like in human hematopoietic stem and progenitor cells, in morphologically polarized CLL cells CXCR4 was redistributed to the leading edge and CD50 to the uropod. Coupled to the increased frequencies of morphologically polarized cells, CpGs type B and C stimulated CLL cells showed higher migration activities in vitro and following intravenous injection higher homing frequencies to the bone marrow of immunocompromised NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. Thus, presumably independent of TLR-9 signaling, CpGs type B and C promote the cellular polarization process of CLL cells and their ability to migrate in vitro and in vivo.

Single-cell RNAseq/VDJseq of tumor cells and normal residual B (NRB) cells from peripheral blood of chronic lymphocytic leukemia (CLL) patients identified three distinct tumor subsets according to phenotype, transcriptome, and immunoglobulin-V-gene (IgV)-mutations. Two major subsets share a typical CLL phenotype but differ in signaling, metabolism and cell cycle control, indicating that the circulating CLL pool is shaped by two states of activity. The third CLL subset shows the phenotype, proliferation capacity and extensive IgV-mutation diversity of normal CD5+ memory B cells. This previously unrecognized CLL tumor subset, which intermingles with NRB cells, was confirmed in 33 IgV-mutated (M-CLL) and IgV-unmutated (U-CLL) cases. Longitudinal IgV-mutation phylogenetics suggest that these NRB-associated CLL cells are generated pathogenetically early, mostly in germinal center reactions, and archive the individual IgV-diversification program, which is conserved throughout CLL course. Our study suggests that diversity is established early in CLL, that each tumor is composed of multiple subclonal expansions, and subclonal evolution can be depicted by IgV mutation phylogenetics.