Quantitative Detection of NPM1 Mutations as Marker of Minimal Residual Disease (MRD) in the Large Majority of AML with Normal Karyotype.
Authors: Saglio Giuseppe , Gorello Paolo , Cazzaniga Giovanni , Gottardi Enrico , Dell’Oro Maria Grazia , Rosati Roberto , Roti Giovanni , Alberti Federica , Diverio Daniela , Martelli Massimo Fabrizio , Lo Coco Francesco , Biondi Andrea , Falini Brunangelo , Mecucci Cristina 
Published: October 2019

Abstract
Normal cytogenetics in AML is a major drawback to detect minimal residual disease. We have identified heterozygous NPM1 mutations as the most frequent genetic lesion in AML with normal karyotype (NEJM2005:352,254). Mutations A, B and D, all characterized by tetranucleotide insertions, account for more than 90% of all mutations. NPM1 mutations are new important prognostic markers in AML with normal cytogenetics, being predictive of successful response to induction treatment (NEJM2005:352,254) and longer overall survival in cases without FLT3 mutations (Dohner K et al, and Schnittger S et al, Blood 2005, on line). Their clinical impact prompted us to develop a Real Time Quantitative PCR assay for the identification and monitoring of NPM1 mutants.
Tests were set up using either cDNA (13 adult cases with mutation A and 1 with mutation B) or genomic DNA (gDNA) (7 pediatric and 8 adult patients).
cDNA RQ-PCR . Forward primer was designed in exon 11, probe in exon 11/exon 12 junction (5′-FAM-3′-MGB) and reverse primers in exon 12. Samples were analyzed in ABI PRISM 7700 Sequence Detection System (Applied Biosystems). ABL1 gene was used as control. For the absolute quantitative assessment of NPM1 mutation A copies a standard curve with serial dilutions of a plasmid containing the target sequences was used. The highest reproducible sensitivity of this RQ-PCR assay was 10 molecules. The highest reproducible sensitivity of the relative quantification RQ-PCR assay for mutation A and B was 10−4. Thirteen AML NPM mutation A patients were monitored at diagnosis and over follow up. The number of mutated copies was high in all cases at diagnosis and significantly decreased after induction treatment in all cases with complete hematological remission. Only a slight decrease was observed in the case who did not reach remission. Patients with a complete hematological remission but MRD decrease <3log showed a higher risk of relapse.
gDNA RQ-PCR . specific forward primers for six different mutations were designed using Primer Express software to anneal to the mutated region of NPM exon 12. Reverse primers were designed on NPM1 exon 12. The TaqMan core reagent kit (Applied Biosystems) was used for RQ-PCR. The albumin gene was used as control. A reproducible sensitivity of 10−4 was reached in all but one case. The mean Ct of undiluted DNA samples was 23.3 (range 22.1–24.8). The mean slope of the dilution curves was 3.6 (2.9–4.0). High correlation coefficients (0.99 in all but one) were obtained.
Using both cDNA and gDNA we set up sensitive and reproducible systems to detect minimal residual disease in 60% of AML with normal karyotype and NPM gene mutations. While gDNA RQ-PCR has the advantage to be directly related to the number of residual leukemic cells, cDNA RQ-PCR can be easily applied in samples collected in the routine diagnostic testing for common translocations. Large prospective studies are necessary to clarify the clinical impact of NPM1-based MRD monitoring of AML with normal karyotype.