Molecular gene panel analysis in aplastic anaemia: Diagnostic utility and clinical implications Free

Background Aplastic anaemia (AA) is an acquired immune-mediated bone marrow failure and pathogenic somatic variants are rarely present. If present, these are often linked to age-related clonal haematopoiesis (CH), and can be crucial for differential diagnosis or monitoring of clonal evolution.

Methods We retrospectively reviewed patients diagnosed with AA, hMDS, inherited marrow failure syndromes (telomeropathies, Fanconi anaemia), PRCA, or acquired amegakaryocytic thrombocytopenia at a tertiary centre (Aug 1998–Jun 2025). Clinical, haematological, molecular, treatment and outcome data were collected. NGS-based somatic myeloid gene panel (MGP, 51 genes, Illumina NextSeq 500, >5% sensitivity) was performed at diagnosis or during follow-up.

Results Among 759 patients, 370 (48%) underwent MGP testing, with a median follow-up of 38 months (IQR 35-144). Pathogenic variants were found in 85 (23%). At diagnosis, 271 were tested, with 36 (13%) showing mutations. Most frequent mutated genes were BCOR (10), TET2 (5), ASXL1 (4); 1 had low-VAF (7%) TP53. Median VAF was 14% (3–51), with 6 patients having multiple mutations. Pathogenic variants (MGP+ patients) were more frequent in >60 years (18% vs 11%, ns) and in those with hMDS (26% vs 11%, p=0.02). PNH clones were more frequent in MGP+ (55% vs 46%, ns), 1 progressed to haemolytic PNH after losing the mutation; MGP+ also had higher MDS/AML progression rate (14% vs 4%, p=0.04).

Among 204 AA patients, 20 (10%) had a pathogenic variant. BCOR was most frequent (9/20), others included DNMT3A (4), TET2 (4), ASXL1 (2), SF3B1, CUX1, RAD21; 2 had multiple mutations. Median VAF 12.5% (4–50). Median age was 60y (22–84), with male predominance. Baseline parameters showed Hb 80g/L (60–140), reticulocytes 23x10⁹/L (9–126), platelets 16x10⁹/L (2–118), neutrophils 1x10⁹/L (0–2). Fourteen patients received ATG-based treatment, half of which responded, while 2 underwent allo-SCT. Cytogenetics by conventional karyotyping or SNP-array was normal in 80% and a PNH clone detected in 70% (median size 1.05%, 0.1–25). Only 1 patient progressed to haemolytic PNH and 1 to MDS. The latter had BCOR mutation which was lost at progression with gain of RUNX1+U2AF1. Typically, BCOR-mutated patients were younger (median 36y, p=0.04), and more often had a PNH clone (8/9, p=0.05), though none progressed to haemolytic PNH. Compared to MGP+, 184 MGP- AA patients had similar features, except for younger age (median 50y), lower prevalence of PNH clones (47% vs 70%, p=0.04), and trend towards more ATG cycles (median 1, 0–2 vs 1, 0–1, p=0.06). Conversely, MGP analysis in hMDS patients demonstrated a different pattern with no mutations in BCOR, mutations in RUNX1 (2), and a higher proportion of multiple mutations (3/9); median VAF was slightly higher (17.5%, 3–64, ns).

During follow-up, 172 patients had MGP testing at median 46 months from diagnosis, 58 (33%) had somatic mutations. In 73 with prior MGPs, the panel remained negative in 48 (67%). In 3, baseline mutations (ASXL1, BCOR, TET2+GATA2) disappeared. Thirteen with prior negative MGPs acquired mutations, most commonly DNMT3A (4), ASXL1 (3), RUNX1 (3), at a median VAF of 46% (2–57). Only one patient acquired BCOR, while one acquired two TP53 mutations (VAF 45% each), and progressed to MDS. Nine patients remained MGP+ at follow-up, with persistent clones in 7 and shifting mutations in 2 cases: BCOR to RUNX1+U2AF1 and SF3B1 to ASXL1. One patient with KIT, IKZF1, WT1 at baseline acquired ETV6 before AML progression. Of 99 newly tested during follow-up, 37% were MGP+, median VAF 33.5% (5–83). 16 progressed to MDS/AML, 7 (44%) with multiple mutations including EZH2 (2), RUNX1, SETBP1, ETV6, NRAS. Among non-progressors, most common mutations were CH (15/21): ASXL1 (7), DNMT3A (5), TET2 (3), BCOR in 1.

Conclusion MGP+ at diagnosis was rare (~10%) in a real world clinical setting, supporting the immune, non-clonal nature of AA. When present, mutations often reflect age-related CH or a diagnosis of hMDS. In acquired AA the dominating mutation is BCOR, often associated with younger age and PNH positivity, and with low transformation risk unless new mutations appear. Higher MGP+ observed during follow-up (~30%) likely reflects targeted testing driven by clinical suspicion, and presents multiple, non-CH mutations

at progression. Serial testing revealed clonal dynamics, supporting its role in long-term surveillance and risk stratification.