This investigator-initiated study was approved by the institutional ethics committee of Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (IIT2023038-EC-2) and registered on ClinicalTrials.gov (NCT05975996). All aspects of this study—including manuscript drafting, data acquisition, and statistical analysis—were exclusively performed by the authors. The data and its interpretation are fully validated, and protocol adherence was rigorously maintained throughout the research. Ethical compliance was ensured through written informed consent from every patient or their legal guardian. This research complied with the Declaration of Helsinki.

This was a single-center, non-randomized, phase II clinical trial initiated by investigators. This study employed Simon’s two-stage optimal design to estimate the required sample size, with the 3-month ORR as the primary endpoint. The null hypothesis (H₀) assumed a 3-month ORR of 45% for the standard IST and hetrombopag regimen, while the alternative hypothesis (H₁) projected an ORR of 65%. Using R software (version 4.0.2) with a one-sided significance level (α) of 0.05 and a target power ≥80%, the two-stage design yielded a total sample size of 43 patients. Enrollment would be halted for futility if less than 9 responses were observed in the first 22 patients (stage 1).

From July 2023 to September 2024, a total of 43 patients were enrolled in this study (Figure 1A). The inclusion criteria for this study were as follows: (1) patients aged ≥12 years; (2) confirmed diagnosis of treatment-naïve acquired severe or very severe aplastic anemia; (3) ineligibility or unwillingness to undergo allogeneic hematopoietic stem cell transplantation; (4) Eastern Cooperative Oncology Group (ECOG) performance status score ≤2; and (5) ability to fully understand the study protocol and voluntarily provide written informed consent. Patients were excluded from the study if they met any of the following criteria: (1) prior treatment with TPO-RA for >4 weeks before enrollment; (2) prior immunosuppressive therapy for >4 weeks before treatment initiation; (3) intolerance to cyclophosphamide or hetrombopag; (4) allergy to porcine ATG; (5) uncontrolled active infection; (6) uncontrolled hypertension (≥140/90 mmHg) or diabetes mellitus (fasting blood glucose ≥7.0 mmol/L or random blood glucose ≥11.1 mmol/L); (7) abnormal liver or kidney function, defined as aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >3× the upper limit of normal (ULN), or serum creatinine ≥2.5×ULN; (8) history of chemotherapy/radiotherapy for malignant solid tumors; (9) history of other severe systemic diseases; (10) pregnancy, lactation, or women with childbearing potential; or (11) considered ineligible by investigators due to other factors that might affect study completion.

SAA was defined as bone marrow cellularity of less than 25% and decreased values for at least two of three blood counts (reticulocyte count <20×10⁹ cells/L, platelet count <20×10⁹ cells/L, and neutrophil count<0.5×10⁹ cells/L). Aplastic anemia was considered very severe if the patient met the criteria for SAA and had a neutrophil count less than 0.2 ×10⁹ cells/L. Congenital hematopoietic failure must be excluded. Patients under the age of 50 are required to undergo genetic screening for congenital bone marrow failure-related mutations as part of the diagnostic process. Patients aged 50 and above must test negative in both the mitomycin C (MMC) and comet assays to rule out potential congenital genetic factors before enrollment.

Following enrollment, patients were administered a combined immunosuppressive regimen consisting of porcine ATG at 25 mg/kg/day on days 1–5, CsA 3–5 mg/kg/day maintained throughout the treatment course, and hetrombopag 15 mg/day initiated on day 1 and continued for 6 months. Sequential low-dose CTX (20 mg/kg/day) was subsequently administered on days 29–30 and 43–44 (Figure 1B). Supportive care, including infection prophylaxis and transfusion management, was provided in accordance with institutional guidelines. Infection prophylaxis and management include: for patients with an absolute neutrophil count (ANC) <0.5×10⁹/L, administer granulocyte colony-stimulating factor (G-CSF) at 2–5 μg/kg daily until ANC recovers to >5×10⁹/L, and initiate posaconazole for antifungal prophylaxis. If fever develops during treatment, prompt pathogen-directed testing and early empirical anti-infective therapy should be pursued based on clinical presentation.

The primary endpoint was 3-month overall response rate (ORR), defined as the proportion of patients achieving any of the following hematologic responses by 3 months after treatment initiation: complete response (CR), very good partial response (VGPR), good partial response (GPR), partial response (PR). The secondary endpoints comprised efficacy and safety outcomes. Efficacy endpoints included the 3-month CR rate and superior response rate (CR +VGPR+ GPR), as well as the 6-month ORR, CR rate, superior remission rate, and time to superior remission. Safety endpoints encompassed early all-cause mortality within 3 months, grade ≥3 treatment-related adverse events (per CTCAE v5.0), and clonal evolution (assessed via cytogenetic or molecular profiling).

Efficacy assessment criteria were derived from the Guidelines for the Diagnosis and Management of Adult Aplastic Anemia (1), refined further through our clinical experience. CR was defined as hemoglobin > 10 g/dL, absolute neutrophil count > 1.0×10^9/L, and platelet count > 100×10^9/L. VGPR was defined as hemoglobin > 10 g/dL, absolute neutrophil count > 1.0×10^9/L, and platelet count > 80×10^9/L. GPR was defined as hemoglobin > 8 g/dL, absolute neutrophil count > 1.0×10^9/L, and platelet count > 50×10^9/L. PR was defined as no longer meeting the criteria for SAA and transfusion independence but not meeting any of the response criteria defined above. Continuous transfusion dependency was classified as no response (NR). Superior remission includes CR, VGPR, and GPR, whereas inferior remission encompasses PR.

Both primary and secondary endpoints were analyzed according to the intention-to-treat (ITT) principle, with the analysis population comprising all patients who received at least one dose of the investigational drug.

For descriptive statistical analyses, unless otherwise specified, categorical data will be presented as the number of subjects (n) and percentage (%) for each category level. For continuous data, the number of non-missing subjects (n), mean, standard deviation, median, minimum, and maximum will be reported as appropriate based on the specific context. The endpoints of rate were reported with 95% CIs, calculated with Clopper–Pearson binomial CIs. Time-to-event data were estimated using KaplanMeier analysis and presented with accompanying 95% CI.

Among the 43 enrolled patients, 30.2% (13/43) were diagnosed with VSAA. The baseline characteristics of the cohort are detailed in Table 1. The median follow-up time was 14.9 months (range, 4.6–20.7 months). 45 patients were screened, with two patients withdrawing informed consent prior to treatment initiation. Among the 43 enrolled patients, 40 (93.0%) received four cyclophosphamide infusions per protocol, 2 (4.7%) received two infusions, and 1 (2.3%) received one infusion.

The primary endpoint of this study, the 3-month ORR, was 65.1% (28/43, 95%CI=49.1-79.0). The 3-month superior remission rate and CR rate were 25.6% (11/43, 95%CI=13.5-41.2) and 9.3% (4/43, 95%CI=2.6-22.1), respectively. At the 6-month follow-up, the ORR showed no significant improvement compared to the 3-month assessment (69.8%, 30/43, 95%CI=53.9-82.8). However, the superior remission rate increased to 60.5% (26/43, 95%CI=44.4-75.1), with the CR rate rising to 27.9% (12/43, 95%CI=15.3-43.7) (Figure 2). The median time to first PR was 86 days (95% CI = 74–109), while the median time to first superior remission was 131 days (95% CI = 100–181).

Compared with patients with SAA, those with VSAA demonstrated a significantly lower 3-month ORR (30.8% vs. 80.0%; p = 0.0041). From the hematologic response kinetics (Figure 3), it can be observed that among the 15 patients who showed no response at 3 months, 10 patients (66.7%) still failed to achieve hematologic response by 6 months. In contrast, among the 17 patients who achieved inferior remission at 3 months, 13 patients (76.5%) progressed to superior remission (including 3 CR), 2 patients (11.8%) remained in inferior remission, while 1 patient (5.9%) relapsed and 1 patient (5.9%) died due to an unrelated accident.

The adverse events associated with hetrombopag, p-ATG, and cyclosporine in this study were consistent with standard immunosuppressive therapy (IST), primarily manifesting as serum sickness-like reactions and liver function abnormalities. With the addition of cyclophosphamide, investigator-assessed CTX-related adverse events included alopecia (Grade 1, 48.8%), nausea (Grades 1-2, 100%), neutropenia (Grades 1-4, Grade 3 or higher in 62.8%), and infections (Grades 1-3, Grade 3 in 25.6%), as detailed in Table 2. Neutrophil decline was observed in both CTX treatment cycles (Cycle 1: days 29-30; Cycle 2: days 43-44), with a median neutropenia duration of 6 days (range 0–30 days) in Cycle 1 and 4.5 days (range 0–28 days) in Cycle 2 (Supplementary Figure S1A). Three patients with VSAA experienced persistently low neutrophil counts. However, these patients were already in a state of granulocyte deficiency prior to CTX treatment and remained in a state of persistent neutropenia after therapy. No reduction in hemoglobin or platelets attributable to CTX was observed. The overall infection rate within 3 months of treatment was 60.5%, and 46% (20/43) of patients developed new infections post-CTX, predominantly febrile neutropenia (16%, 7/43), pneumonia (12%, 5/43), and upper respiratory tract infections (9%, 4/43) (Supplementary Figure S1B). No death was observed during the 3-month follow-up.

In the cohort, 20.9% of patients harbored at least one Type I or II gene mutation prior to treatment (Table 1). The top three mutated genes were DNMT3A (25.0%), PIGA (16.7%), and TET2 (16.7%); however, the variant allele frequency (VAF) of these genes across the entire cohort were each below 10% (Figure 4). By the 6-month time point, the proportion of patients harboring Type I/II somatic mutations increased to 51.4% (18/35), with the most frequently mutated genes being PIGA (31.0%), ASXL1 (14.3%), and BCOR (14.3%). In addition to shifts in mutation spectrum, the variant allele frequencies (VAFs) of these mutations also rose compared to baseline levels (Figure 4). No progression to myelodysplastic syndromes or acute myeloid leukemia was observed in any patient as of the last follow-up date.

At baseline, 3 patients exhibited abnormal karyotypes: -Y, del (13) (q12q22), and t(X;19) (p10; p10). By the 6-month follow-up, the patient with -Y achieved normalization of karyotype, while the other two patients retained their original abnormal karyotypes. 18 of 43 patients (41.9%) tested positive for PNH clones at baseline. By 3 months, 16 of 40 patients (40.0%) remained PNH clone-positive, and at 6 months, the positivity rate was 16/38 (42.1%).