Chromosome Study On Chronic Lymphocytic Leukemia Using CpG- Oligodeoxynucleotide as Immunostimulant Agent.

To verify whether CpG-oligodeoxynucleotide (CpG-ODN) can raise the detection rate of the karyotypic abnormalities in chronic lymphocytic leukemia (CLL).

The bone marrow (BM) or peripheral blood (PB) cells from 57 cases of CLL were collected and cultured with CpG-ODN DSP30+interleukin-2 (IL-2), phytohemagglutinin (PHA), pokeweed(PWM) or IL-2, respectively. 5 days later cells were harvested for chromosome preparation. Karyotypic analysis was performed using R banding technique. Panel fluorescence in situ hybridization (FISH) was carried out on 19 cases of CLL with normal karyotype using following probes: Cen12, D13S25, Rb1, ATM, P53, MYB and IgH. Genomic DNA from 21 cases of them was extracted from BM or PB. The immunoglobulin variable heavy chain (IgVH) were amplified by polymerase reaction (PCR) and sequenced. CD38 and ZAP70 expressions in leukemic cells were determined by flow cytometry (FCM).

The detection rate of karyotypic abnormalities in CpG-ODN+IL-2 group (43.85%) was obviously higher than PHA (15.09%), PWM (17.31%) and IL-2 (3.13%) groups (P<0.01). 52 karyotypic abnormalities were found. Among them, trisomy12 (+12) or +12 with other abnormalities were the most common, while translocations were the most frequent structural abnormalities including 3 unbalanced and 11 balanced translocations among which 7 had 14q32 rearrangements. 13 cases showed one or more abnormalities on FISH examination including trisomy12 and P53 deletion each in one case, IgH rearrangement and partial deletion each in one case, 13q14.3 deletion in 11 cases of which 5 cases also had Rb1 deletion, one case had Rb1 partial deletion. No cases with ATM or MYB deletions were found. IgVH mutation was detected in 10/21 cases by PCR and sequencing. FCM showed 10/45 cases expressed CD38, while 11/27 cases expressed ZAP70. Among 26 cases examined for CD38 and ZAP70 expressions simultaneously, 5 cases were CD38+ZAP70+, 13 cases CD38-ZAP70-, 6 cases CD38-ZAP70+, 2 cases CD38+ZAP70-, respectively. Statistics disclosed a correlation between complex karyotype and IgVH without mutation, but no association between karyotype and CD38 or ZAP70 expression.

CpG-ODN immunostimulation can obviously raise the detection rate of abnormal karyotypes, especially balanced and unbalance translocations in CLL. FISH is important complement to conventional karyotypic analysis, the combination of both methods can provide more comprehensive genetic information for CLL.

No relevant conflicts of interest to declare.