Comprehensive Evaluation of Anti-PD-1, Anti-PD-L1, Anti-CTLA-4 and Their Combined Immunotherapy in Clinical Trials: A Systematic Review and Meta-analysis

Immunotherapy with immune checkpoint inhibitor (ICI) drugs is gradually becoming a hot topic in cancer treatment. To comprehensively evaluate the safety and efficacy of ICI drugs, we employed the Bayesian model and conducted a network meta-analysis in terms of progression-free survival (PFS), overall survival (OS) and severe adverse events (AEs). Our study found that treatment with ipilimumab was significantly worse than standard therapies in terms of PFS, whereas treatment with cemiplimab significantly improved PFS. The results also indicated that cemiplimab was the best choice for PFS. Treatment with nivolumab, pembrolizumab and nivolumab plus ipilimumab significantly improved OS compared to standard therapies. In terms of OS, cemiplimab was found to be the best choice, whereas avelumab was the worst. In terms of severe AEs, atezolizumab, avelumab, durvalumab, nivolumab, and pembrolizumab all significantly reduced the risk of grade 3 or higher AEs compared to standard therapy. The least likely to be associated with severe AEs were as follows: cemiplimab, avelumab, nivolumab, atezolizumab, and camrelizumab, with nivolumab plus ipilimumab to be the worst. Therefore, different ICI drug therapies may pose different risks in terms of PFS, OS and severe AEs. Our study may provide new insights and strategies for the clinical practice of ICI drugs.


INTRODUCTION
Immunotherapy has become one of the most important breakthroughs in the treatment of cancer in recent years, and its development has promoted changes in many cancer treatment methods. As a series of co-inhibitory and co-stimulatory receptors and ligands, immune checkpoint inhibitors (ICI) drugs can block negative regulatory factors expressed by immune or tumor cells to enhance their immune function against cancer cells, mainly programmed death-1 (PD-1), programmed death-ligand-1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Rosenberg et al., 2004). In 2011, the CTLA-4 inhibitor ipilimumab was approved by the US Food and Drug Administration for the treatment of advanced melanoma (Hodi et al., 2010). Subsequently, several ICI drugs were also approved for the treatment of cancer (Topalian et al., 2012;Gong et al., 2018). Since then, interest for immunotherapy with ICI drugs has been increasing. Many studies focused on the prognosis and treatment for different cancers (Wu et al., 2015).
Chemotherapy is the first-line treatment for advanced cancer, and patients undergoing chemotherapy often experience severe adverse events (AEs). Although ICI drugs have achieved good anticancer effects in the treatment of many solid tumors, they may still cause severe treatment-related or drug-related AEs. Progression-free survival (PFS) and overall survival (OS) are usually efficacy end-points. In terms of PFS and OS, the therapeutic effects of ICI drugs remain unclear compared with standard therapies. Due to the limitations of randomized clinical trials, the overall safety evaluation of different ICI drugs for cancer treatment is not comprehensive, especially in terms of PFS and OS.
We conducted a systematic review and network meta-analysis of the therapeutic effects of ICI drugs targeting PD-1, PD-L1, and CTLA-4, focusing on PFS, OS and treatment-related severe AEs in patients receiving ICI drug monotherapy, combination therapies and standard therapies (chemotherapy, targeted therapies and their combination therapies included). This study comprehensively evaluated the safety and efficacy of different ICI drugs and their combination therapies, aiming to provide better guidance for the clinical application of various ICI drugs.

Search Methods and Study Selection
We searched PubMed, Embase, and Cochrane Library for English-language studies between January 2000 and September 2021, using keywords such as ipilimumab, tremelimumab, pembrolizumab, nivolumab, cemiplimab, camrelizumab, toripalimab, tislelizumab, spartalizumab, atezolizumab, avelumab, durvalumab, PD-1, PD-L1, and CTLA-4. The search strategy was described in Supplementary Table S1. The study search, selection and data extraction were independently conducted by two reviewers (ZX and ZZ), and discrepancies were evaluated by an independent reviewer (JL). The three authors (ZX, JL and ZZ) reviewed and discussed the full text of studies that may be eligible, and differences of opinions were resolved by consensus.
Only high-quality head-to-head phase 2 and 3 randomized controlled trials (RCTs) comparing two or more treatments including ICI drug monotherapy, ICI drug combination therapies and standard therapies were included. Some RCTs only presented interim results, as insufficient information may affect the final analysis, we selected the most recent results as much as possible. Data provided include at least one of the following: hazard ratios (HRs) of PFS, OS and treatment-related severe AEs. We excluded reviews, conference abstracts and posters. The study was performed based on the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline (Hutton et al., 2015;Wang et al., 2021). This study was approved by International prospective register of systematic reviews (PROSPERO) (registered ID: CRD42021278158).

Data Extraction
The authors (ZX and ZZ) independently extracted data according to the PRISMA guidelines. The first author, year of publication, national clinical trial identification number, trial name, phase, number of patients, type of cancer, drug used, follow-up time, number of severe AEs, HRs, and confidence interval (CI) of PFS and OS were summarized in standardized Tables 1-3. PFS is considered to be the primary endpoint of randomized clinical trials evaluating patients with solid tumors (Korn and Crowley, 2013). OS is defined as the time from the start of treatment to death or the last follow-up. The HRs of PFS and OS represent HRs between treatment 1 and 2. In assessing AEs, we chose treatment-related or drug-related AEs as the main results. If there were no treatment or drug-related AEs in studies, we included all AEs. The classification of AEs is often used to evaluate the type and severity of AEs in clinical trials. According to AE classification, grade 3 or higher AEs are considered as severe AEs. The risk of severe AEs is the focus of the evaluation of therapeutic effectiveness, so the number of AEs surveyed and severe AEs were both extracted (Xu et al., 2018).

Adverse Events Analysis
We used gemtc and pcnetmeta packages in R v4.0.3 and called JAGS v4.3.0 to perform statistical analysis in a Bayesian framework based on Markov Chain Monte Carlo (MCMC) methods, and generated the graph depicting the network geometry .
Firstly, we made a rough comparison between the fit of the consistent model with the inconsistent model. Secondly, the inconsistency on the specific comparison was tested by node splitting analysis. p < 0.05 was considered as indicating a significant inconsistency. Outstanding consistency is the key to robust results, as evidenced by the consistency between direct and indirect results. We compared the results of network metaanalysis (indirect results) with those of pairwise analysis (direct results) to explore the sources of inconsistency. Additionally, if there existed significant heterogeneity, we used the random-effect model. Otherwise, we used the fixed-effect model (Dias et al., 2011). We used non-information prior distributions and overdispersed initial values (scaling 2.5) in 3 chains to fit the model. 56 independent randomized controlled         experiments yielded 100,000 iterations (including 20,000 optimization iterations) with 10 refinement intervals for each chain. This method was used to generate a posterior distribution of model parameters. The convergence of iterations was evaluated by using the Gelman-Rubin-Brooks statistics, all of which converge near 1. Based on the odds ratio (OR) advantage ratio and posterior probability, we ranked probabilities of each treatment as the safest, followed by the second, third and so on.

Progression-free Survival and Overall Survival Analysis
For the consistency and heterogeneity analysis of PFS and OS, we chose to use R's netmeta package in the Frequentist framework to make a preliminary judgment using the traditional frequency method, avoiding the artificial bias caused by complex prior settings, settings of dummy variables and variance-covariance matrices of regression models in Bayesian statistics, which would simplify the operator's parameter setting. The I 2 test was used to evaluate the heterogeneity between studies, with the significance level set as p < 0.05. I 2 greater than 25, 50 or 75% indicated low, medium and high heterogeneity respectively. If significant heterogeneity exists, the random-effect model was used. Otherwise, we employed the fixed-effect model (Higgins and Thompson, 2002).
Since Bayesian statistics are more accurate and the results are highly consistent with those in the frequency model, we subsequently chose the Bayesian framework by using the MCMC method in WinBUGS v1.4.3 for network metaanalysis. We used the consistency model (due to I 2 < 25) to calculate HRs and 95% CIs. We simulated 3 different chains, each with 45,000 built-in samples, resulting in 15 iterations with a refinement rate of 15 (3 different chains with 15,000 iterations and 45,000 burn-in samples and 50 thinning rates). The model fitting was further determined according to the deviation information criterion. The output was a posterior distribution of relative effect size, and we got the estimated average of HR and 95% CI (95% CI as the 2.5th and 97.5th percentiles) (van Valkenhoef et al., 2012). The ranking probability distribution was calculated, ranking the probabilities of each treatment as the safest, followed by the second, third and so on.

Progression-free Survival
In analyzing PFS, no significant heterogeneity (I 2 = 19%) or inconsistency was observed (p = 0.97) ( Supplementary Table  S2). Therefore, the Bayesian fixed-effect model was used. HRs and 95% CI from the network meta-analysis are shown in Figure 3A. Treatment with ipilimumab was significantly worse in terms of PFS than standard therapies, whereas treatment with cemiplimab significantly improved PFS. According to the probability ranking diagram, the results showed that cemiplimab was the best choice in terms of PFS, camrelizumab ranked the second safest and nivolumab plus ipilimumab ranked the third safest, whereas ipilimumab was the worst ( Figure 3B). Additionally, treatment with ipilimumab was significantly worse than most other treatments in terms of PFS. Interestingly, nivolumab plus ipilimumab significantly improved PFS compared to ipilimumab, which suggested that treatment with combinations of ICI drugs may benefit PFS compared to monotherapy. The results calculated according to the frequency model were highly consistent with the results of the Bayesian fixed-effect model. Additionally, we performed subgroup analyses based on treatment of different cancer types, particularly lung cancer and melanoma. The safety profile and probability ranking diagram for lung cancer and melanoma are shown in Supplementary Figures S1, S4 in the Supplement respectively. Cemiplimab was also the best choice in terms of PFS in treating lung cancer, and nivolumab plus ipilimumab ranked the second safest. Compared with standard therapies, HR (95% CI) for cemiplimab was 0.77 (0.61-0.96). Treatment with cemiplimab also significantly improved PFS compared to nivolumab.
Tremelimumab was considered the worst choice in terms of PFS in treating lung cancer. In terms of melanoma, our results showed that nivolumab plus ipilimumab was the best choice for PFS. HR (95% CI) for nivolumab plus ipilimumab was 0.69 (0.52-0.92) compared with standard therapies. In addition, HRs (95% CI) for nivolumab and pembrolizumab were 0.78 (0.65-0.94) and 0.80 (0.64-0.99) respectively. The probability ranking diagram of melanoma indicated that ipilimumab was the worst choice for PFS.

Overall Survival
In analyzing OS, no consistency (I 2 = 0%) or inconsistency (p = 0.60) ( Supplementary Table S2) was observed, and so the Bayesian fixed-effects model was applied. HRs and 95% CI are  shown in Figure 4A. Treatment with nivolumab, pembrolizumab and nivolumab plus ipilimumab significantly improved OS compared to standard therapies. According to the probability ranking diagram, cemiplimab was the best choice in terms of OS, and durvalumab ranked the second safest, whereas avelumab was the worst ( Figure 4B). Of note, nivolumab plus ipilimumab may improve OS compared with nivolumab and ipilimumab monotherapy, which was similar to durvalumab plus tremelimumab compared with durvalumab and tremelimumab monotherapy. The results calculated based on the frequency model were also highly similar to the results of the Bayesian fixed-effect model.
We also conducted subgroup analyses for lung cancer and melanoma. The safety profiles and probability ranking diagrams of lung cancer and melanoma are shown in Supplementary  Figures S5, S8 in the Supplement. For lung cancer treatment, cemiplimab was the best option for OS and durvalumab ranked the second safest. Safety profile of lung cancer suggested that compared with standard therapies, HRs (95% CI) for nivolumab, nivolumab plus ipilimumab and pembrolizumab were 0.91 (0.82-0.99), 0.87 (0.76-0.99), and 0.89 (0.80-0.99) respectively. Standard therapies were considered to be the worst option for lung cancer in terms of OS whereas. For melanoma treatment, nivolumab plus ipilimumab was the best option. Safety profiles showed that HR (95% CI) for nivolumab was 0.84 (0.71-0.99) compared with standard therapies. Our results also indicated that standard therapies were the worst choice for melanoma in terms of OS.

Severe Adverse Events
In the network meta-analyses of severe AEs, high heterogeneity was found (Supplememntary Table S3), and the random-effect model was employed. Safety profile in the consistency model is shown in Figure 5A. Atezolizumab, avelumab, durvalumab, nivolumab, and pembrolizumab all significantly reduced the risk of grade 3 or higher AEs compared to standard therapies.
Through node splitting analysis, significant inconsistency could not be detected for most comparisons (Supplementary Table S4). There was significant inconsistency between ipilimumab and nivolumab plus ipilimumab, and ipilimumab and standard therapies (p < 0.05). The comparison between nivolumab plus ipilimumab and standard therapies also showed a degree of inconsistency (p = 0.08). In the direct comparisons, patients receiving nivolumab plus ipilimumab were more likely to have severe AEs than those receiving ipilimumab, and patients receiving ipilimumab were more likely to have severe AEs than those receiving standard therapies. However, patients receiving standard therapies were more likely to have severe AEs than those receiving nivolumab plus ipilimumab. The comparison between the above three groups may be the main reason for the inconsistency. In the safety profile, efficacy of treatment for grade 3-5 adverse events is represented as odd ratios (ORs) with 95% confidence intervals. All comparisons are made as column versus row. Statistically significant results are in bold. Probability ranking diagram shows the probability of the safety of different therapies ranking the first to the last for severe adverse events.
Frontiers in Pharmacology | www.frontiersin.org We performed subgroup analyses of lung cancer, melanoma, and squamous cell carcinoma treatment. The respective safety profiles and probability ranking diagram are shown in Supplementary Figures S9, S14 in the Supplement. Interestingly, the results suggested that nivolumab was the joint best choice for lung cancer, melanoma and squamous cell carcinoma. Standard therapies, based on the probability ranking diagram, were considered to be the worst for lung cancer and squamous cell carcinoma treatment, and nivolumab plus ipilimumab was the worst for melanoma treatment.

DISCUSSION
In order to comprehensively evaluate the safety and efficacy of ICI drug monotherapy and combination therapies, we conducted a network meta-analysis combining HRs of PFS and OS, and the risk of severe AEs, and performed subgroup analyses particularly for lung cancer and melanoma. The application of bioinformatics is often used to analyze published data (Wu et al., 2017). To our knowledge, this study is the first comprehensive report comparing PFS HRs, OS HRs, and corresponding treatmentrelated severe AEs among ICI drug monotherapy, combination therapies and standard therapies.
In terms of PFS and OS, we first tested the heterogeneity and consistency of network meta-analysis based on the frequency method. No significant heterogeneity and consistency were found, indicating that this network meta-analysis was consistent in PFS and OS. We used the frequency model and the Bayesian model separately. The results of the frequency model and the Bayesian model agree well. In view of the greater accuracy of the Bayesian model, our final results were presented by the Bayesian model.
In terms of PFS, treatment with ipilimumab was significantly worse than standard therapies, whereas treatment with cemiplimab significantly improved PFS. The results also indicated that cemiplimab was the best choice for PFS. Treatment with nivolumab, pembrolizumab and nivolumab plus ipilimumab significantly improved OS compared to standard therapies. For OS, cemiplimab was considered to be the best choice, whereas avelumab was the worst. Since few studies compared cemiplimab and camrelizumab with other therapies, nivolumab plus ipilimumab ranked the third safest in PFS and durvalumab ranked the second safest in terms of OS. In comparing ICI drug combination therapies with monotherapy, we found that nivolumab plus ipilimumab significantly improved PFS compared to ipilimumab. Additionally, nivolumab plus ipilimumab may improve OS compared with nivolumab and ipilimumab monotherapy, similar to durvalumab plus tremelimumab compared with durvalumab, and tremelimumab monotherapy. However, further studies are needed to compare the safety and efficacy of ICI drug combination treatment with monotherapy. Although abundant included studies may lead to low significance of the overall results in terms of PFS and OS, we thought that the results would be more reliable and further performed subgroup analysis.
Severe AEs were examined as the measure of the toxicity of different ICI drug therapies and standard therapies. In terms of severe AEs, there was a large inconsistency in the comparison between the above three groups, which is similar to inconsistency reported by Chen et al (Xu et al., 2018), but the degree of inconsistency was more obvious in the comparison between ipilimumab and standard therapies in this study. We considered the main reasons as follows: In spite that the inclusion and exclusion criteria in our study are similar to those of Chen et al, eligible studies with inconsistent results were relatively abundant, which may lead to higher inconsistency. Of note, Chen et al combined durvalumab plus tremelimumab and nivolumab plus ipilimumab as the two ICI drug group, however, we grouped them in our study to assess the safety and efficacy more precisely.

Strengths and Limitations
The main strengths of our studies are as follows: we used the Bayesian model to conduct network meta-analysis and then employed the frequency model for inconsistency test and result verification in terms of PFS and OS. We found that the results of the frequency model were highly consistent with those of the Bayesian model, and we represented our final results from the Bayesian model, which greatly enhanced the reliability of conclusions. To comprehensively investigate the safety and efficacy of various ICI drugs, we analyzed three different indicators PFS, OS and severe AEs. Of note, we included enough studies to ensure the accuracy of the results. Despite that some studies represent the data of the same RCTs at different times, we chose the most recent results as much as possible.
This study also has several limitations. Firstly, enrolled studies showed high heterogeneity. In order to avoid publication bias, we tested the heterogeneity and used different models accordingly. Secondly, the number of RCTs that meet the requirements for inclusion is different among ICI drugs at present, and there is obvious inconsistency in severe AEs, which require more studies for higher-level verification. Thirdly, despite randomization of the eligible studies, there are still characteristic imbalances between the groups in trials.

CONCLUSION
In the present study, the Bayesian model was used to comprehensively assess survival data and the risk of severe AEs for ICI drugs, which showed that different ICI drug therapies may pose different risks in terms of PFS, OS and severe AEs. Our study may provide new insights and strategies for the clinical practice of ICI drugs.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.