Efficacy and safety of immune checkpoint inhibitors in Proficient Mismatch Repair (pMMR)/ Non-Microsatellite Instability-High (non-MSI-H) metastatic colorectal cancer: a study based on 39 cohorts incorporating 1723 patients

Purpose This study was designed to investigate the efficacy and safety of immune checkpoint inhibitors (ICIs)-based therapy in proficient mismatch repair (pMMR)/non-microsatellite instability-high (non-MSI-H) metastatic colorectal cancer (mCRC). Methods Electronic databases were screened to identify relevant trials. The primary endpoints were pooled objective response rate (ORR) and disease control rate (DCR). Stratified analysis was accomplished on ICIs-based regimens, treatment lines and RAS status. Results Totally, 1723 mCRC patients from 39 cohorts were included. The pooled ORR, DCR, 12-month overall survival (OS) rate and 6-month progression-free survival (PFS) rate of ICIs-based therapy in pMMR/non-MSI-H mCRC were 8.5% (95% CI: 4.4%-13.5%), 48.2% (95% CI: 37.8%-58.6%), 52.3% (95% CI: 46.4%-58.1%) and 32.8% (95% CI: 23.5%-42.7%) respectively. As a whole, no significantly differences were shown between ICIs-based and non-ICIs-based therapy for pMMR/non-MSI-H mCRC in terms of both PFS (HR = 1.0, 95% CI: 0.9–1.1, P = 0.91) and OS (HR = 1.0, 95% CI: 0.9–1.2, P = 0.51). It was worth noting that the addition of ICIs to anti-vascular endothelial growth factor (VEGF) agent plus chemotherapy displayed excellent efficacy in pMMR/non-MSI-H mCRC (ORR = 42.4%, 95% CI: 10.0%-78.6%; DCR = 92.0%, 95% CI: 68.3%-100.0%; 12-month OS rate = 71.4%, 95% CI: 50.0%-89.1%; 6-month PFS rate = 55.2%, 95% CI: 24.8%-83.8%; and PFS (compared with non-ICIs-based therapy): HR = 0.9, 95% CI: 0.8–1.0, P = 0.02), especially served as first-line therapy (ORR = 74.2%, 95% CI: 61.4%-85.4%; DCR = 98.7%, 95% CI: 92.0%-100.0%); and without additional treatment related adverse events (TRAEs) were observed. Conclusions ICIs-based combination therapy, especially the addition of ICIs to first-line anti-VEGF agent plus chemotherapy, is promising in pMMR/non-MSI-H mCRC with good efficacy and controllable toxicity. Supplementary Information The online version contains supplementary material available at 10.1186/s12865-023-00564-1.


Introduction
Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related morbidity and mortality all over the world [1].Despite remarkable improvements have been made in clinical outcomes with the optimization of chemotherapy and targeted therapy, the results continue to fall far short of durable curative treatment of mCRC patients.Consequently, it is crucial to seek a novel approach against mCRC.During the last decade, immune checkpoint inhibitors (ICIs) have made tremendous breakthroughs in the clinical treatment of several hematological and solid tumors, including Hodgkin lymphoma, malignant melanoma, non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), advanced hepatocellular carcinoma (HCC) and microsatellite instability-high (MSI-H) mCRC [2][3][4][5][6][7].However, ICIs remain largely ineffective in the majority of mCRC patients, characterized by proficient mismatch repair (pMMR)/non-MSI-H.
It has been recorded that a lack of efficacy of the anti-PD-1 and a modest clinical benefit of the anti-PD-L1 plus the anti-CTLA-4, reserved only to patients with a tumor mutational burden (TMB) more than 28 mut/ Mb on circulating tumor DNA [8,9].Based on these considerations, accumulating focus has been recently placed on developing effective combination regimens in which ICIs have been incorporated with chemotherapy, radiotherapy and biologic agents with the purpose of reshaping the microenvironment of pMMR/non-MSI-H tumors towards an immune-inflamed/hot phenotype, that may lead to ICIs sensitivity.However, much of these approaches have been largely disappointing [10][11][12].Notably enough, two phase II studies named AtezoTRIBE and MAYA, assessing combinations of ICIs with chemotherapy, have rekindled hope for the use of ICIs in pMMR/non-MSI-H mCRC [13,14].
To overcome the limitations of individual studies and assess the overall benefit, therefore, we conducted a comprehensive survey based on a large sample size (39 cohorts incorporating 1723 individuals), diverse dimensions (including pooled rate, odd ratio (OR), and hazard ratio (HR)), multiple stratifications (based on ICIs-based regimens, treatment lines and RAS status), and various evaluation indicators (incorporating objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS)) to evaluate the efficacy and safety of ICIs-based therapy in pMMR/non-MSI-H mCRC.

Selection of studies
Initially, two investigators performed a screening of titles and abstracts respectively, then examined the full-text of articles to acquire eligible studies.For the duplicate studies based on the same study patients, only the latest or most comprehensive data were recruited.

Inclusion criteria
(1) Prospective or retrospective studies to evaluate the efficacy and safety of ICIs in pMMR/non-MSI-H mCRC; (2) patients pathologically confirmed as CRC; (3) the data (involving any of the following outcomes: ORR, DCR, PFS, OS, 6-month PFS rate and 12-month OS rate) to evaluate the efficacy of ICIs in pMMR/non-MSI-H mCRC could be obtained or calculated from the original literature.

Data extraction
Data extraction was implemented conforming to the PRISMA guidance (Table S1).All eligible studies involved information as follows: the first author's name, publication year, number of pMMR/non-MSI-H mCRC patients, ICIs agent, and endpoints.

Quality assessment
The quality of included studies was assessed independently by two reviewers using the Newcastle-Ottawa Scale (NOS) for case-control and cohort studies.It encompassed three dimensions of selection, comparability, and exposure, with a full score of 9 points.

Statistical methods
The primary endpoints were ORR and DCR measured by pooled rates with corresponding 95% CIs for pMMR/non-MSI-H mCRC.The secondary endpoints were pooled PFS, OS, 6-month PFS rate and 12-month OS rate.Subgroup analysis was accomplished based on various ICIs-based regimens, treatment lines and RAS status.The summary measures of prognostic parameters and adverse events (AEs) were pooled rate, ORs and HRs with 95% CIs.Funnel plots and Egger's test were performed to evaluate publication bias.Statistical analysis was performed with R 4.0 statistical software.Survival data were obtained based on the Kaplan-Meier curves.Heterogeneity was assessed by I-square tests and Chi-square.If P < 0.1 or I 2 > 50%, remarkable heterogeneity existed.A random effect model was adopted to calculate the pooled data when heterogeneity existed, or else, a fixed effect model was employed.

Selection of study
Initially, 421 relevant articles were scrutinized intensively.Of them, 18 were filtered for duplication, and 330 were excluded for digression after screening the titles and abstracts.Then the full text of 73 articles was thoroughly reviewed, and 38 were filtered for: they were not human research, and not in English, commentaries, case reports, review articles, or letters to the editor, and without enough data for calculation.Finally, a total of 35 articles (including 39 cohorts) incorporating 1723 patients were recruited in this study (Table S2).The elaborate procedure was displayed in Fig. 1.

Study traits
Totally, 1723 individuals from the 35 articles (39 cohorts) published until August 15th, 2022 were recruited.The sample size ranged from 6 to 250.Of these studies, 4 were randomized controlled trials (RCTs), and 9 retrospective studies.Meanwhile, all of these studies involved ICIs: ICIs monotherapy, ICIs plus targeted therapy, ICIs Fig. 1 Flowchart on selection including trials in the meta-analysis plus chemotherapy or radiotherapy, ICIs plus both targeted therapy and chemotherapy/radiotherapy. Pooled rate and 95% CIs were used to report the ORR, DCR, 6-month PFS rate, 12-month OS rate, and AEs of ICIs in pMMR/non-MSI-H mCRC; HRs with corresponding 95% CIs were utilized to assess the PFS and OS of ICIs for pMMR/non-MSI-H mCRC.The principal traits were presented in Table 1.

Subgroup analysis was carried out based on various
ICIs-based regimens (Table 2).Obviously, the pooled DCR of ICIs plus anti-VEGF agent and chemotherapy was the best (DCR = 92.0%,95% CI: 68.3%-100.0%),especially when it served as first-line therapy.

The safety of ICIs-based therapy in pMMR/non-MSI-H mCRC
A total of 21 cohorts were included to calculate the safety of ICIs-based therapy in pMMR/non-MSI-H mCRC (Table 4), and the pooled rate of grade 3 or beyond AEs was 31.8% (95% CI: 20.1%-44.8%).Despite the regimen of ICIs plus anti-VEGF agent and chemotherapy revealed higher incidence of grade 3 or beyond AEs, no additional treatment related adverse events (TRAEs) were observed.

Assessment of study quality
The quality assessment of 35 recruited articles was summarized in Table S3 with relatively satisfying results for bias risk assessment.been incorporated with chemotherapy, radiotherapy and anti-VEGF agent in order to transform immunologically "cold" pMMR/non-MSI-H mCRC into responsive "hot" lesions.However, the results of such studies have been inconsistent [9][10][11][12][13][14] and the AEs caused by ICIs cannot be ignored.To overcome the limitations of individual studies, we performed a meta-analysis of relevant trials to investigate the benefit and safety of ICIs-based therapy for pMMR/non-MSI-H mCRC.
Based on the existing studies, the pooled results of our study revealed that the addition of ICIs into anti-VEGF agent plus chemotherapy (especially first-line) is promising in pMMR/non-MSI-H mCRC in terms of ORR, DCR, PFS, 6-monhs PFS rate and 12-month OS rate.At the same time, it has been supported that the potential clinical efficacy of anti-VEGF agent plus ICIs combination was also founded in other tumors such as HCC and NSCLC.For HCC, compared with sorafenib monotherapy, atezolizumab plus bevacizumab (IMbrave 150), and Sintilimab plus bevacizumab (ORIENT-32) were founded to significantly improve PFS and OS [45,46]; with regard to NSCLC, the addition of atezolizumab to bevacizumab plus chemotherapy (IMpower150) significantly improved PFS and OS among patients with metastatic nonsquamous NSCLC, regardless of PD-L1 expression and EGFR or ALK genetic alteration status [47], Sintilimab plus bevacizumab biosimilar IBI305 and chemotherapy (ORIENT-31) improved PFS of patients with EGFRmutated non-squamous NSCLC who progressed on Table 2 The details of pooled ORR and DCR EGFR tyrosine-kinase inhibitor therapy [48].As we know, ICIs can effectively alleviate immune escape [49] and activate the human immune system to kill tumor cells, aims to improve immunity and enhance the antitumor response, then to achieve its anti-tumor effects [50,51].The limited advantage may be attributed to that cancer with pMMR/non-MSI-H has an immunedesert or immune-excluded (or "cold") microenvironment, finally resulting in a blunted immune activation of tumor microenvironment that causes the futility of ICIs in these patients [52].It has been recorded that cytotoxic agents are able to induce immunogenic cell death and activate CD8+ T lymphocytes, favoring an immune enriched microenvironment as the consequence of the release of tumor-associated neoantigens [53].However, there are a lot of neovascularization with special structure in tumor tissue, which makes it difficult for antitumor drugs and immune cells to reach the tumor site.The VEGF pathway plays a pivotal role in establishing and maintaining an immunosuppressive tumor microenvironment.Therefore, the addition of anti-VEGF agent has a consistent vessel fortification effect in pMMR/non-MSI-H cancer, and can establish an immune permissive tumor microenvironment [54].Therefore, the combination of chemotherapy, antiangiogenic and ICIs might have subadditivity, additivity or synergism effects to delays progression in patients achieving tumor shrinkage with subsequent release of neoantigens and immune activation of tumor microenvironment that allows ICIs efficacy [55][56][57].
Although there was no difference between ICIbased therapy and non-ICI-based therapy in both OS and PFS of pMMR/non-MSI-H mCRC on the whole, the subgroup analysis revealed that the addition of ICIs to anti-VEGF agent plus chemotherapy could significantly improve PFS of pMMR/non-MSI-H Table 3 The details of pooled 6-month PFS rate and 12-month OS rate mCRC; moreover, there was no directly correlation between the improvement of PFS and RAS status.At the same time, some other advantages of ICI-based therapy were still founded among these studies.The study conducted by Eng et al. found that although not superior to standard therapy, treatment with the combination of ICIs and MEK inhibitor resulted in equivalent survival without the introduction of any new AEs [10].Besides, Redman et al. found that despite a lack of improvement in clinical outcomes in the experimental arm, the addition of ICIs to chemotherapy was biologically active and produced multifunctional T-cell responses to cascade antigens MUC1 and brachyury [19].
With regard to the safety, the regimens of ICIs plus chemotherapy with/without anti-VEGF agent revealed higher incidence of grade 3 or beyond AEs.Among the included studies, Bocobo et al. found that the grade 3 or beyond TRAEs only occurred in 28% patients, of which less than half (11%) were immunerelated and none was associated with bevacizumab [43]; besides, Redman et al. revealed that no TRAEs were observed outside the expected safety profile with the addition of ICIs to bevacizumab plus chemotherapy, and most TRAEs were chemotherapy-related and controllable [19].
The best strategy and biomarkers of ICIs for pMMR/non-MSI-H mCRC remain to be established.On one hand, in order to seek the best strategy of ICIs-based therapy for pMMR/non-MSI-H mCRC, we are obliged to optimize which regimen is beneficial in combination with ICIs (with maximizing efficacy and minimizing toxicity), facilitate clinical research based on biomarkers, and explore the development of other ICIs drugs and cell-based treatment schemes [58]; on the other hand, in screening the beneficiaries of ICIs for pMMR/non-MSI-H mCRC, we need to further seek appropriate biomarkers (such as TMB, PD-L1 expression, tumor infiltrating lymphocytes (TILs), and status of polymerase ε (POLE), neutrophil to lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR)) [59][60][61][62].Moreover, prospective, larger confirmatory and translational studies are recommended in the future.

Limitations
This study came up with three drawbacks as follows: firstly, there were only 4 RCTs, despite containing 39 cohorts 1723 patients, for analyzing the efficacy and safety of ICIs in pMMR/ non-MSI-H mCRC; secondly, considering the limited number of studies with survival outcomes for ICIs in pMMR/non-MSI-H mCRC patients, we took the ORR and DCR as primary endpoints; finally, only studies published in English were included, which might yield language bias to some degree.

Conclusions
ICIs-based combination therapy, especially the addition of ICIs to first-line anti-VEGF agent plus chemotherapy, is promising in pMMR/non-MSI-H mCRC with good efficacy and controllable toxicity.

Discussion
In the last decade, ICIs has initiated a new era for immunotherapy in oncology by monoclonal antibodies to release the anti-tumor activity of preexisting tumor-specific T-cell immunity, which inspired researchers to focus on the application of ICIs in mCRC.However, a lot of studies have confirmed that ICIs monotherapy has not shown significant clinical activity in pMMR/non-MSI-H mCRC, which was considered with an immune-desert or immuneexcluded (or "cold") microenvironment.Therefore, accumulating studies have been carried out recently focusing on ICIs-based combination regimens in which ICIs have

Fig. 2
Fig. 2 The pooled objective response rate (ORR) of immune checkpoint inhibitors (ICIs) in proficient mismatch repair (pMMR)/non-microsatellite instability-high (non-MSI-H) metastatic colorectal cancer (mCRC): a forest plot and b funnel plot; and the pooled disease control rate (DCR) of ICIs in pMMR/non-MSI-H mCRC: c forest plot and d funnel plot

Table 1
The principal characteristics of eligible articles