Téma: Drug resistance and pharmacology
Číslo abstraktu: P975
Autoři: RNDr. Ludmila Šebejová; MUDr. Vlastislav Šrámek, PhD; Mgr. Zuzana Jašková; RNDr. Jitka Malčíková, Ph.D.; Mgr. Veronika Navrkalová; Mgr. Marek Borský; prof. MUDr. Michael Doubek, Ph.D.; prof. MUDr. Jiří Mayer, CSc.; prof. RNDr. Šárka Pospíšilová, Ph.D.; MUDr. Martin Trbušek, PhD
TP53 and ATM defects have been associated with inferior outcome in chronic lymphocytic leukemia (CLL). The response of corresponding patients to conventional chemotherapy is usually poor. Monoclonal antibodies targeted to leukemic cells are supposed to act primarily independently on apoptosis induction and should, therefore, bypass a block imposed by the TP53 or ATM inactivity. Despite that, treatment outcome using rituximab on its own or in combination with chemotherapy is usually unsatisfactory among TP53-affected patients. This can be accounted either to a higher primary resistance to rituximab or to a faster and more aggressive relapse.
To compare in vitro primary response of CLL cells with well defined TP53 and ATM defects to rituximab.
Deletions 17p and 11q were detected by I-FISH; TP53 mutations were identified by yeast functional analysis and direct sequencing; ATM mutations were captured through resequencing microarray. All selected aberrant samples harbored complete p53 or ATM inactivation as evidenced by defective response to IR and doxorubicin; by contrast, all selected wild-type samples were functioning in these tests. The analysis of CLL cells sensitivity to rituximab was performed on 70 characterized samples using a metabolic WST-1 assay; 20% active human serum was added to allow cell lysis by complement; non-specific immunoglobulin was used as a negative control in all samples. The ability of rituximab to induce apoptosis under our experimental conditions was verified by flow-cytometry using propidium iodide vs. annexin V measurement.
The following main observations have been made: (1) Concentration of 10 μg/ml was sufficient for rituximab in vitro testing; higher concentrations (20 and 30 µg/ml) showed the same effect in all cultures; (2) Final viability of tested cultures ranged from 45% to 100% in comparison with untreated control; all cultures were inert to non-specific immunoglobulin. There were clear differences in sensitivity to rituximab between the tested genetic groups; the TP53-mutated group (n=27 samples) was substantially more resistant than both wild-type samples (n=31) (P=0.04) and especially than ATM-mutated samples (n=12) (P=0,017; Wilcoxon test) (3) rituximab used at concentration of 10 μg/ml (and without active serum) was able to induce apoptosis (annexin V positivity) after 24 h treatment in tested cultures.
Summary / Conclusion:
Our results showing significantly different response to rituximab for CLL cells harboring TP53 mutation vs. those harboring ATM mutation were quite unexpected, especially because active serum was assumed to allow the lysis by complement. This observation indicates that apoptosis might play important role during primary CLL cells response to rituximab. This work was supported by grant NT13519-4/2012 by IGA Ministry of Health, Czech Republic and by project MUNI/A/0723/2012.
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Keywords: Apoptosis, Chronic lymphocytic leukemia, p53, Rituximab
Datum přednesení příspěvku: 15. 6. 2013