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, TP53wt/wt; RAJI, TP53mut/wt; MEC-1 and MAVER1, TP53mut/del; HL-60, TP53del/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 TP53mut/wt, 15 TP53mut/del) with high basal TP53 without increase after treatment (mutant pattern); iii) 19 cases (5 TP53mut/wt; 3 TP53mut/del; 11 TP53wt/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 (TP53mut/del or TP53mut/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 TP53wt/wt, 3 TP53del/wt, 12 TP53mut/del, 12 TP53mut/wt). Conclusions The proposed functional assay may integrate the conventional analyses for the identification of TP53 dysregulated CLL.

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.

POZZATO, GABRIELE;ZAULI, GIORGIO;ZAJA, FRANCESCO
2013

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, TP53wt/wt; RAJI, TP53mut/wt; MEC-1 and MAVER1, TP53mut/del; HL-60, TP53del/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 TP53mut/wt, 15 TP53mut/del) with high basal TP53 without increase after treatment (mutant pattern); iii) 19 cases (5 TP53mut/wt; 3 TP53mut/del; 11 TP53wt/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 (TP53mut/del or TP53mut/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 TP53wt/wt, 3 TP53del/wt, 12 TP53mut/del, 12 TP53mut/wt). Conclusions The proposed functional assay may integrate the conventional analyses for the identification of TP53 dysregulated CLL.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11368/2834399
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 16
  • ???jsp.display-item.citation.isi??? 14
social impact