Blog

Introduction

Globally, hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality, with increasing incidence projected over the next two decades. In China, it is the second most lethal cancer, and the long-term prognosis following curative surgery remains poor due to high recurrence rates, with 5-year recurrence rates reported at approximately 50%–70%.^1–6 Recurrence is most frequent within the first postoperative year and often appears as distant intrahepatic or extrahepatic metastases, thought to originate from undetected micrometastases present at surgery. Tumor-related features such as size, number, differentiation, microvascular invasion (MVI), and elevated alpha-fetoprotein (AFP) are major determinants of early recurrence. Conversely, factors including patient age, sex, underlying liver disease etiology, and cirrhosis are more commonly linked to late recurrence.^7–9 While transcatheter arterial chemoembolization (TACE) is commonly used in this setting, the introduction of immune checkpoint inhibitors (ICIs) offers a novel therapeutic option. Building on recent trials investigating ICIs in advanced or unresectable HCC, such as IMbrave150 and CheckMate 459, attention has shifted to their potential in earlier disease stages. However, limited data exist regarding their application following surgery, especially in patients with MVI. Moreover, the duration of ICI therapy necessary to maximize efficacy remains undefined. This study sought to compare ICI-based therapy with TACE in a real-world postoperative setting and to assess whether extended ICI treatment (≥12 months) offers added benefit in reducing recurrence.

Patients and Methods

Patients and Study Design

To evaluate the comparative efficacy of adjuvant ICI therapy versus TACE in improving RFS among HCC patients with MVI, we conducted a retrospective, multicenter cohort study enrolling patients who underwent curative-intent hepatic resection between January 1, 2017, and March 31, 2024, at Peking Union Medical College Hospital (Beijing), China-Japan Friendship Hospital (Beijing), and Sun Yat-sen University Cancer Center (Guangzhou). Inclusion criteria were: (1) complete (R0) resection; (2) age between 18 and 75 years; (3) pathologically confirmed HCC with MVI; and (4) Eastern Cooperative Oncology Group performance status (ECOG PS) score ≤ 1. Exclusion criteria encompassed: (1) recurrent HCC following prior curative resection; (2) history of spontaneous tumor rupture with hemorrhage; (3) coexistence of other active malignancies, including those in sustained remission; (4) administration of any neoadjuvant treatment modalities for HCC, including TACE, molecular targeted therapy, immunotherapy, or radiotherapy; and (5) death from non-HCC-related causes prior to follow-up.

Treatment and Data Collection

For patients anticipated to undergo extensive hepatectomy, preoperative evaluation of hepatic functional reserve was performed using the indocyanine green (ICG) retention test at 15 minutes. Surgical procedures were standardized across centers in accordance with established protocols previously described in the literature.¹⁰

Postoperative follow-up was routinely conducted 4 to 8 weeks after hepatic resection. For patients with MVI, adjuvant therapy was recommended contingent upon satisfactory general health and absence of contraindications. Given the lack of universally accepted adjuvant standards for MVI-positive HCC, treatment decisions were made based on multidisciplinary clinical evaluation, institutional practice patterns, and physician consensus. Regimen selection followed consistent clinical principles across centers and reflected contemporary real-world management of high-risk HCC. All adjuvant therapies, when applied, were initiated within 4 to 8 weeks following surgery.

Adjuvant TACE was initiated within 4–6 weeks post-resection, involving femoral artery catheterization and infusion of chemotherapeutic agents, as per institutional protocols.

Frequently employed targeted agents included lenvatinib,¹¹ apatinib,¹² donafenib, regorafenib,¹³ bevacizumab,¹⁴ administered once daily with dosage adjusted according to body weight or manufacturer guidelines. Immunotherapeutic agents commonly utilized in the adjuvant setting included sintilimab,¹⁵ carrelizumab,¹⁶ atezolizumab,¹⁴ tislelizumab.¹⁷ Prior to initiation, patients underwent thorough pre-treatment screening comprising blood panels, thyroid function assessment, electrocardiography, and chest CT imaging to ensure eligibility. ICIs were administered intravenously every three weeks in accordance with recommended dosing protocols. Vital signs were closely monitored during infusion and for at least 1hour post-infusion to promptly detect and manage any infusion-related reactions. Although the intended duration of adjuvant ICI therapy was 24 months, actual treatment durations varied in clinical practice. Some patients discontinued treatment within the first few months due to adverse events, financial burden, or poor adherence, even in the absence of tumor recurrence. To reduce immortal time bias in survival comparisons, patients who received ICI therapy for less than 6 months or experienced disease recurrence within 6 months were excluded from the duration-based survival analysis.

Follow-Up

Postoperative surveillance was rigorously implemented. All patients were scheduled for monthly follow-up visits during the first three months after hepatic resection, followed by assessments every three months for the subsequent two years, and semiannually thereafter. Each follow-up visits included evaluation of serum tumor markers, abdominal ultrasonography, and contrast-enhanced abdominal CT or MRI. Additional investigations, such as chest CT, bone scan, or positron emission tomography–CT (PET-CT), were performed when distant metastasis was clinically suspected. Follow-up was continued until patient death or loss to follow-up. The endpoint of the follow-up was September 20, 2024. Treatment of recurrence was personalized according to tumor profile, organ function, and patient status. The primary endpoint was RFS, as it reflects the direct effect of adjuvant therapy on preventing early relapse, particularly relevant in MVI-positive patients. OS was designated as a secondary endpoint, acknowledging the variability introduced by post-recurrence treatment heterogeneity. Of the 319 patients who received postoperative adjuvant therapy, 30 (9.4%) were lost to follow-up. Among the 80 patients under routine surveillance who did not receive adjuvant treatment, 9 (11.2%) were lost to follow-up. These individuals were not excluded from the analysis; instead, the time of their last documented follow-up was incorporated as censored observations in the survival analysis. This censoring strategy, implemented via the Kaplan–Meier method, ensured the inclusion of all available patient data and upheld the statistical robustness and validity of survival estimations.

Statistical Analysis

Continuous variables were summarized as mean ± standard deviation or median with interquartile range (IQR), and compared using Student’s t test or Mann–Whitney U-test, as appropriate. Categorical variables were compared using Pearson’s chi-square or Fisher’s exact test. To adjust for baseline differences between the TACE and immunotherapy groups, 1:1 PSM was performed using a nearest-neighbor algorithm (caliper = 0.2, no replacement). Covariates included AFP grade, HBV DNA, tumor differentiation, surgical margin, tumor number and size, satellite nodules, tumor embolus, MVI, and liver cirrhosis, selected based on clinical relevance and potential influence on treatment assignment. RFS and OS were estimated by Kaplan–Meier analysis and compared using Log rank tests; Cox regression identified independent predictors. Variables with P < 0.05 in univariate analysis, as well as clinically relevant covariates, were entered into the multivariate Cox regression model. To reduce immortal time bias, a 6-month landmark analysis excluded patients with recurrence or death before this point. Analyses were conducted using R (v4.3.1), with p < 0.05 considered significant.

Results

Patient Characteristics

From January 2017 to March 2024, a total of 1526 HCC patients from the three aforementioned centers were initially enrolled. All patients underwent curative liver resection, and postoperative pathology confirmed the diagnosis of HCC. Among them, 1,048 patients without MVI, 46 patients who received neoadjuvant therapy, 23 patients diagnosed with concurrent malignancies, and 10 patients who died from non-HCC-related causes were excluded. Ultimately, 399 patients were included in the final analysis (Figure 1). Among these patients, 132 received TACE alone, 58 received TACE combined with targeted therapy, 68 received TACE combined with targeted immunotherapy, 21 received targeted therapy combined with immunotherapy, 40 received only immunotherapy, and the remaining 80 patients did not receive any form of postoperative adjuvant therapy. Among the 319 patients who received postoperative adjuvant therapy, 30 were lost to follow-up, and among the 80 patients under active monitoring, 9 were lost to follow-up.

Figure 1 Patients flow chart. Abbreviations: MVI, microvascular invasion; HCC, hepatocellular carcinoma; TACE, transcatheter arterial chemoembolization; ICI, immune checkpoint inhibitor.

A total of 129 patients received adjuvant immunotherapy following curative hepatic resection. Among them, 40 received tislelizumab (anti–PD-1), 24 received camrelizumab (anti–PD-1), 21 received sintilimab (anti–PD-1), 11 received atezolizumab (anti–PD-L1), 10 received cadonilimab (a bispecific PD-1/CTLA-4 antibody), 10 received toripalimab (anti–PD-1), 7 received envafolimab (anti–PD-L1), and 6 received pembrolizumab (anti–PD-1). To minimize immortal time bias in the treatment duration analysis, 45 patients were excluded due to tumor recurrence occurring between 2 and 6 months postoperatively or receipt of immunotherapy for fewer than 6 months. The remaining 84 patients were included in the final analysis: 46 received adjuvant immunotherapy for less than 12 months, and 38 received it for 12 months or longer (Figure 1).

Before matching, the TACE and immunotherapy groups were largely comparable in baseline demographics and disease characteristics, except for differences in age, HBV DNA, and AFP levels. After 1:1 propensity score matching, 108 patients were included in each group (Figure 1), with no significant differences observed in baseline characteristics. A summary of baseline characteristics before and after matching is presented in Table 1.

Table 1 Baseline Characteristics of HCC Patients in the TACE and Immunotherapy Groups Before and After PSM

Treatment and Efficacy

During a median follow-up of 18 months (IQR 10–29 months), recurrence or metastasis occurred in 44 patients (34.1%) in the immunotherapy cohort and 86 patients (65.2%) in the TACE cohort. The predominant recurrence sites were the liver, lungs, and bones.

The median RFS was significantly longer in the immunotherapy cohort at 35 months (95% CI, 19–NA), compared with 16 months (95% CI, 10.8–27) in the TACE cohort (HR = 0.50, 95% CI, 0.34–0.72; p = 0.00015; Figure 2a). RFS rates at 12, 24, and 36 months were 75.4%, 53.1%, and 49.6% in the immunotherapy group, versus 54.5%, 42.8%, and 25.9% in the TACE group. Mortality was lower in the immunotherapy cohort (5.4%, 7 patients) compared to the TACE cohort (17.4%, 23 patients). Median overall survival (OS) was not reached in either group, but OS was significantly improved with immunotherapy (HR = 0.34, 95% CI, 0.14–0.80; p = 0.0096; Figure 2b). OS rates at 12, 24, and 36 months were 100.0%, 93.6%, and 86.9% in the immunotherapy cohort, versus 88.6%, 82.8%, and 81.0% in the TACE cohort. After PSM, the RFS benefit associated with immunotherapy remained statistically significant (HR = 0.54, 95% CI, 0.36–0.82; p = 0.0042; Figure 2c), while the OS difference was no longer statistically significant (HR = 0.43, 95% CI, 0.18–1.40; p = 0.060; Figure 2d).

Figure 2 Kaplan-Meier survival curves comparing the adjuvant ICI cohort and the TACE cohort: (a) RFS and (b) OS before PSM; (c) RFS and (d) OS after PSM. Abbreviations: HR, hazard ratio; CI, confidence interval.

Univariate and Multivariate Analyses of RFS and OS

Univariate and multivariate Cox regression analyses identified several independent predictors of poor prognosis (Table 2). For RFS, an advanced CNLC stage was significantly associated with shorter survival. For OS, a resection margin of less than 0.5 cm was identified as independent adverse prognostic factors.

Table 2 Univariate and Multivariate Analysis for RFS and OS of HCC Patients

Adverse Events (AE) in the Immunotherapy Cohort

Among the 129 patients who received immunotherapy, 55 patients (42.6%) experienced at least one treatment-related AE. Grade 1–2 AEs occurred in 51 patients (39.5%), while 13 patients (10.1%) experienced grade 3–4 events. No grade 5 AEs were reported (Supplementary Table 1). The most frequent AEs (any grade) were rash (9.3%), elevated AST (8.5%), ALT (7.8%), thrombocytopenia (7.0%), hypoalbuminemia (6.2%), and hypothyroidism (6.2%). Most events were grade 1–2 in severity. The most common grade 3–4 AEs included rash (5.4%), hypothyroidism (4.7%), and hypertension (3.9%). Other observed toxicities such as increased bilirubin (3.9%), elevated creatinine (3.9%), leukopenia (3.1%), mouth ulcers (3.1%), and fatigue (1.6%) were generally mild and manageable. No treatment-related deaths were observed.

Efficacy of Immunotherapy Duration on Survival Outcomes and AEs

To minimize immortal time bias, we excluded patients with recurrence-free survival less than 6 months and those who received ICI therapy for fewer than 6 months. Among the remaining cohort, patients who received adjuvant ICI therapy for 12 months or longer demonstrated significantly better RFS compared to those treated for less than 12 months (HR: 0.46, 95% CI: 0.21–0.99, p = 0.041; Figure 3a). A similar trend toward improved overall survival was observed, although the difference did not reach statistical significance (HR: 0.19, 95% CI: 0.02–1.59, p = 0.086; Figure 3b).

Figure 3 Survival outcomes of patients receiving adjuvant ICI therapy for ≥12 months versus <12 months: (a) RFS and (b) OS. Abbreviations: HR, hazard ratio; CI, confidence interval.

Among the 129 patients who received ICI-based adjuvant therapy, 68 received TACE combined with targeted immunotherapy, 21 received targeted immunotherapy, and 40 received immunotherapy alone. To account for the potential influence of different treatment regimens on survival outcomes, we conducted a subgroup analysis focusing on the largest group—patients who received postoperative TACE combined with targeted immunotherapy. After excluding those with a DFS less than 6 months and those who received immunotherapy for less than 6 months, we reevaluated RFS and OS. The analysis showed that patients who received ICIs for 12 months or longer had significantly improved RFS compared to those treated for less than 12 months (HR: 0.29, 95% CI: 0.11–0.79, p = 0.011; Figure 4a). While a longer OS was also observed in patients treated for 12 months or more, this difference was not statistically significant (HR: 0.19, 95% CI: 0.02–1.61, p = 0.089; Figure 4b).

Figure 4 Survival outcomes in the subgroup receiving TACE combined with targeted immunotherapy, stratified by ICI treatment duration (≥12 months vs <12 months): (a) RFS and (b) OS. Abbreviations: HR, hazard ratio; CI, confidence interval.

Among patients who received adjuvant immunotherapy, no significant differences were observed in the total number of adverse events between those treated for ≥12 months and those treated for <12 months (84.8% vs 89.5%, p >0.999; Supplementary Table 2). Similarly, the occurrence of grade 3–4 adverse events did not differ significantly between the two groups (26.1% vs 15.9%, p = 0.149; Supplementary Table 2). When comparing specific adverse events, no individual AE type showed a statistically significant difference between the two groups. However, numerically higher rates of hypertension and thrombocytopenia were noted in the ≥12-month treatment group, suggesting a trend toward increased incidence of some events with prolonged immunotherapy. Overall, extended treatment duration was not associated with a significantly increased risk of severe toxicity.

Discussion

Adjuvant therapies such as TACE, targeted agents, and immunotherapy have been associated with improved RFS and OS in HCC patients after curative resection.^18–20 However, the optimal duration of adjuvant immunotherapy for HCC patients with high-risk recurrence factors remains undefined, and real-world evidence on this topic is lacking. Although certain guidelines recommend that adjuvant immunotherapy should not exceed one year, they do not specify a minimum or preferred treatment duration.^21,22 In our study, compared to TACE alone, immunotherapy—either as monotherapy or in combination with TACE or targeted agents—was associated with a significant reduction in recurrence and improvement in OS among HCC patients with MVI who underwent R0 resection. After PSM, the benefit in RFS remained statistically significant, while the difference in overall survival was attenuated and no longer reached statistical significance. Importantly, ICI-based adjuvant therapies did not lead to a significant increase in AEs, with most AEs being grade 1–2, indicating good safety and tolerability. Among patients receiving adjuvant ICIs, a treatment duration of 12 months or longer was associated with significantly improved RFS compared to shorter durations. While a numerically favorable trend in OS was noted in the longer-duration group, the difference was not statistically significant. Therefore, no definitive conclusion regarding OS benefit can be drawn based on the current data, and this observation should be interpreted with caution. Furthermore, the total number of AEs and the incidence of grade 3–4 AEs were not significantly increased with longer treatment durations. Nevertheless, given the limited sample size of our study, larger and more comprehensive trials are needed to validate the safety and efficacy of postoperative adjuvant ICI therapy in this setting.

The clinical efficacy of ICIs was initially established in the advanced or unresectable HCC setting, as demonstrated by trials such as CheckMate 459²³ and IMbrave150.⁷ These studies showed that ICIs enhance antitumor immunity and may eradicate disseminated tumor cells. Extending this principle to earlier disease stages, adjuvant therapy aims to eliminate residual micrometastases and reduce distant recurrence, a major cause of treatment failure in MVI-positive patients. Indeed, several recent trials have tested this hypothesis. IMbrave050 reported an early RFS benefit with atezolizumab plus bevacizumab, though its updated analysis raised concerns about durability.²⁴ In contrast, a Phase II randomized controlled trial investigating adjuvant sintilimab showed a significant improvement in RFS among HCC patients with MVI,¹⁹ aligning with our findings and underscoring that high-risk populations may derive the greatest benefit. Retrospective studies have also suggested that adjuvant ICIs may improve prognosis among patients at high risk of recurrence.^25,26

Although several studies have investigated the safety and efficacy of adjuvant immunotherapy for HCC, the optimal duration of treatment has not been thoroughly explored. Given that treatment duration may critically influence patient outcomes, there is an urgent need for dedicated clinical trials addressing this issue. However, research specifically focused on treatment duration remains scarce. A prospective, multicenter cohort study evaluated the impact of adjuvant ICI treatment duration on RFS and OS in HCC patients at high risk of recurrence.²⁷ The results suggested that patients receiving adjuvant ICI therapy for more than six months tended to achieve better RFS and OS compared to those treated for six months or less, although the differences did not reach statistical significance. Despite the absence of a positive finding, the study indicated that six months of adjuvant ICI therapy might be insufficient and that extended treatment duration could potentially yield greater clinical benefits. Importantly, the design of ongoing Phase III randomized trials also reflects this rationale. Major studies such as CheckMate-9DX, KEYNOTE-937, JUPITER-04, SHR-1210-III-325, EMERALD-2, and DaDaLi have all adopted a 12-month adjuvant ICI regimen as the standard duration,²⁷ underscoring the clinical plausibility of our chosen cutoff. Nevertheless, the optimal duration of adjuvant immunotherapy remains an unresolved issue, not only in HCC but also in other malignancies such as non-small cell lung cancer^28,29 and melanoma, where prolonged ICI therapy has shown improved outcomes in certain settings. Our findings suggest that extending ICI therapy beyond 12 months may confer additional benefits for high-risk HCC patients; however, this hypothesis requires validation in prospective randomized studies. Future research should focus on defining the optimal duration of adjuvant immunotherapy, identifying predictive biomarkers for treatment benefit, and developing combination strategies tailored to individual recurrence risk profiles.

As a retrospective study, our analysis is inevitably subject to inherent biases. We acknowledge that comparisons based on treatment duration are vulnerable to immortal time bias, as longer-lived patients may be more likely to receive prolonged therapy. To mitigate this issue, we excluded patients who experienced recurrence or death within 6 months after surgery and those who received ICI therapy for less than 6 months. By restricting the analysis to patients who survived at least 6 months and initiated ICI treatment early, the impact of immortal time bias was reduced, although residual confounding remains possible. We also recognize that analyses involving secondary endpoints and subgroup comparisons may increase the risk of type I error. Another limitation is that the majority of ICIs used in our cohort were PD-1 inhibitors, with only a minority of patients treated with a bispecific PD-1/CTLA-4 antibody. Consequently, the efficacy and safety of other classes of immunotherapeutic agents, such as PD-L1 inhibitors, CTLA-4 inhibitors, and dual-targeting antibodies, were not assessed and warrant further investigation. Moreover, the heterogeneity of treatment regimens within our cohort may have influenced the outcomes. In addition, most patients in our study had HBV-related HCC, reflecting the epidemiological profile of HBV-endemic regions. Therefore, the generalizability of our findings to populations with HCV-related, alcohol-related, or non-viral HCC remains uncertain. Taken together, these limitations indicate that our conclusions should be interpreted with caution. Nonetheless, our findings provide important insights into the potential inadequacy of immunotherapy durations shorter than one year in high-risk HCC patients and highlight the need for prospective, standardized studies across diverse patient populations to confirm these observations.

Conclusions

This retrospective cohort study suggests that adjuvant ICI therapy following curative resection may improve RFS in HCC patients at high risk of recurrence compared to TACE. Notably, our findings indicate that a treatment duration of 12 months or longer is associated with improved RFS in patients with MVI. However, no statistically significant improvement in OS was observed with longer treatment duration. These results highlight the need to reconsider adjuvant immunotherapy strategies in this population, and underscore the importance of prospective, randomized, and large-scale clinical trials to determine the optimal duration of adjuvant ICI therapy for HCC.

Abbreviations

HCC, Hepatocellular carcinoma; MVI, Microvascular invasion; ICI, Immune checkpoint inhibitor; TACE, Transcatheter arterial chemoembolization; RFS, Recurrence-free survival; OS, Overall survival; PSM, Propensity score matching; AFP, Alpha-fetoprotein; HAIC, Hepatic arterial infusion chemotherapy; ECOG PS, Eastern Cooperative Oncology Group performance status; CT, Computed tomography; MRI, Magnetic resonance imaging; ICG, Indocyanine green; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; CNLC, China Liver Cancer staging system; BCLC, Barcelona Clinic Liver Cancer staging; PET-CT, Positron emission tomography-computed tomography; HR, Hazard ratio; CI, Confidence interval; AE, Adverse event; PD-1, Programmed death-1; PD-L1, Programmed death-ligand 1; CTLA-4, Cytotoxic T-lymphocyte-associated protein 4.

Data Sharing Statement

All data supporting the results of the study can be found in the article. Further inquiries can be directed to the corresponding author.

Statement of Ethics

This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking Union Medical College Hospital (Approval No. I-23PJ964). Informed consent was obtained from all individual participants included in the study.

Acknowledgments

Xiaokun Chen, Jiali Xing, and Baoluhe Zhang are co-first authors for this study. We thank all the patients and the medical staff.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work was supported by the National Natural Science Foundation of China (81972698); the CAMS Innovation Fund for Medical Sciences (CIFMS 2021-I2M-01-014); Changzhou Xi Tai Hu development foundation for frontier cell- therapeutic technology (2024-P-019); the 2024 PhD Short-term Academic Visiting Program of Peking Union Medical College; the Start-up Fund from the Department of Liver Surgery, Peking Union Medical College Hospital; the Central high-level hospital clinical research special key cultivation project (2022-PUMCH-C-047); and 2021 Liver Cancer Diagnosis and Treatment Exchange Fund of Hubei Chen Xiaoping Science and Technology Development Foundation (CXPJJH1200009-01).

Disclosure

The authors have no conflicts of interest to declare in this work.

References

1. Han B, Zheng R, Zeng H, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4(1):47–53. doi:10.1016/j.jncc.2024.01.006

2. Rumgay H, Arnold M, Ferlay J, et al. Global burden of primary liver cancer in 2020 and predictions to 2040. J Hepatol. 2022;77(6):1598–1606. doi:10.1016/j.jhep.2022.08.021

3. Zhou L, Wang S-B, Chen S-G, et al. Risk factors of recurrence and poor survival in curatively resected hepatocellular carcinoma with microvascular invasion. Adv Clin Exp Med. 2020;29(7):887–892. doi:10.17219/acem/76750

4. Lee S, Kang TW, Song KD, et al. Effect of microvascular invasion risk on early recurrence of hepatocellular carcinoma after surgery and radiofrequency ablation. Ann Surg. 2021;273(3):564–571. doi:10.1097/SLA.0000000000003268

5. Straś WA, Wasiak D, Łągiewska B, et al. Recurrence of hepatocellular carcinoma after liver transplantation: risk factors and predictive models. Ann Transplant. 2022;27:e934924. doi:10.12659/AOT.934924

6. Hirokawa F, Hayashi M, Asakuma M, et al. Risk factors and patterns of early recurrence after curative hepatectomy for hepatocellular carcinoma. Surg Oncol. 2016;25(1):24–29. doi:10.1016/j.suronc.2015.12.002

7. Qin S, Chen M, Cheng A-L, et al. Atezolizumab plus bevacizumab versus active surveillance in patients with resected or ablated high-risk hepatocellular carcinoma (IMbrave050): a randomised, open-label, multicentre, Phase 3 trial. Lancet. 2023;402(10415):1835–1847. doi:10.1016/S0140-6736(23)01796-8

8. Pan H, Zhou L, Cheng Z, et al. Perioperative Tislelizumab plus intensity modulated radiotherapy in resectable hepatocellular carcinoma with macrovascular invasion: a phase II trial. Nat Commun. 2024;15(1):9350. doi:10.1038/s41467-024-53704-5

9. Peng N, Mao L-F, Su J-Y, et al. The efficacy and safety of tislelizumab with or without tyrosine kinase inhibitor as adjuvant therapy in hepatocellular carcinoma with high-risk of recurrence after curative resection. Front Immunol. 2025;16:1593153. doi:10.3389/fimmu.2025.1593153

10. Shi M, Guo R-P, Lin X-J, et al. Partial hepatectomy with wide versus narrow resection margin for solitary hepatocellular carcinoma: a prospective randomized trial. Ann Surg. 2007;245(1):36–43. doi:10.1097/01.sla.0000231758.07868.71

11. Kudo M, Finn RS, Qin S, et al. Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet. 2018;391(10126):1163–1173. doi:10.1016/S0140-6736(18)30207-1

12. Qin S, Li Q, Gu S, et al. Apatinib as second-line or later therapy in patients with advanced hepatocellular carcinoma (AHELP): a multicentre, double-blind, randomised, placebo-controlled, phase 3 trial. Lancet Gastroenterol Hepatol. 2021;6(7):559–568. doi:10.1016/S2468-1253(21)00109-6

13. Bruix J, Qin S, Merle P, et al. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet. 2017;389(10064):56–66. doi:10.1016/S0140-6736(16)32453-9

14. Zhu AX, Abbas AR, de Galarreta MR, et al. Molecular correlates of clinical response and resistance to atezolizumab in combination with bevacizumab in advanced hepatocellular carcinoma. Nat Med. 2022;28(8):1599–1611. doi:10.1038/s41591-022-01868-2

15. Ren Z, Xu J, Bai Y, et al. Sintilimab plus a bevacizumab biosimilar (IBI305) versus sorafenib in unresectable hepatocellular carcinoma (ORIENT-32): a randomised, open-label, Phase 2–3 study. Lancet Oncol. 2021;22(7):977–990. doi:10.1016/S1470-2045(21)00252-7

16. Qin S, Chan SL, Gu S, et al. Camrelizumab plus rivoceranib versus sorafenib as first-line therapy for unresectable hepatocellular carcinoma (CARES-310): a randomised, open-label, international phase 3 study. Lancet. 2023;402(10408):1133–1146. doi:10.1016/S0140-6736(23)00961-3

17. Qin S, Kudo M, Meyer T, et al. Tislelizumab vs sorafenib as first-line treatment for unresectable hepatocellular carcinoma: a phase 3 randomized clinical trial. JAMA Oncol. 2023;9(12):1651–1659. doi:10.1001/jamaoncol.2023.4003

18. Wen Y, Lu L, Mei J, et al. Hepatic arterial infusion chemotherapy vs transcatheter arterial chemoembolization as adjuvant therapy following surgery for MVI-positive hepatocellular carcinoma: a multicenter propensity score matching analysis. J Hepatocell Carcinoma. 2024;11:665–678. doi:10.2147/JHC.S453250

19. Wang K, Xiang Y-J, Yu H-M, et al. Adjuvant sintilimab in resected high-risk hepatocellular carcinoma: a randomized, controlled, phase 2 trial. Nat Med. 2024;30(3):708–715. doi:10.1038/s41591-023-02786-7

20. Chen X, Wu X, Peng W, et al. Combined TACE with targeted and immunotherapy versus TACE alone improves DFS in HCC with MVI: a multicenter propensity score matching study. J Hepatocell Carcinoma. 2025;12:561–577. doi:10.2147/JHC.S504016

21. Benson AB, D’Angelica MI, Abrams T, et al. NCCN clinical practice guidelines in Oncology. (NCCN Guidelines^®). Hepatobiliary cancers. 2024 (Version 2.2024).

22. Singal AG, Llovet JM, Yarchoan M, et al. AASLD practice guidance on prevention, diagnosis, and treatment of hepatocellular carcinoma. Hepatology. 2023;78(6):1922–1965. doi:10.1097/HEP.0000000000000466

23. Yau T, Park J-W, Finn RS, et al. Nivolumab versus sorafenib in advanced hepatocellular carcinoma (CheckMate 459): a randomised, multicentre, open-label, phase 3 trial. Lancet Oncol. 2022;23(1):77–90. doi:10.1016/S1470-2045(21)00604-5

24. Yopp A, Kudo M, Chen M, et al. LBA39 Updated efficacy and safety data from IMbrave050: phase III study of adjuvant atezolizumab (atezo) + bevacizumab (bev) vs active surveillance in patients (pts) with resected or ablated high-risk hepatocellular carcinoma (HCC). Ann Oncol. 2024;35.

25. Chen W, Hu S, Liu Z, et al. Adjuvant anti-PD-1 antibody for hepatocellular carcinoma with high recurrence risks after hepatectomy. Hepatol Int. 2023;17(2):406–416. doi:10.1007/s12072-022-10478-6

26. Yang J, Jiang S, Chen Y, et al. Adjuvant ICIs plus targeted therapies reduce HCC recurrence after hepatectomy in patients with high risk of recurrence. Curr Oncol. 2023;30(2):1708–1719. doi:10.3390/curroncol30020132

27. Su J-Y, Liu SP, Xu XL, et al. Treatment duration of adjuvant immune checkpoint inhibitors in hepatocellular carcinoma patients at high risk of recurrence after resection: a prospective, multicentric cohort study. Liver Cancer. 2024;1–18.

28. Waterhouse DM, Garon EB, Chandler J, et al. Continuous versus 1-year fixed-duration nivolumab in previously treated advanced non-small-cell lung cancer: checkMate 153. J Clin Oncol. 2020;38(33):3863–3873. doi:10.1200/JCO.20.00131

29. Zalcman G, Balmaña J, Bober SL, et al. 972O Nivolumab (Nivo) plus ipilimumab (Ipi) 6-months treatment versus continuation in patients with advanced non-small cell lung cancer (aNSCLC): results of the randomized IFCT-1701 phase III trial. Ann Oncol;2022:33. doi:10.1016/j.annonc.2022.10.004

Introduction

Severe pneumonia is a major infectious disease in elderly patients, often characterized by rapid progression, multiple organ involvement, and poor prognosis.^1,2 One of its most severe complications is acute respiratory distress syndrome (ARDS), which arises from diffuse alveolar-capillary damage and manifests as refractory hypoxemia, respiratory distress, and decreased lung compliance.^3–5 Due to age-related immune decline, impaired pulmonary reserve, and high comorbidity burden, elderly patients are particularly vulnerable to developing ARDS after pneumonia, with mortality reaching 30–50%.^6,7 Despite advances in supportive care and precision medicine approaches, early diagnosis and prognosis assessment of ARDS still rely mainly on clinical criteria, imaging, and arterial blood gases,⁸ which lack sensitivity and specificity. This underscores the urgent need for reliable molecular biomarkers to improve early identification, risk stratification, and clinical decision-making.

MicroRNAs (miRNAs) are endogenous, non-coding small RNAs that regulate gene expression at the post-transcriptional level by binding to the 3′-UTR of target mRNAs. They are involved in processes such as inflammation, oxidative stress, apoptosis, and immune regulation.^9,10 miR-27a has been implicated in multiple inflammatory and malignant diseases, where it modulates immune signaling and cellular stress responses.^11,12 In pulmonary studies, miR-27a has been linked to anti-inflammatory and antioxidant effects, suggesting its potential role as a biomarker of lung injury and prognosis.¹³

Forkhead box O3 (FOXO3), a transcription factor downstream of the PI3K/Akt pathway, plays a central role in regulating apoptosis, oxidative stress responses, and inflammatory mediator release.^14,15 Previous reports indicate its involvement in lung tissue injury and ARDS pathogenesis.^16,17 Importantly, FOXO3 is a validated target of miR-27a: downregulation of miR-27a leads to FOXO3 activation, thereby promoting inflammatory cascades and exacerbating tissue damage.¹⁸

However, evidence regarding the specific expression patterns of miR-27a and FOXO3 in elderly patients with severe pneumonia complicated with ARDS remains scarce. In particular, their correlation with oxygenation index and short-term prognosis has not been fully elucidated. Therefore, this study retrospectively analyzed elderly patients with severe pneumonia, aiming to (1) detect serum levels of miR-27a and FOXO3, (2) assess their relationship with ARDS severity and 28-day mortality, and (3) evaluate their predictive value as biomarkers for prognosis. These findings may provide a molecular basis for improved survival assessment and risk stratification in elderly ARDS patients.

Materials and Methods

Study Subjects

This was a retrospective observational study including a total of 189 elderly inpatients (aged ≥60 years) with severe pneumonia admitted to the intensive care unit of our hospital from February 2023 to October 2024. According to whether ARDS was present, patients were divided into two groups: Group A (n=114, with ARDS) and Group B (n=75, without ARDS). Additionally, 70 healthy volunteers undergoing physical examination at the physical examination center during the same period were selected as the healthy control group. Among the 114 patients in Group A, there were 70 males and 44 females, with an average age of (73.16±6.72) years; in Group B, there were 46 males and 29 females, with an average age of (74.11±6.98) years; in the control group, there were 41 males and 29 females, with an average age of (73.24±7.19) years. There were no statistically significant differences in gender and age among the three groups (P>0.05), indicating comparability.

The sample size was estimated based on a preliminary analysis of 40 patients in our institution, where the difference in serum miR-27a expression between ARDS and non-ARDS patients was approximately 0.8 standard deviations. Using a two-sided α=0.05 and power (1–β)=0.80, the minimum required sample size per group was calculated as 64 cases. Considering potential dropouts and missing data, the final enrollment exceeded this requirement, ensuring adequate statistical power for group comparisons.

This study was approved by the Liberation Army General Hospital Medical Ethics Committee (Approval No.: 2024ZZLS12) and conducted in strict accordance with the ethical principles of the Declaration of Helsinki. All participants provided informed consent and signed relevant informed consent forms.

Inclusion and Exclusion Criteria

Inclusion criteria: (1) Age ≥60 years, regardless of gender; (2) First diagnosis of severe pneumonia or severe pneumonia with ARDS; (3) Diagnosis of severe pneumonia meets the criteria of the “Chinese Expert Consensus on Clinical Practice of Severe Pneumonia in Emergency Medicine”;¹⁹ (4) Diagnosis of ARDS conforms to the “Berlin Definition of Acute Respiratory Distress Syndrome”;²⁰ (5) Complete and reliable clinical data available for analysis.

Exclusion criteria: (1) Combined with malignant tumors, tuberculosis, HIV infection, or other immunodeficiency diseases; (2) Severe hepatic or renal failure, or heart failure; (3) Complicated with pulmonary tuberculosis, COPD, congenital lung dysplasia, or other pulmonary diseases; (4) History of immunosuppressant or hormone therapy within the past 6 months; (5) Complicated with severe infection at other sites or multiple organ failure; (6) Incomplete test data or improper specimen storage; (7) Pregnant or lactating women; (8) Considered unsuitable for inclusion by the researchers, such as those with psychiatric disorders or cognitive impairment who cannot cooperate with the study procedures.

Clinical Data

The following clinical data were collected for all enrolled patients: (1) Demographic data: gender; age; body mass index (BMI); smoking history; drinking history; living alone status; (2) Pneumonia-related information: type of pneumonia (community-acquired pneumonia/hospital-acquired pneumonia); whether complicated with underlying pulmonary diseases (eg, COPD, bronchiectasis, interstitial lung disease, etc).; (3) Underlying diseases: hypertension; diabetes; coronary heart disease; chronic liver disease; (4) Laboratory test indicators (within 24 h of admission): white blood cell count (WBC); C-reactive protein (CRP); procalcitonin (PCT); serum creatinine (Scr); blood urea nitrogen (BUN); (5) Mechanical ventilation: duration of mechanical ventilation.

All clinical data were extracted from the standardized hospital electronic medical record system, and only baseline information within 24 hours of admission was included to minimize bias. Data collection was independently performed by two trained researchers in a blinded manner, and any inconsistent entries were rechecked by a third investigator to ensure accuracy and reliability.

Grouping Method

Patients in Group A (with ARDS) were further subdivided according to the oxygenation index [arterial oxygen partial pressure (PaO₂) / fraction of inspired oxygen (FiO₂)] after admission: Mild ARDS subgroup: PaO₂/FiO₂ between 200–300 mmHg (n=28); Moderate ARDS subgroup: PaO₂/FiO₂ between 100–200 mmHg (n=36); Severe ARDS subgroup: PaO₂/FiO₂ <100 mmHg (n=50). Meanwhile, Group A patients were also stratified by their 28-day outcome into: Survival subgroup: patients who survived within 28 days (n=79); Death subgroup: patients who died within 28 days (n=35).

Detection of Serum miR-27a and FOXO3 mRNA

All enrolled patients underwent collection of a fasting early-morning venous blood sample (5 mL) within 24 hours of admission. Blood was placed in anticoagulant-free centrifuge tubes, left to clot at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 10 minutes to isolate serum. The serum was immediately aliquoted into RNase-free centrifuge tubes (free of RNA contamination) and stored at –80°C in an ultra-low temperature freezer to ensure the quality and stability for subsequent RNA extraction. To detect the expression levels of miR-27a and FOXO3 mRNA in serum, qRT-PCR (quantitative real-time polymerase chain reaction) was used. The detection process included three main steps: total RNA extraction, reverse transcription, and real-time PCR amplification, detailed as follows: (1) Total RNA extraction: According to the instructions of the RNA extraction kit produced by Nanjing Vazyme Biotech Co., Ltd. (Product No.: RC112-01), total RNA was extracted from frozen serum. Strict RNase-free procedures were followed throughout. The concentration and purity of RNA samples were assessed using a NanoDrop™ UV spectrophotometer, and samples with A260/A280 between 1.8 and 2.1 were considered qualified. (2) Reverse transcription: RNA samples that passed quality assessment were reverse-transcribed into cDNA using the reverse transcription kit provided by Nanjing Saihongrui Biotech Co., Ltd. (Product No.: DV807A). Specific stem-loop primers were used for miRNA reverse transcription with U6 small nuclear RNA as the internal control, while mRNA reverse transcription was performed using a mixed system of Oligo(dT) and random primers, with GAPDH as the reference gene.(3) Real-time quantitative PCR amplification: The PCR reaction system was constructed based on the kit provided by Yeasen Biotechnology (Shanghai) Co., Ltd. (Product No.: 11203ES03). The total volume per reaction was 20 μL, including: 2× Master Mix buffer 10 μL, forward primer 0.5 μL, reverse primer 0.5 μL, cDNA template 2.0 μL, and RNase-free DEPC-treated water to 20 μL. Amplification was performed on an ABI 7500 real-time PCR system. Reaction conditions were: Pre-denaturation: 95°C for 30 seconds, 1 cycle; Amplification: 95°C for 5 seconds and 60°C for 40 seconds, 40 cycles; Melting curve: fluorescence signals were acquired from 65°C to 95°C, with 0.5°C increments to verify amplification specificity. (4) Data analysis and quality control: Fluorescence signal data were collected and calculated using the instrument’s built-in software. All raw amplification plots and melting curves were manually reviewed to confirm specificity. Samples with ambiguous amplification results were repeated to ensure reproducibility. Relative expression levels of miR-27a and FOXO3 mRNA were calculated using the 2^–ΔΔCt method, with U6 and GAPDH as internal controls for normalization, respectively. All primers were designed based on published sequences, verified in the NCBI database, and synthesized with quality certification by Wuhan GeneCreate Bioengineering Co., Ltd., and the primer sequences are shown in Table 1.

Table 1 Primer Sequence Information

Statistical Analysis

Statistical analysis was performed using SPSS 26.0 software, and figures were generated using GraphPad Prism 9.0. Continuous variables conforming to normal distribution were expressed as (); comparisons between two groups were made using the t-test, and comparisons among multiple groups were performed using analysis of variance (ANOVA). Categorical data were expressed as counts (n) and percentages (%), and comparisons were made using the χ²-test. Correlation analysis was performed using Pearson or Spearman correlation methods. Multivariate analysis used a binary logistic regression model to identify independent risk factors for 28-day mortality. ROC curves were plotted to compare the predictive performance of miR-27a, FOXO3, and their combination for patient mortality. Differences in AUC were compared using the Z-test. The significance level was set at α=0.05, with P<0.05 considered statistically significant.

Results

Comparison of Serum miR-27a and FOXO3 mRNA Levels Among the Three Groups

Serum miR-27a levels were significantly higher in Group B than in Group A, and further elevated in the control group compared with Group B. Conversely, FOXO3 mRNA levels were significantly lower in Group B than in Group A, and further reduced in the control group (F=77.352, 62.956, P<0.001), as shown in Figure 1. This indicates that compared with the control group, serum miR-27a levels were downregulated while FOXO3 mRNA levels were upregulated in the disease groups.

Figure 1 Comparison of serum miR-27a and FOXO3 mRNA levels among the three groups. Notes: *P<0.05 vs Group A; #P<0.05 vs Group B. All comparisons are made with reference to the control group.

Comparison of Serum miR-27a and FOXO3 mRNA Levels in Patients with Different Severities of Severe Pneumonia with ARDS

In patients with severe pneumonia and ARDS, serum miR-27a levels decreased progressively from the mild to moderate and severe subgroups, whereas FOXO3 mRNA levels increased in the same order (F=83.597, 111.834, P<0.001), as shown in Figure 2. These subgroups all belong to the disease group and reflect different severity classifications.

Figure 2 Comparison of serum miR-27a and FOXO3 mRNA levels in patients with different severities of severe pneumonia with ARDS. Notes: aP<0.05 vs mild subgroup; bP<0.05 vs moderate subgroup. All comparisons are relative within the disease group.

Correlation Between Serum miR-27a and FOXO3 mRNA Levels

Pearson correlation analysis showed that serum miR-27a and FOXO3 mRNA levels in elderly patients with severe pneumonia and ARDS were negatively correlated (r=–0.624, P<0.001), as shown in Figure 3.

Figure 3 Scatter plot of correlation between serum miR-27a and FOXO3 mRNA levels.

Spearman correlation analysis showed that the oxygenation index (mild=3, moderate=2, severe=1) was positively correlated with serum miR-27a levels (r=0.635, P<0.001), and negatively correlated with FOXO3 mRNA levels (r=–0.672, P<0.001), as shown in Figure 4. The oxygenation index was expressed in mmHg to ensure clarity.

Figure 4 Scatter plot of correlation between serum miR-27a, FOXO3 mRNA levels and oxygenation index (mmHg).

Comparison of Clinical Data in Patients with Different Prognoses of Severe Pneumonia with ARDS

The 28-day mortality rate in elderly patients with severe pneumonia and ARDS was 30.70% (35/114). The death subgroup had higher age, CRP, mechanical ventilation time, and FOXO3 mRNA levels, and lower oxygenation index and miR-27a levels compared to the survival subgroup (P<0.05). There were no statistically significant differences in other data (P>0.05), as shown in Table 2.

Table 2 Comparison of Clinical Data in Patients with Different Prognoses of Severe Pneumonia with ARDS

Multivariate Logistic Regression Analysis of Prognostic Factors in Elderly Patients with Severe Pneumonia and ARDS

Taking prognosis (survival=0, death=1) as the dependent variable, possible influencing factors from Table 1 were assigned as independent variables (see Table 3). A multivariate logistic regression model was established. Results showed that increased age, prolonged mechanical ventilation time, and elevated FOXO3 mRNA were independent risk factors, while increased oxygenation index and miR-27a levels were independent protective factors, as shown in Table 4.

Table 3 Variable Assignment Table

Table 4 Multivariate Logistic Regression Analysis of Prognostic Factors in Elderly Patients with Severe Pneumonia and ARDS

Predictive Value of miR-27a, FOXO3 mRNA, and Their Combination for Mortality in Elderly Patients with Severe Pneumonia and ARDS

The AUCs for serum miR-27a, FOXO3 mRNA, and their combination in predicting mortality in elderly patients with severe pneumonia and ARDS were 0.775, 0.781, and 0.867, respectively. The combined AUC was superior to each single index (Z_combined–miR-27a=2.557, P<0.05; Z_combined–FOXO3 mRNA=2.974, P<0.05), as shown in Table 5 and Figure 5.

Table 5 Predictive Value of miR-27a, FOXO3 mRNA, and Their Combination for Mortality in Elderly Patients with Severe Pneumonia and ARDS

Figure 5 ROC curves for predictive value of miR-27a, FOXO3 mRNA, and their combination in elderly patients with severe pneumonia and ARDS.

Discussion

This study focused on elderly patients with severe pneumonia complicated by ARDS, systematically analyzing the relationship between serum miR-27a and FOXO3 mRNA expression levels and disease severity and prognosis. Compared with healthy controls and patients with severe pneumonia without ARDS, ARDS patients showed significantly decreased serum miR-27a levels and markedly increased FOXO3 mRNA levels. Moreover, across different oxygenation index strata, miR-27a levels progressively declined with worsening ARDS severity, whereas FOXO3 mRNA levels increased stepwise relative to less severe subgroups. These findings indicate that these alterations are evident not only when compared with non-ARDS populations but also dynamically vary with disease progression, suggesting that both markers are closely associated with the pathophysiology of ARDS.

The observed inverse correlation between miR-27a and FOXO3 mRNA highlights a potential regulatory axis, in which miR-27a may play a protective role while FOXO3 promotes tissue damage. Previous studies^21,22 have confirmed that miR-27a regulates inflammatory, apoptotic, and oxidative stress pathways and plays crucial roles in various pulmonary diseases, including asthma, pulmonary fibrosis, and infections. Mechanistically, miR-27a may inhibit the release of pro-inflammatory mediators, limit oxidative damage, and reduce apoptosis by modulating NF-κB, TGF-β, and PI3K/Akt signaling pathways.^23–25 Downregulation of miR-27a weakens these protective effects, thereby amplifying inflammatory cascades. Conversely, persistent activation of FOXO3 exacerbates oxidative stress, induces mitochondrial dysfunction, and promotes immune imbalance.^26,27

Our results are consistent with the study by Lv et al,²⁸ who reported that downregulation of miR-27a aggravated alveolar injury in a murine ARDS model, whereas miR-27a mimic intervention effectively alleviated inflammation and tissue damage. FOXO3, on the other hand, is recognized as a transcription factor that promotes oxidative stress responses and cellular senescence. Wu et al²⁹ demonstrated that inhibition of FOXO3 could reduce alveolar epithelial apoptosis and preserve lung function. Together, these studies support the hypothesis that an imbalance between miR-27a and FOXO3 signaling contributes to the pathogenesis and progression of ARDS. Recent evidence also indicates that FOXO3 can influence macrophage polarization and T-cell differentiation, leading to immune dysregulation and impaired tissue repair.^30,31 These processes collectively create a vicious cycle of lung injury and inadequate repair, which aligns with the clinical features of refractory hypoxemia in elderly ARDS patients.

Clinically, our study found that the 28-day mortality rate among elderly ARDS patients reached 30.70%, higher than that reported for general ARDS populations,³² reflecting age-related vulnerability and the influence of comorbidities. Notably, multivariate logistic regression analysis indicated that elevated FOXO3 levels were an independent risk factor, whereas miR-27a and oxygenation index served as independent protective factors. ROC curve analysis showed that combined detection of these two markers achieved an AUC of 0.867, outperforming individual markers and providing a practical approach for risk stratification. Zhao et al³³ similarly demonstrated that multi-marker combined detection significantly improves prognostic prediction in ARDS patients. Therefore, this study expands the ARDS biomarker panel in elderly patients and validates the clinical utility of miR-27a and FOXO3.

In terms of novelty, this study has three main contributions. First, it is the first to combine the detection of miR-27a and FOXO3 mRNA in elderly ARDS patients, integrating molecular mechanisms with clinical prognostic assessment. Second, the inclusion of a relatively large cohort with stratification across ARDS severity enhances the clinical representativeness and reliability of the findings. Third, by focusing on elderly patients—a subgroup with poor outcomes that is often underrepresented in biomarker studies—this work fills a critical gap in ARDS research. These findings not only enrich current understanding but also provide a foundation for future therapeutic strategies targeting the miR-27a/FOXO3 signaling pathway.

However, several limitations should be acknowledged. First, as a single-center retrospective study, selection bias cannot be excluded, and multicenter prospective cohort studies are needed for validation. Second, only serum levels were assessed, lacking mechanistic validation in bronchoalveolar lavage fluid, lung tissue, or animal models. Moreover, miR-27a may regulate multiple targets beyond FOXO3, and FOXO3 may be influenced by other miRNAs or upstream signals; therefore, causal relationships remain to be confirmed. Functional experiments and multi-omics approaches could provide deeper insights into these interactions.

In conclusion, this study demonstrates that downregulated serum miR-27a and upregulated FOXO3 mRNA are closely associated with ARDS severity and short-term prognosis in elderly patients with severe pneumonia. Combined detection of these markers enhances predictive accuracy, providing a novel molecular basis for early identification, risk assessment, and potential therapeutic intervention. Future studies should integrate mechanistic validation and dynamic longitudinal monitoring to establish causal roles and explore their feasibility as intervention targets, ultimately advancing personalized management of ARDS.

Conclusion

The results of this study indicate that, compared with healthy controls and elderly patients with severe pneumonia without ARDS, serum miR-27a levels are significantly decreased, whereas FOXO3 mRNA levels are significantly increased in elderly patients with severe pneumonia complicated by ARDS. Within ARDS subgroups stratified by oxygenation index, miR-27a levels progressively decreased from mild to moderate to severe ARDS, while FOXO3 mRNA levels increased stepwise, highlighting their close association with disease severity. Elevated miR-27a may act as a protective factor, whereas elevated FOXO3 mRNA serves as an independent risk factor for poor short-term outcomes. Combined detection of these two biomarkers provides higher predictive efficacy for 28-day mortality than either marker alone, underscoring their potential utility for early risk stratification and clinical intervention. These findings suggest that the imbalance between miR-27a and FOXO3 is not only involved in the pathogenesis of ARDS but also has practical implications as prognostic biomarkers. Future studies should further investigate the molecular mechanisms underlying miR-27a regulation of FOXO3 and related downstream signaling pathways and validate their clinical utility in elderly ARDS populations through multicenter, large-sample prospective studies.

Funding

Project of National Clinical Research Center for Geriatric Diseases, Research on Diagnosis, Treatment and Comprehensive Prevention and Control Measures of Multidrug Resistant Pathogenic Bacteria Infections in Elderly Patients (Project Number: NCRCG-PLAGH-DX-2024002) 2. Military Health Care Project: Research on the Clinical Application Value of the Combined Evaluation Method of Pepsin, amylase and Lipid Cells in Tracheal Aspirates for the Diagnosis of Airway Aspiration (Project Number: 21BJZ24).

Disclosure

The authors report no conflicts of interest in this work.

References

1. Xu Y, Wang XY. [Clinical characters and influencing factors of perioperative pneumonia in elderly patients with hip fractures aged ≥80 years at a tertiary hospital in Beijing City]. Zhonghua Yu Fang Yi Xue Za Zhi. 2025;59(6):916–924. doi:10.3760/cma.j.cn112150-20250330-00257 Wolof

2. Kang M, Li J, Wan Q, et al. [Factors influencing the choice of endotracheal intubation and mechanical ventilation in patients with acute respiratory distress syndrome caused by viral pneumonia]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2022;34(6):586–591. doi:10.3760/cma.j.cn121430-20220607-00549 Dutch

3. Zhang J, Li Y, Li H, et al. [Acute respiratory distress syndrome caused by severe respiratory infectious diseases: clinical significance and solution of maintaining artificial airway closure]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2025;37(3):221–224. doi:10.3760/cma.j.cn121430-20240506-00404 Dutch

4. Tang R, Tang W, Wang D. [Predictive value of machine learning for in-hospital mortality for trauma-induced acute respiratory distress syndrome patients: an analysis using the data from MIMIC III]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2022;34(3):260–264. doi:10.3760/cma.j.cn121430-20211117-01741 Dutch

5. Liu YN, Ma XC. [The precision medicine for the treatment of acute respiratory distress syndrome is based on identification of phenotypes]. Zhonghua Yi Xue Za Zhi. 2024;104(15):1253–1257. doi:10.3760/cma.j.cn112137-20230928-00603 Danish

6. Wu J, Xiao H, Li X, et al. [Evaluation value of sequential organ failure assessment score for predicting the prognosis of patients with acute respiratory distress syndrome due to severe pneumonia]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021;33(9):1057–1062. doi:10.3760/cma.j.cn121430-20210115-00076 Dutch

7. Zhang HL, Shang Y. [The value of point-of-care ultrasound in the diagnosis and management of acute respiratory distress syndrome]. Zhonghua Yi Xue Za Zhi. 2024;104(15):1225–1229. doi:10.3760/cma.j.cn112137-20230906-00407 Danish

8. Chalmers S, Khawaja A, Wieruszewski PM, et al. Diagnosis and treatment of acute pulmonary inflammation in critically ill patients: the role of inflammatory biomarkers. World J Crit Care Med. 2019;8(5):59–71. doi:10.5492/wjccm.v8.i5.74

9. Tian F, Ying H, Liao S, et al. lncRNA SNHG14 promotes the proliferation, migration, and invasion of thyroid tumour cells by regulating miR-93-5p. Zygote. 2022;30(2):183–193. doi:10.1017/S0967199421000319

10. Xing J, Chen LP, Yu WJ. [Non-small cell lung cancer-derived exosomal circular RNA circEZH2 activates fibroblasts by regulating the miR-495-3p / TPD52 axis and NF-κB pathway]. Zhonghua Zhong Liu Za Zhi. 2024;46(12):1176–1186. doi:10.3760/cma.j.cn112152-20230717-00015 Polish

11. Zhang G, Liu R, Dang X, et al. [Experimental study on improvement of osteonecrosis of femoral head with exosomes derived from miR-27a-overexpressing vascular endothelial cells]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2021;35(3):356–365. doi:10.7507/1002-1892.202011026 Danish

12. Xie E, Lin M, Sun Z, et al. Serum miR-27a is a biomarker for the prognosis of non-small cell lung cancer patients receiving chemotherapy. Transl Cancer Res. 2021;10(7):3458–3469. doi:10.21037/tcr-20-3276

13. Fan X, Wang J, Qin T, et al. Exosome miR-27a-3p secreted from adipocytes targets ICOS to promote antitumor immunity in lung adenocarcinoma. Thorac Cancer. 2020;11(6):1453–1464. doi:10.1111/1759-7714.13411

14. Ruan P, Zheng Y, Dong Z, et al. [Research progress in the regulation of autophagy and mitochondrial homeostasis by AMPK signaling channels]. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2024;36(4):425–429. doi:10.3760/cma.j.cn121430-20230302-00132 Dutch

15. Yang F, Zhang H, Zhuo M, et al. [Effects of Peiminine on biological behavior and chemotherapy resistance of ovarian cancer by regulating the FOXO3-FOXM1 signaling axis]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2024;40(11):976–982. Wolof

16. Tao Y, Li -L-L, Liu S-H, et al. [FOXO3a signaling pathway in prostate cancer: progress in studies]. Zhonghua Nan Ke Xue. 2020;26(8):745–750. Basque

17. Wang Y, Liang H-X, Zhang C-M, et al. FOXO3/TRIM22 axis abated the antitumor effect of gemcitabine in non-small cell lung cancer via autophagy induction. Transl Cancer Res. 2020;9(2):937–948. doi:10.21037/tcr.2019.12.33

18. Zhang H, Xia J, Hu Q, et al. Long non‑coding RNA XIST promotes cerebral ischemia/reperfusion injury by modulating miR‑27a‑3p/FOXO3 signaling. Mol Med Rep. 2021;24(2):566.

19. Shang WF, Chen DC. [Prone positioning ventilation therapy in acute respiratory distress syndrome: knowns and unknowns in clinical efficacy]. Zhonghua Yi Xue Za Zhi. 2024;104(15):1236–1241. doi:10.3760/cma.j.cn112137-20231012-00720 Danish

20. Yuan XY, Liu L, Qiu HB. [New 2023 global definition of acute respiratory distress syndrome: progress and limitation]. Zhonghua Yi Xue Za Zhi. 2024;104(15):1216–1220. doi:10.3760/cma.j.cn112137-20231016-00770 Danish

21. Dong M, Wang X, Li T, et al. miR-27a-3p alleviates lung transplantation-induced bronchiolitis obliterans syndrome (BOS) via suppressing Smad-mediated myofibroblast differentiation and TLR4-induced dendritic cells maturation. Transpl Immunol. 2023;78:101806. doi:10.1016/j.trim.2023.101806

22. Sun Y, Jiang R, Hu X, et al. CircGSAP alleviates pulmonary microvascular endothelial cells dysfunction in pulmonary hypertension via regulating miR-27a-3p/BMPR2 axis. Respir Res. 2022;23(1):322. doi:10.1186/s12931-022-02248-7

23. Abd-Elhakim YM, Mohamed AA-R, Khamis T, et al. Alleviative effects of green-fabricated zinc oxide nanoparticles on acrylamide-induced oxidative and inflammatory reactions in the rat stomach via modulating gastric neuroactive substances and the MiR-27a-5p/ROS/NF-κB axis. Tissue Cell. 2024;91:102574. doi:10.1016/j.tice.2024.102574

24. Ong J, Faiz A, Timens W, et al. Marked TGF-β-regulated miRNA expression changes in both COPD and control lung fibroblasts. Sci Rep. 2019;9(1):18214. doi:10.1038/s41598-019-54728-4

25. Zhao XR, Zhang Z, Gao M, et al. MicroRNA-27a-3p aggravates renal ischemia/reperfusion injury by promoting oxidative stress via targeting growth factor receptor-bound protein 2. Pharmacol Res. 2020;155:104718. doi:10.1016/j.phrs.2020.104718

26. Shen J, Wang H, Wang J-S, et al. [The relationship between MicroRNA expression profiling in imatinib-resistant cell line K562/G and potential mechanism through FOXO3/Bcl-6 signaling pathway]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022;30(1):107–112. doi:10.19746/j.cnki.issn.1009-2137.2022.01.017 Hausa

27. Artham S, Gao F, Verma A, et al. Endothelial stromelysin1 regulation by the forkhead box-O transcription factors is crucial in the exudative phase of acute lung injury. Pharmacol Res. 2019;141:249–263. doi:10.1016/j.phrs.2019.01.006

28. Lv X, Zhang X-Y, Zhang Q, et al. lncRNA NEAT1 aggravates sepsis-induced lung injury by regulating the miR-27a/PTEN axis. Lab Invest. 2021;101(10):1371–1381. doi:10.1038/s41374-021-00620-7

29. Wu Z, Wang Y, Lu S, et al. SIRT3 alleviates sepsis-induced acute lung injury by inhibiting pyroptosis via regulating the deacetylation of FoxO3a. Pulm Pharmacol Ther. 2023;82:102244. doi:10.1016/j.pupt.2023.102244

30. Duan XH, Li H, Lyu Y, et al. Regulation of baicalin on growth of extranodal NK/T cell lymphoma cells through FOXO3/CCL22 signaling pathway. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2023;31(3):730–738. doi:10.19746/j.cnki.issn.1009-2137.2023.03.017

31. Wang H, Wang Z. Oroxylin-A attenuates taurocholate-induced lung injury via NF-κB pathway suppression. Clin Mol Epidemiol. 2025;2:5. doi:10.53964/cme.2025005

32. Wang HF, Hu WH, Song QW, et al. [Clinical study on the relationship between the exosomes in bronchoalveolar lavage fluid and plasma and the severity of lung injury and outcome in early acute respiratory distress syndrome patients]. Zhonghua Yi Xue Za Zhi. 2022;102(13):935–941. doi:10.3760/cma.j.cn112137-20211105-02448 Danish

33. Zhao C, Li Y, Wang Q, et al. Establishment of risk prediction nomograph model for sepsis related acute respiratory distress syndrome. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2023;35(7):714–718. doi:10.3760/cma.j.cn121430-20230215-00088