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

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 TP53 wt/wt, 12 TP53 del/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