Monitoring of minimal residual disease in acute myeloid leukemia with frequent and rare patient‐specific NPM1 mutations

Abstract

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...

Improved minimal residual disease detection by targeted quantitative polymerase chain reaction in Nucleophosmin 1 type a mutated acute myeloid leukemia

“…The validation showed a reproducible sensitivity of the NPM1 RQ‐PCR ranging from 0.002% (2 × 10 −5 ) to 0.04% (4 × 10 −4 ) NPM1 type A mutant DNA, which is comparable to levels attained by others using a genomic DNA‐based RQ‐PCR approach for reproducible sensitivity (Gorello et al, ). The results also demonstrated that LOD 50 is near 0.001% (10 −5 ) of NPM1 type A mutant DNA, which also is comparable to levels attained by others (Chou et al, ; Dvorakova et al, ). Even the mere detection of a signal indicates the presence of leukemic cells, which may have a bearing on the risk of relapse (Chou et al, ).…”

“…The key question about the use of these mutated genes as candidates for MRD monitoring regards their stability over the course of disease. NPM1 mutations can be identified in 45%–60% of patients with cytogenetically normal AML (Gorello et al, ; Chou et al, ; Dvorakova et al, ; Falini and Martelli, ; Kristensen et al, ; Buccisano et al, ), and is thus the most frequent genetic change. Many studies indicate that NPM1 mutations are stable at relapse, which makes it an ideal candidate for MRD assessment (Chou et al, ; Falini et al, ; Buccisano et al, ; Hubmann et al, ).…”

Prognostic Value and Correlation Between WT1 Overexpression and NPM1 Mutation in Patients with Acute Myeloblastic Leukemia

“…Вопрос о стабильности мутации NPM1 (сохра-нение клона) при рецидиве остается спорным [14,[37][38][39][40][41]. В нашем исследовании у 5 из 13 пациентов с рецидивами ОМЛ имелась мутация NPM1.…”

“…Значения в разных источниках варьируют от 3 до 5 log [14,30,33]. В настоящее время большинство исследователей рассматривают NPM1 как высокоспецифичный [34][35][36] и стабильный маркер оценки МОБ [14,[37][38][39][40][41]. Тем не менее в от-дельных наблюдениях отмечается утрата экспрессии мутации NPM1 при рецидивах ОМЛ, что ставит под сомнение ее использование для мониторинга эффек-тивности терапии.…”

New Quantitative Method to Identify NPM1 Mutations in Acute Myeloid Leukaemia

“…Most of the NPM1m identified to date, as the type A mutation (75–80% of cases), are exon 12 frameshift mutations [1, 5, 8] leading to an aberrant accumulation of the protein in the cytoplasm [9]. Several protocols and methods have been developed for the detection of NPM1m including DNA sequencing of different mutation-specific RT-PCR assays [10–13], denaturing high-performance liquid chromatography [14], capillary electrophoresis [15], locked nucleic acid-mediated polymerase chain reaction clamping [16], and high-resolution melting analysis [17]. Although these methods possess a high specificity to assess NPM1 mutational status at diagnosis, they always require direct sequencing for NPM1m characterization, a more expensive and time-consuming method.…”