Dear Editor:
We greatly appreciate the insights from your multicenter trial on thymosin α1 for sepsis and seek clarification on two key aspects to deepen our understanding of the findings.

First, regarding the subgroup analysis showing potential differential effects by age (hazard ratio 1.67 for <60 years vs. 0.81 for ≥60 years, P for interaction=0.01) and diabetes (P=0.04) [1], we note that eight subgroups were evaluated. Could you comment on whether adjustments for multiple testing—such as Bonferroni correction—were considered? The reported P values for age and diabetes (P=0.04) may require contextualization within the framework of multiple comparisons, as highlighted in a systematic review of immunomodulatory drugs in sepsis, which emphasized the need for rigorous statistical adjustment in subgroup analyses [3]. Additionally, the post hoc adjustment for baseline organ support in the age subgroup retained a trend (P=0.04) [1]. Would you elaborate on the rationale for this adjustment and its impact on result robustness, as recommended in guidelines for critical care research [4]?

Furthermore, the sample size was powered to detect an 8% absolute mortality difference (27% vs. 35%) based on prior trial data [1], but the observed mortality in your trial was nearly identical (23.4% vs. 24.1%, HR=0.99, 95%CI 0.77–1.27) [1]. Given the median APACHE II score of 14 in your cohort—potentially reflecting less severe illness than in prior studies—could you discuss how the projected effect size was informed? A meta-analysis has shown that inaccurate effect size estimation may lead to inconclusive trial results [4], while integrated genomic analyses in complex diseases highlight the value of subgroup-specific targeting to optimize sample size [5]. Would future research consider focusing on predefined subgroups (e.g., ≥60 years, diabetes) to validate efficacy?

Your clarifications would significantly aid in interpreting the trial’s implications for clinical practice and guide subsequent research directions.

Conflict of Interest: None declared.

References
[1] Wu J, Pei F, Zhou L, et al. The efficacy and safety of thymosin α1 for sepsis (TESTS): multicentre, double blinded, randomised, placebo controlled, phase 3 trial. BMJ. 2025 May 30;389:r1098. doi:10.1136/bmj-2024-082583
[2] Shankar-Hari M, Calandra T, Soares MP, et al. Reframing sepsis immunobiology for translation: towards informative subtyping and targeted immunomodulatory therapies. Lancet Respir Med. 2024;12(4):323-336. doi:10.1016/S2213-2600(23)00468-X
[3] Robey RC, Logue C, Caird CA, et al. Immunomodulatory drugs in sepsis: a systematic review and meta-analysis. Anaesthesia. 2024;79(8):869-879. doi:10.1111/anae.16263
[4] Renard Triché L, Futier E, De Carvalho M, et al. Sample size estimation in clinical trials using ventilator-free days as the primary outcome: a systematic review. Crit Care. 2023;27(1):303. doi:10.1186/s13054-023-04562-y
[5] Torchia J, Golbourn B, Feng S, et al. Integrated (epi)-Genomic Analyses Identify Subgroup-Specific Therapeutic Targets in CNS Rhabdoid Tumors. Cancer Cell. 2016;30(6):891-908. doi:10.1016/j.ccell.2016.11.003