Wild Type-TP53 is more highly expressed in NPM1-mutated AML compared to other AML subtypes
In our clinical diagnostic hematopathology practice we routinely perform screening for TP53 and NPM1 mutations by chromogenic immunohistochemistry (IHC) as a component of new AML evaluations.8,10 In select cases of molecularly-
confirmed NPM1-mutated/TP53-WT AML, we have observed p53 expression at a level often observed in the cases harboring missense mutations in the DNA binding domain of TP53 (Figure 1A, representative case). We hypothesized that the elevated expression of WT-p53 could be a result of increased TP53 gene expression in at least a subset of NPM1-AML cases. Based on an analysis of the BeatAML cohort dataset,6 we found that NPM1-AML cases (n=74) are associated with significantly higher TP53 expression than TP53-WT/NPM1-WT cases (n=190) [p= 0.047, multivariate limma model] (Figure 1B). We considered the possibility that this difference may be driven by mutations with prognostic significance co-occurring with NPM1 (FLT3-ITD, FLT3-TKD, DNMT3A, SF3B1, SRSF2, U2AF1); however, exclusion of these genes by comparing cases of NPM1-AML to NPM1-WT/TP53-WT/gene mutation-positive cases revealed a similar result
[p=0.018, multivariate limma model] (Figure 1C). Furthermore, we observed no effect of specimen type (e.g., peripheral blood, bone marrow, or leukapheresis product) on TP53 expression level (Figure 1D).
p53-associated gene modules are upregulated in NPM1-mutated AML and correlate with wild-type TP53 expression level
To assess the activity of WT-p53 signaling we focused on known p53-associated gene sets. By comparing NPM1-AML with TP53-AML cases (n=17), we first established the pattern of up-/down-regulation for gene sets associated with DNA repair, apoptosis, and cell cycle pathways as a function of TP53 mutation (Figure 1E). Despite the genetic heterogeneity among NPM1-WT cases, relative to NPM1-AML cases the -WT group exhibited a similar gene set enrichment pattern as
seen for TP53-AML cases (Figure 1F). We next performed a similar analysis restricted only to NPM1-AML cases, comparing the uppermost (n=19) and lowermost (n=19) quartiles for TP53 gene expression; we noted a near-complete overlap in the pattern of up- and/or downregulation across DNA repair, apoptosis, and cell cycle gene sets as we observed when comparing NPM1-AML and TP53-AML cases (Figure 1G), suggesting that the activity of these pathways may be directly influenced by WT-p53 dosage within the context of NPM1-AML.
WT-p53 protein is over-expressed in NPM1-mutated cells in AML and correlates with remission status post-induction therapy
Based on our initial findings of increased WT-p53 protein expression in NPM1-mutated cells in a case of AML, and evidence of increased TP53 expression in NPM1-AML cases within the BeatAML cohort, we further assessed p53 protein expression in a cohort of primary human bone marrow tissue samples from our institution (Table 1). Using a multiplex immunofluorescence-based phenotyping assay coupled with digital image analysis, we phenotyped cells in situ at single cell resolution, identifying them as NPM1-mutant or -WT using a mutant protein-specific antibody, and then evaluated the frequency and intensity of p53 expression (Figure 2A). The median number of nucleated cells analyzed per case was 8,957 (range: 1,869 - 31,151) [Supplemental Figure 1]. Within each case, a greater proportion of NPM1-mutated cells were p53-positive, compared to NPM1-WT cells [p<0.001, paired Wilcoxon test] (Figure 2B-C); overall, we found >10% of NPM1-mutant cells to be p53-positive in 24 of 33 cases (73%). Similarly, the mean fluorescence intensity (MFI) of p53 was higher in NPM1-mutant compared to WT cells [p<0.001, paired Wilcoxon test] (Figure 2D-E). We observed no significant difference in p53 proportion between patients above or below the age of 60, in those presenting with or without leukocytosis, or with respect to peripheral blood or bone marrow blast percentage (data not shown). We considered the possibility that p53 expression may simply be associated with a non-G0 state of the leukemic cells; however, we observed no correlation between p53 and Ki67 expression (Supplemental Figure 2). We also wondered if p53 overexpression could be a result of diminished MDM2-mediated degradation due to cytoplasmic sequestration of MDM2 by mutant NPM1; however, an analysis of the few highest p53 co-expressors revealed no significant cytoplasmic MDM2 signal by multiplex immunofluorescence (Supplemental Figure 3). Furthermore, we found no significant difference in p53 co-expression frequency based on presence or absence of common co-mutations (e.g. FLT3-ITD, DNMT3A, IDH1/2) [Supplemental Figure 4]. As a proportion of total nucleated cells, p53 was most frequently detected in TP53-AML cases, as anticipated; NPM1-AML cases included a range of p53 expression frequency, with a subset exhibiting p53 expression near the level found in TP53-AML cases. We observed no significant difference in p53 co-expression among total nucleated cells between NPM1-AML and a small comparison group of normal karyotype NPM1-WT cases (p>0.05) [Supplemental Figure 5]. Similarly, p53 MFI was significantly higher in TP53-mutated versus all TP53-WT cases (Supplemental Figure 6).
Post-induction remission status was available for 27 of the 33 cases analyzed. Interestingly, we found a significantly higher p53-positive proportion among NPM1-mutated cells at diagnosis in patients who achieved complete remission with or without complete count recovery (CR/CRi, n=21) compared to patients with grossly persistent disease (PD, n=6) [median 0.219 vs. 0.086, p=0.018] [Figure 2F]. Of note, we found no difference in the frequency of FLT3-ITD co-mutations in CR/CRi versus PD patients (p>0.05).