Conventional cytogenetics (CC) of B-cell lymphoproliferations remains difficult because of low mitotic in vitro activity of the leukemic cells. Therefore, mitogen stimulation of B cells is required to analyze an adequate number of metaphases. Chromosome abnormalities using CC with 12-0-tetradodecanoyl-phorbol-13-acetate (TPA) can be detected in up to 50% of chronic lymphocytic leukemia (CLL), but the development of interphase fluorescence in situ hybridization (FISH) techniques has allowed the detection of selected chromosome abnormalities in nondividing cells in more than 80% of CLL.1 However, FISH is restricted, as information is available only for the genes/loci for which probes are used. So, for a comprehensive genetic analysis, CC is essential because it provides an overview of all microscopically visible chromosome abnormalities important as prognostic factors.
Bacterial DNA and synthetic oligonucleotides containing a CpG motif (CpG-ODN) can activate cells of the immune system such as monocytes, dendritic cells and B cells.2 Apart from stimulating cells, CpG-ODN have many direct effects on B-CLL cells, including upregulation of costimulatory molecules, cell cycle entry and upregulation of potential target antigens for antibody therapy. The use of the immunostimulatory CpG-oligonucleotide DSP30 to effectively induce cell cycle progression of CLL cells in vitro has been reported.3, 4, 5, 6 This proliferation is markedly enhanced upon the addition of interleukin-2 to cultures.4, 7 Recently, Haferlach et al.5 characterized 506 CLL stimulated with DSP30+IL-2. Metaphases were generated in 98.8% and aberrations detected in 83% of cases. Compared with FISH, chromosome analysis using CC resulted in the detection of additional abnormalities. The identification of subgroups with complex aberrant karyotypes suggested that CC might provide additional prognostic information.
To our knowledge and to date, there has been no direct comparison of classical TPA versus DSP30+IL-2. DSP30+IL-2 stimulation has been successfully tested in CLL but no data are available for other lymphoproliferations. We cultured 132 B-cell lymphoproliferations (80 CLL and 52 B-cell lymphoid neoplasms) in parallel, in the presence of TPA or DSP30+IL-2. The objective of this study was to evaluate the suitability of DSP30+IL-2 as a routine B-cell mitogen for metaphase cytogenetics.
In our cohort of 132 patients, diagnosed between December 2006 and October 2007, 76 patients were classified as CLL according to the World Health Organization Classification and the Matutes scoring system, 4 as atypical CLL, 10 as mantle cell lymphoma (3 in leukemic phase), 5 as follicular lymphoma (2 in leukemic phase), 4 as lymphoplasmacytic lymphoma (3 in leukemic phase), 4 as marginal zone B-cell lymphoma in leukemic phase, 1 as B-cell prolymphocytic leukemia, 1 as splenic lymphoma with villous lymphocytes, 1 as lymphocytosis with binucleated lymphocytes, 1 as hairy cell leukemia and 25 as small B-cell NHL (8 in leukemic phase and 1 in transformation) otherwise not categorized. Biological data are provided in Supplementary Table 1.
Two cultures were carried out for each sample: one with DSP30 (1 μM) plus IL-2 (20 U/ml) and the other with TPA (50 μg/l). The number of metaphases per slide (assessed for an equal concentration of nuclei) was calculated with a metaphase finder (Metafer Msearch; Metasystems, Altlussheim, Germany) and the proportion of metaphases with clonal abnormalities was determined. Karyotypes with less than 20 normal metaphases were not retained (nos. 43 and 48). FISH was performed according to cytogenetics and morphology. Complete results of karyotypes with the two mitogenes and FISH are given in Table 1 and Supplementary Table 2. Data in bold characters show the results obtained with only one technique.
Conventional cytogenetics successfully analyzed 74 of 78 CLL (94.9%). A median of 26 metaphases was analyzed per case (range 19–52, mean 27.4). Failures of karyotype were observed in 13 cases but were more frequent in cultures with DSP30+IL2 (14%) than in those with TPA (4%). For 61 patients with metaphases in the two culture conditions, there were significantly fewer metaphases in DSP30+IL-2 than in TPA spreads (mean of 50 versus 72 metaphases per slide respectively, P=0.0007; Mann–Whitney test). However, the proportion of abnormal metaphases was higher in DSP30+IL-2 (mean of 59%) compared with the TPA cultures (mean of 26%, P=0.0265). Fifty patients (82%) showed chromosomal aberrations (79% with DSP30+IL-2 and 70% with TPA). In seven patients (11%), chromosomal abnormalities were detected only in the DSP30+IL-2 cultures (TPA metaphases were normal), whereas abnormal karyotypes were identified only in the TPA cultures in two patients (3%) (DSP30 metaphases were normal). A comparison of the sidelines observed among the 41 abnormal karyotypes showed that both DSP30+IL2 and TPA cultures allowed to detect additional abnormal subclones (found in seven and five patients, respectively) (bold characters in Supplementary Table 2). FISH detected abnormalities in 80% of CLL (n=49). Four cases with normal FISH results presented an abnormal karyotype, indicating that the total number of abnormal cases was higher than 80%. Details of recurrent abnormalities (13q14 deletion, ATM deletion, trisomy 12 and P53 deletion) are provided in Table 1, as well as the most frequently rearranged chromosomal regions. Three recurrent translocations were observed in both DSP30+IL-2 and TPA cultures: t(14;18)(q32;q21)(IGH-BCL2), t(14;18;22)(q32;q21;q11)(BCL2-IGL) and t(8;22)(q24;q11)(cMYC-IGL). The other chromosomal abnormalities were principally deletions and unbalanced translocations, whereas monosomies and trisomies were seldom observed.
Karyotype was successfully analyzed in 51 of 52 B-cell lymphoid neoplasms (98.1%). A median of 23 metaphases was analyzed per case (range 9–41, mean 24.4). Failures of karyotype were observed in five patients and were more frequent in DSP30+IL2 (6%) than in TPA cultures (4%). For the 46 patients with metaphases in the two culture conditions, fewer metaphases were observed in DSP30+IL-2 than in TPA spreads (mean of 50 versus 71 metaphases per slide respectively, P=0.009). As seen for CLL, the proportion of abnormal metaphases was higher in DSP30+IL-2 (mean of 57%) than in TPA cultures (mean of 33%) (P=0.0065). In this small series, marginal-zone B-cell lymphomas seemed to respond in the same way to TPA or DSP30+IL-2 (based on the number of metaphases as well as on the proportion of abnormal metaphases). However, the proportion of abnormal metaphases with TPA was higher in lymphoplasmacytic lymphomas. Among these B-cell lymphoid neoplasms, 80% of karyotypes (n=37) showed chromosomal aberrations (76% with DSP30+IL-2 and 68% with TPA). In six patients (13%), the chromosomal abnormalities appeared only in DSP30+IL-2 cultures (TPA metaphases were normal) compared with 4% in TPA cultures (n=2). For 29 other abnormal karyotypes, DSP30+IL-2 allowed to detect more additional abnormal subclones (n=11) than TPA cultures (n=9) (bold characters in Supplementary Table 2). The main abnormalities (IGH, 17p, trisomy 12, 13q) as well as the most frequently rearranged chromosomal regions are detailed in Table 1. The main trisomies (total or partial) were +18/18q (n=4, one not detected in the TPA culture), +8/8q (n=3, one not detected in the TPA culture) and +3/3q (n=3, two not detected in the DSP30+IL-2 culture). These findings suggested that responses to mitogens might differ according to the type of lymphoma, but this hypothesis should be corroborated on a larger series of B-cell lymphoid neoplasms. The other abnormalities were principally unbalanced translocations and deletions.
In this study, 94.9% of CLL and 98.1% of B-cell lymphoid neoplasms were successfully analyzed using CC with more than 80% abnormal karyotypes. These data are in agreement with the preliminary study of Dicker et al3 concerning 132 CLL stimulated with DSP30+IL-2 alone. In a more recent study with a larger analyzed pool (n=506),5 98.8% success was observed using CC with 83% aberrations. Our data revealed differences depending on the mitogene used: for both CLL and B-cell lymphoid malignancies, the number of metaphases per slide was higher with TPA stimulation, with a gain of 20 metaphases on the average (P<0.01). The rate of failure was lower with TPA, but the proportion of abnormal metaphases was significantly higher with DSP30+IL-2 (P=0.0065). Stimulation with DSP30+IL-2 allowed the detection of more abnormalities, more abnormal subclones and more complex karyotypes in CLL and in the majority of B-cell lymphoid neoplasms. As in earlier studies,3, 5, 6 we think that CC is complementary to FISH analysis. Though FISH exploration using a large probe panel has yielded valuable results in lymphoproliferative diseases, it underestimates the heterogeneity of chromosomal aberrations. Complexity of chromosomal changes, recently associated with unfavorable outcomes, can only be assessed with CC.5, 6 One important benefit of karyotype is that the prognostic subgroups defined by interphase FISH may be further subdivided if chromosomal rearrangement status is included. For instance, in our cohort, 23 CLL would have been assigned to the prognostically most-favorable group because of a 13q deletion as the sole abnormality detected by FISH, but six of them (10%) presented additional abnormalities on karyotypes, potentially changing their prognosis. Likewise, patients with the 11q deletion, P53 deletion or trisomy 12 could be further subdivided into groups with or without translocations that may significantly change treatment-free survival rates as described by Mayr et al.6
In conclusion, our results in both CLL and B-cell lymphoid neoplasms indicate that the immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for routine chromosomal banding.
Groupe Français de Cytogénétique Hématologique (GFCH). Recommendations for the cytogenetic management of chronic lymphocytic leukemia established by the French Group for Cytogenetic Hematology. Pathol Biol 2004; 52: 254–256.
Decker T, Peschel C . Effect of immunostimulatory CpG-oligonucleotides in chronic lymphocytic leukemia B cells. Leuk Lymphoma 2001; 42: 301–317.
Dicker F, Schnittger S, Haferlach T, Kern W, Schoch C . Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression. Blood 2006; 108: 3152–3160.
Decker T, Schneller F, Kronschnabl M, Dechow T, Lipford GB, Wagner H et al. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype. Exp Hematol 2000; 28: 558–568.
Haferlach C, Dicker F, Schnittger S, Kern W, Haferlach T . Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgV(H) status and immunophenotyping. Leukemia 2007; 21: 2442–2451.
Mayr C, Speicher MR, Kofler DM, Buhmann R, Strehl J, Busch R et al. Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia. Blood 2006; 107: 742–751.
Decker T, Schneller F, Sparwasser T, Tretter T, Lipford GB, Wagner H et al. Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells. Blood 2000; 95: 999–1006.
We thank Mariana Titorov for her editorial assistance.
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Struski, S., Gervais, C., Helias, C. et al. Stimulation of B-cell lymphoproliferations with CpG-oligonucleotide DSP30 plus IL-2 is more effective than with TPA to detect clonal abnormalities. Leukemia 23, 617–619 (2009). https://doi.org/10.1038/leu.2008.252
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