CpG Oligonucleotide Combined with Interleukin-2 Reveals Unexpected Chromosomal Lesions In B-CLL

Abstract 2411

In B-CLL the precise definition of the chromosomal pattern has always been a very difficult goal as leukemic cells poorly respond to the traditionally used set of mitogens. Thus, conventional cytogenetic studies (CC) have always been replaced by interphase FISH (iFISH) analyses. However, recent studies have demonstrated that the CpG oligonucleotide plus interleukin-2 (ODN+IL-2) stimulation can effectively increase the detection rate of chromosomal abnormalities by inducing metaphase spreads from B-CLL leukemic cells. Based on these data, we decided to compare the results obtained by the ODN+IL2 combination, the pokeweed mitogen (PKWM) stimulation and iFISH in ninety-one B-CLL patients diagnosed at our Institution between January 2007 and July 2010. In addition, we evaluated any possible correlation with other prognostic markers and clinical parameters. There were thirty-eight females and fifty-three males with a median age of 64 years (range 42–83). According to Binet, sixty-three patients (69.2%) were considered as stage A, seventeen (18.7%) as stage B and eleven (12.1%) as stage C. PKWM did not provide any mitotic figure in thirteen patients (14.2%) and detected clonal defects in sixteen patients (17.5%). In contrast, the ODN+IL-2 combination was unable to provide metaphase spreads in three patients only (3.3%) and revealed clonal abnormalities in fifty-six patients (61.5%). In eight patients both cultures revealed an equal percentage of cells carrying the same abnormality. In addition, the ODN+IL-2 combination revealed a complex karyotype (≥ three defects) in twelve patients (13.2%), two chromosomal defects in fourteen patients (15.4%)(including five with band 17p13 rearrangement and three with band 11q13 rearrangement) and a single chromosomal defect in thirty patients (32.9%). Fifteen patients harboured various chromosomal rearrangements (16.4%). iFISH was carried out with the B-CLL FISH probe panel (Vysis, Downers Grove, IL, USA) and revealed clonal abnormalities in sixty-two patients (68.1%). The most common defects were: 13q-, +12, 11q- and 17p- having an incidence of 46.1%, 14.2%, 7.7% and 6.5%. Three patients with a normal FISH pattern presented a +12 when analysed with the ODN+IL-2 combination and five, who showed the loss of one p53 signal on iFISH, presented a structural 17p13 defect when investigated with the ODN+IL2 combination. In addition, considering the forty-two patients who harboured a 13q- on iFISH analyses, the ODN+IL2 combination revealed that in five patients this defect was included in a complex karyotype. From a clinical point of view, considering the fifteen patients with various chromosomal rearrangements nine were classified as stage A, four as stage B and two as stage C and considering the twelve patients with a complex karyotype three were considered as stage B and nine as stage C. In conclusion, the ODN+IL2 combination i) allows a precise definition of the chromosomal pattern in B-CLL patients and in our series identified clonal defects in 61% of patients; ii) reveals minor +12 clonal cell populations which may evade iFISH identification; iii) does not always identify a 13q deletion because of the sub-microscopic nature of this chromosomal defect; iv) reveals that the loss of one p53 signal on iFISH analyses is frequently due to an unbalanced rearrangement; iv) is the only technique which allows the identification of complex karyotypes and/or chromosomal translocations both associated with an advanced Binet stage. Thus, the ODN+IL2 combination should be used in conjunction with iFISH to achieve a more accurate karyotype definition in B-CLL patients.