title: 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.
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.
authors.forename: Federico
authors.initials: F
authors.lastname: Pozzo
authors.forename: Michele
authors.initials: M
authors.lastname: Dal Bo
authors.forename: Nadia
authors.initials: N
authors.lastname: Peragine
authors.forename: Riccardo
authors.initials: R
authors.lastname: Bomben
authors.forename: Antonella
authors.initials: A
authors.lastname: Zucchetto
authors.forename: Francesca
authors.initials: F
authors.lastname: Rossi
authors.forename: Massimo
authors.initials: M
authors.lastname: Degan
authors.forename: Davide
authors.initials: D
authors.lastname: Rossi
authors.forename: Annalisa
authors.initials: A
authors.lastname: Chiarenza
authors.forename: Alberto
authors.initials: A
authors.lastname: Grossi
authors.forename: Francesco
authors.initials: F
authors.lastname: Di Raimondo
authors.forename: Francesco
authors.initials: F
authors.lastname: Zaja
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pozzato
authors.forename: Paola
authors.initials: P
authors.lastname: Secchiero
authors.forename: Gianluca
authors.initials: G
authors.lastname: Gaidano
authors.forename: Giovanni
authors.initials: G
authors.lastname: Del Poeta
authors.forename: Giorgio
authors.initials: G
authors.lastname: Zauli
authors.forename: Robin
authors.initials: R
authors.lastname: Fo À
authors.forename: Anna
authors.initials: A
authors.lastname: Guarini
authors.forename: Valter
authors.initials: V
authors.lastname: Gattei
mesh_terms: D015153:Blotting, Western; D015331:Cohort Studies; D016158:Genes, p53; D005838:Genotype; D018922:HL-60 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology*; D011379:Prognosis; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D007093:Imidazoles; D010879:Piperazines; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3