Ultra-Sensitive <i>KIT</i> testing Uncovers Previously Undetected <i>KIT</i> mutations in Patients with Indolent Systemic Mastocytosis: Results from the Pioneer Trial

Introduction : Systemic mastocytosis (SM), including indolent SM (ISM), is a clonal mast cell disease driven by activating mutations in KIT in &amp;gt;95% of cases. A positive test for a KIT-activating point mutation, most commonly at codon 816 (e.g. D816V), is a minor diagnostic criterion for SM (World Health Organization [WHO] 2022). Particularly relevant for ISM, circulating neoplastic cells in peripheral blood (PB) and the associated KIT mutation can be difficult to detect; droplet digital PCR (ddPCR), one of the most sensitive tests in clinical use, has a limit of detection (LOD) ranging from 0.022-0.03% variant allele frequency (VAF). Serum tryptase level &amp;gt;20 ng/mL, another minor diagnostic criterion, may be absent in up to 30% of patients with ISM (Sánchez-Muñoz L et al. Mod Pathol, 2011) and may lead to missed diagnoses. Ultra-sensitive duplex sequencing is designed to address some limitations of current KIT testing by increasing sensitivity for KIT D816V (LOD 0.0013% VAF) and by detecting other exon 17 non-D816V KIT mutations that are known to drive SM. PIONEER (NCT03731260), a randomized, double-blind study, showed the efficacy of avapritinib, a potent, highly selective oral therapy targeting KIT D816V, compared with placebo and best supportive care in patients with ISM. All patients were tested by ddPCR for KIT D816V at baseline; for the minority who were undetectable, KIT mutation status in PB by duplex sequencing was performed. Here, we report the results of ultra-sensitive duplex sequencing in these patients. Methods: ISM diagnoses (1 major + 1 minor or 3 minor criteria per WHO 2016) in PIONEER were confirmed by central pathology review of bone marrow (BM) biopsies and patient-level data. During screening, patients were tested centrally for KIT D816V mutations using ddPCR in PB (LOD 0.022%). Samples from patients with undetectable KIT D816V by ddPCR were retrospectively tested by duplex sequencing. Testing was done with the TwinStrand DuplexSeqTM custom panel for human KIT exon 17 mutations, including KIT D816V (TwinStrand BioSciences, Seattle, WA), with the assay performed per manufacturer's instructions. Results: A total of 251 patients were enrolled in PIONEER and based on PB testing, were divided into 3 mutually exclusive groups. Mean baseline total symptom score, as assessed using the ISM-specific patient-reported outcome tool, ISM-Symptom Assessment Form (©2018 Blueprint Medicines Corporation), was severe (&amp;gt;42) in each group. In the first group, 214 patients (85%) tested positive for KIT D816V by ddPCR in PB during screening. Baseline measures of disease burden on Day 1 of therapy included median serum tryptase of 45.0 ng/mL (range, 4.2-501.6), median BM mast cells of 10.0% (range, 1.0-70.0), and median PB VAF of 0.49% (range, 0.0-41.3). In a second group of patients (n=26, 10%) KIT D816V was not detected by ddPCR of PB at screening but KIT activating mutations were detected by duplex sequencing. In this group, baseline measures of disease burden included median serum tryptase of 23.4 ng/mL (range, 3.6-200.0) and median BM mast cells of 5.0% (range, 1.0-40.0). Notably, 10/26 patients in this group had serum tryptase levels &amp;lt;20 ng/mL and thus would have been missing 2 SM diagnostic criteria on standard PB testing. Median KIT D816V VAF in this group as measured by duplex sequencing was 0.0068% (range, 0.0013-0.0261). This group included 2 patients with dual mutations in KIT (D816I+D816V, C788Y+D816V) and 3 patients with lone non-D816V KIT exon 17 activating mutations (D816I, D816Y; VAF 0.0075-4.5%). In the third and final group (n=11, 4%) KIT mutations were undetectable by both ddPCR and duplex sequencing. In this group, median serum tryptase was 20.8 ng/mL (range, 4.5-288.0) and median BM mast cells were 3.0% (range, 2.0-30.0). Aggregating PB testing results from ddPCR and duplex sequencing, the total number of PIONEER patients that had KIT exon 17 mutations was 240/251 (96%). Conclusion: In total, 15% of patients with ISM in PIONEER (n=37) had samples in which KIT D816V mutations were undetectable by ddPCR. Of these, 26/37 (70%) still harboredactivating KIT mutations when assessed by duplex sequencing. While PB tryptase and ddPCR testing for KIT D816V are important, when these tests are negative the possibility of SM cannot be ruled out. In this setting, BM biopsy should still be performed if SM is suspected and remains the standard for evaluating SM until more sensitive blood tests are widely available.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.