Colorectal cancer (CRC) is the third most commonly diagnosed malignancy worldwide and the fourth leading cause of cancer-related death (1, 2). Its global incidence is expected to rise to 2.5 million new cases by 2035 (3, 4). CRC development results from a complex interplay of genetic and environmental factors, including a sedentary lifestyle, age, obesity, and dietary habits (4–8). Due to its insidious onset, approximately 20% of cases are diagnosed as metastatic colorectal cancer (mCRC) (9), and about 40% of patients with localized disease experience recurrence after prior treatment (10).The high mortality rate in advanced colorectal cancer (aCRC) is largely due to tumor progression and metastasis. Despite advances in treatment, the prognosis for aCRC patients remains poor, with a median overall survival (mOS) of 30 months (11, 12) and a 5-year survival rate of less than 14% (13, 14). Surgery, chemotherapy, and radiotherapy remain the primary therapeutic interventions for CRC, often used in combination depending on disease localization and progression (15–18). Surgery is the cornerstone of curative treatment for localized CRC (stage I–III) (19), and in mCRC with limited metastasis, surgery and chemotherapy may achieve curative outcomes in some cases (14, 19, 20). However, complete cancer cell removal is often unattainable, leading to disease recurrence in many patients (21). Although significant progress has been made in systemic and local therapies for aCRC, it remains rarely curable. Resistance to conventional treatments, systemic toxicity, and low selectivity highlight the need for more effective alternatives (13, 21, 22).

Immunotherapy has emerged as a promising approach, aiming to reverse tumor-induced immune suppression and activate antitumor responses (23). Immune checkpoints, such as programmed cell death 1 (PD-1), its ligand (PD-L1), and cytotoxic T lymphocyte antigen 4 (CTLA-4), have been extensively studied because of their overexpression in many solid tumors and hematological malignancies (24, 25). Blocking the interaction between PD-1/PD-L1 or CTLA-4 and their ligands activates T cells, promoting tumor infiltration and cancer cell death (26, 27). In response, the U.S. Food and Drug Administration (FDA) approved immune checkpoint inhibitors (ICIs) for patients with microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) CRC. These cancers exhibit high mutation rates and a strong neoantigen burden, triggering robust T cell-mediated tumor immune responses (28–30). A recent study showed that 100% of 12 patients with advanced dMMR rectal cancer achieved a complete clinical and pathological response after six months of treatment with dostarlimab (31). However, ICIs have limited efficacy in proficient mismatch repair (pMMR), microsatellite-stable (MSS), or microsatellite instability-low (MSI-L) CRC, which account for 85% of cases (32, 33). Given the biological complexity of the tumor microenvironment (TME) in most solid tumors, a multi-checkpoint inhibition strategy seems logical (34). Since CTLA-4 and PD-1 target non-redundant pathways, combining inhibitors for both may provide additive or synergistic effects. Some clinical trials have reported improved outcomes with combined PD-1/PD-L1 and CTLA-4 blockade compared to monotherapy (35). However, other studies suggest that combining these inhibitors has shown limited success in treating CRC (36). Moreover, combined ICI therapy is associated with a higher incidence of adverse events compared to single-agent treatment (37). To address these uncertainties, we conducted a systematic meta-analysis of the existing literature to evaluate the efficacy and safety of combining PD-1/PD-L1 and CTLA-4 inhibitors in CRC treatment.

Chemotherapy, cytotoxic drugs, and molecular targeted therapies have commonly been used in the treatment of advanced colorectal cancer (aCRC), but their efficacy has plateaued (14, 53). Recently, numerous clinical trials related to immunotherapy have confirmed that it is an encouraging new treatment strategy for colorectal cancer (54–57). The two most widely studied immune checkpoints are PD-1 and CTLA-4. PD-1 is a cell surface receptor commonly found on T cells, B cells, and NK cells. When the PD-1 receptor binds to its ligand, it inhibits cell proliferation, cytokine secretion, and the cytotoxic capabilities of immune cells, thus weakening the immune response (58). CTLA-4 receptors, expressed on activated or regulatory T cells, bind to B7 ligands on antigen-presenting cells with a higher affinity and lower surface density. This binding prevents CD28 receptors from interacting with B7 ligands, leading to a net downregulation of T cell activation (59). A previous meta-analysis indicated that PD-1 inhibitors and combination immunotherapy demonstrate promising clinical responses and overall survival (OS) rates, alongside manageable adverse events (AEs) in the treatment of dMMR colorectal cancer (60). Although single-agent immune checkpoint inhibitors (ICIs) perform well, one study noted that ICI therapy resistance has been observed in up to 50% of patients with microsatellite instability-high (MSI-H) CRC. Furthermore, single-agent ICIs show minimal efficacy in patients with proficient mismatch repair (pMMR) or microsatellite stable (MSS) metastatic CRC (61, 62). To address tumor escape mechanisms, substantial efforts have been made to enhance the clinical efficacy of ICIs in colorectal cancer through combination therapies. Specifically, the combination of CTLA-4 and PD-1/PD-L1 blockade increases T cell activation and treatment efficacy, resulting in better antitumor outcomes (56, 63). Preclinical studies have shown that combined immunotherapy is superior to monotherapy. The dual blockade of PD-1/PD-L1 and CTLA-4 can increase the number of T cells regulated by multiple mechanisms and enhance their effector function (64). Currently, extensive research is being conducted to extend the indications for dual immune checkpoint blockade with PD-1 and CTLA-4 to various solid tumors (64, 65). However, the efficacy and safety of PD-1/PD-L1 and CTLA-4 immune checkpoint inhibitors in the treatment of aCRC remain largely unconfirmed. Twelve clinical trials involving 566 patients with metastatic colorectal cancer (mCRC) have reported results for these agents. Therefore, we conducted a meta-analysis to provide valid and reliable conclusions. We compared the effectiveness and AEs of several therapy regimens involving PD-1/PD-L1 and CTLA-4 immune checkpoint inhibitors for potential clinical application. The pooled objective response rate (ORR), disease control rate (DCR), and overall AE rate were 50%, 33%, 94%, and 44%, respectively. The pooled median overall survival (mOS) and median progression-free survival (mPFS) were 8.81 months and 2.57 months.

In order to further analyze the efficacy and safety of PD-1/PD-L1 and CTLA-4 immune checkpoint inhibitors, we performed subgroup analyses based on country (USA, Other), sample size (≥40, <40), other therapies (None, Yes), types of ICIs (Nivolumab+Ipilimumab, Durvalumab+tremelimumab, QL1706 (PSB205)), and molecular phenotypes (dMMR/MSI-H, pMMR/MSS, NR). In terms of country, the USA exhibited lower ORR and DCR, as well as fewer AEs and grade ≥3 AEs compared to other countries. Regarding sample size, when the sample size is ≥40, this may indicate higher ORR, DCR, and an increased incidence of AEs and grade ≥3 AEs. Concerning ICIs types, Nivolumab+Ipilimumab is considered to have higher efficacy and safety than Durvalumab+tremelimumab and QL1706 (PSB205). From the molecular phenotype perspective, the efficacy of dual ICIs in pMMR/MSS aCRC patients is lower than that in dMMR/MSI-H aCRC patients, while the incidence of adverse events may be higher. This discrepancy arises because MSI-H tumors typically have a high tumor mutation burden (TMB) and a high tumor neoantigen burden (TNB), which can activate the immune system more effectively. In contrast, MSS tumors have a lower mutation burden and generate relatively fewer neoantigens, reducing the likelihood of T cells recognizing and attacking tumor cells (4, 61). Notably, the combination of different treatment modalities is often believed to enhance therapeutic effects. However, our analysis yielded opposite results. When PD-1/PD-L1 and CTLA-4 immune checkpoint inhibitors are combined with other treatments (such as chemotherapy, radiotherapy, etc.), the ORR and DCR are significantly lower, and the risk of adverse effects is higher. This may be due to the limited number of studies that we did not categorize by different treatment regimens. However, a careful review of the literature reveals that the efficacy of PD-1/PD-L1 and CTLA-4 immune checkpoint inhibitors combined with chemotherapy is better than the pooled results. Past studies have demonstrated that cytotoxic chemotherapy could reduce immunosuppressive T-regulatory cells, induce cell surface death receptor density to promote immune killing, and enhance the release of neoantigens in the microenvironment (66–68). In the Thibaudin study, a median PFS of 8.2 months and an objective response rate of 63% were reported for the treatment plan involving durvalumab and tremelimumab combined with mFOLFOX6, which compares favorably with FOLFOX monotherapy (44). This study supports the notion that chemo-immunotherapy could promote immune responses against shared tumor antigens and neoantigens in MSS metastatic CRC, with this immune response associated with therapeutic efficacy. In another study, temozolomide in combination with ipilimumab and nivolumab in patients with microsatellite stabilization achieved encouraging results, with median PFS and mOS of 7.0 and 18.4 months, respectively, and an overall response rate of 45% (47). This provides evidence of the role of temozolomide as an immune-sensitizing agent for MSS and immune-cold aCRCs. However, the combination of radiotherapy with CTLA-4 and PD-1 blockers for the treatment of aCRC was less than satisfactory. Although preclinical data suggest that radiation can induce tumor-specific immune responses (69), increase T-cell infiltration (70), and promote immune-mediated tumor cell death (71), clinical trials to date have not met their intended goals. In a study involving 40 aCRC patients receiving nivolumab and ipilimumab in combination with radiation therapy, Parikh observed a DCR of 25% and an ORR of 10% (48), significantly lower than those reported in previous studies that did not involve radiation therapy. Two other similar clinical trials combining PD-L1 and CTLA-4 inhibitors with radiation therapy in aCRC patients also failed to meet their predefined endpoints, although they noted that this treatment can enhance the immunogenicity of the local and systemic immune microenvironment (36, 49). In the future, more clinical trials are warranted to investigate the timing factors associated with radiation in immunotherapy and to identify appropriate radiation dosages (72, 73).

The results of this meta-analysis demonstrate that the combination of PD-1 inhibitors and CTLA-4 inhibitors has shown promising and profound clinical responses in clinical studies. The synergistic effect of combining PD-1/PD-L1 and CTLA-4 inhibitors surpasses the sum of their individual contributions as monotherapies (74, 75). Thibaudin reported that the median progression-free survival (mPFS) for metastatic colorectal cancer treated with durvalumab and tremelimumab in combination with FOLFOX was 8.2 months, significantly higher than the mPFS of 5-6 months for FOLFOX alone and comparable to the mPFS of 8 months for the bevacizumab-combined chemotherapy regimen (44, 76). A previous meta-analysis of PD-1/PD-L1 inhibitors in the treatment of aCRC suggested that the objective response rate (ORR) for dMMR/MSI-H aCRC was 37%, while that for pMMR/MSS aCRC was 11% (77). In our molecular phenotype subgroup analysis, the ORR for PD-1/PD-L1 inhibitors combined with CTLA-4 inhibitors in dMMR/MSI-H aCRC was 63%, whereas in pMMR/MSS aCRC it was 18%, both significantly higher than the 37% and 11% observed for PD-1/PD-L1 inhibitors alone, respectively. Treatment-related adverse events (TRAEs) are associated with overactivation of the immune system, leading to damage to one or more organs. As expected, we observed that the incidence of TRAEs in patients receiving the combination of anti-PD-1/PD-L1 and anti-CTLA-4 therapy was higher compared to those receiving anti-PD-1/PD-L1 monotherapy (94% vs. 85%) (36, 77). Among the included studies, the highest incidence of grade ≥3 TRAEs was reported by Parikh (48). Notably, despite a high incidence of grade ≥3 TRAEs in this study, only a few patients discontinued treatment due to clinically relevant toxicity. Similar results were observed in other studies. Moreover, we found that grade ≥3 TRAEs often occurred in the early stages of treatment, and most TRAEs could be controlled and resolved with a short interruption of treatment and a brief course of oral steroid therapy (61, 78). Currently, the incidence of TRAEs is common in anti-PD-1/PD-L1 + anti-CTLA-4 combination therapy, although the pathogenesis is poorly understood (79). We believe that the occurrence of grade ≥3 TRAEs may be associated with drug dosage, treatment cycles, and the patients’ physical conditions. In anti-PD-1/PD-L1 + anti-CTLA-4 combination therapy, it is crucial to enhance monitoring of immunotherapy during treatment, pay attention to severe TRAEs, and manage them according to relevant guidelines.

There are several limitations in the current meta-analysis. First, most studies had small sample sizes. Additionally, there is currently limited data on the combination therapy of PD-1 inhibitors and CTLA-4 inhibitors, and few studies are randomized or blinded. Second, we relied on published trial data but did not have access to raw patient data, which may introduce biases in our analysis. Therefore, more large-scale clinical trials are needed to further validate the efficacy and safety of the combination therapy of PD-1 inhibitors and CTLA-4 inhibitors. Third, it is crucial to identify the predominant factors influencing immunosuppressive therapy. Due to the limited experimental data, this study did not analyze the associations between gender, age, tumor mutation burden (TMB), tumor neoantigen burden (TNB), and the site of metastasis with treatment efficacy and safety.

In summary, our meta-analysis demonstrates the efficacy and safety of combination therapy with PD-1 inhibitors and CTLA-4 inhibitors in patients with aCRC, supporting its potential for future clinical application. However, due to the limited clinical data available, further large-scale, multicenter randomized controlled trials (RCTs) are necessary to confirm these findings.