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Brain metastases (BM) affect approximately a third of all cancer patients with systemic disease. Treatment options include surgery, whole-brain radiotherapy, or stereotactic radiosurgery (SRS) while chemotherapy has only limited activity. In cases where patients undergo resection before irradiation, intraoperative radiotherapy (IORT) to the tumor bed may be an alternative modality, which would eliminate the repopulation of residual tumor cells between surgery and postoperative radiotherapy. Accumulating evidence has shown that high single doses of ionizing radiation can be highly efficient in eliciting a broad spectrum of local, regional, and systemic tumor-directed immune reactions. Furthermore, immune checkpoint blockade (ICB) has proven effective in treating antigenic BM and, thus, combining IORT with ICB might be a promising approach. However, it is not known if a low number of residual tumor cells in the tumor bed after resection is sufficient to act as an immunizing event opening the gate for ICB therapies in the brain. Because immunological data on tumor bed irradiation after resection are lacking, a rationale for combining IORT with ICB must be based on mechanistic insight from experimental models and clinical studies on unresected tumors. The purpose of the present review is to examine the mechanisms by which large radiation doses as applied in SRS and IORT enhance antitumor immune activity. Clinical studies on IORT for brain tumors, and on combined treatment of SRS and ICB for unresected BM, are used to assess the safety, efficacy, and immunogenicity of IORT plus ICB and to suggest an optimal treatment sequence.
Brain metastases (BM) are an advanced-stage manifestation of cancer that affect up to a third of patients with systemic disease. BM predominantly originate from primary lung, breast, or gastrointestinal cancers and melanoma. Given the change in demographics in industrialized countries with increasing cancer frequencies, combined with the increase in numbers of long-term survivors owing to improved diagnostics and therapy, the incidence is believed to rise further. Depending on the clinical condition, treatment options for BM include surgery, whole-brain radiotherapy (WBRT), stereotactic radiosurgery (SRS), or a combination of these, while chemotherapy has only limited activity owing to low penetration of the blood–brain barrier (BBB). A considerable proportion of patients undergo upfront surgery for debulking the tumor mass or for the determination of histology and/or mutational status. In such cases, local relapse occurs in roughly 60% of patients 1–6months after surgery alone (1), indicating that tumor stem cells capable of forming recurrent tumors have microscopically invaded the borders of the surgical cavity. While some degree of local control may be achieved by adding WBRT, this is associated with high morbidity and intracranial recurrences are common. Randomized phase III trials did not show improved overall survival by adding adjuvant WBRT (2, 3) and most patients now undergo SRS directed to the tumor bed, a procedure that was proposed and developed even before these trials were done (4, 5). Although level I evidence for this treatment is lacking, initial data suggest a low toxicity profile (6–8). However, even with the best treatment available, the median survival is rarely much longer than 1year and, thus, there is a strong need for improved treatment beyond the BM and the tumor bed around the excised cavity.
Similar to SRS, intraoperative radiotherapy (IORT) to the cavity and margins treats the tumor site while minimizing dose to the surrounding normal tissue. Early clinical studies on IORT after the resection of glioma were conducted especially in Japan and in Germany, typically applying 15–25Gy of high-energy electrons in a single fraction. Results were encouraging, with comparable or better local control and overall survival, and less radionecrosis than after fractionated WBRT with external X-ray beams (9–12). A large retrospective study of IORT as a boost combined with external beam WBRT versus WBRT alone did not show any significant improvement by IORT (13). However, failures were found to be associated with insufficient dose coverage (14) and a case of long-term (9years) survival was indeed observed (15). Because of technical limitations, few centers were able to pursue this treatment at the time, but in the last decade, dedicated mobile machines for delivering IORT by high-energy electrons or low-energy X-rays (LEX) in the operating room have become more widely available.
Compared to SRS for resected metastases, IORT eliminates the healing time between surgery and the beginning of RT during which tumor cells may proliferate and possibly spread beyond the tumor bed. In contrast, IORT requires the total dose to be applied in a single fraction, whereas hypofractionated treatment is optional with SRS (e.g., for larger tumors or cavities). Recently, the potential use of IORT for brain tumors may be supported by encouraging results from a phase I/II trial on IORT with 50kV X-rays for glioblastoma (16), which was found to be safe in these patients (Giordano et al., submitted) and prompted the initiation of a randomized phase III trial (NCT02685605). The rationale for IORT in glioblastoma has been reviewed by Giordano et al. (17). Notably, the treatment of solitary BM with excision and IORT alone using 50kV X-rays has been shown to be feasible with disease-specific outcome comparable to other modalities (18).
It has been suggested that in addition to targeted cell killing induced by conventional fraction sizes, vascular, cohort (bystander), and immune effects may contribute to the biological effect of very large doses per fraction (19–22). In contrast, it has been disputed whether additional effects other than the 5R’s of radiotherapy (reassortment, repair, reoxygenation, repopulation, and radiosensitivity) need to be invoked to explain the successes of SRS, SBRT/SABR, and IORT (23). Nevertheless, there is a strong case that large radiation doses may act as an adjuvant for immunogenic cell death by releasing tumor antigens and danger signals (24). At the same time, the identification and characterization of immune checkpoints has led to a surge in clinical studies on immune therapy using immune checkpoint blockade (ICB) antibodies (frequently termed “checkpoint inhibitors” although to date no small-molecule inhibitors are available). For example, an early phase II study showed dramatic effects in melanomas, which generally are immunogenic tumors (25). Thus, combining RT and ICB is considered to offer potential synergies, in particular since antitumor immune effects outside the irradiated target volume, so-called abscopal effects (26), might help control microscopic systemic disease.
Although the brain has, for decades, been regarded as a “privileged site” that provided limited scope for antitumor immunity, activated T cells are known to be able to cross the BBB (27). While conventional radiotherapy mildly increases BBB permeability (28), SRS disrupts the BBB within hours after application, allowing cells and substances to easily cross into the CNS for a period of roughly a month (29). In the case of BM, early studies suggested improved overall survival rates when ICB was combined with SRS for unresected metastases (30, 31), reaching levels similar to patients without BM (32). In contrast, a study applying ICB in patients previously treated with SRS found no significant difference to SRS alone (33) and ICB combined with conventionally fractionated WBRT after resection also failed to show an effect (34), suggesting that timing and fractionation may be important.
Whereas a potential interaction between SRS and ICB is readily understandable in the case of non-resected metastases where radiation can release tumor antigens, it is not obvious if the irradiation of residual tumor cells in the tumor bed after resection of the tumor is sufficient to elicit a tumor-directed immune response. Since no systematic studies on resected tumors have been published, a rationale for combining IORT with ICB must be based on an understanding of the mechanisms involved. Therefore, the purpose of the present review is to examine the immunological interaction between radiation and ICB to elucidate whether high single doses to the resection cavity and the residual cancer cells within its margins might act as an immunizing event opening the gate for ICB therapies in the brain. Because of the complexity and dynamic nature of this topic, we first give a brief overview of the antitumor immune response and immune checkpoints for the non-expert. We then present the experimental evidence for the interactions between radiation and the immune system. Finally, we review the clinical studies on SRS combined with ICB and discuss the implications and potential for combining IORT with ICB for BM.
The innate immune system acts as a non-specific first-line defense against infection and foreign antigens but also activates the adaptive immune system to provide an antigen-specific response. Upon infection, trauma (including irradiation), or during tumor initiation and progression (35), an inflammatory cascade is induced. In case of an infection, this is initiated by pathogen-associated molecular pattern (PAMP) molecules such as bacterial liposaccharides. Similarly, trauma release damage-associated molecular pattern (DAMP) molecules including proteins such as nuclear high-mobility group box 1 (HMGB1) and endoplasmatic calreticulin (CRT) as well as non-protein molecules adenosine triphosphate (ATP) and mitochondrial peptides and DNA (in the case of necrotic cell death) (36–38).
Soon after the appearance of PAMP or DAMP molecules, neutrophils enter the tissue secreting a large variety of chemokines and cytokines, including pro-inflammatory interleukin (IL)-12 (39), which in turn recruit monocytes and lymphocytes into the inflamed tissue. Depending on the cytokines, monocytes can differentiate into inflammatory or anti-inflammatory macrophages, and dendritic cells (DC). Phagocytes (neutrophils and macrophages) have pre-formed pattern recognition receptors (PRRs), mainly toll-like receptors (TLRs) and receptors for advanced glycation end-products (RAGE) that bind to PAMPs on microbial surfaces or to DAMPs from damaged cells. DAMPs are found on cell membranes, released into the extracellular space, or detected in the cytoplasm by intracellular PRR sensors such as TLR-9, which activates the STING [stimulator of interferon (IFN) genes] pathway (40) inducing the expression of type 1 IFN, e.g., IFNβ.
Natural killer (NK) cells are an important component of immune surveillance that remove cells with low expression of major histocompatibility complex (MHC) class I surface molecules. NK cells are CD3− CD8+ lymphocytes lacking the T-cell receptor (TCR), which CD3+ lymphocytes use for the detection of antigens on MHC. Instead, they express activating receptors belonging to the family of killer-cell immunoglobulin-like receptors (KIRs). The body’s own cells are protected by inhibitory KIRs that recognize MHC class I presenting “self” antigens. Combinations of IL-12 or IL-15 with IL-18 stimulate NK cells activated by target cell recognition to secrete chemotactic cytokines, e.g., macrophage inflammatory protein followed by inflammatory cytokines IFNγ and tumor necrosis factor (TNF)-α in different subpopulations (41).
The adaptive immune system reacts to specific antigens and is made up largely of T and B lymphocytes, which are responsible for the cell-mediated and humoral adaptive immune responses, respectively. This part of the system carries a memory of previous antigens with lymphocytes being distributed between lymph nodes and the body tissues. Antigens need to be presented to lymphocytes by antigen-presenting cells (APCs). Most cell types present a small fraction of degraded proteins as peptide antigens on MHC molecules on their surface. Non-professional APCs (essentially all cell types) present 3–18amino acid (a.a.) peptides from degraded cellular protein on 105–106 MHC I molecules found on each cell (42), while so-called professional APCs (DC found mainly in superficial tissue, macrophages, and B cells) also present peptides on MHC class II molecules. The peptides presented on MHC class II are generated from antigens taken up by endocytosis and can be longer than 18 a.a. but are often degraded by peptidases to approximately 12 a.a. (42). Tumor cells and dying normal cells translocate CRT to the cell surface acting as an “eat me” signal. If CRT is able to overcome the inhibitory “do not eat me” signal from CD47, it will activate TLRs on phagocytes (43, 44). Together with the release of other DAMP molecules, this stimulates phagocytosis by DC or macrophages which process the antigens and present them on MHC class II leading to activation of these APCs (45). Activated professional APCs migrate to the nearest lymph nodes (or via the blood vessels to the spleen) where the MHC:peptide complexes are presented to lymphocytes that recognize specific antigens by their T- or B-cell receptors (BCR). B cells recognize native antigens by their BCR and can internalize, process, and present antigen peptides on their MHC class II molecules to T cells (46). According to the clonal selection theory, the highly variable TCR and BCR give rise to an extremely large number of mature, so-called naive, lymphocytes that each recognize different epitopes made up of antigen peptides presented on MHC molecules. While an adaptive antitumor immune response requires CD8+ and CD4+ T cells, the role of B cells and the humoral adaptive immune response is unclear.
The two major classes of T cells, cytotoxic (“killer”) T cells (Tc) and helper T cells (Th), express different co-receptors, CD8 and CD4, respectively. CD8 on Tc bind to MHC class I (on all cells), while CD4 on Th cells bind to MHC class II (on professional APC). The binding between the Th and professional APCs is reinforced by induced expression of co-stimulatory molecules, mainly CD28, which binds to B7.1 (CD80) and B7.2 (CD86) on APCs, and CD40 ligand (CD40L), which binds to the CD40 receptor. Once a specific MHC II antigen-peptide combination binds the TCR and CD4 co-receptor of a naive Th, co-stimulatory binding results in its activation with clonal expansion and differentiation to a secretory effector Th cell releasing different cytokines.
Subsets of differentiated Th cells mediate either a cytotoxic immune response (mainly Th1cells characterized by secretion of IFNγ) or a humoral immune response (follicular helper, TFH) (47). Th1 cytokine IFNγ stimulates the function of macrophages and the activation of CD8+ T cells, binding to MHC I:peptide complexes. TFH are thought to activate B cells, while Th1, Th2 (characterized by IL-4, IL-5, and IL-13), and Th17 (characterized by IL-17a, IL17b, and IL-22) direct immunoglobulin class switching according to different types of pathogens. Since B cells function as professional APCs they may activate Th cells recognizing the antigen peptides presented on the MHC II molecules of the B cell and the secreted cytokines in turn activate the B cell causing it to proliferate and produce specific antibodies. An overview of the immune activation is shown schematically in Figure Figure11 (48, 49).
Various mechanisms prevent the immune system from attacking its own body cells (autoimmune reactions) and from excessive normal immune reactions. Basically, tolerance to “self”-antigens is induced by the deletion of naive Tc recognizing MHC:peptide complexes that present fragments of the individuals own proteins. In addition, a number of other mechanisms help limit the physiological immune response. A special type of CD4+ regulatory T cells (Tregs, characterized by CD25high and the canonical transcription factor FoxP3) limit or modulate the adaptive immune reaction by a variety of mechanisms [reviewed in Ref. (50)]. Tregs secrete inhibitory cytokines IL-10 and TGF-β1 and express CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) which is a negative regulator competing with CD28 for co-stimulatory binding to the B7 molecule on APCs [reviewed in Ref. (51)]. Tregs constitutively express CTLA-4 (52), but CTLA-4 is also induced during Tc activation, thus providing a feedback mechanism for downregulating APC-mediated Tc activation to prevent an excessive inflammatory reaction (53). Another member of the CD28 family, programmed death-1 (PD-1), is expressed on lymphocytes and inhibits the function of activated T cells, by binding to the B7 family ligand PD-L1. PD-L1 is not expressed in most normal cells but can be induced in tumor cells by IFNγ in the tumor microenvironment (54). PD-L1 can bind to B7.1, and PD-L1 signaling via PD-1 mediates immune suppression by stimulating apoptosis of T cells, inducing IL-10 and inducible Tregs, which contributes to a dysfunctional state termed T-cell “exhaustion” (55). Thus, according to the current model of immune checkpoints, CTLA-4 exerts its action mainly during antigen presentation and Tc activation, i.e., in the afferent arm of the adaptive immune response (leading to the secondary lymphoid tissue). By contrast, PD-L1/PD-1 is considered to act mainly in the efferent arm (leading from the lymph nodes back to the affected tissue) by modulating the cytotoxic action of CD8+ T cells in the tumor, although PD-1 is also expressed on Tregs, NK, and B cells, while PD-L1 is expressed on myeloid cells in tumors (56, 57). In addition to Tregs, myeloid-derived suppressor cells (MDSC; of monocytic and granulocytic lineages) contribute to immune suppression via secretion of immunosuppressive cytokines IL-10, and TGF-β1, and other mechanisms (58). The major immune checkpoints currently exploited in cancer therapy are shown schematically in Figure Figure22.
Because tumor cells arise from the body’s own cells they might be expected to escape immune surveillance. In spite of this inherent tolerance, an immune response may be elicited by overexpressing naturally occurring “self” proteins (tumor-associated antigens), mutated “self” proteins, or foreign proteins such as viral proteins (tumor-specific antigens, TSA) (59). However, genetic and epigenetic changes during tumor progression may select for mechanisms that avoid detection or suppress the immune response. Thus, an inflammatory response in tumors may upregulate PD-L1 and cause tumor-associated macrophages and MDSC to express IL-10 and TGF-β1 (60, 61).
Targeting the immune checkpoints by antibodies against CTLA-4 and PD-1/PD-L1 has recently shown to result in clinically relevant responses in some cancer patients (62–66). Antibodies against CTLA-4 broadly stimulate the adaptive immune response but may be associated with severe side effects, while anti PD-1/PD-L1 therapy may be more specific toward tumors and appears to be better tolerated (51). However, ICB antibodies given alone are effective only if the tumor is immunogenic per se.
Although low doses of radiation are immunosuppressive, it has become clear in the last 10–15years that higher doses may stimulate the antitumor immune response (45, 67, 68). Indeed, some evidence suggests that immunogenic cell death contributes to the efficacy of hypofractionated or single-dose radiotherapy (37, 69, 70). However, data regarding the influence of dose and fractionation are conflicting, thus warranting a critical review of the dose dependence of immune activation.
The first evidence that irradiation releases DAMP molecules was found in murine thymoma cells that released HMGB1 after a dose of 10Gy in an apoptosis-dependent fashion since the release was suppressed by the caspase inhibitor Z-VAD-fmk (71). Golden et al. found that CRT translocation and the release of DAMP molecules ATP and HMGB1 in a murine breast adenocarcinoma cell line were increased by single doses in the range of 2–20Gy (72). The data indicated a quasi-linear increase up to 10Gy, whereas 20Gy produced a moderate further increase for CRT and ATP but only little further increase of HMGB1. Radiation-induced release of DNA into the cytosol (e.g., from the mitochondria) activates the STING pathway leading to the induction of type I IFN, the first step in the inflammatory cytokine cascade (73). Thus, IFNβ was induced after a single dose of 20Gy to B16 melanoma tumors (74). NK cells and lymphocytes are very radiosensitive and undergo apoptosis after doses <2Gy. Furthermore, translocation of nuclear HMGB1 into the cytosol was recently reported after irradiation of human skin fibroblasts with doses in the range 4–12Gy (75). Therefore, it seems a distinct possibility that high-dose irradiation of the normal tissue in the tumor bed may contribute to producing an inflammatory microenvironment conducive of an antitumor immune reaction.
Irradiation induces cytokines in various cell types, most importantly via nuclear factor (NF)-κB [reviewed in Ref. (76)], which can be activated by DNA damage-induced kinases, ATM, and DNA-PKcs (77, 78). Furthermore, HMGB1 is a ligand for RAGE and TLR4 signaling to NFκB, which may contribute to cytokine induction after higher doses (79). NFκB regulates transcription of a large number of cytokine genes, including pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, IL-33, and TNF-α. Thus, expression of IL-1β and TNF-α was induced within a few hours of irradiating macrophages with doses of 3–20Gy in vitro (80–82). Early upregulation of IL-1β was also observed after in vivo irradiation with 18.5Gy (83), whereas a lower dose of 3Gy caused upregulation approximately 5–7days later, during macrophage differentiation preceding regeneration of the spleen (80). Early transcriptional upregulation of a number of cytokines including IL-1β and TNF-α occurred in brain or lung tissue after irradiation with doses of 7–25Gy (84, 85). Thus, robust expression seems to require high single doses although daily fractions of 4Gy also produced sustained expression in lung macrophages (85). Strong, dose-dependent secretion of IL-6 regulated by NFκB and activator-protein-1 was found in HeLa cells 24h after irradiation with 3–20Gy, while no significant increase was observed after 1Gy (86, 87). In another study, secretion of IL1-α, IL-6, and IL-8 over 24h was induced 1.7-, 1.6-, and 2.1-fold, respectively, by a low dose of 1.5Gy in a monocytic cell line but not in A549 adenocarcinoma cells (88). However, irradiation of murine lymphoma with a single high-dose of 30Gy initially decreased IFNγ and TNF-α in splenocytes but expression recovered 7–10days after irradiation (70). A comprehensive review of the inflammatory response to ionizing radiation was given recently by Di Maggio et al. (89).
While it is not surprising that leukocytes express cytokines, it may be important for other cell types that p53 and NF-κB show a reciprocal relationship (90, 91). Veldwijk et al. (22) tested the secretion of 36 cytokines by p53 wild-type MCF7 breast cancer cells over 24h after irradiation with 15Gy. Only six cytokines (CD40L, IFNγ, IL-6, IL-8, IL-23, and Serpine E1) were detectable, and none showed significant upregulation after irradiation. Thus, it is conceivable that radiation-induced p53 may limit induction of the ATM/DNA-PKcs/NFκB pathway in p53 wild-type normal and tumor cells (A549 and MCF7) and that stronger induction in HeLa cells is due to the suppression of p53 by expression of the HPV18 E6 protein. Whereas in vitro induction may require high doses, there is ample evidence for radiation-induced expression of pro-inflammatory cytokines after moderate doses given in vivo (76). For example, dose-dependent upregulation was demonstrated in peritoneal mouse macrophages isolated 16h after whole-body irradiation with 0.075–6Gy with maximum upregulation at 4Gy showing twofold increase for IL-12 and fivefold increase for IL-18 (92). The apparently higher sensitivity in vivo may be explained by additional activation due to lymphocyte apoptosis that may release DAMP molecules in situ including HMGB1 which activates the NF-κB pathway (79, 93).
Tumor cells frequently downregulate MHC surface molecules, thus reducing the opportunity of antigen presentation. However, radiation doses of 10–20Gy upregulated MHC class I expression by ≥10% in 8/23 human colon, lung, and prostate, tumor cell lines tested (94). In a human melanoma cell line, MHC class I was increased in a dose- and time-dependent fashion with maximum expression at 48–72h yielding a twofold increase for 10–25Gy compared to 1.3-fold after 4Gy (95). This study also showed that intracellular peptides for antigen presentation were initially generated by the degradation of existing proteins, but at later time points, novel peptides from new protein synthesis were presented on MHC class I. In a similar system, upregulation of MHC class I appeared to depend partly on radiation-induced IFNγ (96). Further aspects of different radiotherapy schemes on immune stimulation in vitro and in vivo have been reviewed in Ref. (97, 98).
Experiments using a tumor antigen-specific adenoviral vaccine showed that a single, moderate dose of 8Gy given before vaccination produced a synergetic antitumor effect against a murine colorectal tumor, which was also observed when irradiation was given in three fractions of 3.5Gy each (99). Since irradiation after vaccination had no effect, this seems to suggest a role of irradiation as an adjuvant creating a local microenvironment that supports immunization rather than a role in antigen presentation in this setting. Such a model is supported by the strong immunogenic effect of a TLR-7 agonist on a colorectal tumor, which was potentiated by fractionated radiotherapy with 5×2Gy beginning simultaneously with the first application of the agonist but without any further immune therapy (100). However, the complexity and multiple components of the dynamic immune reaction may explain why combining radiotherapy with systemic type I or II IFNs was mostly unsuccessful, while the combination with IL-2 or IL-12 showed only limited effects in early clinical studies [reviewed in Ref. (101, 102)].
Few studies have investigated antitumor immunogenic effects of radiation in vivo without applying immune stimulation or checkpoint inhibitors. Lugade et al. found that a single radiation dose of 15Gy increased the number of APC capable of activating IFNγ-secreting cells in lymph nodes in an experimental mouse model B16 of melanoma genetically modified to express ovalbumin (OVA) as a non-self antigen (67). A fractionated schedule of 5×3Gy showed smaller increases of such APC in the lymph nodes. A similar difference between single and fractionated irradiation was seen for infiltration of the tumor by CD45+, CD4+, CD8+, CD11c, and CD11b immune cells 7days after irradiation, indicating the recruitment of T cells, DC, macrophages, and possibly NK cells (CD8+ but CD3−). Interestingly, the difference between single and fractionated doses was observed for specific T cells, activated by tumor-derived peptide presentation on MHC I but not on MHC II in both lymph nodes and tumors. Infiltration into the tumors was due to lymphocyte trafficking and was dependent on the upregulation of vascular cell adhesion molecule-1 on endothelial cells (96). A study by Lee et al. using unmodified B16 melanoma confirmed a growth inhibitory effect after a single dose of 20Gy when tumors were grown from 2×106 injected cells, and local control was observed after 15Gy when the number of injected cells was reduced to 1×105 (69). In the same study, local tumor control was also observed when an MHC class I-binding peptide (“SIY”) was introduced as antigen and tumors grown from 2×105 injected cells were irradiated with 25Gy. Growth delay for 5×105 injected cells and irradiation with 20Gy was dependent on CD8+ and was not seen for fractionated irradiation with 4×5Gy. The effect of dose and fraction size was studied by Schaue et al. who irradiated B16-OVA tumors with single doses of 5–15 and 15Gy applied in 1, 2, 3, or 5, fractions (103). Inhibition of tumor growth was seen at 7.5–15Gy, whereas no significant effect was seen after 5Gy. Applying a dose of 15Gy in 1, 2, 3, or 5, fractions reduced tumor size and increased antigen-specific IFNγ expressing cells in the spleen for all schedules with a trend for 2×7.5Gy being more effective. Notably, doses of 1×7.5Gy or 2×7.5Gy, but not other doses, also seemed to reduce the number of Tregs in the spleen. Taken together, single doses of 15-25 Gy, or hypofractionated irradiation with large dose fractions (7.5Gy), seem capable of eliciting an immunogenic antitumor response against the primary tumor in the B16 murine melanoma system even without including ICB in the treatment.
The combination of radiotherapy with ICB has shown considerable synergies on local tumor control in experimental systems. Demaria et al. found that a single dose of 12Gy followed by CTLA-4 blockade showed a synergistic growth delay of mammary tumors and two fractions of 12Gy separated by 48h combined with CTLA-4 blockade produced local control in a small number of animals (104). In a study by Dewan et al., a single dose of 20Gy, or daily fractionated irradiation with 3×8Gy or 5×6Gy, caused similar growth delay but adding anti-CTLA-4 antibody 2, 5, and 8days after the first irradiation inhibited growth for all schemes with an apparent, small advantage of 3×8Gy (105). Incidentally, 5×3Gy fractionated irradiation of B16 melanoma produced slightly more tumor infiltration than a single dose of 15Gy for CD8+ T cells activated by tumor-derived peptide presented on MHC class II, whereas 1×15Gy produced higher numbers of cells activated by peptide presentation on MHC class I (67). This would seem consistent with a model in which hypofractionated irradiation combined with CTLA-4 blockade increases MHC class II-mediated antigen presentation by APC, while high single doses may be more efficient in promoting antigen presentation via MHC class I. In a radioresistant triple-negative mammary tumor studied by Verbrugge et al., a single dose of 12Gy combined with anti-CD137 and anti-PD-L1 antibody treatment produced regression with some control of subcutaneous tumors while 4×5Gy daily fractionated irradiation in combination with the same antibodies was effective in orthotopic tumors (106). Sharabi et al. showed regression of murine melanoma and mammary tumors irradiated with a single dose of 12Gy combined with anti-PD-1 treatment (107). Irradiation with five fractions of 2Gy upregulated expression of PD-L1 in colorectal cancer cells isolated from murine tumors but did not control tumors in a study by Dovedi et al. (108). However, concomitant administration of anti-PD-1 or anti-PD-L1 during and after irradiation resulted in 66–80% local control, and significant effects were confirmed in two other tumor lines. Irradiation combined with both anti-PD-1 and anti-PD-L1 showed no further enhancement. While local control was influenced by NK cells, survival was dependent on CD8+ T cells that also induced PD-L1 via IFNγ. Azad et al. irradiated syngenic pancreatic ductal adenocarcinoma (PDAC) tumors combined with anti-PD-L1 antibody therapy (109). In the KPC line, combined treatment produced non-significant growth delays after 1×6Gy or 5×2Gy, while a single dose of 20Gy produced growth inhibition but excessive dermatitis required termination of the experiment. By contrast, combined treatment with a single dose of 12Gy, or 5×3Gy fractionated irradiation, caused significant growth delay in KPC and regression in the Pan02 line. This was associated with an increase in T-cell infiltration and a reduction in myeloid cell numbers and was only seen for simultaneous treatment and not when anti-PD-L1 was started 1week after irradiation. In another study, Twyman-Saint Victor et al. showed that resistance in patients against hypofractionated SBRT combined with anti-CTLA4 was caused by the upregulation of PD-L1. Mimicking this in a mouse model, the resistance could be overcome by combining CTLA-4 and PD-L1 blockade with radiation, thus confirming and exploiting that the two immune checkpoints are non-redundant (110).
An overview of preclinical studies on immune effects in irradiated tumors is given in Table Table1.1. Overall, dose fractions larger than 7–8Gy seem to be more efficient in eliciting an inflammatory response and immune effects in irradiated tumors (67, 69, 103, 109). In many systems, tumor-infiltrating lymphocytes are increased after irradiation and an increase in the CD8+/Treg ratio seems to be associated with a successful immune reaction in some systems (103, 109, 110), although this is not universally found and MDSC reduction in tumors also seems to play a role (57, 110, 111). The question whether high single doses or hypofractionated irradiation with large fraction sizes is more efficient may depend on the tumor system, the role of antigen presentation by MHC class II, and the immune checkpoint being targeted.
Sporadic cases of abscopal effects of radiotherapy were first described in clinical case reports (112–114), but meanwhile, this rare phenomenon is well documented although in some cases it may be associated (or to some extent overlap) with spontaneous regression [reviewed in Ref. (115)]. Experimentally, a non-specific abscopal effect on unirradiated distant tumors (Lewis lung carcinoma or T241 fibrosarcoma) was found by irradiating a non-tumor-bearing leg of mice with five fractions of 10Gy each, whereas a lower dose of 12×2Gy normo-fractionated irradiation was less effective (116). Interestingly, this effect was dependent on wild-type p53 function in the host animal cells. Irradiation and a special form of immunotherapy prevented distant metastases in the lung when primary tumors of a melanoma B16 line overexpressing CC chemokine receptor-7, or the breast cancer cell line 4T1, were irradiated with 2×12Gy followed by adenoviral transduction with LIGHT, a TNF superfamily member, which enhances host immune responses (69). However, the systemic potential of radiation was much clearer when DC were stimulated by a growth factor or an ICB antibody was added (26, 105, 117). An early study achieved 60% long-term survival in a metastatic Lewis lung tumor model by irradiating the primary tumor with a single, very high dose of 60Gy combined with the DC growth factor Flt-3 ligand (Ftl3-L) given for 10days beginning 1day after irradiation (117). Significant growth retardation was also obtained in a mammary tumor model after irradiation of one of the two tumors with a moderate dose of only 2Gy combined with Flt3-L (26). In metastatic mammary tumors, the number of lung metastases was reduced in a CD8+-dependent fashion after 12Gy followed by CTLA-4 blockade (104). Another study compared different fractionation schemes in combination with CTLA-4 blockade in irradiated primary and unirradiated secondary tumors (105). The growth delay in secondary tumors was larger for 3×8Gy, intermediate for 5×6Gy, and smallest for 1×20Gy. For 3×8Gy, delaying the CTLA-4 antibody until 4days after the first fraction (2days after the last fraction) reduced the abscopal effect. The alternative approach of combining radiation with a PD-L1 checkpoint inhibitor was tested using two mouse mammary tumors irradiated with single doses of 12 or 20Gy combined with anti-PD-L1 every third day on days 0–9 (57). After regression of the primary tumor, rechallenge did not result in tumor growth, and furthermore, an abscopal effect on growth delay was seen in unirradiated secondary tumors. Similarly, blocking PD-1 at the time of irradiation showed abscopal effects on the growth of unirradiated secondary tumors (melanoma and renal cell carcinoma) when the primary tumors were irradiated with single fractions of 15Gy (118). A recent study reported an anti-metastatic effect of radiation and anti-PD-L1 after ex vivo irradiation of tumor cells with 12Gy but because no primary tumor was irradiated, this experimental design detected tumor take and not an abscopal effect (109). An overview of preclinical studies on abscopal effects of irradiation is given in Table Table22.
Most studies found that immune effects of RT alone or in combination with ICB were dependent on CD8+ T cells (57, 69, 70, 94, 104, 106, 108). However, there is also evidence on an influence of NK cell (106, 108), though this has been less often tested and was not found in an earlier study (69). The role of CD4+ T cells is more ambiguous with little or even a negative influence in most studies (104, 106, 108), while an important role was reported in a glioma model (119). This variation may be explained by the fact that CD4+ represents not only tumor-reactive Th cells but also Treg cells. Since the latter constitutes a significant but variable fraction, the stimulating effect of Th and the inhibitory effect of Treg may frequently cancel each other. Although PD-L1 may enhance Treg, their number was not affected in the mammary tumors (57). Instead irradiation combined with anti-PD-L1 treatment was found to confer a delayed decrease in immunosuppressive MDSC mediated by TNF secreted by infiltrating Tc cells (57). Similarly, no change in the CD8+/Treg ratio but a late decrease in myeloid cell numbers was observed in PDAC tumors after irradiation with a single dose of 12Gy combined with PD-L1 blockade (109).
In accordance with the stimulating effect of Flt3-L on antigen presentation and the effect of CTLA-4 inhibition on Tc activation and Treg downregulation, these agents were effective when applied concurrently with and immediately after irradiation though full abscopal effects were only manifested several weeks later. Since blocking the PD-1/PD-L1 checkpoint is considered to prevent the exhaustion of cytotoxic Tc lymphocytes infiltrating the tumor in the efferent phase, one might expect a synergistic effect by applying radiation and anti-PD-1/PD-L1 antibody sequentially. However, delaying the beginning of PD-L1 blockade until 6days after irradiation abrogated the synergistic immune effect on irradiated tumors (109). Since four anti-PD-L1 treatments were given in 10days, this seems to imply that irradiation acts on the tumor microenvironment before modulation by ICB, while ICB acts on the inflammatory microenvironment induced by irradiation. This suggests that although the PD-L1/PD-1 checkpoint is considered to be effective mainly in the efferent pathway of the adaptive immune response (120), it may be more important in the afferent pathway (activation and antigen presentation) after irradiation than previously thought. If this finding is confirmed in other systems, it would provide a strong argument for starting ICB immediately after irradiation (which is supported by initial clinical data, see below).
The success of ICB antibodies in preclinical and early clinical trials has prompted a large number of clinical trials applying different ICB antibodies with radiotherapy in different schedules and tumor sites [reviewed in Ref. (121)].
With the discovery of a lymphatic vessel system in the CNS (122), and the knowledge that antigen presentation to T cells occurs in the (deep) cervical lymph nodes (123), it is becoming clear that the immune system of the brain communicates with its systemic counterpart (124). In fact the traditional concept of CNS immune privilege no longer seems appropriate (124, 125). Microglial cells representing CNS innate immune cells perform many functions similar to macrophages, including recognition of DAMP, while DC appear to be important for antigen presentation in the cervical lymph nodes (125). Thus, the general model of immune response and immunosuppression also applies to tumors located in the brain (126).
A series of articles by Lim and colleagues examined the interaction between stereotactic irradiation with a single dose of 10Gy and different ICB antibodies in an intracranial glioma model using a small-animal irradiator. Anti-PD-1 antibody given three times in 4days beginning the day of irradiation produced significant survival at 3months in approximately 28% of the animals (127). Challenging the survivors with glioma cells in the flank demonstrated adaptive immune memory. Triple treatment with a CD137 agonist, an anti-CTLA-4 antibody, and radiation resulted in 50% long-term survival (119). Omitting the CD137 agonist yielded approximately 20% survival for concurrent treatment starting before or on the day of irradiation but only 10% when CTLA-4 inhibition was started 2days after irradiation. Survivors after triple treatment also produced a memory response. A different triple treatment combining anti-TIM-3 and anti-PD-1 ICB antibodies with irradiation achieved 60% survival (128).
These preclinical data are in line with a number of clinical studies that suggested considerably improved overall survival rates by adding the antibody ipilimumab (IPI, anti-CTLA-4) to SRS (30–33, 129–131) (Table (Table3).3). In two of the studies, a median number of two BM was present (32, 131), but generally the number and size of metastases varied over a wide range. In some of the studies, information on prescription dose and fractionation was missing or incomplete but the treatment of individual BM with a single fraction of 20–21Gy (median dose) appeared to be common (129, 131). However, doses and the number of fractions to individual BM varied: 14–24Gy and 1–5 fractions (31), 15–20Gy (129), 15–24Gy in a single fraction (131), or 15–21Gy with 16/20 patients receiving a single fraction and 3–5 fractions given to the last four (33). These early studies used retrospective or prospective series of patients, the sequence of IPI and SRS varied greatly, which may have contribute to the variable outcome, and frequently little detail was given regarding timing. Thus, clearly prospective studies with defined protocols are needed. Nevertheless, some of the studies seem to support the preclinical results that this ICB antibody shows better efficacy when given concurrently or immediately after SRS compared to delayed treatment although differences may exist between the irradiated metastases and abscopal effects on out-of-field disease (31, 33, 129, 131). However, although one trial included four patients who underwent prior resection of metastases before SRS to the cavity plus IPI (131), none have a priori addressed therapy of a purely resected population. Combining SRS with an anti-PD-1 antibody (nivolumab) has only been described in a single study on 73 lesions in 26 patients with median 9.4months follow-up (132), including patients with resected lesions. Overall, local control (82% at 12months) was comparable to conventional treatments, while distant control (53%) was higher than for other treatments. Interestingly, seven patients with resected BM appeared to have superior overall survival with five patients surviving after 24months.
Although the application of radiotherapy during surgery to inactivate any malignant cells remaining after tumor excision is not a new concept, IORT has only become a practical option during the last decade owing to the development of novel, dedicated machines. Thus, mobile linear accelerators producing high-energy electrons, or miniature X-ray machines emitting LEX allow irradiation of the tumor bed in the operating room with minimal radiation protection issues directly after the tumor has been removed (133–135). Different dose distributions can be achieved using special applicators in combination with the type and energy of the beam (136–138). However, IORT differs from conventional adjuvant RT in several aspects that may potentially influence the biological effect [reviewed in Ref. (20, 21)].
Intraoperative radiotherapy is given as a single fraction during surgery, whereas fractionated RT has been the established procedure for decades, applying daily fractions of typically 1.8–2.0Gy. Thus, IORT eliminates the time of some weeks required for wound healing between surgery and the beginning of RT during which residual cancer stem cells may proliferate and increase the number of recurrence-forming cells that need to be inactivated, or possibly spread by migration out of the tumor bed and thus escape focused SRS (139). SRS represents an intermediate between the two since it is usually applied as a single, large-dose fraction a few weeks after surgery. When comparing the biological effects of IORT and conventionally fractionated RT, the radiation quality, distribution of dose, and dose rate must be considered. High-energy electrons show a relative biologic effectiveness (RBE) similar to that of high-energy X-rays (20) and produce a relatively uniform dose distribution at dose rates of 1–5Gy/min. IORT with LEX involves increased RBE values, a non-uniform dose distribution with a steep radial dose gradient, and protracted irradiation with reduced dose rates allowing the repair of sublethal damage during irradiation. The biological implications of these characteristics have been studied by radiobiological modeling and experimental measurements (140–142). Adverse reactions of the normal, healthy tissue are limited to a small volume around the applicator, while the risk of recurrence is predicted to be similar to that of conventional external beam radiotherapy within a spherical shell, the “sphere of equivalence,” thus defining a new target volume for tumor bed irradiation with LEX (140–145).
The treatment of solitary BM by excision and IORT in 23 patients using 50kV X-rays at a dose of 14Gy in 2mm depth yielded a disease-specific outcome at 5-year follow-up that was comparable to other modalities (18). In a large retrospective study from the same institution, localized RT versus WBRT alone or in combination was compared in 212 patients including 37 patients treated with SRS only and 19 patients treated with IORT only (146). The results indicated a slightly higher local recurrence rate for SRS/IORT, though this was not significant (P=0.27). Rates of distant intracranial recurrences were higher than for local recurrences in both groups (WBRT and SRS/IORT) and were significantly higher after SRS/IORT compared with WBRT (P<0.001). In spite of this, overall survival was comparable in the two groups and perhaps even marginally higher for SRS/IORT (P=0.27). These results emphasize that distant recurrence is an issue when treating single lesions, especially with adjuvant localized RT although it may not directly affect overall survival.
At present, no studies combining IORT with ICB have been published. However, IORT differs from single fraction SRS by eliminating the delay between tumor excision and postoperative SRS. Thus, residual tumor cells are irradiated before they can be stimulated by factors released during the wound-healing process. Another important aspect is that the primary tumor is not irradiated but only the tumor bed, consisting mainly of normal brain tissue with an unknown, presumably low number of residual tumor cells. This poses the question whether the radiation-induced immune activity will suffice to elicit a tumor-directed immune response on which an interaction with ICB may be based. In the following, key points relevant to the potential use of ICB in combination with IORT for BM are discussed.
Brain metastases have a high likelihood of local recurrence after resection, but at present, there is no standard radiotherapy technique to boost the surgical cavity. Thus, SRS to a narrow high-dose volume (e.g., by focusing different beam angles and/or by modulating the beam intensities) with Gammaknife or Cyberknife, or a linear accelerator are being used. An intraoperative boost of IORT appears a promising alternative, which does not require irradiating large volumes of healthy tissues or organs and which would eliminate the time required for wound healing (typically 2–4weeks) before SRS is initiated. For both modalities, high single doses may elicit immunological effects that can reach beyond the tumor bed. A review of the mechanisms of radiation-induced immune reactions supports a model in which doses >~8Gy may act as an adjuvant for antitumor immune reactions present before irradiation or enhanced by the release of tumor antigens from irradiated residual cancer cells in the tumor bed and possibly by immunogenic cancer cell death elsewhere. The efficacy of an immune response is supported by retrospective studies on SRS for (mainly) unresected BM combined with ICB antibodies (mostly IPI), suggesting that the antibody must be present at the time of and immediately after irradiation. Recent data on a small number of patients with resected BM indicate that SRS in combination with ICB antibodies, and in particular anti-PD-1, might increase overall survival in these patients, thus supporting the rationale for combining IORT with ICB for resected BM. Since IORT limits the dose to a small volume of normal brain tissue, one might even hypothesize that this approach would not preclude adding SRS in the case of oligometastases. Although these effects need to be more comprehensively understood, a combination therapy of very large dose fractions with ICB antibodies appears to be specifically synergistic, warranting further prospective clinical evaluation.
CH performed the literature search, wrote the manuscript, and drafted the figures. FW included clinical aspects and suggested literature. FG performed the clinical literature search and wrote the manuscript. All authors conceived of the aim of the review and read the final manuscript.
Carl Zeiss Meditec AG, Jena, Germany, and Elekta, Crawley, UK, support training and radiobiological research at Universitaetsmedizin Mannheim. FG serves as a consultant and speaker for Carl Zeiss Meditec AG, NOXXON Pharma AG, Merck Serono GmbH, Roche Pharma AG, and Siemens Healthcare Diagnostics GmbH and holds patents related with Carl Zeiss Meditec AG. All other authors declare no conflict of interest.
The authors acknowledge the financial support of the Deutsche Forschungsgemeinschaft and Ruprecht-Karls-Universität Heidelberg within the funding programme Open Access Publishing. We thank Mr. A. Yazgan for expert graphical assistance.