Chronic lymphocytic leukemia (CLL) is the most common leukemia in Europe and North America. For many years scientists and doctors have been working on introducing the most effective therapy into CLL as prognosis of survival time and the course of the disease differ among patients, which might pose a problem in treating. Nanotechnology is providing new insights into diagnosis and, compared with conventional treatments, more efficient treatments, which might improve patients' comfort by decreasing side effects. Among the various nanoparticles that are available, dendrimers are one of the most promising. The aim of this study was a preliminary assessment of the clinical value of treating CLL patients with fourth generation poly(propylene imine) (PPI) dendrimers-either unmodified (PPI-G4) or approximately 90% maltotriose-modified (PPI-G4-DS-Mal-III). PPI-G4-DS-Mal-III dendrimers have, in contrast to the cationic PPI-G4, a neutral surface charge and are characterized by low cyto-, geno-, and hematotoxicity in vitro and in vivo. For the in vitro study we used blood mononuclear cells collected from both untreated CLL patients and from healthy donors. Apoptosis was measured by an annexin-V (Ann-V)/propidium iodide (IP) assay, and mitochondrial membrane potential was estimated with use of Mito Tracker Red CMXRos. Presented results confirm the influence of dendrimers PPI-G4 and PPI-G4-DS-Mal-III on apoptosis and CLL lymphocytes viability in in vitro cultures. Both tested dendrimers demonstrated higher cytotoxicity to CLL cells than to healthy donors cells, whereas unmodified dendrimers were more hematotoxic. The surface modification clearly makes glycodendrimers much more suitable for biomedical applications than unmodified PPI-G4; therefore further biological evaluations of these nanoparticles are conducted in our laboratories.

The acquisition of new aberrations during the course of chronic lymphocytic leukemia (CLL) named clonal evolution (CE) is usually detected by one of the two methods: chromosome banding analysis (CBA) and interphase fluorescence in situ hybridization (I-FISH). The purpose of this study was to compare the usefulness of FISH and CBA for detecting CE and to evaluate its influence on clinical outcome. FISH and CBA were performed at two time points: baseline and follow-up. Thirty-eight previously untreated patients with CLL were included in this study. CBA and I-FISH revealed CE in 15 (39.5%) and 10 (26.3%) patients, respectively. High-risk CE was detected in six cases by CBA and in five cases by I-FISH. In four cases with CE-dependent 17p abnormalities detected by CBA, metaphase FISH was needed for the confirmation of 17p13.1 deletion. Time from first-line to second-line treatment (TTST) and overall survival (OS) did not differ between patients with and without CE, irrespective of the CE-detecting method used. However, shorter OS (P = 0.043) and TTST (P = 0.006) were observed for the patients with potentially relevant CE (rCE) detected by CBA, in which acquired aberrations were present in at least 20% of undivided cells and/or changed baseline karyotype to abnormal or complex and were not resulting from 13q deletion. Our results suggest that some, but not all, CE-dependent aberrations detected by CBA influence clinical outcome. Moreover, I-FISH, which was aimed at detecting aberrations of prognostic significance, was found to be more precise than CBA in their detection, especially TP53 deletion.

4601 Treatment of chronic lymphocytic leukemia (CLL), including conventional therapy based on alkylating agents, purine nucleoside analogues (cladribine, fludarabine (FA)) or monoclonal antibodies (rituximab (Rit), alemtuzumab), significantly improved overall and complete responses. Although there are many treatment possibilities, this disease still remains incurable. Therefore, searching for new therapeutical strategies in CLL is vital. Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles (polymers) of dendritic architecture, used as carriers of drugs, nucleic acid and photosensitizers for targeted delivery, contrast agents in magnetic resonance imaging (MRI) have already been reported. It seems likely that dendrimers themselves might be damaging for neoplastic cells. The aim of our study was to preliminarly assess the clinical value of treating CLL patients with poly(propylene imine) (PPI) dendrimers. The assessment was based on the in vitro induction of cytotoxicity and apoptosis by fourth generation PPI dendrimers, with the surface amino groups substituted with maltotriose [Mal-III] residues. PPI-G4-OS-Mal-III dendrimers have, in contrast to the parental cationic PPI-G4, a virtually neutral surface charge. Therefore, they are preferentially involved in H-bond driven interactions. The study was conducted in 15 untreated CLL patients (7 men, 8 women) who were diagnosed in accordance with IWCLL criteria and followed at the Hematology Department, Medical University, Lodz, Poland. The Ethics Committee of the Medical University of Lodz, Poland approved the study (RNN/75/10/KE). Informed consent was obtained from all patients involved in the study. The mean age of CLL patients was 63.80 yrs. Peripheral blood mononuclear cells (MNCs) were separated from EDTA fresh blood. MNCs were incubated with dendrimers at concentrations of 4, 6 and 8 mg/ml. The cultures without dendrimers served as controls. Dendrimers in which approximately 35% of peripheral amino groups were coated with Mal-III (Leibniz Institute of Polymer Research, Dresden, Germany) have been defined as PPI-G4-OS-Mal-III. The abbreviation OS describes the open shell structure of carbohydrate-modified dendrimers (Fig. 1). The molar mass of this PPI dendrimer was 31000 g/mol. Apoptosis was measured by the Annexin-V/Propidium Iodide test. A decrease in mitochondrial membrane potential (one of the earliest events in the apoptotic pathway) was evaluated by the CMXRos technique, using flow cytometry. The percentage of cells with lower mitochondrial membrane potential (MMP) (DYmlow/Gly-A−) was determined. The percentage of apoptotic MNCs induced by PPI-G4-OS-Mal-III after 24 h and 48 h incubations was significantly higher than the percentage of spontaneous apoptotic leukemic cells. PPI glycodendrimers induced MNC apoptosis to a greater degree after 48 h than 24 h. The largest differences were observed for Ann-V+JP+ cells at a concentration of 8 mg/ml (p = 0.007). The IC50 value after 48-h-incubation was calculated as 8.24 mg/ml. Moreover, the CMXRos technique revealed apoptosis induction by PPI-G4-OS-Mal-III at each examined concentration in comparison with control cultures (Table 1). Table 1. 24h Control PPI-G4-OS-Mal-III 4 mg/ml PPI-G4-OS-Mal-III 6 mg/ml PPI-G4-OS-Mal-III 8 mg/ml FA 1.6 μM Rit 10 μg/ml X 23.7 51.0 60.1 64.4 62.9 28.0 48h X 28.3 67.8 71.9 79.3 89.0 29.4 X – mean percentage of cells with lower mitochondrial potential (DYmlow/Gly-A− [%]) Finally, PPI-G4-OS-Mal-III was not observed to have any harmful effects on erythrocytes (RBCs) or platelets (PLTs). The studied dendrimer induced PLT aggregation at a concentration of 50 mg/ml. Hemolysis induced by the PPI-G4-OS-Mal-III dendrimer is not important from the biological point of view. To summarise, the PPI-G4-OS-Mal-III dendrimer demonstrated higher cytotoxicity towards CLL cells than healthy donor cells. Its potency to trigger apoptosis is similar to many PNAs and monoclonal antibodies widely used in CLL therapy. Thus, dendrimers are a potential tool for CLL treatment. The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371 from the National Science Centre, Poland. Fig. 1. Fig. 1. Disclosures: No relevant conflicts of interest to declare.

The strongest cytotoxic effect of RIT in the presence of AS suggests that this drug activity towards CLL cells is enhanced by known cytotoxic mechanisms, caspase-dependent apoptotic pathway and possible influence of other extracellular factors present in the patients' sera.