Development of a Closed System qPCR Assay to Detect Mutations in IDH1/IDH2 in Patients with Acute Myeloid Leukemia (AML)
Authors: Baculi Edgar , Kadiyala Vineela , Tan Long , Nguyen Bach , Huynh Nhung , Song Tengyao , Day Gwo-Jen , Yuan Lin , Bates Michael , Wei Huilin 
Published: December 2023

Background:
Acute myeloid leukemia (AML) is a heterogenous disease characterized by clonal proliferation of poorly differentiated myeloid cells and is associated with mutations in several genes including the isocitrate dehydrogenase (IDH) genes IDH1 and IDH2. It has been reported that 20% of AML patients carry IDH1/IDH2 mutations (IDH1 - 8%, IDH2 - 12%) which are strongly associated with disease progression and relapse. Additionally, several IDH1/IDH2 inhibitors such as ivosidenib and enasidenib have been approved by the FDA for treatment of AML patients with IDH1 and IDH2 mutations respectively. Therefore, identification of these single nucleotide polymorphism (SNP) mutations in IDH1/IDH2 can help in disease classification and selection of effective targeted inhibitors.
Objectives:
Our goal is to develop a rapid and user-friendly cartridge-based qPCR assay that can detect 7 IDH1 and 10 IDH2 mutations in human genomic DNA from EDTA whole blood lysate. Using a single cartridge, any of the four codons (IDH1 R132/R100 or IDH2 R140/R172) will be called out when a mutation is detected. This reporting style is more than sufficient in deciding a course of treatment as current therapies only differ between a mutation in either IDH1 or IDH2.
Methods:
Six levels of custom generated plasmids (64%, 16%, 4%, 1%, 0.25%, and 0.0625%) for the four most prevalent IDH1/IDH2 mutations were spiked-into EDTA whole blood lysate and tested with prototype IDH1/IDH2 mutation assay. Standard curves of Ct vs. log % mutation were generated for R132C, R132H, R140Q, and R172K. Custom generated plasmids were also made for the remaining 13 mutations and have been tested around the LoD (1%). In addition to plasmids, human cell line DNA with 14 of the 17 mutations were also spiked into EDTA whole blood lysate and tested around the LoD (1%). A standard curve of Ct vs Log % mutation was generated with R132H human cell line DNA. The remaining 3 mutations were not commercially available as cell line DNA. Specificity of the IDH1/IDH2 mutation assay was assessed by testing EDTA whole blood lysate from 20 individual donors in quadruplicate.
Results:
The prototype IDH1/IDH2 assay demonstrated a dynamic range from 64% to 0.0625% with a limit of detection at 1% for all the mutations that were tested. Figure 1 shows the dynamic range curves for the most prevalent mutations: IDH1 R132C & R132H, and IDH2 R140Q & R172K. These dynamic range curves include data points for both plasmid DNA (pDNA in red) and human cell line DNA (gDNA in blue) to show equivalency between the two test materials. The performance of the remaining 13 mutations in plasmid DNA perform similarly in their respective codons with no more than 2Ct difference at 1% levels. The remaining 10 human cell line DNA mutations perform similarly to plasmid DNA.
Conclusions:
We have prototyped 18-plex assay designed to detect 7 IDH1 and 10 IDH2 mutations and call out its corresponding codon in EDTA whole blood samples using a single cartridge at an estimated LoD of 1%.