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Purpose: There is evidence that vascular endothelial growth factor (VEGF) is a critical microenvironmental factor that exerts angiogenesis-independent effects on the survival of chronic lymphocytic leukemia (CLL) cells. Vatalanib and pazopanib are potent orally available VEGF receptor tyrosine kinase inhibitors. We investigated the efficacy and selectivity of both compounds in CLL cells, simulated potential combination with conventional cytostatics, and tested the effect of both substances on CLL-like tumor xenografts.Experimental Design: Primary CLL and normal peripheral blood cells were tested for viability after incubation with varying concentrations of both inhibitors. Further, phosphorylation status of VEGF receptor on treatment, caspase activation, and poly(ADP-ribose) polymerase cleavage were assessed. Combinations of each inhibitor with fludarabine, vincristine, and doxorubicin were analyzed for possible synergistic effects in vitro. For in vivo testing, mice grafted with the CLL-like cell line JVM-3 were treated orally with each inhibitor.Results: Vatalanib and pazopanib decreased phosphorylation of the VEGF receptor, along with induction of apoptosis in CLL cells in clinically achievable concentrations. Healthy B cells were only mildly affected. Immunoblots showed downregulation of the antiapoptotic proteins XIAP and MCL1, whereas poly(ADP-ribose) polymerase cleavage was increased. Combinations with conventional cytostatic agents resulted in synergistic effects. Treatment of xenografted mice with 100 mg/kg of body weight for 21 days resulted in tumor inhibition rates of 76% (vatalanib) and 77% (pazopanib). In two mice, a total tumor eradication could be observed. No gross systemic toxicity occurred.Conclusion: We conclude that VEGF inhibition is a promising new therapeutic approach in CLL. Vatalanib and pazopanib seem to be effective and safe candidates to be further evaluated for this purpose.

We determined lymphoid enhancer-binding factor-1 (LEF1) mRNA expression in 112 chronic lymphocytic leukemia (CLL) samples and assessed correlations with the prognostic markers ZAP70 and CD38, Binet stages, the percentage of lymphocytes in the peripheral blood, and fibromodulin (FMOD) transcripts. The mean LEF1 relative expression ratios (RER) were 53.72 and 37.10 in ZAP70-positive and ZAP70-negative patients, respectively (P=0.004). However, we did not observe a significant difference in LEF1 expression between CD38-positive and CD38-negative patients. Moreover, patients requiring treatment showed a mean LEF1 RER of 85.61 whereas patients in recently diagnosed Binet A stage had a mean of only 22.01 (P<0.001). We also found significant correlations of LEF1 with the percentage of lymphocytes and FMOD expression. Our results suggest that high LEF1 expression is associated with poor prognosis and disease progression. Thus, LEF1 might be involved in the process of disease progression and possibly can serve as a molecular parameter for risk assessment and/or monitoring of CLL.

New therapeutic strategies developed recently for chronic lymphocytic leukemia (CLL) have led to remarkable treatment response rates and complete hematological remissions. This means highly sensitive and specific techniques are increasingly needed to evaluate minimal residual disease (MRD) in CLL patients. Quantitative MRD levels can be used as prognostic markers, where total MRD eradication is associated with prolonged survival. Nowadays, PCR and flow cytometry techniques used to detect MRD in CLL patients can generate reliable and quantitative results with the highest sensitivity. MRD Flow is based on four-color flow cytometry using specific antibody combinations. For allele specific oligonucleotide real-time quantification (ASO RQ) PCR individual primers are designed to detect a specific immunoglobulin heavy chain (IgH) rearrangement in each patient clone. Five comprehensive studies investigated and compared the sensitivity and specificity of both methods. Groups of patients receiving different therapies were analyzed at different time points to generate quantitative MRD levels and MRD kinetics. All studies confirmed that both methods generate equivalent results with regard to sensitivity and MRD quantification, although each method has advantages and disadvantages in the daily routine of a standard hematological laboratory. Here, we review these investigations and compare their results in the light of modern therapies.

3451 Poster Board III-339 We and others have shown that vascular endothelial growth factor (VEGF) plays a pivotal role in growth, survival and migration of chronic lymphocytic leukemia (CLL). VEGF dependent survival benefits in CLL are thereby mediated via autocrine and paracrine loops. However, so far no approach was studied focusing on the selective inhibition of VEGF in CLL. Vatalanib (PTK787/ZK 222584) and pazopanib (GW786034) are potent orally available, selective VEGF tyrosine kinase inhibitors. The aim of the present investigation was i) to study the efficacy and selectivity of both inhibitors in CLL cells, ii) to simulate potential combination with conventional cytostatics in vitro and iii) to test the effect on CLL like tumor xenografts in a mouse model. Primary CLL as well as healthy B cells were incubated with varying concentrations of both inhibitors for different time periods. Cells were then treated with combinations of each inhibitor and fludarabine, vincristin and doxorubicin, respectively. Apoptosis induction was analysed by flow cytometry (annexin V-FITC/PI staining) and cell survival was additionally investigated by an ATP dependent fluorescence assay. Percentage of surviving cells was defined as double annexin/PI negative cells in flow cytometry analysis. For in vivo experiments, four-week old BALB/c nu/nu mice were grafted with cells of a human chronic B cell leukemia cell line (JVM3). After tumors reached a mean volume of 100 mm3 per group (10 mice), drugs were administered once daily by oral gavage at 100 mg/kg bodyweight. Tumor volume was measured every second day by calliper. Vatalanib and pazopanib effectively induced apoptosis in CLL cells in vitro in a dose and time dependent fashion. During 24h incubation of CLL cells vatalanib showed a 50% lethal concentration (LC50) of 46.7 μM (n=26) and pazopanib of 32.7μM (n=19). In contrast, survival of B cells derived from healthy donors was only slightly affected at high concentrations of both drugs, thereby suggesting a large therapeutic range. Healthy B cells survived 80.5 ± 4.5% after 24h incubation with 100 μM vatalanib. In contrast, CLL cells showed a survival of 27.8 ± 6.0% (n=5; p=0.0001). Survival of healthy B cells treated with 100 μM of pazopanib was 89.1 ± 2.2% whereas survival of CLL cells was only 40.8 ± 2.3% (n=5; p<0.0001). Combination of the low dosed drugs with conventional cytostatics like fludarabin, vincristin and doxorubicin showed synergistic effects and significantly increased apoptosis rates in vitro. Single drug treatment showed survival rates of 80.1 ± 5.7% for 10 μM vatalanib, 86.2 ± 10.1 for 10 μM fludarabin, 69.8 ± 6.1 % for 0.10 μM vincristin and 51.6 ± 5.2% for 10 μM of doxorubicin. The combination of 10 μM vatalanib with 10 μM fludarabin showed survival rates of 59.4 ± 11.6% (p=0.0167), with 0.1 μM vincristin 32.5 ± 7.2% (p=0.0095) and with doxorubicin 26.0 ± 11.8% (p=0.0381). Survival rates for single treatment with 10 μM pazopanib were 84.3 ± 4.3%. The combination of 10 μM pazopanib with 10 μM fludarabin showed survival rates of 55.0 ± 6.1% (p=0.0384), with 0.1 μM vincristin 42.8 ± 5.3 (p=0.0473) and with doxorubicin 41.5 ± 11.8% (n.s.). After three weeks of treatment of the xenograft mice with each inhibitor the mean tumor volume was 645.0 ± 241.2 mm3 with pazopanib (p=0.002), 671.8 ± 198.0 mm3 with vatalanib (p=0.002) and 2,458.3 ± 742.39 mm3 in the vehicle treated group. This translated into a mean tumor inhibition rate of 77.3% for pazopanib and 71.3% for vatalanib after 21 days of treatment. Summing up, specific inhibition of VEGF by vatalanib or pazopanib might be a promising new therapeutic approach in CLL. Both compounds are orally available and showed acceptable in vivo toxicities in clinical trials, promising good compliance for the treatment. Therefore, they are attractive candidates for further testing in CLL. Disclosures Hallek: BayerScheringAG: Honoraria, Research Funding.