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2690 Methodology and Clinical Significance of Longitudinal UBA1 Clonal Burden Evaluation in Vexas Syndrome

Program: Oral and Poster Abstracts
Session: 508. Bone Marrow Failure: Acquired: Poster II
Hematology Disease Topics & Pathways:
Research, Acquired Marrow Failure Syndromes, Translational Research, Bone Marrow Failure Syndromes, Assays, Clinical Research, Diseases, Technology and Procedures, Measurable Residual Disease , Molecular testing
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Carmelo Gurnari, MD, PhD1,2, Elisa Galossi1*, Eleonora Lumia1*, Alfonso Piciocchi3*, Mariadomenica Divona1*, Tiziana Ottone1*, Elisa Casciani1*, Francesca Romano1*, Elisa Diral, MD4*, Alessandro Tomelleri, MD5*, Federico Caroni6*, Antonio Vitale7*, Gregorio Maria Bergonzi, MD4*, Annalisa Condorelli8*, Giorgia Battipaglia, MD9*, Erika Morsia10*, Antonella Poloni, PhD11*, Elena Crisa, MD12*, Daniela Caravelli12*, Paola Triggianese1*, Arianna Savi, MD13*, Chiara Cardamone, MD14*, Matteo Dragani, MD15*, Giulia Rivoli, MD15*, Federica Pilo, MD16*, Davide Firinu17*, Sara Plebani18*, Francesco D'Agostino19*, Alessandro D'Ambrosio, MD20*, Katja Sockel, MD21*, Antonio Curti, MD, PhD22, Cristina Papayannidis, MD, PhD23, Marco Frigeni8*, Attilio Olivieri10*, Fabrizio Pane24, Monica Bocchia, MD6*, Luca Cantarini7*, Alessandro Rambaldi25, Corrado Campochiaro, MD5*, Lorenzo Dagna, MD26*, Raffaella Greco, MD27*, Fabio Ciceri, MD28*, Orietta Spinelli8*, Christian Thiede, MD21 and Maria Teresa Voso, MD1

1Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
2Cleveland Clinic Foundation, Cleveland, OH
3GIMEMA Foundation, Rome, Italy
4Unit of Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy
5Unit of Immunology, Rheumatology, Allergy and Rare diseases, IRCCS San Raffaele Hospital, Milano, Italy
6Hematology, Azienda Ospedaliera Universitaria Senese, University of Siena, Siena, Italy
7Rheumatology Unit, Department of Medical Sciences, Surgery and Neurosciences, University of Siena and Azienda Ospedaliero-Universitaria Senese [European Reference Network (ERN) for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases (RITA) Center, Siena, Italy
8Hematology and Bone Marrow Transplant Unit, ASST Papa Giovanni XXIII, Bergamo, Italy
9Federico II University of Naples, Hematology Department, Naples, Italy, Naples, Italy
10Clinica di Ematologia Azienda Ospedaliero Universitaria delle Marche, Ancona, Italy
11Hematology Unit, Azienda Ospedaliero Universitaria delle Marche, Ancona, Italy
12Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
13University of Turin, Department of Clinical and Biological Sciences, Turin, Italy
14Division of Allergy and Clinical Immunology, University of Salerno, Salerno, Italy
15Hematology and Cellular Therapy Division, IRCCS Ospedale Policlinico San Martino, Genova, Italy
16Hematology and BMT Unit, A.Businco hospital, AOB Cagliari, Italy, Cagliari, Italy
17Department of Medical Sciences and Public Health, University of Cagliari, Cagliari, Italy
18Hematology, San Salvatore Hospital, L'Aquila, Italy
19UOC Hematology, IRCCS Ospedale Casa Sollievo della sofferenza, San Giovanni Rotondo, Italy
20AORN S.Anna e S. Sebastiano, Caserta, Italy
21University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany
22Istituto di Ematologia "Seràgnoli", IRCCS Azienda Ospedaliero–Universitaria di Bologna, Bologna, Italy
23IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, Italy
24Division of Hematology and SCT Unit, Federico II University, Naples, Naples, Italy
25Department of Oncology-Hematology, Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, Italy
26Unit of Immunology, Rheumatology, Allergy and Rare diseases, IRCCS San Raffaele Hospital, Milan, Italy
27Unit of Hematology and Stem Cell Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy
28Hematology and Bone Marrow Transplantation Unit, I.R.C.C.S. San Raffaele Scientific Institute, Milan, Italy

VEXAS is a hemato-inflammatory syndrome genetically defined by somatic mutations in the X-linked UBA1 gene in hematopoietic precursor cells. Val/Thr/Leu substitutions at the Met41 hotspot constitute the molecular underpinnings of VEXAS in more than 90% of patients, typically men during the 6-7th decades of life. Clinical manifestations of the disease are extremely various and tend to relapse after most hematological and rheumatological treatments. Based on patients’ phenotypes, therapy can either target the UBA1 mutant clone (e.g., with azacitidine or allogeneic hematopoietic cell transplantation [allo-HCT]) or control the inflammation and hypercytokynemia germane to the syndrome (e.g., with DMARDs). To date, no guidelines exist for the management of VEXAS, and scarce is the evidence on methodology and clinical significance of longitudinal UBA1 clonal burden evaluation upon therapy. To address this gap in knowledge, we devised a new ddPCR method to quantify UBA1 clonal burden and explored its applicability in patients with VEXAS.

In a first methodological part, 6 serial DNA dilutions (102 to 10-3 plus a set of negative control) for each of the 3 UBA1 Met41 hotspot substitutions (Leu/Val/Thr) were generated in quadruplicates (N=216 samples) and independently analyzed by ddPCR in ours and 2 external laboratories in a blinded fashion. Results were then gathered to evaluate the variability of measurements across different centers, mutations, and dilutions. For each dilution, acceptable lower and upper limits were calculated on the logarithmic scale, with VAF considered out of range if not falling within the defined limits for the respective dilutions. Intraclass correlation coefficient (ICC) was then computed as an index of interrater reliability of VAF data. F-test and confidence interval were provided on the hypothesis (r>0.5).

Overall, we observed a highly significant ICC across centers (p=7.24e-24, 95%CI=0.89-0.95). Such strong significance held true also when looking individually at the three different assays (Met41Leu, p=1.22e-19, 95% CI=0.95-0.99; Met41Thr, p=1.17e-11, 95%CI=0.87-0.96; Met41Val, p=2.85e-06, 95%CI=0.79-0.95). With regards to VAF, the assay reached a sensitivity of 10−2 with loss of significant ICC for the 10-3 dilution. Once assessed the reliability of our method, we asked ourselves whether longitudinal UBA1 clonal dynamics may be clinically useful to unravel interactions with specific hematological and rheumatological treatments, potentially informing therapeutic decision. To do so, we leveraged our established nationwide network and gathered N=64 samples from N=34 patients with VEXAS and the canonical Met41 hotspots at various treatment time points (N=14 onset, N=46 follow-up). All patients were initially diagnosed by means of Sanger sequencing with primers covering the entire UBA1 exon 3 from DNA extracted from either peripheral blood (PB, N=12, median VAF 46.6%, IQR=33.9-69.7) or bone marrow (BM, N=5 median VAF 92.3%, IQR=72.5-94.1). Notably, the lowest VAF in diagnostic samples was 11.9%, still amenable to be detected by means of Sanger sequencing, which in fact had a Positive Predictive Value of 100% in our cohort. However, we noticed that BM yielded higher VAF in a set of 4 patients whose paired BM/PB samples were evaluated at the same timepoint (median VAF 65.7% vs 18.5%, respectively; p=0.2). Finally, we observed highly diverse clonal dynamics according to the therapy received in samples collected longitudinally by quantifying the VAF changes across consecutive timepoints (t) ∑min(VAFt+1-VAFt ⁄ VAFt). Specifically, erythropoietin, steroids, cDMARDS (e.g.,hydroxychloroquine/methotrexate) and bDMARDs (e.g., anti-IL1/6) minimally influenced UBA1 clonality (ΔVAF= -9.4%) while ruxolitinib tended to increase it (ΔVAF= +61%). Conversely azacitdine was able to dramatically abate UBA1 clonality (median VAF 1.3%, IQR 0.3-4.8 after a median of 4 cycles, ΔVAF= -97.5%) and a complete eradication was noticed after allo-HCT, paralleling the clinical reversion of VEXAS phenotypes.

In conclusions, ddPCR for UBA1 Met41 hotspots is a reliable and reproducible method to longitudinally track clonal dynamics of patients with VEXAS. Given the different treatment interactions, this method may allow for informed therapeutic decisions and implementation of personalized strategies in a MRD-like fashion.

Disclosures: Curti: Abbvie: Honoraria; Pfizer: Honoraria, Research Funding; Menarini stemline: Honoraria; Jazz Pharmaceutics: Honoraria. Papayannidis: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Menarini/Stemline: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Servier: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Blueprint: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Delbert Laboratories: Membership on an entity's Board of Directors or advisory committees. Pane: GSK Incyte Amgen BMS Janssen Jazz Novartis Pfizer: Speakers Bureau; GSK Incyte: Consultancy. Bocchia: Novartis: Honoraria, Other: travel grant; Incyte: Honoraria, Other: travel grant; Abbvie: Honoraria, Other: travel grants. Rambaldi: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Kite-Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau. Ciceri: ExCellThera: Membership on an entity's Board of Directors or advisory committees. Voso: Celgene/BMS: Other: Research support, Advisory Board, Speakers Bureau; Abbvie: Speakers Bureau; Syros: Other: Advisory Board; Astellas: Speakers Bureau; Novartis: Other: Research support, Speakers Bureau; Jazz: Other: Advisory Board, Speakers Bureau; Astra Zeneca: Speakers Bureau.

*signifies non-member of ASH