Patient-specific evaluation of p53 status: projection on future possible targeted therapy. Cytogenetics and fluorescence in situ hybridization (FISH) detect 17p⁻ (associated with mutp53 and LOH). SNP-As (single-nucleotide polymorphism arrays), identify CNAs and LOH at a deeper level of detection than conventional cytogenetics and are also used to detect common polymorphisms, including those of TP53. The Sanger sequencing method continues to be a mainstay technology for rapid analysis and sequence determination of relatively small fragments of human DNA, embracing single or few genes at a time. Despite the relatively low of sensitivity of the method (VAF detection lower than ∼20%), it is still considered the gold standard for mutation analysis and is commonly used to confirm NGS results. NGS technology is the current gold standard for comprehensive DNA variant analysis. It enables rapid sequencing of large segments of an individual’s DNA and facilitates precise detection of genetic variations ranging from single-nucleotide substitution to large structural rearrangement, with a VAF detection sensitivity of 2% to 5%. It is based upon preparation of DNA fragment libraries, which are subsequently clonally amplified and sequenced by synthesis in multiple parallel reaction. The generated sequences are aligned and assembled on a human reference genome and computationally analyzed. The most commonly used target enrichment techniques include hybrid capture of target genes or multiplex-based PCR.^17,125  NGS assays (selected either for whole-genome/exome/target sequencing based upon the AML gene panel) identify TP53 mutations and related genomic variations, including LOH and CNAs, and provide quantitative measurement of TP53 mutation abundance. Calculating TP53 mutation variant allele frequency allows one to distinguish an initiator mutation from a cooperating one. Additionally, appropriate bioinformatic methods allow SNP calling from NGS data, enabling distinction between a consequential variant and an inconsequential SNP.¹²⁶  Distinction between somatic and germ line mutations necessitates availability of a matched control tissues sample (eg, patient’s skin cells), which is presumed to retain a germ line configuration. Tag-based methods detect ultrarare TP53 mutations.¹²⁷  p53 protein expression and activity should be evaluated independently of p53 mutational status. p53 protein levels, assessed by western blotting and immunohistochemistry, should be interpreted cautiously, since p53 overexpression is not a surrogate for TP53 mutations (might be caused by numerous nonmutational stimuli), while lack of expression does not rule out their presence (eg, in cases of TP53 nonsense mutations/deletions).^6,128  p53 activity can be evaluated by in vitro (AML blasts) assessment of p53 and its target genes before and after exposure to genotoxic stress induced by Ara-C or anthracyclines. The preferable target genes are p21 and PUMA, whose activation results normally in cell cycle arrest/senescence and apoptosis. These effects are usually assessed by measuring protein levels and by cell cycle/apoptosis and cell viability assays. The HL-60 cell line, characterized by major TP53 deletions, serves as the negative control for p53 functional evaluation.⁹  A discrepancy between wtp53 expression and an attenuated genotoxic stress response can be attributed to wtp53 inactivation. In case of mutp53, no stress response can be attributed to mutp53 LOF or the presence of mutant p53 GOF, which requires further assessment. The suggested therapies represent merely a conceptual model for future potential therapeutic strategies. In Red: current practice and agents that are already in AML clinical trials with reference to p53 status. *With the exception of RETRA and MDR reversal agents (italicized), all the agents mentioned are already in AML clinical trials, though not with reference to p53 status. Pretreatment ex vivo drug sensitivity testing of patient-derived AML cells may predict efficacy of the suggested therapeutic regimen. Pol I inhibitors, which augment p53 activity, are not listed herein, since their efficacy in AML with mutp53 or inactivated wtp53 is currently unknown.