The Efficacy of Immune Checkpoint Inhibitors in Microsatellite Stable Colorectal Cancer: A Systematic Review

Abstract The use of immune checkpoint inhibitors (ICIs) has revolutionized cancer care, particularly in immune-inflamed tumors and tumors with a high mutational burden, like microsatellite instable colorectal cancer (CRC). However, their effectiveness in microsatellite stable (MSS) CRC is limited. This systematic review aims to evaluate the efficacy of ICIs in MSS CRC and explore promising combination strategies. A comprehensive search from the Web of Science, Medline, and Embase databases, for studies published until 14 November 2022, identified 53 clinical trials included in the review. ICI monotherapy or ICI-ICI combinations demonstrated limited clinical activity for patients with MSS CRC, with overall response rates below (ORR) 10% in most studies. The ICI and tyrosine kinase inhibitor (TKI) garnered ORRs ranging from 10% to 40% and indicated a higher benefit for patients, particularly those without active liver metastases. The combination of ICIs with anti-VEGF agents showed modest ORRs, especially in the earlier treatment lines and in combination with chemotherapy. While these combinations could lead to modest improvements, well-defined biomarkers for long-term benefit are yet to be delineated. Combinations involving BRAF inhibitors with ICIs were studied, showing promising responses with combination approaches in molecularly defined subgroups. In conclusion, while ICI monotherapy has limited efficacy in MSS CRC, combination strategies hold promise to enhance survival outcomes. Further research is necessary to identify optimal combination approaches, predictive biomarkers for treatment response, as well as enrollment according to tumor molecular characteristics.


Introduction
Immunotherapy became the fifth pillar of cancer care and significantly changed the landscape of cancer management in the last decade. 1The immune checkpoint inhibitors (ICIs), a class of immunotherapy agents, work by releasing the breaks of immune systems with the modification of regulatory immune checkpoints and paved the way in the immunotherapy era. 2,35][6][7][8] In contrast, the ICIs had limited efficacy for patients with immunecold tumors and tumors with a lower mutational burden. 9,10icrosatellite instability-high (MSI-H) colorectal cancer (CRC) was among the earliest tumors in which a benefit with ICIs was demonstrated.In the seminal paper by Le et al., the response rate to pembrolizumab monotherapy was 40% in mostly pretreated patients with CRC,11 and in the update of this study by expansion cohorts, the response rate was The Oncologist, 2024, Vol. 29, No. 5 e581 over 50% in patients with metastatic MSI-high CRC.This benefit was confirmed in larger-scale studies and in longterm follow-ups, suggesting highly durable disease control for patients with MSI-H CRC with ICIs. 12,13The MSI-H CRC carry a higher tumor mutation burden (TMB) with a median TMB of 46.8 mutations/megabase, and this high TMB due to frameshift alterations leads to increased neoantigen generation and expression representing the hallmark of MSI-H CRC for high ICI efficacy. 14hile the ICIs became the standard of care for patients with MSI-H CRC, earlier studies demonstrated very limited ICI benefits for patients with MSS CRC, especially with ICI monotherapy. 11,15The MSS CRC has distinct molecular pathogenesis and tumor microenvironment (TME) compared to MSI CRC. 16,17The tumor mutational burden is significantly lower in MSS CRC than in MSI-H CRC. 14 Additionally, immunosuppression and immune exhaustion in TME are more prominent in MSS CRC. 18Despite these inherent limitations for the clinical benefit of ICIs for patients with MSS CRC, there is an important body of data in MSS CRC for the use of ICIs, albeit with variable designs, sample sizes, and treatment lines.Several combination strategies with tyrosine kinase inhibitors (TKIs), vascular endothelial growth factor inhibitors (anti-VEGF), and chemotherapy have been tested in clinical trials and yielded varying results.Therefore, in this article, we systemically reviewed recently published clinical trials on the ICI efficacy in MSS CRC to overview the promising combination strategies and review the ongoing studies to give perspectives on how the field could evolve.

Search Strategy
We report this systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) 2020 guideline. 19This protocol was registered with the Open Science Framework at https://doi.org/10.17605/OSF.IO/GZ7RX (accessed on July 11, 2023).We systematically searched the Web of Science (topic), Medline (all fields), and Embase (all fields) databases for studies published until 14 November 2022 for inclusion.The selected MeSH search terms were ("colon cancer" OR "colorectal cancer" OR "rectum cancer" OR "rectal cancer") AND ("immunotherapy" OR "immune checkpoint inhibitor") AND ("microsatellite stable").Additionally, congress abstracts from the ESMO and ASCO congresses were evaluated for inclusion.

Study Selection and Data Extraction
We included clinical trials evaluating the ICI efficacy in advanced MSS CRC.In order to prevent missing any relevant studies, studies without the available full-text publication were included.The exclusion criteria were as follows: duplicated articles, animal studies, commentaries, opinion papers, and reviews, articles not in English, studies without separate data for MSS CRC patients, the inclusion of the patients with localized disease, studies from real-life cohorts, and study protocols of ongoing trials.
DCG and EE independently screened the literature to assess their eligibility for inclusion in the study.Two authors (DCG and EE) independently extracted the following data from the studies following the PRISMA 2020 guidelines: clinical trial number, treatment line, treatment scheme, overall response rate (ORR), disease control rate (DCR), median progression-free survival (PFS), and median overall survival (OS).The ORR was defined as the proportion of patients who had either confirmed complete or partial response, and the DCR was defined as the proportion of patients who had a complete or partial response or stable disease under ICIs.Any disagreements were resolved by discussing with the senior author (IHS).The individual study qualities were evaluated with the MINORS tool for single-arm studies 20 and Risk of Bias (RoB) version 2 for randomized trials. 21

Article Selection
A total of 1507 records were identified with a database search.After removing the duplicates (n = 978), we then excluded 413 records after screening the titles and abstracts.The full texts of the remaining 116 records were evaluated, and additional 74 records were excluded due to the following reasons: study protocols (n = 24), not clinical trials (n = 24), MSI-H tumors (n = 8), outcome other than efficacy (n = 3), immunotherapy other than ICI (n = 12), no separate data for CRC (n = 1), MUTYH-related CRC (n = 1), and re-evaluation of the previously published data (n = 1).Eleven additional studies were retrieved from the citation check of the included studies and from the clinicaltrials.govdatabase.Fifty-three clinical trials were included in the quantitative synthesis.The PRISMA diagram for article selection is summarized in Fig. 1.

Immune Checkpoint Inhibitor Monotherapy or Doublets
A total of 5 studies evaluated the use of ICI therapy alone, either as monotherapy or ICI-ICI combinations in MSS CRC (Table 1).One study was phase I 23 , and the remaining 4 studies were phase II. 11,12,22,24No phase III studies were available.Two studies enrolled patients who had received the 3+-prior lines of therapy.The sample sizes varied between 18 and 180.The ORR was the main outcome in most studies.The ORR was consistently < 10% in all studies except the botensilimab plus balstilimab combination. 23,25The DCR varied between 11% and 73%.ICI monotherapy has not shown any significant clinical activity, and no objective responses were seen in 2 studies with pembrolizumab or nivolumab. 11,22However, the proportion of patients on nivolumab for 18 weeks and beyond was 38% in patients without liver metastases in the ANICCA study testing nivolumab monotherapy in tumor-specific major histocompatibility complex II (tsMHC-II)-positive MSS CRC. 22linical trials assessing dual ICIs have been tested.The ORR and DCR of phase I study of botensilimab (next generation CTLA-4 inhibitor) plus balstilimab (PD-1 inhibitor) combination were 22% and 73%, respectively. 23Importantly, in the updated data from this study, the ORR in patients with or without active liver metastases was 0% and 23%, respectively.In a randomized phase II trial of durvalumab plus tremelimumab combination with best supportive care as the control arm, there was one patient with MSS tumor who showed a partial response (PR) lasting more than 21 months. 24In the same study, patients with high TMB (28 Mb or higher) derived the greatest OS benefit from ICI therapy (HR, 0.34; 90% CI, 0.18-0.63;P = .004).

Immune Checkpoint Inhibitor and Tyrosine Kinase Inhibitor Combination
Ten studies tested the efficacy of the ICI plus TKI combination in MSS CRC (Table 2).Regorafenib was the most commonly used agent in these studies.The recommended phase II dose for regorafenib was 80-120 mg in the available studies.The available studies yielded mixed results.The Regonivo study conducted in Japan included gastric and patients with CRC. 26 For the patients with MSS CRC, the ORR was 33.3%, while it was 7% in a similarly designed study from the US. 31 Additionally, 63.6% of the lung target lesions responded, while the response rate in target liver lesions was 8.3% in the Regonivo study.Similarly designed Regomune study with regorafenib and avelumab reported no response in 48 patients. 33The median PFS varied between 2.3 months in the LEAP-005 study with pembrolizumab and lenvatinib to 7.9 months in the Regonivo study. 26,34Grade 3 or higher adverse events were variable, with studies reporting up to 90% grade 3 or higher adverse events with combination were present.

Immune Checkpoint Inhibitor and Chemotherapy Combinations
Eight studies evaluated the efficacy of chemotherapy and ICI combinations (Table 3).Three studies were conducted in the 2nd line or later with capecitabine or trifluridine-tipiracil (TPI), while 4 studies were conducted in the first line with  chemotherapy combinations.Five of the studies used bevacizumab in combination with chemotherapy and ICIs.While the frontline AtezoTRIBE and Checkmate 9 × 8 studies were randomized phase II studies, the remaining studies were singlearm phase I/II studies. 36,44These randomized studies conducted in the 1st-line included patients with MSI-H and MSS CRC and used PFS as the primary endpoint.Both studies were negative regarding the primary outcome in the MSS subgroup.
In the Checkmate 9 × 8 study, subgroup analyses according to CMS subtypes demonstrated that in patients with CMS1 or CMS3 disease, there was a PFS benefit after 12 months of follow-up.44Lastly, the preliminary results of the MEDITREME trial, in which the combination of FOLFOX with durvalumab and tremelimumab is being investigated as first-line therapy, showed an ORR of 61%, and 8.4 months PFS43 among patients with KRAS-mutated CRC.

Other Combinations
In addition to ICI monotherapy, ICI-TKI, and ICIchemotherapy combinations, several other agents were evaluated in combinations (Table 4).The BRAF or MEK inhibitors were tested in combination with ICIs in 4 studies in patients with MSS CRC.In one of the earliest studies, the combination of encorafenib, cetuximab, and nivolumab was related to an ORR of 45% in patients with chemotherapy-refractory unresectable or metastatic MSS, BRAF-V600E mutated CRC, while the combination of atezolizumab and cobimetinib was related to an ORR of 3% in patients with previously treated metastatic MSS CRC in the IMblaze 370 study. 51,67Five additional studies evaluated the efficacy of radiotherapy plus ICI combinations.The NCT03104439 study evaluating radiotherapy plus nivolumab and ipilimumab in patients with metastatic MSS CRC demonstrated 15% ORR and 2.5 months PFS.The study included heavily pretreated patients, with a median of 2 previous treatment lines. 62In the pooled analysis of the MSS CRC cohort of 2 PraG basket studies, the combination of radiotherapy and anti-PD-1 inhibitor and GM-CSF, the ORR was 25%, and the DCR 62.5%. 66Additionally, one of 9 treated patients had complete response.Several other agents, such as EGFR antagonists, immunomodulatory agents, BITE, and CART therapies, were also tested with ICIs, and the results of these studies are summarized in Table 4.

Ongoing Studies
Our search retrieved a total of 38 clinical trial records (Table 5).Five studies did not start recruiting participants at the time of this review.Twenty of the studies were active and recruiting.Thirteen of the studies were ongoing, and participants were receiving an intervention or being examined, but potential participants were not currently being recruited or enrolled.There were 4, 31, and 3 phases I, II, and III studies, respectively.Seven of these trials were first-line studies.The ICI and TKI combinations and chemotherapy and ICI combinations are being evaluated in 6 and 10 studies, respectively.Additionally, 17 studies evaluated other TME modifiers in combination with ICIs, while RT and ICI combinations were evaluated in 5 studies.The sample sizes varied between 12 and 665, and 7 studies were of the Far East region.

Discussion
Patients with MSS CRC is an important target population for ICI clinical trials due to limited therapeutic options in the  later lines and the high frequency of the disease. 18However, unlike immune hot tumors, the effectiveness of ICI monotherapy in MSS CRC was found to be limited in previous clinical trials, particularly late in the course of the disease.So far, no studies have reported a response rate exceeding 10%. 87While the response to ICI therapy is limited for the majority of MSS CRC, hypermutated tumors with POLE/POLD1 mutations 88 could represent a subgroup of MSS patients who may benefit from ICI-based therapies.Therefore, it would offer limited benefit to pursuing further ICI monotherapy in the MSS CRC in biomarker unselected cohorts, while better biomarker selection and translational research should be pursued in patients with a demonstrated response to ICI monotherapy.
In contrast to limited efficacy with ICI monotherapy and ICI-ICI combinations, the combinations with anti-VEGF agents have revealed varying results in several studies (Table 2).The interest in this area was amplified after the demonstration of a 36% ORR in the RegoNivo study 26 ; however, the same success could not be reproducible in a similar study conducted in the US. 27Furthermore, the recent press release from the phase III LEAP-017 trial comparing pembrolizumab plus lenvatinib versus standard of care in previously treated CRC reported no statistically significant OS benefit. 85,89onetheless, the CAMILLA trial with cabozantinib plus durvalumab reported a 30% ORR 30 and the STELLAR-303 study, a phase III randomized trial investigating XL092 and atezolizumab combination versus regorafenib is currently enrolling based on promising responses seen in the CAMILLA trial (NCT05425940). 86he VEGF pathway is instrumental in creating and perpetuating the immunosuppression in TME.The VEGF overexpression by tumor-associated macrophages leads to increased TGF-β secretion, activated epithelial-mesenchymal transition, and impaired dendritic maturation.1][92] Therefore, combining the anti-VEGF agents and ICIs to create a synergistic immune activation emerged as a tool to improve ICI efficacy. 93These combinations significantly improved the survivals and became among the standard of care options in the treatment of RCC, 94 and cervix cancer. 95It is important to note that although the VEGF pathway contributes significantly to the development and maintenance of immunosuppression in the tumor microenvironment (TME) similar to these cancers, it is relatively less active in CRC compared to RCC, 96 a tumor in which ICI plus anti-VEGF TKI combination became the standard of care.
The intracrine VEGF signaling demonstrated to factor in migration and invasion in CRC cell lines and the VEGF depletion leads to significant decreases in EGFR, cMET, and phosphorylated focal adhesion kinase, critical factors in invasion and motility. 91Furthermore, the increased VEGF levels were correlated with hepatic metastases 97, and the suppression of VEGF attenuates the STAT3 phosphorylation, proliferation, and metastasis. 98The hepatic metastases are the most frequent sites of metastasis in patients with metastatic CRC and were linked to ICI resistance for patients with MSS CRC in several trials. 26,31This indicates that there may be additional biological characteristics of hepatic metastases beyond increased VEGF signaling impacting immune response and abrogating antitumor immunity.Considering these points, targeting the VEGF pathway in addition to ICIs has a strong biological rationale for patients without liver metastasis and more translational research is needed to better understand  the biological underpinnings of ICI resistance to deliver more effective approaches to clinical trials.However, the reason for variable efficacy across different anti-VEGF agents and across different populations is yet to be defined.Whether the cMET inhibition of cabozantinib in addition to VEGF blockade 99 could be the reason for higher ORR in CAMILIA study 30 should be further investigated considering the instrumental role of cMET in mediating VEGF effects in TME.
The combination of ICIs with chemotherapy is another intriguing revenue to explore ICI potential in MSS CRC.The ICI plus chemotherapy combinations created significant success in NSCLC, 100 gastric cancer, 101 and triple-negative breast cancer. 102However, a similar success could not be replicated in patients with CRC in most studies with 5-FU-based therapy and ICIs, 39,103 and the trials with these combinations in the later lines were ceased.In contrast, there is a recent interest in the first-line trials.Oxaliplatin was previously demonstrated to increase the calreticulin and high-mobility group box 1 protein (HMGB1) levels in the murine and human CRC cell lines and corresponding immunogenic cell death (ICD) via apoptosis activation. 104Furthermore, oxaliplatin resulted in ICD, activation in dendritic cells and cytotoxic T cells in a murine lung carcinoma model. 105These immune effects could be amplified by blocking the VEGF pathway via bevacizumab. 106,107Therefore, recent studies tested the benefit of ICIs with 5-FU + oxaliplatin and bevacizumab backbones.However, the Checkmate 9 × 8 study, comparing FOLFOX plus bevacizumab with or without nivolumab was negative for PFS, while overall survival outcomes were more favorable for patients who received nivolumab. 44In contrast, a modest improvement in PFS was present with addition of atezolizumab to FOLFIRINOX plus bevacizumab in AtezoTRIBE study. 36Both the short follow-up times and the lack of precise and well-validated biomarkers for both studies reduced the study results' generalization and pointed out a need for further research on the role of chemotherapy plus ICI combinations in MSS CRC.However, recently published biomarker analyses from the AtezoTRIBE study demonstrated that higher immunoscore values were associated with greater immunotherapy benefits in patients with pMMR CRC. 108,109If similar results are replicated in future clinical trials, the immunoscore could be a biomarker for the use of ICIs in pMMR CRC.
Patients with BRAF-mutated MSS CRC have very poor prognosis despite the recent advances with BRAF inhibitors in second-line therapy. 110,111The activation of the BRAF-MEK-ERK pathway leads to proliferation, survival, and immune escape with widespread effects on the TME, including increased levels of IL-6, IL-10, and VEGF, impaired dendritic cell and T-lymphocyte maturation and increased expression of immunosuppressive immune checkpoints. 112,113onsidering that most of these effects could be reversed by ICIs, combining BRAF/MEK inhibitors with ICIs has significant potential in MSS CRC, similar to melanoma.The combination of encorafenib and cetuximab with nivolumab leads to an ORR of 45% in patients with MSS, 51 BRAFV600E metastatic CRC in a phase I/II trial leading to a randomized phase II. 51,78The same success was not achieved in 2 studies with atezolizumab plus cobimetinib, an MEK inhibitor in an unselected metastatic MSS CRC population. 45,67n addition to further advances in the ICI-VEGF, ICI-Chemotherapy, and ICI plus BRAF inhibitor, there are several other intriguing combinations based on combinations of immune modulators like CAR-T therapies 69 and immune activators 45 and ICI-ICI combinations. 23,24The combination of a next-generation anti-CTLA4 inhibitor, botensilimab, and balstilimab (anti-PD-1) was recently evaluated in 41 patients with heavily pretreated (median of 4 prior treatments) patients with MSS CRC.The ORR and DCR were 24% and 73%, respectively.Additionally, in patients without an active liver metastasis the ORR and DCR was 42% and 96% indicating the benefit is perhaps again limited to patients without active liver metastasis. 23Notably, in this study, no patient with active liver metastasis has responded to this combination.Half of the responding patients had RAS mutations, and only one responding had a high tumor mutational burden.Additionally, the POLE mutation was not present in 10 patients with a response to therapy.No grades 4-5 toxicities were observed. 23nother unmet need for research is the combination of anti-TGF-β inhibitors with ICIs.The TGF-β is the main driver of epithelial-to-mesenchymal (EMT) transition and a potent proliferative and pro-invasive signal in TME.Additionally, TGF-β has pivotal effects on immune surveillance.The TGF-β may have inhibitory effects on T-helper1 phenotype differentiation, CD8 T-cell activity, IL-2 expression, and natural cell killer proliferation and function and increased the percentage of immunosuppressive M2 macrophages and myeloid-derived suppressor cells (MDSC). 114he TGF-β pathway is active starting from the earlier phases of CRC carcinogenesis and is even more prominent in the CRC hepatic metastases.Due to these effects on the immune system and the association between TGF-beta and resistance to ICIs, combining TGF-β inhibitors with ICIs emerged as a feasible strategy to activate immune machinery. 115In a recent phase II open-label trial, the combination of vactosertib (selective TGF-β receptor I kinase inhibitor) and pembrolizumab led to an ORR of 15.2% in patients with MSS CRC who had progression under all available options.The combination was safe with grade 3 or higher treatmentrelated adverse events in 3 of 33 patients. 71While these results hold promise for the future, it should be noted that 82% of the patients had consensus molecular subtype (CMS) 4 disease, 71 a subtype in which TGF-β pathway alterations are pivotal. 116,117Further exploration of TGF-β and ICI combinations is needed and could be especially important for patients with CMS4 disease.Furthermore, patient stratification according to CMS molecular subtypes could be of benefit for treatment individualization and improvement of outcomes.Several other TME modifiers, like maraviroc, and 72 pixatimod were also combined with ICIs, although the success was limited, and no further studies are planned with these agents. 56here are several limitations in the systematic review, mostly inherent to the included studies.Most of the studies included had very limited sample sizes and included heterogeneous populations regarding the treatment line and previous treatments.The available studies mostly used regorafenib as the standard third-line treatment, although the current standard treatment is different in this setting.Additionally, detailed subgroup analyses were absent in most studies, limiting the ability to pool the data regarding possible subgroups with a higher benefit with ICIs.For example, patients without liver metastases seem to benefit more from ICIs; however, reporting for this subgroup of patients was not available in most papers.

Conclusion
In conclusion, in the systematic of ICI studies in MSS CRC, we observed that the ICI monotherapy was significantly limited in efficacy, while the combinations with anti-VEGF TKI, other immune-modulatory agents and BRAF inhibitors may have more efficacy.However, with varying results seen in phase II studies with TKI and ICI therapy combinations with a recently negative phase III trial of LEAP-17, the benefit of this combination in MSS CRC appears to be limited.The data regarding the chemotherapy plus ICI combinations in the first-line treatments reported modest benefits.Further research is needed to delineate the best strategies and best combinations to implement ICIs in the treatment of patients with MSS CRC.

Table 1 .
Immune checkpoint inhibitor monotherapy or dual immune checkpoint inhibitors.

Table 2 .
Immune checkpoint inhibitor and tyrosine kinase inhibitor combination.

Table 3 .
Immune checkpoint inhibitor and chemotherapy combinations.

Table 4 .
Other Combinations