Telomere shortening associated with increased karyotypic complexity in chronic lymphocytic leukemia.

JULIETA PANERO; PATRICIA DOS SANTOS; CARMEN STANGANELLI; ANA TRAVELLA; FLAVIA STELLA; FERNANDO BEZARES; IRMA SLAVUTSKY

Colonia

Workshop; Fifteenth Internacional Workshop on Chronic Lymphocytic Leukemia; 2013

Telomeres are specialized protective structures at the end of eukaryotic chromosomes. Because of the end-replication problem, telomeres progressively shorten with repeated cell division, leading to telomere dysfunction and, ultimately, contributing to tumorigenesis. Telomere length (TL) has been proposed as an emerging prognostic factor in chronic lymphocytic leukemia (CLL) as short telomeres were associated with unmutated status of the immunoglobulin heavy-chain variable region (IGHV) gen, high-risk genomic aberrations and poor outcome. However, the relationship between TL and clonal chromosome alterations observed by conventional cytogenetics has not been explored. In this study, we have analyzed TL in 70 patients with diagnosis of CLL (40 males; mean age: 64.6 years, range: 38-89 years) cytogenetically studied in our Laboratory, and 46 age-matched controls (17 males; mean age: 61,5 years, range: 30-91 years). Patients were diagnosed according to the National Cancer Institute-Working Group guidelines (2008). Most of patients were at early Rai clinical stages. All individuals provided their informed written consent. The study was approved by the Ethics Committee of our Institution. In all CLL patients and controls, absolute TL was analyzed by real-time quantitative PCR (qRT-PCR). Chromosome and FISH (fluorescence in situ hybridization) analyses were performed on peripheral blood lymphocyte cultures. Mutational status and IGHV gene rearrangements were also evaluated. A significant telomere shortening in patients (23.7±2.62 Kb/genome) compared to controls (31.3±2.24 Kb/genome) (p=0.0028) was observed. In line with previous studies TL in CLL patients did not correlate with age or sex (p=0.13), supporting that these features have little or no effect in cancer cells. TL was analyzed taking into account FISH risk groups, IGVH mutational status and karyotypic characteristics. No differences in mean TL between patients with deletion 13q14 as a single alteration (29.73±5.55 Kb/genome) and controls was found, whereas cases with poor-risk abnormalities (12.95±3.43 Kb/genome) had significant telomere reduction compared to both patients with 13q14 (p=0.025) and controls (p=0.009). Significant differences in TL between unmutated (25.27±6.13 Kb/genome) and mutated IGVH CLL patients (47.64±8.85 Kb/genome) was also observed (p=0.0275). Furthermore, we found a positive linear correlation between IGHV gene mutation frequency and the TL (p=0.0029). Interestingly, two out of three cases with IGHV3-21 gene rearrangement, associated to poor outcome, showed very short TL. Among them, the case with the shortest TL (6.54 Kb/genome) showed stereotyped B cell receptor subset #2. Cytogenetic analysis showed 31 (44%) patients with normal karyotype while 39 (56%) cases had clonal chromosome aberrations. Among them, 11 cases (28%) had only numerical alterations and 28 (72%) showed structural abnormalities. No differences between cases with numerical and structural aberrations were found (p=0.25), thus they were evaluated as a whole group. Patients with normal karyotype and those with only one alteration (26.91±3.32 Kb/genome) did not show significant TL differences with respect to controls; however a reduction in TL was observed in those cases with 2 or more abnormalities (16.18±3.78 Kb/genome) (p=0.006). Three patients studied both at diagnosis and during clonal evolution showed a clear reduction in their TL: mean TL 30.75 Kb/genome and 6.86 Kb/genome, respectively. No differences in TLs in cases with two or more anomalies compared to patients with deletions 17p/11q were observed. Although limited number of patients in this study, our results support a novel association between short TL and cytogenetic alterations in CLL. These findings reinforce previous reports showing the adverse prognostic value of chromosome abnormalities in CLL, particularly complex karyotypes, and support a role for telomere shortening at the origin of genomic instability in this pathology. The analysis of a larger series of patients with chromosome alterations will permit to evaluate association with outcome and define the behavior of discordant cases.