Ewelina Ziolkowska

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.

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.

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

Abstract 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.