Organ-Specific Immune-Related Adverse Events Associated With Immune Checkpoint Inhibitor Monotherapy Versus Combination Therapy in Cancer: A Meta-Analysis of Randomized Controlled Trials

Background Although combination therapy with immune checkpoint inhibitors (ICIs) provides a promising efficacy in multiple cancers, their use is facing challenges for a high incidence of adverse effects. This meta-analysis was conducted to compare the risks of organ-specific immune-related adverse events (IRAEs) associated with ICI monotherapy versus combination therapy among cancer patients. Methods Electronic databases were systematically searched to include eligible randomized controlled trials (RCTs). Any-grade and 3-5 grade IRAEs (colitis, pneumonitis, hepatitis, hypothyroidism, hyperthyroidism, and hypophysitis) were extracted for meta-analysis. Two reviewers independently assessed the methodological quality. The RevMan 5.3.5 software was used for meta-analysis. Results A total of 10 studies involving 8 RCTs with 2716 patients were included in this study. The most common any-grade adverse event was colitis (14.5%), followed by hypothyroidism (13.8%), hepatitis (10.4%), hypophysitis (10.0%), hyperthyroidism (9.3%), and pneumonitis (4.6%). Meta-analysis showed that ICI combination therapy significantly increased the risks of any-grade IRAEs in colitis [relative risk (RR), 3.56; 95% confidence interval (CI), 1.56–8.12; p < 0.05], pneumonitis (RR, 2.31; 95% CI, 1.54–3.45; p < 0.05), hepatitis (RR, 2.54; 95% CI, 1.65–3.91; p < 0.05), hypothyroidism (RR, 2.17; 95% CI, 1.71–2.76; p < 0.05), hyperthyroidism (RR, 3.13; 95% CI, 2.08–4.70; p < 0.05), and hypophysitis (RR, 3.54; 95% CI, 2.07–6.07; p < 0.05) compared with ICI monotherapy, as well as 3-5 grade IRAEs in colitis (RR, 2.50; 95% CI, 1.62–3.86; p < 0.05), pneumonitis (RR, 1.99; 95% CI, 1.00–3.93; p = 0.05), and hepatitis (RR, 2.70; 95% CI, 1.29–5.63; p < 0.05). Conclusions This meta-analysis demonstrated that, compared with ICI monotherapy, patients receiving ICI combination therapy significantly increased organ-specific IRAEs in colitis, hypothyroidism, hepatitis, hypophysitis, hyperthyroidism, and pneumonitis. The incidence and severity of organ-specific IRAEs were drug and dose independent.

In recent years, the combined use of PD-1 and CTLA-4 inhibitors has attracted increasing attention for the promising efficacy in the treatment of advanced melanoma, lung cancer, and sarcoma (Larkin et al., 2015;D'angelo et al., 2018;Hellmann et al., 2018a;Hellmann et al., 2018b). In patients with advanced melanoma, combination therapy with nivolumab and ipilimumab had significantly improved clinical outcomes with prolonged progression-free survival (PFS) and higher objective response rate (ORR) compared with ipilimumab alone (Postow et al., 2015;Hodi et al., 2016). Four clinical trials (CheckMate 012/032/227/568) demonstrated a durable response associated with ICI combination therapy among patients with lung cancer (Antonia et al., 2016;Hellmann et al., 2017;Hellmann et al., 2018b;Ready et al., 2019). Although ICI combination has become a significant breakthrough in cancer therapeutics, their use was associated with toxic effects resulting from unbalanced activation of the immune system. To distinguish from other treatment-related side effects, these toxic effects caused by immune activation were specifically termed as immune-related adverse events (IRAEs) (Postow et al., 2018).
IRAEs may occur in almost any organ, such as the colon, lungs, liver, muscle, and thyroid. According to the published study (Baxi et al., 2018), IRAEs were classified into three categories: organ-specific IRAEs (colitis, hepatitis, pnemonitis, etc.), general IRAEs (fatigue, diarrhea, and rash) and musculoskeletal IRAEs (arthritis, arthralgia, back pain, etc.). They demonstrated that the general adverse events are more prevalent, but the organ-specific IRAEs are more clinically important. Yang et al. (Yang et al., 2019) also suggested that oncologists should focus on the organ-specific IRAEs, which are more meaningful in clinical practice. Therefore, the organ-specific adverse event has been a new challenge in the treatment of cancers (Baxi et al., 2018;Martins et al., 2019).
Currently, although several meta-analysis have evaluated the efficacy and safety of ICIs (Wang et al., 2017;Barroso-Sousa et al., 2018;Ma et al., 2018;Wang et al., 2018;You et al., 2018), most studies included chemotherapy as the control group for the analysis, and few studies specifically assessed the safety of ICIs. A published meta-analysis by Wang et al. in 2018 reported fatal toxic effects associated with ICIs. They demonstrated that the organ-specific IRAEs were the most common causes for death: colitis for CTLA-4 (70%, 135/193 deaths), pneumonitis (35%, 115/333 deaths) for PD-1 or PD-L1 inhibitors, and colitis (37%, 32/87) for the combination PD-1 and CTLA-4 (Baxi et al., 2018). However, they failed to provide the detailed data about the incidences of low-grade and high-grade adverse events.
A comprehensive understanding of the epidemiology of the organ-specific IRAEs is essential for clinicians to balance the benefits and risks of ICI combination during cancer treatment (Martins et al., 2019). Therefore, we conducted this metaanalysis based on randomized controlled trials (RCTs) aiming to compare the organ-specific IRAEs of ICI monotherapy versus combination therapy among cancer patients.

MATERIALS AND METHODS
This study was performed based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement.

Inclusion and Exclusion Criteria
The following inclusion criteria were used in this study: (1). types of included studies: randomized controlled trials (RCTs); (2). types of participants: patients over 18 years of age diagnosed with malignancies regardless of region, racial, and gender; (3). interventions: patients received the intervention treatment of either ICI monotherapy or combined therapy with CTLA-4/PD-1/PD-L1 antibodies; (4). types of outcomes: colitis, pneumonitis, hepatitis, hypothyroidism, hyperthyroidism, and hypophysitis. The severity of adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version (CTCAE) 4.0, and grade ≥3 were evaluated as high grade or severe grade.
The exclusion criteria were: (1). types of studies: ongoing trials, quasi-RCT, non-RCT, reviews, commentaries, conference paper, and quality of life studies; (2) interventions: patients treated with placebo, chemotherapy, or chemotherapy plus immunotherapy.

Data Sources and Searches
A literature search was conducted to identify RCTs comparing ICI monotherapy versus combination therapy among cancer patients. Without the restriction on language and publication status, the databases of MEDLINE, EMBASE, and Cochrane databases and ISI Web of Knowledge were searched to determine potentially eligible studies up to May 30, 2019. The following search terms were used: CTLA-4, ipilimumab, tremelimumab, PD-1, nivolumab, pembrolizumab, PDL-1, atezolizumab, avelumab, durvalumab, and checkpoint inhibitors. Additionally, the reference lists of identified studies and Google scholar were checked for other potentially eligible trials.

Data Collection and Quality Assessment
Two blinded authors (Da and Teng) independently extracted data according to a standardized extraction form. Any discrepancy was resolved by discussion with a third author. If insufficient data was reported, efforts were made to contact the authors for the additional information. The methodological quality of the eligible studies was evaluated using the following items recommended by the Cochrane Collaboration: randomization, allocation concealment blinding of participant, blinding of outcome assessors, incomplete outcome data, selective reporting, and other bias (Higgins et al., 2011).

Statistical Analysis
Meta-analysis was conducted using the software Review Manager 5.3.5. Risk ratio (RR) and 95% confidence interval (95% CI) were calculated to estimate the event rates for dichotomous outcomes. Heterogeneity was tested using I 2 index and the Cochran Q statistic (I 2 > 50% indicating significant heterogeneity, and I 2 ≤ 50% indicating no significant heterogeneity). If no heterogeneity (I 2 ≤ 50%) was presented in the meta-analysis, a fixed-effect model was used to estimate the pooled odds ratio and 95% confidence interval, otherwise, a random-effect model was used. Subgroup analyses were performed to explore the sources of heterogeneity according to the different tumors types. Figure 1 showed the flow chart of literature screening. A total of 2,279 records were yielded in the initial search from the database. After removing duplicates, 1,352 studies were assessed for abstract and full-text review. Finally, 10 studies involving eight RCTs were included in this meta-analysis (Larkin et al., 2015;Postow et al., 2015;Antonia et al., 2016;Hodi et al., 2016;Wolchok et al., 2017;D'angelo et al., 2018;Hellmann et al., 2018b;Long et al., 2018;Omuro et al., 2018;Sharma et al., 2019).

The Characteristics and Quality Assessment of Included RCTs
The detailed characteristics of included RCTs were shown in Table 1. A total of 2,716 patients (monotherapy group, 1,315; combination group, 1,401) were included in the analysis. In the combination group, all the patients received intervention with nivolumab and ipilimumab. Three studies compared the efficacy of two different doses of drug combinations: nivolumab 3 mg/kg plus ipilimumab 1 mg/kg (N3I1), or nivolumab 1 mg/kg plus ipilimumab 3 mg/kg (N1I3) (Antonia et al., 2016;Omuro et al., 2018;Sharma et al., 2019). In the monotherapy group, patients received intervention with either ipilimumab (Larkin et al., 2015;Postow et al., 2015;Hodi et al., 2016;Wolchok et al., 2017) or nivolumab alone (Larkin et al., 2015;Antonia et al., 2016;Wolchok et al., 2017;D'angelo et al., 2018;Hellmann et al., 2018b;Long et al., 2018;Omuro et al., 2018;Sharma et al., 2019). The included RCTs involved five kinds of tumors: lung cancer in two studies (Antonia et al., 2016;Hellmann et al., 2018b), melanoma in three studies (Larkin et al., 2015;Postow et al., 2015;Hodi et al., 2016;Wolchok et al., 2017;Long et al., 2018), metastatic sarcoma in one study (D'angelo et al., 2018), urothelial carcinoma (Sharma et al., 2019), and recurrent glioblastoma in one study (Omuro et al., 2018). All the included RCTs used the CTCAE 4.0 to evaluate the severity of IRAEs. The publication date of the included studies was between 2015 and 2018. Additionally, two updated RCTs were included in this meta-analysis without duplicate counting of the sample (Hodi et al., 2016;Wolchok et al., 2017). Table 2 showed the methodological quality of the included studies. The randomization was reported in all the studies, and blinding of outcome assessment was reported in six studies. However, few studies described the allocation concealment and the blinding of participants during trial.

Outcomes of Meta-Analysis
The outcomes of meta-analysis were presented in Table 3, and the forest plots of meta-analysis were attached in Supplementary Materials.

Meta-Analysis of Total Treatment-Related Adverse Events
A total of 2,716 patients were included in 10 studies with 1315 in the monotherapy group (nivolumab, 958; ipilimumab, 357) and 1,401 in the combination group (nivolumab and ipilimumab). A random-effect model was used for the outcome of total 3-5 grade adverse events due to significant heterogeneity among studies (I 2 > 50%). When compared with the monotherapy group, metaanalysis showed that patients in the ICI combination group had significantly higher risks for total treatment-related adverse events in any and 3-5 grade. The RR was 1.68 (95% CI, 1.35-2.08; p < 0.05) and 2.99 (95% CI, 2.00-4.46; p < 0.05) for total any-grade and 3-5 grade hepatitis, respectively.

Different Types of Tumors
As for the insufficient number of included studies on lung cancer, glioblastoma, urothelial carcinoma, and sarcoma, only one subgroup analysis was performed on melanoma. Meta-analysis showed that, the combination therapy significantly increased the risks of total 3-5 grade organ-specific IRAEs (RR, 1.70; 95% CI, 1.25-2.30; p < 0.05) in melanoma patients, but no difference was found in the incidences of total any-grade IRAEs between both groups (RR, 1; 95% CI, 0.99-1.01; p = 0.77).

Publication Bias
The funnel plot was used to explore the potential publication bias. All the included studies showed a symmetric distribution on the funnel plots. No significant publication bias was found in this meta-analysis.

DISCUSSION
In this study, 10 literatures involving 8 RCTs with 2716 patients were included for meta-analysis. The most important finding of this study is that the use of ICI combination (nivolumab and ipilimumab) significantly increased the risks in any-grade IRAEs in colitis, pneumonitis, hepatitis, hypothyroidism, hyperthyroidism, and hypophysitis, as well as the 3-5 grade IRAEs in colitis, pneumonitis, and hepatitis. The rapid development of ICIs has dramatically changed the therapeutic options in numerous cancers. Compared with ICI monotherapy, ICI combination therapy has become a more popular therapeutic way for its superior clinical efficacy. However, few studies are focused on the organ-specific IRAEs. Although a previous meta-analysis by Wang et al. (2018) had assessed the toxic effects caused by ICIs, the authors only reported the mortality related to the ICI toxicity (122 deaths in 19,217 patients). Nevertheless, for a prompt recognition and management of adverse events, we should not only know the epidemiology regarding the fatal events, but also for moderate and severe adverse effects (Martins et al., 2019). Therefore, we designed this study to compare the risks of any-grade and 3-5 grade adverse effects associated with ICI combination therapy with monotherapy.
In our study, colitis and hepatitis were included as ICIinduced gastrointestinal and hepatic injury. The most frequent IRAE associated with combination therapy was colitis (any grade, 14.5%; 3-5 grade: 11.9%), with a significantly higher incidence than that in the monotherapy group (any grade: 5.6%; 3-5 grade: 3.5%). Hepatitis induced by ICI was less frequent compared to colitis, occurring in approximately 10.4% of patients receiving ICI combination therapy, with 3.7% above grade 3. Meta-analysis showed that ICI combination therapy significantly increased risks of colitis and hepatitis than ICI monotherapy. Of note, the increased colitis in the combination therapy group might be mainly contributed to the use of anti-CTLA-4 drugs. Earlier studies demonstrated a higher incidence of gastrointestinal adverse events associated with CTLA-4 inhibitors alone compared with anti-PD-1 therapy. In a largesample phase-3 study with 945 patients, Larkin et al. (2015) compared the safety of nivolumab alone, ipilimumab alone, and nivolumab plus ipilimumab. The results showed that colitis of any grade occurred in 0.6% of the patients in the nivolumab group, 7.7% of those in the ipilimumab group, and 8.3% of those in the nivolumab-plus-ipilimumab group, respectively. This result was consistent with our subgroup analysis, which showed that patients receiving ipilimumab alone (10.6%) more likely experienced any-grade and serious colitis than those who received nivolumab alone (1.6%). Currently, the related pathogenesis of colitis initiated by ipilimumab still remains unclear. Histopathologic features might be related with an increase in intraepithelial lymphocytes for CTLA-4 inhibitors (Gupta et al., 2015;Weidner et al., 2015). For the organ-specific IRAEs in thyroid dysfunction, we included the outcomes of hypothyroidism and hyperthyroidism in this study. Meta-analysis showed that the combination group had significant high risks in any-grade hypothyroidism and hyperthyroidism compared with the monotherapy group. In terms of 3-5 grade adverse events, prior studies demonstrated that ICI therapy rarely resulted in serious thyroid dysfunction (Morganstein et al., 2017;Zhang et al., 2018). This study revealed that only 3 (0.4%, 3/1401) patients had serious hypothyroidism in combination therapy group, while 1 (0.1%, 1/958) and 5 (0.4%, 5/ 958) patients had serious hyperthyroidism in nivolumab and combination groups, respectively. Meta-analysis showed no differences between the monotherapy and combination groups. Hypophysitis was regarded as the most frequent endocrine dysfunction caused by ICI therapy. Notably, we found that the variation tendency of hypophysitis was similar with that of thyroid dysfunction, which showed that the combination group had statistically higher incidence in all-grade events, but no difference in serious events between two groups. A possible explanation was that the thyrotropin hormone was affected by hypophysitis, thus resulting in thyroid disorders (Barroso-Sousa et al., 2018;Zhang et al., 2018).
Pneumonitis was a relatively rare adverse event during checkpoint inhibition therapy, which appeared more prevalent in lung cancer patients (Spain et al., 2016). Low-grade pneumonitis was commonly manageable with treatment discontinuation, but serious pneumonitis is potentially lifethreatening (Martins et al., 2019;Rahouma et al., 2019). In this study, meta-analysis revealed that ICI combination therapy was associated with a significantly higher risk of pneumonitis compared with monotherapy. Subgroup analysis revealed that the rates of all-grade pneumonitis were 4.6%, 2.3% and 1.4% in patients receiving nivolumab plus ipilimumab, nivolumab alone, and ipilimumab alone, respectively. Interestingly, unlike colitis, pneumonitis was more frequent among patients receiving anti-PD-1/PD-L1 therapies as opposed to those receiving anti-CTLA4 therapies (Martins et al., 2019). Analysis based on the drug types also showed that patients receiving anti-PD-1 inhibitors (nivolumab) experienced more high-grade pneumonitis than those receiving anti-CTLA4 inhibitors (ipilimumab), and the incidences were 0.8% and 0.3%, respectively. Moreover, 1 (1.1%) case and 3 cases (2.3%) of pneumonitis-related death associated with nivolumab were reported by Postow et al. (2015) and Gettinger et al. (2015), respectively. Therefore, despite a relatively low incidence of pneumonitis, this adverse effect should be closely followed up by clinicians, in particular when anti-PD-1/PD-L1 inhibitors are being used.
For the subgroup analysis, we found that the organ-specific IRAEs appeared to be drug-and dose-dependent. Regarding the drug dependent, the risks of colitis and hypophysitis appeared to be more related to the CTLA-4 antibodies (ipilimumab); the pneumonitis and hepatitis appeared to be more related to the PD-1 antibodies (nivolumab). Regarding the dose dependent, we compared two different doses in drug combinations (nivolumab 3 mg/kg pus ipilimumab 1 mg/kg versus nivolumab 1 mg/kg plus ipilimumab 3 mg/kg). The result showed that nivolumab 3 mg/ kg plus ipilimumab 1 mg/kg significantly increased the total 3-5 grade IRAEs.

STUDY STRENGTHS AND LIMITATIONS
The most important strength of this study is that all the included studies are RCTs with detailed registration information in ClinicalTrials.gov. Meanwhile, there are also several limitations in this study. First, the number of the included RCTs was small (10 studies involving 8 RCTs and 2,716 patients), which limited us to perform subgroup analysis. Further high-quality RCTs with large sample sizes are needed to verify our conclusion. Second, some definitions of adverse events were not uniform. For example, immune-related hepatitis was reported as hepatitis or increased aspartate transaminase/alanine transaminase, and immune-mediated colitis was reported as colitis or diarrhea, which might lead to incomplete data collection. This should be standardized in the future study. Third, patients with various cancers were included, which might have bias in the incidence of some adverse effects. For example, lung cancer was related with a high risk of developing pneumonitis from previous lung disease, radiotherapy, and smoking history. Subgroup analysis was not done based on different types of cancers due to the insufficient studies on lung cancer, sarcoma, glioblastoma, and urothelial carcinoma. Fourth, heterogeneity was found in the outcomes of colitis and total adverse events among the included studies. The heterogeneity might have resulted from the differences of cancer types, follow-up time, drug dose, and so on.

CONCLUSIONS
This meta-analysis demonstrated that, compared with ICI monotherapy, combination therapy with ICI drugs significantly increased the risk of organ-specific IRAEs in colitis, hypothyroidism, hepatitis, hypophysitis, hyperthyroidism, and pneumonitis. The incidence and severity of organ-specific IRAEs were drug-and dose-independent. Although the incidence of high-grade organ-specific IRAEs was relatively low, clinicians should be aware of these adverse effects so that patients can be promptly managed.