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 the RIMA (Rete Italiana MAstocitosi) Association
Authors: Soverini Simona , Monaldi Cecilia , De Santis Sara , Mancini Manuela , Albano Francesco , Anelli Luisa , Bruno Loredana , Brusca Ignazio , Fazio Grazia , Corral Abascal Lilia Maria , Guerrini Francesca , Bono Clara , Iaquinta Giovanni , Grammatico Paola , Novella Elisabetta , Perbellini Omar , Spinelli Orietta , Salmoiraghi Silvia , Baldoni Stefano , Persico Alessia , De Luca Giuseppa , Lauricella Calogero , Piras Giovanna , Palmas Angelo D. , Bruno Samantha , Mosca Cristina , Sartor Chiara , Papayannidis Cristina , Guglielmelli Paola , Vannucchi Alessandro M. , Bonadonna Patrizia , Mannelli Francesco , Cross Nicholas C. P. , Cavo Michele , Zanotti Roberta , Elena Chiara 
Published: December 2024

The recent introduction of targeted therapies for Systemic Mastocytosis (SM) has raised the awareness about the importance of timely and accurate diagnosis- that, outside reference centers, is hampered by the rarity of this malignancy, by the heterogeneity of clinical symptoms and presentation and by the need to integrate the specific expertise of different healthcare professionals. Molecular testing plays a key role in the diagnosis, classification and management of SM: detection of KIT D816V activating mutation, underlying approx 95% of SM cases, is among the diagnostic criteria and quantitation of the allele burden (AB) helps in subtype classification, provides prognostic information and might serve, in future, for minimal residual disease monitoring. Screening for KIT D816V requires sensitive PCR-based methods, since disease burden, especially in the indolent forms, may be very low.
The importance of reliable and accurate molecular testing in SM prompted the Rete Italiana MAstocitosi (RIMA) to undertake, with the sponsorship of the GIMEMA Working Party on Chronic Myeloproliferative Neoplasms, an initiative aimed to i) map the status of KIT D816V mutation testing in Italy; ii) foster the creation of a network of specialized, reference laboratories available to support clinicians in the diagnosis of SM.
A survey was conducted among 35 molecular biology labs at major hematological centers all across Italy to assess whether and with which methodology KIT D816V mutation testing was offered. Twenty-nine labs (83%) declared they were routinely performing KIT mutation testing, either by Sanger sequencing (n=4), NGS (n=4), digital PCR (with either home-brew assays or commercial kits; n=17), semi-quantitative real time PCR (either home-brew or commercial; n=2), quantitative ARMS-PCR (home-brew; n=1), or qualitative ASO-PCR (home-brew; n=1). After a pilot experience that involved 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) selected among those who reported to be using a quantitative assay on a digital PCR platform (BioRad QX200 or QX600, n=8; Thermo Fisher QuantStudio Absolute Q, n=1; Qiagen QIAcuity One, n=1).
KIT D816V-mutated and wild-type DNAs isolated from the HMC-1.2 and HL-60 cell lines, respectively, were mixed in variable proportions to mimic different ABs (5%; 0.5%; 0.3%; 0.1%; 0.05%; 0.02%; 0%) and used to prepare 10 identical batches of 7 blinded vials. Dilutions were first externally assessed and validated by the UK Wessex Genomics Laboratory Service (WGLS) using an accredited droplet dPCR assay with an LoD=0.01% and then shipped to the participating labs, where they were analyzed according to local procedures. Labs were asked to score each sample as positive or negative for KIT D816V and to provide a quantitative estimate of the AB. Agreement between measurements was assessed using Weighted Deming Linear Regression and Bland-Altman bias analyses.
The vial containing no KIT D816V DNA was scored negative by 9/10 labs. The 0.02% vial was scored positive for the D816V by 5 labs, borderline by one 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 correlation coefficients ranging from 0.9997 to 0.9999 and Bland-Altman plots yielding a mean bias ranging from -0.17 to 0.02.
Overall, our experience shows that the rarity of the disease and the lack of awareness about the diagnostic challenges in detecting KIT D816V result into routine use of an array of heterogeneous and sometimes inadequately sensitive methods (like Sanger sequencing and NGS, that may yield false-negative results leading to missed/delayed SM diagnosis in a not negligible proportion of patients). Our control round demonstrates that digital PCR, regardless of the platform, provides a reliable and reproducible option for KIT D816V mutation detection and quantitation, although further efforts should be made to enhance LoD in some cases. Our experience also shows that lab networking and exchange of positive controls and expertise plays a critical role in guaranteeing high standards of molecular testing in rare diseases like SM.
Supported by Istituto Gentili.