IDH1/2 Mutation Testing with TRUPCR®: A Practical Guide for Glioma and AML Labs

Why IDH1/2 Status Matters

A single amino-acid change in IDH1 or IDH2 can reshape how a brain tumour or a leukaemia is classified, discussed and managed. That is why IDH mutation status has moved from a research marker to a routine question in neuro-oncology and haemato-oncology laboratories.

This article explains what IDH1 and IDH2 mutations are, where they matter clinically, and how the TRUPCR® IDH1/2 Detection & Differentiation Kit brings this testing to laboratories equipped with a real-time PCR instrument.

What Are IDH1 and IDH2 Mutations?

IDH1 and IDH2 encode isocitrate dehydrogenase enzymes. IDH1 is located on chromosome 2q33.3 and functions in the cytoplasm, while IDH2 is located on chromosome 15q26.1 and functions in the mitochondria.

Cancer-associated mutations affect a small number of active-site residues: IDH1 R132 and R100, and IDH2 R140 and R172. These mutations give the enzyme a new function. Instead of following its normal metabolic pathway, the mutant enzyme produces the oncometabolite D-2-hydroxyglutarate (2-HG).

2-HG inhibits several alpha-ketoglutarate-dependent enzymes, including histone demethylases and TET proteins. The resulting changes in histone and DNA methylation contribute to tumour development.

Where IDH Mutations Matter: Glioma and AML

Glioma. IDH mutations are found in more than 80% of low-grade gliomas and secondary glioblastomas. They are frequent in adult WHO grade II and III gliomas and grade IV secondary glioblastomas. Beyond their diagnostic value, IDH1/2 mutations are associated with a more favourable prognosis in patients with glioma.

Acute myeloid leukaemia (AML). AML is the most common acute leukaemia in adults, and around half of patients have a normal karyotype. In these patients, molecular markers carry much of the diagnostic and prognostic information. Somatic IDH1 mutations occur in approximately 7–14% of AML cases, while IDH2 mutations occur in approximately 8–19%.

In both settings, knowing not only whether an IDH mutation is present but also which mutation is present provides a clearer molecular picture.

How the TRUPCR® IDH1/2 Detection & Differentiation Kit Works

The TRUPCR® IDH1/2 Detection & Differentiation Kit detects and differentiates 15 IDH1/2 hotspot mutations using eight real-time PCR tubes per sample. Each mutation assay is detected in the FAM channel, while an endogenous internal control is detected in the VIC/HEX channel. The internal control helps assess DNA quality, extraction and possible PCR inhibition that could contribute to false-negative results.

The Workflow

1. Sample: EDTA blood, EDTA bone marrow or FFPE tissue. For FFPE samples, a pathologist should confirm that the selected sections contain at least 30–50% tumour cells.
2. DNA extraction: Purified genomic DNA with an A260/A280 ratio of 1.7–1.9, for example using the TRUPCR® Blood DNA Extraction Kit or TRUPCR® FFPE Tissue DNA Extraction Kit.
3. Real-time PCR: The reaction is optimised for 50 ng of DNA across eight tubes, with a positive control and a no-template control included on every plate.
4. Result: Each mutation tube is compared with the reference tube from the same sample to determine mutation status.

The kit is compatible with Applied Biosystems™ 7500, QuantStudio® 3 and 5, Rotor-Gene Q, and Bio-Rad CFX96 and CFX384 real-time PCR instruments.

Key Features and Clinical Performance

In a blinded clinical correlation involving 137 samples and a reference method, the kit demonstrated 96.15% sensitivity and 98.82% specificity.

          ·    Detection and differentiation: 15 IDH1/2 mutations, with R132H, R132C, R100Q and R172K reported individually.

          ·    Three sample types: EDTA blood, EDTA bone marrow and FFPE tissue.

          ·    Built-in quality check: An endogenous internal control in every tube, with positive and negative controls included in the kit.

          ·    Ready-to-use components: Hot-start multiplex master mix and all primer-probe mixes are included.

          ·    CE-IVD certified: Manufactured by an ISO 13485:2016 certified company.

Specificity was also evaluated using DNA from 20 healthy whole-blood samples and 10 FFPE tumour tissues, all of which met the wild-type criteria.

Ordering Information

Store all kit components at −20 °C.

Bring IDH1/2 Testing In-House

With one kit, eight tubes and a standard real-time PCR instrument, laboratories can detect and differentiate key IDH1 and IDH2 mutations in DNA extracted from blood, bone marrow and FFPE tissue.

Want to learn more about TRUPCR® IDH1/2 testing? Visit the TRUPCR® IDH1/2 Detection & Differentiation Kit page or write to info@3bblackbio.com.

References:

1. Dohner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373:1136 1152. doi:10.1056/NEJMra1406184 2. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of theWorldHealthOrganization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and importantchanges. Blood. 2009;114:937–951. doi:10.1182/blood-2009-03-209262 3. Rowe JM. AML in 2017: advances in clinical practice. Best Pract Res ClinHaematol. 2017;30:283 286. doi:10.1016/j.beha.2017.09.010 [PubMed] [CrossRef] [Google Scholar]

2. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of theWorldHealthOrganization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and importantchanges. Blood. 2009;114:937–951. doi:10.1182/blood-2009-03-209262 3. Rowe JM. AML in 2017: advances in clinical practice. Best Pract Res ClinHaematol. 2017;30:283 286. doi:10.1016/j.beha.2017.09.010 [PubMed] [CrossRef] [Google Scholar]

3. Rowe JM. AML in 2017: advances in clinical practice. Best Pract Res ClinHaematol. 2017;30:283 286. doi:10.1016/j.beha.2017.09.010 [PubMed] [CrossRef] [Google Scholar]