Aurora-A Kinase Expression In Untreated Patients With Chronic Lymphocytic Leukemia

Aurora-A kinase is a cell-cyle regulating kinase required for chromosomal segregation. Overexpression of Aurora-A kinase has been detected some solid tumors and hematological malignancies such as multiple myelomai non-Hodgkin's lymhoma, and acute leukemia. But there are only two studies in chronic lymphocyic leukemia (CLL). This prospective study was approved by Ethical Committee of University. We investigated Aurora-A kinase in bone marrow of 41 untreated patients (22 male and 19 female with mean age of 70±10 years) with CLL and 19 patients (8 male and 11 female with mean age of 54±20 years) with anemia such as megaloblastic, autoimmune hemolytic, and iron deficiency anemia using a quantitative reverse transcriptase-PCR (RT-PCR) method. β-Actin and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA was used as the internal controls. Total RNA was extracted from bone marrow cells using the Trizol method (High Pure Isolation Kit, Roche Diagnostics). cDNA was prepared with the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). Aurora-A cDNA was quantified using TaqMan Universal PCR Mastermix (Applied Biosystems) and the Aurora-A TaqMan Gene Expression Assay. β-Actin was assayed using TaqMan Universal PCR Mastermix with forward (CCCTGGCACCCAGCAC) and reverse (GCCGATCCACACGGAGTAC) primers at 400nM each and probe (fam-ATCAAGATCATTGCTCCTCCTGAGCGC-bhq) at 100nM concentrations. Real-time quantitative RT-PCR was performed in LightCycler 480II (Roche Diagnostics). Relative RNA level was reported via standard delta delta Ct (dd Ct). Immunhistochemical analysis was performed using formalin-fixed, parafin-embedded sections of bone marrow biopsy specimens from patients and controls. Tissue sections were incubated for 60 minutes with Aurora-A (Novus Biologicals Inc. Littleton CO, 1:100 dilution). Aurora-A kinase is establish as positive if exist >10% in the cytoplasms and nuclei of the neoplastic cells in all cases. If this staining is 0-10% of cells, it is accepted as slight positive. If these cells is never (0%) stained, it is negative. For the comparison of values, Mann-Whitney-U, Chi-square, and One-Way ANOVA tests were used by SPSS 15.0 for Windows. With FISH method, 17p and 13q deletions were detected in 10% and 37% of the patients CLL, respectively. There was trisomy 12 in 7% of teh patients. 68% of the patients with CLL were in Binet-A stage. By immunohistochemical analysis, while Aurora-A kinase was positive in 61% of the patients, it was negative in 72% and slight positive 28% of controls. These positivity was statistically significant (p<0.001). By RT-PCR, β-Actin and GAPDH mRNA values were 3.85±2.61 and 3.49±2.32 in the patients with CLL, respectively. These values were 3.80±2.73 and 4.34±2.36 in controls, respectively. There was no difference for both β-Actin and GAPDH mRNA values between two groups (p>0.05). According to Binet classification, there was no difference for Aurora-A kinase expression with RT-PCR and immunohistochemical staining (p>0.05). Moreover there was no difference for both expression of Aurora-A kinase and immunhistochemical staining in between the patients with chromosmal ambnormalities and without (p>0.05). Although overexpression of Aurora-A kinase expression was not detected, significant immunohistochemical staining represented that Aurora-A kinase can be potential therapeutic target in CLL.