Rituximab senzitizes some B-CLL samples to fludarabine and chlorambucil in vitro, regardless of p53/ATM status
| Authors | |
|---|---|
| Year of publication | 2007 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Background: Aberrations of two co-operating genes, the p53 and ATM, significantly deteriorate prognosis and treatment options for B-CLL patients. Monoclonal antibody rituximab (anti-CD20) is preferably used in combination regimens in B-CLL, often in those containing fludarabine. Very few in vitro data exist, however, showing an effect of such common treatment on B-CLL cells with aberrant p53 and/or ATM. In this respect, the data are also missing for a potential application of rituximab with chlorambucil. Aims: The aims were to assess the in vitro effect of the above mentioned combinations of drugs on B-CLL cells with various p53/ATM status. Methods: An interphase FISH was used for a determination of p53 and ATM deletions. Functional FASAY analysis coupled to sequencing was employed to supplement the screening also for p53 mutations. A metabolic WST-1 assay monitored the drugs effect on cell viability. An in vitro system lacking active human plasma was used, thus omitting the CDC pathway. After rituximab pre-treatment (10ug/ml, 72h) the chemotherapeutics were applied in four concentrations for additional 48h (F: 25 - 0.4ug/ml; CLB: 50 - 6.25 uM). Two-way analysis of variance (ANOVA) was used for determination of rituximab pre-treatment significance. Results: For the rituximab/fludarabine combination we tested forty samples having a median 85% of B-CLL lymphocytes, with the following characteristics: 13 were wild-type, 10 harbored ATM deletion (median 83% of deleted cells) and 17 exhibited p53 defects of various complexity - both alleles inactivation (del/mut and mut/mut) as well as the separate (one allele) aberrations (del or mut). The sensitivity to fludarabine was determined for the concentration 1.6ug/ml, which provided significant differences among the samples. The sensitivity was assessed as follows: resistant - viability over 60%; medium - viability between 60% and 40%; sensitive - viability under 40%. The p53-affected samples were mostly resistant (71%) and none were sensitive. Among ATM deleted samples, on the contrary, 40% were sensitive, what was more than in wild-type subgroup (23%). Rituximab alone slightly increased a metabolic activity in most of samples, usually to 110-130% compared to untreated controls, while rarely a decrease was also noted (up to 80%). When the viability of fludarabine-tretead and rituximab/fludarabine-treated samples was assessed in relation to fully untreated control, the positive sensitization effect of rituximab pre-treatment (P = 0,05) was noted as follows: within the p53-affected as well as ATM-deleted subgroups in 30% of samples and within the wild-type subgroup in 62% of samples. For the rituximab/chlorambucil testing, which was performed as a pilot study in the same manner in eight samples, the positive effect of antibody pre-treatment was also noted in some samples of all the three subgroups. Summary/Conclusions: Our results indicate that the p53/ATM status is critical for the sensitivity of B-CLL cells to fludarabine. Regardless of the p53 and ATM aberrations, some samples are available for the rituximab-mediated sensitization to this agent. Our pilot data also support a warranty of testing a combined regimen containing rituximab and chlorambucil. Supported by grant IGA MH CR No. 8445-3/2005. |