Identification of 17 Distinct IDH1/IDH2 Mutations from Acute Myeloid Leukemia Peripheral Blood Using a Multiplex Real-Time PCR Closed Cartridge System

Background:

Acute myeloid leukemia (AML) is characterized by clonal expansion of poorly differentiated “blast cells” in peripheral blood and bone marrow causing ineffective erythropoiesis and bone marrow failure. It has been demonstrated that mutations in isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) are associated with disease relapse and reduced survival. There are 17 AML-related IDH1/IDH2 mutations located at R100, R132, R140, and R172 that are targeted by different IDH1/IDH2 selective inhibitors. The sensitivity of detecting IDH1/IDH2 needs to be improved for early cancer detection. Current PCR-based assays cannot detect all 17 IDH1/IDH2 mutations, and next generation sequencing approaches take longer, use complicated workflows, and require costly reagents as well as substantial data analysis. Therefore, the rapid identification of patients with AML who harbor IDH1/IDH2 mutations could improve the efficient selection of targeted inhibitors for these patients.

Aim:

To create a multiplex PCR assay that detects 17 IDH1/IDH2 mutations from peripheral blood with high sensitivity and specificity.

Method:

We will highlight five technical aspects of this qPCR assay that contribute to its high sensitivity and specificity: 1. the “anchor - bridge - foot” design of SuperSelective (SS) primers that can detect different mutations within the same codon. 2. the amplification-refractory mutation system (ARMS) primer design strategy was also tested which has comparable effects to SS primer. 3. a two-step nested PCR to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. 4. a short (20-25nt) blocking oligonucleotide (wild-type blocker, WTB) to reduce SS and ARMS primer binding at the wild-type codon site. 5. four probes carrying different fluorescence reporters were used to identify the four IDH1/IDH2 mutation codons.

Results:

We synthesized 17 plasmid DNA (pDNA) templates and 14 CRISPR-engineered cell lines, each carrying one IDH1 or IDH2 mutation and the housekeeping gene ABL. SS and ARMS primer were screened with 5·10 ⁴ cps/test of mutation template in a whole blood background. 6 mutations of R132 Codon (R132C, G, H, L, S, V), R100Q, 4 mutations of R140 (R140Q, L, G, W), and 6 mutations of R172 (R172K, M, G, S1 [516G>T], S2 [516G>C], W) can successfully be identified from whole blood lysate at the LoD level (1%) as shown in Figure 1 in red. Moreover, adding WTB to the PCR reaction improved the specificity with a reduced background signal. Importantly, in addition to the plasmid, cell line genomic DNA (gDNA) that was spiked into blood lysate at the LoD level (1%) also performed similarly to plasmid DNA as shown in Figure 1 in blue.

Summary/Conclusion:

We present a prototype assay with design intention to detect 17 mutations of IDH1/IDH2 from the peripheral blood of AML patients with excellent sensitivity and specificity and a strong potential to outperform any products on the market. Utilizing our Xpert Instrument System a fully automated, single-use cartridge product with pre-loaded sample-processing reagents, the assay aims to report results in less than 3 hours, including hands-on time.