Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the non-genotoxic drug Nutlin-3: a proposal for clinical applicationReportar como inadecuado




Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the non-genotoxic drug Nutlin-3: a proposal for clinical application - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Journal of Hematology and Oncology

, 6:83

First Online: 05 November 2013Received: 03 September 2013Accepted: 30 October 2013DOI: 10.1186-1756-8722-6-83

Cite this article as: Pozzo, F., Dal Bo, M., Peragine, N. et al. J Hematol Oncol 2013 6: 83. doi:10.1186-1756-8722-6-83

Abstract

BackgroundTP53 defects, i.e. 17p13 deletion and-or nucleotide mutations, associate with short survival and chemorefractoriness in chronic lymphocytic leukemia CLL. In this context, since direct sequencing of the TP53 gene does not evaluate TP53 functionality, a functional assessment of TP53 pathway may be of interest to identify high risk CLL. By taking advantage of a training cohort of 100 CLL and a validation cohort of 40 CLL with different patterns of TP53 mutation-deletion by FISH and sequencing, we propose an in-vitro assay in which the modulation of TP53 protein and CDKN1A mRNA were investigated upon 24-hour exposure of CLL cells to Nutlin-3.

MethodsThe functional assay was set-up on cell lines recapitulating all TP53 genotypes EHEB, TP53; RAJI, TP53; MEC-1 and MAVER1, TP53; HL-60, TP53 and evaluated in two multi-institutional cohorts, purposely enriched in CLL bearing TP53 disruption: a training cohort of 100 cases and a validation cohort of 40 cases, both characterized by FISH and TP53 direct sequencing. Cells were exposed to 10 μM Nutlin-3 for 24 hours; TP53 accumulation was evaluated by Western blotting; TP53 transcriptional activity was determined by quantitative realtime PCR qRT-PCR of the TP53 target gene CDKN1A.

ResultsAccording to TP53 protein modulation, in the training cohort we identified: i 63 cases 51 TP53, 12 TP53 with absence of basal TP53 and induction after treatment normal pattern; ii 18 cases 3 TP53, 15 TP53 with high basal TP53 without increase after treatment mutant pattern; iii 19 cases 5 TP53; 3 TP53; 11 TP53 with basal TP53 that increases upon treatment intermediate pattern. Evaluation of CDKN1A mRNA levels upon Nutlin-3 exposure showed that the 26 TP53 mutated TP53 or TP53 cases had lower induction levels than the majority 57-63 of cases with normal pattern, and 10-12 cases with intermediate pattern without evidence of TP53 derangement by FISH and sequencing. These results were confirmed in the independent validation cohort of 40 cases 13 TP53, 3 TP53, 12 TP53, 12 TP53.

ConclusionsThe proposed functional assay may integrate the conventional analyses for the identification of TP53 dysregulated CLL.

KeywordsCLL TP53 Prognosis AbbreviationsFISHFluorescence in situ hybridization

qRT-PCRQuantitative real time PCR

FACSFluorescence activated cell sorting

GEPGene expression profiling

7-AAD7-amino-actinomycin.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-8722-6-83 contains supplementary material, which is available to authorized users.

Federico Pozzo, Michele Dal Bo contributed equally to this work.

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