Analysis of cKIT mutations in Acute Myeloid Leukemia and systemic mastocytosis by multiplex SuperRCA® technology Free

Introduction Alterations in cKIT residue D816 are the hallmark of systemic mastocytosis (SM) and are typically associated with poor prognosis in acute myeloid leukemias (AMLs), most notably the core binding factor-related AMLs. Although the D816V mutation is the most prevalent, other variants at the D816 residue are known: D816Y, D816I, and D816H. The “real world” sensitivity of current clinical laboratory methodologies for detecting cKIT D816 mutations, particularly in SM, is often limited by a relatively small proportion of tumor cells in clinical samples. Searching for these mutations may require an extensive and costly NGS sequencing approach, which can significantly prolong treatment decisions for patients.

The SuperRCA®assay can be performed on DNA extracted from the remnant samples used for the typical diagnostic assessment of the patient (blood, bone marrow, FFPE tissues etc) and can be completed within 1 to 2 days of the sample submission, which may lead to more timely disease prognosis and treatment intervention decisions. Here, we assessed if SuperRCA®technology can detect all 4 variants of the cKIT D816 mutation in a single patient sample.

Methods The SuperRCA® uses 2 consecutive rolling circle amplification (RCA) reactions, which enable highly specific genotyping and accurate quantification of low-frequency variants. Since the RCA products are large enough particles (on the order of microns in diameter) to be evaluated by routine flow cytometry, in any given assay the proportions of wild-type and mutated forms of the gene of interest are easily detected by differential fluorescence. No wash or purification steps are required, only addition of reagent to 96-well plate and incubation. In our experiments, limits of detection for KIT variants were assessed by using various de-identified clinical samples as well as a quantitative, serial dilution (“spiking”) of synthetic controls into normal blood. Numerical positive signals detected using flow cytometry provided a highly quantitative assessment of the assay reportable range. Assay specificity was assessed by comparing with sensitivity of the alternative approaches, such as ddPCR, as well as by the number of signals from normal individuals' DNA. Reproducibility was assessed on the same samples, by performing the assay in triplicate, at different days and by different operators.

Results For the cKIT D816V mutation, linear correlation was observed between differentially spiked samples, and the assay could detect low copy numbers with low overall and inter-operator CVs in both spiked and clinical samples. The assay was capable of detecting rare D816V variant of cKIT mutations at a sensitivity of 0.01% VAF down to 0.001% VAF. The number of positive signals was essentially zero for healthy donor samples, indicating an extremely low limit of blank and thus high specificity. Both experienced users and new users were able to obtain accurate and precise results within 10% CV. For the alternative cKIT D816 mutations, the assay showed similar sensitivity, specificity and reproducibility.

Conclusions These results demonstrate that SuperRCA®can be used to detect clinically relevant cKIT D816 variants with high specificity, sensitivity and reproducibility, which may offer a powerful tool for improving the diagnosis and treatment management of multiple hematological diseases.