Correlation of Low Variant Allele Frequency STAT3 Gain-of-Function Mutations and T-Cell Clonality with Cyclic Thrombocytopenia

Background: Cyclic thrombocytopenia (CTP) is a rare disease characterized by periodic platelet count oscillations ranging from thrombocytopenic nadirs to normal levels or to thrombocytotic peaks. The etiology of CTP remains unclear, and may involve various pathogenic predisposing risk factors compromising platelet homeostasis and external triggers initiating the cycling phenotype [Zhang et al, 2024. PMID: 38083878]. We characterized 2 CTP patients possessing STAT3 gain-of-function (GOF) mutations at low variant allele frequency (VAF) and clonal T cells [Zhang, et al, 2023. PMID: 35381066]. Patient CT1 has 2 STAT3 mutations, Y640F (2% VAF) and D661Y (1% VAF), and patient CT2 has STAT3 D661Y (6% VAF). These data suggested that low VAF STAT3 GOF mutations and clonal T cells could be risk factors for CTP. Further evaluation of these risk factors may inform CTP pathophysiology and illuminate treatment options. Here we characterized an additional patient with clinical CTP, and searched the Stanford NGS database to identify patients with low VAF (<10%) STAT3 gain-of-function mutations. Clinical data of these patients was reviewed for evidence of cyclical thrombopoiesis and T-cell clonality.

Methods: Heme-STAMP is a clinically validated hybrid capture 200 gene NGS panel with digital error suppression and an average sequencing depth of 2000. The Stanford NGS clonality assay utilizes commercially available primer sets to characterize the TRG and TRB loci (Invivoscribe). Bioinformatics pipelines for both assays were developed at Stanford and clinically validated. Referred patients were characterized by available clinical information and testing with the above assays. Database review was performed by screening for patients with known pathogenic STAT3 GOF mutations with VAF <10% in peripheral blood or bone marrow samples. These patients were manually curated for clinical presentation, treatment history and evidence of cyclical thrombopoiesis and T-cell clonality.

Results: In addition to the two CTP patients (CT1 and CT2) we have previously reported, a third CTP patient (CT3) was referred, who showed platelet count cycling between 30 x10⁹/L and 1,700 x10⁹/L over a period of 4-6 weeks as well as T-cell clonality. Heme-STAMP analysis revealed a STAT3 H410R GOF mutation with VAF of 1.4%.

Heme-STAMP database screening identified 34 additional patients with STAT3 GOF mutations of less than 10% VAF. Of those, 3 patients lacked any platelet count data. Of the remaining 31 patients, 4 (12.9%) demonstrated platelet count cycling: 1 patient with STAT3 Y640F (1% VAF) had clinically significant platelet count cycling ranging from 5 x10⁹/L to over 1000 x10⁹/L; 3 patients had cycling within or near the normal range, and they each had one STAT3 mutation: D661Y (1.6% VAF), Y640F (8.3% VAF), and N647I (1.5% VAF), respectively. T cell clonality assay was performed on 3 of the 4 patients, and all 3 had clonal TCR. 12 patients had infrequent platelet count measurements and could not be evaluated for platelet cycling.

Conclusions: This study provides additional evidence that subclonal (VAF<10%) STAT3 GOF mutation and T-cell clonality correlate with CTP. While clinically significant CTP requiring interventions is rare, our database review suggests that clinically silent perturbations of thrombopoiesis in these patients are more prevalent than previously appreciated. Identification of these patients can be challenging as they require sensitive (high depth) NGS methods to reliably detect low VAF STAT3 mutations and clonal T-cell populations as well as frequent (at least weekly) platelet counts to reliably identify cycling.

For patients requiring therapy, JAK-STAT inhibitors may be considered as an investigational targeted intervention.

Connors:Abbott: Honoraria; Anthos: Honoraria; Bristol Myers Sqibb: Honoraria; Bayer: Honoraria; Janssen: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Werfen: Honoraria; Perosphere: Honoraria. Shomali:Blueprint Medicines: Research Funding; Incyte Inc: Consultancy, Research Funding. Gotlib:Incyte: Consultancy, Honoraria, Research Funding. Bussel:RallyBio: Consultancy, Research Funding; Alpine-Vertex: Consultancy; Sobi: Consultancy; UCB: Membership on an entity's Board of Directors or advisory committees; Janssens: Consultancy; Argenx: Consultancy.