13q Deletion Size Predicts Disease Progression and Response to Treatment in Patients with Chronic Lymphocytic Leukaemia.

Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease characterised by a variable clinical course. Chromosomal abnormalities involving 13q14 are the most common genetic alterations in CLL, occurring in ∼60% of cases and when identified as a sole abnormality, are associated with a favourable clinical outcome. A minimally deleted region (MDR), found in most cases, encompasses DLEU2, DLEU1, miR15a and miR16-1. However, recent SNP profiling data showed that 13q deletions extends beyond the MDR and exhibit considerable heterogeneity both in size and location (Ouillette et al, Cancer Res 2008 68(4);1012). Furthermore, this study showed that larger genomic deletions were associated with a higher Rai stage and were enriched in treated patients. To explore this association further, we performed an initial analysis on samples taken at diagnosis from 100 Stage A patients, of whom 50% had stable disease for over 10 years, and on pre-treatment samples from 100 patients enrolled on the LRF CLL4 trial who had either a complete or partial response to fludarabine and cyclophosphamide [n=200] using the Affymetrix SNP6.0 microarray platform. Deletions of 13q were identified in 99/200 patients (49.5%), the majority (94%) of which included a mono- or biallelic deletion of the MDR. The smallest deletion in this series was 130Kb, while the largest was an 82Mb terminal deletion (mean deletion size 7.25Mb). Furthermore, we identified 12 cases with acquired copy number neutral LOH affecting 13q, exclusive in the presence of a bi-allelic deletion of the MDR. In agreement with the previous study we demonstrated that the size and genomic location of the deletions were highly heterogeneous. Common deletion breakpoints within a 1.9Mb region on 13q between genomic locations 48.7 and 50.6Mb defined 42 cases with small deletions. The remaining 57 cases (58%) showed deletions extending further towards the telomere or centromere and defined those cases with a large deletion. The presence of a large 13q deletion at diagnosis was associated with disease progression (p<0.03) and in cases with unmutated VH genes, a median treatment free survival of 3 months compared to 18 months for cases with small deletions (p<0.003). In the CLL4 trial samples, large deletions were associated with partial rather than complete response to treatment (p<0.04). Interestingly, the deletion sub-types defined by Ouillette et al were not associated with disease progression or response to treatment in our series. To extend this observation we designed a series of custom 13q oligonucleotides for multiplex ligation-dependant probe amplification (MLPA) and are in the process of screening an unselected cohort of 500 CLL patients. Of the 50 patients currently screened with MLPA, five cases analyzed with both the SNP6.0 platform and MLPA showed 100% concordance. The remaining 45 cases had a 13q deletion identified by FISH and confirmed the association between 13q deletion size and clinical outcome (Figure, p<0.03). Furthermore, this analysis shows that this association is independent of VH gene mutational status and the presence of an 11q or 17p deletion. These findings suggest that whilst 13q abnormalities are generally associated with a favourable outcome, aberrant expression of gene(s) on 13q outside of the MDR adversely affect outcome. We are currently refining the 13q region associated with adverse outcome to identify causative gene loci. In conclusion, 13q deletion size represents a new biomarker for predicting outcome of CLL, whose target gene(s) could provide new therapeutic strategies.