Changes In Apoptotic Gene Expression Profile In CLL Patients Treated with Cladribine, Cyclophosphamide and Rituximab (CCR)

Abstract 2469

Genetic mechanisms leading to the development and progression of chronic lymphocytic leukemia (CLL) are mainly unknown. The key to understanding the disease is apoptotic gene expression and a lot of important genetic markers have already been identified. Application of monoclonal antibodies has led to considerable progress in CLL treatment. Although, rituximab as a single-agent has limited activity as first line therapy, it has synergistic activity when combined with chemotherapy. Currently, the combination of cladribine, cyclophosphamide and rituximab (CCR) is the most effective with acceptable toxicity as the first line therapy. The microarray technique represents one of the most innovative and powerful tools, providing a lot of information on genes expression. The aim of our study was to investigate the apoptotic gene expression profile in cells of CLL patients before and after CCR treatment. The study was conducted in 10 previously untreated CLL patients. All of them were diagnosed and followed at the Department of Hematology, Medical University of Lodz, Poland, acc. to the WHO classification. The study received approval of the Medical University of Lodz Ethical Committee (RNN/196/07/KE). Informed consent was obtained from all the patients. Fresh blood samples were collected before and after 2 weeks of the 1st cycle of CCR treatment (rituximab 375mg/m² D1, cladribine 0.12mg/kg D 1–3, cyclophosphamide 600mg/m² D 1–3; q 4 wks × 6). Mononuclear cells (MNCs) were separated and used for further studies. The TaqMan^® Low Density Array (Human Apoptosis Panel)(Applied Biosystems, USA) with 96 transcripts (93 examined, 3 controls) was used to analyse gene expression profiling. Microarray analysis was performed using 0,5 mg of RNA. cDNA samples were subjected to real-time PCR in duplicate in an Abi Prism 7900HT Sequence Detection System (Applied Biosystems). Comparisons of the respective intensities of hybridization signals between microarrays were analysed. The relative expression of each gene was quantified by the comparative cycle threshold (Ct) method (DDCt), using 18S as an endogenous control. Fold change (RQ) for each gene was evaluated. Ratios of hybridization signal intensities of either a decrease of 50% or greater (downregulated) or an increase of twofold or greater (upregulated) were considered to be significant and reported. Data analysis pointed 15 out of 93 examined apoptotic genes whose expression was significantly important. Details concerning genes description and mean values of RQ are collected in the table. Our results show genes expression changes mostly regard to the intrinsic pathway of apoptosis, whereas only 4 genes represent the extrinsic pathway (TNFRSF21, TNFRSF25, DAPK1, and FAS). The greatest differences in genes expression before, against, after treatment demonstrate antiapoptotic genes such as: BCL2L1, BIRC8, BIRC1, BIRC5, BCL2, and FAS, whose expression decreases. However, two proapoptotic genes: NOXA and CASP10 as well as inhibitor of transcriptional factor NF-kappa B (NFKBIZ ) are highly overexpressed. Our data suggest that CCR regimen triggers multifactorial stimulation of gene expression which are involved in proliferation and apoptosis processes. Nevertheless, further studies including clinical usefulness of this observation for the development of new therapeutical strategies especially for p53 family genes are in process. This study was supported in part by Grant No PBZ/MNiSW/07/2006/28 from the Polish Ministry of Science and Higher Education, Poland.