Progressive telomere shortening is part of the natural history of chronic lymphocytic leukaemia and impacts clinical outcome: evidences from long term follow‐up

Many biological markers at diagnosis have improved the prediction of outcome in chronic lymphocytic leukaemia (CLL) (Nabhan et al, 2015). Previous studies on telomere length (TL) have demonstrated that shorter telomeres at diagnosis are associated with poor outcome, along with unmutated IGHV status, high levels of CD38, CD49d and ZAP70 (Grabowski et al, 2005; Roos et al, 2008; Lin et al, 2014; Dos Santos et al, 2015). Moreover, our previous study, on a large CLL population (n = 401), showed that short TL (<5000 bp) at diagnosis is an independent predictor of shorter overall survival (OS), treatment-free survival (TFS) and progression to Richter syndrome (Rossi et al, 2009). Here we report TL dynamics over time and its predictive value in 90 CLL patients with a longer follow-up. Patients from the previous cohort that were willing to donate further samples were analysed. Diagnosis and treatment of CLL were managed according to institutional guidelines, based on the National Cancer Institute (NCI) Working Group (Hallek et al, 2008). Clinical and biological data were recorded (Table SI). All patients provided informed consent. TL was assessed at two time-points; telomere loss over time was calculated in terms of absolute loss, defined as the loss of telomeric DNA (in base pairs, bp) between the first and second determination of TL, and then adjusted for time, as Yearly Loss [YL = absolute loss (bp)/time (months)], in order to limit the bias of different timing in the second sample acquisition among patients. The second TL analysis was performed on peripheral blood mononuclear cells, recovered using density gradient stratification procedure (Ficoll-Hypaque, GE Healthcare, Buckinghanshire, UK), as previously described (Rossi et al, 2009). Genomic DNA was extracted using DNAzol (Invitrogen, Carlsbad, CA, USA). DNA yields and quality were measured by Nanodrop2000 (ThermoScientific, Waltham, MA, USA). TL was determined by Southern blot analysis. Primary end-point was TFS, defined as the time from the first TL evaluation to the time of first treatment. For univariate analyses, the TFS curve was estimated by the Kaplan– Meier method and compared using the log-rank test. TFS was then analysed by the Cox proportional hazards model comparing, by the Wald test, relevant risk factors (Table I). All reported P-values were two-sided, at the 5% significance level. Data were analysed as of June 2016 by R v.3.3.0 (R Foundation for Statistical Computing, Vienna, Austria). Ninety CLL patients from the original series were clinically monitored for a median time of 128 months (range, 21– 336 months). The first TL determination (baseline TL) was assessed at early stage of the disease, while the second, follow-up, determination (FU TL) was not uniform for all patients, with a median time between the two TL measures of 44 months (range: 11 5–231 months). At the time of the second determination, 26 patients had already relapsed after a first-line treatment, while 64 patients were still in Watch and Wait (WW). Among these, 33 progressed and required a treatment between FU TL and the last follow-up. Telomeres were shorter at FU TL compared to baseline TL (median YL 137 bp; range +174 to 1906 bp, P < 0 001). This was particularly evident in IGHV-mutated patients, as compared to unmutated (median YL 205 bp vs. +63 bp; P < 0 05). Patients with longer telomeres at baseline showed