Digital Droplet PCR is a Specific and Sensitive Tool for Detecting IDH2 Mutations in Acute Myeloid LeuKemia Patients.

Susanna Grassi,Riccardo Puccetti,Elisa Mazzantini,Matteo Pelosini,Serena Salehzadeh,Maria Rita Metelli,Alessia Di Vita,Francesca Guerrini,Francesco Mazziotta,Enrico Orciuolo,Sara Galimberti,Elena Ciabatti,Pietro Rossi,Francesco Caracciolo,Mario Petrini,Cristiana Domenichini

doi:10.3390/cancers12071738

Susanna Grassi, Riccardo Puccetti + Show 14 more

Open Access

https://doi.org/10.3390/cancers12071738

Copy DOI

Abstract

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) interfere with cellular metabolism contributing to oncogenesis. Mutations of IDH2 at R140 and R172 residues are observed in 20% of acute myeloid leukemias (AML), and the availability of the IDH2 inhibitor Enasidenib made IDH2 mutational screening a clinical need. The aim of this study was to set a new quantitative polymerase chain reaction (PCR) technique, the drop-off digital droplet PCR (drop-off ddPCR), as a sensitive and accurate tool for detecting IDH2 mutations. With this technique we tested 60 AML patients. Sanger sequencing identified 8/60 (13.5%) mutated cases, while ddPCR and the amplification refractory mutation system (ARMS) PCR, used as a reference technique, identified mutations in 13/60 (21.6%) cases. When the outcome of IDH2-mutated was compared to that of wild-type patients, no significant difference in terms of quality of response, overall survival, or progression-free survival was observed. Finally, we monitored IDH2 mutations during follow-up in nine cases, finding that IDH2 can be considered a valid marker of minimal residual disease (MRD) in 2/3 of our patients. In conclusion, a rapid screening of IDH2 mutations is now a clinical need well satisfied by ddPCR, but the role of IDH2 as a marker for MRD still remains a matter of debate.

Highlights

Acquired somatic mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2), have been reported in acute myeloid leukemia (AML) [1], myelodysplastic syndromes (MDS), and chronic myeloproliferative neoplasms (MPN) [2,3]
We evaluated different experimental conditions, such as increasing DNA quantities, different annealing temperatures, and variable primers/probes concentrations
In order to try to better understand why five cases resulted in wild-type by Sanger but mutated by ddPCR, we focused on their mutational burden: As expected, these discordant cases showed low mutational levels, ranging from

Summary

Introduction

Acquired somatic mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2), have been reported in acute myeloid leukemia (AML) [1], myelodysplastic syndromes (MDS), and chronic myeloproliferative neoplasms (MPN) [2,3]. They occur into conserved active sites and mutant enzymes cannot still catalyze the oxidative decarboxylation. IDH2 mutations are found in about 20% of de novo AMLs, mainly in patients with normal karyotype.

Objectives

Methods

Results

Discussion

Conclusion