Influence of Nucleophosmin (NPM1) Genotypes on Outcome of Patients With AML: An AIEOP-BFM and COG-SWOG Intergroup Collaboration

Abstract

Purpose: Several genomic subsets of NPM1 mutations with varying sequences (type A, B, D, etc) have been identified. Despite molecular heterogeneity, NPM1 mutations cumulatively portend a more favorable outcome, but biology and prognostic implications of different genomic subsets have not been extensively studied. In this multicentric study, we investigated the impact of NPM1 genotypes on patient's outcomes and interrogated the underlying biology of the different subtypes.

Materials and methods: Of more than 4,000 patients enrolled in multiple pediatric cooperative (AIEOP, BFM, ELAM02, NOPHO, DCOG, and COG trials), or adult (SWOG) trials, 348 pediatric and 75 adult AML patients with known NPM1 genotype and available outcome were selected for this study. Diverse NPM1 variants were correlated with the probabilities of overall survival (OS) and event-free survival. Nuclear localization and translational efficiency of the NPM1 variants was studied.

Results: Evaluation of clinical outcome on the basis of NPM1 genotypes showed that patients with type A, B, and other rare variants had similarly favorable outcomes, whereas those with type D had a significantly worse outcome (OS of 63% for type D v 86% for type non-D, P = .005). Multivariate analysis confirmed type D as an independent prognostic factor associated with inferior OS (hazard ratio, 3; P = .005). In vitro, we demonstrated that in type D versus type A synonymous variants, codon optimality plays major roles in determining gene expression levels, and translation efficiency, which resulted in a more expressed NPM1-D mRNA and protein, mediating peculiar mitochondrial gene expression.

Conclusion: The evaluation of specific NPM1 genotypes identified AML patients with type D mutations being significantly associated with inferior outcomes, suggesting a reclassification of D cases to higher-risk groups.

Figure 1.. NPM1 diverse mutations in AML pediatric cohorts and survival.

A) Frequency of NPM1 variants, classified as type A, B, D, and rare variants including type A-like and non-A-like, in the merged pediatric cohort (big circular graph), and graphical representation of NPM1 variants distribution in each pediatric cohort (small circular graphs). B-C) Kaplan-Meier curves for event-free survival (EFS, B) and overall survival (OS, C) among AML pediatric patients according to different NPM1 variants.

Figure 2.. Influence of type-D NPM1 on Survival in AML pediatric and adult cohorts.

A-B) Kaplan-Meier curves for event-free survival (EFS) and overall survival (OS) of type-D vs type non-D NPM1 AML pediatric patients (p=0.0449 and p=0.0053 in EFS and OS, respectively, A), and including AML pediatric patients with wild type (WT) NPM1 (p<0.0001 in EFS and OS, B). C) Kaplan-Meier curves for EFS and OS among type-D, type non-D NPM1 AML and NPM1-WT, merging pediatric (n=2359) and adult (n=351) patients (p<0.0001 in EFS and OS). D) Incidence of death among patients with relapsed AML in the childhood cohorts (type-D: 4 dead out of 7 relapsed patients, 57%; type non-D: 23 dead out of 68 relapsed patients, 34%), and in all cohorts including adults (type-D: 6 dead out of 9 relapsed patients, 67%; type non-D: 48 dead out of 104 relapsed patients, 46%).

Figure 3.. Impact of FLT3-ITD on Survival and Oncoprint in NPM1-mutated AML pediatric patients.

A) Kaplan-Meier curve for overall survival (OS) among NPM1-mutated AML pediatric patients with or without FLT3-ITD (p=0.0068). B) Kaplan-Meier curve for OS according to NPM1-type-D or type non-D, and the presence or absence of FLT3-ITD. C) Kaplan-Meier curve for OS among NPM1-type non-D without FLT3-ITD versus type-D cases merged with type non-D with FLT3-ITD (p=0.0008). D) Oncoprint analysis: rows represent individual genomic lesions, and columns represent NPM1-mutated patients of the COG cohort classified for NPM1 genotype, according to color legend (type A n=90, type A-like n=25, type B n=40, type D n=14, type non-A-like n=11). Histogram shows the frequency of all variant types detected in the reported genes according to NPM1 variants (* p<0.05).

Figure 4.. mRNA and protein stability of NPM1-type-D vs type-A.

A) Kaplan-Meier curve for overall survival (OS) in type-A vs type-D NPM1 AML pediatric patients (p=0.0204). B) Scheme of factors influencing variation in gene expression and protein translation. C-D) Quantitative measurement of NPM1 mutated transcript by Rq-PCR in patients of the AIEOP (type-A: n=10, type-D: n=3, C) and BFM (type-A: n=21, type-D: n=3, D) cohorts. E) Quantitative measurement of NPM1 mutated transcript by Rq-PCR in HEK293T 40 hours post transfection with EGFP-NPM1typeA or EGFP-NPM1typeD vectors. F) Western blot showing mutated-NPM1, GFP, NPM1 wt and GAPDH protein levels 40 and 64 hours post transfection. Histograms show the quantification of mutated-NPM1 and GFP normalized to GAPDH housekeeping. G) Histogram showing the ratio of mean fluorescence intensity (MFI) of HEK293T cells transfected with EGFP-NPM1typeA vector relative to EGFP-NPM1typeD one at 40, 48 and 64 hours post transfection. H) Relative quantitative measurement of NPM1 mutated transcript by Rq-PCR in HEK293T transfected with EGFP-NPM1typeA or EGFP-NPM1typeD vectors, 8 and 30 hours post Actinomycin D (ActD) treatment. I) Western blot showing mutated-NPM1, GFP, wt NPM1 and GAPDH protein levels at 40 hours post transfection (corresponding to 0 hours of treatment), 4, 8 and 30 hours post Actinomycin D treatment. Histograms show the quantification of mutated-NPM1 and GFP normalized to GAPDH housekeeping. L) Representative immunofluorescence images of NPM1 wt, type-A or type-D AML primary cells stained with NPM1-mutant antibody (green) and diamidino-2-phenylindole (DAPI; blue) as the nuclear counterstain. Data are the mean ± SEM. *p< 0.05; **p< 0.01; ***p< .001; Original magnification ×40. Bar represents 10 μm.

Figure 5.. Gene expression profile of NPM1-mutated AML pediatric patients.

A) Hierarchical and k-means clustering of transcriptomes in 116 NPM1-mutated patients of the COG cohort. B) Unsupervised analysis considering the 90 differentially expressed genes between type-D and others NPM1 patients (p<0.1). C) Kaplan-Meier curve for event-free survival (EFS) in cluster 4 vs others NPM1 patients (p=0.009). D) Incidence of death among patients with relapsed AML between cluster 4 and others (cluster 4: 6 dead out of 9 relapsed patients, 67%; others: 48 dead out of 104 relapsed patients, 46%). E) Gene-set enrichment analysis based on the 128 over-represented genes in type-D vs other NPM1, showing gene sets derived from the GO Cellular component ontology.