P53 Protein Expression Levels Are Prognostic in AML and Predict for Mutational Status.

Mutation of the P53 gene in AML confers an adverse prognosis and is associated with unfavorable cytogenetics typically with complex karyoptyes. Since P53 function can also be modulated by MDM2 associated, ubiquitin dependent, destruction, the amount of P53 protein may also be relevant to leukemia blast biology. Previous studies have focused on the presence or absence of mutations, but have not examined the relevance of protein expression levels. We evaluated the levels of expression of P53 using high-throughput reverse phase protein array technology (RPPA) on 537 leukemia cell enriched samples from 423 patients (258 newly diagnosed, 47 primary refractory, 118 relapse 1, 2 or refractory). Levels of P53 were lower, equal and higher than that of normal CD34+ cells in 57%, 37% and 16% of cases, respectively. Almost all cases with very high P53 had unfavorable cytogenetics with complex karyotypes (15/16 newly diagnosed), and very high P53 was observed in 13% of patients with unfavorable cytogenetics compared to <1% with favorable or intermediate cytogenetics. Changes involving chromosome 5 (66% vs. 9%) and 7 (50% vs. 11%) were very common in those with very high P53 compared to those with normal or low P53. Levels of expression were similar regardless of disease status; newly diagnosed, primary refractory or relapsed We tested the hypothesis that patients with very high P53 were likely to have mutations by performing RT-PCR sequencing, covering exons 5–9 in 23 very high, and 11 low P53 expressing patients. Among very high expressers 11 missense mutations were found in 9 patients (39% overall), including 7 of 13 newly diagnosed cases (54%) compared to 0 of 11 low expressers (p=0.02 all cases, 0.05 newly diagnosed). Mutations occurred at known hot spots: including 5 in exon 8 [3 at codon 273], 3 in exon 7 and 2 in exon 5 and 6. High P53 expression was inversely correlated with PTEN expression (P=0.001, see accompanying abstract). Expression was unaffected by FLT3-ITD mutation status. For outcome analysis, martingale residuals were utilized to define an optimal cutpoint for dichotomization into groups with Higher (n=57, 50 treated) vs. lower (n=201, 165 treated) P53. Patients with higher P53 were significantly more likely to have an antecedent hematological disorder (37% vs. 17%, P=0.002) and unfavorable cytogenetics (65% vs. 39%, P=0.0002). Response to therapy and outcome were significantly worse in those with higher P53. The complete remission rate was significantly lower in those with higher P53 (46% vs. 66%, P=0.01). The relapse rate was significantly higher (74% vs. 47%, P=0.02) and the median remission duration was significantly shorter (26 vs. 43 weeks, P=0.02). The combination resulted in a significantly shorter median survival (26 vs. 44 weeks, P=0.0003). Analysis of MDM2 expression in these same cases is underway. This data demonstrates that cases of AML with very high levels of P53 protein expression, measured by RPPA, are frequently found in those with P53 mutations. Not all patients with high P53 levels have mutations, but high levels of P53 protein carry an adverse prognosis, regardless of mutation status.