Chromosome Banding Analysis of Peripheral Blood Lymphocytes Stimulated with IL‑2 and CpG Oligonucleotide DSP30 in Patients with Chronic Lymphocytic Leukemia

Authors: K. Štěpanovská 1;  G. Vaňková 1;  V. Némethová 1;  L. Tomášiková 1;  P. Šmuhařová 1;  E. Divíšková 1;  V. Vallová 2;  P. Kuglík 2;  K. Plevová 1;  A. Oltová 1;  M. Doubek 1,3;  Š. Pospíšilová 1,3;  J. Mayer 1,3
Authors‘ workplace: Interní hematologická a onkologická klinika, Centrum molekulární bio­logie a genové terapie, LF MU a FN Brno2 Oddělení lékařské genetiky, laboratoř molekulární cytogenetiky, LF MU a FN Brno3 CEITEC –  Středoevropský technologický institut, MU, Brno 1
Published in: Klin Onkol 2013; 26(4): 263-270
Category: Original Articles


Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics.

Patients and Methods:
In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22–23, CEP12 and 17p13.

Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients’ karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis.

Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.

Key words:
cytogenetics – chronic lymphocytic leukemia – interleukin-2 – CpG-ODN DSP30 – fluorescent in situ hybridization – cultivation – chromosome aberration


1. Dicker F, Schnittger S, Haferlach T et al. 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(9): 3152– 3160.

2. Swerdlow SH, Campo E, Harris NL et al. WHO Classification of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon: IARC 2008.

3. Adam Z, Šmardová J, Krejčí M et al. Chronická B‑lymfatická leukemie. In: Adam Z, Krejčí M, Vorlíček J et al (eds). Hematologie –  přehled maligních hematologických nemocí. 2. vyd. Praha: Grada 2008: 177– 196.

4. Malciková J, Smardová J, Rocnová L et al. Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage. Blood 2009; 114(26): 5307– 5314.

5. Juliusson G, Oscier D, Gahrton G et al. Cytogenetic find­ings and survival in B‑ cell chronic lymphocytic leukemia –  2nd IWCCLL compilation of data on 662 patients. Leuk Lymphoma 1991; 5(1): 21– 25.

6. Haferlach C, Dicker F, Schnittger S et al. Comprehensive genetic characterization of CLL: a study on 506 cases analysed with chromosome banding analysis, interphase FISH, IgVH status and immunophenotyping. Leukemia 2007; 21(12): 2442– 2451.

7. Haferlach C, Bacher U. Cytogenetic methods in chronic lymphocytic leukemia. In: Campbell LJ (ed). Cancer cytogenetics: Methods and protocols. 2nd ed. New York: Humana Press 2011: 119– 130.

8. Shaffer L, Slovak ML, Campbell L. ISCN 2009: An International System for Human Cytogenetic Nomenclature (2009). 1st ed. Basel: Karger 2009.

9. Swansbury J, Min T, Aruliah S. Fluorescence in situ hy­bridization methods and troubleshooting applied to fixed cell suspensions. In: Campbell LJ (ed). Cancer cytogenetics: Methods and protocols. 2nd ed. New York: Humana Press 2011: 13– 32.

10. Wren C, Moriarty H, Marsden K et al. Cytogenetic investigation of chronic lymphocytic leukemia. Cancer Genet Cytogenet 2010; 198(2): 155– 161.

11. 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 2009; 23(3): 617– 619.

12. Put N, Konings P, Rack K et al. Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin‑2 stimulation: A Belgian multicentric study. Gene Chromosomes Cancer 2009; 48(10): 843– 853.

13. Buhmann R, Kurzeder CH, Rehklau J et al. CD40L stimulation enhances the ability of conventional metaphase cytogenetics to detect chromosome aberrations in B‑ cell chronic lymphocytic leukaemia cells. Br J Haematol 2002; 118(4): 968– 975.

14. Heerema NA, Byrd JC, Dal Cin PS et al. Stimulation of chronic lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent karyotypic results among laboratories: A CLL Research Consortium (CRC) Study. Cancer Genet Cytogenet 2010; 203(2): 134– 140.

15. Stilgenbauer S, Bullinger L, Benner A et al. Incidence and clinical significance of 6q deletions in B cell chronic lymphocytic leukemia. Leukemia 1999; 13(9): 1331– 1334.

16. Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2012) [homepage on the Internet]. Mitelman F, Johansson B and Mertens F (eds). [updated 2012 Aug; cited 2012 Aug 20]. Available from: http:/ / Chromosomes/ Mitelman.

17. Pospisilova H, Baens M, Michaux L et al. Interstitial del(14q) involving IGH: a novel recurrent aberration in B‑ NHL. Leukemia 2007; 21(9): 2079– 2083.

18. Sen F, Lai R, Albitar M. Chronic lymphocytic leukemia with t(14;18) and trisomy 12. Arch Pathol Lab Med 2002; 126(12): 1543– 1546.

19. Lu G, Kong Y, Yue C. Genetic and immunophenotypic profile of IGH@ rearrangement detected by fluorescence in situ hybridization in 149 cases of B‑ cell chronic lymphocytic leukemia. Cancer Genet Cytogenet 2010; 196(1): 56– 63.

20. Winter JN, Gascoyne RD, Van Besien K. Low‑ grade lymphoma. Hematology Am Soc Hematol Educ Program 2004: 203– 220.

21. Cuneo A, Bigoni R, Negrini M et al. Cytogenetic and interphase cytogenetic characterization of atypical chronic lymphocytic leukemia carrying BCL1 translocation. Cancer Res 1997; 57(6): 1144– 1150.

22. De Angeli C, Gandini D, Cuneo A et al. BCL‑ 1 rearrangements and p53 mutations in atypical chronic lymphocytic leukemia with t(11;14)(q13;q32). Haematologica 2000; 85(9): 913– 921.

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