Diagnostic relevance of SH2B3 mutations in suspected myeloid malignancies: Insights from a large-scale NGS-based screening study

Abstract

Abstract INTRODUCTION The SH2B3 gene encodes the cytoplasmic adaptor protein LNK that acts as a negative regulator of hematopoietic progenitor cell expansion and self-renewal, particularly through its interaction with JAK2 and regulation of the JAK-STAT pathway. In humans, SH2B3 alterations have been identified across a spectrum of hematologic malignancies, especially myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), MDS/MPN overlap syndromes including chronic myelomonocytic leukemia (CMML), and acute lymphoblastic leukemias (ALLs). Despite increasing recognition, their precise clinical relevance and pathogenic role remain incompletely understood. Here, we retrospectively investigated a cohort of 17 833 patients referred for suspicion of a myeloid neoplasm or acute leukemia. Through systematic assessment of SH2B3 as part of routine next-generation sequencing (NGS)-based diagnostic workflows, we aimed to delineate the mutational spectrum and clinical implications of SH2B3 variants. METHODS DNA was extracted from bone marrow or peripheral blood samples and analyzed using a custom targeted NGS panel covering the entire coding region of SH2B3, along with 125 other genes recurrently mutated in hematological malignancies.Clinical and biological data were retrospectively collected from medical records and communication with the referring physicians who ordered the molecular analyses. RESULTS Among the 17 833 individuals tested, 13 423 (75.3%) carried at least one SH2B3 variant. The vast majority (n = 11 589, 86.3%) harbored only the common p.W262R polymorphism (rs3184504).The remaining 1 830 individuals carried 415 unique SH2B3 variants, which were classified hierarchically into four tiers of pathogenicity based on variant type, minor allele frequency in gnomAD v4.1.0 and variant allele frequency in patient samples. This led to the following distribution: 169 individuals with Tier I variants, 86 with Tier II variants, 167 with Tier III variants and 1 451 individuals with Tier IV (other than p.W262R) variants. Overall, 246 unique individuals (~1.4% of the total 17 833) with Tier I and/or Tier II SH2B3 variants were identified, with notable enrichment in CMML (7.2%) and MPN cases (5.6%). Null variants were observed throughout the entire protein, without restriction to specific domains, suggesting they are loss-of-function alterations, likely through protein instability or complete loss of expression. By contrast, missense variants exhibited a non-random distribution, with clear enrichment in the PH and SH2 domains, which are essential for LNK's membrane localization and interaction with JAK2. In the cohort of 246 patients carrying Tier I/II SH2B3 variants, the retained diagnosis was as follow: AML (n=61), MDS (n=56), CMML (n=33), essential thrombocythemia or idiopathic thrombocytosis (n=31), myelofibrosis (n=17), polycythemia vera or idiopathic erythrocytosis (n=17), BCP-ALL (n=4), MDS/MPN with ring sideroblasts and thrombocytosis (n=4), T-cell ALL (n=4), and clonal cytopenia of undetermined significance (n=9). Diagnosis remained undetermined in 10 cases. The overall median age of this cohort was 72 years (IQR 61–78). Among them, 212 SH2B3-mutated patients (86.2%) harbored additional mutations, with a median of 3 co-mutations per patient. Mutations in TET2 (47%), TP53 (10%), and SF3B1 (17%) were associated with significantly lower hemoglobin levels, while RUNX1 (13%) and CBL (9%) mutations were associated with decreased platelet counts. Additionally, 54 patients (22%) carried canonical MPN driver mutations, including 40 with JAK2 V617F, 10 with CALR mutations, and 5 with MPL W515L. These included 15 patientswith AML (25% of SH2B3-mutated AML), suggesting an enrichment of secondary AML transformed from a previous MPN. By contrast, SH2B3 mutation as the sole aberration was associated with higher hemoglobin concentrations (β = 1.95; 95% CI: 0.87–3.02) and platelet counts (β = 0.23 log; 95% CI: 0.05–0.41). Individuals with isolated SH2B3 mutations were often younger and had higher SH2B3 variant allele frequencies. Familial co-segregation was confirmed in three families. CONCLUSION SH2B3 alterations are enriched in CMML and MPNs, including triple-negative cases. These findings underscore the biological and clinical relevance of SH2B3 in myeloid disorders and support its inclusion in diagnostic screening panels, particularly in cases of unexplained thrombocytosis or erythrocytosis.