A newly developed high-sensitivity test substantially improved detection of the KIT D816V mutation in systemic mastocytosis (SM) and is recommended for routine diagnostic use, particularly in patients with low disease burden or unclear results, according to a study published recently in the International Journal of Molecular Sciences.
The KIT D816V mutation, which promotes mast cellMast cell A type of white blood cell produced in the bone marrow. They help defend against infections and play a key role in allergic reactions. In SM, mast cells become overactive and build up throughout the body. survival and growth, is a key driver of SM. Detecting this mutation is essential for diagnosis, but standard quantitative polymerase chain reaction (PCR) tests often struggle when mutation levels are very low.
Researchers developed a multiplex mRNA-based droplet digital PCR assay that is designed to overcome these limitations. The conventional testing method uses genomic DNA — someone’s total genetic information. The new method tests RNA via testing complementary DNA to reduce the amount of material being tested and better detect the mutation even when it occurs in low numbers. The test measures how often alleles carry the mutation using two reference genes, ABL1 and KIT.
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In validation studies, the assay showed very low background noise and high precision, with limits of detection as low as 0.062% and 0.027%, depending on the reference gene used. Its dynamic range spanned five orders of magnitude, indicating strong performance across a wide range of mutation levels.
“Although both assays demonstrated similar high analytical performances, the [complementary] DNA measurements proved superior to the [genomic] DNA results, as observed by finding a median 57-fold higher abundance of mutant KIT cDNA transcripts compared to mutant KIT [genomic] DNA alleles in [bone marrow] samples of SM patients,” the study’s authors said.
The 57-fold more mutant copies using RNA compared with DNA testing was tested in bone marrow samples from 79 patients with SM. This higher sensitivity translated into meaningful clinical gains. Among 18 patients suspected of having the disease but testing negative with DNA-based methods, 15, or 83%, were found to have detectable KIT D816V expression using the new assay.
Read more about the testing and diagnosis of SM
The study also showed that mutation signals may be present in cell types beyond mast cells, a finding linked to worse prognosis. Correlations between expressed allele burden, variant allele frequency and the percentage of excess mast cells suggested broader involvement of the mutation across blood cell lineages in some patients.
For patients, this approach could mean earlier and more accurate diagnosis, especially when disease levels are low or symptoms are unclear. It may also help clinicians better understand disease severity and progression. While the technology requires specialized equipment and may be more costly than standard tests, its improved sensitivity offers a meaningful step forward in identifying and managing SM.
