C081 | OPTIMIZING MOLECULAR DIAGNOSIS OF SYSTEMIC MASTOCYTOSIS IN THE ERA OF KIT TYROSINE KINASE INHIBITORS: RESULTS OF A SURVEY AND OF A NATIONWIDE PROFICIENCY CONTROL ROUND OF KIT D816V MUTATION TESTING PROMOTED BY RIMA (RETE ITALIANA MASTOCITOSI)

Molecular testing plays a key role in the diagnosis and classification of SM and requires sensitive PCR-based methods, since disease burden, especially in the indolent forms, may be very low. The importance of reliable and accurate testing prompted RIMA to undertake, with the sponsorship of GIMEMA MPN WP, an initiative aimed to i) map the status of KIT D816V testing in Italy; ii) build a network of specialized labs available to support clinicians in the diagnosis of SM. A survey was conducted among 35 molecular biology labs across Italy to assess whether and with which method KIT D816V testing was offered: 29 labs (83%) declared they were routinely performing testing, either by Sanger sequencing (n=4), NGS (n=4), digital PCR (n=17), semi-quantitative real time PCR (n=2), qualitative (n=1) or quantitative ARMS-PCR (n=1). After a pilot experience with 7 labs in 2023, a second control round of proficiency testing was conducted among 10 labs (Milan, Monza, Bergamo, Vicenza, Pisa, Pescara, Rome, Bari, Palermo, Nuoro) using digital PCR (BioRad QX200/600, n=8; Thermo Fisher QuantStudio Absolute Q, n=1; Qiagen QIAcuity One, n=1). KIT D816V-mutated and wild-type DNAs were mixed to mimic different ABs (5%; 0.5%; 0.3%; 0.1%; 0.05%; 0.02%; 0%) and used to prepare identical batches of 7 blinded vials. Dilutions were first validated by the UK Wessex Genomics Laboratory Service (WGLS) and then shipped to the participating labs, where they were analyzed according to local procedures. Labs were asked to score each vial for KIT D816V and to estimate the AB. Agreement between measurements was assessed using Weighted Deming Linear Regression and Bland–Altman bias analyses. The vial with no KIT D816V DNA was scored negative by 9/10 labs. The 0.02% vial was scored positive by 5 labs, borderline by 1 lab and negative by 4 labs (in accordance with their reported LoDs of 0.1%, 0.1%, 0.06% and 0.04%). The remaining dilutions were correctly scored positive by all labs. In these samples, quantitation of AB showed excellent correlation and concordance among labs and between labs and the WGLS, with Pearson R coeffs ranging from 0.9997 to 0.9999 and Bland-Altman plots yielding mean bias ranging from -0.17 to 0.02. Our results demonstrate that digital PCR, regardless of the platform, provides reliable and reproducible KIT D816V detection and quantitation. Lab networking is the key to guarantee high standards of molecular testing in rare diseases like SM. Supported by Istituto Gentili.