Ultra-Sensitive Duplex Sequencing Improves Diagnostic Capabilities in Indolent Systemic Mastocytosis

Advanced diagnostic tools like ultra-sensitive duplex sequencing are overcoming challenges detecting KIT mutations in indolent systemic mastocytosis (ISM), where mutations that occur at low variant allele frequencies (VAF) are often missed by standard techniques, or where serum tryptase levels are absent, according to Tracy I. George, MD. She added that this technology is expected to play a critical role in improving diagnostic accuracy and capturing the full spectrum of disease heterogeneity.

Findings from an analysis of patients in the phase 2 PIONEER trial (NCT03731260), which evaluated avapritinib (Ayvakit) in ISM, highlight the utility of duplex sequencing. Among 251 patients, 96% harbored KIT exon 17 mutations when combining droplet digital PCR (ddPCR) and duplex sequencing results. A total of 85% of patients tested positive for KIT D816V via ddPCR in peripheral blood. For the remaining patients with undetectable mutations by ddPCR, duplex sequencing revealed KIT mutations, including low mutational VAF (median 0.0068%, range 0.0013%-0.0261%) and other non–KIT D816V exon 17 mutations such as D816I and D816Y. This group also included patients with dual KIT mutations and those who lacked serum tryptase levels less than 20 ng/mL. Four percent had no detectable KIT mutations by either method.

“By doing [ultra-sensitive duplex sequencing], especially in peripheral blood, which is much easier to access, we’re going to increase diagnostic sensitivity, [thereby] identifying more patients with ISM who can then qualify for therapy,” George stated.

In an interview with OncLive®, George highlighted the limitations of current diagnostic tools in detecting low-level KIT D816V mutations in ISM, discussed how ultra-sensitive duplex sequencing enhances detection and identifies additional mutations that inform treatment decisions in this setting, and emphasized the need for broader adoption and validation of this technology to improve diagnostic accuracy and access to effective therapies.

George serves as the chief scientific officer (CSO) and president of the Innovation Business Unit at ARUP Laboratories and a professor of pathology at Spencer Fox Eccles School of Medicine, University of Utah, in Salt Lake City.

OncLive: What are the key limitations of current diagnostic tools in detecting KIT D816V mutations in patients with indolent systemic mastocytosis?

George: The key limitations of the current assays like ddPCR 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 ISM. In patients with systemic mastocytosis, we find that approximately 30% are going to have serum tryptase levels less than 20 ng/mL. That’s one of the minor diagnostic criteria used for the diagnosis of systemic mastocytosis.

How might ultra-sensitive duplex sequencing address these challenges?

ddPCR is great compared with next-generation sequencing, which has detection limits of 1% to 3% VAF. With ddPCR, we can get [the detection limit] down to approximately 0.022% to 0.03% [VAF], which is better. We’re finding that in patients with very low levels of KIT mutations, duplex sequencing [allows us to bring detection sensitivity] down to the order of 0.001%. That’s essentially 1 mutation in 100,000 events. This is providing us with a whole different level of magnitude to detect the KIT mutation [using] ultra-sensitive techniques. We’re able to do that because with duplex sequencing, we’re labeling both strands of complementary DNA and then comparing them. Using informatics, we’re able to eliminate the strands that don’t have matching alterations in DNA. That virtually eliminates all the mistakes. It’s a neat method for [achieving] ultra-sensitive detection. Importantly, we can not only detect low levels of KIT D816V mutation but can discover other non-D816V exon 17 KIT mutations.

Did the use of ultra-sensitive duplex sequencing improve diagnostic accuracy when applied to the patient population in the PIONEER trial?

We developed the technique and then we applied it to patients in the phase 2 PIONEER study. These are well-described patients with ISM who are symptomatic despite best supportive care. We detected the KIT mutation in 85% of patients. [However], by using duplex sequencing, we were able to detect the KIT mutation in 96% of patients. Then we looked at the characteristics of those patients from the PIONEER trial. This is quite interesting, because the median basal serum tryptase value in those patients who were [subsequently] detected with duplex sequencing was 23 ng/mL compared with median basal serum tryptase levels of 45 ng/mL in those ddPCR-positive for KIT D816V mutations. The median mast cell burden in those biopsies went from 10% mast cells to 5% mast cells in bone marrow biopsies, and the median KIT VAF went from 0.49% detected via ddPCR to 0.0068% by duplex sequencing. [With this novel technique] we’re essentially detecting patients with very early ISM who have very low burdens of disease but are still symptomatic.

What is the clinical significance of this assay in ISM, and how might it improve patient care and access to effective therapies?

The significance is that we’re able to detect more patients with the KIT D816V mutation. If we review what’s out there in the literature, we find that the detection of the KIT mutation is the single best criterion that we have for detecting systemic mastocytosis. It is even better than the major criterion of multifocal dense mast cell aggregates. [Additionally], when we looked at the patients who had non–KIT D816V mutations that were identified in the [PIONEER] study, they also responded to avapritinib.

When the National Comprehensive Cancer Network guidelines were developed based on the European Competence Network on Mastocytosis-American Initiative in Mast Cell Diseases guidelines, they included the detection of KIT mutations using sensitive techniques. At that time, they were not commercially available in the United States. In my role as CSO at ARUP Laboratories, I brought on this clinical testing and validated it. That forced other reference laboratories to also offer this testing or to send [information] to us, and that increased access to getting diagnosed with systemic mastocytosis across the US. [Ultra-sensitive duplex sequencing] is the next generation of sequencing and is going to be the next step to identify these patients. Now we have effective therapies, which is exciting, but we have to get these diagnostic techniques out into the general population. That means getting them out into reference laboratories or academic laboratories.

Apart from efforts to address logistical challenges, what additional clinical research is needed to refine the application of this technology?

We have validation in one clinical trial. When we’re [evaluating] a lab-developed test, we need to apply it to real-world samples obtained outside of a clinical trial and see how it performs in the wild, so to speak. When we develop these assays, we also have to bring them to scale. We test hundreds of patients a day, so we have to make sure that the techniques are robust, that we’ve got a supply chain established, and that we harden the assay so that anyone can run it, not just one person in a research laboratory. That’s what we do in clinical labs. Now in the US, with rulemaking by the FDA, you have to submit your laboratory-developed tests either to the FDA or through a surrogate, such as the Wadsworth Center of the New York State Department of Health. It’s quite a bit of a process if we’re not dealing with a FDA kit.

How do you envision the use of ultra-sensitive duplex sequencing for myeloproliferative neoplasms (MPNs) evolving beyond ISM?

We’re going to be seeing this technique used for common mutations in MPNs, [such as] JAK2 V617F, and other exon 17 mutations. People are going to want to be able to detect them in blood, which we can do with this technique. Because of the levels that we’re seeing now, this is in the realm of minimal residual disease [MRD] testing for myeloid neoplasms such as acute myeloid leukemia. These techniques are already being used in other studies for MRD.

Reference

Radia DH, Tashi T, Alvarez-Twose I, et al. Ultra-sensitive KIT testing uncovers previously undetected KIT mutations in patients with indolent systemic mastocytosis: results from the Pioneer trial. Blood. 2024;144(suppl 1):3164. doi:10.1182/blood-2024-207798