RADIATION RESEARCH

182, 259–272 (2014)

0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13740.1

REVIEW Radiation Biology in the Context of Changing Patterns of Radiotherapy David Murray,a,1 William H. McBrideb and Jeffrey L. Schwartzc a Department of Oncology, Division of Experimental Oncology, University of Alberta, Edmonton, Alberta, Canada; b Department of Radiation Oncology, David Geffen School of Medicine at UCLA, Los Angeles, California; and c Department of Radiation Oncology, University of Washington, Seattle, Washington

fractionation were enshrined by Rod Withers (1) in ‘‘The Four R’s of Radiotherapy’’, namely: repair, repopulation, redistribution and reoxygenation. Repair here refers to the sparing effect when the radiation dose is delivered as a series of fractions over a period of time. The total dose needed to achieve the same (iso-)effect as the fraction size changes is typically modeled using the Linear-Quadratic (LQ) equation, which usefully fits responses around the 2 Gy dose fractions used in ‘‘conventional’’ RT. The a/b ratio is the most useful clinical parameter. The dogma is that the preferential repair/sparing of complications in late responding relative to early responding normal tissues is associated with a low a/b ratio (2). Tumors typically behave like earlyresponding normal tissues with high a/b ratios, with some slow growing exceptions such as prostate, melanoma, soft tissue sarcoma, liposarcoma and possibly breast where the differential between tumor and surrounding normal tissues might be too small to yield much advantage to fractionation. The LQ philosophy through about the year 2000 led clinicians to be aware of the problem of complications in late-responding normal tissues, to a desire for field homogeneity, and to the use of low fraction size to magnify the therapeutic index. The number of fractions was limited by time in the form of the threat of accelerated tumor repopulation. Trials of hyper- and accelerated fractionation and combinations thereof were initiated to test the hypothesis, with results generally in accord with modeling predictions (3). Increases in early normal tissue complications were predicted in hyperfractionated and overly aggressive accelerated regimens, and this proved to be the case (4). The advent of precision inverse-planned tumortargeting RT techniques, allowing a sharp dose fall-off in normal tissues combined with improved imaging to minimize margins, especially when allied with recognition of the low a/b ratios in some cancers, caused a resurgence of interest in the use of ‘‘moderate’’ hypofractionation [e.g., 3–7 Gy in 20 to 8 fractions (5)]. Fewer sessions and a decreased overall treatment time had obvious advantages for patients and clinicians. Some concern was expressed about possible late complications and inflammation at high doses

Murray, D., McBride, W. H. and Schwartz, J. L. Radiation Biology in the Context of Changing Patterns of Radiotherapy. Radiat. Res. 182, 259–272 (2014).

The last decade has witnessed a revolution in the clinical application of high-dose ‘‘ablative’’ radiation therapy. Initially this approach was limited to the treatment of brain tumors, but more recently we have seen its successful extension to tumors outside the brain, e.g., for small lung nodules. These advances have been driven largely by improvements in image-guided inverse treatment planning that allow the dose per fraction to the tumor to be increased over the conventional 2 Gy dose while keeping the late normal tissue complications at an acceptable level by dose limitation. Despite initial concerns about excessive late complications, as might be expected based on dose extrapolations using the linear-quadratic equation, these approaches have shown considerable clinical promise. Our knowledge of the biological consequences of high-doses of ionizing radiation in normal and cancerous tissues has lagged behind these clinical advances. Our intent here is to survey recent experimental findings from the perspective of better understanding the biological effects of high-dose therapy and whether they are truly different from conventional doses. We will also consider the implications of this knowledge for further refining and improving these approaches on the basis of underlying mechanisms. Ó 2014 by Radiation Research Society

BACKGROUND: CHANGING PATTERNS OF RADIATION THERAPY

The last decade has witnessed major advances in the clinical practice of radiation therapy (RT). Early clinical and laboratory studies led to a straightforward set of radiobiological guidelines for the use of conventional fractionated RT to treat human cancer. The advantages of dose 1 Address for correspondence: Department of Oncology, Division of Experimental Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada; e-mail: [email protected].

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per fraction, but moderate hypofractionation has generally proven efficacious for sites such as prostate and breast (6). The optimal size of the dose per fraction in such regimens is not precisely known. The refinement of modern precision techniques such as intensity-modulated radiation therapy (IMRT) and imageguided radiation therapy (IGRT) enabled oncologists to further increase the dose per fraction and reduce the number of fractions. By analogy with the single-dose gamma knife stereotactic radiosurgery (SRS) for brain lesions introduced by Leksell in the 1980s (7), the technique became known as either stereotactic body radiation therapy (SBRT) or stereotactic ablative radiotherapy (SABR); examples include the 18 Gy 3 3 protocol used in the RTOG 0236 trial for inoperable stage I non-small cell lung cancer (NSCLC) (8) or the 12 Gy 3 4, 10 Gy 3 5 or 34 Gy 3 1 protocols used in some clinics (9). Clinical experience with SBRT and single-dose radiation therapy (SDRT) suggests improved therapeutic ratios for some tumor sites. There is an ongoing debate as to the limitations of the LQ model for predicting the biological effectiveness of SBRT (10, 6), principally because: (i) the LQ model may overestimate radiation cell killing after high-dose fractions as it fits a continuously downward-bending survival curve (11); and (ii) the biology underlying low-dose versus highdose fractionated exposures may differ substantially, as will be discussed later. Although the LQ-derived biologically equivalent dose (BED) can be used in a relative sense to compare dose schedules in some tumor sites such as NSCLC (6), this might not be the case for other cancers (12). This mathematical modeling controversy is not the purview of the current article. Rather, our focus is on how radiobiological effects change with low to moderate to high fraction sizes, knowledge that will be essential for improving RT in the long term. INTRACELLULAR OR ‘‘CELL-AUTONOMOUS’’ RESPONSES TO DNA DAMAGE: INFORMATION FLOW IN RESPONSE TO DNA DOUBLE-STRAND BREAKS (DSBS)

The anticancer activity of RT is ascribed largely to free radicals damaging tumor cell DNA, although reactive oxygen species (ROS) generated by other pathways can lead to the activation of redox-sensitive pathways with multiple consequences, including cell death. The major signaling response expressed after irradiation is the highly coordinated DNA damage response (DDR). ATM/p53 proteins are key participants in the DDR as master regulators of both pro-survival and pro-death responses. The DDR is responsible for cell cycle checkpoint activation that leads to transient arrest in damaged cells, enabling time for decisions on DNA repair, cell survival or elimination of cells with high levels of genomic injury. Failure to properly activate any aspect of the DDR can lead to genomic

instability, a risk factor for carcinogenesis and for the development of tumor resistance to therapy. The most biologically significant DNA lesions induced by ionizing radiation are DSBs. DSBs are repaired by nonhomologous end joining (NHEJ) and homologous recombination repair (HRR) processes (13). The proper repair of a DSB involves the spatiotemporal coordination of a large number of proteins involved in functions that range from the initial recognition of the DSB lesion through to the final chromatin reorganization and reassembly that occurs when repair is completed. An obvious question with respect to nonconventional dose fractions is whether higher radiation doses might differentially affect DSB formation or DDR pathways. Although this phenomenon has not been studied systematically, there is some evidence that DSB repair may be less efficient after high single doses, at least in some cell lines [e.g., see ref. (14)]. TISSUE REPAIR AND THE IRRADIATED MICROENVIRONMENT

The effects of DDRs are not limited to an individual cell. DDRs integrate with other cellular processes to optimize tissue repair/wound healing while minimizing genomic instability and carcinogenesis. In recent years, multiple radiation-induced responses have emerged as key determinants of in vivo responses to RT. One of these is a downstream facet of the DDR: the DNA-damage secretory program or ‘‘DDSP’’. The challenge now is to understand the impact of these events in the context of the tumor and tissue milieu and the implications for therapeutic gain and modulation, as outlined in an excellent review of this subject (15). The tumor microenvironment is home not only to cancer cells but also to many types of normal cells such as vascular endothelial cells (VECs) and fibroblasts as well as to resident and infiltrating inflammatory cells. Tumor cell products are thought in most cases to dictate the nature of infiltrating host cells and the interactive tumor microenvironment. Molecules associated with ‘‘damage-associated molecular patterns’’ or ‘‘DAMPs’’ (16, 17) (nucleosides, cytokines, chemokines, adhesion molecules, growth factors, proteases, etc.) act locally and systemically to create the tumor microenvironment. RT will damage all cell types, with prolonged courses affecting infiltrating cells more than short courses. Expression of numerous cell adhesion molecules is upregulated in normal tissues after radiation exposure so that the site can be recognized immunologically, tipping the immune balance towards inflammatory and subsequent adaptive immune responses. In an evolutionary context, this can be regarded as preparing the tissue for pathogen elimination and includes considerable self-inflicted cell death. In time, this segues into cell reprogramming with increasing ‘‘stemness’’, angiogenesis, proliferation and wound healing.

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FIG. 1. Key features of the DNA damage response (DDR) after exposure to a single dose of ionizing radiation. The indicated chain of events is triggered by radiation-induced damage to a cell, notably genomic DNA double-strand breaks (DSBs). The sequential recognition and processing of the DSB involves sensor, mediator, transducer and effector proteins. Whether or not the cell survives will depend on the balance between pro-survival (transient cell cycle checkpoint activation, DNA repair) and pro-death (e.g., apoptosis) or stressinduced premature senescence (SIPS) responses. Cellular damage also results in the secretion of diverse factors including cytokines, growth factors and proteases, a response known collectively as the DNA-damage secretory program or ‘‘DDSP’’. In the subset of senescent cells, this secretory response has been termed the ‘‘senescenceassociated secretory phenotype’’ or ‘‘SASP’’.

These events are summarized in Fig. 1. A major question is how the irradiated tumor microenvironment responds to RT, where tumor-specific products, hypoxia, deranged vasculature and altered redox status, as well as immunosuppressive cells and their products, might have a major impact and even enhance tumor cell proliferation (repopulation) and aggressiveness. Since many of these secreted factors are known modulators of cellular sensitivity or resistance to RT and are disseminated outside the radiation field regionally and systemically, understanding the irradiated normal and tumor microenvironments are key to better understanding the biology of differing fraction sizes and their therapeutic exploitation. It is important to recognize that the LQ model ignores most of this complex underlying biology and just because a model ‘‘fits’’ data, it does not imply biological mechanism, nor should we assume that it will fit all doses and responses for all tumors and tumor types. The LQ model, as it is used in RT, assumes that equal dose fractions have the same effect. For conventional RT, hypofractionation and SBRT, each dose of radiation

subsequent to the first fraction is delivered into a dynamic background that changes as therapy progresses. The types of changes observed will depend on the size, number and timing of the fractions. This complexity involves many local and systemic influences with different triggering thresholds and may in part be reflected in the four R’s of RT.’’ Even the four R’s are at odds with the assumption that there is an equal effect per fraction. There is an implicit expectation of nonlinearity with dose. CELL DEATH IN TUMOR AND NORMAL TISSUE RESPONSES TO RADIATION THERAPY

Changing the size of the radiation dose is likely to modify the rate of radiation-induced cell death and whether death is by apoptosis, autophagy, mitotic catastrophe, necrosis or a hybrid form of these. This is in part because different cell types sense the extent to which they are damaged in different ways and can vary in their preferred response pathway. Mode of cell death might depend on what pathways are activated or blocked. For example cells that

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have a functional loss of p53 tend not to undergo apoptosis in the same way as their wild-type counterparts. Apoptosis in its purest form follows either the intrinsic (mitochondrial) pathway mediated by DNA damage or the extrinsic pathway mediated by membrane death receptors of the tumor necrosis factor receptor (TNFR) family (18, 19), although these pathways do crosstalk. Alternative pathways act through acid sphingomyelinase (ASMase)/ceramide or direct mitochondrial damage (20, 21). ASMase and TNFRdependent pathways may be particularly important for the induction of vasculature damage, and both require fairly high doses of radiation on the order of 7–10 Gy to be induced. At least in some cells, high radiation doses can change the form of cell death from, for example, mitotic to apoptotic death (22). Autophagy is a cell survival response to stress that involves internal self-digestion of organelles (23). It may become hyperactivated by many signals, including RT and ultimately lead to cell death (24). Higher doses of radiation seem to promote autophagy in some cell types at the expense of mitotic death (25). Mitotic catastrophe, the ‘‘classic’’ form of cell death after irradiation, was originally described by Puck and Marcus. It is a failure to undergo proper mitosis after DNA injury (26) and it is the primary mode of cell death in tumor cells that lack functional p53 (24, 26). In certain cell types, mitotic catastrophe ultimately leads to apoptotic, necrotic or other modes of cell death (27, 28). Puck and Marcus (29) also showed that multinucleated/polyploid giant cells may be generated during mitotic catastrophe and that following doses less than 8 Gy, cells could go through several cell division cycles. This mechanism of escape from mitotic death may therefore be more important after low-dose versus high-dose fractions. Necrosis has historically been regarded as the type of cell death seen in pathological specimens in vivo. It is considered to be a passive process, but recent observations suggest that it might have a programmed component as well (30). Radiation-induced programmed necrosis has been reported in some thyroid and adrenocortical carcinoma cell lines (31), a process that is sometimes referred to as ‘‘necroptosis’’ because it has some mechanistic overlap with apoptosis (32). Since increased membrane permeability is a feature of high-dose-irradiated cells, one might expect higher doses to be superior at inducing necrosis. In fact, it is commonly thought that necrosis requires very high/ supralethal doses of radiation (e.g., ;30 Gy) whereas apoptosis can be triggered by quite low doses (33). Stress-induced premature senescence (SIPS), which is sometimes referred to as permanent growth arrest or therapy-induced cellular senescence, results in cells remaining viable for extended periods without undergoing cell division. SIPS is the favored response to in vitro irradiation of human fibroblasts and of many (but not all) wild-type p53 solid tumor-derived cell lines [(34) and references therein].

Except for a few reports in selected cell types, there is little systematic knowledge of the contribution of these various modes of cell death to the shapes of the doseresponse survival curves and of the effect of fractionation and fraction size thereon. Clonogenic assays focus on surviving (rather than dead) cells and the form of death is not considered, even though it might be critical with respect to the ‘‘danger’’ signals described above. In general, cells with a predisposition to die by early-onset apoptosis appear more radiosensitive, and if the apoptotic fraction is high there will be little shoulder on the survival curve. What is clear is that radiation-induced cell death is a pathological signal and that higher doses enhance expression of immunologically relevant molecules and immune responses, irrespective of the mode of cell death. Radiation-induced apoptosis, unlike natural programmed apoptosis, is not necessarily immunologically ‘‘silent’’ (24, 35). We will next consider SIPS in a little more detail because, unlike cells that undergo other modes of cell death, these cells remain metabolically active for relatively long times, so they could have continuing roles in determining fractionated RT outcomes. SIPS: KEY RADIOBIOLOGICAL ISSUES RELEVANT TO CANCER RADIATION THERAPY

An important question to address is whether and under what circumstances RT-induced SIPS promotes tumor control or leads to tumor recurrence. Early articles tended to focus on its contribution to loss of clonogenicity and thus to tumor control (36–39). However, SIPS is essentially a cytostatic mechanism that is likely to give a slow response to RT and there are some major unresolved issues. First, it remains to be seen whether SIPS is truly a state of ‘‘permanent’’ growth arrest or whether some cells might eventually reprogram to regenerate in vivo. There is some evidence for radiation-induced reprogramming of cells in vitro (40, 41). Thus, an initial robust SIPS response was seen in MCF-7 breast cancer cells after a 10 Gy exposure, but this was followed by the emergence of a proliferating cell population beginning at about 12 days (41). A similar pattern of SIPS followed by recovery was seen in MCF-7 cells after a fractionated (2 Gy 3 5) radiation exposure (42). There is convincing evidence for radiation induction of reprogramming of non-stem cells into a stem cell phenotype (43). Whether this reprogramming is SIPS-dependent is still not clear, but it raises concern as to whether this is a major cause of tumor recurrence in RT. Furthermore, if the reemergent clones have increased genomic instability, the result could be therapy resistance. Such an outcome has been suggested in a study of the effect of doxorubicin on HCT116 cells which resulted in SIPS in the majority of the cells but also led to the appearance of proliferating aneuploid ‘‘giant’’ cells (44), a phenomenon also seen in some other stem cell reprogramming studies (43). Several

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other reports suggest that some cancer cells might give rise to aneuploid progeny that can re-enter the cell-division cycle (45–47). How the long-term sustainability of the SIPS phenotype is regulated needs to be understood with some urgency. One demonstrated mechanism of escape involves deregulation of CDK1 and its downstream effectors (48, 40) and the emergence of classic pluripotent stem cell markers, such as Oct4, SOX2, Nanog and nestin, in irradiated cultures (43). Even if they undergo permanent arrest, both malignant and nonmalignant cells within the tumor microenvironment could represent an obstacle to therapeutic success because they will continue to have an active DDSP; this response has been coined the ‘‘senescence-associated secretory phenotype’’ (SASP) (49). SASP-secreted proteins can promote tumor growth and progression (50, 15) as well cellular reprogramming and epithelial-mesenchymal transition (EMT), which could further drive cancer progression (51–53). Activation of SASP could have a negative impact by the secretion of factors that evoke radioresistance in the tumor cells. For example, fibroblasts that undergo SIPS in response to radiation exposure were shown to promote the proliferation of and induce radioresistance in co-cultured breast cancer cells (54). One would expect radioresistant tumor-associated macrophages to be another powerful source of tumor growth-promoting cytokines and growth factors. Such macrophages accumulate in hypoxic areas created by radiation-damaged vasculature in some tumors (55). The precursors of these suppressor macrophages can be generated by RT and are a possible target for intervention (56). Recently, Wang et al. (57) reported that RT-induced tumor invasiveness was linked to SDF-1-regulated macrophage mobilization and vasculogenesis, and that in the irradiated microenvironment different subpopulations of tumor-associated macrophages inhabited different niches. It should be stressed that the nature of these vascular and macrophage changes are highly tumor-dependent. In light of what was discussed in the Tissue Repair and the Irradiate Microenvironment section for the more generalized DDSP response, along with the fact that during fractionated RT additional doses are being superimposed over a period of several weeks onto ongoing responses, it is reasonable to expect fraction sizes and numbers to influence these mechanisms. Simplistically, ablative RT given in one or a few fractions should generate less complex interactivity than conventional fractionated RT, whether this is correct should become evident as we learn more about the underlying biology of these ablative RT techniques. With respect to SIPS and clinical outcomes, the literature for RT is less mature than it is for chemotherapy, where SIPS in tumor tissue was identified as a possible negative prognostic factor in pilot studies of NSCLC (40) and mesothelioma (58). The presumption is that this is due to promotion of tumor recurrence. This knowledge gap for RT and particularly the effect of dose fraction size, needs to be addressed.

NONTARGETED TUMOR CELL KILLING BY RADIATION

Among the phenomena that do not fit so neatly into the direct cell kill model of radiation effects are: 1. Radiation-induced bystander effects, where cells that have not themselves been directly ‘‘hit’’ by an ionization track show evidence of DNA damage caused by irradiated cells that are in proximal communication with them (59). These effects are largely mediated by signaling pathways involving MAP kinase and NF-jB pathways, and are at least in part similar to those invoked by DDR/DDSP, inflammatory/immune cytokines and DAMPS acting through Toll-like receptor pathways (60, 61). These can be can be growth inhibitory or stimulatory (62, 63). 2. Immunological effects, in which local RT to the tumor and adjacent normal tissue generates ‘‘danger’’ signals (16, 17), including DDSP/SASP components (24, 62), which can generate a microenvironment that attracts and activates inflammatory and immune cells such as M1 macrophages and cytotoxic T lymphocytes (CTLs) (64, 15) that can kill cancer cells or damage the vasculature through antigen-specific or innate immune mechanisms. The extent to which this occurs relative to the regeneration of the original microenvironment with M2 growth-stimulatory macrophages and lymphocytes has yet to be determined. The generation of a dangerous microenvironment by radiation involves alteration of the expression profiles of tumor and normal cells so as to increase their immune recognition (27). In this microenvironment, maturation of dendritic cells and the presentation of processed antigen to specific T cells are promoted (10). The generation of such a microenvironment has the potential to impact both local tumor control and distant masses outside the radiation field (i.e., abscopal effects; see below). 3. Abscopal effects, in which localized RT to a cancer evokes a distal inhibitory response in tumors outside the radiation field. Abscopal effects have often been associated with stimulation of the immune system (65– 67), but they could also involve effects on tumor angiogenesis. 4. Vasculature-mediated effects, in which direct effects of RT on vascular components such as VECs have downstream consequences for the irradiated tumor and/or its microenvironment (see section Indirect Vascular-Mediated Effects on Tumor Control in Relationship to Dose and Fractionation). Some of these phenomena may be interrelated or at least involve common mechanisms. As will be seen below, there is some evidence to suggest that the success of high dose per fraction treatments may be due in part to these nontargeted or indirect radiation effects in addition to the classical radiobiological mechanisms of direct tumor cell

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killing. Such nontargeted and indirect effects of radiation do not necessarily show LQ dose-response characteristics. IMMUNE RESPONSES TO LOCALIZED TUMOR RADIATION THERAPY AND DOSE RELATIONSHIPS

FIG. 2. Dose responses for the induction of: (panel A) nuclear factor jB) DNA-binding activity in ECV304 endothelial-like human cells at 3 h after exposure to c rays. Significant increases (*) were seen only after 8 Gy (P , 0.01) and 20 Gy (P , 0.001) (73). Panel B: mRNAs encoding the proinflammatory cytokines tumor necrosis factor a (TNF-a), interleukin b (IL-1b), intercellular adhesion molecule 1 (ICAM-1) and interleukin a (IL-1a) in the cerebrum of C3Hf/Sed/Kam mice 4–8 h after exposure to X rays (74). Panel C: Antitumor-specific interferon-c-producing splenocyte levels in the B16-OVA/C57BL/6 mouse model, at 1 week after exposure to X rays. *Indicates P , 0.05 vs. 0 Gy from ref. (72). Panel B is redrawn and republished with permission and panel C is republished with

The relationship between local radiation exposures to a tumor and the host immune system is a complex one. It was shown many years ago that the dose needed to achieve 50% tumor control was significantly higher in mouse models that lack T cells, suggesting that active host antitumor immune responses can be an important contributor to tumor control and metastasis (68). There are also reports in some (but not all) model systems that local RT can act as an immune adjuvant and, through this mechanism, impact the growth of both the irradiated tumor and nonirradiated distant tumors (i.e., abscopal effects) (69, 70). An extensive amount of literature now exists on radiation and immune effects (71), a comprehensive review of which is beyond the scope of this article. Rather, we will focus here on selected studies related to the impact of dose and fractionation on such effects, what we might learn from these in the context of SBRT and what we need to know going forward. Although the activation of elements of antitumor immune responses by single-radiation exposures are loosely described as ‘‘dose-dependent,’’ there has been no real consensus as to the actual shape of the dose-response curve (71). In general, higher doses of radiation generate more danger signals and antitumor immune responses (72, 67), but the optimal size of dose per fraction in a clinical protocol still needs to be defined. Significant data suggest that proinflammatory and immune responses to ionizing radiation are nonlinear with dose and that there may be an important transition in the 7–8 Gy dose range. An example is shown in Fig. 2A for induction of the classic proinflammatory transcription factor NF-jB in ECV304 endothelial-like human cells (73). In this example, NF-jB activity was increased only after doses of 8 Gy and higher (indicated by the gray arrow), an observation that is consistent with a number of studies of NF-jB activation by radiation in other model systems [reviewed in ref. (73)]. As shown in Fig. 2B, the early transcription of NF-jBdependent genes that encode proinflammatory cytokines such as TNF-a after radiation exposure is also typically nonlinear, the example shown is for the C3H mouse brain (74). TNF-a, IL-1b and IL-1a transcript levels were increased only above 7 Gy (e.g., see gray arrow for TNFa) in a generally radiation dose-dependent manner, although ICAM-1 did appear to be induced after lower doses. In the context of cancer RT, these proinflammatory responses mark the tumor with danger signals that promote immune

permission, Schaue D, et al. Int J Radiat Oncol Biol Phys 2012; 83:1306–10 and Hong JH, et al. Int J Radiat Oncol Biol Phys 1995; 33:619–26, respectively.

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FIG. 3. Primary and abscopal tumor growth inhibition in TSA mammary tumor-bearing BALB/C mice exposed to single or fractionated doses of radiation (RT), with or without CTL activation with the 9H10 monoclonal antibody. RT (either 20 Gy 3 1, 8 Gy 3 3 or 6 Gy 3 5) was given to the primary tumor only. Bars indicate the weight of the primary (left panel) and secondary (right panel) tumors at day 35 post-inoculation. The fraction shown above each bar represents the number of mice in that group that showed complete tumor regression. Reproduced with permission, Dewan et al. (65) Clin Cancer Res 2009; 15:5379–88. (65).

recognition, triggering the translocation of CD8þ CTLs and other immune cells to the tumor microenvironment (64). An example of the dose dependency for a particular in vivo immune response to radiation-induced danger signals, the induction of antitumor-specific interferon-c-producing T cells in the spleen, is shown in Fig. 2C for the B16-OVA melanoma/C57BL/6 mouse model (which expresses the target antigen, ovalbumin) at 7 days post-RT (72). As indicated by the gray arrow, there was again a dose transition with only single doses of 7.5 Gy and above being immunostimulatory. The common observation of a dose threshold in the vicinity of 7–8 Gy for activation of various aspects of the inflammatory and immune responses after single doses of RT is consistent with radiation switching the tumor microenvironment to a proinflammatory state that is critical for generating antitumor immune responses, as shown above for the key regulator NF-jB (72). DOSE FRACTIONATION AND IMMUNE RESPONSES

Several research groups have attempted to compare the relative ability of single versus fractionated local doses of radiation to stimulate antitumor immune responses. Because the RT regimens used in these various studies rarely overlap in terms of doses and timing, we will frame this discussion in terms of 3 arbitrary windows: fractionated low doses 4 Gy (conventional/moderate hypofractionation); fractionated high doses 5 Gy (corresponding roughly to SBRT); and single high doses 15 Gy (SDRT). A study done by Dewan et al. (65) used two syngeneic mouse tumor models, TSA mammary carcinoma in BALB/ C hosts and MCA38 colon carcinoma in C57BL/6 hosts, both of which are weakly immunogenic, plus a CTL-

activating antibody 9H10 (which overcomes T-cell tolerance by blocking the CTLA-4 inhibitory receptor) to derepress antitumor immune responses. Each animal was set up with 2 tumors, one of which (the primary) was irradiated and the other (the secondary) was not irradiated, such that both in-field and out-of-field (i.e., abscopal) responses to therapy could be evaluated. Key findings were as follows: (i) 20 Gy 3 1, 8 Gy 3 3 and 6 Gy 3 5 inhibited growth of the primary tumor to a similar degree; (ii) all combination RT þ 9H10 treatments inhibited growth of the primary tumor more than RT alone. For fractionated RT, this enhancement was statistically significant and for SDRT it was not. The improvement from adding 9H10 was greater for the 8 Gy 3 3 (Fig. 3, left panel, gray arrow) than for the 6 Gy 3 5 group; (iii) as a single modality, neither fractionated RT nor SDRT evoked a significant abscopal effect; and (iv) for the combined RT þ 9H10 treatments, abscopal inhibition of the secondary tumor was most dramatic with 9H10 plus 8 Gy 3 3 (Fig. 3, right panel, gray arrow), followed by a modest effect for 9H10 plus 6 Gy 3 5, but there was little effect with the 9H10 þ SDRT combination. Thus, when combined with CTL activation, the 8 Gy 3 3 regimen was the most potent for inducing antitumor immunity. We should be cautious in over-interpreting these data because the numbers of animals used per group was quite small, but these findings do suggest feasibility for the concept of combining RT, especially fractionated RT with high doses per fraction, with immunologic modifying agents that can enhance the potency of tumor-specific Tcell responses and thereby increase antitumor activity (66). Schaue and colleagues (72), using the B16-OVA/C57BL/6 mouse model, reached a similar conclusion on the impact of dose fractionation on the levels of antitumor-specific

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interferon-c-producing T cells as well as immune-suppressive regulatory T cells (Tregs) in the spleen at 1 week postRT. A total dose of 15 Gy was given in 1, 2, 3 or 5 fractions. A key consideration in the study design and interpretation was that B16-OVA cells have a high a/b ratio of 36 Gy, and the interfraction interval was only 6 h, so there should have been little impact of repair or repopulation on tumor control. Key observations were: (i) all four RT regimens provided significant tumor growth inhibition (Fig. 4A); and (ii) each RT regimen increased OVA-specific tumor-reactive T-cell levels in the spleen (Fig. 4B). Although these effects were significant (P , 0.05) in comparison to nonirradiated controls, no statistical comparisons among the different fractionation regimens were made, nonetheless, the 7.5 Gy 3 2 and 5 Gy 3 3 regimens appeared somewhat more effective in generating immune responses than 15 Gy SDRT or 3 Gy 3 5. Thus, in this model, tumor control and splenic tumor-specific T-cell levels were greatest for the 7.5 Gy 3 2 regimen and levels of splenic Tregs were the lowest for this regimen, although not significantly so (Fig. 4C). Although the differential responses among the various RT groups were modest, fractionation did appear to be a factor with higher fraction sizes of ;7.5 Gy resulting in the best tumor control, apparently invoking the strongest tumor-reactive Tcell responses while not inducing Tregs. A study by Lee and colleagues (70) used B16 melanoma and its immunogenic derivative B16-SIY growing in wildtype C57BL/6 or nude (T-cell deficient) mice to compare the effects of SDRT and fractionated RT on primary tumor and immune response. Important findings were: (i) after 20 Gy SDRT, B16 tumors in wild-type mice showed a much stronger growth inhibition than the same tumors in nude mice; (ii) tumors in the wild-type hosts showed increased levels of infiltrating CTLs after local RT; and (iii) for B16 tumors in wild-type mice, the therapeutic effect of 15 Gy SDRT was largely reversed by post-RT administration of aCD8 antibody that depletes CD8þ CTLs. In this model, then, immunodeficiency was associated with a reversal of the primary antitumor effect of SDRT, and CD8 þ CTLs appeared to be a critical element of the therapeutic response. The mechanism of this effect in what is regarded as a poorly immunogenic model appears to involve the ability of ablative SDRT to change the status of the tumor microenvironment from immune suppressive to immune promoting, resulting in the activation and maturation of dendritic cells and the priming and expansion of effector T cells. The Lee et al. study (70) also showed that 20 Gy SDRT strongly inhibited the growth of B16-SIY melanoma tumors in wild-type mice (Fig. 5). As expected, this effect was reversed by anti-CD8 antibody. The effect was also reversed by dose fractionation, with the schedule of 5 Gy 3 4 delivered over 2 weeks causing only a small and transient delay in tumor growth. This may reflect a direct cytotoxic effect of the local RT on infiltrating CTLs during fractionation, although these regimens were designed to be isodose (20 Gy), rather than isoeffective; the impact of

FIG. 4. Impact of dose fractionation on the antitumor-specific immune response to X rays in the B16-OVA melanoma/C57BL/6 mouse model. Tumor size and splenic responses were measured at 7 days postirradiation. For each irradiated group the total dose was 15 Gy and the interfraction interval was 6 h. *Indicates a significant difference (P , 0.05) vs. 0 Gy. Panel A: Tumor size. Panel B: Number of antitumor-specific interferon-c (IFNc)-producing T cells in the spleen. Panel C: Number of CD4þ/CD25high/Foxp3þ regulatory T cells (Tregs) in the spleen. Republished with permission, Schaue D, et al. (72) Int J Radiat Oncol Biol Phys 2012; 83:1306–10.

repair and repopulation in the fractionated schedule is unknown but was considered to be small because delivering 3 additional fractions caused no improvement in tumor control. The authors concluded that activation of a strong antitumor immune response was probably the major determinant of the superiority of SDRT over fractionated RT and that at least part of the reason for this was that fractionation resulted in the suppression/loss of RT-initiated antitumor immunity. Thus, among several studies that used various murine model systems and fractionation schedules, the common finding is that localized RT to the tumor does evoke

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267 RELEVANCE OF STUDIES OF ANTITUMOR IMMUNITY IN MICE TO THE TREATMENT OF CANCER PATIENTS?

FIG. 5. Effect of X rays on the growth of the immunogenic B16SIY melanoma model in wild-type C57BL/6 mice. Tumors were either sham irradiated (0 Gy) or exposed to a single dose of 20 Gy or to 4 fractions of 5 Gy per fraction delivered over 2 weeks. The data represent the mean tumor volume 6 standard deviation at 20 days post-RT. The dashed line represents the average tumor size at 4 days post-RT. The right-hand bar shows the reversal of the growthinhibitory effect of the 20 Gy single dose after CTL depletion by postirradiation administration of aCD8 antibody. Created using data from Lee et al. 2009 (70).

antitumor immune responses that can contribute somewhat or greatly to the growth inhibition of the primary tumors and sometimes to tumors outside of the RT field. Whether there is an optimal size of dose/dose per fraction for RT to be effective as an immune adjuvant, as suggested in some studies, cannot be generalized at this point. Such discrepant findings are perhaps not surprising, given several considerations: (i) interesting observations have typically been reported using a single model system chosen on the basis of some favorable experimental characteristic; (ii) even the same model may not generate equivalent data when used in different laboratories; (iii) these diverse tumor models differ in key features, notably their immunogenicity in the particular host; and (iv) even in the relatively controlled scenario afforded by mouse models, local RT to a tumor can exert either immune-suppressive or -stimulatory responses depending on the model used (69, 70). A study by Lugade et al. (35) reminds us that another important factor may be the impact of the specific fractionation protocol (and particularly of one of the four R’s: repair) on direct tumor cell killing, which would in turn impact the extent of tumor antigen generation and uptake by antigen-presenting cells. In other words, the magnitude of these responses may largely track with the degree and type of tumor cell death. This line of inquiry clearly requires systematic study with defined models and end points.

Although studies on the impact of fraction size on antitumor immune responses are in their infancy, they clearly point to an intriguing story in terms of their clinical potential and the possibility of using immunotherapy in combination with RT. Clearly these studies highlight an important knowledge gap in radiobiology. A key question is whether local tumor RT of some optimal fraction size or schedule, in addition to its direct cytotoxic effect on the tumor, might act as an immune adjuvant in the clinic by creating an immunologically permissive environment that can increase the likelihood of controlling local and/or disseminated cancer (75, 17). Tumor-specific CD8þ T-cell immune responses have been reported in cancer patients who received conventional RT, so repeated 2 Gy fractions do seem to be active in this context (76), whether this immunity contributes to cure is still uncertain. There are issues with the extrapolation from animal tumor models, which are rarely rigorously tested for immunogenicity but which are considered in general to be more immunogenic than human tumors. Immunogenicity not only predisposes towards responsiveness but also alters the nature of the negative regulatory barriers that would have to be overcome to generate a significant immune response during RT (17). The dogma is that only a fraction of human cancers (perhaps ;15%) are sufficiently immunogenic to elicit immune responses, although this can be increased by the use of agents that remove immune checkpoint controls, such as CTLA4. Under conventional conditions, then, RT might fail to generate a clinically meaningful level of antitumor immunity. It could even be suppressive (69). One clinical opportunity based on the studies described above might be to combine immunomodulatory molecules with RT in general and with SBRT in particular to provide the signal strength necessary to generate antitumor T cells and recruit them to the tumor (77, 67, 35). One reason for anticipating a better result with SBRT is simply that, for fractionated RT, repeated damage to immune cells within the tumor microenvironment could limit the development of these immune responses (35). Such a strategy was seen in the preclinical study by Dewan et al. (65) outlined in the Dose Fractionation and Immune Responses section, in which the antibody 9H10 was used to potentiate immune responses by blocking the CTLA-4 inhibitory receptor on T cells. Indeed, the combination of RT with the anti-CTLA-4 antibody ipilimumab has recently been evaluated in a Phase I/II study in advanced prostate cancer patients and is now entering Phase III trials (77). The findings described in the Dose Fractionation and Immune Responses section, suggest that successfully combining immune modifiers with RT may be highly dependent on the specific RT regimen used. Indeed, an anecdotal report indicated a marked abscopal response in a melanoma patient treated with local RT (9.5

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Gy 3 3) and ipilimumab (78), a regimen similar to the above mentioned 8 Gy 3 3 plus antibody regimen reported by Dewan et al. (65) to be optimal in their mouse tumor models (71). Agents targeting PD1 and PDL1, the former expressed by T cells and the latter by many tumor cells, may be an even more effective strategy for alleviating immune checkpoint control. Many other immunomodulatory strategies are at various stages of development, but their discussion is beyond the scope of this review. INDIRECT VASCULAR-MEDIATED EFFECTS ON TUMOR CONTROL IN RELATIONSHIP TO DOSE AND FRACTIONATION

Another nontumor cell targeted mechanism that might differentially influence tumor control after conventional versus high doses per fraction RT involves damage to the tumor stroma. An important study on the role of tumor stroma in RT response (79) used MCA/129 fibrosarcoma and B16F1 melanoma tumors growing in wild-type C57BL/ 6 host mice or in their acid sphingomyelinase (ASMase)deficient counterparts. As noted in the Cell Death in Tumor and Normal Tissue Responses to Radiation Therapy section, ASMase is an enzyme that can trigger apoptosis in certain cell types. It should be emphasized that in these models the ASMase defect is restricted to the host and is selective to the VECs by virtue of their high ASMase levels. Key findings of this study (79) include: (i) whereas the growth of tumors in ASMaseþ/þ hosts after a single dose of 10 or 15 Gy was strongly inhibited, growth of the same tumors in ASMase–/– hosts continued unabated after these doses; (ii) VECs in tumor sections from ASMase þ/ þ mice that had been irradiated with 15 Gy in vivo showed much more extensive apoptosis than those from ASMase–/– mice at up to 6 h postRT as indicated by the TUNEL assay; and (iii) purified tumor-derived VECs from ASMaseþ/þ mice irradiated in vitro were also markedly more susceptible to apoptosis than those from ASMase–/– mice, indicating that this is a cellautonomous effect. Strikingly reminiscent of the data discussed in the Immune Responses to Localized Tumor Radiation Therapy and Dose Relationships section for activation of inflammatory and immune responses, there was a clear dose threshold for VEC apoptosis at 8 h postRT, with no response observed below ;11 Gy (Fig. 6). In these models, then, single doses of RT above ;8–10 Gy appeared to cause a wave of apoptosis of tumor endothelial cells peaking several hours after irradiation, followed by tumor regression over a period of days. Such doses are typical of those used in SBRT at many cancer centers, although a fractionated isoeffective dose was not used, so whether an accumulated fractionated dose would do the same is not known. In a TRAMP tumor model (55), 20 Gy 3 1 and 2 Gy 3 10 both caused vascular loss that did not seem strikingly different. Although the above findings were provocative, aspects of their interpretation have

FIG. 6. Dose response for apoptosis in purified tumor vascular endothelial cells isolated from MCA/129-bearing ASMaseþ/þ (black bars) and ASMase–/– (gray bars) mice, irradiated ex vivo and assayed at 8 h postirradiation. Apoptosis was assessed using the TUNEL assay. From Garcia-Barros et al. (79) Science 2003; 300(5622):1155–9.

proven controversial (80). The essence of the initial report (79) focused on the implication that apoptosis of VECs is the dominant factor in tumor response to high single doses of RT. The opposing perspective raised the issue of how these findings might be rationalized with the known major role for direct tumor cell killing in local control by RT. Recent data (81)2 suggest that these indirect effects on the tumor stroma can in fact be rationalized with classical tumor radiobiology. After high doses, ceramide-dependent early damage to the tumor microvasculature may actually be coupled to clonogenic ‘‘death’’ of irradiated tumor cells. The premise is that the early (within the first 30 min postRT) ceramide-driven vascular dysfunction, which is highly selective for ASMaseþ/þ hosts, leads to some form of hypoxia-reperfusion injury. An important downstream consequence is the suppression of DSB repair activity (specifically of the HRR pathway) in the irradiated tumor cells, but only in the ASMaseþ/þ background. These events should in turn lead to enhanced death of the irradiated tumor clonogenic cells in the ASMaseþ/þ hosts.2 It should be stressed that the effects of RT on tumor vasculature appear to depend on the tumor type, and more scenarios need to be examined before generalizations can be made. Also, rapidly growing murine tumors may be a poor model for human tumors and may have a more radiosensitive vasculature. When angiogenesis is blocked, vasculogenesis restores tumor growth and this is a less efficient process, as is seen in the phenomenon of the tumor bed effect where tumors 2 Kolesnick R, Haimovitz-Friedman A, Fuks Z. Coupling of endothelial dysfunction to tumor stem cell demise. Abstract S102. Proceedings of the 58th Annual Meeting of the Radiation Research Society; 2012 Sep 13–Oct 3; San Juan, Puerto Rico.

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grow slower in preirradiated sites. The dependence of these processes on fraction size needs further investigation. So why might vascular effects be differentially involved in tumor responses to conventional versus high-dose RT in animal models? Radiation effects on the vascular endothelium are clearly involved in the tumor response to conventional fractionated RT, as illustrated in mouse xenograft studies (82). However, the implications of these effects appear to be quite different than for SDRT. The indirect effect of vascular dysfunction on tumor cell radiosensitivity seen with SDRT might simply not occur with conventional fractionated RT schedules because the individual doses are too low to trigger such events, particularly if they are related to an underlying acute inflammatory response. Moeller and colleagues (82) showed that in response to a schedule of 3 Gy 3 3 delivered over 3 days tumor cells actually radioprotect their vascular endothelium by the generation of HIF-1 and the subsequent secretion of HIF-1-regulated radioprotective cytokines such as VEGF. Thus VEC damage can occur after fractionated low dose RT, but unlike after SDRT this might not enhance tumor cell death by inhibiting DSB repair, possibly because the signal is suppressed by the DDSP, and specifically by activation of tumor cell HIF-1 (83). Damage to the tumor vasculature may also have different consequences for SDRT versus fractionated RT because the latter might allow time for the replacement of VECs from circulating cells (84). SUMMARY AND FUTURE OPPORTUNITIES

Although the basic principles of the four R’s outlined by Withers (1) several decades ago may not be totally violated by SBRT or SDRT, it is clear that aspects of the radiobiology of large dose fractions are quite different from those of conventional dose fractions. We need to better understand the reasons for and implications of these differences to fully exploit or modify this biology for therapeutic advantage. A major challenge to translating this knowledge will be to ask relevant questions with appropriate biological models that apply to clinical practice with human patients. All of the above-mentioned studies of RT-induced immune effects used syngeneic mouse models of cancer. Demonstrating that similar principles apply to human tumors is an essential step, but conventional human tumor xenograft models would be of limited value because, by their very nature, they utilize immune-deficient mouse strains. However, there are potential ways to circumvent this problem. For example, Lee et al. (70) developed a partially immune-competent A549 human lung cancer/CTLþ xenograft model that recapitulates key aspects of the CTL response. This model allowed the authors to show that, as in their syngeneic mouse model described above, the response of a human tumor model to RT was also highly dependent

on the presence of an active CTL population. Thus, tumor growth was inhibited only when RT (20 Gy single dose) was administered in the presence of CD8þ T cells. These data therefore highlight the need to revisit earlier conclusions based on xenograft models that used only immunodeficient mice. Another issue is that preclinical studies relevant to the clinical practice of SDRT/SBRT should use dose-delivery capabilities analogous to those used in the clinic. To date, animal studies with ‘‘local’’ tumor RT, such as those described above, typically used crude shielding techniques and no imaging or treatment planning. To irradiate the whole tumor, which is critical for end points based on tumor growth or animal survival, broad radiation fields were typically used that subject unrealistically large volumes of normal tissue to high doses, thus evoking extensive and unrealistic DDSP/SASP, inflammatory, immune, vascular and wound-healing responses. Recently developed small animal IGRT platforms better simulate the clinical situation for in vivo research on such responses or on the combination of RT with modifiers such as immunotherapy (85) and offer great opportunity in this regard. In summary, many fascinating observations have been reported in individual models, but it remains unclear which of these reflect some common underlying mechanism as opposed to a model-specific effect. There are clearly some important differences in the biological response of tumors to low doses versus high doses per fraction. What is less clear is how much these differences contribute to RT outcomes. In regards to the question of whether there is a need to invoke new biology to explain the clinical effectiveness of SDRT/SBRT the answer appears to be both no and yes. Even if the LQ model does adequately model outcomes in some data sets involving the use of high doses per fraction (6) there is clearly some very exciting and novel radiobiology to understand and exploit, as noted previously (84). ACKNOWLEDGMENTS This work was supported by grants from the Canadian Breast Cancer Foundation-Prairies/NWT region and the Canadian Association of Radiation Oncology. Received: March 18, 2014; accepted: May 15, 2014; published online: July 16, 2014

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Radiation biology in the context of changing patterns of radiotherapy.

The last decade has witnessed a revolution in the clinical application of high-dose "ablative" radiation therapy. Initially this approach was limited ...
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