Hematological Malignancies Nucleophosmin Mutation Testing in Acute Myeloid Leukemia

The nucleophosmin (NPM) protein shuttles between the nucleus and cytoplasm and is involved in ribosomal regulation, centrosome duplication and regulation of apoptosis.  Mutations of this gene have been discovered in 35% of all AML cases and approximately 50% of AML cases with normal cytogenetics.¹  The mutation is >97% of the time a four base pair insertion near the C-terminus between nucleotide 960-961 or 964-5 in exon 12.  Other rare reported mutations include a 9bp insertion between 965-966 (2 cases out of 166), two 2bp insertions (1 case out of 166) between 960-961 and 962-963², and a 13bp insertion with a 9bp deletion (1 case out of 408) at position 965-973³.

The mutation is always heterozygous and results in a frameshift.  The mutation results in a longer protein due to the creation of a new stop codon further downstream.  The mutated protein is retained in the cytoplasm.  The mutation is associated with significantly better complete remission rates in AML.  

Clinical Utility:
All patients with a new diagnosis of acute myeloid leukemia will be assayed for NPM mutations, and the NPM sequencing analysis will be part of an AML panel.  A knowledge of the NPM mutation status in combination with cytogenetic data, other gene mutations, and clinical correlations including, but not limited to WBC, platelet count, bone marrow blasts count and immunophenotype are part of an oncologist’s assessment on management strategy, treatment response, risk of relapse, and disease-free interval.  Also, NPM status may impact the decision regarding marrow transplant.

This test is performed by PCR-based Next Generation Sequencing of DNA extracted from formalin fixed paraffin embedded (FFPE) tissue or fresh tissue including peripheral blood and bone marrow.  Hotspot C-terminal exon 11 in NPM1 is sequenced using massively parallel sequencing (next-generation sequencing) with a combination of multiplexed PCR (customized QIAseq Targeted DNA panel) and sequencing on an Illumina platform.  An in-house bioinformatics analysis pipeline has been used that employs multiple established variant calling tools (FreeBayes, MuTect2 and Scalpel) and variant annotation tools.  The genomic variants have been interpreted in accordance with the 2017 guideline recommendations by AMP/ASCO/CAP (PMID: 27993330).  The assay is validated in accordance with the AMP guidelines (PMID: 28341590).

For samples sent as formalin-fixed paraffin-embedded tissue (FFPE), the tissue was subjected to pathologist review and, if needed, enrichment (macrodissection) of involved areas of tissue – prior to extraction

• 5-10 mL of blood or bone marrow — yellow (ACD) or purple (EDTA) tube. 
• Formalin-fixed paraffin-embedded (FFPE) tissue blocks
• 10 unstained slides (4-5 microns)
• DNA: 10µg at a minimum of 100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS)
• If sample cannot arrive at laboratory within 24 hours of draw, referigerate until sample can be transported.

Contact Client Services for shipping kit and instructions at (855) 535-1522. 

A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES.  Please include detailed clinical information.

Weekly

7-10 days

• Package and ship specimen to remain cold, but not frozen. 
• Ship via overnight express, using the FedEx priority overnight label provided. 
• Contact Client Services for shipping kits and instructions at (855) 535-1522.

1. Mrozek K., et al.  Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification? Blood 2007, 109(2): 431-448.
2. Schnittger S., et al.  Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype.  Blood 2005, 106(12): 3733-3739.
3. Thiede C., et al.  Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia. Blood 2006, 107(10): 4011-4020.