Blood and bone: The story of KIT D816V testing
Another key tool in the haematological assessment toolkit is testing for KIT D816V. The presence of this somatic mutation in clonal cells is common in SM. However, the question of which tissue type should be tested for KIT D816V to yield the highest sensitivity result is only just now being answered. Navarro-Navarro et al. recently employed several elegant assays to explore whether different sets of blood or bone marrow samples produce more accurate results for KIT D816V positivity in SM, with the aim of optimising the test’s sensitivity as well as exploring its potential for prognostics.1
Introduction
90% of patients living with systemic mastocytosis (SM) have KIT D816V mutation in their bone marrow (BM) samples, serving as one of the World Health Organization’s (WHO) minor diagnostic criterion for SM.2 Given the inherent convenience and ubiquity of blood testing in haematology though, non-invasive blood screening has increased.3 However, no studies have characterised the accuracy of blood versus bone marrow KIT D816V testing in mastocytosis. KIT D816V positivity in blood is held to be a sensitive surrogate for BM involvement in patients with advanced SM (AdvSM), though not in indolent SM (ISM) or BM mastocytosis (BMM).1
In this study, Navarro-Navarro et al. set out to compare, for the first time, KIT D816V positivity in paired samples of blood and BM from patients with mast cell activation syndrome (MCAS) and mastocytosis, analyse potential prognostic information from these data, and investigate next generation KIT D816V testing methods.1
Concordance between blood and bone marrow tests
The authors used a high-sensitivity PCR technique to quantify genomic DNA: allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR).1,4 At present, both high-sensitivity PCR methods ASO-qPCR and droplet digital PCR (ddPCR) demonstrate comparable sensitivity levels (∼0.01%), whereas the widely used next-generation sequencing (NGS) assay has insufficient sensitivity (∼1-5%) to detect KIT D816V in most patients with SM.5
Navarro-Navarro et al. assayed genomic DNA derived from 268 patients with MCAS and mastocytosis, of whom 86% (230/268) were KIT D816V-positive based on BM samples. The frequency of mutated cells in blood samples versus BM samples was highly concordant with 85%–100% concordance across ISM, smouldering SM (SSM) and AdvSM subtypes. However, there was lower concordance for ISM and especially BMM patients (Figure 1), which may merit other sensitive diagnostic methods being operationalised such as ddPCR (another high throughput and cost-effective PCR method, with the added advantage of not requiring a calibrator qPCR reaction), and purified sampling, which is explored later in this study.1,5
Figure 1. Concordance rates between blood and BM samples in different diagnostic subtypes of mastocytosis1
Prognostics
Consistent with other published studies, Navarro-Navarro et al. found that a frequency of >3.5% mutated cells (variant allele frequency [VAF] of >1.75%) was the most accurate cut-off for predicting 10-year progression-free survival. The study also suggested a novel use for KIT mutation analysis, by examining the variability in frequency of mutated cells over time: patients with stable mutation status (51%) consistently showed no disease progression at 5 and 10 years. However, those with unstable mutation status (49%), regardless of the subtype or specific diagnosis, had lower 5-year progression-free survival rates (Figure 2), highlighting a potential new utility of follow-up KIT testing to identify patients with progressive disease.1
Figure 2. Prognosis of SM and a subset of ISM/SSM patients based on their KIT mutation stability measured over time1
Figure from Navarro-Navarro P, et al. Allergy. 2023;78(5):1347–1359, open access article under the terms of the Creative Commons Attribution-NonCommercial License
Next-generation methods
Whole BM samples failed to detect positive KIT D816V in 31/268 patients but they were successfully detected using a purified BM method. Using a novel blood purification method in 28 patient samples, the authors showed that 61% (17/28) of samples became positive, with higher numbers of mutated basophils and eosinophils compared with other cell lineages. This highlights the importance of further evaluating patients with suspicion of SM using an enriched cell population in the future to further reduce false negative results.1
Conclusion
While there is high concordance between BM and blood KIT D816V testing, as revealed in this first ever evaluation, a negative KIT result does not rule out SM (e.g. in 15% of ISM cases), underscoring the need for continued monitoring for MCAS and mastocytosis patients. Among available testing methods, it is already known that ddPCR and ASO-qPCR methods demonstrate better performance versus NGS in detecting KIT D816V mutation. The present work further confirms the high accuracy in quantifying KIT mutation with ASO-qPCR in blood samples in most diagnostic subtypes, and the prognostic value of high KIT mutation burden on 10-year survival. Moreover, the authors highlight a possible new avenue to utilise stability of KIT mutation status as a predictor of progression-free survival, though further studies are warranted before this prognostic tool can become part of routine clinical practice.1,5
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