Colorectal cancer (CRC) represents the second most common cancer in Europe with marked differences in prognosis and response to treatments. In the past years research showed emerging interest in genomic and immunologic fields. The clinical heterogeneity, that occurs during the pathogenesis of CRC, is driven by chromosomal alterations and defective function of DNA mismatch repair genes. CRC is classified in four consensus molecular subtypes (CMS) with different immunogenic characteristics and prognosis. CMS1 microsatellite instable (MSI)-like and CMS4, both characterized by high levels of immune infiltration, are recognized as the most immunogenic subtypes, even though functional characteristic leading to different prognosis are reported. In particular, MSI tumors have been identified as the best candidates for immunotherapy treatment and a number of studies have evaluated the efficacy of anti-programmed cell death ligand-1 (PDL-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in this setting. However, literature data show that the majority of patients with CRC have microsatellite stable (MSS) tumors and this status seems related to lower response to PDL-1/programmed cell death-1 or CTLA4 blockade. The aim of this paper is to investigate the role of immunotherapy in MSI and MSS CRC.
Colorectal cancer (CRC) represents the second most common cancer in Europe with significant heterogeneity in prognosis and response to treatment. Prognostic factors include stage of disease, site of metastasis, and type of treatment given. Tumor genetic mutations gained a pivotal role as the prognostic factor. To date, the median overall survival (OS) for patients with metastatic CRC is about 30 months[
In the past years, research on CRC has shown an emerging interest in genomic and immunologic fields. The clinical heterogeneity that occurs during the pathogenesis of CRC is driven by chromosomal alterations and defective function of DNA mismatch repair (MMR) systems[
Microsatellites are defined as areas within the DNA sequence where a single nucleotide (mononucleotide) or units of two or more nucleotides are repeated in genome. They are usually located in the introns of genes and the number of repeats contained in every microsatellite is usually preserved in every single cell of the body[
Clinical and biologicals differences between dMMR and pMMR are well established. Specifically, dMMR causes genetic instability (aneuploidy, allelic losses, amplifications, translocations, and chromosomal gains) that influences the expression of genes leading to CRC carcinogenesis[
Conventionally, clinical and pathological features, along with tumor characteristics, are known to define cancer aggressiveness. Nevertheless, in the past years, tumor microenvironment (TME) has shown to play an important role in tumor growth and metastatic potential. TME is composed of epithelial cells, blood and lymphatic vessels, stromal cells, and infiltrating immune cells, including T lymphocytes, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages, and granulocytes. Each tumor displays a specific composition of TME and CRC shows a high degree of immune cell infiltration and high presence of mesenchymal stromal cells[
Studies in this field highlighted that different constituents of TME may influence tumor proliferation, infiltration and metastatic spread in different ways. Cancer growth or inhibition represents the result of the interplay between tumor cells and TME. Immune system has been demonstrated to be a key-mechanism of tumor regulation.
Immune system recruits, in cancer surveillance, the coordinated and balanced activation of both innate immune cells [such as macrophages, neutrophils, myeloid derived suppressor cells (MDSC), mast cells, eosinophils, and antigen-presenting cells (APCs)] and adaptive immune cells (NK cells, T and B lymphocytes cells)[
At first, innate immune system is recruited by abnormal cells without specific antigen recognition and inflammatory response is activated promoting angiogenesis and tumor cells proliferation. Later, adaptive immune response is triggered by interaction and recognition between non-self-antigens and peptides presented by the major histocompatibility complexes (MHC) of APCs and T cells[
Immune system cells play different roles during tumor immune response. CD4+ cells sustain inflammatory response by secreting a variety of cytokines such as interferon γ, tumor necrosis factor α, interleukin-2 (IL-2), and IL-17. CD4+ cell activation promotes proliferation and function of a specific subgroup of CD8+ cells called cytotoxic T lymphocytes, that are capable of direct lysis of tumor cells. CD8+ cells can also secrete cytokines causing cytotoxic response. NK cells are involved in antibody-dependent cell-mediated cytotoxicity and natural cytolytic activity against tumor cells. Macrophages destroy cancer cells through phagocytosis and release matrix-degrading substances (metalloproteinases and cysteine cathepsin proteases). Consequently, high levels of metalloproteinase represent an important factor to predict CRC prognosis and metastasis[
Part of the cells described above make up tumor-infiltrating lymphocytes (TILs) that showed to have a prognostic role in cancer treatment and appeared often to be associated with better clinical outcomes[
Mesenchymal stem cells (MSC) are non-hematopoietic stromal cells with proliferative potential, immunosuppressive properties, and ability to differentiate into several cell types. Their immunosuppressive function is releasing of proinflammatory factors, inhibiting lymphocyte proliferation and DCs maturation, promoting the production of macrophages, and regulating T cells (Treg). MSC are also involved in tumor initiation, angiogenesis, resistance to chemotherapy, invasion and metastatic process.
Criteria such as composition, density and location of TILs have shown to correlate with different prognosis indicators. Notably, in CRC the number and location of cytotoxic and memory T lymphocytes can predict tumor recurrence and prognosis in early-stage CRC[
Tumors cells are well known to develop strategies of immune escape. Indeed, they may show genetic alterations that enhance the expression of mesenchimal transition or immunosuppressive genes along with chemokines responsible for immune suppressive cells recruitment, conferring to cancer cells innate resistance to anti-programmed cell death-1 (PD-1) drugs. Different mutations might be responsible for resistance acquired after an initial benefit out of immunotherapy; during clonal expansion a resistant clone develops high proliferation potential and drives resistance advance.
For example, loss-of-function mutations in Janus Kinases 1/2 (JAK 1/2) might be responsible both for primary and adaptive resistance to immunotherapy. These inactivating mutations affect interferon gamma signaling rendering cancer cells unable to respond to interferon gamma by expressing programmed cell death ligand-1 (PDL-1) and other interferon-stimulated genes, and patients with such tumors became unlikely to respond to PD-1 blockade therapy. This mechanism has already been described in melanoma patients. Zaretsky
Another mechanism that has been accounted for acquired resistance to immunotherapy in melanoma is inactivation of beta-2-microglobulin (B2M), a fundamental component of the antigen-presenting MHC I. Le
The tight interaction between tumor and immune system has driven to the hypothesis of cancer immunoediting. This concept reinvented tumor immunosurveillance taking into account the dual role played by immune responses as host-protective and tumor-promoting. According to immunoediting cancer growth is structured in three different phases: elimination, equilibrium and escape. In the elimination phase immune system engages both innate and adaptive response to eliminate developing tumors before they become clinically evident. If this phase is satisfactorily fulfilled and the tumor results fully eradicated, the whole process might be considered completed. However if a single cancer cell variant escapes the elimination phase it proceeds to the equilibrium phase. During the second phase clonal growth of selected cell variant is prevented by immune system, but those cells still survive in a state of dormancy. Notably, adaptive responses are engaged in the equilibrium phase which is also the time of cancer immunoediting. Also equilibrium might be the end of the entire process whether the immune system keeps under control the “survivor cells” for the lifetime of the host. Nevertheless, the continuous immune pressure on tumor cells may lead them to enter the escape phase. In this third phase tumor variants elude immune system with different mechanisms and they outgrow to clinically apparent cancer[
As previously reported CRC clinical pathological characteristics and tumor TMN stage largely affect CRC prognosis and drive treatment choices along with mutation in
Furthermore, an international consortium of experts has introduced a gene expression and immune -based classification system: the “consensus molecular subtypes” of CRC, providing new prognostic and predictive tools[
Becht
Regarding the others subgroups, CMS2 and CMS3 that occur approximately in 50% of CRC, have low immune and inflammatory infiltration and, intermediate prognosis[
Also tumor genetic signature has a strong prognostic value. It is reported that stromal composition might strongly affect tumor transcriptional profile hiding tumor cell intrinsic transcriptional traits, especially in those tumors whose gene expression is largely sustained by stromal cells. Using patient-derived xenografts, Isella
Many of these traits differ from those reported in other transcriptional classification, confirming the strong influence of stromal contexture. CRIS grouping may be applied both to primary and metastatic CRC with low overlap on previous transcriptional classifications. Interestingly, CRIS subtypes were demonstrated to have new prognostic and predictive potentials[
In the past years, research on immunology and molecular biology fields has clarified the role of the immune system in cancer growing and metastatic potential of tumors. Interestingly, MSI tumors show a marked predisposition to express a wide variety of neoantigens reflecting a significantly high mutational burden [20 fold higher compared to microsatellite stable (MSS)], due to dMMR. The load of neoantigens and the pronounced expression of T-cell recruiting chemokines cooperate to sustain an active immune TME characterized by diffuse immune infiltrate. This explains why CMS1 subtype is recognized as highly immunogenic. This consideration builds up a strong rationale for the use of immunotherapy in MSI CRC. Furthermore, Llosa
Immune system defends our bodies from non-self antigens activating immune response. However, it is pivotal that immune defenses arise at the appropriate time and are limited when they are no more requested in order to prevent chronical inflammation and autoimmune disease. A variety of co-inhibitory checkpoints are engaged to balance activation signals.
One of the most important immune checkpoints is represented by PD-1 and PDL-1. PD-1 is expressed on activated T-cells while PDL-1 is usually expressed on APCs’ surface and their interaction mediates a co-inhibitory stimulus that limits excessive immune responses in peripheral tissues ensuring the maintenance of peripheral tolerance
Interactions between cancer cells and T-cells and the role of PD-1/PDL-1 and CTLA4. PD-1: programmed cell death-1; PDL-1: programmed cell death ligand-1; CTLA4: cytotoxic T-lymphocyte-associated protein 4; APC: antigen-presenting cell; MHC: major histocompatibility complexes
The biological significance of PD-1/PDL-1 and CTLA4 suggests a therapeutic role of blockade of these pathways in different types of cancer, including CRC[
In particular, the immune-related objective response rate (ORR) and immune-related 6-month progression-free survival (PFS) were 40% and 78% respectively, for dMMR CRC patients (cohort A) and 0% and 11% for pMMR CRC patients (cohort B). The median PFS was 2.2 months (95% CI, 1.4-2.8) and OS was 5 months (95% CI, 3.0 to not estimable) in the cohort with pMMR CRC. The median PFS and OS were not reached in the cohort with dMMR CRC. Indeed, the authors revealed 1782 somatic mutations per tumor in dMMR compared with 73 in pMMR tumors (
Clinical trials of immunotherapy in colorectal cancer
Trial | Phase | Drugs | Setting | PFS months | OS months | ORR | Number of identifier | |
---|---|---|---|---|---|---|---|---|
PD-1 Blockade in Tumors with Mismatch-Repair Deficiency | ||||||||
dMMR CRC | II | Pembrolizumab 10 mg/kg each 14 days | 21 | NR | 40 | |||
pMMR CRC | II | Pembrolizumab 10 mg/kg each 14 days | 11 | 2.2 (95% CI, 1.4 to 2.8) | 5.0 (95% CI, 3.0 to not estimable) | 0 | ||
Other dMMR non-CRC | II | Pembrolizumab 10 mg/kg each 14 days | 9 | 5.4 (95% CI, 3 to not estimable) | NR | 71 | ||
CheckMate 142 |
II | Nivolumab 3 mg/Kg with Ipilimumab 1 mg/Kg every 3 weeks for 4 doses followed by Nivolumab 3 mg/Kg every 2 weeks until progression | Pre-treated | NE* |
55%*° | NCT02060188 | ||
A Phase 2 Study of Pembrolizumab (MK-3475) in Combination With Azacitidine in Subjects With Chemo-refractory Metastatic Colorectal Cancer AAM 2017 n 3054 |
II | Pembrolizumab 200 mg on day 1 of every 21 day cycle |
31 | Pre-treated | 2.1 m (1.8 to 2.8) | 6.2 m (3.5 to 8.7) | 3% CI (1-17) | NCT02260440 |
*Preliminary data
CheckMate142 investigated efficacy of both nivolumab monotherapy and nivolumab plus ipilimumab combination therapy in MSI CRC. In the monotherapy cohort, seventy-four pretreated dMMR/MSI-H metastatic CRC patients were treated with nivolumab 3 mg/kg every 14 days. Nivolumab provided evidence of benefit in previously treated patients with dMMR CRC, with an ORR of 34% (95% CI, 23.2-45.7) with a disease control rate (DCR) of 62% (95% CI, 50.1-73.2). Interestingly, durable responses were observed and 64% of patients had response lasting more than 12 months. Median PFS was 6.6 months and 12 months OS was 72% (95% CI, 60.0-80.9) with a median follow up of 21 months[
CheckMate 142 combination cohort evaluated nivolumab plus ipilimumab, an anti-CTLA4 antibody. Indeed, nivolumab and ipilimumab can act synergistically to promote T cell antitumor activity. In this cohort, one hundred ninety-nine previously treated patients with metastatic or recurrent dMMR CRC were treated with 4 doses of combination immunotherapy with nivolumab and ipilimumab followed by nivolumab. At median follow-up of 13.4 months, primary endpoint ORR was 55% (95% CI, 45.2-63.8) and DCR for 12 weeks or more was 80%. PFS rates were 76% at 9 months and 71% at 12 months while OS rates were 87% and 85%, respectively. Responses were observed irrespective of PDL-1 expression,
Although the comparison is only indirect, these results suggest that a double-blockade might improve clinical outcomes, thus becoming a promising treatment option for MSI CRC[
Ongoing clinical trials of immunotherapy in colorectal cancer
Trial | Phase | Drugs | Setting | Number of identifier |
---|---|---|---|---|
MK-3475-177/KEYNOTE-177 |
III | Pembrolizumab 200 mg each 21 days for up to 35 treatments |
1st line | NCT02563002 |
A Study to Investigate Efficacy and Safety of Cobimetinib Plus Atezolizumab and Atezolizumab Monotherapy |
III | Regorafenib (160 mg days 1-21 every 28 days) |
3rd line | NCT02788279 |
A Phase 2 Study With Safety Lead-in, Evaluating TAS-102 Plus Nivolumab in Patients With Microsatellite Stable Refractory Metastatic Colorectal Cancer |
II | TAS102 plus Nivolumab | 3rd line | NCT02860546 |
MK-3475-158/KEYNOTE-158 |
II | Pembrolizumab 200 mg every 3 weeks for up to 35 administrations | Pre-treated | NCT02628067 |
Phase 2 Study of MK-3475 in Patients With Microsatellite Unstable (MSI) Tumors |
II | -MSI Negative Colorectal Cancer: Pembroluzumab |
Pre-treated | NCT01876511 |
A Phase I, Open-Label, Multi-Centre Study to Assess the Safety, Tolerability and Preliminary Anti-tumour Activity of Ascending Doses of Selumetinib (AZD6244 Hyd-sulfate) in Combination With MEDI4736 and Selumetinib in Combination With MEDI4736 and Tremelimumab in Patients With Advanced Solid Tumours |
I | Selumetinib + MEDI4736 |
Pre-treated | NCT02586987 |
An Open-label, Phase II Basket Study of a hypoMEThylating Agent Oral Azacitidine and DURvalumab (MEDI4736) (Anti-PDL1) in Advanced Solid Tumors (METADUR) |
II | Azacitidine 300 mg daily for 14 consecutive days of every 28 days cycle for 3 cycles. PLUS |
Pre-treated | NCT02811497 |
Evaluate the Efficacy of MEDI4736 in Immunological Subsets of Advanced Colorectal Cancer |
II | subjects will receive MEDI4736 for 12 months, or until PD, initiation of alternative cancer therapy, unacceptable toxicity. Following the 12-month treatment period, subjects without evidence for PD or other reason to discontinue treatment will be monitored without further treatment. Upon evidence of PD during the monitoring period, administration of MEDI4736 may resume at the Q2W schedule, for up to another 12 months |
3rd line | NCT02227667 |
*Preliminary data; MSI: microsatellite instable; MSS: microsatellite stable; TAS: Trifluridine/Tipiracil; PD: progressive disease; PDL-1: programmed cell death ligand-1; CRC: colorectal cancer
Albeit dMMR tumors proved to be responsive to immune-checkpoint inhibition, the majority of patients with CRC have pMMR tumors and this status was related to lower response to PDL-1/PD-1 or CTLA4 blockade. Hence, other molecular subtypes require different strategies. Theoretically, immunotherapy could be useful for all CRC if it was possible to convert the tumor towards a “CMS1-like” immune phenotype. CMS4 tumors (which showed the worse prognosis in terms of overall and relapse-free survival), for example, are characterized by an unfavorable, inflammed immune phenotype. They revealed high expression of mesenchymal genes, stromal cell infiltration and an angiogenic microenvironment.
Vascular endothelial growth factor-A (VEGF-A), a proangiogenic molecule produced by the tumors, has a crucial role in the development of the immunosuppressive microenvironment[
Another key aspect of the TME of CMS4 tumors is represented by activation of TGF-β signaling. Using a preclinical model of CT26 colon carcinoma cells, Triplett
CMS2 and CMS3 are considered as “cold” tumors, meaning that they lack immune cell infiltration. The level of expression of immunosuppressive genes is low, thus suggesting different mechanisms of immune escape. For example, the downregulation of MHC class I observed in these tumors, results in reduced presentation of tumor-associated antigens[
Other approaches that are being tested to improve immunotherapy response among CMS subtypes are represented by cytokine treatment, cancer vaccination and passive immunotherapy with adoptive T cell transfer or monoclonal antibody targeting tumor-associated antigens. Klein
Likewise, other malignancies, combining immunotherapy with conventional chemotherapeutic strategies or with radiotherapy (RT) might represent an useful and practical means to stimulate immune cell infiltration and elicit immune response. To this purpose, clinical trials testing the combination of anti-PDL-1/PD-1 treatment with RT or modified FOLFOX are ongoing (NCT02437071, NCT02375672). In the first one is a phase II study to evaluate the safety and abscopal effect of pembrolizumab after palliative RT or ablation in pts with unresectable/recurrent pMMR metastatic colorectal cancer, who have received ≥ 2 standard therapies, with ORR in a non-targeted lesion as primary objective[
A different strategy that is currently under evaluation to improve efficacy of immunotherapy in MSS/pMMR CRCs is combination of histone deacetylase inhibitor and PD-1 inhibitors. Entinostat, an oral, class I-selective histone deacetylase inhibitor is able to enhance anti-PD-1 activity by downregulation of immunosuppressive cell types in the TME[
Preliminary results of a phase II study of entinostat in combination with pembrolizumab have been recently presented at ASCO 2018 annual meeting. Sixteen pretreated MSS/pMMR CRC patients were enrolled and at data cut-off 6 patients remained on study (1 PR, 6 stable disease). The treatment showed acceptable safety with common adverse events including fatigue (37.5%), arthralgia (18.8%), and increased alkaline phosphatase (18.8%). These results can be viewed as promising, considering that have been obtained in a patient population in which objective responses have not been reported with anti-PD-1 monotherapy[
In addition to immune strategies focused on PD-1/PDL-1 axis and CTLA4 and against cancer immunotolerance, a series of different approaches (albeit still on the side of immunotherapeutic approaches) are recently been investigated in CRC. T lymphocytes engineered to express chimeric antigen receptors (CAR-T cells) have been tested for their potential role as therapeutic agents in CRC. In a recent paper of Magee
However, in another paper of Huang
Albeit manipulation of the mutational load of CRC patients is a mere piece of science fiction, it is well-known that, for treatments that are focused on PD-1/PDL-1 axis, mutational load might represent the best way to identify those patients who could benefit from this kind of strategy (more than the simplistic way of assessment of patients as in microsatellite stable/unstable). In particular, in a recent paper of Fabrizio
These data suggest that, at least in the foreseeable future, more data are needed to further assess the clinical impact of these treatment approaches in everyday practice, as there are a few crucial topics still to be addressed (namely the fitness of T cells, how to increase sensitivity of the TME towards T cell mediated killing and the selection of patients that benefit best from these treatment approaches).
In the past few years, introduction of new therapeutic approaches and better selection of patients have significantly changed treatment strategy of CRC and definitely improved patient outcome.
Immunotherapy has been the most important revolution in cancer treatment of recent years and it continues to show impressive results in lethal malignancies such as melanoma or lung cancer. Still, results observed in CRC with checkpoint inhibitors immunotherapy are modest if compared to other tumor entities and limited to a small subset of patients with MSI. In this context, a better knowledge of tumor immune microenvironment is essential to developing effective therapeutic strategies and overcoming resistance.
Interestingly, molecular characterization of CRC has shown that CMSs are associated with specific immune infiltration profiles corresponding with characteristic mechanisms of immune escape.
In particular, CMS1 subtype presents the most favourable situation for immunotherapy efficacy with high immune infiltration rich in Th1 cells and TILs, explaining the efficacy of checkpoint inhibitors in this subtype. CMS4 also presents high immune infiltrate but with an unfavourable, inflamed molecular orientation characterized by intratumoral MDSC, M2-macrophages and B-cells associated with pro-inflammatory gene expression, including myeloid chemokines, immune suppressive molecules and complement factors. In this situation, the combination of checkpoint inhibitors with TGF pathway inhibition represents a promising strategy as well as the use of angiogenesis inhibitors or anti-MDSCs treatment. On the contrary, CMS2 and CMS3 are poorly immunogenic tumors with scarce immune infiltrate. In this context, combination of checkpoint inhibitors with MEK-inhibition or anti-EGFR monoclonal antibodies could allow to overcome resistance. In addition, monoclonal antibodies targeting tumor-associated antigens, such as CEA, engineered with IL-2 may be able to increase immune infiltration and activates NK and T cells also in tumors with poor immune infiltration. Other strategies which may be effective in the setting of CMS2 and CMS3 are the combination of chemotherapy and immune checkpoint inhibitors or passive immunotherapy treatments as cancer vaccines with primed DCs.
In conclusion, the development of new effective immunotherapeutic strategies in CRC should be driven by a better knowledge of mechanisms of resistance to current treatments and take in account differences in immune microenvironment between different molecular subtypes to find the best treatment for each patient.
Responsible for the paper: Berardi R
Concept, design, definition of intellectual content: Bittoni A
Literature search: Meletani T, Sotte V, Cantini L
Manuscript preparation: Meletani T, Sotte V, Cantini L, Giampieri R
Manuscript editing: Meletani T, Sotte V, Cantini L, Giampieri R, Bittoni A
Manuscript revision: Berardi R, Bittoni A, Meletani T, Sotte V, Cantini L, Giampieri R
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All authors declared that there are no conflicts of interest.
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© The Author(s) 2018.