The Use of Tetradenoylphorbol Acetate Stimulated Peripheral Blood Cells for Interphase Fluorescence In-Situ Hybridization Analysis May Enhance Its Prognostic Value for Patients with Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by a variable clinical course. Genomic aberrations, as identified by interphase fluorescent in situ hybridization (I-FISH), have a remarkable predictive power in terms of response to therapy, time to first treatment and overall survival of these patients. I-FISH studies can be performed either on unstimulated peripheral blood mononuclear cells (I-FISH-PBMC) or on tetradecanoylphorbol acetate (TPA) stimulated peripheral blood cells (I-FISH-TPA). We have previously observed that I-FISH-TPA could identify genomic abnormaties that might be overlooked with I-FISH-PBMC. The aim of the study was to evaluate whether this increased detection of genomic abnormalities, as identified by I-FISH-TPA, was clinically relevant for a group of consecutive CLL patients. Blood samples from 47 CLL patients were stimulated with TPA and cultured in RPMI medium supplemented with fetal calf serum. Peripheral blood mononuclear cells were isolated using density-gradient centrifugation and fixed according to standard methods. I-FISH was performed on both TPA stimulated and unstimulated cells using 11q22.3 (ATM), 13q14 (13S272), 17p13.1 (p53), and centromeric 12 (D12Z3) probes. 200 nuclei were evaluated for each probe, and cut-off points were set at 6%, 7%, 5% and 2% for del(11q), del(13q), del(17p) and trisomy 12, respectively. Metaphase FISH and conventional cytogenetics were also performed in selected cases. For all patients, chemotherapy was initiated according to Cheson criteria and treatment-free and overall survival curves were plotted using SPSS software. Following a modified version of Dohner’s hierarchical model, patients were divided in those with del(17p) and/or del(11q) and those with other or no genomic abnormalities. Fourteen cases with negative or bordeline results with I-FISH-PBMC became positive with I-FISH-TPA for del(11q) (2 cases), del(13q) (9 cases) and trisomy 12 (3 cases). In all but one patient, either conventional karyotyping or metaphase FISH confirmed these abnormalities. I-FISH-TPA provided a better prediction of treatment-free interval compared to I-FISH-PBMC (P= 0.002 vs 0.019, see Figures 1 and 2). In particular, two patients with no cytogenetic abnormalities detected by I-FISH-PBMC required chemotherapy 3 and 11 months after diagnosis, more in keeping with the presence of del(11q) found using I-FISHTPA. Furthermore, I-FISH-TPA also improved the overall survival prediction compared to I-FISH-PBMC (P= 0.036 vs 0.042). In summary, I-FISH-TPA increased the detection rate and had an improved prognostic value compared to I-FISH-PBMC. Further studies with larger numbers of patients are warranted.