Label Name: NPM1 MUTN
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6705 
Last Review:  3/17/2017 10:05:06 AM
Specimen Type
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 3  ML
Label Reminders
  Be sure to include patient's name, history #, date and time of collection, and collector's initials.
Collection Notes
  • Bone Marrow: One lavender-top EDTA tube (minimum of 1 ml) is required for testing. Forward unprocessed bone marrow promptly at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.

    Peripheral Blood: One lavender-top EDTA tube (minimum of 3 mls) is required for testing. Forward unprocessed peripheral blood promptly to the laboratory at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.
  Deliver peripheral blood and bone marrow specimens to lab at ambient temperature. If there is a delay of more than 24 hours in delivery, refrigerate the sample. DO NOT FREEZE.

Turn Around Time -  Routine: 7 days   Stat: N/A
Reference Values


No Reference Values
  This assay uses PCR with Locked Nucleic Acid (LNA) clamping followed by amplicon nucleotide length analysis to detect insertion or insertion/deletion mutations that cause a global 4 bp change in allele length and a resulting frame shift in exon 12 of the NPM1 gene. Genomic DNA is first purified from a peripheral blood or bone marrow specimen. PCR is then performed using fluorescently tagged oligonucleotide primers that specifically amplify exon 12 and an adjacent segment of intron 11 of the NPM1 gene. An LNA probe is included to suppress amplification of wild type sequences. The nucleotide length of the fluorescently tagged NPM1 amplicon is determined by capillary electrophoresis on the ABI 3130 or ABI 3500 Genetic Analyzer.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
Clinical Significance and Interpretive Data

Acute myelogenous leukemia (AML) is a relatively common malignancy with approximately 13,000 new cases diagnosed each year in the United States alone. Even with recent advances in our understanding of the molecular etiology of AML, mortality rates remain high and in the US, AML causes 9,000 deaths each year. While most patients with AML ultimately succumb to their disease, a significant proportion can be treated and effectively cured. This is in part due to heterogeneity in the underlying molecular events causing any individual patient’s AML.

Previously, AML was sub-typed based on histology and immunophenotype alone. In this classification scheme, categories of AML ranged from AML with minimal differentiation to AML with maturation towards any one of the specific myeloid lineages. Recently the WHO has proposed a revised classification scheme that includes specific recurrent genetic abnormalities which confer either a good (t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q22;a12)) or average (11q23 rearrangements) prognosis. More recently, mutations have been reported in the FMS-like tyrosine kinase 3 (FLT3), nucleophosmin (NPM1), c-KIT, CEBPA and MLL genes in patients with AML. Like the cytogenetic abnormalities in the WHO classification scheme, evaluation of mutations in these individual genes can provide significant prognostic information.

NPM1 Mutations in AML: NPM1 is a 32.5 kDa RNA binding protein which is localized to the nucleolus and is involved in pre-ribosomal assembly, ribonucleoprotein transport, centrosome duplications and DNA repair. Heterozygous NPM1 mutations are found in a subset of patients with AML. These mutations are localized to exon 12 and are almost exclusively small frameshift mutations. These mutations disrupt the carboxy terminal several amino acids of NMP1, leading to a newly created nuclear export signal. This results in aberrant cellular localization of NPM1 and a subsequent loss of function. NPM1 mutations are common in AML and particularly common in AML cases with a normal karyotype. In children with AML, the incidence of NPM1 mutations ranges from approximately 2% to 6.5%. If one considers only cases with a normal karyotype, the incidence increases to 9% to 27%. In adults with AML, the frequency of NPM1 mutations ranges from 25% to 35% or in cases with a normal karyotype, from 46% to 54%.

NPM1 mutations are found more frequently in acute myelomonocytic leukemia and acute monoblastic and monocytic leukemia. However, NPM1 mutations have been reported in most sub-types of AMLs even those of erythroid and megakaryocytic lineage. Most cases of AML which have cytoplasmic NPM1 (a surrogate marker for NPM1 mutations) are CD34 negative. NPM1 mutations are associated with a greater percentage of bone marrow blasts and a higher circulating leukocyte and platelet count. Although NPM1 mutations occur more frequently in AML cases with these characteristics, the presence of NPM1 mutations cannot be inferred from histology, immunophenotype, the patient’s disease presentation or clinical picture.


Patients with AML with a normal karyotype and a mutated NPM1 gene show a significantly higher complete remission rate after induction chemotherapy, longer overall survival and longer disease free survival than similar patients without NPM1 mutations. This favorable prognosis is modified, in part, by mutations in the FLT3 gene. Generally, activating mutations in the oncogene FLT3 are associated with a worse prognosis in AML patients. Forty percent of patients with mutations in NPM1 also harbor mutations in FLT3. In this patient population, the poor outcome of patients with FLT3 mutations was dominant over the beneficial effects of NPM1 mutations. Thus, the favorable prognosis of NPM1 mutations is only seen in the context of wild-type FLT3 alleles and testing for NPM1 mutations has the greatest impact when done in conjunction with FLT3 mutation analysis.


The diagnosis and sub-typing of AML based on underlying molecular etiology has become the standard of care for this disease. This is most evident by the WHO’s adoption of cytogenetic analysis (FISH and conventional karyotype) for recurrent genetic abnormalities in the classification of AML. In the case of t(15;17), the genetic abnormality dictates the therapy best suited for this disease. However, in the case of inv(16), t(8;12) and 11q23 abnormalities, this classification scheme was designed to reflect the different prognoses of patients with these different cytogenetic abnormalities. Mutation testing of individual genes including NPM1 and FLT3 represents the logical extension of this prognosis-based classification scheme. This mutation data can be used to help stratify patients based on risk and has the potential to guide decisions about treatment options for the individual patient.

Identifying patients with NPM1 mutations may also help in evaluating response to therapy. Monitoring of minimal residual disease (MRD) in an AML patient with a normal karyotype is difficult due to a lack of molecular markers. Since NPM1 mutations occur in 50% to 60% of AML, these mutations make an attractive target for MRD detection. This assay is not appropriate for this indication due to its relatively low analytic sensitivity. However, effective MRD testing could (and likely will) be developed using techniques such as quantitative PCR which offer a better dynamic range and analytical sensitivity. Knowing a patient’s NPM1 status prior to the initiation of therapy would allow for an assay such as this to be used in their care in the future.


This assay uses PCR with Locked Nucleic Acid (LNA) clamping followed by amplicon nucleotide length analysis to detect insertion or insertion/deletion mutations that result in the loss of 4 bps and a resulting frame shift in exon 12 of the NPM1 gene.

Variants other than a 4 bp insertion/insertion-deletion within NPM1 exon 12 have been cited and may be detected by this assay. Confirmation and reporting of such variants will be handled on a case by case basis at the discretion of the Laboratory Director.


Boissel N, Renneville A, Biggio V, et al. Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype. Blood. 2005;106:3618-3620.

Cazzaniga G, Dell’Oro MG, Mecucci C, et al. Nucleophosmin mutations in childhood acute myelogenous leukemia with normal karyotype. Blood. 2005;106:1419-1422.

Chou WC, Tang JL, Lin LI, et al. Nucleophosmin mutations in de novo acute myeloid leukemia: the age-dependent incidences and the stability during disease evolution. Cancer Res. 2006;66:3310-3316.

Dohner K, Schlenk RF, Habdank M, et al. Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations. Blood. 2005;106:3740-3746.

Falini, B., et al., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med, 2005. 352(3): p. 254-66.

Falini, B., et al., Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features. Blood, 2007. 109(3): p. 874-85.

Schneider et al. NMP1 but not FLT3-ITD mutations predict early blast cell clearance and CR rate in patients with normal karyotype AML (NK-AML) or high-risk myelodysplastic syndrome (MDS). Blood. 2009; 113:5250-5253.

Schnittger S, Schoch C, Kern W, et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood. 2005;106:3733-3739.

Suzuki, T., et al., Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia. Blood, 2005. 106(8): p. 2854-61.

Thiede, C., et al., Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood, 2006. 107(10): p. 4011-20.

    This assay can be used for:

• Prognostic testing in patients with acute myelogenous leukemia (AML).
    This assay only detects the presence or absence of insertion or insertion/deletion mutations in exon 12 of NPM1 that result in differences in gene size. This assay does not detect any other changes in the NPM1 gene.

• For detection by this assay, the NPM1 mutant population must comprise at least 1% of total cells. Smaller populations may not be detected.

• This assay is subject to interference by various factors such as PCR inhibitors, poor DNA quality, insufficient DNA quantity, or the presence of genetic variants in the primer binding regions of NPM1.

• The prognostic impact of NPM1 mutations may be further influenced by the subtype of AML, mutations in other genes, chromosomal aberrations or patient specific clinical factors. Thus, this assay is intended for use as an aid in developing patient specific prognostic predictions but is not a substitute for a complete pathologic and clinical evaluation, or physician's judgment and clinical experience
Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432

 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: