Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the ... View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2013-12

AUTHORS

Federico Pozzo, Michele Dal Bo, Nadia Peragine, Riccardo Bomben, Antonella Zucchetto, Francesca Maria Rossi, Massimo Degan, Davide Rossi, Annalisa Chiarenza, Alberto Grossi, Francesco Di Raimondo, Francesco Zaja, Gabriele Pozzato, Paola Secchiero, Gianluca Gaidano, Giovanni Del Poeta, Giorgio Zauli, Robin Foà, Anna Guarini, Valter Gattei

ABSTRACT

BACKGROUND: TP53 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. METHODS: The functional assay was set-up on cell lines recapitulating all TP53 genotypes (EHEB, TP53(wt/wt); RAJI, TP53(mut/wt); MEC-1 and MAVER1, TP53(mut/del); HL-60, TP53(del/del)) 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. RESULTS: According to TP53 protein modulation, in the training cohort we identified: (i) 63 cases (51 TP53wt/wt, 12 TP53del/wt) with absence of basal TP53 and induction after treatment (normal pattern); (ii) 18 cases (3 TP53(mut/wt), 15 TP53(mut/del)) with high basal TP53 without increase after treatment (mutant pattern); (iii) 19 cases (5 TP53(mut/wt); 3 TP53(mut/del); 11 TP53(wt/wt)) 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(mut/del) or TP53(mut/wt)) 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(wt/wt), 3 TP53(del/wt), 12 TP53(mut/del), 12 TP53(mut/wt)). CONCLUSIONS: The proposed functional assay may integrate the conventional analyses for the identification of TP53 dysregulated CLL. More... »

PAGES

83

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1756-8722-6-83

DOI

http://dx.doi.org/10.1186/1756-8722-6-83

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1006296969

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24283248


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