Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin‐2 stimulation: A Belgian multicentric study

Abstract

We performed a multicentric study to assess the impact of two different culture procedures on the detection of chromosomal abnormalities in 217 consecutive unselected cases with chronic lymphocytic leukemia (CLL) referred for routine analysis either at the time of diagnosis (n = 172) or during disease evolution (n = 45). Parallel cultures of peripheral blood or bone marrow were set up with the addition of either the conventional B-cell mitogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or a combination of CpG oligonucleotide (CpG) and interleukin-2 (IL-2). Cytogenetic analyses were performed on both cultures. Clonal abnormalities were identified in 116 cases (53%). In 78 cases (36%), the aberrant clone was detected in both cultures. Among these, the percentages of aberrant metaphases were similar in both conditions in 17 cases, higher in the CpG/IL-2 culture in 43 cases, and higher in the TPA culture in 18 cases. Clonal aberrations were detected in only one culture, either in CpG/IL-2 or TPA in 33 (15%) and 5 (2%) cases, respectively. Taken together, abnormal karyotypes were observed in 51% with CpG/IL-2 and 38% with TPA (P < 0.0001). Application of FISH (n = 201) allowed the detection of abnormalities not visible by conventional cytogenetic analysis in 80 cases: del(13q) (n = 71), del(11q) (n = 5), +12 (n = 2), del(14q) (n = 1), and del(17p) (n = 1). In conclusion, our results confirm that CpG/IL-2 stimulation increases the detection rate of chromosomal abnormalities in CLL compared with TPA and that further improvement can be obtained by FISH. However, neither conventional cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques.

Genetics of Chronic Lymphocytic Leukemia: Practical Aspects and Prognostic Significance

“…CpG stimulation of CLL and normal B-cells induces expression of AID, an enzyme that is linked to the development of genetic abnormalities (Capolunghi et al, 2008). However, culturing B-cells of healthy blood donors with CpG/IL-2 did not induce clonal abnormalities, thus validating CpG/IL-2 as a tool for the cytogenetic analysis of CLL (Dicker et al, 2006;Put et al, 2009a;Wu et al, 2008).…”

“…The addition of the cytokine IL-2 to TPA stimulated CLL cell cultures was reported to directly stimulate CLL proliferation, even in absence of T-lymphocytes (Touw and Lowenberg, 1985). Although the latter findings provide evidence for the addition of IL-2 to TPA cultures, it is not mandatory for successful CCA (Put et al, 2009a;Struski et al, 2009). …”

“…an increase of 9-13% of cases with aberrations) compared with TPA stimulated cultures (Put et al, 2009a;Struski et al, 2009). Moreover, the detection of translocations and del(13q) in particular, has been found to be superior after CpG/IL-2 stimulation compared with TPA (Put et al, 2009a).…”

“…The influence of CpG/IL-2 on quality of banding and metaphase generation is not clear (Put et al, 2009a;Struski et al, 2009).…”

“…Recently, improved culture methods have been introduced, i.e. CD40 ligand (CD40L)-induced cell cycle stimulation, and the immunostimulatory CpG oligonucleotide (DSP30) (Dicker et al, 2006;Haferlach et al, 2007;Mayr et al, 2006;Put et al, 2009a;Struski et al, 2009). …”