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: firstname.lastname@example.org
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.
1. Van Cutsem E, Cervantes A, Nordlinger B, Arnold D, ESMO Guidelines Working Group. Metastatic colorectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2014;25 Suppl 3:iii1-9.
2. Cronin KA, Lake AJ, Scott S, Sherman RL, Noone AM, Howladeret N, et al. Annual report to the nation on the status of cancer, part I: National cancer statistics. Cancer 2018; 124(13):2785-800.
3. Goodwin RA and Asmis TR. Overview of systemic therapy for colorectal cancer. Clin Colon Rectal Surg. 2009;22(4):251-6.
4. Stein A, Moehler M, Trojan J, Goekkurt E, Vogel A. Immuno-oncology in GI tumours: Clinical evidence and emerging trials of PD-1/PD-L1 antagonists. Crit Rev Oncol Hematol. 2018;130:13-26.
5. Battaglin F, Naseem M, Lenz HJ, Salem ME. Microsatellite instability in colorectal cancer: overview of its clinical significance and novel perspectives. Clin Adv Hematol Oncol. 2018;16(11):735-745.
6. Pagès F, Kirilovsky A, Mlecnik B, Asslaber M, Tosolini M, Bindea G, et al. In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. J Clin Oncol. 2009;27(35):5944-51.
7. Mlecnik B, Bifulco C, Bindea G, Marliot F, Lugli A, Lee JJ, et al. Multicenter International Society for Immunotherapy of Cancer Study of the Consensus Immunoscore for the Prediction of Survival and Response to Chemotherapy in Stage III Colon Cancer. J Clin Oncol. 2020;38(31):3638-51.
8. Angell HK, Bruni D, Barrett JC, Herbst R, Galon J. The Immunoscore: Colon Cancer and Beyond. Clin Cancer Res. 2020;26(2):332-9.
9. Inamura K. Colorectal Cancers: An Update on Their Molecular Pathology. Cancers (Basel). 2018;10(1):26.
10. Chowdhury S, Hofree M, Lin K, Maru D, Kopetz S, Shen JP. Implications of Intratumor Heterogeneity on Consensus Molecular Subtype (CMS) in Colorectal Cancer. Cancers (Basel). 2021;13(19):4923.
11.Yamamoto H, Imai K. Microsatellite instability: an update. Arch Toxicol. 2015;89(6):899-921.
12. Li K, Luo H, Huang L, Luo H, Zhu X. Microsatellite instability: a review of what the oncologist should know. Cancer Cell Int. 2020;20:16.
13. Dedeurwaerdere F, Claes KB, Van Dorpe J, Rottiers I, Van der Meulen J, Breyne J, et al. Comparison of microsatellite instability detection by immunohistochemistry and molecular techniques in colorectal and endometrial cancer. Sci Rep. 2021;11(1):12880.
14. Zhu L, Huang Y, Fang X, Liu C, Deng W, Zhong C, et al. A Novel and Reliable Method to Detect Microsatellite Instability in Colorectal Cancer by Next-Generation Sequencing. J Mol Diagn. 2018;20(2):225-31.
15. Svrcek M, Lascols O, Cohen R, Collura A, Jonchère V, Fléjou JF, et al. MSI/MMR-deficient tumor diagnosis: Which standard for screening and for diagnosis? Diagnostic modalities for the colon and other sites: Differences between tumors. Bull Cancer. 2019;106(2):119-28.
16. Ganesh K, Stadler ZK, Cercek A, Mendelsohn RB, Shia J, Segal NH, et al. Immunotherapy in colorectal cancer: rationale, challenges and potential. Nat Rev Gastroenterol Hepatol. 2019;16(6):361-75.
17. Lichtenstern CR, Ngu RK, Shalapour S, Karin M. Immunotherapy, Inflammation and Colorectal Cancer. Cells. 2020;9(3):618.
18. Bouzourene H, Hutter P, Losi L, Martin P, Benhattar J. Selection of patients with germline MLH1 mutated Lynch syndrome by determination of MLH1 methylation and BRAF mutation. Fam Cancer. 2010;9(2):167-72.
19. Chalmers ZR, Connelly CF, Fabrizio D, Gay L, Ali SM, Ennis R, et al. Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden. Genome Med. 2017;9(1):34.
20. Schrock AB, Ouyang C, Sandhu J, Sokol E, Jin D, Ross JS, et al. Tumor mutational burden is predictive of response to immune checkpoint inhibitors in MSI-high metastatic colorectal cancer. Ann Oncol. 2019;30(7):1096-103.
21. Chan TA, Yarchoan M, Jaffee E, Swanton C, Quezada SA, Stenzinger A, et al. Development of tumor mutation burden as an immunotherapy biomarker: utility for the oncology clinic. Ann Oncol. 2019;30(1):44-56.
22. Innocenti F, Ou FS, Qu X, Zemla TJ, Niedzwiecki D, Tam R, et al. Mutational Analysis of Patients With Colorectal Cancer in CALGB/SWOG 80405 Identifies New Roles of Microsatellite Instability and Tumor Mutational Burden for Patient Outcome. J Clin Oncol. 2019;37(14):1217-27.
23. Palles C, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, et al. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet. 2013;45(2):136-44.
24. Mur P, García-Mulero S, Del Valle J, Magraner-Pardo L, Vidal A, Pineda M, et al. Role of POLE and POLD1 in familial cancer. Genet Med. 2020;22(12):2089-100.
25. Picard E, Verschoor CP, Ma GW, Pawelec G. Relationships Between Immune Landscapes, Genetic Subtypes and Responses to Immunotherapy in Colorectal Cancer. Front Immunol. 2020;11:369.
26. Tarhini AA, Gogas H, Kirkwood JM. IFN-α in the treatment of melanoma. J Immunol. 2012;189(8):3789-93.
27. Marcus A, Eshhar Z. Allogeneic chimeric antigen receptor-modified cells for adoptive cell therapy of cancer. Expert Opin Biol Ther. 2014;14(7):947-54.
28. Thomas S, Prendergast GC. Cancer Vaccines: A Brief Overview. Methods Mol Biol. 2016;1403:755-61.
29. Gulley JL, Borre M, Vogelzang NJ, Ng S, Agarwal N, Parker CC, et al. Phase III Trial of PROSTVAC in Asymptomatic or Minimally Symptomatic Metastatic Castration-Resistant Prostate Cancer. J Clin Oncol. 2019;37(13):1051-61.
30. Marin-Acevedo JA, Kimbrough EO, Lou Y. Next generation of immune checkpoint inhibitors and beyond. J Hematol Oncol. 2021;14(1):45.
31. Yan Y, Zhang L, Zuo Y, Qian H, Liu C. Immune Checkpoint Blockade in Cancer Immunotherapy: Mechanisms, Clinical Outcomes, and Safety Profiles of PD-1/PD-L1 Inhibitors. Arch Immunol Ther Exp (Warsz). 2020;68(6):36.
32. Kwok G, Yau TC, Chiu JW, Tse E, Kwong YL. Pembrolizumab (Keytruda). Hum Vaccin Immunother. 2016;12(11):2777-89.
33. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, et al. PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome. Front Pharmacol. 2017;8:561.
34. Hodi FS, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Cowey CL, et al. Nivolumab plus ipilimumab or nivolumab alone versus ipilimumab alone in advanced melanoma (CheckMate 067): 4-year outcomes of a multicentre, randomised, phase 3 trial. Lancet Oncol. 2018;19(11):1480-92.
35. Motzer RJ, Rini BI, McDermott DF, Arén Frontera O, Hammers HJ, Carducci MA, et al. Nivolumab plus ipilimumab versus sunitinib in first-line treatment for advanced renal cell carcinoma: extended follow-up of efficacy and safety results from a randomised, controlled, phase 3 trial. Lancet Oncol. 2019;20(10):1370-85.
36. Lipson EJ, Drake CG. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma. Clin Cancer Res. 2011;17(22):6958-62.
37. Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013;369(2):122-33.
38. Postow MA, Chesney J, Pavlick AC, Robert C, Grossmann K, McDermott D, et al. Nivolumab and ipilimumab versus ipilimumab in untreated melanoma. N Engl J Med. 2015;372(21):2006-17.
39. Villani A, Ocampo-Garza SS, Potestio L, Fabbrocini G, Ocampo-Candiani J, Ocampo-Garza J, et al. Cemiplimab for the treatment of advanced cutaneous squamous cell carcinoma. Expert Opin Drug Saf. 2022;21(1):21-9.
40. Wang L, Peng Y, Zeng X, Peng L, Li S, Qin S, et al. Cost-Effectiveness Analysis of Cemiplimab Versus Chemotherapy as First-Line Treatment in Advanced NSCLC with PD-L1 Expression Levels of at Least 50. Adv Ther. 2021;38(8):4354-65.
41. Rischin D, Gil-Martin M, González-Martin A, Braña I, Hou JY, Cho D, et al. PD-1 blockade in recurrent or metastatic cervical cancer: Data from cemiplimab phase I expansion cohorts and characterization of PD-L1 expression in cervical cancer. Gynecol Oncol. 2020;159(2):322-8.
42. Herbst RS, Giaccone G, de Marinis F, Reinmuth N, Vergnenegre A, Barrios CH, et al. Atezolizumab for First-Line Treatment of PD-L1-Selected Patients with NSCLC. N Engl J Med. 2020;383(14):1328-39.
43. Mansfield AS, Każarnowicz A, Karaseva N, Sánchez A, De Boer R, Andric Z, et al. Safety and patient-reported outcomes of atezolizumab, carboplatin, and etoposide in extensive-stage small-cell lung cancer (IMpower133): a randomized phase I/III trial. Ann Oncol. 2020;31(2):310-7.
44. Paz-Ares L, Dvorkin M, Chen Y, Reinmuth N, Hotta K, Trukhin D et al. Durvalumab plus platinum-etoposide versus platinum-etoposide in first-line treatment of extensive-stage small-cell lung cancer (CASPIAN): a randomised, controlled, open-label, phase 3 trial. Lancet. 2019;394(10212):1929-39.
45. Powles T, Park SH, Voog E, Caserta C, Valderrama BP, Gurney H, et al. Avelumab Maintenance Therapy for Advanced or Metastatic Urothelial Carcinoma. N Engl J Med. 2020;383(13):1218-30.
46. Balar AV, Galsky MD, Rosenberg JE, Powles T, Petrylak DP, Bellmunt J, et al. Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial. Lancet. 2017;389(10064):67-76.
47. Kaufman HL, Russell J, Hamid O, Bhatia S, Terheyden P, D’Angelo SP, et al. Avelumab in patients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 2 trial. Lancet Oncol. 2016;17(10):1374-85.
48. Bodor JN, Boumber Y, Borghaei H. Biomarkers for immune checkpoint inhibition in non-small cell lung cancer (NSCLC). Cancer. 2020;126(2):260-70.
49. Stenehjem DD, Tran D, Nkrumah MA, Gupta S. PD1/PDL1 inhibitors for the treatment of advanced urothelial bladder cancer. Onco Targets Ther. 2018;11:5973-89.
50. Gavrielatou N, Doumas S, Economopoulou P, Foukas PG, Psyrri A. Biomarkers for immunotherapy response in head and neck cancer. Cancer Treat Rev. 2020;84:101977.
51. Joshi SS, Maron SB, Catenacci DV. Pembrolizumab for treatment of advanced gastric and gastroesophageal junction adenocarcinoma. Future Oncol. 2018;14(5):417-30.
52. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med. 2015;372(26):2509-20.
53. Le DT, Kim TW, Van Cutsem E, Geva R, Jäger D, Hara H, et al. Phase II Open-Label Study of Pembrolizumab in Treatment-Refractory, Microsatellite Instability-High/Mismatch Repair-Deficient Metastatic Colorectal Cancer: KEYNOTE-164. J Clin Oncol. 2020;38(1):11-9.
54. André T, Shiu KK, Kim TW, Jensen BV, Jensen LH, Punt C, et al. Pembrolizumab in Microsatellite-Instability-High Advanced Colorectal Cancer. N Engl J Med. 2020;383(23):2207-18.
55. Andre T, Amonkar M, Norquist JM, Shiu KK, Kim TW, Jensen BV, et al. Health-related quality of life in patients with microsatellite instability-high or mismatch repair deficient metastatic colorectal cancer treated with first-line pembrolizumab versus chemotherapy (KEYNOTE-177): an open-label, randomised, phase 3 trial. Lancet Oncol. 2021;22(5):665-77.
56. Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz HJ, Morse MA, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol. 2017;18(9):1182-91.
57. Morse MA, Overman MJ, Hartman L, Khoukaz T, Brutcher E, Lenz HJ, et al. Safety of Nivolumab plus Low-Dose Ipilimumab in Previously Treated Microsatellite Instability-High/Mismatch Repair-Deficient Metastatic Colorectal Cancer. Oncologist. 2019;24(11):1453-61.
58. Lenz HJ, Van Cutsem E, Luisa Limon M, Wong KYM, Hendlisz A, Aglietta M, et al. First-Line Nivolumab Plus Low-Dose Ipilimumab for Microsatellite Instability-High/Mismatch Repair-Deficient Metastatic Colorectal Cancer: The Phase II CheckMate 142 Study. J Clin Oncol. 2022;40(2):161-70.
59. Kim JH, Kim SY, Baek JY, Cha YJ, Ahn JB, Kim HS, et al. A Phase II Study of Avelumab Monotherapy in Patients with Mismatch Repair-Deficient/Microsatellite Instability-High or POLE-Mutated Metastatic or Unresectable Colorectal Cancer. Cancer Res Treat. 2020;52(4):1135-44.
60. Østrup O, Dagenborg VJ, Rødland EA, Skarpeteig V, Silwal-Pandit L, Grzyb K, et al. Molecular signatures reflecting microenvironmental metabolism and chemotherapy-induced immunogenic cell death in colorectal liver metastases. Oncotarget. 2017;8(44):76290-304.
61. Walle T, Martinez Monge R, Cerwenka A, Ajona D, Melero I, et al. Radiation effects on antitumor immune responses: current perspectives and challenges. Ther Adv Med Oncol. 2018;10:1758834017742575.
62. Osada T, Chong G, Tansik R, Hong T, Spector N, Kumar R, et al. The effect of anti-VEGF therapy on immature myeloid cell and dendritic cells in cancer patients. Cancer Immunol Immunother. 2008;57(8):1115-24.
63. Mettu N, Twohy E, Ou F, Halfdanarson T, Lenz H, Breakstone R, et al. BACCI: A phase II randomized, double-blind, multicenter, placebo-controlled study of capecitabine (C) bevacizumab (B) plus atezolizumab (A) or placebo (P) in refractory metastatic colorectal cancer (mCRC): An ACCRU network study. Ann Oncol. 2019;30:v203.
64. Grothey A, Tabernero J, Arnold D, De Gramont A, Ducreux M, O’Dwyer P, et al. Fluoropyrimidine (FP) + bevacizumab (BEV) + atezolizumab vs FP/BEV in BRAFwt metastatic colorectal cancer (mCRC): Findings from Cohort 2 of MODUL – a multicentre, randomized trial of biomarker-driven maintenance treatment following first-line induction therapy. Ann Oncol. 2018;29:viii714-viii715.
65. Eng C, Kim TW, Bendell J, Argilés G, Tebbutt NC, Di Bartolomeo M, et al. Atezolizumab with or without cobimetinib versus regorafenib in previously treated metastatic colorectal cancer (IMblaze370): a multicentre, open-label, phase 3, randomised, controlled trial. Lancet Oncol. 2019;20(6):849-61.
66. Ren C, Mai ZJ, Jin Y, He MM, Wang ZQ, Luo HY, et al. Anti-PD-1 antibody SHR-1210 plus apatinib for metastatic colorectal cancer: a prospective, single-arm, open-label, phase II trial. Am J Cancer Res. 2020;10(9):2946-54.
67. Li J, Cong L, Liu J, Peng L, Wang J, Feng A, et al. The Efficacy and Safety of Regorafenib in Combination With Anti-PD-1 Antibody in Refractory Microsatellite Stable Metastatic Colorectal Cancer: A Retrospective Study. Front Oncol. 2020;10:594125.
68. Fukuoka S, Hara H, Takahashi N, Kojima T, Kawazoe A, Asayama M, et al. Regorafenib Plus Nivolumab in Patients With Advanced Gastric or Colorectal Cancer: An Open-Label, Dose-Escalation, and Dose-Expansion Phase Ib Trial (REGONIVO, EPOC1603). J Clin Oncol. 2020;38(18):2053-61.
69. Fakih M, Raghav K, Chang D, Bendell J, Larson T, Cohn A, et al. Single-arm, phase 2 study of regorafenib plus nivolumab in patients with mismatch repair-proficient (pMMR)/microsatellite stable (MSS) colorectal cancer (CRC). J Clin Oncol. 2021;39(15_suppl):3560.
70. Troiani T, Martinelli E, Ciardiello D, Zanaletti N, Cardone C, Borrelli C, et al. Phase II study of avelumab in combination with cetuximab in pre-treated RAS wild-type metastatic colorectal cancer patients: CAVE (cetuximab-avelumab) Colon. J Clin Oncol. 2019;37(4_suppl):TPS731.
71. Stein A, Binder M, Goekkurt E, Lorenzen S, Riera-Knorrenschild J, Depenbusch R, et al. Avelumab and cetuximab in combination with FOLFOX in patients with previously untreated metastatic colorectal cancer (MCRC): Final results of the phase II AVETUX trial (AIO-KRK-0216). J Clin Oncol. 2020;38(4_suppl):96.
72. Segal N, Kemeny N, Cercek A, Reidy D, Raasch P, Warren P, et al. Non-randomized phase II study to assess the efficacy of pembrolizumab (Pem) plus radiotherapy (RT) or ablation in mismatch repair proficient (pMMR) metastatic colorectal cancer (mCRC) patients. J Clin Oncol. 2016;34(15_suppl):3539.
73. National Library of Medicine (U.S.), https://clinicaltrials.gov/ Accessed: 29th of December 2021.
74. Kopetz S, Grothey A, Yaeger R, Van Cutsem E, Desai J, Yoshino T, et al. Encorafenib, Binimetinib, and Cetuximab in BRAF V600E-Mutated Colorectal Cancer. N Engl J Med. 2019;381(17):1632-43.
75. Wu S, Li X, Gao F, de Groot JF, Koul D, Yung WKA. PARP-mediated PARylation of MGMT is critical to promote repair of temozolomide-induced O6-methylguanine DNA damage in glioblastoma. Neuro Oncol. 2021;23(6):920-31.
76. Chow L, Gainor J, Lakhani N, Lee K, Chung H, Lee J, et al. A phase I study of ALX148, a CD47 blocker, in combination with standard anticancer antibodies and chemotherapy regimens in patients with advanced malignancy. J Clin Oncol. 2020;38(15_suppl):3056.
77. Abdul Razak AR, Mau-Soerensen M, Gabrail NY, Gerecitano JF, Shields AF, Unger TJ, et al. First-in-Class, First-in-Human Phase I Study of Selinexor, a Selective Inhibitor of Nuclear Export, in Patients With Advanced Solid Tumors. J Clin Oncol. 2016;34(34):4142-50.
78. Chalabi M, Fanchi LF, Dijkstra KK, Van den Berg JG, Aalbers AG, Sikorska K, et al. Neoadjuvant immunotherapy leads to pathological responses in MMR-proficient and MMR-deficient early-stage colon cancers. Nat Med. 2020;26(4):566-76.
79. Jung SY, Yug JS, Clarke JM, Bauer TM, Keedy VL, Hwang S, et al. Population pharmacokinetics of vactosertib, a new TGF-β receptor type Ι inhibitor, in patients with advanced solid tumors. Cancer Chemother Pharmacol. 2020;85(1):173-83.