Trial Watch: Immunotherapy plus radiation therapy for oncological indications

ABSTRACT

Malignant cells succumbing to some forms of radiation therapy are particularly immunogenic and hence can initiate a therapeutically relevant adaptive immune response. This reflects the intrinsic antigenicity of malignant cells (which often synthesize a high number of potentially reactive neo-antigens) coupled with the ability of radiation therapy to boost the adjuvanticity of cell death as it stimulates the release of endogenous adjuvants from dying cells. Thus, radiation therapy has been intensively investigated for its capacity to improve the therapeutic profile of several anticancer immunotherapies, including (but not limited to) checkpoint blockers, anticancer vaccines, oncolytic viruses, Toll-like receptor (TLR) agonists, cytokines, and several small molecules with immunostimulatory effects. Here, we summarize recent preclinical and clinical advances in this field of investigation.

KEYWORDS:

• Danger-associated molecular patterns
• immunogenic cell death
• ipilimumab
• nivolumab
• pembrolizumab
• TGFβ1

Abbreviations

CTL	=	cytotoxic T lymphocyte
CTLA4	=	cytotoxic T lymphocyte associated protein 4
DC	=	dendritic cell
EBRT	=	external beam radiation therapy
FDA	=	Food and Drug Administration
GM-CSF	=	granulocyte macrophage colony-stimulating factor
HNSCC	=	head and neck squamous cell carcinoma
ICD	=	immunogenic cell death
IDH	=	isocitrate dehydrogenase (NADP+) 1, cytosolic
IDO1	=	indoleamine 2,3-dioxygenase 1
IL	=	interleukin
mAb	=	monoclonal antibody
NK	=	natural killer
NSCLC	=	non-small cell lung carcinoma
TAA	=	tumor-associated antigen
TAM	=	tumor-associated macrophage
TGFβ1	=	transforming growth factor β1
TNF	=	tumor necrosis factor
TLR	=	Toll-like receptor

Introduction

Ionizing irradiation constitutes one of the pillars of modern cancer therapy.^1-4 According to current estimates, indeed, at least 50% of subjects with cancer (all confounded) have received or will receive radiation therapy in the course of their disease.^5,6 For a long time, radiation therapy was believed to operate in a merely cell-intrinsic manner, i.e., by promoting the death or permanent proliferative arrest of malignant cells upon the establishment of oxidative damage to macromolecules including DNA.^7-12 More recently, however, it has become clear that the antineoplastic effects of ionizing irradiation also involve a considerable cell-extrinsic component. Irradiated cancer cells release a wide panel of biologically active mediators that act locally to promote the death of bystander cells.^13-15 These factors include not only reactive oxygen and nitrogen species,^16-18 but also various potentially cytotoxic (and immunomodulatory) cytokines such as interleukin (IL)-6,¹⁹ IL-8,²⁰ transforming growth factor β1 (TGFβ1),^21-24 and tumor necrosis factor (TNF).²⁵ Moreover, radiation therapy can promote a particularly immunogenic form of cell death that eventually stimulates the activation of a tumor-targeting immune response with systemic therapeutic potential.^26-32 The capacity of ionizing irradiation to stimulate anticancer immunity upon the induction of immunogenic cell death (ICD) explains the so-called abscopal or out-of-field effect, i.e., the relatively rare but sometimes very pronounced clinical response to radiation therapy that can manifest in distant, non-irradiated lesions.^33-38 Finally, some forms of radiation therapy promote the normalization of the tumor vasculature, hence improving the access of chemotherapeutic agents and immune effector cells to malignant lesions.^39-41

For the purpose of this Trial Watch, radiation therapy can be broadly subdivided into two major therapeutic paradigms: external-beam radiotherapy (EBRT) and internal radiotherapy.^3,4 In the former setting, malignant lesions are treated across the intact skin, according to collimation procedures that can concentrate the irradiation energy on very specific areas of the tumor.^42,43 In the latter setting, radionuclides are brought in direct contact with transformed cells, either as pellets that are deposited within the tumor mass (a strategy that is known as brachytherapy), or upon conjugation with (or encapsulation within) tumor-targeting agents, including monoclonal antibodies (mAbs).^44-46 Both types of radiation therapy are associated with acute and chronic side effects.^47-50 Acute side effects stem from the unavoidable (but ever more limited, thanks to the technological advances in modern irradiators for clinical use) damage temporarily imposed by irradiation on particularly radiosensitive healthy tissues (like the skin) and often resolve in a few days/weeks after interruption.^44,51 On the contrary, the chronic toxicity of radiation therapy originates from the permanent damage possibly imposed by considerable radiation doses to stem cell compartments like intestinal crypts,^44,51 coupled to the establishment of dysregulated chronic inflammatory processes.⁵² Moreover, radiation therapy has been linked to a small but non-negligible increase in incidence of secondary, treatment-related malignancies later in life.^53-55

Throughout the past five decades, several strategies have been conceived to improve the therapeutic index of radiation therapy by either improving efficacy (radiosensitization) and/or by selectively limiting toxicity to normal tissues (radioprotection).^2,56-58 Multiple molecules have been shown to mediate consistent radiosensitization or radioprotection in rodent models of radiation therapy.⁴² However, the antioxidant amifostine (also known as Ethyol®) remains the only agent that is licensed by the US Food and Drug Administration (FDA) for use as a radioprotector in humans.^59-63 One of the most common practices in radiation oncology is dose fractionation, i.e., the delivery of the total irradiation dose in multiple fractions (therapy sessions spaced by at least 6 h) over several days or weeks.^64,65 Fractionation exploits the improved capacity of normal over malignant tissues to repair the damage imposed by irradiation, hence maximizing its therapeutic window.^64,65 Importantly, total dose and delivery schedule have a prominent impact on the ability of radiation therapy to promote ICD and hence drive the establishment of a therapeutically relevant anticancer immune response.^28,64,66,67

Classically, radiation therapy has been employed in the context of combinatorial treatment regimens (involving surgery and chemotherapy), either with a curative objective (i.e., with the aim to eradicate primary neoplasms or prevent recurrence) or with a palliative intent (i.e., to limit the pain/discomfort caused by malignancies at specific anatomical locations).^5,6 Along with the recognition that radiation therapy can mediate potent immunostimulatory effects, considerable interest has been attracted by combinatorial regimens involving EBRT plus one (or more) immunotherapeutic agent(s),^68-71 including checkpoint blockers,^72-75 immunostimulatory antibodies,^72,76 recombinant cytokines,^77-79 anticancer vaccines,^80-84 indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors,^85,86 adoptively transferred cells,^87-89 oncolytic viruses,^90-93 Toll-like receptor (TLR) agonists,^94,95 and various small molecules that operate on the immunological tumor microenvironment. In this Trial Watch, we summarize recent preclinical and clinical advances in the development of combinatorial anticancer regimens based on EBRT plus immunotherapy.

Published literature—highlights

On 2016 May 1st, querying PubMed with the string “cancer AND radiation therapy AND (2014 OR 2015 OR 2016)” returned more than 23,000 entries, which gives a good indication on the continuous interest of scientists and clinicians in radiation oncology (source http://www.ncbi.nlm.nih.gov/pubmed). Obviously, a considerable fraction of such an extremely abundant literature deals with the cancer cell-intrinsic effects of radiation therapy.

Among these reports, we found of particular interest (and at least partially related to immunotherapy) the works of: (1) Boelens and collaborators (from the University of Pennsylvania, Philadelphia, PA, US), who identified an exosome-dependent mechanism linked to antiviral signaling⁹⁶ whereby stromal cells improve the resistance of breast cancer cells to radiation therapy;⁹⁷ (2) Leder and colleagues (from the University of Minnesota, Minneapolis, MN, US), who developed a mathematical model of platelet-derived growth factor (PDGF)-driven glioblastoma that allowed for the identification of optimal radiation dosing schedules;⁹⁸ (3) Tavora et al. (from the Queen Mary University, London, UK), who identified protein tyrosine kinase 2 (PTK2; best known as FAK)⁹⁹ within the endothelial (not malignant) tumor compartment as a prominent player in the resistance of neoplasms of DNA-damaging agents including radiation therapy;¹⁰⁰ (4) Tollini and co-workers (from the University of North Carolina at Chapel Hill, Chapel Hill, NC, US), who demonstrated that the capacity of MDM2 to tag tumor protein p53 (TP53; best p53)^101-103 for proteasomal degradation is dispensable during embryogenesis and development, but essential for normal cellular responses to DNA damage;¹⁰⁴ (5) Ceccaldi and collaborators (from the Harvard Medical School, Boston, MA, US), who identified in polymerase (DNA) theta (POLQ)¹⁰⁵ a key regulator or DNA repair in homologous recombination (HR)-deficient tumors;¹⁰⁶ (6) Moding and colleagues (from the Duke University Medical Center, Durham, NC, US), who showed that ATM (a kinase with a key role in the DNA damage response)¹⁰⁷ in malignant cells, but not in endothelial cells, is required for the eradication of experimental sarcomas by stereotactic body radiation therapy;¹⁰⁸ (7) Osswal et al. (from the University Hospital Heidelberg, Heidelberg, Germany), who identified cellular networks involving malignant astrocytes that underlie (at least in part) the pronounced radio- and chemoresistance of astrocytomas;¹⁰⁹ (8) Reid and coworkers (University of California at San Diego, La Jolla, CA, US), who demonstrated that the radiosensitizer RRx-001 (a hypoxia-inducible agent)¹¹⁰ is well tolerated by patients with advanced solid tumors and appears to mediate clinical activity (at least to some extent);¹¹¹ (9) Tarish and collaborators (Karolinska Institute, Stockholm, Sweden), who demonstrated that the response of prostate cancer patients to radiation therapy is exacerbated by chemical castration¹¹² (at least in part) as a consequence of deficient DNA repair in malignant cells;¹¹³ and (10) Zhang and colleagues (University of Michigan, Ann Arbor, MI, US), who reported that the haploinsufficient tumor suppressor F-box and WD repeat domain containing 7 (FBXW7)^114-116 may constitute a promising target for radiosensitization owing to its role in non-homologous end-joining¹¹⁷ DNA repair.¹¹⁸

Moreover, approximately 600 PubMed entries of those mentioned above contained the keyword “immunotherapy,” dealing (from an experimental or theoretical perspective) with the possibility to combine radiation therapy with anticancer immunotherapy in vitro, in vivo or in patients (source http://www.ncbi.nlm.nih.gov/pubmed). Of these studies, we found of special interest the work of: (1) Deng and colleagues (from the University of Chicago, Chicago, Illinois, US), who not only demonstrated that radiation therapy and checkpoint blockade with antibodies specific for CD274 (best known as PD-L1)¹¹⁹ synergize to promote antitumor immunity in mice, but also reported that transmembrane protein 173 (TMEM173; best known as STING)^120-122 signaling in dendritic cells (DCs) is essential for the elicitation of antitumor immune responses by radiation therapy;^123,124 (2) Denham and collaborators (from the University of Newcastle, Newcastle, Australia), who showed that zoledronic acid, an immunostimulatory agent that targets immunosuppressive tumor-associated macrophages (TAMs),^125-129 synergizes with radiation therapy and intermediate-term androgen deprivation in the treatment of patients with locally advanced prostate carcinoma;¹³⁰ (3) Vantourout et al. (from the King's College, London, UK), who confirmed that irradiation increases the immunological visibility of tumors also by promoting the upregulation of killer cell lectin-like receptor K1 (KLRK1; best known as NKG2D)^131-134 ligands in epithelial cells, hence favoring natural killer (NK) cell activation;^135,136 (4) Surave and colleagues (from the University of Zurich, Zurich, Switzerland), who involved the complement system in radiation therapy-driven anticancer immune responses;¹³⁷ and (5) Twyman-Saint Victor and collaborators (University of Pennsylvania, Philadelphia, PA, US), who identified in the upregulation of PD-L1 a common mechanism whereby human and murine tumors become resistant to radiation therapy plus checkpoint blockers specific for cytotoxic T lymphocyte-associated protein 4 (CTLA), and demonstrated that anti-PD-L1 antibodies can be efficiently employed to revert resistance (at least in mice).¹³⁸ Moreover, one of our laboratories provided proof-of-principle clinical evidence in support of the possibility to combine local radiation therapy with recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) to increase the incidence of therapeutically relevant abscopal effects in patients with advanced solid tumors.¹³⁹ Finally, we demonstrated that the so-called immunoscore (a multiparametric biomarker conveying quantitative and spatial information on the immunological tumor infiltrate)¹⁴⁰ not only conveys prognostic information for patients with rectal carcinoma treated by primary surgery, but also predicts clinical response to preoperative chemoradiation.¹⁴¹

Besides unveiling parts of the mechanism whereby cancer cells may become resistant to the cytostatic and cytotoxic effects of irradiation, these findings lend additional support to the notion that radiation therapy and immunotherapy may be conveniently combined to improve disease outcome in cancer patients.

Ongoing studies

In the period of time elapsing since the publication of the latest Trial Watch dealing with topic (2014 July 1^st)⁴² through 2016 May 1st, no less than 620 clinical studies testing the safety and efficacy of anticancer therapeutic regimens based on (or at least involving) EBRT have been initiated (source: https://clinicaltrials.gov/). Nearly one-third of these studies (210 trials) investigates the clinical profile of EBRT as a standalone therapeutic intervention, in particular among patients affected by breast carcinoma (34 studies), prostate cancer (44 studies), non-small cell lung carcinoma (NSCLC; 15 studies), and hepatocellular carcinoma (14 studies). Some additional 220 trials initiated between 2014 July 1st and 2016 May 1st assess the safety and efficacy EBRT in combination with various chemotherapeutic regimens, for the most part among individuals with head and neck cancer (34 studies), esophageal cancer (32 studies), pancreatic carcinoma (25 studies), and NSCLC (19 studies). Finally, approximately 70 of these trials evaluate the therapeutic profile of EBRT combined with targeted anticancer agents, including tumor-targeting mAbs such as the epidermal growth factor receptor (EGFR)-specific molecule cetuximab,^142-145 or with various alternative non-immunotherapeutic interventions, like hyperthermia or nanoparticles. Since all these studies do not involve bona fide immunotherapeutic agents, we will not discuss them in further detail here. Rather, we will focus on 95 clinical trials initiated between 2014 July 1st and 2016 May 1st that aim to evaluate the safety and efficacy of EBRT combined with immunomodulatory mAbs including checkpoint blockers (66 studies), adoptive cell transfer (4 studies), TLR agonists (4 studies), DC-based vaccination (5 studies), recombinant cytokines (4 studies), peptide-based vaccines (3 studies), oncolytic virotherapy (2 studies), or other immunostimulatory agents (10 studies) (source: https://clinicaltrials.gov/).

The safety and efficacy of EBRT combined with the FDA-approved CTLA4-targeting checkpoint blocker ipilimumab^36,146,147 alone or with ipilimumab plus the experimental TLR9 agonist SD-101^94,148-150 is being assessed in cohorts of melanoma patients (NCT02406183, NCT02662725), NSCLC patients (NCT02221739),¹⁵¹ lymphoma patients (NCT02254772),¹⁵² and individuals with advanced solid tumors (NCT02239900). EBRT is being tested together with nivolumab, an FDA-approved checkpoint blocker targeting programmed cell death 1 (PDCD1; best known as PD-1),^153-155 alone or in combination with cytotoxic chemotherapy or targeted anticancer agents, in patients with breast carcinoma (NCT02499367), glioblastoma (NCT02617589, NCT02667587), head and neck squamous cell carcinoma (HNSCC) (NCT02684253, NCT02764593), melanoma (NCT02716948), and NSCLC (NCT02768558). In addition, EBRT plus a combined immunotherapeutic regimen involving both ipilimumab and nivolumab is being assessed for safety and efficacy in individuals affected by melanoma (NCT02659540) or intracranial metastases originated from NSCLC (NCT02696993). The clinical profile of EBRT given in combination with yet another FDA-approved PD-1-targeting checkpoint blocker, i.e., pembrolizumab,^156-160 generally alone or in the context of conventional chemotherapeutic regimens, is being investigated among patients with HNSCC (NCT02289209, NCT02296684, NCT02402920, NCT02586207, NCT02609503, NCT02641093, NCT02707588, NCT02759575, NCT02775812, NCT02777385), lung carcinoma (NCT02444741, NCT02492568, NCT02621398, NCT02658097), bladder carcinoma (NCT02560636, NCT02621151, NCT02662062), brain tumors (NCT02530502, NCT02313272), colorectal carcinoma (NCT02437071, NCT02586610),¹⁶¹ gastroesophageal cancer (NCT02730546), breast carcinoma (NCT02303366, NCT02730130), endometrial cancer (NCT02630823), melanoma (NCT02562625), pancreatic carcinoma (NCT02305186), renal cell carcinoma (NCT02599779), thoracic tumors (NCT02587455), or advanced solid tumors of multiple derivation (NCT02303990, NCT02318771, NCT02407171, NCT02608385). In addition, EBRT plus pembrolizumab-based immunotherapy is being tested in combination with a genetically modified allogenic cancer cell-based vaccine (GVAX)^162-164 in subjects with pancreatic cancer (NCT02648282), or together with intratumoral autologous DCs^165-171 in patients with lymphoma (NCT02677155) (Table 1).

Table 1. Clinical trials recently started to investigate the safety and efficacy of EBRT plus immunostimulatory antibodies in cancer patients^*.

Antibody	Indication(s)	Phase	Status	Type of RT	Notes	Ref.
Adalimumab	Thyroid cancers	I	Recruiting	EBRT	Combined with chemotherapy	NCT02516774
AMP-224^**	Colorectal carcinoma	I	Active, not recruiting	SBRT	Combined with cyclophosphamide	NCT02298946
Atezolizumab	NSCLC	0	Recruiting	HIGRT	None	NCT02463994
		I	Not yet recruiting	SBRT	None	NCT02400814
		I	Not yet recruiting	SBRT	None	NCT02599454
		II	Not yet recruiting	EBRT	Combined with carboplatin and paclitaxel	NCT02525757
Avelumab	Merkel cell carcinoma	I/II	Recruiting	EBRT	Combined with recombinant IFN-β and polyclonal autologous CD8^+ CTLs	NCT02584829
Combo	Melanoma	I	Not yet recruiting	EBRT	Ipilimumab and/or nivolumab	NCT02659540
	Pancreatic cancer	I	Recruiting	SBRT	Tremelimumab and/or MEDI4736	NCT02311361
	SCLC	II	Recruiting	HRT, SBRT	Durvalumab and/or tremelimumab	NCT02701400
	Solid tumors	I	Recruiting	HRT	Durvalumab and/or tremelimumab	NCT02639026
		I/II	Not yet recruiting	SRS, WBRT	Ipilimumab and/or nivolumab	NCT02696993
Durvalumab	Esophageal carcinoma	I/II	Not yet recruiting	EBRT	Combined with CAPOX	NCT02735239
	Glioblastoma	II	Recruiting	EBRT	None	NCT02336165
Fresolimumab	NSCLC	I/II	Not yet recruiting	SABR, SBRT	None	NCT02581787
IPH2201	HNSCC	I/II	Recruiting	EBRT	None	NCT02331875
Ipilimumab	Lymphoma	I/II	Recruiting	EBRT	Combined with SD-101 (TLR9 agonist)	NCT02254772
	Melanoma	I	Recruiting	SBRT	None	NCT02406183
		II	Completed	SRS	None	NCT02662725
	NSCLC	II	Active, not recruiting	3D-EBRT, IMRT	None	NCT02221739
	Solid tumors	I/II	Recruiting	SBRT	None	NCT02239900
Nivolumab	Breast carcinoma	II	Recruiting	EBRT	None	NCT02499367
	Glioblastoma	II	Recruiting	EBRT	Combined with temozolomide	NCT02667587
		III	Not yet recruiting	EBRT	Combined with temozolomide	NCT02617589
	HNSCC	I	Not yet recruiting	IMRT	Combined with cetuximab and/or cisplatin	NCT02764593
		II	Recruiting	SBRT	None	NCT02684253
	Melanoma	I	Recruiting	SBRT	None	NCT02716948
	NSCLC	III	Not yet recruiting	3D-EBRT, IMRT	Combined with cisplatin and etoposide	NCT02768558
Pembrolizumab	Bladder carcinoma	I	Not yet recruiting	EBRT	None	NCT02560636
		II	Not yet recruiting	EBRT	Combined with gemcitabine	NCT02621151
		II	Not yet recruiting	EBRT	Combined with cisplatin	NCT02662062
	Breast carcinoma	I	Recruiting	SABR	None	NCT02303366
		II	Not yet recruiting	EBRT	None	NCT02730130
	Colorectal carcinoma	II	Not yet recruiting	EBRT	Combined with capecitabine	NCT02586610
		II	Recruiting	EBRT	None	NCT02437071
	Endometrial carcinoma	0	Not yet recruiting	EBRT	Combined with carboplatin and paclitaxel	NCT02630823
	Gastroesophageal carcinoma	0	Not yet recruiting	HDRB	None	NCT02642809
		I/II	Not yet recruiting	EBRT	Combined with carboplatin and paclitaxel	NCT02730546
	Glioblastoma	I/II	Recruiting	Focal RT	Combined with temozolomide	NCT02530502
	Glioma	I	Recruiting	HSBRT	Combined with bevacizumab	NCT02313272
	HNSCC	I	Not yet recruiting	IMRT	Combined with cisplatin	NCT02775812
		I	Recruiting	EBRT	Combined with cisplatin	NCT02586207
		I/II	Recruiting	EBRT	Combined with cisplatin	NCT02759575
		II	Not yet recruiting	IMRT	None	NCT02609503
		II	Not yet recruiting	EBRT	Combined with cetuximab	NCT02707588
		II	Not yet recruiting	IMRT	Combined with cisplatin	NCT02777385
		II	Recruiting	EBRT	None	NCT02289209
		II	Recruiting	IGRT, IMRT	Combined with cisplatin	NCT02296684
		II	Recruiting	EBRT	Combined with cisplatin	NCT02641093
	Lymphoma	II	Recruiting	EBRT	Combined with autologous DCs	NCT02677155
	Melanoma	II	Not yet recruiting	EBRT	None	NCT02562625
	NSCLC	I	Not yet recruiting	3D-EBRT, IMRT	Combined with carboplatin and paclitaxel	NCT02621398
		I/II	Recruiting	SBRT, WFRT	None	NCT02444741
		II	Not yet recruiting	SFRT	None	NCT02658097
		II	Recruiting	SBRT	None	NCT02492568
	Pancreatic carcinoma	I/II	Recruiting	EBRT	Combined with capecitabine	NCT02305186
		II	Not yet recruiting	SBRT	Combined with a genetically-modified allogenic cancer cell-based vaccine and cyclophosphamide	NCT02648282
	Renal cell carcinoma	II	Not yet recruiting	SBRT	None	NCT02599779
	SCLC	I	Recruiting	EBRT	Combined with multimodal chemotherapy	NCT02402920
	Solid tumors	I	Not yet recruiting	SBRT	None	NCT02608385
		I	Recruiting	HRT	None	NCT02303990
		I	Recruiting	EBRT	None	NCT02318771
		I/II	Recruiting	SBRT	None	NCT02407171
	Thoracic tumors	I	Not yet recruiting	Palliative RT	None	NCT02587455
REGN2810	Solid tumors	I	Recruiting	HRT	Combined with cyclophosphamide	NCT02383212
Tremelimumab	Breast carcinoma	EBRT	Recruiting	SRS WBRT	None	NCT02563925
Varlilumab	Prostate cancer	I	Recruiting	SBRT	None	NCT02284971

Abbreviations: 3D-CRT, 3D conformal radiotherapy; CAPOX, capecitabine plus oxaliplatin; CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBRT, external beam radiation therapy; HIGRT, hypofractionated image-guided radiotherapy; HNSCC, head and neck squamous cell carcinoma; HRT, hypofractionated radiation therapy; HSRT, hypofractionated stereotactic radiation therapy; IFN-β, interferon β; IMRT, intensity-modulated radiation therapy; NSCLC, non-small cell lung carcinoma; SART, stereotactic ablation radiation therapy; SBRT, stereotactic body radiation therapy; SCLC, small cell lung carcinoma; SRS, stereotactic radiosurgery; WBRT, whole brain radiation therapy; WFRT, wide-field radiation therapy.

* initiated between 2014, July 1st and 2016, May 1st;

** anti-PD-1 fusion protein.

Additional (hitherto experimental) checkpoint blockers that are being investigated for their capacity to synergize with EBRT include: (1) atezolizumab, a mAb specific for PD-L1,^119,172-175 which is administered together with EBRT alone or with EBRT plus conventional chemotherapy to NSCLC patients (NCT02400814, NCT02463994, NCT02525757, NCT02599454); (2) avelumab, another PD-L1-targeting mAb,^175-177 which is given to Merkel cell carcinoma patients in combination with EBRT (as a measure to upregulate MHC Class I expression by cancer cells) and optionally autologous T lymphocytes genetically redirected against tumor-associated antigens (TAAs) (NCT02584829); (4) the PD-L1-specific mAbs durvalumab,¹⁷⁸ which is tested in combination with EBRT alone, EBRT plus chemotherapy, or EBRT plus the CTLA4-targeting agent tremelimumab^179,180 in patients with esophageal cancer (NCT02735239), glioblastoma (NCT02336165) small cell lung carcinoma (NCT02701400), and advanced solid tumors (NCT02639026), and MEDI4736,^181-184 which is studied in combination with EBRT plus tremelimumab in subjects with unresectable pancreatic cancer (NCT02311361); (5) a new mAb specific for PD-1, namely, REGN2810, whose safety and efficacy in combination with EBRT plus cyclophosphamide-based chemotherapy and recombinant GM-CSF are assessed in patients with advanced solid neoplasms (NCT02383212);¹⁸⁵ (6) a novel fusion protein-targeting PD-1 (called AMP-224; source http://www.cancer.gov/publications/dictionaries/cancer-drug?cdrid=700595), which is tested together with EBRT in colorectal carcinoma patients (NCT02298946); (7) tremelimumab, whose clinical profile in combination with EBRT is investigated in breast carcinoma patients (NCT02563925); and IPH2201, a mAb specific for killer cell lectin-like receptor C1 (KLRC1; an inhibitory NK-cell receptor best known as NKG2A),^186-188 which is studied together with EBRT in HNSCC patients (NCT02331875) (Table 1).

The following immunostimulatory antibodies that do not operate as checkpoint blockers are also being evaluated for their safety and efficacy when administered in combination with EBRT: (1) the FDA-approved mAb adalimumab, an inhibitor of TNF and hence of immunosuppressive TAMs,^189-193 which is tested together with EBRT in patients with anaplastic thyroid tumors (NCT02516774); (2) fresolimumab, a mAb that neutralizes TGFβ1,^{22,66,194-197} which is studied in combination with EBRT in individuals with NSCLC (NCT02581787); and (3) varlilumab, an immunostimulatory mAb specific for CD27,^198-201 which is assessed for its capacity to improve the efficacy of EBRT in subjects with prostate carcinoma (NCT02284971) (Table 1).

As for immunotherapies not based on checkpoint blockers and other immunostimulatory antibodies, EBRT is being evaluated in combination with: (1) autologous DCs expanded ex vivo in the presence of tumor cell lysates^202,203 in children with advanced solid tumors (NCT02496520) or in Grade IV glioma patients (NCT02772094); (2) unmodified autologous DCs re-infused upon expansion ex vivo, in subjects with NSCLC concurrently receiving standard-of-care platinum-based chemotherapy^204,205 (NCT02662634); (3) an autologous DC-based vaccine specific for mutant isocitrate dehydrogenase (NADP⁺) 1, cytosolic (IDH1)^206-208 in glioma patients bearing IDH1^R132H (NCT02771301); and (4) vaccines based on TAA-derived peptides or heat-shock protein (HSP)-enriched preparations of tumor lysates²⁰⁹ in glioma patients (NCT02287428, NCT02722512) or women with cervical carcinoma concurrently receiving cisplatin-based chemotherapy (NCT02722512); (5) FDA-approved^90,210 or experimental²¹¹ oncolytic viruses in individuals with soft tissue sarcoma (NCT02453191) or children with brain malignancies (NCT02457845) (Table 2).

Table 2. Clinical trials recently started to investigate the safety and efficacy of EBRT plus other forms of immunotherapy in cancer patients^*.

Immunotherapy	Indication(s)	Phase	Status	Type of RT	Notes	Ref.
ACT	Colorectal carcinoma	II	Active, not recruiting	EBRT	DC-CIK cells combined with FOLFOX regimen	NCT02202928
	Hepatocellular carcinoma	III	Active, not recruiting	RFA	Highly-purified autologous CD8^+ CTLs	NCT02678013
	Merkel cell carcinoma	I/II	Recruiting	EBRT	Polyclonal autologous CD8^+ CTLs combined with recombinant IFN-β and an anti-PD-L1 mAb	NCT02584829
	Sarcoma	I	Recruiting	Palliative EBRT	Autologous NY-ESO-1-specific CD8^+ CTLs	NCT02319824
Celecoxib	HNSCC	II	Recruiting	EBRT	COX2 inhibitor	NCT02739204
Cytokines	Glioblastoma	II	Recruiting	HIMRT	GM-CSF combined with temozolomide	NCT02663440
	Merkel cell carcinoma	I/II	Recruiting	EBRT	Recombinant IFN-β combined with polyclonal autologous CD8^+ CTLs and an anti-PD-L1 mAb	NCT02584829
	NSCLC	II	Not yet recruiting	SBRT	L19-IL2 immunocytokine	NCT02735850
	Renal cell carcinoma	II	Recruiting	SBRT	High-dose IL-2	NCT02306954
DC-based interventions	Glioma	n.a.	Recruiting	EBRT	Combined with chemotherapy	NCT02771301
	Glioma	II	Active, not recruiting	EBRT	Combined with temozolomide	NCT02772094
	Lymphoma	II	Recruiting	EBRT	Combined with pembrolizumab	NCT02677155
	NSCLC	I/II	Recruiting	EBRT	Combined with chemotherapy	NCT02662634
	Solid tumors	I/II	Recruiting	EBRT	Combined with chemotherapy	NCT02496520
Indoximod	Brain tumors	I	Recruiting	CRT	IDO1 inhibitor, combined with temozolomide	NCT02502708
LY2157299	Breast carcinoma	II	Recruiting	EBRT	TGFBR1 inhibitor	NCT02538471
	Rectal carcinoma	II	Not yet recruiting	EBRT	TGFBR1 inhibitor, combined with chemotherapy	NCT02688712
Oncolytic virotherapy	Brain tumors	I	Not yet recruiting	EBRT	Oncolytic HSV-1 (G207)	NCT02457845
	Soft tissue sarcoma	I/II	Recruiting	EBRT	Talimogene laherparepvec	NCT02453191
Thymalfasin	Colorectal carcinoma	II	Not yet recruiting	EBRT	Unspecific immunostimulatory agent, aimed at improving abscopal effects	NCT02535988
	Esophageal carcinoma	II	Not yet recruiting	SBRT	Unspecific immunostimulatory agent, aimed at improving abscopal effects	NCT02545751
	NSCLC	II	Not yet recruiting	EBRT	Unspecific immunostimulatory agent, aimed at improving abscopal effects	NCT02542930
	SCLC	II	Not yet recruiting	EBRT	Unspecific immunostimulatory agent, aimed at improving abscopal effects	NCT02542137
TLR agonists	Lymphoma	I/II	Recruiting	EBRT	SD-101 (TLR9 agonist), combined with ipilimumab	NCT02254772
	I/II	Recruiting	EBRT	SD-101 (TLR9 agonist)	NCT02266147
	Melanoma	III	Not yet recruiting	EBRT	Imiquimod (TLR7 agonist)	NCT02394132
	Soft tissue sarcoma	I	Recruiting	EBRT	GLA-SE (TLR4 agonist)	NCT02180698
Trabectedin	Soft tissue sarcoma	I/II	Recruiting	3D-CRT	TAM-targeting agent	NCT02275286
Vaccination	Cervical cancer	II	Not yet recruiting	EBRT	Peptide-based vaccine, combined with cisplatin	NCT02501278
	Glioblastoma	I	Recruiting	EBRT	Peptide-based vaccine	NCT02287428
	Glioma	I	Not yet recruiting	Focal RT	HSP-based vaccine	NCT02722512
	Pancreatic carcinoma	II	Not yet recruiting	SBRT	Allogenic cancer cell-based vaccine, combined with cyclophosphamide and pembrolizumab	NCT02648282
Zoledronic acid	NSCLC	IV	Not yet recruiting	EBRT	TAM-targeting agent	NCT02480634

Abbreviations: 3D-CRT, 3D conformal radiotherapy; ACT, adoptive cell transfer; CIK, cytokine induced killer; CRT, conformal radiotherapy; CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBRT, external beam radiation therapy; FOLFOX, folinic acid plus 5-fluoruracil plus oxaliplatin; GLA-SE, glucopyranosyl lipid adjuvant in stable emulsion; GM-CSF, granulocyte macrophage colony-stimulating factor; HIMRT, hypofractionated intensity-modulated radiation therapy; HNSCC, head and neck squamous cell carcinoma; HSP, heat-shock protein; HSV-1, herpes simplex virus type 1; IDO1, indoleamine 2,3-dioxygenase 1; IFN-β, interferon β; IL-2, interleukin-2; mAb, monoclonal antibody; NSCLC, non-small cell lung carcinoma; RFA, radiofrequency ablation; RT, radiation therapy; SBRT, stereotactic body radiation therapy; SCLC, small cell lung carcinoma; TAM, tumor-associated macrophage; TGFBR1, transforming growth factor β receptor 1; TLR, toll-like receptor.

* initiated between 2014, July 1st and 2016, May 1st.

In addition, the safety and efficacy of EBRT combined with immunotherapy is being assessed in the context of (1) adoptive cell transfer,^87,212 in colorectal cancer patients receiving autologous DCs plus cytokine induced killer (CIK) cells along with FOLFOX (folinic acid plus 5-fluoruracil plus oxaliplatin) chemotherapy (NCT02202928), sarcoma patients treated with autologous CD8⁺ cytotoxic T lymphocytes (CTLs) genetically modified to recognize the TAA NY-ESO-1 (NCT02319824), and hepatocellular carcinoma patients receiving highly purified autologous CD8⁺ CTLs (NCT02678013); (2) TLR stimulation,⁹⁴ in soft tissue sarcoma patients receiving the experimental TLR4 agonist glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE)^213,214 (NCT02180698), lymphoma patients concurrently administered with the experimental TLR9 agonist SD-101^215,216 (NCT02266147), and melanoma patients co-treated with the FDA-approved TLR7 agonist imiquimod^217-221 (NCT02394132); and (3) relatively unspecific immunostimulation with recombinant IL-2 or GM-CSF in patients with renal cell carcinoma (NCT02306954), glioblastoma (NCT02663440), and NSCLC (NCT02735850), with thymalfasin (a recombinant version of the human T_H1-skewing peptide thymosin α1)²²² in colorectal cancer patients (NCT02535988), lung cancer patients (NCT02542137, NCT02542930), and esophageal cancer patients (NCT02545751), with TAM-targeting agents like trabectedin^223-225 or zoledronic acid^128,226 in subjects with soft tissue sarcoma (NCT02275286) or metastatic NSCLC (NCT02480634), with a chemical inhibitor of IDO1 (i.e., indoximod)^196,227 in children with brain tumors concurrently receiving temozolomide-based chemotherapy (NCT02502708), with chemical inhibitors of the TGFβ1 receptor^228-230 in breast carcinoma patients (NCT02538471) and rectal carcinoma patients concurrently treated with standard-of-care chemotherapy (NCT026887129), and with celecoxib, an inhibitor of the immunosuppressive enzyme prostaglandin-endoperoxide synthase 2 (PTGS2; best known as COX2),^231,232 in HNSCC patients (NCT02739204) (Table 2).

With a single exception, all these studies are ongoing (i.e., they are listed as “Active, not recruiting,” “Not yet recruiting” or “Recruiting” by official sources). NCT02662725, a Phase II clinical trial testing stereotactic radiosurgery plus ipilimumab-based immunotherapy in melanoma patients with brain metastases, appears as “Completed.” To the best of our knowledge, however, he results of this study have not yet been disseminated (sources: https://clinicaltrials.gov/; http://www.ncbi.nlm.nih.gov/pubmed; and http://meetinglibrary.asco.org/abstracts;).

Concluding remarks

Total-body irradiation has been extensively employed in the clinic as a myelo- and lymphoablating measure to pre-condition hematopoietic stem cell transplantation recipients.²³³ Nonetheless, it is now well established that the localized, targeted irradiation of malignant lesions in the context of dose fractionation within the standard therapeutic range promotes direct antineoplastic effects while eliciting a therapeutically relevant anticancer immune response.²³⁴ Thus, radiation therapy currently stands out as an accessible and promising tool for improving the efficacy of immunotherapeutic agents as diverse as checkpoint blockers, immunostimulatory antibodies, anticancer vaccines, oncolytic viruses, recombinant cytokines, TLR agonists, and small molecules that repolarize the tumor microenvironment. The clinical activity of all these immunotherapeutic interventions (and presumably that of many chemotherapeutic agents as well)²⁹ relies indeed on the activation of a robust and polyclonal tumor-specific immune response, and radiation therapy has been convincingly demonstrated to promote such a response by favoring the release of immunostimulatory signals by dying cancer and stromal cells, hence improving their adjuvanticity.^31,235 Intriguingly, fractionated radiation appears to be superior to single-dose radiation therapy in its capacity to trigger anticancer immune responses in vivo.^64,236 This has been linked to improved capacity of fractionated radiation (as compared to single-dose radiation therapy) to induce the release of damage-associated molecular patterns (DAMPs) by the tumor.^237,238 In addition, it may reflect (at least in part) the capacity of fractionated (but not single-dose) radiation to temporarily allow for the survival of malignant cells accumulating genetic and genomic defects that result in exacerbated antigenicity.^239-241 This intriguing hypothesis has not yet been formally addressed. Irrespectively, by virtue of its well-established efficacy and safety profile, radiation therapy lies together with chemotherapy and immunotherapy at the core of a multimodal therapeutic regimen that holds great promise for the future of clinical tumor immunology.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

FA is supported by Sara Borrell fellowship CD15/00016 from Instituto de Salud Carlos III. GK is supported by the French Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; ANR under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Institut National du Cancer (INCa); Institut Universitaire de France; Fondation pour la Recherche Médicale (FRM); the European Commission (ArtForce); the European Research Council (ERC); the LeDucq Foundation; the LabEx Immuno-Oncology; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM); and the Paris Alliance of Cancer Research Institutes (PACRI). SD is supported by the National Institutes of Health R01 CA201246 and R01 CA198533, the Breast Cancer Research Foundation, and the Chemotherapy Foundation. SCF is supported by the National Institutes of Health R01 CA161891, the USA Department of Defense Breast Cancer Research Program (W81XWH-11-1-0530); and the Breast Cancer Research Foundation.