Overall survival according to time-of-day of combined immuno-chemotherapy for advanced gastric cancer: a propensity score-matched analysis

Circadian rhythms constitute endogenous timing systems in organisms, characterized by 24-hour oscillations in physiological processes (1). These evolutionarily conserved mechanisms maintain organism-environment homeostasis by coordinating essential functions including sleep-wake cycles, energy metabolism, and immune responses (2–4). Chronotherapy, also known as circadian medicine, leverages endogenous circadian rhythms through administering treatments during biologically optimal time windows, thereby maximize therapeutic outcomes (5).

Previous studies have gradually elucidated the mechanisms through which circadian rhythms influence chemotherapy toxicity and the efficacy of therapeutic cytokines (6, 7). In recent years, the regulatory relationship between circadian rhythms and immune function has become increasingly well-defined. Research demonstrates that various immune cells, including monocytes, macrophages, neutrophils, and natural killer cells, possess intrinsic circadian clocks and through these regulatory mechanisms play vital roles in both innate and adaptive immune responses (8–11).Preclinical studies have demonstrated significant circadian fluctuations in the number of peripheral blood lymphocytes in healthy adults, with secretion patterns of various pro-inflammatory cytokines closely linked to cancer progression and treatment response (12, 13). CD8⁺ T cells, which are key targets of immune checkpoint inhibitors (ICIs), have recently been shown to possess intrinsic circadian rhythms, with their immune response intensity demonstrating significant diurnal variation and peaking within specific time windows (14). Collectively, these findings underscore that synchronizing immunotherapy with the body’s intrinsic circadian rhythms is critical for maximizing therapeutic efficacy.

A single-center cohort study published in 2021(MEMOIR) was the first to establish a significant correlation between the time-of-day administration (ToDA) of ICIs and patient outcomes (15). In melanoma patients, those who received ≥20% of their ICI infusions after 1630h had significantly worse survival compared to those who received <20% of infusions after this time. This finding has since been corroborated in a variety of solid tumors, including lung cancer, head and neck cancer, and renal cell carcinoma (RCC) (16–26). Real-world data from eighteen retrospective studies indicated that early ToDA of ICIs could improve progression-free survival (PFS) and overall survival (OS) by up to fourfold compared to late ToDA (27). However, research on advanced gastric cancer (GC) has been limited to nivolumab in later-line therapies (28, 29).

Given that combination chemotherapy and ICIs have been incorporated into first-line treatment guidelines for advanced GC, clarifying the impact of ICI infusion timing on survival outcomes with this specific regimen is of substantial clinical significance. The findings are expected to provide evidence-based insights for optimizing clinical dosing schedules.

This retrospective study demonstrates a significant association between the timing of ICI administration and survival outcomes in patients with advanced GC receiving first-line immunotherapy combined with chemotherapy. The primary analysis revealed that in the unmatched cohort, patients who received ≥20% of their ICI infusions after 1630h had significantly shorter overall survival. This finding remained consistent after controlling for potential confounding factors through propensity score matching.

Sensitivity analyses further confirmed the robustness of our findings. When using 1630h as the reference time point with different thresholds (30%, 40%, and 50%), all thresholds demonstrated significant associations with both OS and PFS in the pre-matched cohort. These associations remained statistically significant in the matched cohort. Furthermore, analyses using alternative time cut-offs (1530h, 1600h, and 1700h) revealed significant associations with OS in the matched cohort. It should be noted that for the 1700h cut-off, the substantial sample size disparity in the pre-matched cohort and the subsequent reduction in effective sample size after 1:2 propensity score matching warrant cautious interpretation. The generalizability of these particular findings requires validation in prospective studies.

The biological plausibility of our findings is supported by a growing body of evidence on circadian immunology. Previous studies have demonstrated that the timing of vaccination could influence the magnitude of antibody responses; for instance, morning administration of influenza or COVID-19 vaccines has been associated with a more robust antiviral antibody response (32, 33). The circadian clock regulates innate immunity by controlling the rhythmic production and transport of cytokines and chemokines, as well as the maturation and tissue infiltration of immune cells. Within 24 hours following antibody infusion, tumor-infiltrating CD8⁺ T cells exhibit significant time-dependent phenotypic alterations, indicating their participation in the primary circadian response. It is noteworthy that the antitumor effect was significantly higher in the morning compared to the afternoon (34). Studies in immunodeficient mice have shown that dendritic cells (DCs) rhythmically migrate to tumor-draining lymph nodes, thereby orchestrating the circadian rhythm of tumor antigen-specific CD8⁺ T cell responses (35). This mechanism indicates that synchronizing immunotherapy with the peak functional phase of DCs could significantly enhance its antitumor efficacy. Conversely, disruption of the epithelial cell circadian clock alters cytokine secretion, exacerbates inflammatory responses, promotes neutrophil infiltration, and can ultimately lead to the expansion of myeloid-derived suppressor cells (MDSCs) (36). Importantly, the therapeutic efficacy of PD-L1 inhibitors appears to be optimal when administered in alignment with the circadian fluctuations of MDSCs (36). Consequently, strategically timing immune system activation based on circadian principles may not only maximize the intensity of immune responses but also optimize antitumor treatment outcomes.

Two previous retrospective studies investigating nivolumab as a later-line treatment for GC both showed superior survival outcomes for patients receiving morning infusions (28, 29). Following the publication of the CheckMate649 trial, the era of first-line chemotherapy combined with immunotherapy became the standard of care for advanced GC (37). This regimen has improved mOS to approximately 15 months, compared to about 10 months with chemotherapy alone. Subsequent trials, including ORIENT-16, RATIONALE-305, and GEMSTONE-303, have further established the efficacy of Sintilimab, Tislelizumab, and Sugemalimab in first-line setting (30, 38, 39). Given the prevalent use of sintilimab in our clinical practice and study cohort, we performed a subgroup analysis focusing on patients receiving sintilimab plus chemotherapy. In the unmatched sintilimab subgroup, a significant association between infusion timing and OS was observed, which persisted after multivariable adjustment. However, in the propensity score-matched sintilimab subgroup, univariate analysis indicated only a borderline significant association (HR:1.53, 95% CI: 0.94-2.49, P = 0.088), which lost statistical significance after multivariable adjustment (HR:1.50, 95% CI: 0.92-2.45, P = 0.101).This discrepancy may be attributed to several factors: First, while propensity score matching balanced known prognostic variables, there may be unmeasured confounding variables, unmeasured confounders including tumor microenvironment heterogeneity, genomic profiles, or lifestyle differences may persist. The influence of such residual confounding can be magnified in subgroup analyses, potentially obscuring a true treatment effect. Second, the reduced sample size post-matching limited statistical power, increasing the risk of a Type II error. Notably, the hazard ratios from both univariate and multivariate analyses consistently ranged between 1.50 and 1.53, suggesting a stable, albeit not statistically significant, effect size. Although the P-values slightly exceeded the conventional threshold, this consistent trend may still hold clinical relevance, particularly in light of established circadian regulation of immune function.

Previous studies have reported circadian rhythm and gender differences in immune therapy responses, with female patients showing better prognoses than males (26, 40, 41). For instance, studies of hepatitis A and influenza vaccines have shown that female individuals often mount stronger peak antibody responses (42). This heightened response may be partly attributed to sex-specific resilience to immune dysfunction induced by sleep disturbances and psychological stress (43). Additionally, women tend to display more pronounced circadian rhythmicity, characterized by higher plasma levels of melatonin and cortisol (44, 45). In contrast, a separate systematic review and meta-analysis demonstrated that while ICIs improve overall survival in patients with advanced cancers like melanoma and non-small cell lung cancer, the survival benefit was more pronounced in male patients (46). In our study, we found no significant association between patient sex and survival outcomes. Further research is warranted to clarify the potential role of sex as a biological variable and its interaction with circadian timing in shaping immunotherapy efficacy.

Our study has several limitations. First, although we observed a significant benefit in OS, no between-group differences were observed in PFS or ORR. This discrepancy may be explained by several factors. Patients in the control group might have received subsequent immunotherapy or other effective treatments after disease progression. Such crossover could mitigate between-group differences in PFS while concurrently improving long-term survival in the control group. Additionally, pseudoprogression or delayed clinical responses may not be accurately captured by conventional RECIST criteria, thereby influencing PFS and ORR assessments. The relatively limited sample size, coupled with closely matched median PFS between groups, may also have reduced the statistical power to detect a significant difference in PFS. Second, thresholds for defining early and late ICI infusion vary across previous studies, with cutoff times ranging from 1137h to 1630h and infusion proportion thresholds from 20% to 75% (27, 33). The 1630h cut-off adopted in our primary analysis is the most commonly applied, potentially due to its alignment with findings from earlier vaccination studies. To better identify optimal daily infusion timing, Catozzi et al. proposed a circadian mortality risk model; however, this model was derived from a heterogeneous cohort encompassing multiple cancer types and treatment lines (26). While our sensitivity analyses investigated various infusion cut-offs, they were insufficient to pinpoint a single optimal administration time. This highlights the need for future research specifically designed to identify the most therapeutic time window. Previous studies often determined assessment timing based on initial imaging or overall patient condition and typically excluded patients receiving fewer than four ICI doses (47, 48). In our study, considering standard efficacy assessment practices in China and the generally good performance status (ECOG score ≤1) of enrolled patients, we included all patients who received at least two infusions. The persistence of therapeutic antibody effects over weeks suggests that circadian regulation during the initial ICI cycles may be clinically more impactful than in later cycles. Thus, our study design is particularly relevant for evaluating the effect of infusion timing during the early phase of immunotherapy. Including patients with fewer infusion cycles also improves the real-world applicability of our findings, as early treatment discontinuation due to toxicity or progression is common in clinical practice. We nevertheless acknowledge that this approach introduces limitations, including relatively limited treatment exposure and potential heterogeneity in immune responses, which may affect the robustness of the results.

Adjusting the administration timing of ICIs to the biologically favorable morning window may not only enhance antitumor efficacy but also improve healthcare resource allocation and patient throughput. In accordance with our institutional treatment pathway, an “ICI-first” sequence is generally adopted, wherein immunotherapy is administered prior to chemotherapy. This approach aims to fully activate the immune system before it is subjected to chemotherapy-induced suppression, thereby potentially improving the body’s tolerance to and recovery from the immunotoxic effects of subsequent chemotherapy. However, uniformly concentrating all ICI infusions in the morning hours presents two major challenges. First, it fails to account for individual differences in circadian rhythm phenotypes, which may diminish the potential benefits of chronotherapy for some patients. Second, such highly consolidated scheduling can lead to overutilization of infusion center capacity, nursing staff, and pharmacy resources during the morning, while resulting in underutilization in the afternoon, ultimately undermining sustained gains in operational efficiency. Therefore, future implementation of chronotherapy-based ICI administration requires careful consideration of both individual chronobiological profiles and institutional resource management to optimize therapeutic outcomes while maintaining healthcare system sustainability.

In conclusion, our study demonstrates a significant link between earlier ICI infusion timing and improved survival in advanced gastric cancer patients treated with first-line chemoimmunotherapy. Confirming this association in prospective trials and elucidating the specific circadian mechanisms involved are critical next steps.