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Published on: September 20, 2016
Nucleophosmin mutation analysis in acute myeloid leukaemia: Immunohistochemistry as a surrogate for molecular
Anita Chopra1, Sushant Soni1, Haraprasad Pati2
1Department of Laboratory Oncology, Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India.
Background & Objectives:
Mutation of nucleophosmin (NPM1) gene in the absence of FLT3-ITD (FMS related tyrosine kinase 3 - internal tandem duplications) mutation carries a good prognosis in cytogenetically normal acute myeloid leukaemia (AML). NPM1, a multifunctional nucleolar phosphoprotein that shuttles between nucleus and cytoplasm, gets trapped in the cytoplasm when mutated. Immunohistochemical (IHC) demonstration of its aberrant cytoplasmic location (NPMc+) has been suggested as a simple substitute for the standard screening molecular method. This study was aimed to assess the diagnostic utility of IHC on formalin fixed bone marrow biopsies in comparison with the reference molecular method (allele specific oligonucleotide - polymerase chain reaction; ASO-PCR) to predict NPM1 mutation status in AML patients.
Methods:
NPM protein IHC was performed using mouse anti-NPM monoclonal antibody on 35 paraffin-embedded bone marrow biopsies of patients with primary AML of any French-American-British (FAB) subtype. Results of IHC were compared with those of ASO-PCR.
Results:
Of the 35 AML patients, 21 (60%) were positive for NPM1 exon 12 gene mutation by ASO-PCR, 19 (90.47%) of these 21 were NPMc+. Thirteen of the 35 patients were negative by both the methods. One NPMc+ patient was not detected by ASO-PCR. IHC had a sensitivity and specificity of 90 and 93 per cent, respectively, compared to the molecular screening gold standard.
Interpretation & Conclusions:
Mutation of NPM1 determined by the widely available and inexpensive IHC agrees closely with results of the standard molecular methods. Thus, technically and financially not well endowed laboratories can provide the prognostically and potentially therapeutically important information on NPM1 mutation using IHC.
Insights
Immunohistochemistry (IHC) for cytoplasmic nucleophosmin (NPMc+) is a reliable method for detecting NPM1 gene mutations in acute myeloid leukaemia (AML). This cost-effective IHC test closely matches standard molecular methods, aiding prognostication.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- NPM1 gene mutations are crucial prognostic markers in cytogenetically normal acute myeloid leukaemia (AML).
- Mutated NPM1 protein mislocalizes to the cytoplasm (NPMc+), unlike its normal nuclear localization.
- Immunohistochemistry (IHC) is proposed as a simpler alternative to molecular methods for detecting NPMc+.
Purpose of the Study:
- To evaluate the diagnostic accuracy of IHC for NPM1 mutation detection in AML.
- To compare IHC results with the gold standard molecular method (ASO-PCR).
Main Methods:
- NPM protein IHC was performed on 35 bone marrow biopsies from AML patients.
- IHC results were compared against allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) results.
Main Results:
- NPM1 exon 12 mutations were detected in 21 (60%) patients by ASO-PCR.
- Of these, 19 (90.47%) showed cytoplasmic NPM localization (NPMc+) by IHC.
- IHC demonstrated a sensitivity of 90% and specificity of 93% compared to ASO-PCR.
Conclusions:
- IHC for NPMc+ is a highly accurate method for predicting NPM1 mutation status in AML.
- This inexpensive and accessible IHC technique can be utilized by laboratories with limited resources.
- IHC provides crucial prognostic and potentially therapeutic information regarding NPM1 mutations.
