ACHAIKI IATRIKI | 2022; 41(3):126-134
Geroge Zarkavelis1,2, Ioanna Gazouli1, Stefania Gkoura1, Nantezda Torounidou1, Aristeides Gogadis1, Eleftherios Kampletsas1,2, Davide Mauri1,2
1Department of Medical Oncology, University General Hospital of Ioannina, 45500 Ioannina, Greece
2Society for Study of Clonal Heterogeneity of Neoplasia (EMEKEN), 45444 Ioannina, Greece
Received: 13 Jan 2022; Accepted: 18 May 2022
Corresponding author: George Zarkavelis, MD, MSc, PhD, Medical Oncologist, University Hospital of Ioannina, E-mail: email@example.com
Key words: Colorectal cancer, metastatic, immunotherapy, microsatellite instability, pembrolizumab, nivolumab
The landscape of contemporary cancer therapeutics has changed significantly with the advent of immunotherapy. The constantly expanding indications of immune checkpoint inhibitors have resulted in improved clinical outcomes including colorectal cancer patients. Colon cancer is listed among the most common neoplasms with a quarter of newly diagnosed patients presenting with metastatic disease while a significant proportion of localized cases will eventually develop metastatic lesions. Apart from classic cytotoxic chemotherapy, targeted therapies based on tumor molecular profiling are the mainstay in colon cancer therapeutics. Immunotherapy is incorporated in the treatment algorithms for patients with advanced colorectal cancers whose tumors are found to be microsatellite unstable or mismatch repair (MMR) deficient with significant clinical benefit. On the other hand, patients with MMR proficient/microsatellite stable tumors do not seem to respond as well to immunotherapy. Clinical trials are underway to identify potential mechanisms for improving colorectal cancer patients’ outcomes, further deploy immune checkpoint inhibitors application and assess a variety of combinations of targeted therapies and immunotherapy either in the adjuvant or metastatic setting of the disease.
Colorectal cancer is among the most frequently diagnosed cancer types with high mortality rates [1,2]. Despite the efforts for early detection through screening programs, a quarter of all colorectal cancer patients present with metastatic disease at initial diagnosis while approximately 50% of patients will eventually develop metastases. The prognosis for advanced disease remains unfavorable despite the deployment of therapies . During the last two decades, cytotoxic chemotherapy has been the backbone of treatment, while the addition of targeted therapies based on molecular profiling and identification of actionable mutations of the tumors led to increased survival rates . Thus, novel therapies are under investigation to fulfill the unmet need for effective treatments for patients with advanced disease.
Without a doubt, immunotherapy changed the therapeutic landscape in oncology. Immunotherapy integration in contemporary therapeutics first took place in solid tumors like melanoma and lung cancer where it managed to achieve significantly improved response rates and longer survival. It has also proved to be effective in gastrointestinal cancers, especially hepatocellular and esophageal carcinoma, renal and urothelial cancer, squamous head and neck cancers while indications continue to expand to several neoplastic diseases .
In 2017, immunotherapy was approved by the FDA for the treatment of microsatellite instability high (MSI-h) or mismatch-repair-deficient (dMMR) metastatic colorectal cancer. This population represents only a small fragment of all metastatic colorectal cancer patients . On the other hand, patients with pMMR tumours do not seem to gain a similar benefit. The field of immunotherapy in colorectal cancer either as monotherapy or in combination remains challenging. This review aims to summarize the current status of immunotherapy application in CRC through the existing literature and appose future perspectives.
Rationale for immunotherapy application in colorectal cancer
Colorectal cancer not being conventionally regarded as an immune sensitive tumor, it is worth to investigate the primary implications of applying immunotherapy against it. It has been more than a decade since the significance of cytotoxic and helper immune T cells infiltrating the tumor microenvironment, has been recognised as a major prognostic factor of recurrence risk in patients with early-stage colorectal cancer . Sequentially, this led to the establishment of “Immunoscore”, an immunohistochemical assessment of the proportion of co-stimulatory CD3 and cytotoxic CD8 T lymphocytes present within the tumor microenvironment. Immunoscore was thereafter investigated as a prognostic marker of the recurrence probability of early-stage colorectal cancer after therapeutic surgery, as well as a probable predictive marker of adjuvant chemotherapy benefit , but it was not incorporated in routine clinical practice. Nonetheless, these observations set the pathway towards the deployment of the immune system against colorectal cancer .
In the era of gene-expression based research, molecular subtyping has been applied to colorectal cancer, identifying four subtypes: CMS1 or MSI-Immune, CMS2 or Canonical, CMS3 or Metabolic and CMS4 or Mesenchymal. CMS1, accounting for 14% of colorectal cancers, is characterized by a higher level of immune activation, probably associated with the molecular phenomenon of microsatellite instability, compared to the rest three, microsatellite stable types CMS 2, 3 and 4 [9,10]. This identification set a rational basis of employing immunotherapeutic approaches in colorectal cancer treatment, as well as for using microsatellite instability as a predictive biomarker of any probable clinical benefit.
Microsatellite instability (MSI) is a molecular characteristic implying defective DNA damage repair mechanisms, resulting in a disruption of repetitive DNA sequences, known as DNA microsatellites. The underlying mechanism is the loss or silencing of genes encoding four enzymes involved in the mismatch repair machinery, MLH1, MSH2, MSH6 and PMS2, a phenomenon described as mismatch repair deficiency (dMMR) [11,12]. MSI may be detected by PCR (Polymerase Chain Reaction) or NGS (Next Generation Sequencing) in either blood or paraffin tissue specimen, while dMMR is examined by applying immunohistochemistry on the tumor specimen, in order to check for the presence of all four mismatch repair enzymes. Although MSI/dMMR was originally identified among Lynch syndrome carriers, there has been evidence that these genetic characteristics may also arise from somatic tumor mutations and may be present in non-Lynch syndrome patients as well [13-15].
Genomic instability is thought to give rise to neoplastic neoantigens, prone to be detected and activate antigen presenting and cytotoxic immune cells, thus supporting the emerging role of pharmaceutical immune activators, in the treatment of MSI-high/dMMR CRC. Consequently, MSI/dMMR have been used as predictive biomarkers, promising to distinguish CRC patients more probable to benefit from immunotherapy agents, such as the widely employed immune checkpoint inhibitors [16,17].
MSI may as well be the result of epigenetic silencing of the involved repair genes [11,12]. Specifically, methylation of the promoter of the MLH1 gene, may result into genomic instability, due to reduced MLH1 production, without loss or mutation of the coding area, often co-existing with BRAF V600E mutation . Similarly, deletion of the EPCAM (Epithelial cell adhesion molecule) protein may result to MSH2 epigenetic methylation and silencing, thus leading to genomic instability and subsequent higher tumor immunogenicity [11,12].
Tumor mutational burden (TMB), stands out as a distinctive hallmark of tumor genomic instability and the basis for increased neoantigens variability, also emerging as an alternative predictive immunotherapy biomarker, detectable with molecular sequencing. Indeed, colorectal carcinomas with high mutational load have been shown to be more responsive to immunotherapy [19-22]. Although microsatellite instability and high tumor mutational burden both account for tumors rich in neoantigens, thus easily perceived by the hosts’ immune system and susceptible to immunotherapeutic agents, they should not be regarded as one and the same. In fact, increased tumor mutational burden, may issue from genetic and molecular deficits, other than mismatch repair deficiency, such as mutations in the exonuclease domain of DNA polymerases POLE and POLD1. Such mutations, also involved in familial colorectal and endometrial cancer cases, due to their high penetrance, compromise the proofreading capacity of the mutated enzymes, leading to accumulation of DNA misallied nucleotides during the DNA duplication phase [23,24]. Evidently, POLE and POLD1 mutations may give rise to microsatellite stable but hypermutated tumors; consequently, TMB and MSI/dMMR may be regarded as distinctive hallmarks of tumor neoantigen enrichment and may be independently examined as two separate predictive biomarkers of immunotherapy susceptibility .
Multiple immunotherapeutic strategies have been investigated so far, including interferon administration , CAR T-cells engineering (Chimeric antigen/antibody receptors T-cells) , vaccination with antigen presenting cells exposed to tumor neoantigens  or with viral vectors transporting genes of immunostimulatory molecules  and, remarkably, immune checkpoint inhibitors (ICIs). Indeed, ICIs have been successfully incorporated in everyday practice of clinical oncology during the last decade, providing realistic therapeutic solutions against solid tumors and hematologic malignancies, insensitive to traditional chemotherapeutic approaches [30,31].
Monoclonal antibodies targeting PD-1 (Programmed Death 1) and PD-L1 (Programmed Death ligand 1), are the most widely applied, because of their effectiveness and their manageable toxicity profile. Pembrolizumab  and nivolumab , both bind and inhibit PD-1, a receptor found on cytotoxic T-lymphocytes, which, when activated, suppresses T-lymphocyte expansion and activation; thus, its inhibition by anti-PD-1 monoclonal antibodies unleashes the cytotoxic potential of T-lymphocytes, against cancer cells. Especially nivolumab, is often co-administered with an older checkpoint inhibitor, ipilimumab [34,35]; this latter, blocks another T-lymphocyte molecular brake, a surface molecule named CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) and it has been the first immune checkpoint inhibitor ever put into clinical practice . As anti-CTLA-4 monotherapy with ipilimumab had a satisfactory effectiveness level only at high doses, at the cost of severe toxicities, its administration at lower doses, combined with nivolumab, has been established as a preferable strategy [37,38]. Cemiplimab is another anti-PD-1 monoclonal antibody, which has shown to be active against squamous cell carcinomas, non-small cell lung cancer and cervical cancer [39-41].
The anti-PD-L1 antibodies, such as durvalumab, atezolizumab and avelumab were later developed, targeting the PD-1 ligand, PD-L1, a molecule often located on the surface of tumor cells. It seems that PD-L1 binds with PD-1, activating the downregulation of cytotoxic lymphocytes, impeding their antineoplastic activity . Their efficacy has also been proven in several clinical trials, mostly in combination with chemotherapy [42,43], as maintenance treatments after response to first line treatment chemotherapy [44,45], as an alternative to chemotherapy when the latter is contraindicated due to patient comorbidities  or even as first line treatment options in non-chemosensitive neoplasms .
Based on the above, immunohistochemistry for PD-L1, either solely on cancer cells or in both immune and tumor cells, is employed as a predictive biomarker for ICIs against NSCLC , urothelial cancer , head and neck malignancies  and upper gastrointestinal tract tumors , despite its many controversies. Nonetheless, in colorectal cancer it is substituted by dMMR/MSI and TMB, which have been employed in the clinical trials of ICIs against colorectal malignancies, as predictive biomarkers.
Immunotherapy in the treatment of dMMR/MSI-high colorectal cancer
Given that MSI-high/dMMR has been established as an efficient predictive marker of immunotherapy benefit in colorectal cancer patients, it has served as a major patient recruitment criterion in pivotal immunotherapy trials.
The anti-PD-1 agent pembrolizumab, managed to induce objective responses in 40% of the MSI-high colorectal cancer patients, with a subsequent 20-week PFS of 78%, in a phase II single arm trial . In KEYNOTE-164, monotherapy with pembrolizumab at 200mg every three weeks, induced objective responses in pretreated patients with MSI-high colorectal cancers ranging from 21 up to 46% as a second or further line of treatment; responses were durable, as the median duration of response was not reached, during a follow up lasting up to 35.6 months. Severe adverse events of grade 3 or greater, affected about 13-16% of patients .
In the practice-changing clinical trial KEYNOTE-177 , pembrolizumab (200mg every 3 weeks), managed to induce superior clinical outcome in MSI-high colorectal cancer, treatment naïve patients, compared with 5-fluorouracil based chemotherapy, with or without anti-VEGF and anti-EGFR targeted agents. Pembrolizumab monotherapy reduced the probability of disease progression by 40%, prolonging median PFS from 8 to 16.5 months, and OS from 11 to 13.7 months, while it induced an overall response rate of 44% versus 33% for traditional antineoplastic treatment. 22% of patients on pembrolizumab monotherapy experienced severe adverse events, as opposed to two thirds of patients in the chemotherapy arm . A later assessement of quality of life of this study population determined that patients on pembrolizumab were twice as probable to maintain their level of physical and social activities, compared to patients receiving chemotherapy . Based on the above, pembrolizumab is now the recommended choice of treatment in the first line setting of metastatic patients with MSI-high colorectal cancer, being both tolerable and effective in this population.
Nivolumab has also shown significant clinical activity against MSI-high colorectal cancer. It has been examined as a second line treatment of metastatic pretreated patients, as a sole agent , as well as in combination with the anti-CTLA-4 agent ipilimumab , in the trial Checkmate 142. As monotherapy, (at 3mg/kg every 2wks), it managed to induce responses in one third of the patients, with most of them lasting beyond 3 months, with a PFS of 1 year, and a manageable toxicity profile (grade >3 AEs in up to 8% of patients) . When co-administered with ipilimumab (1 mg/kg every 3weeks) for the first 4 cycles, overall response rate increased up to 52 and 57%, for patients not experiencing and experiencing immunotherapy related toxicity, respectively. Severe AEs associated with the combination were observed in 32% of patients . More interestingly, in a third, more recent checkmate-142 cohort, nivolumab was administered to treatment naïve MSI-high colorectal cancer patients, achieving an ORR of 69%, with complete responses in 13% of patients, and a disease control rate of 84%. Median duration of response was not reached, while 74% were free of disease progression at 2 years of treatment . Although not yet head-to-head compared to chemotherapy, the combination of ipilimumab with nivolumab is now considered as a safe and effective option for the treatment of metastatic MSI-high colorectal cancer, even in the first line setting.
As for the anti-PD-L1 agents, avelumab has been explored as a second line treatment of MSI-high/dMMR colorectal cancer, including also tumors hosting POLE mutation. It was used at 10mg/kg every 2 weeks, resulting in an ORR of 24%, with median response duration of 14 months and a median PFS of 8.1 months among MSI-high cancer patients . So far, no anti-PD-L1 antibody has gained approval against MSI-high colorectal cancer.
Immunotherapy in patients with pMMR/MSS colorectal cancer
Although patients with dMMR/MSI-H colorectal cancer, experience durable responses and prolonged survival rates, patients with pMMR disease do not seem to benefit from these therapies, either as monotherapy application or as double inhibition. Extensive research has been done so far to better comprehend the profile of pMMR colorectal cancer. The main goal is to increase tumor immunogenicity to achieve responses to immunologic therapies. Most trials investigating immunotherapy in MSS and/or mixed population mainly focus on combinations of immune checkpoint inhibitors with standard chemotherapy (5-fluorouracil, oxaliplatin, irinotecan), radiotherapy, or targeted therapies and explore potential biomarkers, other than MSI. There is evidence that chemotherapy alters the intratumoral environment through the induction of immunogenic cell death. . Radiotherapy is also related to induced immunogenic cell death; it increases the number of infiltrating T cells while also having the abscopal effect . Moreover, targeted therapies for metastatic CRC such as anti – EGFR and anti-VEFG antibodies (cetuximab and bevacizumab respectively), seem to enhance the immunotherapeutic effects .
Several international studies have been conducted to evaluate the efficacy of PD-L1 agent combined with antiangiogenesis. The researchers of the BACCI trial, a placebo-controlled randomised phase II study, assessed the efficacy of atezolizumab combined with capecitabine and bevacizumab in metastatic colorectal cancer. The study population consisted mainly of MSS metastatic colorectal cancer patients. The entire study population reached a better PFS with the addition of atezolizumab. Especially, regarding the pMMR population, the PFS benefit from atezolizumab was notable, however RR and OS remained almost the same .
Another study aiming to evaluate the use of bevacizumab and atezolizumab in this setting based on biomarkers was the MODUL trial, a randomised phase III international umbrella trial. Patients with wild-type BRAF colon cancers underwent therapy with FOLFOX and bevacizumab followed by maintenance therapy of fluorouracil and bevacizumab either with or without atezolizumab as first-line treatment for mCRC. This study was a negative trial as PFS and OS were similar in both study arms .
The IMBlaze 370 trial assessed the efficacy of atezolizumab in addition to the MEK inhibitor cobimetinib. The study included 363 patients previously treated for metastatic colorectal cancer, stratified into three arms. The first one received the combination atezolizumab – cobimetinb, the second arm received atezolizumab alone and the third was on regorafenib. Most of the patients harbored microsatellite stable tumors. The median overall survival was 8.9 months for the atezolizumab – cobimetinib arm, similar to regorafenib which was 8,5 months. Atezolizumab monotherapy failed to improve mOS. In general, none of the three arms achieved significant differences in terms of OS, PFS, OR . Moreover, in a small study combining the anti-PD-1 agent SHR-1210 with apatinib, in MSS mCRC patients, no benefit was achieved, either in OS or PFS .
Anti-PD-1 agent nivolumab has been tested in different combinations, in a series of clinical trials. As mentioned above, in the CheckMate 142 phase II study, the nivolumab plus ipilimumab combination achieved objective response rates as high as 69%, among MSI high mCRC patients [56-58]. As for MSS/pMMR mCRC, Li J. et al, in a retrospective review of 23 pretreated patient cases of MSS/pMMR mCRC, noticed that the combination of variable anti-PD1 monoclonic antibodies, with the VEGFR inhibitor regorafenib, induced a disease control rate of 78.3%, although without any benefit in terms of overall response rate and a modest median PFS of 3 months . Furthermore, the Japanese REGONIVO phase Ib study showed that the combination of nivolumab and regorafenib had emboldened results in response rate . On the contrary, Fakih M et al recently reported the results of a single-arm phase II study, where the same combination, resulted in worse outcomes in the North American population .
Moreover, a significant number of clinical trials is evaluating the synergistic effects of immunotherapy and anti-EGFR antibodies combination in MSS CRC patients. The CAVE colon phase II trial analyzed the effectiveness of avelumab combined with cetuximab as a rechallenge in pretreated, RAS wild type, pMMR metastatic colorectal cancer patients. The trial met the primary endpoint reaching a median OS of 11,6 months, suggesting that the combination represents an active, well-tolerated therapeutic option . Similarly, in the AVETUX trial, mFOLFOX6 combined with cetuximab and avelumab was tested in patients with RAS/BRAF wild type, mCRC. From a total study population of 43, 40 patients harbored pMMR tumors. The results of this single-arm phase II study, indicate a high response rate in MSS patients .
Preclinical data indicating a potential synergistic effect between immunotherapy and radiotherapy application led to the investigation of the combination in small scale clinical trials. Published results suggest a manageable toxicity profile and noticeable responses in patients with advanced pretreated pMMR metastatic disease and guarantee the further exploration of this strategy .
Currently, numerous clinical trials are exploring the possibilities of immunotherapy in the treatment of metastatic colorectal cancer . Tolerability and efficacy of administrating immune check point along with targeted treatments, is still under investigation, in phase I/II trials, such as NCT03657641, combining pembrolizumab with regorafenib, in colorectal cancer patients beyond the 2nd line of treatment. The MAP kinases pathway inhibitors encorafenib (BRAF inhibitor) and binimetinib (MEK inhibitor), have already proven their value, showing clinical benefit in patients carrying the BRAF V600E mutation, resulting in the recent approval of combination of the anti-EGFR monoclonal antibody cetuximab and encorafenib for the treatment of BRAF mutated colorectal cancer . At present, co-administration of encorafenib and binimetinib along with nivolumab (NCT04044430) is under examination, in MSS stable, BRAF V600E mutation carriers.
Chemotherapy in combination with immunotherapy is also an intriguing option; temozolomide is a well-established alkyliotic agent, applied against glioblastoma multiform. It seems that resistance against temozolomide is mediated by a DNA repair enzyme, known as O6-methylguanine DNA methyltransferase (MGMT), whose methylation and epigenetic silencing confer susceptibility to temozolomide . Building on that, temozolomide is now tested in combination with nivolumab and ipilimumab, against MSS stable but MGMT methylated metastatic colorectal cancer (NCT03832621). Similarly, the combination of avelumab with irinotecan and cetuximab, (NCT03608046) as well as of pembrolizumab with oxaliplatin, capecitabine and bevacizumab are also under investigation (NCT04262687), against treatment refractory, pMMR stable colorectal cancer.
Immune checkpoint inhibitors may also be combined with novel agents; ALX148 is a new immune checkpoint inhibitor, binding on CD47, a molecule found on cancer cells, serving to the suppression of immunostimulatory potential of myeloid cells . Phase II trial NCT05167409 is using triple blockade with AXL148 (anti-CD47), pembrolizumab (anti-PD-1) and cetuximab (anti-EGFR), against chemotherapy refractory, MSS stable colorectal cancer. More recently, the comparative study NCT04854434, has started recruiting colorectal cancer patients carrying RAS mutations, aiming to assess the potential benefits of combined treatment with selinexor and pembrolizumab. Selinexor is a novel oral agent, inhibiting exportin 1, an intracellular protein involved in the transport of oncogenic mediators from the nucleus to the cytosol, promoting oncogenesis, already used against hematologic malignancies .
Immunotherapy has not yet been incorporated in the adjuvant/neoadjuvant treatment setting of colorectal cancer. Nonetheless, a recent, small exploratory phase I trial , showed that the administration of ipilimumab plus nivolumab, was associated with pathologic response rates of 100% and 27% among MSI-high and MSS stable patients, respectively. Moreover, pembrolizumab, together with vactosertib, an inhibitor of the TGF-beta oncogenic pathway , is now examined in patients having undergone hepatic metastasectomy (NCT03844750), in addition to classic perioperative chemotherapy.
Colorectal cancer still imposes major therapeutic challenges on patients and physicians, regardless of the broad variety of antineoplastic treatment choices nowadays available. Up to date, microsatellite instability has served as the cornerstone of applying immunotherapy against colorectal cancer, without providing realistic solutions for pMMR, non-hypermutated colorectal cancer types. Ongoing research aims to overcome this barrier, as well as to provide clinicians with proficient, evidence-based treatment algorithms, so that immunotherapeutic, targeted, and cytotoxic agents may be administered, sequentially or contemporarily, in a way that maximizes clinical benefit.
Conflict of interest disclosure
None to declare
Declaration of funding sources
None to declare
All authors contributed equally to the current work.
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