BioMed Research International
Volume 2017 (2017), Article ID 4346576, 17 pages
https://doi.org/10.1155/2017/4346576

Review Article

Immunotherapy in Gastrointestinal Cancers

Letizia Procaccio,1,2 Marta Schirripa,¹ Matteo Fassan,³ Loredana Vecchione,⁴ Francesca Bergamo,¹ Alessandra Anna Prete,1,5 Rossana Intini,1,2 Chiara Manai,1,5 Vincenzo Dadduzio,1,6 Alice Boscolo,1,2 Vittorina Zagonel,¹ and Sara Lonardi¹

¹Division of Medical Oncology 1, Istituto Oncologico Veneto, IRCCS, Padova, Italy
²Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy
³Department of Medicine, Surgical Pathology & Cytopathology Unit, University of Padova, Padova, Italy
⁴Division of Molecular Carcinogenesis, Cancer Genomics Center Netherlands, The Netherlands Cancer Institute, Amsterdam, Netherlands
⁵Department of Radiological, Oncological and Pathological Sciences, Policlinico Umberto I University Hospital, Rome, Italy
⁶Department of Medical Oncology, Fondazione Policlinico Universitario Agostino Gemelli, Università Cattolica del Sacro Cuore, Rome, Italy

Correspondence should be addressed to Sara Lonardi; sara.lonardi@iov.veneto.it

Received 7 April 2017; Accepted 18 May 2017; Published 3 July 2017

Academic Editor: Carmen Criscitiello

Copyright © 2017 Letizia Procaccio et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Gastrointestinal cancers represent a major public health problem worldwide. Immunotherapeutic strategies are currently under investigation in this setting and preliminary results of ongoing trials adopting checkpoint inhibitors are striking. Indeed, although a poor immunogenicity for GI has been reported, a strong biological rationale supports the development of immunotherapy in this field. The clinical and translational research on immunotherapy for the treatment of GI cancers started firstly with the identification of immune-related mechanisms possibly relevant to GI tumours and secondly with the development of immunotherapy-based agents in clinical trials. In the present review a general overview is firstly provided followed by a focus on major findings on gastric, colorectal, and hepatocellular carcinomas. Finally, pathological and molecular perspectives are provided since many efforts are ongoing in order to identify possible predictive biomarkers and to improve patients’ selection. Many issues are still unsolved in this field; however, we strongly believe that immunotherapy might positively affect the natural history of a subgroup of GI cancer patients improving outcome and the overall quality of life.

1. Gastrointestinal Cancers: Where Do We Stand?

Gastrointestinal (GI) cancers, including colorectal cancer (CRC), gastric cancer (GC), pancreatic cancer, and cancers of the liver (HCC) and of the biliary tract, are among the most frequent malignancies diagnosed annually in Europe and represent a major public health problem worldwide [1].

Although early-stage GI cancers are amenable to surgical resection with curative-intent, the overall 5-year relapse rate remains high. As a matter of fact, the addition of neoadjuvant or adjuvant chemotherapy and radiation therapy, when indicated, only modestly improves the overall long-term survival. Unfortunately, a large proportion of patients present with unresectable disease at the time of diagnosis: approximately 25% of GI cancers are diagnosed at advanced stage, whereas another 25 to 50% of patients will develop metastases during the course of the disease [2, 3]. In the last decade, meaningful improvement in the prognosis of patients with metastatic GI cancers derived from the development of new intensive and/or tailored therapies, which incorporated cytotoxic drugs and targeted therapies (cetuximab, panitumumab, bevacizumab, aflibercept, and regorafenib for mCRC; trastuzumab and ramucirumab for mGC; and sorafenib for HCC), and from the integration of medical treatments with more and more effective locoregional and surgical approaches [4]. Despite these advances, GI cancers are still a leading cause of cancer death [4]; thus, it is imperative to develop novel therapeutic approaches for patients affected by those cancers.

In recent years, we assisted in a paradigmatic shift in the treatment of both solid tumours, such as melanoma, non-small cell lung cancer, and genitourinary cancers, as well as hematologic malignancies, thanks to the striking results with long lasting responses and increased overall survival (OS) obtained with immunotherapy-based agents [5–7]. In parallel, the clinical and translational research on immunotherapy for the treatment of GI cancers started firstly with the identification of immune-related mechanisms possibly relevant to GI tumours and secondly with the development of immunotherapy-based agents in clinical trials.

Undoubtedly, the progress made towards the development of effective antitumour immunotherapies for GI cancers has been relatively slow: the first practice changing clinical data came out only in 2015 and the most part of immunotherapies are still in early phase clinical testing. The main reason for having GI cancers as a kind of Cinderella in the landscape of tumoural immunotherapy resides in the lack of their effector T cell responses and in the their well-known poor immunogenicity [8]. Immunotherapy against cancer has been assumed to be beneficial mainly in tumours with high immunogenicity by nature [9]. However, some approaches to circumvent immunosuppression including programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) blockade were successful to achieve significant response, also in cancers that hardly retain immunogenic nature [10].

This article highlights the state of the art of immunotherapy in GI deepening recent scientific evidence regarding anti-PD-1/PDL-1 and anti-CTLA4 monoclonal antibodies, peptide based vaccine, DNA based vaccine, and pulsed dendritic cells (DC), also outlining current clinical trials and finally suggesting areas for future research.

2. The Rationale for Immunotherapy in GI Cancers

Accumulating evidences indicate that a dynamic cross-talk between tumours and the immune system can regulate tumour growth and metastasis [10]. The increased understanding of the biochemical nature of tumour antigens and of the molecular mechanisms responsible for innate and adaptive immune cell activation has revolutionized the fields of tumour immunology and immunotherapy.

The first notion of a role of immunity in cancer was postulated in 1909 by Ehrlich, speculating that the immune system could repress the growth of carcinomas recognising tumour cells as foreign. About 50 years later, the theory of tumour immune surveillance was proposed by Burnet [11]. However, this theory has been recently completed with the identification of the so-called immunoediting proposed by Schreiber et al. The immunoediting progresses through 3 main phases: (1) the elimination phase (or immunosurveillance), when the innate and adaptive immune cells remove the proliferating cells, thus protecting the host against cancer; (2) the equilibrium phase, when the tumour growth and the immunosurveillance enter into a dynamic balance; in this genetically instable phase, the increase of mutational load and the emergence of resistant clones among tumour cells lead to (3) the escape phase; at this point, tumour variants are able to avoid immune-mediated destruction and speed up tumour progression and clinical expression [12, 13].

A role for the immunoediting in gastroenteropancreatic tumour pathogenesis was suggested since the first observations that T cells infiltration was linked to a more favorable outcome in pancreatic cancer, CRC, and GC [13, 14]. The following studies regarding the molecular basis and regulation of immunoediting have identified the tumour cells, the tumour microenvironment, and the immune system as the key players of a complex network [15]. Defining the relationships between these key players has been critical in facilitating the development of successful immunotherapies.

(A) Tumour cells have developed several mechanisms that directly or indirectly block the activity of effector antitumour CD4+ and CD8+ T cells dampening local tumour-infiltrating immune responses [16, 17]. Examples include (1) the secretion of soluble immunosuppressive factors (TGF-beta, IL-10, VEGF, and indoleamine 2,3 dehydrogenase) [18, 19]; (2) the activation of negative costimulatory signals in the tumour microenvironment such as PD-L1 [20, 21]; (3) tumour-induced impairment of the antigen presentation machinery due to the accumulation of point mutations in the cell surface not recognised by cytotoxic T cells [22]; and finally (4) the downregulation of the major histocompatibility complex (MHC) class I expression which plays a crucial role in tumour antigens presentation to T cells [22].

(B) Mechanisms explaining the tumour microenvironment role in immunoediting are best illustrated in studies on human and mouse pancreatic cancer models, since desmoplasia is the pathologic hallmark of pancreatic cancer [23, 24]. This inflammatory environment consists of regulatory immune cells, extracellular matrix proteins, and all the above fibroblasts (cancer-associated fibroblasts, CAFs) [25]. These stroma players in turn secrete tumour-promoting factors that contribute to tumour invasion and neoangiogenesis [26, 27]. Interestingly, CAFs have a critical role in CRC immunosuppression [28]: their activity in RAS mutant tumours overcome effector T cells signalling leading to tumour progression thanks to the activation of epithelial mesenchymal transition and TGF-beta/SMAD signalling [28]. Actually, high levels of CAFs markers correlated with poor prognosis in CRC [29].

(C) The immune system plays a critical role in immunoediting thanks to the involvement of several innate and adaptive effectors such as myeloid-derived suppressor cells (MDSCs), mast cells, tumour associated macrophages (TAMs), mesenchymal stem cells (MSCs), CD4+/CD25+ regulatory T cells (Tregs), and DCs [30]. By modulating the tumour microenvironment through the secretion of selected chemokines, cancer cells can actively prevent the induction of antitumour immunity through the differentiation, expansion, and/or recruitment of Treg [30, 31]. It has been reported that a low percentage of Tregs in the circulation 1 year after resection of pancreatic cancer correlates with improved survival [32, 33]. In addition, DCs are critically important for the generation and the maintenance of a specific adaptive antitumour immune response [33, 34]. Data from many laboratories obtained during past few years indicate that defects in DCs are among the main factors responsible for tumour escape [35].

Among immunosuppressive mechanisms the immune checkpoint modulation mediated by cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and Programmed Death-1 (PD-1) plays a crucial role. In the normal host setting, immune checkpoint molecules modulate the T-cells response to antigens by either upregulating costimulatory pathways or downregulating coinhibitory pathways of immune signalling. CTLA-4 is an inhibitory receptor expressed by T cells. It can bind to CD80 or CD86 on DCs and inhibit their capabilities to activate T cells. CTLA-4 plays a critical role in the initial phase of immune response. PD-1 is a cell surface coinhibitory receptor that downregulates T cell activity in peripheral tissues during inflammation, thus preventing increased collateral tissue damage during an immune response and the development of autoimmunity. PD-1 is widely expressed on T cells, B cells, monocytes, and natural killer cells and plays a critical role in subsequent phases of immune responses compared to CTLA-4. It has two known ligands, PD-L1 and PD-L2, which are both upregulated during an inflammatory response. Tumour cells of various malignancies have been shown to upregulate PD-L1 as a mechanism that dampens the local T cell response by decreasing cytokine production and T cell proliferation. In GI malignancies, PD-L1 upregulation has been demonstrated to occur in pancreatic, GC, and CRC [34, 35], thus correlating with poor prognosis [35].

Moving from such complex background, immunotherapeutic strategies in GI cancers have been developed and are described in the following paragraphs. In particular, a general overview is firstly provided followed by a focus on major findings on GC, CRC, and HCC. Finally, a pathological and a molecular perspective are provided.

3. Immunotherapeutic Strategies: A General Overview

Activation of immune system against cancer might derive from active immunotherapeutic strategies, such as the adoption of cytokines, cancer vaccines, and immune checkpoints inhibitors or from a passive immunization mediated by adoptive cellular therapy (ACT) or monoclonal antibodies [14, 36]

The first attempts of active host immunity stimulation were based on the adoption of cytokines, in particular interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-10, or GM-CSF. However, cytokines-based strategies are not adopted in clinical practice since results of trials are dated and inconclusive [37].

Cancer vaccines, as active immunotherapy, were firstly investigated 30 years ago. They are designed to activate and expand tumour-specific T cells with the potential to produce a persistent or even permanent anticancer effect. The ideal vaccine is easy to administer, offers prolonged protection, and induces relatively low toxicity. Although many trials investigated the possible role of peptide, protein, whole tumour cells, or DC-based vaccines in GI cancers [37], to date none entered the clinical practice. However, a renewed enthusiasm derived from a new class of recombinant immunogenic protein fused with a novel cell-penetrating peptide (Z12). This compound is able to promote efficient protein loading into the antigen processing machinery of DC and to lead to multiepitopic MHC class I and II restricted presentation. This novel vaccine elicited an integrated and multiepitopic immune response with persistent CD8+ and CD4+ stimulation in different tumour models [38] and will be soon investigated in human GI models.

Undoubtedly, immune checkpoints inhibitors are the real game changers. The anti-CTLA-4 ipilimumab and the anti-PD-1 mAbs, pembrolizumab and nivolumab, were firstly approved by the US FDA for the treatment of metastatic melanoma in 2011 and 2014, respectively [39, 40]. Several trials investigated or are currently investigating such mAbs in GI cancer with very promising preliminary results. As an example, pembrolizumab received the breakthrough therapy designation in mCRC cancers with microsatellite instability in November 2015 [41]. Despite some practice changing results have been obtained, many efforts are currently made in order to identify subgroup of patients benefitting from these agents and to design newer strategies involving the association of standard treatment with immunotherapy. Moreover, new molecules are under investigation such as the anti-PD-L1 avelumab and atezolizumab in GC and the anti-CTLA-4 mAb tremelimumab in HCC patients.

Among passive immunization strategies, adoptive cell therapy (ACT) is based on the passive transfer of tumour-specific T cells into a tumour-bearing host for the direct destruction of tumours. Briefly, T cells are collected from the tumour, draining lymph nodes or peripheral blood, and are activated and expanded in vitro. The first clinical trial of ACT in advanced cancers adopted lymphocytes-activated killer (LAK) cells. Since then, the innovative ACT with tumour-infiltrating immune cells (TILs) has been developed taking advantage of lymphocytes with demonstrated ability to recognise the tumour. ACT with TILs isolated from resected tumours, expanded ex vivo, and administered to patients in combination with IL-2 has demonstrated a 50% response rate in patients with metastatic melanoma [42, 43]. Since TILs have been isolated from a variety of GI cancers, this approach is currently under investigation in the metastatic setting [44]. The most recent ACT treatment adopts engineered T cells able to express chimeric antigen receptors (CARs) specific for CEA. CARs engage their target independently from antigen processing process and from MHC. Thus, CAR therapy is advantageous when MHC class I is downregulated [45]. Since T cells are ubiquitously expressed, targeting self-antigens might cause serious immune-related toxicities and safety concerns are still unsolved [45].

Finally, monoclonal antibodies (mAbs) commonly adopted in GI cancers represent the most relevant example of passive immunotherapy strategy. However, a wide body of literature is already available regarding this topic and it is not discussed in the present review.

4. Focus on Gastric, Esophageal, and Pancreatic Cancers

The first promising data about immunotherapy in GC or gastroesophageal junction cancer (GEJC) came from anti-PD-1 agents. The phase Ib study KEYNOTE-012 was designed to assess the safety and activity of pembrolizumab in GC and GEJC and the predictive role of PD-L1 expression in those malignancies. Primary endpoints were safety and response rate (RR). Toxicity profile was manageable; among 36 evaluable patients, RR was 22% (95% CI 10–39). No association between PD-L1 expression and clinical responses to pembrolizumab was observed [46]. Moving from the promising KEYNOTE-012 results, two trials are currently ongoing: the KEYNOTE 061 is evaluating pembrolizumab versus paclitaxel after progression to a first-line platinum-based therapy [47] and the KEYNOTE 062 is randomizing patients to receive pembrolizumab as monotherapy or platinum and 5-FU in association with pembrolizumab or placebo in the first-line setting [48] (see Table 3).

Pembrolizumab as single agent was also tested in esophageal cancer (EC) in the multicohort, phase Ib KEYNOTE-028 trial. In this study, 23 pretreated patients with either squamous cell carcinoma (SCC) or adenocarcinoma of the esophagus or GEJC were treated. Encouraging results were reported: the ORR was 30.4% and 52.2% in SCC and adenocarcinoma patients, respectively. Six- and 12-month progression free (PF) rates were 30.4 and 21.7%, respectively [49].

In the randomized phase III trial ONO-4538/BMS-936558, the anti-PD-1 nivolumab was tested as monotherapy versus placebo in advanced GC and GEJC after second or later lines. This study met all its endpoints. In detail, mOS was 5.3 versus 4.1 months (HR = 0.63, 95% CI 0.50–0.78, ) and mPFS was 1.61 versus 1.45 months (HR = 0.60, 95% CI 0.49–0.75, ) in the nivolumab and in the placebo arm , respectively [50]. The shape of the curve shows that only a subgroup of patients derives benefit from the treatment reaching a long lasting disease control and response to treatment.

Nivolumab has also been tested in 65 patients affected by advanced esophageal SCC in a Japanese single-arm phase II trial. Patients received one or more previous treatment and were not preselected by PD-L1 status. The preliminary results showed durable activity with a manageable safety profile, with median OS of 12.1 months in 64 evaluable patients [51].

Another promising immunotherapy agent is the anti-PD-L1 avelumab, which has been tested in patients with GC or GEJC in the phase Ib trial JAVELIN. Patients were eligible if treated with a first-line chemotherapy based regimen and grouped by progression status after first line: patients achieving disease control during first line received avelumab as switch maintenance and those with progressive disease after chemotherapy received it as second line . Primary endpoint was safety. An acceptable safety profile was shown. ORR was 9.0% and 9.7% in the 2 subgroups, respectively [52]. Given the promising results of this trial, JAVELIN Gastric 100 and JAVELIN Gastric 300 phase III trials are now ongoing [53, 54].

Less encouraging results come from anti-CTLA-4 agents, which showed higher toxicity and lower efficacy than anti-PD-1 in gastric and esophageal malignancies. The reasons for these differences are still debated. No objective responses were observed with the anti-CTLA-4 tremelimumab, tested as second-line treatment in a phase II trial in advanced GC and EC [55]. Similarly, ipilimumab was compared to best supportive care (BSC) in a randomized phase II trial, in pretreated patients with metastatic or locally advanced GC or GEJC and survival parameters were similar between the two arms [56].

In order to enhance the activity of anti-CTLA-4 antibodies, combination treatments with anti-PD-1 have been tested. The checkMate-032 is a phase I/II multicohort trial that randomized 160 pretreated patients to receive (1) nivolumab alone 3 mg/kg, (2) nivolumab 3 mg/kg plus ipilimumab 1 mg/kg, or (3) nivolumab 1 mg/kg plus ipilimumab 3 mg/kg. A notable RR was seen in each arm, with an overall DCR of 38%. Of interest, the ORR in patients with PD-L1-positive (≥1%) and PD-L1-negative (<1%) tumours was 27% and 12%, respectively, suggesting that PD-L1 expression may increase response rates. The highest ORR (26%) and mOS (6.9 months) were observed in arm 3 (nivolumab 1 mg/kg and ipilimumab 3 mg/kg) [57]. Given these interesting findings, the phase III trial CheckMate-649 investigating nivolumab plus ipilimumab versus FOLFOX/XELOX in untreated patients is ongoing. Table 1 shows ongoing trials in this setting.

Table 1: Ongoing studies on gastric, gastroesophageal junction, and esophageal cancers.

NCT identifier
Setting
Phase
Study interventions
Number of patients
Primary endpoint

Checkpoint inhibitors

 NCT02689284
Metastatic HER2+ GC/GEJC
Ib/II
Margetuximab+ pembrolizumab
52
MTD and MAD for margetuximab; duration of response; 12-month ORR

 NCT02563548
Metastatic GC after 1st line
Ib
PEGPH20 +pembrolizumab
81
DLT; 18-month ORR

 NCT02443324
Metastatic GC/GEJC and other tumours
I
Ramucirumab + pembrolizumab
155
DLT

 NCT02589496
Metastatic GC/GEJC after first line
II
Pembrolizumab
40
2-year RR

 NCT02901301
First-line HER2 + GC
Ib/II
Pembrolizumab + trastuzumab + capecitabine + cisplatin
49
RP2D; 6-week ORR

 NCT02954536
First-line HER2+ GC/GEJC/EC
II
Pembrolizumab + trastuzumab + capecitabine + cisplatin
37
6-month PFS

 NCT02318901
Unresectable HER2 + GC/GEJC
II
Pembrolizumab + ado-trastuzumab emtansine
90
RP2D

 NCT02559687
EC (adenocarcinoma or squamous cell)/GEJC after 2nd line
II
Pembrolizumab
100
2-year ORR

 NCT02494583
First-line GC/GEJC
III (random)
Pembrolizumab versus pembrolizumab + cisplatin + 5-fluorouracil or capecitabine versus placebo + cisplatin + 5-FU or capecitabine
750
44-month PFS and OS

 NCT02370498
Second-line GC/GEJC
III (random)
Pembrolizumab versus paclitaxel
720
PFS, OS

 NCT02564263
EC (adenocarcinoma or squamous cell) /GEJC after 1st line
III (random)
Pembrolizumab versus investigator's choice of standard therapy (paclitaxel, docetaxel, or irinotecan)
600
3-year PFS and OS

 NCT02872116
Unresectable GC/GEJC
III (random)
Nivolumab + ipilimumab versus nivolumab + oxaliplatin + fluoropyrimidine versus oxaliplatin + fluoropyrimidine
1266
40-month OS in patients PD-L1 +

 NCT02864381
Metastatic GC/GEJC
II (random)
GS-5745 + nivolumab versus nivolumab alone
120
2-year ORR

 NCT02340975
Pretreated metastatic/GC/GEJC
Ib/II (random)
MEDI4736 + tremelimumab versus MEDI4736 versus tremelimumab
135
Phase Ib: DTL,
Phase II: ORR and 6-month PFS

 NCT02625623
3rd-line GC/GEJC
III (random)
Avelumab+ BSC versus chemotherapy (paclitaxel or irinotecan)+BSC or BSC alone
330
2-year OS

 NCT02625610
1st-line GC/GEJC
III (random)
Maintenance with avelumab versus continuation of 1st-line chemotherapy
666
3-year OS and PFS

Immunotherapy + radiotherapy

 NCT02642809
1st-line EC
I
Pembrolizumab + brachytherapy
15
Tolerability and toxicity

 NCT02830594
Pretreated EC/GC(GEJC
II
Pembrolizumab + external beam palliative radiation therapy
14
Biomarkers

 NCT02735239
Metastatic EC
I/II
Durvalumab + oxaliplatin/capecitabine
75
AE, DLT, laboratory evaluations

Vaccines

 NCT02276300
Metastatic HER 2 + GC
I
HER2-derived peptide vaccination
12
Safety and tolerability

 NCT02317471
Stage III gastric cancer
I/II
Vaccination with autologous tumour derived heat shock protein gp96
45
DFS

 NCT02795988
Metastatic HER 2 + GC/GEJC
Ib/II
IMU-131 HER2/Neu peptide vaccine+ cisplatin and either 5-FU or capecitabine chemotherapy
18
RP2D, AE

Cytokines

 NCT01691664
Locally advanced EC
NS (random)
Radiation therapy alone or with DC-CIK cellular therapy
40
DFS

 NCT01691625
Locally advanced EC
NS (random)
Concurrent chemoradiation with or without DC-CIK
50
Quality of life

 NCT02504229
Metastatic refractory GC
II
(random)
Chemotherapy with or without DC-CIK
80
PFS

 NCT01783951
Metastatic refractory GC
I/II
S-1 with or without DC-CIK
30
PFS

CAR-T cells

 NCT02713984
Metastatic refractory HER 2 + GC
I/II
Anti-HER2 CAR-T cells
60
Toxicity

 NCT02725125
Metastatic refractory GC
I/II
EPCAM-targeted CAR-T cells
19
DCR

 NCT02617134
Metastatic refractory MUC1+ GC
I/II
Anti-MUC1 CAR-T cells
20
Toxicity

 NCT02349724
Metastatic refractory CEA+ GC
I
Anti-CEACAR-T cells
75
Toxicity

 NCT02862028
Metastatic refractory EGFR+ GC
I/II
Anti-PD-1CAR-T cells
20
ORR, DCR, OS, PFS

 NCT03013712
Metastatic refractory EpCAM+ GC/EC
I/II
Anti-EpCAMCAR-T cells
60
Toxicity

GC, gastric cancer; GEJC, gastroesophageal junction cancer; EC: esophageal cancer; NA, not assessed; MTD, maximum tolerated dose; MAD, maximum administered dose; DTL, dose limiting toxicity; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; DFS, disease-free survival; NS, not specified; RP2D, recommended dose of phase II; AE, adverse events; DCR, disease control rate; BSC, best supportive care; DCR disease control rate.

Pancreatic cancer models have been widely adopted in order to identify the immunotherapeutic rationale in GI cancer; however, data derived from early phase clinical trials yielded no benefit in pancreatic cancers. In particular, negative results derived from checkpoint inhibitor and vaccination trials [58]. Future clinical trials will test combination approaches in order to overcome immunosuppressive intratumour mechanisms and/or to increase the immunogenicity of microenvironment.

5. Focus on Colorectal Cancer

The first data on immunotherapy in CRC came from 1981, when the role of vaccines as immunotherapy was explored, based on the rationale of activating host defense against tumour-specific or tumour-associated antigens by means of the injection of autologous tumour cells with an immunomodulator (Bacillus Calmette-Guérin (BCG)). Preclinical models showed that the injection of BCG and tumour cells (OncoVAX®) was able to activate systemic immunity and stop the tumour burden [59].

The efficacy of OncoVAX was subsequently evaluated in the adjuvant setting in three phase III clinical trials, where patients were randomized to receive surgical resection only or surgical resection plus vaccination. The first study (8102) was initiated in 1981 and enrolled 98 patients with stages II and III CRC. The primary endpoints, OS and disease-free survival (DFS), were not reached (HR for OS = 1.75, ; HR for DFS = 1.58, ). However, in the subgroup analyses a significant benefit of OncoVAX was seen in patients with colon cancer (HR for OS = 2.83, ; HR for DFS = 2.67, ) and not in those with rectal cancer (HR for OS = 1.13, ; HR for DFS = 1.05, ) [60]. The phase III 5283 trial enrolled 412 colon cancer patients with stages II and III; no differences in OS and DFS were observed [61]. Lastly, in the phase 8701 III trial, 254 patients with stages II and III colon cancer patients were enrolled; the vaccine was centrally manufactured and was administered 4 times instead of 3. A 44% risk reduction for disease recurrence was observed in patients treated with OncoVAX (). In the subgroup analyses, the efficacy was only observed in stage II patients (61% risk reduction for disease recurrence) [62].

A meta-analysis including the 3 above reported trials showed an improvement in recurrence-free interval by OncoVAX with an annual odds reduction of % (). The subgroup analysis by stage showed a predominant improvement in stage II patients () [61]. According to these promising data, a multicenter phase III trial with OncoVAX in stage II patient is currently ongoing (Table 1).

Virus modified vaccines were also investigated in CRC. In particular, the Newcastle disease virus-infected (NDV) autologous modified vaccine, obtained admixing a nononcolytic strain Ulster of NDV with irradiated autologous tumour cells was tested in patients undergoing radical liver resection [63]. The results of a randomized phase III trial with NDV autologous modified vaccine in patients who undergone radical resection of CRC liver metastases were published at the end of 2000s. In this study, 51 patients were enrolled. No differences in OS (primary endpoint) and in DFS (secondary endpoint) were detected. However, in the subgroup analyses, a significant advantage was observed in patients with colon cancer with respect to OS (HR 3.3,   ) and DFS (HR 2.7,   ) but not in those with rectal cancer [64].

Data emerging from cancer vaccines have not yet altered the clinical practice. Several trials are currently ongoing in adjuvant and in metastatic settings with the aim of improving vaccines immunogenicity and of identifying a subset of patients amenable of this kind of treatments.

Conversely, the striking results obtained from immune checkpoint inhibitors trials lead to the introduction of new therapeutic options in mCRC. The first data regarding initial success of immune checkpoint inhibitors in mCRC were presented in mid-2015, when the results of the phase II KEYNOTE 016 trial with pembrolizumab in patients with refractory metastatic tumours were published. In this study, three cohorts of patients were recruited: (1) cohort A: patient with high microsatellite instability (MSI-H) or deficient mismatch repair (dMMR) mCRC (); (2) cohort B: patients with microsatellite stability (MSS) or proficient (p)MMR mCRC (); and (3) cohort C: patients with MSI-H non-mCRC cancers (). Immune-related objective response (iORR) rates were 40%, 0%, and 71% in the 3 groups, respectively; the median PFS and OS were not reached in cohort A; 2.2 and 5.0 months, respectively, in cohort B (HR for PFS = 0.10, , HR for OS = 0.22, ) [41]. For the first time the activity of an anti-PD1 was demonstrated in patients with MSI-H while no effect was observed in MSS mCRC patients. As possible explanation of such results, it was demonstrated that tumours with MSI-H are characterized by a high burden of somatic mutations that can be recognised by the patient’s immune system. As a supplementary proof, MSI-H tumours were found to be characterized by a dense immune infiltration and a cytokine-rich environment [65].

Based on these results, on November 2, 2015, the FDA granted “breakthrough therapy designation” for pembrolizumab in advanced CRCs with high microsatellite instability (MSI-H). To further explore this strategy, the KEYNOTE 164 trial was planned [66]; in this trial, pretreated MSI-H mCRC patients are candidate to receive pembrolizumab 200 mg every 3 weeks. Moreover, a phase 3 study of pembrolizumab versus investigator choice chemotherapy for MSI-H mCRC in first line is ongoing (KEYNOTE 177) [67].

One year later, at the 2016 ASCO Annual Meeting, several encouraging preliminary data on immune-checkpoint inhibitors in the treatment of mCRC were presented, including the update of the KEYNOTE 016 trial [68], a new treatment strategy adopting a combination of anti-CTLA4 and anti PD1 (the CHECKMATE 142 trial) [69], and a phase Ib study combining a MEK inhibitor and an anti-PD-L1 in patients with microsatellite stable (MSS) tumours [70].

The phase II CHECKMATE 142 trial investigates nivolumab plus or minus ipilimumab in patients with MSS and MSI-H mCRC patients in advanced lines of treatment. In the MSI-H cohort, ORR was 25,5% in patients receiving nivolumab () and 33,3% in those receiving ipilimumab plus nivolumab (). Data presented at ASCO GI 2017 on 72 patients treated with nivolumab showed encouraging results for ORR, 12-month PF rate, and 12-month survival rate (31%; 48,4%; and 73.8%, resp.). Responses were observed regardless of tumour or immune cell PD-L1 expression, BRAF, KRAS mutation status, or clinical history of Lynch syndrome. Centrally revised data identified 2 patients experiencing complete response. This data represents a big step forward in the treatment of advanced mCRC and we perfectly agree with the conclusion of the authors stating that nivolumab should be considered a new standard of care for patients with previously treated MSI-H advanced CRC [70]. A new cohort of the trial is evaluating the activity of nivolumab and ipilimumab as first-line treatment (Table 1).

Data presented so far are highly significant in the subgroup of MSI-H patients while results in MSS cases are disappointing. RRs in patients with MSS treated with nivolumab or ipilimumab plus nivolumab were 10% and 0%, respectively, with overall poor PFS and OS [70]. Thus, many efforts are ongoing in order to identify possible immunotherapeutic strategies in MSS. In preclinical models, MEK inhibition alone increased the tumour-infiltrating CD8⁺ T cells and induced MHC-I upregulation, the combination of MEK inhibition with an anti-PD-L1 resulted in synergistic and durable tumour regression [71]. In a cohort of 23 mCRC patients receiving the MEK inhibitor cobimetinib and the anti-PD-L1 antibody atezolizumab, the ORR was 17% with 4 partial responses and 5 disease stabilizations. Among responders 3 out of 4 were MSS, thus leading to hypothesizing a possible effect for such strategy in this group of patients. A phase III trial is currently investigating atezolizumab and cobimetinib versus regorafenib in refractory mCRC [71, 72]. Other association strategies of checkpoints inhibitors with chemotherapy or anti-VEGF are also under investigation in mCRC patients irrespective of MSI status. All immune strategies under investigation in CRC are summarized in Table 2.

Table 2: Ongoing studies on colorectal cancers.

NCT identifier
Setting
Phase
Study interventions
Number of patients
Primary endpoint

Checkpoint inhibitors

 NCT03026140
Early stage colon cancer
II
Nivolumab + ipilimumab versus nivolumab + ipilimumab + celecoxib
60
Safety

 NCT02260440
Chemorefractory mCRC
II
Pembrolizumab + azacitidine
40
ORR

 NCT02997228
MSI-H mCRC
III
mFOLFOX6 bevacizumab versus atezolizumab versus atezolizumab + mFOLFOX6 bevacizumab
439
PFS

 NCT02870920
Chemorefractory mCRC
II
BSC + durvalumab + tremelimumab versus BSC
180
OS

 NCT02788279
Chemorefractory mCRC
III
Atezolizumab versus cobimetinib + atezolizumab versus regorafenib
360
OS

 NCT02991196
mCRC
I
DS-8273a + nivolumab
20
DLTs; MTD; ORR; DCR; TTP; PFS;

 NCT02713373
Unresectable mCRC
I-II
Cetuximab + pembrolizumab
42
PFS; ORR; safety and tolerability

 NCT02981524
mCRC
II
CY/GVAX + pembrolizumab
30
ORR

 NCT02754856
mCRC with resectable CLM
I
Tremelimumab + MEDI4736 + FOLFOX bevacizumab
35
Feasibility

 NCT02933944
RAS mutmCRC
I
TG02 versus TG02+ pembrolizumab
20
Safety; irORR

 NCT02860546
MSS mCRC
II
TAS-102 + nivolumab
35
irORR

 NCT02948348
Locally advanced RC
I-II
Chemoradiotherapy with capecitabine → nivolumab → surgical therapy
50
PCR

 NCT02437071
mCRC

II
Pembrolizumab+ radiotherapy versus pembrolizumab+ ablation
48
ORR

NCT02060188 (CheckMate 142)
mCRC
II
Nivolumab/nivolumab + ipilimumab/nivolumab + ipilimumab 1st line/nivolumab + ipilimumab + cobimetinib/nivolumab + BMS-986016/nivolumab + daratumumab
260
ORR

 NCT02512172
mCRC
I
Oral CC, 486 & MK-3475 versus romidepsin & MK-3475, versus oral CC, 486 & romidepsin & MK-3475
30
Degree of change in TIL

 NCT02227667
mCRC
II
MEDI4736
48
ORR

NCT02563002 (KEYNOTE 177)
MSI-H mCRC
III
Pembrolizumab versus
investigator's choice of standard of care
270
PFS

Vaccines

 NCT02448173
Stage II
III
OncoVAX + surgery versus surgery
550
DFS

 NCT01890213
Stage III
I
AVX701
12
AE

 NCT02718430
mCRC with CLM
I
VXM01
24
Safety and tolerability

 NCT01741038
mCRC
II-III
AlloStim® + cryoablation versus AlloStim + physician's choice (PC)
450
OS

 NCT02615574
Refractory mCRC
II
áDC1 vaccine+ CKM
44
OS

Cytokines

 NCT02415699
Stage III

II-III
DC-CIK + chemotherapy versus chemotherapy
100
DFS

 NCT02280278
Stage III
III
Adjuvant CT → CIKCC versus adjuvant CT
550
DFS

 NCT01929499
Stages II-III
II
Adjuvant CT + synchronous CIKCC versus adjuvant CT → CIKCC versus adjuvant CT
210
DFS

 NCT02466906
Stage III
II
RhGM-CSF versus placebo
60
DFS

Oncolytic virus

 NCT01274624
KRAS
II
REOLYSIN® + FOLFIRI, bevacizumab
32
DLTs

 NCT01622543
mCRC
II
FOLFOX + bevacizumab + reolysin versus FOLFOX + bevacizumab
109
PFS

Adoptive cell therapy study

 NCT03008499
mCRC
I-II
High-activity natural killer versus no special treatment
18
Relief degree of tumours evaluated by RECIST

 NCT02577588
mCRC
I
Reactivated T cells
10
DLTs

Adjuvant therapy

NCT01545141
Resectable CRC

I-II
Surgery versus chemokine modulatory regimen (a combination of IFN, celecoxib, and rintatolimod prior to surgery
50
Change in the number of tumour-infiltrating CD8+ cells

Immune modulators therapy

NCT02077868 (IMPALA)
mCRC
III
Maintenance versus MGN1703
540
OS

NCT02413853 (PRIMIER)
mCRC
II
PRI-724 + mFOLFOX6/bevacizumab versus mFOLFOX6/bevacizumab
100
PFS

GC, gastric cancer; GEJC, gastroesophageal junction cancer; EC: esophageal cancer; NA, not assessed; MTD, maximum tolerated dose; MAD, maximum administered dose; DTL, dose limiting toxicity; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; DFS, disease-free survival; NS, not specified; RP2D, recommended dose of phase II; AE, adverse events; DCR, disease control rate; BSC, best supportive care; DCR disease control rate.

Table 3: Ongoing studies on hepatocellular carcinoma.

Trial (ClinicalTrials.gov identifier)
Setting
Phase
Study interventions
Number of patients
Primary endpoint

Checkpoint inhibitors

NCT02576509 CheckMate459
1st line
III
Nivolumab versus sorafenib in first line
726
OS, ORR

NCT02702414 Keynote 224
2nd line
II
Pembrolizumab in second line
100
ORR

NCT02702401 Keynote 240
2nd line
III
Pembrolizumab versus BSC in second line
408
PFS, OS

NCT01658878 CheckMate 040

I/II
Nivolumab versus nivolumab + Ipilimumab nivolumab
620
AEs, SAEs, ORR

 NCT02519348
Advanced
II
Tremelimumab + MEDI4736 versus tremelimumab versus MEDI4736
144
AEs, SAEs, DLTs

Vaccines

 NCT01974661
Advanced
I
COMBIG-DC (allogeneic dendritic cells) cancer vaccine
18
Safety and tolerability

Cytokines

 NCT02632188
Resected
I/II
SURGERY → DC-PMAT treatment
60
PFS

 NCT02873442
Advanced
I/II
TACE versus TACE + precision cell immunotherapy
40
PFS, OS

 NCT02487017
Advanced
II
TACE versus TACE + DK-CIK

OS

CAR-T cells

 NCT02729493
Advanced
II
EPCAM-targeted CAR-T cells
25
DCR

6. Focus on Hepatocellular Carcinoma

Over the last decades, recombinant human interferon-alfa (IFN-α) has been extensively studied in patients with HCC, due to its previous use as immune-stimulatory antiviral agent. Both adjuvant and advanced settings have been investigated [73, 74].

Given the association of a specific HCC-directed immune response with a prognosis improvement, various targets among tumour associated antigens (TAA) or neoantigens have been investigated. Peptide based, DNA/RNA based, and DCs based vaccines have been tested in clinical setting, but efficacy data have been to date disappointing [75, 76]. In a phase 1 study published in 2012, two glypican-3 (GPC3) derived peptides restricted for HLA-A phenotypes induced specific CD8+ cells tumour infiltration; a peptide-specific cytotoxic T response was associated with longer OS, but only one out of 33 treated patients reached an objective response [77]. The same treatment was investigated in a more recent phase II single-arm study in the adjuvant setting after resection or RFA: among 41 patients evaluable, 31 (75,6%) experienced a recurrence, with a mOS of 20.1 months [78]. Telomerase-derived peptide [79], DNA, RNA, and DCs based vaccines have also been studied with overall negative results [80–86]. A new phase I clinical trial to evaluate the safety of an allogenic dendritic cell vaccine-COMBIG-DC in HCC patients is now recruiting participants (NCT01974661).

As for other GI cancers, most exciting results derived from data on immune checkpoint inhibitors. The first report came in 2013 from a pilot phase II study of tremelimumab, an anti-CTLA-4 mAb, in 20 patients with HCC and chronic HCV. Efficacy data showed a good antitumour activity, with a 17.6% RR, 76.4% DCR, and a median PFS of 6.5 months [87]; these results are remarkable when considering that the majority of patients were pretreated with sorafenib and had a Barcelona Clinic Liver Cancer (BCLC) stage C and 43% had a Child-Pugh stage B.

Several phase II/III trials with anti-PD-1/PDL-1 agents, alone or in combination with other compounds, are ongoing, with some preliminary data already reported. The most robust data presented so far are about the anti-PD-1 nivolumab, investigated alone or in combination with ipilimumab in a multicohort phase I/II study opened in 2012 for advanced HCC patients (CHECKMATE040). Four out of 5 scheduled cohorts have completed enrollment for a total of 576 HCC patients treated. Among 262 patients treated with nivolumab monotherapy, across dose escalation () and dose expansion cohorts (), [88] a 20% RR was observed irrespective of dose, HCV, or HBV infection status and PDL-1 expression on tumour cells. Median duration of response was 9.9 months; median OS was 15.0 months and 13.2 months in dose escalation and expansion cohort, respectively. These data are very promising, especially considering the overall poor prognosis of HCC patients. In the Sorafenib Hepatocellular Carcinoma Assessment Randomised Protocol (SHARP) RR, survival rate at 12 months was 2% and 44%, respectively, and mOS was 10.7 months [89, 90]. Thus, a phase III study of nivolumab versus sorafenib in treatment-naïve patients has been planned and is already ongoing (CHECKMATE 459) [91].

The anti-PDL-1 durvalumab demonstrated clinical activity in several solid tumours, including 19 HCC patients, in a phase 1 study published in 2014 [92]; a randomized open-label phase II study is currently ongoing with durvalumab, tremelimumab, or the combination of the two compounds in patients with unresectable HCC. Pembrolizumab is also under investigation in HCC: a phase II open-label study just completed the enrollment with sorafenib intolerant or progressed patients (Keynote 224), and a phase III study planning to enroll 408 second-line patients is recruiting patients (Keynote 240). Ongoing studies are summarized in Table 3.

7. Possible Biomarkers for Immunotherapy: The Pathologist Perspective

The introduction of immunotherapies as possible treatment options in GI cancers made the assessment of MSI status (especially for CRC) and PD-L1 expression crucial in the pathologic assessment of GI cancers. Overall, an adequate characterization of the immune microenvironment in cancer samples emerged as the driver diagnostic element for the identification of patients likely to benefit from specific immunotherapies [93, 94].

Among the others, the greatest focus has been on PD-L1 expression [95]. Increasing evidences pointed out to the association between PD-L1 and a higher burden of disease, more extensive metastatic involvement of lymph nodes, and poorer survival, in both esophageal and gastric cancers. Although PD-L1 testing by immunohistochemistry has been associated with a significant enrichment for populations with clinical benefit to anti-PD1 or anti-PDL1 therapies, no conclusive date have been reported so far [46, 57] and several factors are limiting its use in the clinical practice. Above all, different threshold levels have been adopted for the identification of positive samples in different tumour types [96]. Several reports pinpointed the predictive value of PD-L1 expression on infiltrating immune cells instead of tumour cells [97]. Most companies have developed their own companion PD-L1 immunohistochemistry diagnostic assay characterized by different antibody-specific features. Of course, this diversified request for immunohistochemical testing and the related need of antibody/company-specific immunostainers is inconsistent with the current practice of most surgical pathology laboratories.

From a general perspective, the identification of consistent biomarkers to be introduced into clinical practice is affected by (i) the inherent biological heterogeneity of tumour microenvironment; (ii) the complexity of novel immunotherapeutic regimens and the combination of immunotherapy with other target therapeutics; (iii) the variability on molecular biology testing; (iv) the inconsistent aptitude of formalin-fixed paraffin-embedded (FFPE) preparations with many downstream molecular biology techniques [98]; (v) the significant discrepancies in the proposed biomarker evaluation systems [99]; (vi) the need of integrated diagnostics (i.e., histology, immunophenotyping, and molecular profiling), not always available in “spoke” surgical pathology units.

Similar considerations might be drawn for MSI status assessment that represents the only well-established predictive biomarker for immunotherapy response in mCRC. MSI status is assessed by means of immunohistochemistry evaluating altered expression of mismatch repair proteins (i.e., MLH1, PMS2, MSH2, and MSH6) or by means of PCR techniques detecting mutations on BAT25, BAT26, D2S123, D5S346, and D17D250, according to the Bethesda panel guidelines [100].

Due to the association of MSI status with a higher mutational and neoantigen burden, also more sophisticated next-generation sequencing approaches have been successfully applied in the evaluation of mutational load in immunotherapy clinical trials, and these methods allow the identification of other hypermutated tumour classes such as those characterized by dysfunctions in DNA polymerases (POLE) [101]. However, even these promising data, neither mutational load analysis nor the evaluation of the mismatch repair machinery status, have been included in the clinical selection of the patients undergoing immunotherapy, so far.

Both PD-L1 expression and MSI might play a role as positive predictive factors for GC and immunotherapy according to the data from the Cancer Genome Atlas (TCGA) Research Network project [22].

The presence of tumour-infiltrating lymphocytes (TILs) is an indirect sign of disease control through immune mechanisms and has been evaluated as a predictive biomarker for checkpoint inhibitor immunotherapy [102, 103]. Beside these therapeutic implications, the landmark studies of Jérôme Galon identified the prognostic value of the global assessment of the immune infiltrate (also known as immunoscore) in colorectal cancer and in other solid tumours [104, 105]. However, infiltrating lymphocytes evaluation still lacks intralaboratory and intrapathologist standardization and is not yet a widespread practice among the pathologists’ community.

Because of all these challenging problems in the definition of ultimate optimized model for predicting tumour response to anti-PD1 or anti-PD-L1-based therapies, more technically complex combined biomarker strategies and/or comprehensive immune gene signatures have been also successfully tested. The limited amount of analysable material in preneoadjuvant biopsy specimens and the use of FFPE samples are, however, currently affecting the improvement of these approaches in the clinical setting.

Overall these data underline that an adequate personalized immunotherapy will be obtained only with the integration of traditional microscope-based biomarkers (such as the immunoscore) to more advanced FFPE-compatible genetic, genomic, and expression profiling strategies. A new revolutionizing era of diagnostic surgical pathology has started.

8. Back to the Bench: Biomarkers and Genetic Signatures as Predictive Factors

As stated above, traditional microscope-based techniques are not adequate to comprehensively assess the intriguing landscape of tumour benefitting from immunotherapy. Genetic signatures might be useful tools to identify predictive biomarkers able to help patients’ selection.

Among proposed biomarkers, MSI-H status represents the only validated positive predictive factor for immunotherapy response in mCRC; however, it occurs in only 6% of patients. Given the high benefit deriving from immune checkpoint inhibitors in this setting in terms of OS, responses, and symptoms relief, recently, several papers have pointed out the importance of using gene expression profile to better identify those tumours that behave as MSI-H.

Tian et al. [106] adopted full genome expression data of stage II and stage III CRC to identify genes that correlate with MSI status. An MSI gene signature was developed and further validated in other external data set with an overall accuracy of about 90.6%. The strength of the MSI-signature is that it can identify the true MSI-H patients as well as a group of patients that are not MSI by conventional clinical tests but they are by signature. Those patients are defined as MSI-like and share the hypermutated status as pure MSI-H patients. Furthermore, they seem to not respond to 5-FU regimen as stage II MSI-H patients. If MSI-H mCRC patients benefit from immunotherapy, we can assume that also MSI-like patients will. Indeed, this is the rationale of one of the trials that will soon be run in the frame of the MoTriColor consortium (http://www.motricolor.eu/).

In line with these findings, more recently, Mlecnic et al. [107] performed a comprehensive analysis of the tumour microenvironment, immune gene expression, and mutational status in CRC, so-called immunoscore. They identified a high number of genes upregulated in MSI tumours (high immunoscore) versus MSS (low immunoscore). These genes were mainly associated with INFγ signalling, Th1 related cytokines, antigen presentation pathways, chemokine receptors, and chemokine and leucocyte migration. However, a high immunoscore was identified also in a subset of MSS tumours and was not observed in a certain number of MSI tumours. Both MSI-H and high immunoscore predicted favorable prognosis among CRC patients; however, data derived from multivariate analyses identified the immunoscore as a stronger predictor of good CRC patients’ survival than MSI and proposed it as a stronger predictor of immunotherapy response than MSI.

Although those two studies do not question the role of MSI status in CRC in terms of increased immune infiltrates, higher frequencies of frame shift mutations, and favorable outcome, still they demonstrate that the canonical MSI tests may be not sufficient to fish out all the patients that have a common “MSI phenotype” and could benefit from immunotherapy. Interestingly enough, Zhao et al. [108] also identified a MSI-H mutation signature by using whole genome and whole exome sequencing. They confirmed this signature to be similar to germline DNA, thus meaning that a fraction of genetic variations arises through mutations escaping MSI. Most importantly, they identified a large number of recurrent indels that can be used to detect MSI and that are currently under implementation for clinical application. Moreover, they found that recurrent indels are enriched for the double-strand break repair (DBS) by homologous recombination (HR) pathway. All in all, these data indicate that the MMR pathway is not yet completely known and that in the future new biomarkers belonging to this pathway will need to be validated and used as predictive of response to immunotherapy. Moreover, the importance of those gene signatures has been shown only in CRC. Thus, further studies will be required to know if this applies to other tissues types that show microsatellite instability, such as gastric cancer, genitourinary tract malignancies, and esophageal cancer. Since MSI-H tumours are not that frequent especially in the metastatic setting, the use of gene expression profile could help in enlarging the group of patients who will benefit from such treatment. At the same time, this will also avoid that useless toxicity will be given to patients who seem to carry an MSI-H tumours but that by gene expression it is not defined as MSI-like or immunoscore positive or positive for the MMR-deficient mutation signature. A proof that a response to immunotherapy can also be observed in MSS tumours is provided by the case report published by Chen et al. [109]. Authors report indeed the case of a 64-year-old man who received pembrolizumab as second-line treatment for a HER2 positive metastatic gastric cancer. Clinical tests reported the tumour to be MSS and Epstein Barr negative and to not carry any mutations in the POLE gene, thus meaning carrying all the biomarkers that so far have been identified as negative predictors of response to immunotherapy. Although the authors did not investigate other biomarkers to understand why the patient responded to immunotherapy, based on the data here summarized, we can hypothesize that MSI clinical tests are not sensitive enough and that the integration of multiple tumour and immune response parameters such as protein expression, genomics, and transcriptomics may be necessary for accurate prediction of clinical benefit.

Finally, response to immunotherapy might not only be driven by the “genetic makeup” of the tumour and the way how the immune system reacts to it, but also by its regulation via other mechanisms such as the gut microbiota. Indeed two recent papers show its role in modulating the anticancer activity of CTLA4 and PD1 blockade. Vétizou et al. [110] elegantly showed that the gut microbiota can itself reduce the tumour volume and when combined with immunotherapy it further reduces the tumour size. This effect is driven only by certain microbiota composition, like the Bacteroides spp., which seem to be also regulated by ipilimumab itself. Life style and immunotherapy could change the gut microbiota. This in turn affects interleukin 12 dependent Th1 immune response which facilitates tumour control both in mice and in patients while sparing intestinal integrity. Moreover, the oral administration of Bifidobacterium associates with tumour effect and when combined with anti-PDL1 therapy nearly abolishes tumour outgrowth. Whether the role of the microbiota in modulating the response to immunotherapy is tissue specific is not yet clear. Thus, further investigation is required. If those data will be confirmed, we might consider in the future the use of stool to identify biomarkers and fecal transplantation to modulate the immune response.

9. Future Perspectives

Recently, ASCO proclaimed immunotherapy against cancer as “the advance of the year.” In particular, immune checkpoint blockade was heralded as a major breakthrough in cancer therapy in the last years [111]. However, there are still many challenges that must be overcome; in particular, in GI cancer many drugs are still in the early phase of development.

First of all, biomarkers identification and validation represent a major issue as discussed in the last 2 paragraphs of the review.

Secondly, clinicians still need to learn how to deal with response assessment in patients receiving immunotherapy. Conventional and nonconventional responses have been reported. As an example, patients experiencing a rapid disease progression need to be carefully evaluated with an expert radiologist, to exclude the occurrence of a pseudoprogression, identified as the burning of an inflammatory response that can simulate the onset of new lesions. Moreover, we have to be aware that a RECIST response might be observed after more than three months of treatment but can be persistent after occurrence [112]. It has also been proposed that RECIST criteria might not be adequate to assess immune response; although immune-related response criteria have been developed [112], they are still not universally adopted especially in clinical trials on GI immunotherapy [112].

Toxicity profile of immunotherapeutic agents represents another thorny issue. GI oncologists involved in clinical trials are facing a different adverse event scenario compared to the traditional chemotherapy one and need to improve their knowledge and skills to treat immune-related toxicities in a subset of patients who may already have baseline GI, liver function, and endocrine abnormalities from their underlying cancer or as complications from prior treatments. [113].

Finally the most efforts are focusing on the development of novel approaches to enhance this innovative strategy. All ongoing trials are shown in Tables 1–3. Promising trials have been evaluating innovative combination treatments (so-called “combo-immunotherapy”), that is, PD-1 or PD-L1 blockade in combination with (1) anti-CTLA4, (2) adaptive immunotherapy such as anti-LAG3, (3) innate immunotherapy such as TLRs agonists, (4) chemo- or radiotherapy, (5) drugs able to increase antigen presentation such as the COX-2, JAK1/2 inhibitor or the MEK inhibitor cobimetinib, and (6) targeted therapy (anti-HER2, anti-VEGFR2) [69, 114, 115].

From a clinician perspective, the use of immunotherapies in recent clinical trials gave us the opportunity to contribute to a paradigmatic shift in the treatment of GI cancers. We are glad to observe highly pretreated patients experiencing a dramatic clinical benefit after treatment start, with symptoms relief, long lasting disease stabilization, and an overall manageable safety profile. We are really feeling a revolution in the daily life of our patients. Every day we ask questions about future availability of clinical trials involving immunotherapeutic agents for GI cancers from our new and historical patients. We strongly believe that further steps of drugs development such as larger phases II and III clinical trials are warranted in order to answer unsolved question and to establish the efficacy of immunotherapeutic agents. A wide international involvement of experienced centers in the next clinical trials will break a potential unequal distribution of immunotherapeutic resources.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors’ Contributions

Letizia Procaccio and Marta Schirripa equally contributed as first authors. Vittorina Zagonel and Sara Lonardi equally contributed as senior authors.

References

P. G. Toomey, N. A. Vohra, T. Ghansah, A. A. Sarnaik, and S. A. Pilon-Thomas, “Immunotherapy for gastrointestinal malignancies,” Cancer Control, vol. 20, no. 1, pp. 32–42, 2013.

S. Pernot, M. Terme, T. Voron et al., “Colorectal cancer and immunity: what we know and perspectives,” World Journal of Gastroenterology, vol. 20, no. 14, pp. 3738–3750, 2014.

M. D. Vesely and R. D. Schreiber, “Cancer immunoediting: Antigens, mechanisms, and implications to cancer immunotherapy,” Annals of the New York Academy of Sciences, vol. 1284, no. 1, pp. 1–5, 2013.

T. J. Zumwalt and A. Goel, “Immunotherapy of Metastatic Colorectal Cancer: Prevailing Challenges and New Perspectives,” Current Colorectal Cancer Reports, vol. 11, no. 3, pp. 125–140, 2015.

K.-W. Jung, Y.-J. Won, H.-J. Kong, C.-M. Oh, D. H. Lee, and J. S. Lee, “Prediction of cancer incidence and mortality in korea, 2014,” Cancer Research and Treatment, vol. 46, no. 2, pp. 124–130, 2014.

L. Shi, S. Chen, L. Yang, and Y. Li, “The role of PD-1 and PD-L1 in T-cell immune suppression in patients with hematological malignancies,” Journal of Hematology & Oncology, vol. 6, no. 1, p. 74, 2013.

E. Q. Han, X.-L. Li, C.-R. Wang, T.-F. Li, and S.-Y. Han, “Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges,” Journal of Hematology and Oncology, vol. 6, article 47, 2013.

M. Bonotto, S. K. Garattini, D. Basile et al., “Immunotherapy for gastric cancers: emerging role and future perspectives,” Expert Review of Clinical Pharmacology, vol. 10, no. 6, pp. 609–619, 2017.

D. O. Croci, M. F. Zacarías Fluck, M. J. Rico, P. Matar, G. A. Rabinovich, and O. G. Scharovsky, “Dynamic cross-talk between tumor and immune cells in orchestrating the immunosuppressive network at the tumor microenvironment,” Cancer Immunology, Immunotherapy, vol. 56, no. 11, pp. 1687–1700, 2007.

L. Chen and D. B. Flies, “Molecular mechanisms of T cell co-stimulation and co-inhibition,” Nature Reviews Immunology, vol. 13, no. 4, pp. 227–242, 2013.

F. M. Burnet, “The concept of immunological surveillance,” Progress in Tumor Research Home, vol. 13, pp. 1–27, 1970.

L. Thomas, “Cellular and humoral aspects of the hypersensitive states,” Journal of Internal Medicine, vol. 170, no. 1, p. 128, 1961.

M. J. M. Gooden, G. H. de Bock, N. Leffers, T. Daemen, and H. W. Nijman, “The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis,” British Journal of Cancer, vol. 105, no. 1, pp. 93–103, 2011.

J. Galon, F. Pagès, and F. M. Marincola, “Cancer classification using the Immunoscore: a worldwide task force,” Journal of Translational Medicine, vol. 10, p. 205, 2012.

B. Lakshmi Narendra, K. Eshvendar Reddy, S. Shantikumar, and S. Ramakrishna, “Immune system: A double-edged sword in cancer,” Inflammation Research, vol. 62, no. 9, pp. 823–834, 2013.

R. D. Schreiber, L. J. Old, and M. J. Smyth, “Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion,” Science, vol. 331, no. 6024, pp. 1565–1570, 2011.

K. Sideras, H. Braat, J. Kwekkeboom et al., “Corrigendum to "Role of the immune system in pancreatic cancer progression and immune modulating treatment strategies" [Cancer Treat. Rev. 40 (2014) 513-522],” Cancer Treatment Reviews, vol. 40, no. 7, p. 892, 2014.

N. Martínez-Bosch, M. G. Fernández-Barrena, M. Moreno et al., “Galectin-1 drives pancreatic carcinogenesis through stroma remodeling and hedgehog signaling activation,” Cancer Research, vol. 74, no. 13, pp. 3512–3524, 2014.

G. Brandacher, A. Perathoner, R. Ladurner et al., “Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: Effect on tumor-infiltrating T cells,” Clinical Cancer Research, vol. 12, no. 4, pp. 1144–1151, 2006.

T. Nomi, M. Sho, T. Akahori et al., “Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer,” Clinical Cancer Research, vol. 13, no. 7, pp. 2151–2157, 2007.

Q. Gao, X.-Y. Wang, S.-J. Qiu et al., “Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma,” Clinical Cancer Research, vol. 15, no. 3, pp. 971–979, 2009.

Cancer Genome Atlas Research Network, “Comprehensive molecular characterization of gastric adenocarcinoma,” Nature, vol. 513, pp. 202–209, 2014.

A. Maitra and R. H. Hruban, “Pancreatic cancer,” Annual Review of Pathology: Mechanisms of Disease, vol. 3, pp. 157–188, 2008.

L. M. Coussens and Z. Werb, “Inflammation and cancer,” Nature, vol. 420, no. 6917, pp. 860–867, 2002.

F. Ghiringhelli, C. Ménard, M. Terme et al., “CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-β-dependent manner,” Journal of Experimental Medicine, vol. 202, no. 8, pp. 1075–1085, 2005.

M. Waghray, M. Yalamanchili, M. P. D. Magliano, and D. M. Simeone, “Deciphering the role of stroma in pancreatic cancer,” Current Opinion in Gastroenterology, vol. 29, no. 5, pp. 537–543, 2013.

J. Haqq, L. M. Howells, G. Garcea, M. S. Metcalfe, W. P. Steward, and A. R. Dennison, “Pancreatic stellate cells and pancreas cancer: Current perspectives and future strategies,” European Journal of Cancer, vol. 50, no. 15, pp. 2570–2582, 2014.

M. M. Mueller and N. E. Fusenig, “Friends or foes—bipolar effects of the tumour stroma in cancer,” Nature Reviews Cancer, vol. 4, no. 11, pp. 839–849, 2004.

M. Herrera, A. Herrera, G. Domínguez et al., “Cancer-associated fibroblast and M2 macrophage markers together predict outcome in colorectal cancer patients,” Cancer Science, vol. 104, no. 4, pp. 437–444, 2013.

R. H. Vonderheide, D. L. Bajor, R. Winograd, R. A. Evans, L. J. Bayne, and G. L. Beatty, “CD40 immunotherapy for pancreatic cancer,” Cancer Immunology, Immunotherapy, vol. 62, no. 5, pp. 949–954, 2013.

A. E. Moran, M. Kovacsovics-Bankowski, and A. D. Weinberg, “The TNFRs OX40, 4-1BB, and CD40 as targets for cancer immunotherapy,” Current Opinion in Immunology, vol. 25, no. 2, pp. 230–237, 2013.

T. Yamamoto, H. Yanagimoto, S. Satoi et al., “Circulating CD4 +CD25 + regulatory T cells in patients with pancreatic cancer,” Pancreas, vol. 41, no. 3, pp. 409–415, 2012.

G. P. Dunn, L. J. Old, and R. D. Schreiber, “The immunobiology of cancer immunosurveillance and immunoediting,” Immunity and Immunoediting, vol. 21, no. 2, pp. 137–148, 2004.

S. L. Topalian, C. G. Drake, and D. M. Pardoll, “Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity,” Current Opinion in Immunology, vol. 24, no. 2, pp. 207–212, 2012.

C. Blank, T. F. Gajewski, and A. Mackensen, “Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: Implications for tumor immunotherapy,” Cancer Immunology, Immunotherapy, vol. 54, no. 4, pp. 307–314, 2005.

J. L. Markman and S. L. Shiao, “Impact of the immune system and immunotherapy in colorectal cancer,” Journal of Gastrointestinal Oncology, vol. 6, no. 2, pp. 208–223, 2015.

M. G. Hanna Jr., H. C. Hoover Jr., J. B. Vermorken, J. E. Harris, and H. M. Pinedo, “Adjuvant active specific immunotherapy of stage II and stage III colon cancer with an autologous tumor cell vaccine: First randomized phase III trials show promise,” Vaccine, vol. 19, no. 17-19, pp. 2576–2582, 2001.

M. Derouazi, W. Di Berardino-Besson, E. Belnoue et al., “Novel cell-penetrating peptide-based vaccine induces robust CD4+ and CD8+ T cell-mediated antitumor immunity,” Cancer Research, vol. 75, no. 15, pp. 3020–3031, 2015.

C. Robert, J. Schachter, G. V. Long et al., “Pembrolizumab versus ipilimumab in advanced melanoma,” The New England Journal of Medicine, vol. 372, no. 26, pp. 2521–2532, 2015.

M. E. Valsecchi, “Combined nivolumab and ipilimumab or monotherapy in untreated melanoma,” New England Journal of Medicine, vol. 373, no. 13, p. 1270, 2015.

T. Aparicio, “PD-1 blockade in tumors with mismatch-repair deficiency: Le DT (2015) N Engl J Med May 30,” Colon and Rectum, vol. 9, no. 3, pp. 182–184, 2015.

M. J. Besser, R. Shapira-Frommer, A. J. Treves et al., “Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients,” Clinical Cancer Research, vol. 16, no. 9, pp. 2646–2655, 2010.

S. A. Rosenberg, “Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy,” Clinical Cancer Research, vol. 17, no. 13, pp. 4550–4557, 2011.

A. Amedei, E. Niccolai, and M. M. D'Elios, “T cells and adoptive immunotherapy: recent developments and future prospects in gastrointestinal oncology,” Clinical and Developmental Immunology, vol. 2011, Article ID 320571, 17 pages, 2011.

R. A. Morgan, J. C. Yang, M. Kitano, M. E. Dudley, C. M. Laurencot, and S. A. Rosenberg, “Case report of a serious adverse event following the administration of t cells transduced with a chimeric antigen receptor recognizing ERBB2,” Molecular Therapy, vol. 18, no. 4, pp. 843–851, 2010.

K. Muro, H. C. Chung, V. Shankaran et al., “Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trial,” The Lancet Oncology, vol. 17, no. 6, pp. 717–726, 2016.

https://clinicaltrials.gov/ct2/show/NCT02370498, ?term=keynote+061rank=1.

https://clinicaltrials.gov/ct2/show/NCT02494583, ?term=keynote+062rank=1.

T. Doi, S. A. Piha-Paul, S. I. Jalal et al., “Updated results for the advanced esophageal carcinoma cohort of the phase Ib KEYNOTE-028 study of pembrolizumab (MK-3475),” Journal of Clinical Oncology, vol. 34, no. 4, 7 pages, 2016.

Y. Kang, T. Satoh, M. Ryu et al., “Nivolumab (ONO-4538/BMS-936558) as salvage treatment after second or later-line chemotherapy for advanced gastric or gastro-esophageal junction cancer (AGC): a double-blinded, randomized, phase III trial,” Journal of Clinical Oncology, vol. 35, abstract 2, no. 4, 2 pages, 2017.

T. Kojima, H. Hara, K. Yamaguchi et al., “Phase II study of nivolumab (ONO-4538/BMS-936558) in patients with esophageal cancer: preliminary report of overall survival,” Journal of Clinical Oncology, vol. 34, supplement 4, TPS175 pages, 2016.

D. Oh, A. C. Lockhart, D. J. Wong et al., “Avelumab (MSB0010718C), an anti-PD-L1 antibody, as a third-line treatment in patients with advanced gastric or gastroesophageal junction cancer: a phase Ib JAVELIN Solid Tumor trial,” Journal of Clinical Oncology, vol. 34, supplement 4, 2016.

https://clinicaltrials.gov/ct2/show/NCT02625610, ?term=javelin+gastricrank=2.

https://clinicaltrials.gov/ct2/show/NCT02625623, ?term=javelin+gastricrank=1.

C. Ralph, E. Elkord, D. J. Burt et al., “Modulation of lymphocyte regulation for cancer therapy: a phase II trial of tremelimumab in advanced gastric and esophageal adenocarcinoma,” Clinical Cancer Research, vol. 16, no. 5, pp. 1662–1672, 2010.

M. H. Moehler, J. Y. Cho, Y. H. Kim et al., “A randomized, open-label, two-arm phase II trial comparing the efficacy of sequential ipilimumab (ipi) versus best supportive care (BSC) following first-line (1L) chemotherapy in patients with unresectable, locally advanced/metastatic (A/M) gastric or gastro-esophageal junction (G/GEJ) cancer,” Journal of Clinical Oncology, vol. 34, 2016.

Y. Y. Janjigian, J. C. Bendell, E. Calvo et al., “CheckMate-032: Phase I/II, open-label study of safety and activity of nivolumab (nivo) alone or with ipilimumab (ipi) in advanced and metastatic (A/M) gastric cancer (GC),” Journal of Clinical Oncology, vol. 34, 2016.

E. Knudsen, P. Vail, U. Balaji et al., “Stratification of pancreatic ductal adenocarcinoma: combinatorial genetic, stromal, and immunological markers,” Clinical Cancer Research, 2017.

H. C. Hoover Jr., L. C. Peters, J. S. Brandhorst, and M. G. Hanna Jr., “Therapy of spontaneous metastases with an autologous tumor vaccine in a guinea pig model,” Journal of Surgical Research, vol. 30, no. 4, pp. 409–415, 1981.

H. C. Hoover Jr., J. S. Brandhorst, L. C. Peters et al., “Adjuvant active specific immunotherapy for human colorectal cancer: 6.5-year median follow-up of a phase III prospectively randomized trial,” Journal of Clinical Oncology, vol. 11, no. 3, pp. 390–399, 1993.

J. E. Harris, L. Ryan, H. C. Hoover Jr. et al., “Adjuvant active specific immunotherapy for stage II and III colon cancer with an autologous tumor cell vaccine: Eastern Cooperative Oncology Group study E5283,” Journal of Clinical Oncology, vol. 18, no. 1, pp. 148–157, 2000.

J. B. Vermorken, A. M. E. Claessen, H. Van Tinteren et al., “Active specific immunotherapy for stage II and stage III human colon cancer: A randomised trial,” Lancet, vol. 353, no. 9150, pp. 345–350, 1999.

W. Liebrich, P. Schlag, M. Manasterski et al., “In vitro and clinical characterisation of a newcastle disease virus-modified autologous tumour cell vaccine for treatment of colorectal cancer patients,” European Journal of Cancer and Clinical Oncology, vol. 27, no. 6, pp. 703–710, 1991.

T. Schulze, W. Kemmner, J. Weitz, K.-D. Wernecke, V. Schirrmacher, and P. M. Schlag, “Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: Results of a prospective randomized trial,” Cancer Immunology, Immunotherapy, vol. 58, no. 1, pp. 61–69, 2009.

N. J. Llosa, M. Cruise, A. Tam et al., “The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints,” Cancer Discovery, vol. 5, no. 1, pp. 43–51, 2015.

https://clinicaltrials.gov/ct2/show/NCT02460198, ?term=keynote+164rank=1.

https://clinicaltrials.gov/ct2/show/NCT02563002, ?term=keynote+177rank=1.

D. T. Le, “Programmed death-1 blockade in mismatch repair deficient colorectal cancer,” Journal of Clinical Oncology, vol. 34, no. 29, pp. 3502–3510, 2016.

M. Overman, S. Kopetz, S. Lonardi et al., “Nivolumab ± ipilimumab treatment (Tx) efficacy, safety, and biomarkers in patients (Pts) with metastatic colorectal cancer (mCRC) with and without high microsatellite instability (MSI-H): results from the CheckMate-142 study,” Annals of Oncology, vol. 34, supplementry 6, 2016.

J. C. Bendell, T. W. Kim, B. C. Goh et al., “Clinical activity and safety of cobimetinib (cobi) and atezolizumab in colorectal cancer (CRC),” Journal of Clinical Oncology, vol. 34, 2016.

M. J. Overman, S. Lonardi, F. Leone et al., “Nivolumab in patients with DNA mismatch repair deficient/microsatellite instability high metastatic colorectal cancer: update from CheckMate 142,” Journal of Clinical Oncology, vol. 35, supplementry 4, 519 pages, 2017.

P. J. R. Ebert, J. Cheung, Y. Yang et al., “MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade,” Immunity, vol. 44, no. 3, pp. 609–621, 2016.

J. M. Llovet, M. Sala, L. Castells et al., “Randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma,” Hepatology, vol. 31, no. 1, pp. 54–58, 2000.

L.-T. Chen, M.-F. Chen, L.-A. Li et al., “Long-term results of a randomized, observation-controlled, phase III Trial of Adjuvant Interferon alfa-2b in hepatocellular carcinoma after curative resection,” Annals of Surgery, vol. 255, no. 1, pp. 8–17, 2012.

L. H. Butterfield, A. Ribas, W. S. Meng et al., “T-cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer,” Clinical Cancer Research, vol. 9, p. 5902, 2003.

L. H. Butterfield, A. Ribas, V. B. Dissette et al., “A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four α-fetoprotein peptides,” Clinical Cancer Research, vol. 12, no. 9, pp. 2817–2825, 2006.

Y. Sawada, T. Yoshikawa, D. Nobuoka et al., “Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival,” Clinical Cancer Research, vol. 18, no. 13, pp. 3686–3696, 2012.

Y. Sawada, T. Yoshikawa, K. Ofuji et al., “Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients,” OncoImmunology, vol. 5, no. 5, Article ID e1129483, 2016.

T. F. Greten, A. Forner, F. Korangy et al., “A phase II open label trial evaluating safety and efficacy of a telomerase peptide vaccination in patients with advanced hepatocellular carcinoma,” BMC Cancer, vol. 10, article no. 209, 2010.

Y.-H. Lan, Y.-G. Li, Z.-W. Liang et al., “A DNA vaccine against chimeric AFP enhanced by HSP70 suppresses growth of hepatocellular carcinoma,” Cancer Immunology, Immunotherapy, vol. 56, no. 7, pp. 1009–1016, 2007.

S. Q. Li, J. Lin, C. Y. Qi et al., “GPC3 DNA vaccine elicits potent cellular antitumor immunity against HCC in mice,” Hepatogastroenterology, vol. 61, no. 130, pp. 278–84, 2014.

L. H. Butterfield, J. S. Economou, T. C. Gamblin, and D. A. Geller, “Alpha fetoprotein DNA prime and adenovirus boost immunization of two hepatocellular cancer patients,” Journal of Translational Medicine, vol. 12, no. 1, article no. 86, 2014.

W. C. Lee, H. C. Wang, C. F. Hung, P. F. Huang, C. R. Lia, and M. F. Chen, “Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial,” Journal of Immunotherapy, vol. 28, no. 5, pp. 496–504, 2005.

D. H. Palmer, R. S. Midgley, N. Mirza et al., “A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma,” Hepatology, vol. 49, no. 1, pp. 124–132, 2009.

M. El Ansary, S. Mogawer, S. A. Elhamid et al., “Immunotherapy by autologous dendritic cell vaccine in patients with advanced HCC,” Journal of Cancer Research and Clinical Oncology, vol. 139, no. 1, pp. 39–48, 2013.

F. Tada, M. Abe, M. Hirooka et al., “Phase I/II study of immunotherapy using tumor antigen-pulsed dendritic cells in patients with hepatocellular carcinoma,” International Journal of Oncology, vol. 41, no. 5, pp. 1601–1609, 2012.

B. Sangro, C. Gomez-Martin, and M. de la Mata, “A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C,” Journal of Hepatology, vol. 59, no. 1, pp. 81–88, 2013.

I. Melero, B. Sangro, T. C. Yau et al., “Nivolumab dose escalation and expansion in patients with advanced hepatocellular carcinoma (HCC): the CheckMate 040 study,” Journal of Clinical Oncology, vol. 35, supplementry 4, 226 pages, 2017.

J. M. Llovet, S. Ricci, V. Mazzaferro et al., “Sorafenib in Advanced Hepatocellular Carcinoma,” New England Journal of Medicine, vol. 359, no. 23, pp. 2497–2499, 2008.

J. Bruix, J.-L. Raou, M. Sherman et al., “Efficacy and safety of sorafenib in patients with advanced hepatocellular carcinoma: subanalyses of a phase III trial,” Journal of Hepatology, vol. 57, no. 4, pp. 821–829, 2012.

B. Sangro, J.-W. Park, C. M. Dela Cruz et al., “A randomized, multicenter, phase 3 study of nivolumab vs sorafenib as first-line treatment in patients (pts) with advanced hepatocellular carcinoma (HCC): CheckMate-459,” Journal of Clinical Oncology, vol. 34, 2016.

N. Segal, O. Hamid, W. Hwu et al., “A phase I multi-arm dose-expansion study of the anti-programmed cell death-ligand-1 (PD-L1) antibody MEDI4736: preliminary data,” Annals of Oncology, vol. 25, supplementry 4, 2014.

S. B. Lovitch and S. J. Rodig, “The Role of Surgical Pathology in Guiding Cancer Immunotherapy,” Annual Review of Pathology: Mechanisms of Disease, vol. 11, pp. 313–341, 2016.

G. T. Gibney, L. M. Weiner, and M. B. Atkins, “Predictive biomarkers for checkpoint inhibitor-based immunotherapy,” The Lancet Oncology, vol. 17, no. 12, pp. e542–e551, 2016.

M. A. Postow, M. K. Callahan, and J. D. Wolchok, “Immune checkpoint blockade in cancer therapy,” Journal of Clinical Oncology, vol. 33, no. 17, pp. 1974–1982, 2015.

S. P. Patel and R. Kurzrock, “PD-L1 expression as a predictive biomarker in cancer immunotherapy,” Molecular Cancer Therapeutics, vol. 14, no. 4, pp. 847–856, 2015.

R. S. Herbst, J. C. Soria, and M. Kowanetz, “Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients,” Nature, vol. 515, no. 7528, pp. 563–567, 2014.

M. Fassan, R. Baffa, and A. Kiss, “Advanced precancerous lesions within the GI tract: The molecular background,” Best Practice and Research: Clinical Gastroenterology, vol. 27, no. 2, pp. 159–169, 2013.

B. Eric, “Next-generation sequencing and immunotherapy biomarkers: A medical oncology perspective,” Archives of Pathology and Laboratory Medicine, vol. 140, no. 3, pp. 245–248, 2016.

A. Umar, “Revised bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability,” Journal of the National Cancer Institute, vol. 96, no. 18, pp. 1403-1404, 2004.

Y. Khagi, R. Kurzrock, and S. P. Patel, “Next generation predictive biomarkers for immune checkpoint inhibition,” Cancer and Metastasis Reviews, pp. 1–12, 2016.

T. F. Gajewski, H. Schreiber, and Y. X. Fu, “Innate and adaptive immune cells in the tumor microenvironment,” Nature Immunology, vol. 14, pp. 1014–1022, 2013.

C. Solinas, G. Pusole, L. Demurtas et al., “Tumor infiltrating lymphocytes in gastrointestinal tumors: controversies and future clinical implications,” Critical Reviews in Oncology/Hematology, vol. 110, pp. 106–116, 2017.

R. L. Ferris and J. Galon, “Additional support for the introduction of immune cell quantification in colorectal cancer classification,” Journal of the National Cancer Institute, vol. 108, no. 8, Article ID djw033, 2016.

J. Galon, B. Mlecnik, G. Bindea et al., “Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours,” Journal of Pathology, vol. 232, no. 2, pp. 199–209, 2014.

S. Tian, P. Roepman, V. Popovici et al., “A robust genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency,” Journal of Pathology, vol. 228, no. 4, pp. 586–595, 2012.

B. Mlecnik, G. Bindea, H. K. Angell et al., “Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability,” Immunity, vol. 44, no. 3, pp. 698–711, 2016.

H. Zhao, B. Thienpont, B. T. Yesilyurt et al., “Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks,” eLife, vol. 3, no. 2014, Article ID e02725, pp. 1–26, 2014.

K.-H. Chen, C.-T. Yuan, L.-H. Tseng, C.-T. Shun, and K.-H. Yeh, “Case report: Mismatch repair proficiency and microsatellite stability in gastric cancer may not predict programmed death-1 blockade resistance,” Journal of Hematology and Oncology, vol. 9, no. 1, article no. 29, 2016.

M. Vétizou, J. M. Pitt, R. Daillère et al., “Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota,” Science, vol. 350, no. 6264, pp. 1079–1084, 2015.

H. Lote, C. Cafferkey, and I. Chau, “PD-1 and PD-L1 blockade in gastrointestinal malignancies,” Cancer Treatment Reviews, vol. 41, no. 10, pp. 893–903, 2015.

J. D. Wolchok, A. Hoos, S. O'Day et al., “Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria,” Clinical Cancer Research, vol. 15, no. 23, pp. 7412–7420, 2009.

C. Ciccarese, S. Alfieri, M. Santoni et al., “New toxicity profile for novel immunotherapy agents: Focus on immune-checkpoint inhibitors,” Expert Opinion on Drug Metabolism and Toxicology, vol. 12, no. 1, pp. 57–74, 2016.

C. Robert, L. Thomas, and I. Bondarenko, “Ipilimumab plus dacarbazine for previously untreated metastatic melanoma,” The New England Journal of Medicine, vol. 364, no. 26, pp. 2517–2526, 2011.

F. S. Hodi, J. Chesney, A. C. Pavlick et al., “Combined nivolumab and ipilimumab versus ipilimumab alone in patients with advanced melanoma: 2-year overall survival outcomes in a multicentre, randomised, controlled, phase 2 trial,” The Lancet Oncology, vol. 17, no. 11, pp. 1558–1568, 2016.