Combined KIT D816V and HαT blood testing: Development and validation of a targeted non-invasive work-up to guide invasive testing for systemic mastocytosis Free

Background: Systemic Mastocytosis (SM) is a rare myeloid neoplasm (prevalence of 0.01%) characterized by a clonal mast cell expansion and mast cell mediator related symptoms such as anaphylaxis, flushing, gastrointestinal or neuropsychiatric complaints. Even indolent forms can cause considerable symptom burden, while advanced SM may lead to severe organ damage and reduced survival. Given the efficacy of KIT D816V-targeted therapies (driver mutation in > 95% of patients), accurate diagnosis is essential.

SM is routinely screened by measuring baseline serum tryptase (BST) levels, which also serve as a minor criterion (BST ≥20 µg/L). However, BST elevation lacks specificity, as Hereditary Alpha-Tryptasemia (HαT; prevalence of 5-7%) – caused by an amplification of α-tryptase on the TPSAB1 gene – independently elevates BST levels while mimicking SM symptoms. Thus, diagnosis is challenging in patients with moderately elevated BST, as HαT may lead to unnecessary bone marrow biopsies and SM may be missed in HαT-negative individuals with BST levels <20 µg/L.

This study aimed to develop and validate a non-invasive diagnostic work-up integrating BST, HαT genotyping and KIT D816V blood testing to guide bone marrow biopsy (BMB) decisions.

Methods: An exploratory cohort of 228 KIT D816V-negative patients (tested via highly sensitive digital droplet PCR, LOD 0.009%) with BST ≥8 µg/L from the allergy department was analyzed for BST distribution by HαT genotype. The 8 µg/L cut-off was chosen based on literature showing HαT is rarely found below this level. BST distributions were normalized using logistic transformation and 67%- and 99%-confidence intervals (CIs) were used to define decision thresholds, balancing sensitivity and specificity.

The resulting algorhythm was retrospectively validated in an independent multicenter cohort of 142 patients from three different hematology departments. All patients had moderately elevated BST levels (mean BST 19.8 µg/L, range 8–36 µg/L) and known SM status. Blood samples were tested for HαT and KIT D816V and the proposed work up was applied.

Results: The calculated thresholds from the exploratory cohort were at 11.5 µg/L (67%-CI) and 15 µg/L (99%-CI) in HαT-negative patients (n=94), and 17 µg/L and 32 µg/L (99%-CI) in HαT-positive patients with one additional TPSAB1 copy (n=116).

In the validation cohort, BMB was recommended in HαT-negative patients with BST levels ≥15 µg/L or in those with BST levels between 11.5–14.9 µg/L if KIT D816V was detected in peripheral blood. In HαT-positive patients with one additional TPSAB1 copy, KIT D816V testing was recommended for BST levels between 17–32 µg/L, with BMB performed only in case of KIT positivity. In patients with more than one additional copy, KIT testing was recommended in BST levels ≥30 µg/L; however, due to the small sample size (n=18), these patients were not formally included in the algorithm. In patients with BST levels below the 67%-CI thresholds, KIT D816V blood testing was only recommended in patients with cutaneous mastocytosis (CM) or severe Hymenopteran venom anaphylaxis (HVA), as these conditions are associated with a high risk of SM independent of the BST levels.

The proposed algorithm achieved a sensitivity of 88% (84/96) and specificity of 96% (44/46). Importantly, 41 patients who did not meet the WHO BST ≥20 µg/L minor criterion were correctly identified as SM cases. Conversely, 17 patients with BST ≥20 µg/L avoided unnecessary invasive diagnostics due to negative KIT testing and confirmed HαT genotype. The algorithm yielded a positive predictive value of 98% (84/86) and a negative predictive value of 78% (44/56).

Conclusion:The combination of HαT genotyping and KIT D816V blood testing significantly improves the diagnostic accuracy of SM. Our proposed algorithm achieved high sensitivity (87%) and specificity (97%) and outperformed the use of the BST ≥20 µg/L minor criterion for SM (Sensitivity 26%; specificity 80%). Importantly, the work up identified a substantial number of SM cases missed by standard thresholds and prevented unnecessary bone marrow biopsies in patients with HαT-related tryptase elevation. By integrating molecular diagnostics with genotype-stratified BST interpretation, this non-invasive approach optimizes patient selection for invasive diagnostics and may serve as a new standard for early and accurate SM screening.