Dr George on the Limitations of Current Diagnostic Tools in Detecting KIT D816V Mutations in ISM

"The key limitation of the current assays, like droplet digital PCR and serum tryptase, is false negative results that you get in the peripheral blood. That's due to the KIT mutation having very low levels in the peripheral blood of patients, specifically with indolent systemic mastocytosis."

Tracy I. George, MD, chief scientific officer, president, Innovation Business Unit, ARUP Laboratories; professor, pathology, Spencer Fox Eccles School of Medicine, University of Utah, discusses the key limitations of current diagnostic tools, such as droplet digital PCR (ddPCR) and serum tryptase levels, in detecting KIT D816V mutations in patients with indolent systemic mastocytosis (ISM), and how ultra-sensitive duplex sequencing may address these challenges.

Systemic mastocytosis, including ISM, is a clonal mast cell disease driven primarily by activating mutations in the KIT gene in more than 95% of cases. The most common mutation is at codon 816; this is used as a minor diagnostic criterion for systemic mastocytosis according to the World Health Organization 2022 classification. However, detecting this KIT mutation in ISM can be challenging as circulating neoplastic mast cells in peripheral blood may not always be present at detectable levels, resulting in missed diagnoses, George explains.

Traditional diagnostic methods like ddPCR are sensitive and capable of detecting low levels of KIT mutations, George continues. ddPCR has a limit of detection of approximately 0.022% to 0.03% variant allele frequency (VAF), which makes it one of the most sensitive tests available. However, ddPCR still has difficulty with extremely low levels of mutation and cannot always detect KIT mutations in patients with very low VAF, she notes. Additionally, serum tryptase levels, another minor diagnostic criterion for systemic mastocytosis, may be absent in up to 30% of patients with ISM, George adds. This further complicates the diagnostic process.

Ultra-sensitive duplex sequencing is emerging as a novel solution to overcome these limitations, George states. With a detection sensitivity of 0.0013% VAF, duplex sequencing is much more sensitive than ddPCR and next-generation sequencing (NGS), which typically has detection limits of 1% to 3% VAF, George details. This ultra-sensitive method works by labeling both strands of complementary DNA and using advanced informatics to compare them, she explains. Such an approach significantly reduces errors, allowing for highly accurate detection of the KIT D816V mutation, as well as other non-D816V exon 17 KIT mutations that can also drive systemic mastocytosis, she says. This increased sensitivity is critical for detecting low-level mutations, improving diagnostic accuracy, and enhancing understanding of the disease, George concludes.