Chromosome Study On Chronic Lymphocytic Leukemia Using CpG-ODN As Immunostimulant Agent

【Background】Chronic lymphocytic leukemia (CLL) is a clonal hematological malignancy arising from mature lymphocytes in final phase. Because of its very low spontaneous mitotic index and very poorly stimulated, cytogenetic analysis often showed normal karyotype with phytohemagglutinin (PHA) as immunostimulant agent. Cytogenetic analysis with polyclonal B-cell activator as immunostimulant agent could detect abnormal karyotype in about half of CLL patients. Since 1990s, the detecting rate of karyotypic abnormalities in CLL could be increased to 80%, when panel fluorescence in situ hybridization (FISH) was used. Recently German scholars used CpG-oligodeoxynucleotide (CpG-ODN) as immunostimulant agent in cytogenetic analysis of CLL patients. As a result, the CpG-ODN had further raised the detecting rate of karyotypic abnormalities, especially different translocations associated with poor prognosis.【Objective】To confirm whether the CpG-ODN as immunostimulant agent can raise the detecting rate of karyotypic abnormalities in Chinese CLL patients, we performed a clinical and experimental study on 57 CLL patients.【Materials and Methods】CpG-ODN DSP30 combined with IL-2, PHA, pokeweed(PWM) and IL-2 were used to stimulate the proliferation of the bone marrow or peripheral cells of CLL patients, respectively. RHG banding technique was used for karyotypic analysis. A set of probes including Cen-12, D13S25(13q14.3), Rb1(13q14), ATM(11q22.3), P53(17p13.1), MYB(6q23) and IgH gene rearrangement(14q32) were used to perform FISH assay on cell suspensions from direct method in 19 cases with normal karyotype. The DNAs were extracted and amplified using specific immunoglobulin variable heavy chain (IgVH) gene primers to investigate the IgVH somatic mutation status and identify the location of the mutation. The expressions of CD38 and ZAP70 were determined by flow cytometry. All patients were followed up and statistic analysis was performed to test the significance of differences.【Results】(1)The conventional cytogenetic analysis (CC) with four groups of immunostimulant agent to stimulate the cultured cells of CLL showed that chromosomal aberrations were detected in 43.85% (25/57) cases stimulated by CpG-ODN DSP30 and IL-2 with a mean abnormal cell rate 72.98% including 52 chromosomal aberrations, in 15.09% (8/53) cases stimulated by PHA with a mean abnormal cell rate 6.25%, in 17.31% (9/52) cases stimulated by PWM with a mean abnormal cell rate 7.22%, in 3.13% (1/32) case stimulated by IL-2, respectively. The comparison of the detecting rate of abnormal karyotypes between CpG-ODN DSP30+IL-2 group and other groups (PHA, PWM or IL-2) showed obvious statistic significance (P<0.0001). Among the numerical chromosomal abnormalities, sole trisomy 12 or trisomy 12 with other abnormalities was the most common, seen in 9 times. Forty structural chromosomal abnormalities were detected. Of them, translocation was the most frequent, seen in 14 cases including the balanced translocations in 11(78.5%) cases and unbalanced translocations in 3 (21.43%) cases. 14q32 rearrangement was the most common translocation in this series including t(14;18)(q32;q21) in 3 cases, t(14;19)(q32;q13) in 2 cases, t(11;14)(q13;q32) and t(11;14)(q23;q32) one case each. The other structural abnormalities are comprised of inversion, duplication, isochromosome and marker chromosome. The partial deletion of chromosomes, such as 1q-, 2p-, 3p-, 6p-, 6q-, 9q-, 11q-, 13q- and 15q-, were seen. Among them, 6q- was found in 3 cases, 11q- and 13q- each in 2 cases, respectively. Complex abnormalities were seen in 9 cases. The chromosomal abnormalities detected by CpG-ODN DSP30 +IL-2 stimulation were confirmed in 8 cases by PHA stimulation, in 9 cases by PWM stimulation and in 1 cases by IL-2 stimulation, respectively.(2)Among 19 cases with normal karyotype, FISH detected trisomy 12 in 1 case, deletion of 13q14.3 in 11 cases in whom 6 cases were accompanied by the deletion of Rb1, deletion of P53, IgH gene arrangement and the partial deletion of IgH gene each in one case, no case with deletion of ATM or MYB was found. In a word, of the 19 cases analysed by FISH, 13 (68.42%) cases had one or more abnormalities.(3)PCR analysis for IgVH gene mutation was performed in 18 cases. A mutation of IgVH was found in 55.55% (10/18) cases with the mean mutation rate 5.88%. The mainly mutated IgVH families were in turn VH3, VH4 and VH1. However, an unmutation of IgVH was seen in 8(44.42%)cases. 3/10 (30%) cases with mutation of IgVH had karyoytpic abnormalities. However, 6 out of 8 cases with unmutatioin of IgVH had karyotypic abnormalities including 3 cases with complex karyotypic abnormalities. The difference between two groups with and without mutation of IgVH had no statistic significance (P>0.05).(4)Flow cytometry analysis revealed that 22.22% (10/45) cases had CD38 positive expression, 40.74% (11/27) cases had ZAP70 positive expression, 77.78% (35/45) cases had negative CD38 expression, 59.25% (16/27) cases had negative ZAP70 expression, 19.23% (5/26) cases had positive CD38 and ZAP70 expressions simultaneously, 50% (13/26) cases had negative CD38 and ZAP70 expression, 7.69% (2/26) cases had positive CD38 expression and negative ZAP70 expression, 23.08% (6/26) cases had positive ZAP70 expression and negative CD38 expression. 50% (5/10) cases with CD38 expression and 40% (14/35) cases with negative CD38 expression had karyotypic abnormalities. 36.3% (4/11) cases with positive ZAP70 expression and 50% (8/16) cases with negative ZAP70 expression had karyotypic abnormalities. However, statistic analysis didn't find any association between abnormal karyotype and CD38 or ZAP70 expressions.【Conclusion】①C pG-ODN is a simple and efficient stimulus for cells of CLL. It may obviously increase the detecting rate of abnormal karyotypes;②CpG-ODN could significantly raise the detecting rate of different balance translocations and unbalance translocations;③FISH and CC is complimented each other, FISH in combination with CC could provide more comprehensive genetic information for CLL patients.