Nucleophosmin (NPM1) mutations in exon 12 are the most common genetic alternation in cytogenetically normal AML (CN-AML). Although mutation types A, B, and D represent the majority of cases, rare mutation variants of the NPM1 gene in individual patients do occur. In this study, we have evaluated a novel, DNAbased real-time quantitative polymerase chain reaction (RQ-PCR) for the detection of three of the most commonly occurring mutations and for six rare patient-specific mutation types, which represent 28% of all of the NPM1 mutations in our group of 25 CN-AML patients. Furthermore, the prognostic relevance of NPM1-based monitoring of minimal residual disease (MRD) in peripheral blood (PB), bone marrow (BM), and in specific cell subsets (CD34 1 , CD34 2 , CD34 dim ) of BM were evaluated. In 80% of the evaluable patients, a molecular relapse preceded a hematological relapse. Moreover, in this subset of patients, the molecular relapse occurred at a median of 97 days before the hematological relapse. Our compartment analysis showed a strong correlation between BM and PB (r 5 0.907, P < 0.001) as well as a high copy number of mutated NPM1 in CD341 BM cells.In conclusion, we have demonstrated applicability of our presented RQ-PCR method for a large percentage of mutated NPM1 patients with CN-AML as well as the usefulness for long-term follow-up monitoring of MRD and the prediction of hematological relapse. Am. J. Hematol. 85:926-929, 2010. V V C 2010 Wiley-Liss, Inc. IntroductionThe Nucleophosmin (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 35% of AML patients at the time of diagnosis and in approximately 60% of adult cytogenetically normal AML (CN-AML). On the basis of its unique molecular, genotypic, immunophenotypic, and prognostic features, AML with an NPM1 mutation was included as a separate provisional entity in the 2008 World Health Organization classification of myeloid neoplasms [1].Somatically acquired mutations in the NPM1 gene usually occur in exon 12, which encodes the COOH-terminal region, and in most cases results in the net insertion of four base pairs. More than 50 molecular variants of mutant NPM1 have been identified to date [2]. The most common variant, Type A, represents approximately 80% of all cases, and Types B and D account for 10% and 5% of cases, respectively. Other mutations rarely occur and vary from case to case. NPM1 mutations appear to be stable, since in the majority of patients, the same mutation is detectable at the time of diagnosis and at relapse. Molecular monitoring using real-time quantitative polymerase chain reaction (RQ-PCR) in a group of CN-AML patients in complete remission has the potential for detecting subclinical levels of residual disease to allow for early treatment intervention and prevent an overt hematological relapse.We developed DNA-based RQ-PCR with mutation-specific reverse primers and a common minor groove binding (MGB) probe for nine different types of NPM1 mutations. Therefore, we can moni...

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Within this paper, we emphasize the importance of microchimerism monitoring as a reliable indicator of incipient AML relapse, especially in patients where no other specific molecular marker is available.

The DNA methyl-transferase 3A gene (DNMT3A) is the third most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML) patients (20-30 %), who belong to a group of patients with intermediate risk. About 60 % of mutations in this gene have been identified in the arginine codon R882. To date, there is no consensus on whether these mutations can be used as biomarkers for monitoring of minimal residual disease and management of preemptive AML therapy. We studied the occurrence of mutations in the DNMT3A gene in our cohort of patients and their persistence during AML treatment. Using next-generation sequencing, we identified four mutations in 11/25 of our analyzed patients--frequent R882C and R882H mutations, rare Y735S mutation, and a novel L347P mutation. Mutation R882C was detected in 5/11, R882H in 4/11 patients, and Y735S and L347P in one patient each. In 4/7 patients initially carrying mutations in the R882 codon, we found the persistence of mutations also during complete remission with, however, no correlation to AML kinetics. Our findings suggest that mutations in the DNMT3A gene can only be used as a biomarker for those AML patients in whom DNMT3A mutation is lost after therapy.

The engraftment of AML was influenced by mutation-interactions and other AML characteristics, rather than by single mutated genes, and engraftment intensity best reflected clinical penetrance of AML.

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Summary In this multi‐centre study, we analysed the prognostic impact of mutations in 19 genes associated with myeloid malignancies in 258 newly diagnosed acute myeloid leukaemia patients (aged 19–70 years) undergoing intensive therapy. We identified five patient groups with different prognostic risks and different benefits from allogeneic hematopoietic stem cell transplantation (alloHSCT) within the intermediate cytogenetic risk group patients (n = 184). The most adverse prognosis was observed in patients with DNMT3A and FLT3‐ITD co‐mutation, whose survival could be significantly improved with alloHSCT. In contrast, the most favourable prognosis without any further benefit from alloHSCT was identified in patients with mutations in NPM1 or CEBPA, after exclusion of the unfavourable prognostic groups defined by mutations in DNMT3A, RUNX1 or genes from chromatin/spliceosome group. An additional analysis of 113 diagnosis‐remission paired samples revealed that persistence of non‐DNMT3A mutations (above 2% VAF) represented a further negative prognostic factor. The proposed model offers a possible molecular stratification and treatment guidance for intermediate cytogenetic risk group patients.

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