Nucleophosmin npm1 is a multifunctional, highly conserved protein found most frequently in nucleoli. Minimal residual disease diagnostics in patients with acute myeloid leukemia in the posttransplant period. Chromosomal aberrations involving npm1 were found in patients with nonhodgkin lymphoma, acute promyelocytic leukemia, myelodysplastic syndrome, and acute myelogenous leukemia. Simultaneous detection of npm1 and flt3itd mutations by. Our general targeting strategy was similar to a published knockin model of humanized npm1 floxca 28. Mutant nucleophosmin npm1 predicts favorable prognosis in. In this report, we describe a simple molecular test for the detection of npm1 exon 12 mutations in patients with aml using polymerase chain reaction amplification of genomic dna followed by the analysis of amplification. Npm1 mutation type npm1 mt patients had 2fold higher odds of achieving complete remission compared with npm1 wildtype npm1 wt. Comparison of three techniques sanger sequencing, pyrosequencing, and real time pcr. Npm1 mutations referred to as npm1 c disrupt tryptophan residues and generate an additional nuclear export signal leading to aberrant localization of npm1 c in the cytoplasm.
The kits are for realtime pcr on the rotorgene q and other realtime pcr instruments. The ipsogen npm1 mutascreen kit is intended for research use only and is not for use in diagnostic procedures. Npm1 mutation type npm1 mt patients had 2fold higher odds of achieving complete remission compared with npm1 wildtype npm1. Flt3 and npm1 testing in acute myeloid leukaemia aml. Neolab npm1 mutation analysis liquid biopsy neogenomics. Twoway direct sequencing and gene mapper version 4. Npm1 gene mutation is a founding event in acute myeloid leukaemia. Npm1 dna analysis description insertion mutations in exon 12 of the npm1 gene on chromosome 5 cause abnormal cytoplasmic localization of the npm1 protein and have been identified in 3550% of adult acute myeloid leukemia aml and in 50 60% of aml cases having normal karyotype amlnk. Statistical analyses were performed using stata 12 software. A new dnabased test for detection of nucleophosmin exon. Patients with npm1 mutations typically show a good response to induction therapy. The gene view histogram is a graphical view of mutations across npm1. Research article mutation analysis of jak2v617f, flt3itd.
Mutations in nucleophosmin npm1 exon 12 are thought to be the most common genetic event in acute myelogenous leukemia aml and to confer favorable clinical prognoses. Therefore, a meta analysis of nine studies including a total of 4509 subjects was performed. Six different sequence variants were observed, all leading to a frame shift in the region encoding the cterminal of the npm1 protein. According to the who 2017, the mutation status of npm1, cebpa and runx1.
Aml panel acute myeloid leukaemia test guide eurofins biomnis. Eight samples were randomly selected from 34 positive samples for sequence analysis of npm1 mutation. Npm1 mutation analysis is prognostic in nature, especially in cases with a normal karyotype. Rapid detection of nucleophosmin npm1 mutations in acute. A sample of dna of 50 ng was amplified using an applied biosystems. Detection of npm1 mutations in acute myeloid leukemia aml is important for risk stratification, treatment decision, and therapeutic monitoring. Mutation of npm1 and flt3 genes in acute myeloid leukemia. Thus, in patients with newly diagnosed aml, those with normal karyotype, no flt3 mutation, and a npm1 mutation are considered to have a better prognosis than patients in the same group with neoplasms lacking a npm1 mutation.
Npm1 mutation was detected in 34 out of 161 cases 21%. High npm1mutant allele burden at diagnosis predicts. Probe and primers were designed by primer express software. Acute myeloid leukemia aml with mutated npm1 is a newly recognized separate entity in the revised 2016 world health organization classification and is associated with a favorable prognosis. Prism 3xl genetic analyzer, applied biosystems clearly showed a large. The assay will be interpreted as positive, low positive, or negative for the npm1 mutation. Nucleophosmin shuttles back and forth between the nucleus and the fluid surrounding it the cytoplasm. Prognostic significance of npm1 mutations in acute myeloid.
The kit is for realtime pcr on the rotorgene q and other realtime pcr instruments. The kit provides reagents optimized for reliable and sensitive detection allowing both qualitative analysis of npm1 mutational status mutated or wildtype and identification of npm1 mutation. Detects flt3 and npm1 mutations, which are commonly found in acute myeloid leukemia aml. For this reason, we performed an updated meta analysis of 9 published studies in order to investigate the prognostic significance of npm1 mutations. Finally, in multivariable analysis npm1 mutations express independent. Mutant enrichment with 3modified oligonucleotides memo. Npm1 mutation analysis by sanger sequencing analysis of npm1 exon 12 mutations was done as described by falini et al.
Nucleophosmin npm1 mutations, generally consisting of a four. Cnaml that have cebpa mutations show favorable outcome compared to other groups of cnaml. Sensitive npm1 mutation quantitation in acute myeloid. In patients with newly diagnosed acute myelogenous leukemia, a normal karyotype, and no flt3 mutation, the presence of npm1 mutation is an indicator of a more favorable prognosis. Npm1 gene is upregulated, mutated and chromosomally translocated in many tumor types. Hence, in addition to conventional karyotyping and rtpcr of fusion genes, combined analysis of both flt3 and npm1 mutations will be increasingly relevant in the genetic diagnosis work. These mutations are displayed at the amino acid level across the full length of the gene by default. Npm1 dna analysis description insertion mutations in exon 12 of the npm1 gene on chromosome 5 cause abnormal cytoplasmic localization of the npm1 protein and have been identified in 3550% of. Since the discovery of jak2v617f tyrosine kinaseactivating mutation, several genes have been found mutated in myeloproliferative neoplasms mpns. Mutations in the nucleophosmin npm1 gene are the most common genetic abnormality in aml, occurring in approximately 30% of all amls and in 50% of cnaml 37,38. Cebpa mutation analysis the cebpa gene, a myeloid transcription factor, is mutated in a subset of acute myeloid leukemia aml, particularly those with chromosome analyses showing normal diploid karyotype, cytogenetically normal cn. Mutation analysis of jak2v617f, flt3itd, npm1, and dnmt3a in chinese patients with myeloproliferative neoplasms minwang,nahe,tiantian,luliu,shuangyu,anddaoxinma department of. Clonal evolution patterns in acute myeloid leukemia with npm1. Data were analyzed with kaluza analysis software beckman coulter.
Npm1 acts as a molecular chaperone and is thought to participate in preribosome maturation and centrosome duplication. Npm1 is predominantly localized in the nucleolus and is thought to function as a molecular chaperone of proteins, facilitating the transport of ribosomal proteins through the nuclear membrane. Nucleophosmin npm1 mutation analysis, varies sparrow. Detection of flt3 mutations and overexpression of the wilms tumor wt1. Mutation analysis for runx1, mll ptd, flt3 itd, npm1. Mutations in nucleophosmin npm1 in acute myeloid leukemia. All samples were tested in duplicate, and an average variant allele frequency vaf was calculated. Within this group, flt3 mutations are considered indicators of poor prognosis. In aml cells, npm1 mutations result in abnormal cytoplasmic localization of the mutant protein. Dna samples were also quantified by droplet digital. Positive results are reported quantitatively as percent abnormal dna. In all aml patients carrying npm1 mutations at diagnosis, cdna rqpcr. Therefore, we sought to search for mutations in jak2v617f, flt3itd, npm1.
Flt3itd, npm1, and dnmt3a mutations frequently occurred in aml patients and have been found conferred with myeloproliferative neoplasms in mouse model. Pdf npm1 mutation analysis in acute myeloid leukemia. Npm1 mutations in aml have been reported to occur in about half of. Terminator chemistry and an abi 3100 genetic analyzer applied biosystems, inc. Favorable outcomes in aml cases with npm1 mutations have been previously. Npm1 mutation detection by rtpcr, quantitative arup lab. Quantitative assessment of minimal residual disease in acute.
Six different sequence variants were observed, all leading to a frame shift in the region encoding the cterminal of the npm1. Sequentially inducible mouse models reveal that npm1. Genetic testing for flt3 tyrosine kinase domain flt3tkd mutations is considered. Npm1 mutation analysis acute myeloid leukemia aml, hematologic diseases, molecular exon 12 of npm1 is analyzed by polymerase chain reaction pcr and fluorescent fragment size analysis to detect small insertion mutations. The nucleophosmin npm1 gene is located on chromosome 5q35 and encodes for a protein. Establishment of a quenching probe method for detection of npm1. Npm1 mutation analysis clinical application aml with mutated nucleophosmin npm1. Limitations this assay has a sensitivity to detect approximately the 5% population of cells containing the 4 base pair insertion at nucleotide position 959 exon 12 in a background of nonmutant cells. Acute myelogenous leukemia aml is a heterogenous group of neoplasms. Npm1 mutation has a favorable effect on the outcome for aml 16,1922. While cytogenetic aberrations detected at the time of diagnosis are the most commonly used prognostic feature, approximately 20% of aml cases show a normal karyotype, which is considered an intermediaterisk feature. Npm1 mutations in absence of flt3itd identify a prognostically favorable subgroup. A total of 123 patients with aml treated at the national cancer institute, cairo university were examined for mutations in dnmt3a, flt3, and npm1 using polymerase chain reaction pcr for detecting flt3 internal tandem duplication itd and allelespecific pcr to detect dnmt3a and npm1a mutations. Flt3 itd and tkd regions are analyzed by polymerase chain reaction pcr and fluorescent fragment size analysis.
Clinical effect of combined mutations in dnmt3a, flt3itd. Mutation analysis of jak2v617f, flt3itd, npm1, and dnmt3a. Npm1 nucleophosmin mutation is one of the most common recurring genetic lesions in acute myeloid leukemia aml. Npm1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia aml. A new dnabased test for detection of nucleophosmin exon 12. Cebpa mutation analysis test detail quest diagnostics. Minimal residual disease levels assessed by npm1 mutation specific rqpcr provide important prognostic information in aml. Pcr and fragment analysis of exon 12 of the npm1 gene to detect small insertion mutations specific to aml. The npm1 gene provides instructions for making a protein called nucleophosmin, which is found in a small region inside the nucleus of the cell called the nucleolus. In patients with newly diagnosed acute myelogenous leukemia, a normal karyotype, and no flt3 mutation, the presence of npm1 mutation. Npm1 and flt3itd mutations were detected by fragment analysis. Results were analyzed using bioedit sequence analysis software. Genetic testing for flt3 and npm1 mutations in acute.
Genetic testing for flt3 internal tandem duplication flt3itd and npm1 mutations is considered. The statistical analyses were performed with the statistical software package r, version. Sensitive npm1 mutation quantitation in acute myeloid leukemia. We have devised a pcrbased assay for npm1 exon 12 mutations. Multivariable analysis for os revealed combined npm1mutatedflt3. Flt3 and npm1 mutation detection assays test for two different mutations in the flt3 gene and an insertion in exon 12 of the npm1 gene, all of which are somatic mutations associated with acute. Mutations in exon 12 of the nucleophosmin gene npm1 that cause the encoded protein to abnormally relocate to the cytoplasm are found at diagnosis in about 50% of karyotypically normal acute myeloid leukemias and are associated with a more favorable outcome. It is thought to play a part in many cellular functions, including processes involved in protein formation, dna replication. As a prognostic indicator in patients with newly diagnosed acute myelogenous leukemia with normal karyotype and no flt3 mutation npm1 overview. The signal ratio of the flt3 itd region compares the signal intensity of the mutation to the wildtype. Limitations this assay has a sensitivity to detect approximately the 5% population of cells containing the 4 base pair.