Can immunostimulatory agents enhance the abscopal effect of radiotherapy?

European Journal of Cancer 62 (2016) 36e45

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.ejcancer.com

Review

Can immunostimulatory agents enhance the abscopal effect of radiotherapy? Antonin Levy a,b,c, Cyrus Chargari a,b,c,d, Aurelien Marabelle e, Jean-Luc Perfettini b,c,f, Nicolas Magne´ g, Eric Deutsch a,b,c,e,* a Department of Radiation Oncology, SIRIC SOCRATES Gustave Roussy Cancer Campus, 114 rue E. Vaillant, 94800 Villejuif, France b Universite´ Paris-Saclay, Faculte´ de Me´decine, 94270 Kremlin Biceˆtre, France c INSERM U1030, Molecular Radiotherapy, Gustave Roussy Cancer Campus, Villejuif, France d Institut de Recherche Biome´dicale des Arme´es, Bre´tigny sur Orge, France e DITEP, Gustave Roussy Cancer Campus, 114 rue E. Vaillant, 94800 Villejuif, France f Cell death and Aging team, Gustave Roussy Cancer Campus, 114 rue E. Vaillant, 94800 Villejuif, France g Institut de Cance´rologie Lucien Neuwirth, Saint Priest en Jarez, France

Received 13 February 2016; received in revised form 14 March 2016; accepted 15 March 2016

KEYWORDS Irradiation; Immune checkpoint modulator; Early clinical trial; Immunity

Abstract Ionising radiation (IR) may harm cancer cells through a rare indirect out-of-field phenomenon described as the abscopal effect. Increasing evidence demonstrates that radiotherapy could be capable of generating tumour-specific immune responses. On the other hand, effects of IR also include inhibitory immune signals on the tumour microenvironment. Following these observations, and in the context of newly available immunostimulatory agents in metastatic cancers (anti-cytotoxic T lymphocyte-associated antigen 4 and programmed cell death protein-1 or -ligand 1 [PD1 or PDL-1]), there is a remarkable potential for synergistic combinations of IR with such agents that act through the reactivation of immune surveillance. Here, we present and discuss the pre-clinical and clinical rationale supporting the enhancement of the abscopal effect of IR on the blockade of immune checkpoints and discuss the evolving potential of immunoradiotherapy. ª 2016 Elsevier Ltd. All rights reserved.

* Corresponding author: Department of Radiation Oncology, Gustave Roussy, 114 rue Edouard Vaillant, 94850 Villejuif, France. Tel.: þ 33 1 42 11 65 73; fax: þ 33 1 42 11 52 53. E-mail address: [email protected] (E. Deutsch). http://dx.doi.org/10.1016/j.ejca.2016.03.067 0959-8049/ª 2016 Elsevier Ltd. All rights reserved.

A. Levy et al. / European Journal of Cancer 62 (2016) 36e45

1. Introduction Radiation therapy (RT) is a pivotal treatment in oncology. It is estimated that more than half of patients will receive RT during the course of their disease. The therapeutic potential of RT has long been exclusively ascribed to its ability to mediate antiproliferative and cytotoxic effects. In fact, after exposure to ionising radiation (IR), various macromolecules in tumour cells sustain multiple injuries, notably DNA which is the main target for RT. This damage is prominently consecutive to the undirected establishment of oxidative stress within the tumour cells or to direct interaction between IR and chromosomes. During the past two decades, the accumulated preclinical data suggest that the clinical efficacy of RT also involves mechanisms of interaction between the tumour cell and the stroma. Tumour cells exposed to IR and on the verge of dying release a wide panel of mediators with biological effects that are also involved in cell signalling or inflammation. They include reactive oxygen species, as well as several cytotoxic cytokines, such as transforming growth factor (TGF) b1 and also tumour necrosis factor (TNF) a. Our understanding of the death mechanisms involved in the anti-tumour effect of RT has significantly improved over the last few years. It has now become clear that upon lethal stimulation (such as apoptotic or necrotic signals), irradiated cancer cells can release immunostimulatory molecules leading to a phenomenon called immunogenic cell death (ICD) [1,2]. Indeed, these molecules, also called damage-associated molecular patterns (DAMPs), give rise to the recruitment of antigenpresenting cells in the tumour bed, and the elicitation of a general adaptive anti-tumour immune response. Still, although RT is used routinely as a local treatment in numerous clinical situations, only a few distant responses in unirradiated sites have been reported [3,4]. This phenomenon, described as the “abscopal effect,” purports that RT exerts out-of-field activity. New insights into the mechanism of action of RT brought to the light the fact that exposure to RT induces antigen release from the tumour, thereby activating both the host’s innate and adaptive immune system. Consequently, RT might help reverse the tolerance to weakly immunogenic tumour-associated antigens in order to elicit an anticancer immune response [5,6]. However, the abscopal effect has remained a rare clinical event when RT is used alone. Indeed, the frequency of in-field and distant relapses in locally advanced tumours treated with irradiation alone suggests that this radiationinduced anti-tumour immunity is inadequate or at least remains to be improved in order to maintain a long-term anti-tumour effect. Within the last 5 years, immune checkpoint inhibitors have demonstrated impressive efficacy in various advanced tumour locations [7e9]. Interestingly, there is

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now rationale that RT can positively interact with immunotherapies in inducing a sustained abscopal effect. We review here the preclinical and clinical evidence for the abscopal effect and highlight the therapeutic prospects of combining RT with immune-modulating agents, with special emphasis on immune checkpoint inhibitors. 2. Immune effects of RT: preclinical and clinical evidence 2.1. The immune response as part of the local and distant effect of RT The primary objective of RT is to achieve local tumour control, which ultimately may translate into enhanced survival [10]. Beyond its direct local effects, RT also amplifies tumour immunogenicity by inducing tumour cell death and the subsequent release of proinflammatory cytokines. Since the early 2000s, irradiation has been shown to be capable of inducing or potentiating a systemic anti-tumour immune response culminating in ICD. Schematically, RT triggers tumour antigen release and modulates the tumour cell phenotype, activating immune responses and increasing immune recognition [11,12]. The adaptive and innate immune response induced by RT is part of the antitumour effect in irradiated volumes but also out of field. When the local efficacy of RT is examined, there appears to be intratumour activation of the immune response, characterised by the recruitment of T lymphocytes and the secretion of Th1 cytokines. Furthermore, there is a positive correlation between the intensity of this response and patient outcomes [13,14]. Thus, the shortterm ablation of CD4þCD25þFOXP3þ regulatory T(reg) cells was shown to increase the therapeutic effect of RT [15]. The leukaemia inhibitory factor, which is involved in the terminal differentiation of immunological cells, may also be another key factor that has been observed during the acquisition of a radioresistant phenotype in nasopharyngeal cancer models [16]. The biological mechanisms of the abscopal effect are thought to rely on the ability of RT to elicit an immune response. This was more thoroughly reviewed elsewhere [17]. Briefly, irradiated tumours release danger signals (DAMPs; e.g. heat shock proteins or high-mobility group protein B1 [HMGB1] alarmin protein detected by the toll-like receptor (TLR) 4 on dendritic cells [DC]) that lead to DC activation. RT can also induce a dosedependent increase in MHC class I presentation in human tumour cells leading to tumour recognition. Activated DCs can then prime T cells and cause an appropriate level of CD8þ T lymphocyte cytotoxicity [18,19]. The Fms-like tyrosine kinase receptor 3 ligand (Flt3-L), which activates the production of DC, induced abscopal effects on a moderately immunogenic syngeneic tumour (mouse mammary carcinoma 67NR) in a T-cell-dependent manner [20]. Lee et al. also proposed

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that the abscopal effect could be CD8þ T-cell dependent. RT increased T-cell priming in draining lymphoid tissues, leading to the reduction/eradication of the primary tumour or distant metastases [21]. Local administration of interleukin-2 that can activate CD8þ T lymphocytes, also induced abscopal effects in a Balb/c mouse model of a simultaneous subcutaneous rectal tumour and liver metastasis [22]. Inversely, the absence of type I interferon (IFN) signalling abrogates the efficacy of RT in immunocompetent mice [23]. It is moreover admitted that IR then leads to an increase in tumour-infiltrating lymphocytes [20]. RT facilitates the recruitment of effector T cells to tumours through the induction of chemokines, such as CXCL16 and vascular normalisation [24]. RT promotes immune cell extravasation by increasing cell adhesion molecules (such as ICAM-1) [25]. Finally, the tumour phenotype modifications after IR (e.g. increase of FAS) also promote cytotoxic T lymphocyte-mediated ICD [26]. Altogether, these observations strongly support the immune role of the abscopal effect and the importance of T-cell populations. 2.2. Clinical evidence of abscopal effects The abscopal effect with RT alone is a rare phenomenon and only case reports have been published. In a recent review, Reynders et al. ultimately retrieved 23 case reports (1973e2013) on perceived abscopal effects after RT alone. The median age was 64.5 years (28e83 years), renal cell carcinoma was the most common histology (but non-immunologically prominent tumour types, such as cervical cancer, were also described), and irradiation was delivered to both primary tumours (35%) or metastases (65%), at a median dose of 32 Gy (12e60.75 Gy), in 1.2e26 Gy fractions. The median

time to a documented abscopal response after IR was 5 months (<1e24 months) with a median time of 13 months before the end of follow-up or disease progression [3]. The development of immunotherapies is opening up new prospects to promote abscopal effects. Postow et al. were the first to report an abscopal effect in a patient with metastatic melanoma who experienced disease progression during ipilimumab (a monoclonal antibody that inhibits an immune checkpoint on T cells, the cytotoxic T lymphocyte-associated antigen 4 [CTLA-4]) maintenance. Moreover, the authors were able to demonstrate temporal associations between tumour shrinkage and immune antibody responses (NY-ESO-1 increased and other antigens), and changes in peripheral blood immune cells (myeloid-derived suppressor cells and CD8þ and CD4þ T-cell activation) [27]. Since then, several reports have declared the existence of abscopal effects in melanoma patients receiving ipilimumab (Table 1). Altogether, the time interval between the first ipilimumab injection and IR ranged from 1.5 to 18 months, and the time to onset after IR was 1e6 months [27e31] Stamell et al. [29] also observed an increase in anti-melanoma antibodies (concomitant increase in MAGEA3 titers and a new response to the cancer antigen PASD1) during an abscopal effect. 3. Pharmacological modulation of the abscopal effect 3.1. Immune checkpoint inhibitors IR may also induce immunosuppressive responses in the microenvironment (including an increase of TGFb or inhibitory immune cells including Tregs, alternatively activated macrophages, and myeloid-derived suppression cells [MDSCs], cf. below) that act against the

Table 1 Clinical evidence of abscopal effects in cancer patients treated with immune checkpoint blockade antibodies.c Authors (year) No. of Abs Primary site RT location E/No. of pts

Time interval RT dose b/w first Ipi (Gy)/No. of and RT (m) fractions

Abscopal location

Time to onset Duration of after RT (m) response (m)

Postow (2012) 1

Upper back Paraspinal Mþ

18

4

6

Hiniker (2012) 1 Stamell (2013) 1 Grimaldi 11/21 (52%) (2014)

Arm Scalp NA

Splenic Mþ Hilar LN Cutaneous Mþ Nodal Mþ Various

6 NA 1a (1e4)

6 NA NA OS: 22.4 (versus 8.3 if no Abs E) NA

Chandra (2015)

16/65 (25%)b NA

Hepatic Mþ 1.5 Brain Mþ 8 62% Intracranial 5a (4e8) 28% Extracranial

Various

3a

28.5/3 54/3 ND Various

26a (8e68)/median Various fraction size: 4 Gy (1.8e25 Gy) (NA)

NA

Abbreviations: No. pts: number of patients; between: b/w; RT: radiotherapy; m: months; NA: not available; ND: not determined; Ipi: ipilimumab; Mþ: metastasis, LN: lymph node(s); OS: overall survival; Abs E: abscopal effect. a Median. b 16 Abscopal response/65 radiation course (n Z 47 patients). RT improved response rate of out-of-field lesions response in 68% of cases. c Clinical cases of patient starting simultaneously Ipi and RT are not reported as Ipi alone might be responsible for patients’ response.

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induction of efficient anti-tumour immunity [32]. This, therefore, reinforces the rationale for combining RT with the blockade of immune checkpoints. 3.1.1. Anti-CTLA-4 The key functions of the immune checkpoint CTLA-4 in adaptive immunity and negative regulation of T-cell functions makes it an attractive pharmacological target with which to optimise the abscopal effect. The benefit of ipilimumab, a CTLA-4-targeted antibody, has been demonstrated in several randomised phase III studies [33e35]. This drug is also associated with the most frequent and best documented cases of radiationinduced abscopal effects [27]. In a systemic analysis of abscopal responses in consecutive patients with metastatic melanoma treated with the CTLA-4 inhibitor and receiving 65 palliative courses of RT, out-of-field responses were observed in 11% prior to RT versus 25% after RT, including lesions progressing prior to irradiation. Fraction sizes of 3 Gy were associated with a favourable index response [31]. Grimaldi et al. examined the outcome of patients with advanced melanoma irradiated while progressing after ipilimumab (n Z 21). Abscopal responses were observed in 11 patients (52%). The median time interval between RT delivery and the evidence of an abscopal effect was 1 month. The authors found a significant correlation between the occurrence of an abscopal effect and survival, which was 22.4 months in case of an abscopal effect versus 8.3 months in patients who did not experience this effect. Local responses predicted the abscopal effects observed in 11 in 13 (85%) patients with a local response versus none in those who did not respond locally [30]. Some data suggest a correlation between ipilimumab doses and the probability of an anti-tumour effect. In an analysis of 29 patients who had received 33 courses of extracranial irradiation between their first and last dose of ipilimumab, the frequency of immune-related adverse side effects was 43% at 10 mg/kg and 2% at 3 mg/kg. Toxicity rates were in the range of what had previously been reported in studies of ipilimumab as a single agent [36]. Gerber et al. retrospectively investigated the safety/efficacy of whole-brain irradiation delivered within 30 days of ipilimumab treatment in 13 patients with metastatic melanoma. Responses were observed in 5 patients according to immune-related response criteria and one patient experienced grade IIIeIV in-field toxicity [37]. Other data suggested that ipilimumab could improve survival in patients receiving stereotactic radiotherapy (SRT) for a metastatic brain melanoma [38]. Almost a decade before these retrospective studies were conducted, preclinical data had shown the sound rationale of combining primary RT with CTLA-4 blockade for eliciting an anti-tumour immune response leading to out-of-field responses. Demaria et al. used a poorly immunogenic model of metastatic mouse mammary carcinoma 4T1. The animals were randomised into

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four treatment groups: (1) control IgG (IgG), (2) RT þ IgG, (3) anti-CTLA-4 antibody, and (4) RT þ antiCTLA-4. The irradiation schedule consisted of one or two fractions of 12 Gy. The monoclonal antibody had no significant effect given as a single agent but the combined group had a statistically significant survival advantage, correlated with an inhibition of lung metastasis formation in a CD8þ cell-dependent mechanism [39]. The effect of dose fractionation has also been investigated in a model of subcutaneously grown TSA mouse breast cancer. It showed a synergistic combination between anti-CTLA-4 antibody and irradiation at the irradiated site and also generated an abscopal effect, but only with a fractionated protocol [40]. In murine mesothelioma models, RT increased the population of tumour-infiltrating T cells, while blockade of CTLA-4 led to a significant decrease in Tregs and an increase in cytotoxic cells [41]. Several early-phase studies are prospectively investigating the inhibition of CTLA-4 with conventional RT or SRT. In metastatic prostate cancer, a phase I/II study explored ipilimumab as a single agent and combined with RT (single fraction of 8 Gy) in patients with castration-resistant disease. Ipilimumab (10 mg/ kg)  RT was active, with PSA declines of 50% in 8 of 10 patients, one complete response (CR) and stable disease in six cases [42]. The CA184-043 trial investigated the benefit of ipilimumab as adjuvant therapy after RT in patients with metastatic castration-resistant prostate cancer. Patients with at least one bone metastatic target and who had progressed after docetaxel received bone-directed irradiation (8 Gy in one fraction) followed by either ipilimumab 10 mg/kg or a placebo every 3 weeks for up to four doses. There were no differences in overall survival but signs of drug activity were detected [43] Further trials are ongoing in other tumour types. 3.1.2. Anti-PD-1/anti-PDL-1 The PD-1 receptor and its ligands negatively regulate the immune response of T cells. Among various inhibitors of this pathway that have been developed, the anti-PD-1 monoclonal antibody nivolumab is the most advanced in terms of clinical development. Three recent randomised phase III studies published in 2015 demonstrated the benefit of nivolumab in melanoma patients [44e46]. A rationale exists to associate PD-1 inhibitors with irradiation, although clinical data are still lacking. Liang et al. investigated host immune responses and showed a crucial role of immune cells and their cytokines in suppressing tumour cell repopulation. Depletion of T cells or inhibition of IFN-g abrogated radiation efficacy. The T-cell response was potentiated by blockade of the PDL-1/PD-1 axis, thereby increasing the probability of tumour control [47]. Deng et al. studied whether the pharmacological blockade of PDL1 could enhance T-cell effector function in inflammatory

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tissues. The authors demonstrated that irradiation upregulated PDL-1 in the tumour microenvironment. PDL-1 upregulation on tumour cells after fractionated RT was also demonstrated by another team. This effect may furthermore rely on the IFN-g produced by CD8(þ) T cells [48]. Anyhow, synergistic activity (radiosensitising immunotherapy) was exhibited between irradiation and anti-PDL-1 in terms of tumour shrinkage. This effect was associated with a decrease in local accumulation of tumour-infiltrating myeloidderived suppressor cells which function as inhibitors of T-cell function and contribute to altering the tumourimmune microenvironment [49]. Zeng et al. examined the preclinical activity of anti-PD-1 immunotherapy combined with SRT in a mouse intracranial orthotopic model of glioblastoma. Survival was improved in animals receiving anti-PD-1 therapy plus irradiation, compared with either therapeutic modality alone. Only animals receiving the combination experienced longterm survival and some of them were still alive 180 days after treatment. There was a correlation between the effect of the combination and immunological changes: an increase in tumour infiltration by cytotoxic T cells (CD8þ/IFN-gþ/TNFþ) and a decrease in regulatory T cells (CD4þ/FOXP3) [50]. Increased longterm survival was confirmed in vivo by another team when IR was combined with anti-PD-1 or anti-PDL-1 treatments [49]. In a model of triple-negative breast cancer, the concomitant use of monoclonal antibodies stimulating immunity (a-CD137 or CD40) and irradiation was not effective in mice bearing orthotopic C57BL/6-derived AT-3 tumours and PD-1 signalling was a critical factor limiting the therapeutic efficacy of costimulatory factors given alone or combined with irradiation. However, the adjunction of the anti-PD-1 to a-CD137 (a member of the TNF receptor family acting as a co-stimulatory immune checkpoint molecule) cured all tumours exposed to single- or low-dose fractionated irradiation. After irradiation, tumours were enriched with functionally active tumour-specific effector cells [51]. Finally, an abscopal effect was reported with the combination of high-dose IR (SRT) and PD-1 blockade (66% reduction in the secondary tumour size) in wildtype mice bearing B16-OVA (melanoma) or RENCA (renal) tumours. In that setting, PD-1 blockade alone or SRT plus control Ig treatment had no effect on the secondary tumour outgrowth [52]. SRT was delivered on day 8 and anti-PD-1 was administered between days 7 and 16 following tumour cell inoculation. This abscopal effect was tumour specific and there was no secondary breast tumour shrinkage when SRT was delivered to the primary renal cancer in conjunction with anti-PD-1. In larger tumours, only the triple combination of SRT, anti-PD1, and anti-CTLA-4 induced regression of the secondary tumour (no data on the sequence of administration). Twyman-Saint Victor et al. also demonstrated higher responses with the triple

combination in a melanoma model. They first observed that localised radiation (given before or concurrently) with CTLA4 blockade induced higher distant responses than each treatment delivered alone. Conversely, the overall response rate was low (17%) and resistance was common. The authors were able to decipher that upregulation of PDL-1 on tumour cells and T-cell exhaustion (minority of cells expressing Ki67 and the cytotoxic protein GzmB) were the cause of resistance. Adding anti-PDL-1 then reversed the resistance (and T-cell exhaustion) by increasing the CD8/Treg ratio and oligoclonal T-cell expansion [53]. The activity and toxicity profile of the RT and anti-PD-1 þ anti-CTLA4 combination are being investigated in several early-phase clinical trials, whose results are awaited (Fig. 1) 3.2. Other targets The granulocyteemacrophage colony-stimulating factor (GM-CSF) is a potent stimulator of dendritic cell maturation. In a prospective pilot study published in 2015, patients with a stable or progressive metastatic solid tumour on single-agent chemotherapy or hormonal therapy and with at least three measurable tumour sites were treated with concurrent RT to one site plus GM-CSF. Then, the course was repeated, targeting a second metastatic site. A total of 11 of 41 (6.8%) accrued patients experienced an abscopal effect [54]. In addition to immune modulators and checkpoint inhibitors, several promising immune-modulating pathways are currently being investigated, in combination with RT, including anti-tumour vaccines and peptides but are mostly still at the preclinical stage. We recently showed that combining a specific anti-tumour vaccine with RT not only maximised the local response to RT (immune-driven radiosensitisation) but also induced long-lasting control of distant disease [55]. Vaccination against tumour-associated antigens could also enhance radiation-induced abscopal effects. Thus, Hodge et al. showed that the antigen response induced by a systemic vaccination directed against a tumour-associated antigen (carcinoembryonic antigen [CEA]) led to regression of antigen-negative metastases at distant out-of-field tumour sites. A comparison of the immune response showed significantly higher CEA-specific IFN-g production when the vaccine and irradiation were combined compared with the control group [56]. In a mouse model of a subcutaneous tumour and liver metastasis from colon cancer [26], the intra-tumour injection of interleukin (IL)-2 enhanced the anti-tumour effects of RT, yielding a CR of both the subcutaneous tumour and the liver metastasis. An analysis of the animal’s splenocytes showed that mice receiving the irradiation þ intra-tumour IL-2 contained a higher percentage of CD4þ T cells but a lower percentage of Tregs and of MDSC. The authors also studied human

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Fig. 1. Anti-tumour T-lymphocyte activation with radiotherapy-elicited abscopal effect and checkpoint inhibitors. Ionising radiation (IR) elicits tumour antigen release and modulates the tumour cell phenotype, leading to both activate immune responses and increased immune recognition. Irradiated tumours release danger signals that trigger DC activation. IR can also induce a dose-dependent increase in MHC class I presentation in human tumour cells leading to tumour recognition. Engagement of PD-1 and CTLA-4 receptors on activated T cells by their ligands (PDL-1 and B7, respectively) results in negative T-cell regulation, which protects cancer cells from immune elimination. Danger signals, activated DC and immune checkpoint inhibitors all produce appropriate anti-tumour CD8þ T lymphocyte cytotoxicity. HSP70: heat shock protein 70; DAMPS: damage-associated molecular patterns; DC: dendritic cells; MHC: major histocompatibility complex (MHC).

rectal cancer and reported a correlation between infiltration of CD8þ cells in rectal tumours and both a lower frequency of distant metastases and a better histological response after irradiation [22]. In a pilot study of extracranial SRT followed by high-dose IL-2, conducted in untreated patients with metastatic melanoma or renal cell carcinoma, CR or partial responses were reported in 8 of 12 patients (66.6%). There was a correlation between the tumour response and the frequency of proliferating CD4þ T cells with early activation of the effector memory phenotype in the peripheral blood [57]. The immune response modification afforded by the TLR7 agonist imiquimod was investigated in a model of skin-involving TSA mouse breast carcinoma, injected subcutaneously into syngeneic mice. The authors applied the topical TLR7 agonist imiquimod or a placebo cream to the skin overlying tumours three times/week and local irradiation was delivered to the tumour (3 consecutive fractions of 8 Gy). At the same time, mice received one intraperitoneal injection of cyclophosphamide, 2 mg/animal. Topical applications of imiquimod inhibited tumour growth and this effect was associated with an accumulation of CD11cþ, CD4þ and CD8þ cells. Depletion of CD8þ cells abolished the effect. Furthermore, adding topical

imiquimod led to out-of-field anti-tumour effects. Finally, the authors demonstrated that only animals with a longterm immunologic memory remained tumour free [58]. In a prospective study, imiquimod was shown to stimulate local anti-tumour immunity in 10 patients with skin metastases from breast cancer. Treatment was well tolerated and two partial responses were observed. Tumor response correlated with immunological patterns: the presence of a tumour lymphocytic infiltrate and local production of cytokines [59]. Another team also combined a local TLR9 agonist injection with low-dose RT on a single tumour in patients with mycosis fungoides. Five of 15 patients had a distant abscopal response associated with a reduction of CD25(þ) and Foxp3(þ) T cells in the immunised sites [60]. Inhibition of the TGFb type I receptor kinase proved effective in radiosensitising breast cancer and glioblastoma cell lines [61,62]. Inhibition of TGFb during RT was able to trigger a CD8þ T-cell response to various tumour antigens, leading to out-of-field responses. While the upregulation of PD-1 in the tumour could limit this effect over time, combined inhibition of PD-1 and TGFb plus irradiation was promising [63]. An injection of ECI301, a mutant derivative of macrophage inhibitory protein-1a, after intra-tumour injection of

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tumour cell lysates, was also able to restore tumour immunosurveillance capabilities against tumour cells [64]. Klug et al. [65] showed that irradiation delivered at a low dose (either endogenous or following adoptive transfer) could stimulate the differentiation of iNOSþ M1 macrophages from an immunosuppressive state to one generating an efficient tumour cytotoxic lymphocyte-mediated response. The fact that these socalled “RT-reprogrammed” macrophages are able to promote tumour regression when administered to other tumour-bearing mice suggests their potential implication as co-mediators of the abscopal effect seen after RT. On the other hand, this may not be true in the clinic because metabolically active irradiated macrophages remain viable after cumulative IR doses of up to 10 Gy. In a report by Teresa Pinto et al., macrophages exhibited a pro-inflammatory profile (M1 like) after 10 Gy but were still able to promote tumour cell invasion and angiogenesis (M2 like) [66]. Chiang et al. showed the accumulation of pro-tumourigenic M2 macrophages in areas of hypoxia present in irradiated tumours [67], potentially promoting anti-tumour immunity after RT. This may be the rationale for targeting macrophage differentiation as a complementary strategy to improve RT efficacy [68,69]. Several other pharmacological interventions triggering the microenvironment were shown to be effective for potentiating the abscopal effect: the induction of IFN-g-producing T cells, cell metabolism modulators, an adjuvant intra-tumour injection of bone marrowderived dendritic cells directly into the irradiated lesion, or the use of immunostimulatory factors [40,70,71]. Other potential target/developing immunotherapies that may be combined with IR are summarised in Table 2.

4. The evolving place of radiotherapy Knowledge of the importance of the anti-tumour immune response as part of radiation efficacy has led physicians to rethink the place of irradiation in this setting. Several issues still remain unanswered. The total dose and fractionation seem to be important parameters determining the modulation of the immune response by irradiation. In the preclinical studies described earlier, the dose per fraction was decisive in the therapeutic effect when irradiation was combined with inhibitors of CTLA-4 [40]. There is also a degree of uncertainty regarding the existence of a dose threshold. In contrast to these data, SRT induces the expression of MHC I molecules, inflammatory cytokines, cellecell adhesion molecules, heat shock proteins, and death receptors. Although controversial, it is suggested that SRT could be the most appropriate RT modality to be combined with immunotherapy since it could lead to a more robust immune response than conventionally

Table 2 Immunotherapies with potential synergy with radiotherapy. Immune checkpoint targeted mAbs for adaptive immune cells Anti-B and T-cell lymphocyte attenuator (BTLA) Anti-V-domain Ig suppressor of T-cell activation (VISTA) Anti-T cell immunoreceptor with Ig and ITIM domains (TIGIT) Anti-T-cell immunoglobulin and mucin domain 3 (TIM-3) Anti-lymphocyte activation gene 3 (LAG3) Anti-4-1BB (CD137) Anti-OX40 (CD134) Anti-GITR (CD357) Anti-ICOS (inducible T-cell co-stimulator) Anti-CD40 (tumour necrosis factor receptor) Vaccines Dendritic cell-based vaccines Autologous granulocyteemacrophage colony-stimulating factor (GM-CSF)-transfected Viral vector vaccines mRNA-based vaccines Multipeptide-based vaccine Locally released virotherapy Oncolytic viruses Talimogene (TVEC) Pexa-Vec (JX594) Adoptive T-cell therapy Chimeric antigen receptor (CAR) T cells Tumor-infiltrating lymphocytes (TILs) Immune checkpoint targeted drugs for innate immune cells Anti-inhibitory killer cell immunoglobulin-like receptors (anti-KIR) and anti-NKG2A anti-CSF-1R and CCL2 inhibitors for macrophages () Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, Inducible nitric oxide synthase (iNOS) inhibitors Toll-like receptor agonists PGE2 inhibitors

fractionated RT. Preclinical data obtained in a breast cancer model using CTLA-4 blockade suggested that fractionated RT would be superior to single-fraction RT for inducing a systemic immune response. In a model of mice bearing B16-OVA murine melanoma, Schaue et al. showed a correlation between tumour control and the size of the radiation dose. Fractionated RT with a dose of 7.5 Gy yielded significant results in terms of tumour immunity, whereas 5 Gy doses failed to do so [72]. Other authors observed that a single high-dose fraction or conventionally fractionated schedules (1.8e2.2 Gy) may also trigger immune responses [21,48,73]. Altogether, caution should be exercised when considering these data because they were generated across a large number of tumour models and irradiation schemes. In order to do so, it will be necessary to optimise preclinical models that would better take this aspect into consideration (immunocompetent syngeneic and orthotopic models, in addition to confirmatory autochthonous models and/or genetically engineered models). For example, the tumour suppressor gene p53 was also suggested to be implicated in the abscopal effect in nude mice. Inhibition of distant tumour growth was observed in wild-type p53 but not in p53-null HCT116 human colon cancer-derived xenograft models [74].

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There is also a need for easily accessible surrogate markers to predict or assess the immune response. Survivin is a protein member of the inhibitor of apoptosis family that is highly expressed in most human tumours and implicated in resistance to therapy [75]. Survivin is released into the blood and may thus represent a promising non-invasive immune biomarker after IR. Survivin-specific CD8þ T cells were isolated in the peripheral blood of colorectal and prostate cancer patients and were increased in patients demonstrating tumour downstaging after chemoradiotherapy [72]. Conventional Response Evaluation Criteria in Solid Tumors criteria, as well as overearly response evaluation in patients treated with checkpoint inhibitors may lead to hasty discontinuation of treatment and immune-related response criteria have, therefore, been proposed [76]. Gene expression-based signatures for the immune response are under investigation for initial patient selection. Genome-wide somatic neoepitope analysis and patient-specific HLA typing could be used to identify candidate melanoma neoantigens for each patient [77]. High infiltration of CD3 and CD8 lymphocytes in initial tumour biopsies may also be associated with downstaging of the tumour after chemoradiotherapy [78]. Finally, PDL-1-“positive” (the exact cutoff remains to be determined) tumours have shown trends towards increased rates of response to PD-1 blockade but its practical use still remains debated [79]. The best timing of IR and immunotherapy should further be assessed. This could be a crucial issue as radiosensitising immunotherapy could also contribute to improvements in locoregional control in the earlier stages of disease [80e82]. The sequencing mode led to comparable results when hypofractionated SRT was associated with ipilimumab in a melanoma model in vivo [53]. However, another preclinical study with anti-PDL-1 suggested that concurrent IR delivery seems more efficient than sequential schemes [47]. These important aspects will have to be evaluated in clinical trials. Importantly, for clinicians, a major point to be examined is the potential toxicity of combination therapies, particularly during concurrent schemes. In fact, acute radiation-induced toxicity is mediated by an inflammatory cascade, which could potentially be increased by systemic activation of the immune system. Immune-related adverse events (e.g. increased hepatitis or colitis with abdominal irradiation) could then be increased after localised irradiation. Radiation oncologists should be aware of the practical guidelines which have been published for the management of such toxicities [83]. Conflict of interest statement None declared.

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Acknowledgements The authors thank Lorna Saint Ange for editing.

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