The Immunology of Ablative Radiation Byron Burnette, PhD, and Ralph R. Weichselbaum, MD Radiation has been a staple of cancer therapy since the early 20th century and is implemented in nearly half of current cancer treatment plans. Originally, the genotoxic function of radiation led to a focus on damage and repair pathways associated with deoxyribonucleic acid as important therapeutic targets to augment radiation efficacy. However, in recent decades, the participation of endogenous immune responses in modifying radiation effects have been widely documented and exploited in both preclinical and clinical settings. In particular, preclinical studies have highlighted the capacity of hypofractionated–radiation dose schedules to modify endogenous immune responses raising interest in the use of hypofractionation in the clinical setting to harness the indirect immune effects of radiation and improve clinical responses. We review the current literature regarding the immunomodulatory effects of hypofractionated “ablative” radiation with a primary focus on the preclinical literature but also highlight examples from the clinical literature. Semin Radiat Oncol 25:40-45 C 2015 Published by Elsevier Inc.
Introduction
R
adiation has been a staple of cancer therapy since the early 20th century and is implemented in nearly half of current cancer treatment plans. Originally, the genotoxic function of radiation led to a focus on damage and repair pathways associated with deoxyribonucleic acid as important therapeutic targets to augment radiation efficacy. However, in recent decades, the participation of endogenous immune responses in modifying radiation effects has been widely documented and exploited in both preclinical and clinical settings. In particular, an early study by Stone et al1 was the first to document the effect of immune status (sufficient or deficient due to experimental manipulation) on tumor dose responses in syngeneic mouse fibrosarcoma. This observation was largely ignored until substantial work during the last decade expanded our understanding of how the immune system, and in particular T lymphocytes (T cells), participate in the host response to tumor radiation.2-5 Given the well-known sensitivity of lymphocytes to radiation-mediated apoptosis and potential for unintended immunosuppression arising from suboptimal dose and fractionation schedules, application of local radiation in
Department of Radiation and Cellular Oncology, The Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL. The authors declare no conflict of interest. Address reprint requests to Ralph R. Weichselbaum Department of Radiation and Cellular Oncology, The Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL. E-mail: [email protected]
40
http://dx.doi.org/10.1016/j.semradonc.2014.07.009 1053-4296/& 2015 Published by Elsevier Inc.
hypofractionated ablative doses has been pursued as a means to capture both the direct cytotoxic and indirect immuneactivating effects of radiation. Regarding immunostimulation, there is still a paucity of data on the specific dose and fractionation schedules. The effects of dose and fractionation on tumor-specific immune responses have been reviewed elsewhere6 and are outside the scope of this review; however, we review some of the basic applications of stereotactic body radiation therapy (SBRT) and the preclinical data that support SBRT as an immune modifier. The reader is directed to reviews published in this issue and elsewhere for more comprehensive discussion of some of the basic immunologic mechanisms highlighted in this article.7-9 In addition, for the purposes of this review and for lack of sufficient preclinical and clinical data for a rigorous comparison, we draw little distinction between the varying hypofractionated or single high-dose radiation treatment approaches, such as intensity-modulated radiation therapy, image-guided radiation therapy, and SBRT, despite acknowledgment of the important differences in clinical application.
General Remarks About Preclinical Animal Models of SBRT Most of what we know about the immunomodulation of hypofractionated radiation doses (sometimes referred to “ablative” doses) characteristic of SBRT comes from preclinical animal models that use subcutaneous inoculation of tumor cell
The Immunology of Ablative Radiation lines in syngeneic mice. The strategic advantage of this experimental setup is the ability to achieve relatively complete systemic shielding of the animal with a stationary, single radiation source. The tumor is usually exposed to the beam path by passing the tissue (skin and enclosed tumor) through a narrow orifice in the shielding that is oriented perpendicular to the beam path, thus eliminating all but the small amount of side scatter that can pass into the directly adjacent normal tissue traversing the orifice. It is worth noting that this experimental system, while a close approximation, does not replicate the extent of normal tissue exposure that occurs during the execution of a typical multi-beam SBRT treatment plan. More importantly, considering the translational relevance of these studies, it is prudent to discuss the well-known, but rarely discussed, fact that inoculation of tumor cells lines in syngeneic animals results in the induction of innate and adaptive immune responses. For tumors that are capable of progressive growth in syngeneic animals, the immune response that is induced on inoculation fails to eliminate the deposited cancer cells, thereby resulting in rapid death of the host, usually within 3-6 weeks because of excessive tumor burden. The failure of the immune system to successfully combat the initial tumor challenge results from the formation of a well-documented immunosuppressive tumor microenvironment that thwarts immune-mediated attack during initial adaptation of the tumor cells to in vivo growth. This localized suppression is followed closely by systemic immune suppression accompanying progressive tumor growth. Studies published by Paul Ehrlich in 1906 and Ernest Bashford10 in 1908 demonstrated that the immune response generated from transplantable tumor challenge could successfully reject a second inoculum of the same tumor that was given within a short window following the primary inoculum. Rejection of the secondary challenge despite continued growth of the primary inoculum was a seemingly paradoxical observation that Bashford termed concomitant immunity and demonstrated the vaccine-like function of tumor challenge and the importance of local and systemic immune suppression in syngeneic “progressor” tumors. The time-dependent erosion of concomitant immunity with tumor progression was demonstrated to result from the induction of a systemic suppressor T-cell population11 that was later demonstrated to be regulatory T cells.12 Importantly, as noted by Vaage,13 “once immune resistance is evoked the resistance factors are never absent but may be depressed and not revealed, depending upon the conditions of the tests and strength of the antigens.” Experimental demonstration of the preservation of immune resistance despite systemic suppression comes from studies that “unmasked” antitumor immunity to established tumors by systemic11,12 or local14 depletion of regulatory T cells. Considering these longstanding historical observations, it is fair to say that experimental systems using transplantable tumor cell lines are probably a more appropriate model for how hypofractionated or ablative radiation might augment existing T-cell immunity rather than initiating (or priming) de novo T-cell responses. This conceptual framework bears relevance when
41 considering the widely popularized notion that local radiation of an established tumor can function as an in situ vaccine through the induction of immunogenic cell death and activation and maturation of antigen-presenting cells.7-9 In this review, we focus mainly on the experimental data regarding immunomodulation of ablative radiation in the setting of existing T-cell responses and discuss the potential application and relevance to several clinical treatment scenarios involving SBRT.
Effects of Ablative Radiation Within the Target Volume Direct Sensitization of Tumor Cells to T-cell–Mediated Killing To date, many studies have documented the myriad mechanisms through which ablative radiation doses can increase the sensitivity of tumor cells to direct T-cell–mediated killing. These mechanisms include upregulation of the antigenpresentation machinery through increased expression and cell surface localization of major histocompatibility complex proteins,3,15,16 increased expression of immunogenic tumor antigens,17,18 upregulation of T-cell co-activating ligands,19 and even sensitization of tumor cells to antigen-independent cell death through the Fas receptor.20 These and other mechanisms have been reviewed elsewhere.21 More importantly, an emerging trend in the most current research has been an understanding of the mechanisms through which the tumor stroma modulates ablative radiation and vice versa. The tumor stroma is a relatively loosely defined term that encompasses all cells within the tumor mass that are not neoplastic, including vascular endothelial cells, fibroblasts, hematopoietic cells of lymphoid and myeloid origin, and acellular components such as the extracellular matrix. Pioneering work by Hans Schreiber and Rolf Zinkernagel established the importance of the tumor stroma in immunologic rejection of immunogenic tumors, demonstrating that inoculation of tumor cells with tumor stroma could drastically enhance their tumorigenicity and in some cases facilitate the growth of tumors normally rejected as cell suspensions in syngeneic mice.22,23 Further studies by Spiotto and Schreiber25 demonstrated that presentation of tumor antigens by both cancer cells and the stroma was required for complete elimination of antigenic tumors and tumors harboring antigen-negative clones (antigen-loss variants).24 Given the importance of stromal antigen presentation, Zhang et al26 demonstrated that a single dose of 10 Gy of local radiation was sufficient to sensitize antigenic tumors to T-cell–mediated rejection through “loading” of the tumor stroma with tumor antigens. Studies from our group demonstrated a similar phenomenon, wherein local radiation of established B16 melanoma tumors with a single ablative dose of 20 Gy facilitated cross-presentation of tumor antigens by dendritic cells in the tumor stroma.27 From the standpoint of local radiation, it is unclear what pathways control the transfer of antigen from tumor cells to the
42 surrounding stroma but direct tumor cell death is implicated. Notably, deficiency in type I interferon receptor abrogated tumor antigen cross-presentation by stromal dendritic cells,27 and our group recently demonstrated that type I interferon is a critical mediator/sensitizer of tumor cells to initial cytotoxicity in response to local irradiation.28 Antigen transfer could sensitize protumorigenic stromal cells to T-cell–mediated killing, and this mechanism is supported by several studies that demonstrate T-cell–mediated elimination of critical protumorigenic and immunosuppressive stromal cell subtypes following local ablative radiation. First, a study by our group demonstrated that the combination of ablative radiotherapy and T-cell checkpoint blockade with anti–programmed death ligand-1 led to elimination of stromal myeloid-derived suppressor cells (MDSCs) through T-cell–derived production of tumor necrosis factor.29 A study by Wu et al30 made a similar observation regarding selective elimination of MDSCs using local ablative radiation with intratumoral injection of recombinant tumor-specific antigen to load the tumor stroma. Interestingly, local, but not systemic, injection of antigen facilitated a systemic vaccine response in addition to the effects on MDSCs. Novel strategies, including engineered bacterial delivery systems,31 have been successfully developed to facilitate stromal loading of tumor antigens to sensitize the stroma to T-cell–mediated recognition. Collectively, these studies demonstrate that there is a critical role for stromal antigen presentation following local ablative radiation in both altering the tumor microenvironment and likely promoting a systemic T-cell response. An additional consideration for the differential efficacy of ablative radiation compared with traditional fractionation is the potential role that direct cytotoxicity might play in the tumor stroma, which is by definition “normal” tissue embedded among neoplastic cells. Traditional fractionation schedules have been empirically developed to specifically limit normal tissue toxicity by exploiting the differential mitotic rate and deoxyribonucleic acid repair capacity of neoplastic and normal cells. Notably, fractionated radiation was developed to reduce toxicity that results from normal tissue effects outside of the intended target neoplastic tissue, whereas stereotactic ablative radiation virtually eliminates toxicity from normal tissue exposure and instead the only “normal” tissues targeted are the stroma and vasculature within the target volume. Given the critical role that the stroma plays in regulating the balance of protumor and antitumor influences, direct genotoxic or cytotoxic effects (or a combination of both) of local radiation on the tumor stroma should be considered. Almost nothing is known about the radiosensitivity of the hematopoietic cell types present in the tumor and how their response to radiation might differ from cells of comparable lineage isolated from non-neoplastic tissue. Studies from Bill McBride’s group suggest that exposure of lymphoid organs to fractionated radiation or scatter from local ablative radiation might increase the percentage of regulatory T cells in the periphery because of the relative radioresistance of regulatory T cells compared with conventional T cells.32,33 However, the relative abundance of naïve, and historically radiosensitive, T cells present in lymphoid organs is likely a key determinant
B. Burnette and R.R. Weichselbaum of the effect of radiation on the enrichment of regulatory T cells. An interesting observation that was made by Dunn and North34 several decades ago concerns the relatively rapid development of radioresistance among activated tumorspecific T cells and relative radiosensisitvity of suppressor T cells in the dose range of hypofractionated radiation.35 These studies suggest that effector T cells in the tumor microenvironment might not be rapidly eliminated by hypofractionated or ablative doses, which has been a reasonable concern for maximizing tumor-specific T-cell immunity through application of optimal dose and timing to avoid local T-cell depletion. Further studies on the sensitivity of tumor-infiltrating T cells are warranted to address these questions. A specific instance where there is some mechanistic basis for the differential effects of ablative vs fractionated doses on the tumor stroma is in the sensitivity of the tumor vasculature to radiation cytotoxicity. Studies by Garcia-Barros36 demonstrated that endothelial cell apoptosis within the tumor microenvironment is a rapid event that occurs in the first several hours after radiation exposure. Notably, tumors were treated with ablative doses of 15 Gy, known to induce surface translocation and enzymatic activity of acid sphingomyelinase, leading to the production and release of ceremide and consequently, the rapid apoptosis of endothelial cells (reviewed in Ref. 37). The vasculature is a widely recognized target in cancer therapy and emerging data suggest that T-cell–mediated effects on the tumor and stroma might be similarly linked to effects on the vessels, in part, through the production of cytokines.38,39
Effects of Ablative Radiation Outside the Target Volume Clinical Observations of Abscopal Responses The term “abscopal effect” was originally coined by Mole40 to describe the still poorly understood capacity of radiation to mediate effects on tumors outside the treatment field. A heavily favored theory suggests that the abscopal effect of radiation is mediated by augmented immune function, either cellular or cytokine mediated.41 Abscopal regression has been reported in a variety of tumor types treated with radiation as a single agent including lymphoma,42 papillary adenocarcinoma,43 melanoma,44,45 metastatic cervical carcinoma,46 and lung metastases from primary hepatocellular carcinoma.47,48 However, the report by Stamell et al45 was the only case where hypofractionated radiation was utilized, with the remaining cases using standard fractionation. Given the widely accepted notion that immunologic mechanisms underlie the abscopal effect, treatment combinations employing radiation and immunotherapy are predicted to increase the frequency of abscopal responses. Notably, 2 case reports of combination intensity-modulated radiation therapy and ipilimumab (anti–CTLA-4) have demonstrated abscopal effects in metastatic melanoma49 and non–small cell lung cancer.50 Furthermore, a phase I study of SBRT and high-dose IL -2 (a T-cell growth factor) in metastatic melanoma and
The Immunology of Ablative Radiation metastatic renal cell carcinoma demonstrated impressive abscopal responses in several patients.51 Finally, a small proof of a principle trial of hypofractionated radiation and systemic granulocyte-macrophage colony–stimulating factor administration in patients with tumors of mixed histology reported abscopal responses in 4 of the 12 patients enrolled.52 Lending credence to the role of immunity in the abscopal effect, Postow et al49 observed an increase in circulating antibodies to the tumor-associated antigen NY-ESO-1 (circulating antibodies are a surrogate for T-cell immunity, given the assumption that antibody production is T-cell-dependent in many cases), and Seung et al51 demonstrated elevated proliferation of circulating peripheral blood CD4þ T cells with an effector memory phenotype in patients who responded to treatment compared with nonresponders. Further investigation is warranted, but the results from these small trials employing radiation and immunotherapy are certainly promising.
Preclinical Models of Abscopal Response Following Ablative Radiation Data from preclinical models of radiation-mediated abscopal responses provide important information about the potential mechanism of the abscopal effect. One of the earliest studies by Camphausen et al53 demonstrated that the abscopal effect is dose-dependent using the Lewis lung carcinoma cell line. Notably, the abscopal effect of hypofractionated radiation (10 Gy 5) was more pronounced than that of traditional radiotherapy dose schedules (2 Gy 12), although a formal isodose was not determined for the small dose fractions. In addition, p53 was determined to be essential for the abscopal effect, as treatment with pifthrin-α eliminated the abscopal effect. Demaria et al2 used local radiation on transplantable mammary carcinoma cell lines with systemic treatment with Fms-like tyrosine kinase receptor 3 ligand (Flt3-L), a dendritic cell proliferation and maturation factor. The particular tumor cell line used exhibited marked sensitivity to radiation with a single dose of 2 Gy, producing significant growth delay. In these studies, local radiation alone failed to demonstrate an abscopal effect on a contralateral tumor of the same size, whereas combination therapy led to a significant growth delay of the contralateral tumor. Beyond the successful demonstration of abscopal regression, Demaria et al went on to demonstrate that the abscopal effect observed with local radiation and Fms-like tyrosine kinase receptor 3 ligand was both specific and T-cell-mediated through experiments where the abscopal effect was lost when the contralateral tumor was unrelated (antigenically different) or T cells were systemically depleted, respectively. A second study by Dewan et al54 demonstrated a similar abscopal response from combination therapy with radiation and CTLA-4–blocking antibodies in a contralateral flank trichostatin A murine breast cancer model. Interestingly, they reported that hypofractionation with a schedule of 8 Gy 3 yielded the most significant abscopal responses compared with a slightly different, 6 Gy 5, fractionation or 20-Gy ablative radiation, which correlated with increased infiltration and production of interferon gamma by CD8þ T cells. A study from our
43 laboratory using a contralateral murine flank model of Her2þ breast cancer also demonstrated significant abscopal effects with combined local radiation and T-cell checkpoint blockade with anti–programmed death ligand-1.29 A common feature of the experimental systems that model the abscopal effect is the empirical determination that the effect of radiation on the abscopal tumor, as with therapy to the primary tumor, depends heavily on the tumor size. Partly, this observation can be explained by a sheer numerical disadvantage of systemic immunity to increasing tumor burden. However, we offer another plausible hypothesis that the status of the tumor microenvironment dictates whether secondary tumors are amenable to abscopal effects. Similar to the susceptibility of recently injected tumors to concomitant immunity, tumor size can be thought of as a proxy for the establishment of an immunosuppressive stroma. Whether local therapy with ablative radiation mediates systemic effects through the reawakening of concomitant immune mechansims remains to be determined, but we suspect this to be the case. Regardless, defining the cellular and molecular mechanisms that dictate amenability of secondary lesions to abscopal effects is of great interest, especially in the clinical setting of multiplicity of lesions, wherein the physician must determine lesions warranting intervention with local therapy such as SBRT or other ablative radiation delivery techniques.
Concluding Remarks Clinical and experimental data summarized herein demonstrate the now well-established reciprocal capacity of immunity and radiation to augment each other. Optimal integration of radiation and immunotherapy is yet to be achieved and will depend on a concerted effort between scientists in clinical and basic research to address critical questions that will instruct the rationale and design of combination therapies. Direct tumor cell radiosensitivity is deeply entrenched in the historical application of radiation oncology, but how tumor cell radiosensitivity affects the immunologic aspects of radiation therapy is entirely unknown. Certainly, killing tumor cells is still the goal of therapeutic intervention, but it is conceivable that the magnitude of tumor cell death might be less important than the optimal engagement of immunity. Additionally, we suggest that consideration of the stromal component of the tumor as a therapeutic target is equally legitimate, given the critical role that the stroma plays in therapeutic susceptibility. Presently, the published data offer an assortment of single-dose and hypofractionation schemes without a clear mechanism for integration. Animal models offer appropriate and tractable systems to dissect these mechanisms, but a more thorough examination of the dose and timing of radiation, though scientifically less satisfying, should be integrated into the experimental plans to provide a firm base for the construction of clinical protocols.
References 1. Stone HB, Peters LJ, Milas L: Effect of host immune capability on radiocurability and subsequent transplantability of a murine fibrosarcoma. J Natl Cancer Inst 63:1229-1235, 1979
44 2. Demaria S, Ng B, Devitt ML: Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys 58:862-870, 2004 3. Lugade AA, Moran JP, Gerber SA: Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. J Immunol 174:7516-7523, 2005 4. Lee Y, Auh SL, Wang Y: Therapeutic effects of ablative radiation on local tumor require CD8þ T cells: Changing strategies for cancer treatment. Blood 114:589-595, 2009 5. Gough MJ, Crittenden MR, Sarff M: Adjuvant therapy with agonistic antibodies to CD134 (OX40) increases local control after surgical or radiation therapy of cancer in mice. J Immunother 33:798-809, 2010 6. Demaria S, Formenti SC: Radiation as an immunological adjuvant: Current evidence on dose and fractionation. Front Oncol 2:153, 2012 7. Formenti SC, Demaria S: Systemic effects of local radiotherapy. Lancet Oncol 10:718-726, 2009 8. Burnette B, Weichselbaum RR: Radiation as an immune modulator. Semin Radiat Oncol 23:273-280, 2013 9. Golden EB, Pellicciotta I, Demaria S: The convergence of radiation and immunogenic cell death signaling pathways. Front Oncol 2:88, 2012 10. Bashford EF, Russell B: Further evidence on the homogeneity of the resistance to the implantation of malignant new growths. Proceedings of the Royal Society of London Series B. Containing Papers of a Biological Character 298–306, 1910 11. DiGiacomo A, North RJ: T cell suppressors of antitumor immunity. The production of Ly-1-,2þ suppressors of delayed sensitivity precedes the production of suppressors of protective immunity. J Exp Med 164: 1179-1192, 1986 12. Turk MJ, Guevara-Patiño JA, Rizzuto GA: Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J Exp Med 200:771-782, 2004 13. Vaage J: Concomitant immunity and specific depression of immunity by residual or reinjected syngeneic tumor tissue. Cancer Res 31:1655-1662, 1971 14. Yu P, Lee Y, Liu W: Intratumor depletion of CD4þ cells unmasks tumor immunogenicity leading to the rejection of late-stage tumors. J Exp Med 201:779-791, 2005 15. Reits EA, Hodge JW, Herberts CA: Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J Exp Med 203:1259-1271, 2006 16. Wan S, Pestka S, Jubin RG: Chemotherapeutics and radiation stimulate MHC Class I expression through elevated interferon-beta signaling in breast cancer cells.. PLoS ONE 7(3):e32542, 2012 17. Santin AD, Hermonat PL, Ravaggi A: Radiation-enhanced expression of E6/E7 transforming oncogenes of human papillomavirus-16 in human cervical carcinoma. Cancer 83:2346-2352, 1998 18. Sharma A, Bode B, Wenger RH: γ-Radiation promotes immunological recognition of cancer cells through increased expression of cancer-testis antigens in vitro and in vivo. PLoS ONE 6(11):e28217, 2011 19. Gasser S, Orsulic S, Brown EJ: The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 436:1186-1190, 2005 20. Chakraborty M, Abrams SI, Coleman CN: External beam radiation of tumors alters phenotype of tumor cells to render them susceptible to vaccine-mediated T-cell killing. Cancer Res 64:4328-4337, 2004 21. Burnette B, Fu Y-X, Weichselbaum RR: The confluence of radiotherapy and immunotherapy. Front Oncol 2:143, 2012 22. Singh S, Ross SR, Acena M: Stroma is critical for preventing or permitting immunological destruction of antigenic cancer cells. J Exp Med 175: 139-146, 1992 23. Ochsenbein AF, Klenerman P, Karrer U: Immune surveillance against a solid tumor fails because of immunological ignorance. Proc Natl Acad Sci U S A 96:2233-2238, 1999 24. Spiotto MT, Rowley DA, Schreiber H: Bystander elimination of antigen loss variants in established tumors. Nat Med 10:294-298, 2004 25. Spiotto MT, Schreiber H: Rapid destruction of the tumor microenvironment by CTLs recognizing cancer-specific antigens cross-presented by stromal cells. Cancer Immun 5:8, 2005
B. Burnette and R.R. Weichselbaum 26. Zhang B, Bowerman NA, Salama JK: Induced sensitization of tumor stroma leads to eradication of established cancer by T cells. J Exp Med 204:49-55, 2007 27. Burnette BC, Liang H, Lee Y: The efficacy of radiotherapy relies upon induction of type i interferon-dependent innate and adaptive immunity. Cancer Res 71:2488-2496, 2011 28. Widau RC, Parekh AD, Ranck MC: RIG-I-like receptor LGP2 protects tumor cells from ionizing radiation. Proc Natl Acad Sci U S A 111: E484-E491, 2014 29. Deng L, Liang H, Burnette B: Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest 124:687-695, 2014 30. Wu CY, Yang LH, Yang HY: Enhanced cancer radiotherapy through immunosuppressive stromal cell destruction in tumors. Clin Cancer Res 20:644-657, 2014 31. Binder DC, Engels B, Arina A: Antigen-specific bacterial vaccine combined with anti-PD-L1 rescues dysfunctional endogenous T cells to reject longestablished cancer. Cancer Immunol Res 1:123-133, 2013 32. Schaue D, Ratikan JA, Iwamoto KS: Maximizing tumor immunity with fractionated radiation. Int J Radiat Oncol Biol Phys 83:1306-1310, 2011 33. Kachikwu EL, Iwamoto KS, Liao Y-P: Radiation enhances regulatory T cell representation. Int J Radiat Oncol Biol Phys 81:1128-1135, 2011 34. Dunn PL, North RJ: Adoptive immunotherapy of established tumors. Acquisition of radioresistance by tumor-specific T cells after passive transfer into tumor-bearing recipients. Int J Cancer 57:592-597, 1994 35. North RJ: Radiation-induced, immunologically mediated regression of an established tumor as an example of successful therapeutic immunomanipulation. Preferential elimination of suppressor T cells allows sustained production of effector T cells. J Exp Med 164:1652-1666, 1986 36. Garcia-Barros M: Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science 300:1155-1159, 2003 37. Gulbins E, Kolesnick R: Raft ceramide in molecular medicine. Oncogene 22:7070-7077, 2003 38. Schietinger A, Arina A, Liu RB: Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer. Oncoimmunology 2:e26677, 2013 39. Arina A, Schreiber K, Binder DC: Adoptively transferred immune T cells eradicate established tumors despite cancer-induced immune suppression. J Immunol 192:1286-1293, 2014 40. Mole RH: Whole body irradiation; radiobiology or medicine? Br J Radiol 26:234-241, 1953 41. Kaminski JM, Shinohara E, Summers JB: The controversial abscopal effect. Cancer Treat Rev 31:159-172, 2005 42. Nobler MP: The abscopal effect in malignant lymphoma and its relationship to lymphocyte circulation. Radiology 93:410-412, 1969 43. Ehlers G, Fridman M: Abscopal effect of radiation in papillary adenocarcinoma. Br J Radiol 46:220-222, 1973 44. Kingsley DP: An interesting case of possible abscopal effect in malignant melanoma. Br J Radiol 48:863-866, 1975 45. Stamell EF, Wolchok JD, Gnjatic S: The abscopal effect associated with a systemic anti-melanoma immune response. Int J Radiat Oncol Biol Phys 85:293-295, 2012 46. Takaya M, Niibe Y, Tsunoda S: Abscopal effect of radiation on toruliform para-aortic lymph node metastases of advanced uterine cervical carcinoma—A case report. Anticancer Res 27:499-503, 2007 47. Okuma K, Yamashita H, Niibe Y: Abscopal effect of radiation on lung metastases of hepatocellular carcinoma: A case report. J Med Case Rep 5:111, 2011 48. Ohba K, Omagari K, Nakamura T: Abscopal regression of hepatocellular carcinoma after radiotherapy for bone metastasis. Gut 43:575-577, 1998 49. Postow MA, Callahan MK, Barker CA: Immunologic correlates of the abscopal effect in a patient with melanoma. N Engl J Med 366:925-931, 2012 50. Golden EB, Demaria S, Schiff PB: An abscopal response to radiation and ipilimumab in a patient with metastatic non-small cell lung cancer. Cancer Immunol Res 1:365-372, 2013
The Immunology of Ablative Radiation 51. Seung SK, Curti BD, Crittenden M, et al: Phase 1 study of stereotactic body radiotherapy and interleukin-2—Tumor and immunological responses. Sci Transl Med 4:137-174, 2012 52. Formenti SC, Friedman K, Chao K: Abscopal response in irradiated patients: Results of a proof of principle trial. Int J Radiat Oncol Biol Phys 72:S6-S7, 2008
45 53. Camphausen K, Moses MA, Ménard C: Radiation abscopal antitumor effect is mediated through p53. Cancer Res 63:1990-1993, 2003 54. Dewan MZ, Galloway AE, Kawashima N: Fractionated but not singledose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody. Clin Cancer Res 15:5379-5388, 2009
Introduction
R
adiation has been a staple of cancer therapy since the early 20th century and is implemented in nearly half of current cancer treatment plans. Originally, the genotoxic function of radiation led to a focus on damage and repair pathways associated with deoxyribonucleic acid as important therapeutic targets to augment radiation efficacy. However, in recent decades, the participation of endogenous immune responses in modifying radiation effects has been widely documented and exploited in both preclinical and clinical settings. In particular, an early study by Stone et al1 was the first to document the effect of immune status (sufficient or deficient due to experimental manipulation) on tumor dose responses in syngeneic mouse fibrosarcoma. This observation was largely ignored until substantial work during the last decade expanded our understanding of how the immune system, and in particular T lymphocytes (T cells), participate in the host response to tumor radiation.2-5 Given the well-known sensitivity of lymphocytes to radiation-mediated apoptosis and potential for unintended immunosuppression arising from suboptimal dose and fractionation schedules, application of local radiation in
Department of Radiation and Cellular Oncology, The Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL. The authors declare no conflict of interest. Address reprint requests to Ralph R. Weichselbaum Department of Radiation and Cellular Oncology, The Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL. E-mail: [email protected]
40
http://dx.doi.org/10.1016/j.semradonc.2014.07.009 1053-4296/& 2015 Published by Elsevier Inc.
hypofractionated ablative doses has been pursued as a means to capture both the direct cytotoxic and indirect immuneactivating effects of radiation. Regarding immunostimulation, there is still a paucity of data on the specific dose and fractionation schedules. The effects of dose and fractionation on tumor-specific immune responses have been reviewed elsewhere6 and are outside the scope of this review; however, we review some of the basic applications of stereotactic body radiation therapy (SBRT) and the preclinical data that support SBRT as an immune modifier. The reader is directed to reviews published in this issue and elsewhere for more comprehensive discussion of some of the basic immunologic mechanisms highlighted in this article.7-9 In addition, for the purposes of this review and for lack of sufficient preclinical and clinical data for a rigorous comparison, we draw little distinction between the varying hypofractionated or single high-dose radiation treatment approaches, such as intensity-modulated radiation therapy, image-guided radiation therapy, and SBRT, despite acknowledgment of the important differences in clinical application.
General Remarks About Preclinical Animal Models of SBRT Most of what we know about the immunomodulation of hypofractionated radiation doses (sometimes referred to “ablative” doses) characteristic of SBRT comes from preclinical animal models that use subcutaneous inoculation of tumor cell
The Immunology of Ablative Radiation lines in syngeneic mice. The strategic advantage of this experimental setup is the ability to achieve relatively complete systemic shielding of the animal with a stationary, single radiation source. The tumor is usually exposed to the beam path by passing the tissue (skin and enclosed tumor) through a narrow orifice in the shielding that is oriented perpendicular to the beam path, thus eliminating all but the small amount of side scatter that can pass into the directly adjacent normal tissue traversing the orifice. It is worth noting that this experimental system, while a close approximation, does not replicate the extent of normal tissue exposure that occurs during the execution of a typical multi-beam SBRT treatment plan. More importantly, considering the translational relevance of these studies, it is prudent to discuss the well-known, but rarely discussed, fact that inoculation of tumor cells lines in syngeneic animals results in the induction of innate and adaptive immune responses. For tumors that are capable of progressive growth in syngeneic animals, the immune response that is induced on inoculation fails to eliminate the deposited cancer cells, thereby resulting in rapid death of the host, usually within 3-6 weeks because of excessive tumor burden. The failure of the immune system to successfully combat the initial tumor challenge results from the formation of a well-documented immunosuppressive tumor microenvironment that thwarts immune-mediated attack during initial adaptation of the tumor cells to in vivo growth. This localized suppression is followed closely by systemic immune suppression accompanying progressive tumor growth. Studies published by Paul Ehrlich in 1906 and Ernest Bashford10 in 1908 demonstrated that the immune response generated from transplantable tumor challenge could successfully reject a second inoculum of the same tumor that was given within a short window following the primary inoculum. Rejection of the secondary challenge despite continued growth of the primary inoculum was a seemingly paradoxical observation that Bashford termed concomitant immunity and demonstrated the vaccine-like function of tumor challenge and the importance of local and systemic immune suppression in syngeneic “progressor” tumors. The time-dependent erosion of concomitant immunity with tumor progression was demonstrated to result from the induction of a systemic suppressor T-cell population11 that was later demonstrated to be regulatory T cells.12 Importantly, as noted by Vaage,13 “once immune resistance is evoked the resistance factors are never absent but may be depressed and not revealed, depending upon the conditions of the tests and strength of the antigens.” Experimental demonstration of the preservation of immune resistance despite systemic suppression comes from studies that “unmasked” antitumor immunity to established tumors by systemic11,12 or local14 depletion of regulatory T cells. Considering these longstanding historical observations, it is fair to say that experimental systems using transplantable tumor cell lines are probably a more appropriate model for how hypofractionated or ablative radiation might augment existing T-cell immunity rather than initiating (or priming) de novo T-cell responses. This conceptual framework bears relevance when
41 considering the widely popularized notion that local radiation of an established tumor can function as an in situ vaccine through the induction of immunogenic cell death and activation and maturation of antigen-presenting cells.7-9 In this review, we focus mainly on the experimental data regarding immunomodulation of ablative radiation in the setting of existing T-cell responses and discuss the potential application and relevance to several clinical treatment scenarios involving SBRT.
Effects of Ablative Radiation Within the Target Volume Direct Sensitization of Tumor Cells to T-cell–Mediated Killing To date, many studies have documented the myriad mechanisms through which ablative radiation doses can increase the sensitivity of tumor cells to direct T-cell–mediated killing. These mechanisms include upregulation of the antigenpresentation machinery through increased expression and cell surface localization of major histocompatibility complex proteins,3,15,16 increased expression of immunogenic tumor antigens,17,18 upregulation of T-cell co-activating ligands,19 and even sensitization of tumor cells to antigen-independent cell death through the Fas receptor.20 These and other mechanisms have been reviewed elsewhere.21 More importantly, an emerging trend in the most current research has been an understanding of the mechanisms through which the tumor stroma modulates ablative radiation and vice versa. The tumor stroma is a relatively loosely defined term that encompasses all cells within the tumor mass that are not neoplastic, including vascular endothelial cells, fibroblasts, hematopoietic cells of lymphoid and myeloid origin, and acellular components such as the extracellular matrix. Pioneering work by Hans Schreiber and Rolf Zinkernagel established the importance of the tumor stroma in immunologic rejection of immunogenic tumors, demonstrating that inoculation of tumor cells with tumor stroma could drastically enhance their tumorigenicity and in some cases facilitate the growth of tumors normally rejected as cell suspensions in syngeneic mice.22,23 Further studies by Spiotto and Schreiber25 demonstrated that presentation of tumor antigens by both cancer cells and the stroma was required for complete elimination of antigenic tumors and tumors harboring antigen-negative clones (antigen-loss variants).24 Given the importance of stromal antigen presentation, Zhang et al26 demonstrated that a single dose of 10 Gy of local radiation was sufficient to sensitize antigenic tumors to T-cell–mediated rejection through “loading” of the tumor stroma with tumor antigens. Studies from our group demonstrated a similar phenomenon, wherein local radiation of established B16 melanoma tumors with a single ablative dose of 20 Gy facilitated cross-presentation of tumor antigens by dendritic cells in the tumor stroma.27 From the standpoint of local radiation, it is unclear what pathways control the transfer of antigen from tumor cells to the
42 surrounding stroma but direct tumor cell death is implicated. Notably, deficiency in type I interferon receptor abrogated tumor antigen cross-presentation by stromal dendritic cells,27 and our group recently demonstrated that type I interferon is a critical mediator/sensitizer of tumor cells to initial cytotoxicity in response to local irradiation.28 Antigen transfer could sensitize protumorigenic stromal cells to T-cell–mediated killing, and this mechanism is supported by several studies that demonstrate T-cell–mediated elimination of critical protumorigenic and immunosuppressive stromal cell subtypes following local ablative radiation. First, a study by our group demonstrated that the combination of ablative radiotherapy and T-cell checkpoint blockade with anti–programmed death ligand-1 led to elimination of stromal myeloid-derived suppressor cells (MDSCs) through T-cell–derived production of tumor necrosis factor.29 A study by Wu et al30 made a similar observation regarding selective elimination of MDSCs using local ablative radiation with intratumoral injection of recombinant tumor-specific antigen to load the tumor stroma. Interestingly, local, but not systemic, injection of antigen facilitated a systemic vaccine response in addition to the effects on MDSCs. Novel strategies, including engineered bacterial delivery systems,31 have been successfully developed to facilitate stromal loading of tumor antigens to sensitize the stroma to T-cell–mediated recognition. Collectively, these studies demonstrate that there is a critical role for stromal antigen presentation following local ablative radiation in both altering the tumor microenvironment and likely promoting a systemic T-cell response. An additional consideration for the differential efficacy of ablative radiation compared with traditional fractionation is the potential role that direct cytotoxicity might play in the tumor stroma, which is by definition “normal” tissue embedded among neoplastic cells. Traditional fractionation schedules have been empirically developed to specifically limit normal tissue toxicity by exploiting the differential mitotic rate and deoxyribonucleic acid repair capacity of neoplastic and normal cells. Notably, fractionated radiation was developed to reduce toxicity that results from normal tissue effects outside of the intended target neoplastic tissue, whereas stereotactic ablative radiation virtually eliminates toxicity from normal tissue exposure and instead the only “normal” tissues targeted are the stroma and vasculature within the target volume. Given the critical role that the stroma plays in regulating the balance of protumor and antitumor influences, direct genotoxic or cytotoxic effects (or a combination of both) of local radiation on the tumor stroma should be considered. Almost nothing is known about the radiosensitivity of the hematopoietic cell types present in the tumor and how their response to radiation might differ from cells of comparable lineage isolated from non-neoplastic tissue. Studies from Bill McBride’s group suggest that exposure of lymphoid organs to fractionated radiation or scatter from local ablative radiation might increase the percentage of regulatory T cells in the periphery because of the relative radioresistance of regulatory T cells compared with conventional T cells.32,33 However, the relative abundance of naïve, and historically radiosensitive, T cells present in lymphoid organs is likely a key determinant
B. Burnette and R.R. Weichselbaum of the effect of radiation on the enrichment of regulatory T cells. An interesting observation that was made by Dunn and North34 several decades ago concerns the relatively rapid development of radioresistance among activated tumorspecific T cells and relative radiosensisitvity of suppressor T cells in the dose range of hypofractionated radiation.35 These studies suggest that effector T cells in the tumor microenvironment might not be rapidly eliminated by hypofractionated or ablative doses, which has been a reasonable concern for maximizing tumor-specific T-cell immunity through application of optimal dose and timing to avoid local T-cell depletion. Further studies on the sensitivity of tumor-infiltrating T cells are warranted to address these questions. A specific instance where there is some mechanistic basis for the differential effects of ablative vs fractionated doses on the tumor stroma is in the sensitivity of the tumor vasculature to radiation cytotoxicity. Studies by Garcia-Barros36 demonstrated that endothelial cell apoptosis within the tumor microenvironment is a rapid event that occurs in the first several hours after radiation exposure. Notably, tumors were treated with ablative doses of 15 Gy, known to induce surface translocation and enzymatic activity of acid sphingomyelinase, leading to the production and release of ceremide and consequently, the rapid apoptosis of endothelial cells (reviewed in Ref. 37). The vasculature is a widely recognized target in cancer therapy and emerging data suggest that T-cell–mediated effects on the tumor and stroma might be similarly linked to effects on the vessels, in part, through the production of cytokines.38,39
Effects of Ablative Radiation Outside the Target Volume Clinical Observations of Abscopal Responses The term “abscopal effect” was originally coined by Mole40 to describe the still poorly understood capacity of radiation to mediate effects on tumors outside the treatment field. A heavily favored theory suggests that the abscopal effect of radiation is mediated by augmented immune function, either cellular or cytokine mediated.41 Abscopal regression has been reported in a variety of tumor types treated with radiation as a single agent including lymphoma,42 papillary adenocarcinoma,43 melanoma,44,45 metastatic cervical carcinoma,46 and lung metastases from primary hepatocellular carcinoma.47,48 However, the report by Stamell et al45 was the only case where hypofractionated radiation was utilized, with the remaining cases using standard fractionation. Given the widely accepted notion that immunologic mechanisms underlie the abscopal effect, treatment combinations employing radiation and immunotherapy are predicted to increase the frequency of abscopal responses. Notably, 2 case reports of combination intensity-modulated radiation therapy and ipilimumab (anti–CTLA-4) have demonstrated abscopal effects in metastatic melanoma49 and non–small cell lung cancer.50 Furthermore, a phase I study of SBRT and high-dose IL -2 (a T-cell growth factor) in metastatic melanoma and
The Immunology of Ablative Radiation metastatic renal cell carcinoma demonstrated impressive abscopal responses in several patients.51 Finally, a small proof of a principle trial of hypofractionated radiation and systemic granulocyte-macrophage colony–stimulating factor administration in patients with tumors of mixed histology reported abscopal responses in 4 of the 12 patients enrolled.52 Lending credence to the role of immunity in the abscopal effect, Postow et al49 observed an increase in circulating antibodies to the tumor-associated antigen NY-ESO-1 (circulating antibodies are a surrogate for T-cell immunity, given the assumption that antibody production is T-cell-dependent in many cases), and Seung et al51 demonstrated elevated proliferation of circulating peripheral blood CD4þ T cells with an effector memory phenotype in patients who responded to treatment compared with nonresponders. Further investigation is warranted, but the results from these small trials employing radiation and immunotherapy are certainly promising.
Preclinical Models of Abscopal Response Following Ablative Radiation Data from preclinical models of radiation-mediated abscopal responses provide important information about the potential mechanism of the abscopal effect. One of the earliest studies by Camphausen et al53 demonstrated that the abscopal effect is dose-dependent using the Lewis lung carcinoma cell line. Notably, the abscopal effect of hypofractionated radiation (10 Gy 5) was more pronounced than that of traditional radiotherapy dose schedules (2 Gy 12), although a formal isodose was not determined for the small dose fractions. In addition, p53 was determined to be essential for the abscopal effect, as treatment with pifthrin-α eliminated the abscopal effect. Demaria et al2 used local radiation on transplantable mammary carcinoma cell lines with systemic treatment with Fms-like tyrosine kinase receptor 3 ligand (Flt3-L), a dendritic cell proliferation and maturation factor. The particular tumor cell line used exhibited marked sensitivity to radiation with a single dose of 2 Gy, producing significant growth delay. In these studies, local radiation alone failed to demonstrate an abscopal effect on a contralateral tumor of the same size, whereas combination therapy led to a significant growth delay of the contralateral tumor. Beyond the successful demonstration of abscopal regression, Demaria et al went on to demonstrate that the abscopal effect observed with local radiation and Fms-like tyrosine kinase receptor 3 ligand was both specific and T-cell-mediated through experiments where the abscopal effect was lost when the contralateral tumor was unrelated (antigenically different) or T cells were systemically depleted, respectively. A second study by Dewan et al54 demonstrated a similar abscopal response from combination therapy with radiation and CTLA-4–blocking antibodies in a contralateral flank trichostatin A murine breast cancer model. Interestingly, they reported that hypofractionation with a schedule of 8 Gy 3 yielded the most significant abscopal responses compared with a slightly different, 6 Gy 5, fractionation or 20-Gy ablative radiation, which correlated with increased infiltration and production of interferon gamma by CD8þ T cells. A study from our
43 laboratory using a contralateral murine flank model of Her2þ breast cancer also demonstrated significant abscopal effects with combined local radiation and T-cell checkpoint blockade with anti–programmed death ligand-1.29 A common feature of the experimental systems that model the abscopal effect is the empirical determination that the effect of radiation on the abscopal tumor, as with therapy to the primary tumor, depends heavily on the tumor size. Partly, this observation can be explained by a sheer numerical disadvantage of systemic immunity to increasing tumor burden. However, we offer another plausible hypothesis that the status of the tumor microenvironment dictates whether secondary tumors are amenable to abscopal effects. Similar to the susceptibility of recently injected tumors to concomitant immunity, tumor size can be thought of as a proxy for the establishment of an immunosuppressive stroma. Whether local therapy with ablative radiation mediates systemic effects through the reawakening of concomitant immune mechansims remains to be determined, but we suspect this to be the case. Regardless, defining the cellular and molecular mechanisms that dictate amenability of secondary lesions to abscopal effects is of great interest, especially in the clinical setting of multiplicity of lesions, wherein the physician must determine lesions warranting intervention with local therapy such as SBRT or other ablative radiation delivery techniques.
Concluding Remarks Clinical and experimental data summarized herein demonstrate the now well-established reciprocal capacity of immunity and radiation to augment each other. Optimal integration of radiation and immunotherapy is yet to be achieved and will depend on a concerted effort between scientists in clinical and basic research to address critical questions that will instruct the rationale and design of combination therapies. Direct tumor cell radiosensitivity is deeply entrenched in the historical application of radiation oncology, but how tumor cell radiosensitivity affects the immunologic aspects of radiation therapy is entirely unknown. Certainly, killing tumor cells is still the goal of therapeutic intervention, but it is conceivable that the magnitude of tumor cell death might be less important than the optimal engagement of immunity. Additionally, we suggest that consideration of the stromal component of the tumor as a therapeutic target is equally legitimate, given the critical role that the stroma plays in therapeutic susceptibility. Presently, the published data offer an assortment of single-dose and hypofractionation schemes without a clear mechanism for integration. Animal models offer appropriate and tractable systems to dissect these mechanisms, but a more thorough examination of the dose and timing of radiation, though scientifically less satisfying, should be integrated into the experimental plans to provide a firm base for the construction of clinical protocols.
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