Clonal evolution in CLL patients as detected by FISH versus chromosome banding analysis, and its clinical significance.

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.

Cytotoxic and apoptosis-inducing effects of bendamustine used alone and in combination with rituximab on chronic lymphocytic leukemia cells in vitro.

AIM: The aim of our study was to compare the cytotoxic effects of bendamustine (BENDA) and rituximab (RIT) used either alone or in combination and to evaluate the influence of the above mentioned drugs on apoptosis measured as changes in mitochondrial transmembrane potential (Δψm), expression of caspases and selected apoptosis-regulating proteins in freshly isolated peripheral blood mononuclear cells of chronic lymphocytic leukemia (CLL) patients. MATERIALS/METHODS: Cytotoxic effect of tested drugs, as well as induction of apoptosis, drop in Δψm and expression of selected proteins involved in regulation of apoptosis were assessed in 48 hour cultures containing autologous serum (AS) using flow cytometry. BENDA was used at the concentration of 40 µg/ml and RIT at the concentration of 10 µg/ml. Control cultures were incubated without drugs. RESULTS: BENDA used either alone or in combination with RIT strongly induced apoptosis as well as enhanced expression of selected apoptotic proteins, especially those involved in the intrinsic apoptotic pathway: P53, PUMA and BAX, which cause mitochondrial transmembrane potential changes leading to activation of caspase-9 and -3. CONCLUSIONS: Our results indicate that both BENDA and RIT participate in the induction of apoptosis of CLL lymphocytes in vitro in the presence of AS in the culture medium. The drug-induced apoptosis occurs mainly via intrinsic pathway and activation of P53 and PUMA proteins, however the extrinsic pathway is likely to be involved as well. We also found that the combination of these drugs induces the expression of P53, caspase-8 and -9 more potently than either of them used separately.

The influence of maltotriose-modified poly(propylene imine) dendrimers on the chronic lymphocytic leukemia cells in vitro: dense shell G4 PPI.

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.

Type of serum influences the rituximab dependent cytotoxicity and apoptosis of chronic lymphocytic leukemia cells in vitro.

PURPOSE: The aim of the study was to compare the influence of types of serum on the in vitro viability and on either spontaneous or rituximab (RIT)-induced apoptosis of chronic lymphocytic leukemia (CLL) cells. METHODS: The influence of fetal calf serum (FCS), patients' autologous serum (AS) and human AB-serum (ABS), used alone and in combinations consisting of two of them (v/v-1:1), on RIT-dependent cytotoxicity, apoptosis, detection of active forms of caspases-3,-9,-8 and disruption of mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. RIT was used at the concentration of 10 µg/ml. The spontaneous apoptosis was assessed in culture without RIT. RESULTS: AS revealed the protective action on CLL cells, however this serum added in vitro to the culture either alone or in combination with FCS was the only one to allow RIT to exert its cytotoxic action against CLL cells. RIT-induced apoptosis involved changes in ΔΨm and activation of caspases-3,-8,-9 when AS+FCS was applicated. Drug induced apoptosis (DIA) was 6.02 and 0.34, when FCS+AS and FCS alone were used, respectively (p<0.01). The RIT-dependent cytotoxic effect decreased when FCS+AS or FCS+ABS were used, as compared to effect of AS used separately. The cytotoxic effect of RIT did not depend on drug concentration, but on the type of serum added to the culture. CONCLUSIONS: 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.

The influence of venetoclax, used alone or in combination with cladribine (2-CdA), on CLL cells apoptosis in vitro: Preliminary results.

BACKGROUND: Venetoclax (VEN), a highly selective BCL-2 inhibitor, is successfully used in the treatment of chronic lymphocytic leukemia (CLL). The purine analogue - cladribine (2-CdA) - is also administered to CLL patients, especially as a part of chemoimmunotherapy. OBJECTIVES: To compare the effects of the VEN+2-CdA regimen with that of the 2 drugs used alone on the apoptosis of CLL lymphocytes in vitro. MATERIAL AND METHODS: Mononuclear cells were collected from 103 previously untreated CLL patients. They were incubated with VEN (40 nM) or/and 2-CdA (16 µM) for 48 h. Cytotoxicity, overall apoptosis, mitochondrial transmembrane potential changes (ΔΨm), and expression of selected apoptosis-involved proteins were measured. RESULTS: The cytotoxicity, overall apoptosis, caspase-3 or caspase-9 expression, and ΔΨm were significantly higher after VEN+2-CdA addition compared to both drugs used alone, with a very strong synergistic effect observed. The percentage of BCL-2-positive cells decreased after VEN and VEN+2-CdA addition compared to controls. The TP53-expressing cells increased under the influence of all tested regimens. The VEN+2-CdA increased the expression of BIM, BAX and NOXA compared to either controls or VEN or 2-CdA alone. Similar increases in PUMA expression were observed after VEN, 2-CdA and VEN+2-CdA addition. The FAS-associated death-domain protein (FADD) expression was significantly higher after 2-CdA and 2-CdA+VEN addition as compared to control. CONCLUSIONS: Our results confirm the involvement of both VEN and 2-CdA in the intrinsic apoptotic pathway. They also demonstrate that these agents have a synergistic effect on CLL cells in vitro. Further studies are needed to assess the influence of VEN+2-CdA on the expression of apoptosis-involved genes.

Bendamustine alone or with rituximab modifies expression of apoptosis-regulating genes and proteins of CLL cells, depending on IGVH mutational status.

We studied whether bendamustine (BENDA) alone or with rituximab (RIT) modifies in vitro expression of apoptosis-involved genes and proteins of chronic lymphocytic leukemia (CLL) cells depending on IGVH mutational status. Circulating lymphocytes from 34 untreated patients (18 IGVH-MUT and 16 IGVH-UNMUT) were incubated with above drugs to evaluate proteins expression. Microarray analysis of 93 genes was performed in 14 patients. BENDA and BENDA + RIT increased expression of BAX and BBC3 in IGVH-MUT and IGVH-UNMUT groups, and significant differences in expression of above genes after BENDA + RIT were observed between both groups. Additionally, BENDA + RIT decreased NFκB and BCL-2 genes in IGVH-UNMUT patients and increased expression of P53, BAX and PUMA proteins in IGVH-MUT and UNMUT subjects. However, no significant differences were found between these groups. In conclusion, BENDA + RIT modified gene expression profile in CLL cells and affected expression of some apoptosis-regulating proteins in vitro. Expression of BAX and BBC3 depends on action of drugs and IGVH mutational status.

Glycodendrimer PPI as a Potential Drug in Chronic Lymphocytic Leukaemia. The Influence of Glycodendrimer on Apoptosis in In Vitro B-CLL Cells Defined by Microarrays.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) cells are characterized by failures in the apoptosis pathway and increased proliferation, resulting in the progressive accumulation of B-lymphocytes in blood. Despite the wide range of antileukaemic drugs, CLL remains an incurable disease. However, a breakthrough is expected which will allow more effective treatment. OBJECTIVE: The study investigates the influence of poly(propyleneimine) (PPI) dendrimer with peripheral amino groups, 30% of which were coated with maltotriose (PPI-G4-OS-Mal-III), on CLL cells, and demonstrates that it acts through the induction of the apoptotic mechanism. It is important to note that the dendrimer was used as a drug itself and not as a drug carrier. METHOD: CLL and normal lymphocytes were treated in vitro with the dendrimer, either alone or in combination with fludarabine (FA). The percentages of apoptotic and necrotic cells, and the protein expression, were checked using a flow cytometer. Gene expression was screened using a two-colour microarray with 60-mer probes. RESULTS: The results confirm that PPI-G4-OS-Mal-III influences the viability of CLL cells in vitro and does not exert any significant harmful effect on normal lymphocytes. The dendrimer appears to significantly influence gene and protein expression in CLL cells. CONCLUSION: Since dendrimers can be specifically targeted, they may be very effective in CLL therapy, especially since in vitro PPI-G4-OS-Mal-III has been found to have stronger effect than fludarabine.