NPM1 mutations in therapy-related acute myeloid leukemia with uncharacteristic features.

Frameshift mutations of the nucleophosmin gene (NPM1) were recently reported as a frequently occurring abnormality in patients with de novo acute myeloid leukemia (AML). To evaluate the frequency of NPM1 mutations in patients with therapy-related myelodysplasia (t-MDS) and therapy-related AML (t-AML), and their possible association to type of previous therapy and to other gene mutations, 140 patients with t-MDS or t-AML were analyzed for mutations of NPM1. NPM1 mutations were observed in 7 of 51 patients presenting as overt t-AML, as compared to only 3 of 89 patients presenting as t-MDS (P=0.037). The mutations were not related to any specific type of previous therapy, but they were significantly associated with a normal karyotype and mutations of FLT3 (P=0.0002 for both comparisons). Only 1 of 10 patients with NPM1 mutations presented chromosome aberrations characteristic of therapy-related disease, and 7q-/-7, the most frequent abnormalities of t-MDS/t-AML, were not observed (P=0.002). This raises the question whether some of the cases presenting NPM1 mutations were in fact cases of de novo leukemia. The close association to class I mutations and the inverse association to class II mutations suggest mutations of NPM1 as representing a class II mutation-like abnormality in AML.

Genetics of therapy-related myelodysplasia and acute myeloid leukemia.

Myelodysplasia (MDS) and acute myeloid leukemia (AML) are heterogeneous, closely associated diseases arising de novo or following chemotherapy with alkylating agents, topoisomerase II inhibitors, or after radiotherapy. Whereas de novo MDS and AML are almost always subclassified according to cytogenetic characteristics, therapy-related MDS (t-MDS) and therapy-related AML (t-AML) are often considered as separate entities and are not subdivided. Alternative genetic pathways were previously proposed in t-MDS and t-AML based on cytogenetic characteristics. An increasing number of gene mutations are now observed to cluster differently in these pathways with an identical pattern in de novo and in t-MDS and t-AML. An association is observed between activating mutations of genes in the tyrosine kinase RAS-BRAF signal-transduction pathway (Class I mutations) and inactivating mutations of genes encoding hematopoietic transcription factors (Class II mutations). Point mutations of AML1 and RAS seem to cooperate and predispose to progression from t-MDS to t-AML. Recently, critical genetic effects underlying 5q-/-5 and 7q-/-7 have been proposed. Their association and cooperation with point mutations of p53 and AML1, respectively, extend the scenario of cooperating genetic abnormalities in MDS and AML. As de novo and t-MDS and t-AML are biologically identical diseases, they ought to be subclassified and treated similarly.

Alternative genetic pathways and cooperating genetic abnormalities in the pathogenesis of therapy-related myelodysplasia and acute myeloid leukemia.

Alternative genetic pathways were previously outlined in the pathogenesis of therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML) based on cytogenetic characteristics. Some of the chromosome aberrations, the recurrent balanced translocations or inversions, directly result in chimeric rearrangement of genes for hematopoietic transcription factors (class II mutations) which disturb cellular differentiation. Other genetic abnormalities in t-MDS and t-AML comprise activating point mutations or internal tandem duplications of genes involved in signal transduction as tyrosine kinase receptors or genes more downstream in the RAS-BRAF pathway (class I mutations). The alternative genetic pathways of t-MDS and t-AML can now be further characterized by a different clustering of six individual class I mutations and mutations of AML1 and p53 in the various pathways. In addition, there is a significant association between class I and class II mutations possibly indicating cooperation in leukemogenesis, and between mutations of AML1 and RAS related to subsequent progression from t-MDS to t-AML. Therapy-related and de novo myelodysplasia and acute myeloid leukemia seem to share genetic pathways, and surprisingly gene mutations were in general not more frequent in patients with t-MDS or t-AML as compared to similar cases of de novo MDS and AML studied previously.

Mutations of genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal transduction pathway in therapy-related myelodysplasia and acute myeloid leukemia.

Mutations of the FLT3, c-KIT, c-FMS, KRAS, NRAS, BRAF and CEBPA genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal-transduction pathway are frequent in acute myeloid leukemia (AML). We examined 140 patients with therapy-related myelodysplasia or AML (t-MDS/t-AML) for point mutations of these seven genes. In all, 11 FLT3, two c-KIT, seven KRAS, eight NRAS and three BRAF mutations were identified in 29 patients (21%). All but one patient with a FLT3 mutation presented with t-AML (P=0.0002). Furthermore, FLT3 mutations were significantly associated with previous radiotherapy without chemotherapy (P=0.03), and with a normal karyotype (P=0.004), but inversely associated with previous therapy with alkylating agents (P=0.003) and with -7/7q- (P=0.001). RAS mutations were associated with AML1 point mutations (P=0.046) and with progression from t-MDS to t-AML (P=0.008). Noteworthy, all three patients with BRAF mutations presented as t-AML of M5 subtype with t(9;11)(p22;q23) and MLL-rearrangement (P=0.01). In t-AML RAS/BRAF mutations were significantly associated with a very short survival (P=0.017). Half of the patients with a mutation in the RTK/RAS-BRAF signal-transduction pathway (denoted 'class-I' mutations) simultaneously disclosed mutation of a hematopoietic transcription factor (denoted 'class-II' mutations) (P=0.046) suggesting their cooperation in leukemogenesis.

Amplification or duplication of chromosome band 21q22 with multiple copies of the AML1 gene and mutation of the TP53 gene in therapy-related MDS and AML.

Amplification or duplication of the AML1 gene at chromosome band 21q22 was detected by FISH using a locus-specific probe in three out of 171 unselected patients with therapy-related myelodysplasia (t-MDS) or t-AML (1.7%). In two patients AML1 signals were located tandemly on derivative chromosomes, in one patient on a dic(9;21) and in the the other patient on a derivative chromosome 18 made up of interchanging layers of material from chromosomes 9, 14, 18, and 21. In the third patient three single supernumerary copies of AML1 were located on derivatives of chromosomes 19 and 21. All three patients were older, had previously received therapy with alkylating agents without topoisomerase II inhibitors, had complex karyotypes including abnormalities of chromosomes 5 or 7, and presented acquired point mutations of the TP53 gene. No point mutations of the AML1 gene were observed. The results support a pivotal role of impaired TP53 function in the development of gene amplification or duplication in t-MDS and t-AML.

Methylation of p15INK4B is common, is associated with deletion of genes on chromosome arm 7q and predicts a poor prognosis in therapy-related myelodysplasia and acute myeloid leukemia.

The p14(ARF), p15(INK4B), and p16(INK4A) genes are important negative cell-cycle regulators often inactivated by deletions, mutations, or hypermethylation in malignancy. Hypermethylation of the three genes was studied in 81 patients with therapy-related myelodysplasia (t-MDS) or acute myeloid leukemia (t-AML) by methylation-specific PCR, and p15 methylation additionally by bisulfite genomic sequencing. In all, 55 patients disclosed p15 methylation, five patients showed p16 methylation, whereas p14 methylation was not observed. Methylation of p15 was closely associated with deletion or loss of chromosome arm 7q (P=0.0006). In t-MDS, the p15 methylation frequency and the p15 methylation density both increased significantly by stage (P=0.004 and 0.0002), and p15 methylation frequency increased with an increasing percentage of myeloblasts in the bone marrow (P=0.006). In a two-variable Cox model including the percentage of myeloblasts, p15 methylation was an independent prognostic factor (P=0.005). Methylation of p15 was less common in t-AML of subtype M5 than in other FAB subtypes (P=0.03). Methylation of p15 was unrelated to type of previous therapy, to latent period from start of therapy, to platelet count, and to p53 mutations. Inactivation of p15 and deletion of genes on chromosome arm 7q possibly cooperate in leukemogenesis.

Causality of myelodysplasia and acute myeloid leukemia and their genetic abnormalities.

New insights into causative factors for the development of myelodysplasia (MDS) and acute myeloid leukemia (AML), with associations to specific cytogenetic and genetic abnormalities have been obtained primarily from studies of patients with the therapy-related subsets of the two diseases. Current knowledge now makes it possible to distinguish between at least seven major genetic subgroups of MDS and AML, and has directed research towards more specific causative factors also for de novo MDS and AML.