In Depth Characterization of CLL with Normal Karyotype By Array CGH and Mutation Screening

Background: CLL is characterized by a distinct pattern of cytogenetic abnormalities. The most frequent aberrations are deletions of 13q, 11q, 6q and 17p and trisomy 12. However, based on chromosome banding analysis complemented by interphase FISH no abnormalities are identified in approximately 15-20% of cases. In these cases either no cytogenetic aberrations are present or these may be missed by chromosome banding analysis (CBA) due to insufficient cell division in vitro or to too low resolution of chromosome banding analysis (10 MB). On the other hand by FISH a respective abnormality can only be detected if it is covered by the applied probe panel.

Aims: 1. Apply array CGH and molecular mutation screening to characterize CLL cases in which CBA and FISH both did not reveal any cytogenetic abnormalities. 2. Determine prognostic factors in this CLL subset.

Patients and Methods: Diagnosis of CLL was based on cytomorphology and immunophenotyping. All cases showed at least 15% of CLL cells. The median age was 67 years (range: 40-84, mean 64 years). Overall survival (OS) at 10 years was 81% and median time to treatment (TTT) was 8.9 years. 136 CLL patients were selected based on a normal karyotype in CBA and no abnormalities in interphase FISH with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), the centromeric region of chromosome 12 and t(11;14)(q13;q32) (IGH -CCND1). For all 136 patients the IGHV mutation status was determined and array CGH (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) was performed. Further, mutation analysis by DNA sequencing was performed in the following genes: TP53 (n=106), SF3B1 (n=106), MYD88 (n=83), XPO1 (n=83), NOTCH1 (n=83), FBXW7 (n=83), BIRC3 (n=45) and ATM (n=44).

Results: In total 55 abnormalities were detected in 26/136 (19%) patients by array CGH. Of these 25 were deletions (size of 17 deletions was <10MB and 8 were >10MB), 23 were gains (17 <10MB; 6 >10MB) and 7 were CN-LOH (2 <10MB; 5 >10MB). The following recurrent abnormalities were identified: deletions of 13q14 (n=3); 1q42.12 (n=4), 4p16.3 (n=2), 7p14 (n=3); gains of Xp22.31 (n=2), 3q26-28 (n=2); and CN-LOH 17q (n=2). A mutated IGHV status was present in 68% of cases. Mutations were observed in SF3B1 (19%), NOTCH1 (7%), ATM (5%), XPO1 (4%), TP53 (3%), MYD88 (2%), FBXW7 (1%) and no mutation in BIRC3. Compared to a cohort of 1,115 CLL with aberrant karyotype by CBA/FISH, in the present CLL cohort with normal karyotype SF3B1 mutations were significantly more frequent (19% vs 8%, p=0.001), while TP53 mutations tended to be less frequent (3% vs 8%, p=0.07). In the 26 patients with normal karyotype by CBA/FISH but aberrant karyotype by array CGH (CGHpos) SF3B1 mutations were even more frequent than in cases with normal karyotype by both CBA/FISH and array CGH (CGHneg) (33% vs 14%, p=0.043). A mutated IGHV status was found in 71% of CGHneg patients compared to 58% of CGHpos cases (n.s.). Only age (relative risk (RR): 1.16 per decade, p=0.006) and percentage of CLL cells as determined by flow cytometry (% CLL cells) (RR: 1.36 per 10% increase) were significantly associated with OS and the impact of both parameters was independent of each other. TTT was significantly influenced by the following parameters: CGHpos (RR: 2.4, p=0.017), unmutated IGHV (RR: 4.7, p<0.0001), SF3B1 mutation (RR: 2.9, p=0.006), % CLL cells (RR: 1.32 per 10% increase, p<0.0001), and leucocyte count (RR: 1.043 per 10,000 increase, p=0.031). Multivariate Cox regression analysis revealed an independent impact on TTT for an unmutated IGHV status (RR: 4.7, p<0.0001), mutated SF3B1 (RR: 2.9, p=0.006), and % CLL cells (RR: 1.32 per 10%, p<0.0001). The median TTT was significantly shorter in patients with unmutated IGHV status and/or SF3B1 mutation (n=55) as compared to those without (n=57) (5.1 years vs not reached, p<0.0001).

Conclusions: 1. CLL with normal karyotype as determined by chromosome banding analysis and FISH is characterized by a high frequency of SF3B1 mutations (19%). 2. Array CGH detects abnormalities in 19% of CLL with normal karyotype by CBA/FISH. 3. In CLL with normal karyotype by CBA/FISH a negative effect on TTT was found for the presence of any abnormalities detected by array CGH, SF3B1 mutations, an unmutated IGHV status, and the percentage of CLL cells. Thus, in younger patients the analysis of these parameters should be discussed to better define prognosis.