Purpose: Chronic lymphocytic leukemia (CLL) remains incurable despite advances in therapy. In this study, we characterize the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibition by FK866 in primary CLL cells from patients with various clinical prognostic markers.Experimental Design: CLL cells were treated with FK866 to assess viability by Annexin V/PI staining. Functional analysis of FK866 included time-and concentration-dependent evaluation of cellular NAD, ATP, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and apoptotic signaling. Chemosensitization potential by FK866 to fludarabine was also assessed. Prognostic markers were correlated with drug response.Results: FK866 induced CLL cell death by depleting cellular NAD content by day 1, followed by a drop in ATP on day 2. We observed loss of MMP, ROS increase, and induction of apoptotic signaling at day 3. Ontarget activity of FK866 was confirmed by NAD-mediated rescue of NAD and ATP loss, apoptotic signaling, and viability. The response to FK866 was independent of most prognostic markers. Higher doses were required with short lymphocyte doubling time and positive CD38 status, whereas CLL cells resistant to fludarabine in vitro and from patients with del17p13.1 were equally sensitive to FK866. FK866 did not upregulate the p53-target p21, nor did the p53 activator Nutlin improve FK866-mediated cell death. Furthermore, fludarabine and FK866 were synergistic at clinically relevant concentrations.Conclusions: NAMPT inhibition by FK866 may be a potential treatment for CLL, including patients with del17p13

Evolutionary studies in lichen associations follow that of the fungal symbiont (mycobiont), which is the symbiont after which the lichen is named and forms the majority of the thallus. However, evolution of the algal partner (photobiont) is important to maintain compatibility between symbionts and to optimize productivity of the lichen association. The internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (rDNA) were examined for primary DNA sequence patterns and for patterns in the secondary structure of the rRNA transcripts in both symbionts of the genus Cladonia. Fungal and algal symbionts show opposite trends in rates of evolution and fragment lengths. Both symbionts showed stronger conservation of the ITS2 structure than the ITS1 structure. Homology was evident in the secondary structures between the two highly divergent chlorophyte and ascomycete taxonomic groups. Most fungal species and all species complexes were polyphyletic. The ITS rDNA of the natural lichen algae from Manitoba and four known algal species is highly similar. The natural lichen algae segregate into highly supported clades by environmental features, suggesting that algae that are already adapted to the environment may associate with germinating fungal propagules in the genus Cladonia. Fungal plasticity may allow the mycobiont to adapt to the environment of the photobiont producing variation in lichen morphology. This might explain the incongruence of phylogenetic patterns between the algal and fungal partners tested and the polyphyly of the fungal species.

Background:Chronic lymphocytic leukaemia (CLL) patients have an increased risk of other malignancies. This may be due to surveillance bias, treatment or immunosuppression.Methods:Cohort study of 612 consecutively diagnosed CLL patients in a Canadian province, with comparisons to follicular lymphoma (FL) patients.Results:Treated CLL patients had a 1.7-fold increased risk of second cancers compared with untreated CLL patients. As compared with untreated FL patients, untreated CLL patients had a two-fold increased incidence of second malignancies.Conclusion:Chronic lymphocytic leukaemia patients have an inherent predisposition to second cancers and the incidence is further increased by treatment.

The immunoglobulin heavy chain (IGH) gene rearrangement in chronic lymphocytic leukemia (CLL) provides a unique molecular signature; however, we demonstrate that 26/198 CLL patients (13%) had more than one IGH rearrangement, indicating the power of molecular technology over phenotypic analysis. Single-cell PCR analysis and next-generation immuno-sequencing identified IGH-defined clones. In 23% (18/79) of cases whose clones carried unmutated immunoglobulin heavy chain variable (IGHV) genes (U-CLL), IGH rearrangements were bialleic with one productive (P) and one non-productive (NP) allele. Two U-CLL were biclonal, each clone being monoallelic (P). In 119 IGHV-mutated (M-CLL) cases, one had biallelic rearrangements in their CLL (P/NP) and five had 2–4 distinct clones. Allelic exclusion was maintained in all B-clones analyzed. Based on single-cell PCR analysis, 5/11 partner clones (45%) reached levels of >5x109 cells/L, suggesting second CLL clones. Partner clones persisted over years. Conventional IGH characterization and next-generation sequencing of 13 CLL, 3 multiple myeloma, 2 Waldenstrom’s macroglobulinemia and 3 age-matched healthy donors consistently identified the same rearranged IGH sequences. Most multiple clones occurred in M-CLL, perhaps indicative of weak clonal dominance, thereby associating with a good prognosis. In contrast, biallelic CLL occurred primarily in U-CLL thus being associated with poor prognosis. Extending beyond intra-clonal diversity, molecular analysis of clonal evolution and apparent subclones in CLL may also reflect inter-clonal diversity.

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