Study Of Molecular Cytogenetics In Chronic Lymphocytic Leukemia

Objective To establish the incidence of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) patients using conventional cytogenetics (CC) and interphase fluorescence in situ hybridization (FISH) and to evaluate the prognostic implications of chromosomal abnormalities. Then to correlate cytogenetic abnormalities with clinicobiological parameters such as age, Binet stage, serum lactate dehydrogenase(LDH), serumβ2-microglobulin (β2-MG), ZAP-70 and CD38 expression levels.Methods Bone marrow or peripheral blood cultures were performed using PHA as mitogen, harvested after 72 hours and CC study was carried out in 60 cases with CLL. Whenever possible, 20 metaphases were analyzed. Karyotypes were described according to ISCN 1995. SpectrumOrange^TM labeled sequence-specific DNA probe D13S319 for 13q14, SpectrumOrange^TM labeled sequence-specific DNA probe p53 and CEP 17 SpectrumGreen^TM probe for 17p13, CEP 12 SpectrumGreen^TM probe for 12, LSI ATM SpectrumOrange^TM probe for 11q22, LSI MYB SpectrumAqua^TM probe for 6q23 and LSI IGH Dual Color, Break Apart Rearrangement Probe and interphase fluorescence in situ hybridization (I-FISH) were applied to detect de1(13q14) in 61 patients with B-CLL. The expression level of CD38 and ZAP-70 in fresh bone marrow or peripheral blood of 58, 57 cases respectively with CLL was analyzed by four-color flow cytometry.Results Of the 61 patients analyzed, chromosomal abnormalities were found in 11.7% (7/60) with CC and 80.3% (49/61) with FISH. The frequencies of abnormalities were as follows: 13q14 deletion, 55.7%; trisomy 12, 21.3%; ATM deletion, 11.5%; p53 deletion, 21.3%; 6q23 deletion, 4.9%. Among 50 patients in CLL with normal karyotype using CC, chromosomal abnormalities were found in 41 patients with FISH; Among 7 patients with chromosomal abnormalities using CC, 3 cases were normal karyotype with FISH. Del(17p13), del(11q22) were more frequrntly observed in patients with absolute lymphocyte counts≥50×10⁹/L, high levels of LDH,β2-MG and CD38, however normal karyotype and sole del(13q14) were more frequently found in absolute lymphocyte counts≤50×10⁹/L, normal levels of LDH andβ2-MG, CD38 negative expression. The Kaplan-Meier method was used to construct survival curves, and the log-rank statistics was used to compare these curves. In the present study, patients without del(13q14) showed shorter survival than those with del(13q14) (166.83 months±13.85 months vs 86.18 months±7.55 months), but the difference was not statistically significant (P=0.053); patients with del(17p13) showed shorter survival than those without del(17p13) (65.11 months±13.45 months vs 175.30 months±6.53 months) there were significant difference between patients with and without del(17p13) (P=0.000); The suivival was not associated with del(11q22) (P=0.479), IGH rearrangement (P=0.287) or del(6q23) (P=742).Conclusion CC technique in CLL is hampered by the low mitotic index of the neoplastic cells, in the present study, using PHA as mitogens, but frequencies of chromosome abnormalities were low. The introduction of interphase cytogenetics using fluorescent in situ hybridization (FISH) has greatly increased the sensitivity of cytogenetic analyses compared to CC, and it was a rapid and sensitive technique in analysis of chromosomal abnormalities in CLL. Deletion of 13q14 was the most frequent abnormality. FISH abnormalities were correlated with age, sex, peripheral blood lymphocyte count, serumβ2-MG, serum LDH levels, CD38 expression level, Binet stage and overall survival. Sole del(13q14) is associated with the absolute lymphocyte counts below 50×10⁹/L, the normal level of LDH andβ2-MG, and CD38 negative expression; however del(11q22) and del(17p13) are associated with poor prognosis, and there were significant difference between patients with and without del(17p13).Considering that clinical progression of CLL is slower than other hematological diseases, follow-up was short, Further studies including more CLL patients will be required to know more comprehensive pattern of genetic changes and prognosis in Chinese CLL patients.