Mutation in the Nucleophosmin Gene (NPM1) Is a Stable Marker for Minimal Residual Disease Monitoring in Acute Myeloid Leukemia Patients with Increased Sensitivity and Specificity Compared to Expression of the Wilms Tumor (WT1) Gene.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1602-1602
Author(s):  
Thomas Kristensen ◽  
Birgitte Strange Preiss ◽  
Lone Friis ◽  
Michael B. Møller
Keyword(s):  
Gene Expression ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Wilms Tumor ◽  
Normal Karyotype ◽  
Leukemia Cells ◽  
Mutational Load ◽  
Wt1 Gene ◽  
Npm1 Mutation ◽  
Minimal Residual

Abstract Abstract 1602 Poster Board I-628 Mutation in exon 12 of the nucleophosmin (NPM1) gene occurs in approximately 60% of acute myeloid leukemia (AML) patients with normal karyotype. To date, molecular minimal residual disease (MRD) monitoring in this patient group has primarily been based on expression of the Wilms tumor gene (WT1), although expression of WT1 in non-leukemia cells limits the specificity of this marker. Mutation in the NPM1 gene is potentially a superior MRD marker compared to WT1 gene expression by being specific to the malignant clone. The use of NPM1 mutation as a MRD marker would furthermore be in line with the widespread use of leukemia cell specific fusion-genes as MRD markers in AML patients with balanced translocations. In the present study, we therefore evaluated NPM1 mutation as a MRD marker with respect to stability, sensitivity and specificity in direct comparison to WT1 gene expression. A total of 13 relapsed AML patients with normal karyotype that were positive for mutation in NPM1 and WT1 gene expression at the time of diagnosis were included in the study. The NPM1 mutational load and WT1 gene expression was analyzed by real-time qPCR in up to 22 peripheral blood mononuclear cell samples per patient from the time of primary diagnosis to latest follow-up to compare the kinetics of the two markers during periods of morphological remission and relapse events. The 13 patients experienced a total of 18 morphological relapses which were all accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of NPM1 mutation during relapse in the present material. During periods of complete morphological remission, the NPM1 mutational load was below detection limit (< 1/1000 cells) in all samples. In contrast, WT1 gene expression was detectable in 70% of these samples, thus demonstrating the limited specificity of this marker. This background WT1 expression in non-leukemia cells reached levels of up to 1% of the levels detected at the time of diagnosis thus limiting the de facto MRD marker sensitivity of WT1. All samples with detectable levels of NPM1 mutation after a period of complete molecular remission were followed by a morphological relapse within weeks. The present study therefore shows that mutation in NPM1 is a stable and more sensitive and specific, and therefore superior, molecular MRD marker than WT1. Disclosures No relevant conflicts of interest to declare.

Significance of Minimal Residual Disease Monitored by Quantitative Assessment of WT1gene in Patients in First Remission of Acute Myeloid Leukemia Before Allogeneic Stem Cell Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4485-4485
Author(s):  
Veronika Válková ◽  
Jaroslav Polak ◽  
Marketa Markova ◽  
Hana Hájková ◽  
Antonin Vitek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Conditioning Regimen ◽  
Wt1 Gene ◽  
Significant Difference ◽  
Induction Failure ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Abstract 4485 Purpose Thanks to the development of knowledge in the field of molecular biology, the great progress has been done in risk stratification of patients with acute myeloid leukemia (AML) at diagnosis, in recent years. Based on the recommendations of international expert groups there were identified the patients who may benefit from the allogeneic stem cell transplantation (allo-SCT) as a consolidation of first complete remission (CR). In the absence of an universal marker for minimal residual disease (MRD) measurements, there is still little information about the importance of MRD prior to allo-SCT. Our department has a very good experience with quantitative monitoring of WT1 gene expression as a marker of MRD during treatment of AML. The aim was to retrospectively evaluate the significance of MRD in patients indicated for allo-SCT in 1.CR. Patients and methods Overall 35 patients (pts) in the first morphological CR were transplanted from April 2005 - July 2011. Median age was 46 years (range; 20–63), mens 14, women 21, three good risk, intermediate risk 23, high risk 7 (NA 3). A total of 19 pts achieved CR after second induction (salvage), 11 pts were in 1st iCR. Induction 3+7 was given to 31 pts (4x other), as consolidation has been used HIDAC in 28 pts (7x other). As the graft, peripheral blood stem cells were used in 27 pts, bone marrow in 8 pts. The donor was identical sibling in 15 pts (1x mismatched sibling), matched unrelated donor (MUD) in 10 pts and mismatched UD in 9 pts. Conditioning regimen was myeloablative in 29 pts, reduced-intensity in 6 pts. Median follow-up was 18 months (range; 2–56). The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood according to the European Leukemia Net recommendations. The WT1 expression was related to the expression of a reference gene and the results were calculated with a number of WT1 copies related to 104 copies of ABL gene. The upper limit of normal WT1 expression was set as 50 copies of WT1 to 104 copies of ABL. Before allo-SCT, 25 pts were WT1-negative, ten pts were WT1-positive. Results When comparing the two groups according the MRD status, there was not significant difference in terms of age, risk groups, first induction failure, number of iCR, induction or consolidation type. Also, type of graft, conditioning regimen, or HSCT-CI was not significantly different. The group of WT1-positive pts had more unrelated donors, more aGVHD and shorter follow-up. In terms of cGVHD, the groups were comparable. When comparing the overall survival (OS) and cumulative relapse incidence (RI) of the entire group in terms of: risk group, first induction failure, iCR, consolidations number and incidence of aGVHD, we found no significant difference. Pts with cGVHD had a better OS, lower RI with comparable non-relapse mortality (NRM). In contrast, the MRD status measured by WT1 gene expression appears as clearly significant factor. The outcome of WT1-positive pts is significantly worse in terms of OS (55% vs 83% at 3 years, p = 0.03), RI (50% vs 11% at 3 years, p = 0.008), and there is a trend toward higher NRM (23% vs 5% in 3 years, p = 0.08). Conclusion Our results show that MRD status measured by WT1 gene expression in patients with AML in 1.CR significantly affects their future prognosis. Opportunities to influence the unfavorable prognosis of MRD-positive patients may be more intensive pre-transplantation therapy or earlier immunomodulatory intervention after allo-SCT (pre-emptive DLI). The larger prospective studies are necessary to confirm this hypothesis. The study was supported by scientific project MZ 00023736 granted by the Ministry of Health, Czech Republic. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Presence of Minimal Residual Disease, As Determined By Highly Sensitive Quantitation of NPM1-Mutatation, Provided Powerful Prognostic Information in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5097-5097
Author(s):  
Atsushi Marumo ◽  
Hiroki Yamaguchi ◽  
Yuho Najima ◽  
Kensuke Usuki ◽  
Shinichi Kako ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Normal Karyotype ◽  
Npm1 Mutation ◽  
Minimal Residual ◽  
Hematological Remission ◽  
Acute Myeloid

Background: As recurrence of acute myeloid leukemia (AML) is difficult to predict, it is important to detect it by measuring minimal residual disease (MRD). PML-RARA, RUNX-RUNX1T1, CBFB-MYH11 are regarded as the reliable MRD markers. However, in AML with normal karyotype and many other forms, no MRD markers have been established. NPM1 mutations, occurring in approximately 30% of adult AML cases, and 50-60% of AML cases with normal karyotype, represent one of the most frequent mutations in AML. Recently, NPM1 mutation is reported to be useful in assessing MRD. We undertook a retrospective and prospective investigation of the usefulness of NPM1 mutation as an MRD marker in Japanese patients with AML. Methods: The subjects were 38 NPM1-mutated AML patients with first hematological remission at several hospitals related to our institution between 2001 and 2018. This study was approved by the ethics committee of Nippon Medical School and the informed consents were obtained from all patients, according to the Declaration of Helsinki. We analyzed peripheral blood cells or bone marrow cells at diagnoses, and evaluated only bone marrow cells after diagnoses. Detection of NPM1 mutation was carried out using allele-specific real time PCR following creation of a complementary primer. After dilution of the samples, sensitivity to TCTG, CATG, and CCTG was found to be 0.001%. The NPM1 mutant copies were qualified only at successful amplification of internal control. Results: The median age of the patients was 58 years (18-79 years). There were 32 cases with intermediate cytogenetic prognosis and 6 cases with unclear chromosomal profile. Of the 38 cases, 14 cases (37%) were FLT3-ITD-positive and allogeneic hematopoietic stem cell transplantation was carried out in 14 cases (37%). The base sequence was TCTG in 36 cases and CCTG in 2 cases. Persistence of NPM1-mutatation was present in 25 patients with first hematological remission (66%). Compared with patients with MRD negative, patients with MRD positive were associated with DNMT3A mutation (MRD positive 12/25 vs MRD negative 0/13, p=0.003). The rate of relapse in patients with MRD positive was significantly higher than those of in patients with MRD negative (MRD positive 76% vs MRD negative 23%, p=0.004). The rates of relapse free survival (RFS) and overall survival (OS) in patients with MRD positive were significantly lower than those in patients with MRD negative (RFS at 2 years: MRD positive 14% vs MRD negative 86% p=0.003; Figure 1, OS at 2 years: MRD positive 25% vs MRD negative 93%, p<0.001). In FLT3-ITD negative group, the rates of RFS in patients with MRD positive were significantly lower than those in patients with MRD negative. (RFS at 2 years: MRD positive 21% vs MRD negative 92% p=0.001; Figure 1). Conclusion: The presence of MRD with NPM1 mutation is significantly associated with relapse and it is useful to decide their treatment strategy. Especially, there is the usefulness of NPM1 mutation as an MRD marker in NPM1 positive Flt3-ITD negative AML patients who are generally classified as favorable risk. According to previous reports, it is known that NPM1-mutated AML sometimes relapse with losing NPM1 mutations. However, in this study, all NPM1-mutated AML relapse without losing NPM1 mutations. We need to collect more patients and are going to confirm whether there are patients who relapse with losing NPM1 mutations or not. We plan to analyze the genetic background of MRD positive and negative patients with next-generation sequencing. We are going to announce the genetic characteristics in addition to this result at ASH. Disclosures Usuki: Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Kako:Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

Gene Expression Profiling Can Identify Novel MRD Markers for AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2354-2354
Author(s):  
Eleanor L. Woodward ◽  
Amanda F. Gilkes ◽  
Val Walsh ◽  
Steve J. Austin ◽  
Sarah B. Daly ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Minimal Residual Disease ◽  
Expression Profile ◽  
Expression Profiling ◽  
Residual Disease ◽  
Gene List ◽  
Normal Karyotype ◽  
Unique Genes ◽  
Minimal Residual

Abstract In AML, the majority of patients &lt;60 years of age will enter remission but at least 50% will subsequently relapse, therefore the monitoring of minimal residual disease (MRD) during treatment has become an important issue. Current molecular markers for MRD are mainly limited to the RT-PCR detection of the fusion genes resulting from recurrent translocations which paradoxically are mostly limited to favourable risk groups who are least likely to relapse leaving the majority of the patients, including those with a normal karyotype, without a molecular marker suitable for monitoring. Two hundred and twenty patients have been assessed by gene expression profiling using Affymetrix U133A chips and the data analysed with the aim of identifying novel MRD markers for patients who do not currently have a suitable marker. As an initial “proof of principle”, we have identified possible MRD markers for patients with either t(15;17), t(8;21) or inv(16) and correlated with changes in expression of these markers with clinical changes as measured by established molecular MRD markers (PML-RARα or WT1). Of the expression profile from 22,283 probe sets in 29 cases of t(15;17), 20 genes were identified which had at least a two fold over expression which was unique to the t(15;17) subgroup. Of these several of the probe sets were related to the same gene, but from the reduced gene list 2 (HGF and ILGF binding protein) were selected for quantitation by quantitative PCR. Similarly the expression profile identified 20 genes which were unique to the 15 cases of t(8;21), and 20 genes which were unique to the 19 cases of inv(16). These included ETO and MYH11 representing the respective 3′ end of the respective fusion transcript. Three other genes (PRAME, POU4F1, and IL5RA) were selected for the t(8;21) cases and ST18, CLIP-170 and MNI for the inv(16) cases. When the relative quantitative expression of each of these “unique” genes was correlated with the expression of the established markers of minimal residual disease (PML-RARα or WTI) there was good correlation. These data suggest that gene expression profiling can identify ‘unique’ genes which can be used to develop specific markers for minimal residual disease monitoring for a larger proportion of cases of AML than is currently available.

Evaluation of Minimal Residual Disease Based on NPM1 Mutations in AML with Intermediate Risk Cytogenetics: A Prospective Study of 36 Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2847-2847
Author(s):  
Aline Renneville ◽  
Florence Pasquier ◽  
Selim Corm ◽  
Nathalie Philippe ◽  
Charikleia Kelaidi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Genomic Dna ◽  
Residual Disease ◽  
Normal Karyotype ◽  
Intermediate Risk ◽  
Npm1 Mutation ◽  
Log Reduction ◽  
Minimal Residual

Abstract Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in approximately 50% of adult acute myeloid leukemia (AML) with normal karyotype. More than 40 mutant variants have been identified. Most of these mutations consist of a 4-bp insertion, which can be used as a target for minimal residual disease (MRD) monitoring. We previously checked the stability of NPM1 mutations at relapse in 21 NPM1-mutated patients at initial diagnosis. In this prospective study, we evaluated MRD by real-time quantitative PCR (RQ-PCR) in 36 NPM1-mutated AML patients corresponding to 33 adult and 3 pediatric cases, treated according to the French ALFA9801 or ALFA9802 and ELAM02 protocols, respectively. Out of these patients, 31/34 (91%) had normal karyotype, 13/33 (39%) had a high initial white blood cell count, and 10/36 (28%) were FLT3-Intern Tandem Duplication (FLT3-ITD) positive. 28 (78%) patients carry NPM mutation A, 3 (8%) mutation B and 5 (14%) other rare variants. RQ-PCR assays using a mutation-specific primer were performed on cDNA for mutation A and B and on genomic DNA for other NPM1 mutants. In our experiments, the maximal reproductible sensitivity of NPM1-based MRD detection is about 10−4 on genomic DNA and 10−5 to 10−6 on cDNA. The median follow-up was 260 days [40–791]. 2 to 9 follow-up samples from bone marrow and/or peripheral blood were analysed per patient. No correlation was found between leukocytosis at diagnosis and initial expression ratio of NPM1 mutation. The study of MRD log reduction after induction therapy shows that molecular responses are very heterogeneous (from 4.10−2 to more than 1.10−5), but 50% of patients reach at least a 4 log reduction in NPM1 levels. Patients with FLT3-ITD tend to have lower log reduction after induction than patients without FLT3-ITD, although not statistically significant (P=0.07). The analysis of NPM1-MRD in bone marrow and in peripheral blood at the same follow-up time-points shows that NPM1 levels can be until 1 log higher in bone marrow. This indicates that the evaluation of NPM1-MRD in bone marrow is more informative than in peripheral blood. We found all relapses had NPM1-MRD levels comparable to those observed at diagnosis. Among the 5 patients who relapsed so far, 2 were predictable by increasing MRD levels 1 to 4 months before hematological relapse. In 29 out of 36 cases, we could monitor MRD by both NPM1 mutation and WT1 gene expression. The comparison of the MRD profiles obtained by these two approaches reveals some discordant results, which can be, at least in part, explained by difference in the sensitivity of the RQ-PCR techniques, since the sensitivity of WT1 expression as MRD target is generally not higher than 10−3. In conclusion, NPM1 mutations are very specific and sensitive markers for MRD monitoring in AML. Further studies are required to determine if NPM1-MRD provides an independent prognostic factor and may be useful for therapeutic stratification in AML patients with intermediate risk cytogenetics.

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