Detection of IDH1 and IDH2 Mutations in Patients with Acute Myeloid Leukemia Using Novel, Highly Sensitive Real-Time PCR Assays with Rapid Turnaround Time

Dash DP^1*, Wise L¹, Knier A¹, Boretsky M¹, Simons J¹, Berchanskiy D¹, Indig MA¹ and Joseph AM^2
*Correspondence: Dash DP, Blood Center of Wisconsin (Versiti), Milwaukee, Wisconsin, USA, Tel: 414-937-6076, Fax: 414-937-6202, Email:

Keywords

IDH1; IDH2; Mutation; Isocitrate dehydrogenase; Acute myeloid leukemia; Real-time; Next generation sequencing

Introduction

Genetic profiling of acute myeloid leukemia (AML) reveals distinct molecular subgroups that inform disease classification and prognostic stratification [1]. Dysregulated metabolism is one of the most common features of cancer cells [2]. The isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) play prominent roles in cellular metabolism. IDH1 is present in the cytoplasm and peroxisome and is involved in lipid metabolism and glucose sensing [3]. IDH2 resides in the mitochondria and is a component of the Krebs cycle, involved in the regulation of oxidative respiration [3]. IDH1 and IDH2 enzymes catalyze oxidative decarboxylation of isocitrate to alpha-ketoglutarate (αKG). Mutations in genes encoding IDH1 and IDH2 proteins are typically mutually exclusive, likely due to their common underlying biochemical mechanism and physiological consequences [4]. Both IDH1 and IDH2 mutations produce neomorphic activity resulting in the conversion of αKG to an oncometabolite, 2-hydroxyglutarate (2-HG) [5]. IDH1 and IDH2 mutations can co-operate with other gene mutations and molecular defects to promote leukemogenesis [6]. Expression of mutant IDH1 or IDH2 proteins is sufficient to block differentiation of hematopoietic cells in vitro and in vivo [7-9]. Differentiation block can be reversed by hindering these mutations [7,9,10]. IDH mutations are acquired early in AML pathogenesis and may exist in the founding clone [6]. IDH1 mutations affect codon 132 and have been reported to occur in 7-14% of adult and 1% of pediatric AML cases [11-13]. IDH2 mutations affect codon 140 or codon 172 and are observed in 8-19% of adult and 1-2% of pediatric AML cases [12-14].

Recent regulatory approvals of targeted therapies for treatment of AML highlight the importance of detecting pathogenic mutations that can drive disease progression and lead to relapse. Ivosidenib (TIBSOVO^®; Agios Pharmaceuticals, Inc., Cambridge, MA) and enasidenib (IDHIFA^®; Celgene Corporation, Summit, NJ) are approved for treatment of adult patients with relapsed or refractory (R/R) AML with an IDH1 or IDH2 mutation, respectively. A recent study of 80 patients with mutant-IDH1 or -IDH2 AML in morphologic remission after standard chemotherapy showed 40% of patients had persistent IDH1 or IDH2 mutations during remission, and those patients had an increased risk of relapse at one year of follow-up compared with patients without persistent IDH mutations after chemotherapy (59% vs. 24%, respectively; p<0.01) [15]. Importantly, in that study, IDH mutational burden during remission was not significantly associated with AML relapse: Patients with IDH1/IDH2 variant allele frequencies (VAFs) below 10% had similar risk of relapse as those with higher mutational burden [15].

Timeliness is key to choosing appropriate and optimal treatments for patients with AML [16]. While most AML patients start therapy within four days after initial diagnosis, treatment decisions for patients with R/R AML may require even more rapid therapeutic decision making [16].

Two new companion diagnostic tests, the Abbott Real-Time IDH1 and Abbott Real-Time IDH2 assays (Abbott Molecular, Inc.; Des Plaines, Illinois), were recently developed for detection of IDH1 and IDH2 mutations, to identify patients with AML who may benefit from treatment with targeted IDH inhibitors. The Abbott Real-Time assays are qualitative in vitro polymerase chain reaction (PCR) tests that detect single nucleotide variants (SNV) coding IDH1 and IDH2 mutations.

The objectives of the current analyses were to comprehend the turnaround time (TAT) for results of the three different mutation testing modalities for IDH1 and IDH2 (Abbott Real-Time PCR-based IDH1 and IDH2 assays, Sanger sequencing, and next generation sequencing [NGS]) via workflow analysis. Additionally, we compared the sensitivity of these three methods for detecting mutant IDH1 and IDH2.

Methods

This study was approved by the Medical College of Wisconsin Institutional Review Board. Blood or bone marrow aspirates from 100 patients with AML were received by the Blood Center of Wisconsin (BCW, now Versiti; Milwaukee, Wisconsin) as part of a diagnostic workup. Samples were analyzed for presence of IDH mutations using the Abbott Real-Time IDH1 and IDH2 assays, or BCW PCR-based Sanger sequencing assay, and a 30-gene NGS HemeOnc panel (BCW).

The Abbott Real-Time IDH assays target five IDH1 mutations at codon 132 (R132H, R132C, R132L, R132G, and R132S) and nine IDH2 mutations at codons 140 and 172 (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S, and R172W). The BCW PCR-based Sanger sequencing assay and NGS panel detect mutations in exon 4 of IDH1 and IDH2 with VAF sensitivity limits of 20% and 10%, respectively. NGS was utilized to resolve discordant results between the Real-Time and Sanger methods.

Clinical specimens were blinded (ie, the type of IDH mutation was not identified) to study investigators. DNA was extracted from 100 clinical specimens using the sample preparation system (Abbott Molecular, Inc.) or a QIAGEN DNA extraction kit (QIAGEN; Venlo, Netherlands). A set of commercially available controls (IDH1-R132H/ IDH2-R172K, IDH1-R132C/IDH2-R140Q) with different mutant VAF levels (20%, 5%, 1% and 0.25%) were also tested using the four assays mentioned above.

Abbott real-time IDH1/IDH2 reagent preparation and reaction plate assembly

Details regarding sample handling and preparation and assay procedures are available at https://www.accessdata.fda.gov/cdrh_docs/ pdf17/P170041C.pdf, https://www.accessdata.fda.gov/cdrh_docs/ pdf17/P170005C.pdf.

Briefly, the Abbott Real-Time IDH1 or IDH2 oligonucleotide reagents were each manually combined with DNA polymerase and an activation reagent to create unique “master mixes” that amplify and detect two or three IDH1/IDH2 amino acid mutations, and can detect sequences in regions outside of codon 132 (IDH1) or codons 140 and 172 (IDH2), to serve as endogenous internal controls (IC) (Table 1) [17,18]. Detection of the IC ensures that enough target material is available (target adequacy) and appropriate amplification of the IC ensures that the process has been performed correctly (process control). These master mixes are added to two separate wells of a 96-well optical reaction plate along with aliquots of the extracted DNA sample. The plate is transferred to the Abbott m2000 Real-Time instrument (Abbott Molecular, Inc.).

Target DNA is amplified by DNA polymerase in the presence of the activation agent, which includes primers, deoxyribonucleoside triphosphates (dNTPs), and magnesium chloride (MgCl₂). Amplification of IDH1, IDH2, and IC targets takes place simultaneously. IDH1/IDH2 are detected during the annealing/extension step by measuring the real-time fluorescence signals of the mutant-IDH1, mutant-IDH2, and IC-specific probes, which are labelled with different fluorophores to allow their signals to be distinguishable in a single PCR well.

Positive and negative controls are used in each run to verify that sample processing, amplification, and detection are performed correctly. Positive controls are formulated with DNA containing IDH1-R132H, IDH1-R132L, IDH1-R132G, IDH2-R140Q, IDH2- R140W, IDH2-R172K, or IDH2-R172W mutations, along with the IC signals, which should be detected in the positive controls. The negative controls are formulated with DNA containing only IC sequences, and thus only the IC signal should be detected in these samples.

Sanger and NGS testing

The BCW Sanger sequencing and NGS assay methods followed standard laboratory-developed test procedures. BCW Sanger sequencing follows the PCR-based bidirectional Sanger sequencing targeting IDH1 exon 4 including mutations at codon 132 (R132H, R132C, R132L, R132G, and R132S) and IDH2 exon 4 including mutations at codons 140 and 172 (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S, and R172W). The sensitivity, specificity, and TAT based on workflow analysis for the BCW Sanger sequencing assay are 20%, 99%, and eight business days, respectively. The BCW NGS HemeOnc panel contains 30 genes, including IDH1 and IDH2, targeted for myeloid malignancies; however, for this study, the data were analyzed only for IDH1 and IDH2. The sensitivity, specificity, and TAT based on workflow analysis for the NGS HemeOnc panel are 10%, 99%, and 15 business days, respectively.

Workflow analysis

The workflow analysis to determine TAT of the Abbott Real- Time IDH1 and IDH2 assays included assessment of the times, from the receipt of the specimen, to perform assay procedures (eg, DNA extraction, amplification, detection, data collection), lab reporting, and delivery of test results to a treating physician.

Results

The Abbott Real-Time IDH1 and IDH2 assays demonstrated 100% sensitivity and 95% specificity for detecting IDH1 and IDH2 mutations compared to the BCW Sanger sequencing assay. Five of 100 samples with low-level IDH2 mutations (< 20% VAF) were missed by the BCW Sanger sequencing assay but were detected as mutation-positive in the Abbott Real-Time IDH2 assay. These five discordant samples were subsequently tested by NGS, which detected IDH2 mutations in two of the samples (Table 2).

The Abbott Real-Time IDH1 and IDH2 assays detected all of the commercially available IDH1 and IDH2 mutation controls down to the 1% level (with some variability at the 0.25% level) (Table 2). In contrast, the Sanger method was sensitive to 20% mutant-IDH2 VAF..

TAT workflow analysis showed that the results from the Abbott Real-Time IDH1/IDH2 assays were available in three business days from the time of sample receipt in a clinical testing lab, compared with eight business days for the Sanger sequencing assay and 15 business days for the NGS assay (Figure 1). The longer TATs for the latter two assays mainly reflect additional time needed for sequential steps involving amplification, thermocycling/detection, and data collection and filtering; whereas, the Abbott Real-Time assays perform these processes simultaneously.

Discussion

The availability of effective treatments for a disease like AML with few therapeutic options reinforces the need for rapid diagnosis of pathogenic targets that are treatable. Indeed, the prognostic and therapeutic implications of IDH mutations in AML led to the recent recommendation by the College of American Pathologists and the American Society of Hematology to test for IDH1 and IDH2 mutations during the diagnostic workup for AML [19]. Moreover, paired diagnosis and relapse samples from patients with newly diagnosed mutant-IDH AML show these mutations can persist after induction chemotherapy [20-22]. The presence of persistent IDH mutations as measurable residual disease (MRD) during morphologic remission is a harbinger of relapse and is associated with poorer overall survival [15,23].

Targeted therapy with Ivosidenib and Enasidenib has shown promising results in patients with mutant-IDH R/R AML in clinical trials [24,25]. In a phase 1/2 trial of Enasidenib monotherapy, median overall survival among 214 patients with mutant-IDH2 R/R AML treated with Enasidenib was 8.8 months, which compares favorably to median OS in similar patients treated with a variety of other salvage treatment regimens (~3.5 months) [26,27]. Similarly, in a phase 1/2 study of Ivosidenib monotherapy in 125 patients with mutant-IDH1 R/R AML, ivosidenib was associated with a median overall survival of 8.8 months [25].

Conclusion

Our results demonstrate that the Abbott Real-Time IDH1/IDH2 assays reliably detect IDH1 and IDH2 mutations at VAF levels as low as 1% in patients with R/R AML. The Abbott Real-Time IDH1/IDH2 assays were more sensitive than the PCR-based Sanger sequencing assay and NGS panel, which had sensitivity limits of 20% and 10%, respectively. As noted, IDH1/IDH2 VAFs less than 10%, which is below the limits of detection with Sanger and NGS methodologies, can significantly increase relapse risk and may warrant treatment with an IDH inhibitor. The higher sensitivity of the Abbott Real-Time IDH1/ IDH2 assays could potentially improve the ability to diagnose new and relapsed patients with AML who have a lower IDH mutational burden. Moreover, the more rapid TAT for the Abbott Real-Time IDH assay results could help clinicians to make quicker decisions regarding optimal therapeutic treatment options for patients with AML.

Acknowledgments

Funding and Abbott Real-Time IDH assay reagents for the study were provided by Abbott Molecular Inc. Editorial support was provided by Sheila Truten and Kelly Dittmore of Medical Communication Company, Inc (Wynnewood, PA), funded by Celgene Corporation.

Declaration of Interest

Dash DP, Wise L, Knier A, Boretsky M, Simons J, Berchanskiy D and Indig MDA have no relevant financial interest to report. Joseph AM is employed by Abbott Molecular, Inc.

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