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Test Code NPM1Q Nucleophosmin (NPM1) Mutation Analysis, Varies


Shipping Instructions


1. Refrigerated specimens must arrive within 5 days of collection, and ambient specimens must arrive within 3 days of collection.

2. Collect and package specimen as close to shipping time as possible.



Necessary Information


The following information is required:

1. Pertinent clinical history

2. Clinical or morphologic suspicion

3. Specimen source (blood or bone marrow)



Specimen Required


Submit only 1 of the following specimens:

 

Specimen Type: Blood

Container/Tube: Lavender top (EDTA) or yellow top (ACD-B)

Specimen Volume: 10 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send whole blood specimen in original tube. Do not aliquot.

3. Label specimen as blood.

 

Specimen Type: Bone marrow

Container/Tube: Lavender top (EDTA) or yellow top (ACD-B)

Specimen Volume: 4 mL

Collection Instructions:

1. Invert several times to mix bone marrow.

2. Send bone marrow specimen in original tube. Do not aliquot.

3. Label specimen as bone marrow.


Forms

1. Hematopathology Patient Information (T676)

2. If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.

Secondary ID

604418

Useful For

As a prognostic indicator in patients with newly diagnosed acute myelogenous leukemia with normal karyotype and no FLT3 variant and as a leukemia-specific marker of minimal residual disease

Testing Algorithm

The assay is composed of 2 parts:

-RNA-based, sensitive quantitative real-time, reverse transcription polymerase chain reaction (RT-PCR) that detects and quantifies the most common altered NPM1 messenger RNA transcripts (A, B, D forms) in acute myeloid leukemia (AML)

-DNA-based qualitative NPM1 exon 12 variant screening by fragment analysis that detects essentially all altered forms reported in AML, including the rare non-A, B, D forms (with lower sensitivity at the DNA level)

Method Name

RNA: Reverse-Transcription Quantitative PCR (RT-qPCR)

DNA: Polymerase Chain Reaction (PCR) with Fragment Analysis by Capillary Gel Electrophoresis

Reporting Name

NPM1 Mutation Analysis, V

Specimen Type

Varies

Specimen Minimum Volume

Blood: 8 mL; Bone marrow: 2 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Varies Refrigerated (preferred) 5 days
  Ambient  72 hours

Reject Due To

Gross hemolysis Reject
Bone marrow biopsies
Paraffin-embedded bone marrow clots
Slides
Paraffin shavings
Moderately to severely clotted		 Reject

Clinical Information

Acute myeloid leukemia (AML) is a genetically heterogeneous group of neoplasms. While cytogenetic aberrations detected at the time of diagnosis are the most used prognostic feature, approximately 50% of AML cases show a normal karyotype, which is considered an intermediate-risk feature. Within this group, FLT3 variants are considered indicators of poor prognosis. However, in the absence of a FLT3 variant, the presence of a NPM1 variant is associated with a more favorable prognosis. A NPM1 alteration is a common finding in de novo AML (25%-30% of cases) and consists of small insertion (typically 4 base pairs) or deletion/insertion events involving exon 12. Three variants are highly recurrent, termed types A, B, and D, and together account for approximately 90% of NPM1 alterations in de novo AML. Thus, in patients with newly diagnosed AML, those with normal karyotype, no FLT3 variant, and a NPM1 alteration are considered to have a better prognosis than patients in the same group with neoplasms lacking a NPM1 alteration. Furthermore, the presence of a NPM1 alteration serves as a sensitive marker for evaluating minimal disease and therapeutic response following treatment.

Reference Values

An interpretive report will be provided.

Interpretation

The assay incorporates 2 parts: a qualitative screen for exon 12 NPM1 alterations and a quantitative reverse transcription polymerase chain reaction (RT-PCR) assay to determine the copy number of NPM1 transcripts (relative to ABL1 reference messenger RNA [mRNA]). This strategy will allow for identification of the NPM1 alteration at diagnosis, as well as a high sensitivity method to monitor patients who are post-therapy for minimal residual disease. Results will therefore be interpreted with integration of the quantitative and qualitative test results in the context of NPM1 alteration type identified at the time of AML diagnosis if available. Because the quantitative RT-PCR component only reliably detects and quantifies the 3 most common variant types (A, B, D), there is a very small possibility that the qualitative assay may indicate the presence of an NPM1 alteration, but the quantitative assay will be (falsely) negative. In patients with newly diagnosed acute myeloid leukemia, a normal karyotype, and no FLT3 variant, the presence of an NPM1 alteration is an indicator of a more favorable prognosis. Similarly, following chemotherapy, the presence, relative quantity, and trend of change of NPM1 mRNA transcript is associated with risk of disease relapse.

Cautions

Because of the design of this assay, a very small number of NPM1 alterations at diagnosis may not be detected by the more targeted quantitative polymerase chain reaction component. In that setting, the qualitative part of the test can be used for limited minimal residual disease assessment, although the sensitivity is much lower (approximately 5% at the DNA level).

Clinical Reference

1. Heath EM, Chan SM, Minden MD, Murphy T, Shlush LI, Schimmer AD. Biological and clinical consequences of NPM1 mutations in AML. Leukemia. 2017;31(4):798-807. doi:10.1038/leu.2017.30

2. Kronke J, Schlenck RF, Jensen KO, et al. Monitoring of minimal residual disease in NPM1-mutated acute myeloid leukemia: a study from the German-Austrian acute myeloid leukemia study group. J Clin Oncol. 2011;29(19):2709-2716. doi:10.1200/JCO.2011.35.0371

3. Ivey A, Hills RK, Simpson MA, et al. Assessment of minimal residual disease in standard-risk AML. N Engl J Med. 2016;374(5):422-433. doi:10.1056/NEJMoa1507471

4. Shayegi N, Kramer M, Bornhauser M, et al. The level of residual disease based on mutant NPM1 is an independent prognostic factor for relapse and survival in AML. Blood. 2013;122(1):83-92. doi:10.1182/blood-2012-10-461749

Method Description

RNA is extracted from blood or bone marrow and reverse transcription is performed. Real time quantitative polymerase chain reaction (PCR) is performed from complementary DNA template using the LC480 instrument platform (Roche). This assay targets the most common recurrent NPM1 alterations in acute myeloid leukemia (A, B, and D insertion variants). The quantitative value of NPM1 messenger RNA copy number is determined relative to ABL1 as the reference transcript using the delta-delta CT method. The reproducible analytical sensitivity (limit of detection) of this part of the assay is approximately 0.01%.

 

DNA is extracted from blood or bone marrow, and a PCR assay is performed using primers that amplify a fragment of NPM1 DNA containing the region susceptible to insertion variant. One of the PCR primers contains a fluorescent label. The amplified fragments are size separated by capillary electrophoresis. Wild type NPM1 produces a fragment length of 187 base pairs (bp). PCR fragments containing an insertional variant are observed as larger fragments, most typically 191 bp, as the majority of alterations are 4 bp insertions. The analytical sensitivity (limit of detection) of this part of the assay is approximately 5%.(Unpublished Mayo method)

Day(s) Performed

Monday through Saturday

Report Available

10 to 14 days

Specimen Retention Time

Blood/bone marrow: 2 weeks; Extracted DNA and RNA: 3 months

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

81310-NPM1 (nucleophosmin) (eg, acute myeloid leukemia) gene analysis; exon 12 variants

LOINC Code Information

Test ID Test Order Name Order LOINC Value
NPM1Q NPM1 Mutation Analysis, V 54448-6

 

Result ID Test Result Name Result LOINC Value
MP053 Specimen Type 31208-2
605098 Interpretation 59466-3
605262 Signing Pathologist 19139-5