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We performed single-molecule telomere length and telomere fusion analysis in patients at different stages of chronic lymphocytic leukemia (CLL). Our work identified the shortest telomeres ever recorded in primary human tissue, reinforcing the concept that there is significant cell division in CLL. Furthermore, we provide direct evidence that critical telomere shortening, dysfunction, and fusion contribute to disease progression. The frequency of short telomeres and fusion events increased with advanced disease, but importantly these were also found in a subset of early-stage patient samples, indicating that these events can precede disease progression. Sequence analysis of fusion events isolated from persons with the shortest telomeres revealed limited numbers of repeats at the breakpoint, subtelomeric deletion, and microhomology. Array-comparative genome hybridization analysis of persons displaying evidence of telomere dysfunction revealed large-scale genomic rearrangements that were concentrated in the telomeric regions; this was not observed in samples with longer telomeres. The telomere dynamics observed in CLL B cells were indistinguishable from that observed in cells undergoing crisis in culture after abrogation of the p53 pathway. Taken together, our data support the concept that telomere erosion and subsequent telomere fusion are critical in the progression of CLL and that this paradigm may extend to other malignancies. (Blood. 2010;116(11): 1899-1907) IntroductionNonreciprocal translocations (NRTs) are considered to be key mutational events that can drive many types of malignancy. 1 The underlying mechanisms that result in these types of events can include, among others, deficiencies in double-strand break repair, 2 mitotic checkpoints, 3,4 and telomere dysfunction. 5 Telomeres play a key role in upholding genomic integrity; in the context of DNA damage checkpoint defects, cells in culture undergo crisis and have extensive telomere erosion, chromosomal fusion, and genomic rearrangements. 6,7 NRTs, as well as localized gene amplification, 8 can arise as a consequence of cycles of anaphase-bridging, breakage, and fusion initiated by the formation of dicentric chromosomes after telomere fusion. 9 This paradigm is exemplified in vivo by telomerase knockout mice, where short telomeres appear to drive the formation of tumors containing NRTs. 5 However, evidence for this phenomenon in humans is circumstantial. Numerous malignancies, including breast, prostate, colorectal, and chronic lymphocytic leukemia (CLL), [10][11][12][13][14][15] have been documented to exhibit shorter telomeres compared with normal tissues. These data are consistent with the expected levels of cell division during the progression to malignancy but do not indicate that telomeres become short enough to lose their end-capping function. Telomere fusion, as well as other chromosomal defects, can lead to the formation of anaphase bridges; in situ data show an increase in anaphase bridges, often interpreted as a surrogate marker for telomere f...

In this study, we characterized nuclear factor B (NF- B IntroductionB-cell chronic lymphocytic leukemia (CLL) is a malignancy characterized by the accumulation of CD5, CD19, and CD23 positive lymphocytes. Diagnosis is aided by the CLL immunophenotyping score which includes assessment of CD5 and CD23, FMC7, CD79b, and surface IgM. 1 Although CLL is the commonest leukemia in the Western world, it manifests a very heterogeneous clinical course, with some patients having normal age-adjusted survival, whereas the median survival for those patients with advanced stage disease is only 3 years. 2 The factors that contribute to the pathogenesis and progression of this disease are poorly understood, but decreased susceptibility to apoptosis 3 and dysregulated proliferation have been implicated. 4 Clinical studies have shown that high ZAP-70 expression, high CD38 expression, unmutated V H genes, and cytogenetic abnormalities (especially deletions of 11q and 17p) are all associated with a poor prognosis. [5][6][7][8][9] Nuclear factor B (NF-B) is a collective name for a group of inducible homodimeric and heterodimeric transcription factors made up of members of the Rel family of DNA-binding proteins. In humans, this family is composed of c-Rel, Rel B, p50, p52, and Rel A (p65) which, when bound in the cytoplasm to inhibitor of NF-B (IB) proteins, are inactive. 10,11 Various factors, including ligation of CD40 or the B-cell receptor (BCR), result in proteosomal degradation of IB releasing NF-B, which then translocates to the nucleus. 10,11 Once in the nucleus, NF-B can enhance survival by inducing apoptosis inhibitory proteins, including inhibitor of apoptosis proteins (IAPs), Fas-associated death domain (FADD)-like interleukin (IL)-1␤-converting enzyme (FLICE), and FADD-like IL-1␤-converting enzyme-inhibitory protein (FLIP). 12-14 CLL cells have been reported to exhibit high constitutive NF-B activation compared with normal B lymphocytes. [15][16][17] Although the exact factors responsible for the constitutive expression of NF-B are not fully resolved, many factors, including Akt activation, BCR signaling, CD40 ligation, IL-4, and B-cell activating factor (BAFF), have been shown to increase NF-B activity and enhance CLL cell survival, with members of the Bcl-2 family being principal transcriptional targets. [18][19][20][21][22] Several recent studies have demonstrated the proof of concept of the effectiveness of targeting NF-B in hematologic malignancies, including CLL 23,24 and acute myeloid leukemia. 25,26 In this study, we first set out to determine the range of constitutive DNA binding of NF-B within our patient cohort and to characterize the specific subunits of NF-B in these primary CLL cells. We then went on to investigate the ability of freshly isolated CLL cells to induce NF-B expression in response to BCR Submitted November 20, 2007; accepted January 25, 2008. Prepublished online as Blood First Edition paper, January 28, 2008; DOI 10.1182 DOI 10. /blood-2007 An Inside Blood analysis of this article appears at ...

SummaryDefining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high-resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere 'fusogenic' range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P < 0Á0001; Hazard ratio [HR] = 13Á2, 95% confidence interval [CI] = 11Á6-106Á4) and this was preserved in early-stage disease patients (P < 0Á0001, HR=19Á3, 95% CI = 17Á8-802Á5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high-resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL.

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