Radiation therapy and PD-1/PD-L1 blockade: the clinical development of an evolving anticancer combination

Radiation therapy and PD-1/PD-L1 blockade: the clinical development of an evolving anticancer... Several inhibitors of programmed cell death-1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved as a form of immunotherapy for multiple cancers. Ionizing radiation therapy (RT) has been shown to enhance the priming and effector phases of the antitumor T-cell response rendering it an attractive therapy to combine with PD-1/PD-L1 inhibitors. Preclinical data support the rational combination of the 2 modalities and has paved way for the clinical development of the combination across a spectrum of cancers. In this review, we highlight the preclinical and clinical development of combined RT and PD-1/PD-L1 blockade to date. In addition to a comprehensive evaluation of available safety and efficacy data, we discuss important points of consideration in clinical trial design for this promising combination. Keywords: Radiation therapy, PD-1, PD-L1, Clinical trials, Preclinical, Antitumor, Immune response, Checkpoint inhibitor Background tumor response with associated regression of untreated Early preclinical evidence demonstrated that activation metastases outside of the radiation field has been of the programmed cell death 1 (PD-1) and programmed reported and was first described as the abscopal effect death ligand 1 (PD-L1) axis suppressed the activation [44]. Increasing evidence supports that the abscopal ef- and proliferation of tumor antigen-specific T-cells and fect is likely immune-mediated – largely, in a T-cell promoted tumorigenesis [1, 2]. These processes were dependent manner with a complex interplay between reversed with PD-1/PD-L1 blockade and supported proimmunogenic and proinflammatory factors [45–53]. the concept of PD-1/PD-L1 blockade as a potential Over time, recognition of the immunomodulatory prop- form of anti-cancer immunotherapy. The first agents erties of radiation has led to the integration of RT with in the family of PD-1/PD-L1 inhibitors to be ap- immune-modulating agents including immune check- proved by the Food and Drug Administration (FDA) point inhibitors to potentially develop a combination were the humanized monoclonal IgG4 antibodies, therapy with enhanced or synergistic anticancer activity pembrolizumab and nivolumab, that targeted PD-1 in (Fig. 1). unresectable or advanced melanoma [3–10]. There are Indeed, an initial preclinical study showed that com- currently 5 PD-1/PD-L1 inhibitors approved by the bining RT (1–2 fractions of 12 Gray (Gy) to the primary FDA for the treatment of a number of solid tumors tumor) with an anti-cytotoxic T lymphocyte-associated and hematologic malignancies [11–43]. antigen-4 (CTLA-4) monoclonal antibody resulted in Ionizing radiation therapy (RT) is widely used in the synergistic antitumor activity in a poorly immunogenic definitive and metastatic setting for local tumor control; metastatic mammary carcinoma mouse model when however, the ability of radiation to elicit a systemic CTLA-4 blockade by itself was ineffective [54]. Enhanced antitumor responses have also been observed across several preclinical animal models treated with * Correspondence: richard.tuli@cshs.org combined RT and CTLA-4 blockade [55–58]. Since the Departments of Radiation Oncology and Biomedical Sciences, Samuel first preclinical studies that highlighted the synergistic Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, AC 1023, Los Angeles, CA 90048, USA antitumor activity of combination RT and CTLA-4 Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 2 of 17 Fig. 1 Proposed mechanisms of synergy between RT and PD-1/PD-L1 inhibitors. Emerging evidence demonstrates that immune modulation from PD-1/PD-L1 inhibitors and RT through nonredundant pathways contributes to synergistic antitumor activity, thereby forming the basis for the rationale combination of the two modalities. RT, radiation therapy; PD-1, programmed cell death 1 receptor; PD-L1, programmed death ligand 1; IFN-γ, interferon-γ; cGAS, cyclic GMP-AMP (cGAMP) synthase; STING, stimulator of interferon genes; MHC, major histocompatibility complex; TCR, T-cell receptor; TILs, tumor-infiltrating lymphocytes, Tregs; regulatory T cells; MDSCs, myeloid-derived suppressor cells blockade, several prospective clinical trials have re- Preclinical studies ported on the activity of RT and ipilimumab in ad- The efficacy of combination RT and checkpoint blockade vanced solid tumors [59–66]. Similarly, there are is associated with modulation of immune parameters numerous ongoing clinical trials investigating the within the tumor microenvironment combination of RT and CTLA-4 blockade that have Early investigations in mouse models of solid and been extensively reviewed and are beyond the scope hematologic malignancies showed enhanced antitumor of this manuscript [67, 68]. Herein, we review in effects when treated with PD-1 or PD-L1 blockade in detail the preclinical and clinical development of the combination with in-field RT, sublethal total body combination of RT and PD-1/PD-L1 inhibitors in can- irradiation (TBI), or stereotactic radiosurgery (SRS) cer therapy. compared to single modality treatment (Table 1)[69–85]. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 3 of 17 Table 1 Preclinical studies demonstrating antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade Cell line Experimental model RT dose PD-1/PD-L1 dose Ref. B16-D5 (melanoma) Mice subcutaneous TBI 600 cGy PD-L1 mAb 20 mg/kg IP starting on [69] (1 fraction) day 4 then every 3–4 days +1X10 gp100 or OVA pulsed dendritic 257–264 cell vaccine SC on day 4 and 11 ± 1X10 pmel T-cells (adoptive transfer) IV on day 4 after inoculation AT.3 (triple-negative Mice xenograft 12 Gy (1 fraction) or PD-1 mAb 100 μg + CD137 mAb [70, 71] mammary) 4–5 Gy (4 fractions) 100 μg IP on days 0, 4, 8, and 12 of RT GL261 (glioma) Mice xenograft 10 Gy (1 fraction) PD-1 mAb 10 mg/kg IP on days 10, [72] 12, and 14 of RT B16-SIY (melanoma) TUBO Mice subcutaneous 25 Gy (2 fractions) PD-L1 mAb 200 μg IP every 3 days [92] (mammary) 15 Gy (1 fraction) for 4 doses starting 3 weeks after RT 5 T33 (myeloma) Mice intravenous TBI 500 cGy PD-L1 mAb 200 μg IP on days 12, 14, [75] A20 (B-cell lymphoma) (1 fraction) 17, 19, 21, 26, and 28 after C1498 (leukemia) inoculation 5 T33 (myeloma) Mice intravenous TBI 1100 cGy HSCT on day 0 + PD-L1 mAb 200 μg [73] (1 fraction) IP on days 3, 5, 10, 12, 17, and 19 after HSCT ± vaccine (irradiated 5 T33 cells or 5 T33 cells transfected with empty vectors) on days 3, 10, and 17 after HSCT 5 T33 (myeloma) Mice intravenous TBI 500 cGy PD-L1 mAb 200 μg IP on days 12, 14, [74] (1 fraction) 17, 19, 21, 26, and 28 after inoculation ± LAG-3, TIM-3, or CTLA- 4 mAbs 200 μg IP on same days V600E CT26 (colon 4434 (BRAF - Mice subcutaneous 10 Gy (5 fractions) PD-1 or PD-L1 mAb 10 mg/kg IP 3 [86] mutant melanoma) times weekly up to 3 weeks starting 4 T1 (triple-negative on day 1 of RT mammary) TUBO (mammary) Mice subcutaneous 12 Gy (1 fraction) PD-L1 mAb 200 μg IP every 3 days [76] MC38 (colon for 4 doses starting on day 0 or 1 of RT TSA (mammary) Mice subcutaneous 24 Gy (3 fractions) PD-1 mAb (dose NR) starting on day [77] 15 after inoculation and every 4 days thereafter B16-OVA (melanoma) Mice subcutaneous 15 Gy (1 fraction) PD-1 mAb 10 mg/kg ± CTLA-4 mAb [87] RENCA (renal) 10 mg/kg IP on days 7, 9, 11, 14, and 16 following tumor cell inoculation B16-OVA (melanoma) Mice subcutaneous 12 Gy (1 fraction) PD-1 mAb 200 μg IP every 3 days for [79] 4 T1-HA (mammary) 3 doses starting 1 day prior to RT PyMT (mammary) Mice subcutaneous 12 Gy (1 fraction) PD-1 mAb dose NR + single dose of [78] CTLA-4 mAb (dose NR) 3 days prior to PD-1 and RT B16-F10 (melanoma) Mice subcutaneous 20 Gy (1 fraction) PD-L1 mAb 200 μg + CTLA-4 mAb [61] 200 μg IP every 3 days for 3 doses starting 5 or 9 days after inoculation Meer (head and neck Mice subcutaneous 1, 6, 10 Gy fractions PD-L1 antibody dose NR [82] squamous) Adeno-Cre viral vector (lung) GEMM intrathoracic 8.5 Gy twice daily PD-1 mAb 200 μg IP 3 times weekly [81] injection over 2 days starting 6 h after second RT dose MB49 (bladder) Mice xenograft 12 Gy (1 fraction) PD-L1 mAb 250 μg IP twice weekly [84] for 4 doses starting 1 day prior to RT MC38 (colon Mice subcutaneous 24 Gy (3 fractions) PD-1 mAb ± CD137 mAb 5–10 mg/ [83] 4 T1 (mammary) B16F10-OVA kg IP on days 13, 15, and 17 after (melanoma) inoculation Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 4 of 17 Table 1 Preclinical studies demonstrating antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade (Continued) Cell line Experimental model RT dose PD-1/PD-L1 dose Ref. 4-hydroxytamoxifen GEMM topical 14 Gy (1 fraction) PD-1 + CD137 or PD-1 + CTLA-4 mAb [99] induction (BRAF-mutant, induction 100 μg IP twice weekly for 4 doses PTEN-deficient melanoma) on day 1 of RT 344SQ (lung) Mice subcutaneous 36 Gy (3 fractions) PD-1 mAb 10 mg/kg IP starting on [91] day 1 of RT and continued for additional 3–4 doses ARK (esophageal squamous) Mice subcutaneous 20 Gy (10 fractions) PD-1 mAb (dose NR) starting 2 days [85] before RT and every 3 days thereafter ± carboplatin and paclitaxel IP (dose NR) on day 1 of RT and every 3 fractions GL261 (glioma) Mice xenograft 10 Gy (1 fraction) PD-1 mAb 200 μg IP on days 10, 12, [90] and 14 of RT ± TIM-3 mAb 250 μgIP days 7, 11, and 15 of RT V600E CT26 (colon 4434 (BRAF - Mice subcutaneous 10 Gy (5 fractions) PD-1 or PD-L1 mAb 10 mg/kg IP 3 [88] mutant melanoma) times weekly for 1 week starting on day 1 of RT TSA (mammary) Mice subcutaneous 24 Gy (3 fractions) on PD-1 mAb 200 μg IP on days 12, 15, [89] days 12, 13 and 14 19, 22 and 26 after inoculation after inoculation Hep-55.1c (hepatocellular) Mice orthotopic 30 Gy (3 fractions) PD-1 mAb 250 μg IP on days 7, 14, [96] and 21 after inoculation KPC and Pan02 (pancreatic) Mice subcutaneous 6, 12, or 20 Gy PD-L1 mAb 10 mg/kg IP on days 4, [95] (1 fraction) 7, 10, and 13 after inoculation + 10 Gy (5 fractions) gemcitabine 100 mg/kg IP on days 0 15 Gy (5 fractions) and 3 of inoculation HCa-1 (hepatocellular) Mice intramuscular 10 Gy (1 fraction) PD-L1 mAb 10 mg/kg IP every [97] 3 days for 4 doses starting on day 1 of RT LM8 (osteosarcoma) Mice subcutaneous 10 Gy (1 fraction) PD-L1 mAb 150 μg + CTLA-4 mAb [98] 150 μg IP every 3 days for 3 doses starting on days 9, 12, and 15 after inoculation CT26 (colon Mice intradermal RFA 17-gauge single PD-1 mAb 200 μg IP every 3 days for [94] ablation electrode for 4 doses 3.5–4.5 min at target temperature of 70 degrees C RT radiation therapy, TBI total body irradiation, cGy centigray mAb monoclonal antibody, IP intraperitoneal, SC subcutaneous, IV intravenous, Gy Gray, HSCT hematopoietic stem cell transplantation, LAG-3 lymphocyte-activation gene 3, TIM-3 T-cell immunoglobulin mucin-3, NR not reported, GEMM genetically engineered mouse model, RFA radiofrequency ablation Combined modality therapy was associated with higher Combination modality-induced immune profile changes levels of CD8+/interferon-γ (IFNγ)+/tumor necrosis may be time-dependent factor-α (TNFα) + cytotoxic T-cells, increased PD-1, T-cell Early syngeneic mouse tumor models demonstrating immunoglobulin mucin-3 (TIM-3), lymphocyte-activation significant improvements in survival and tumor volume gene 3 (LAG-3), and 2B4 (immune checkpoints) expres- reduction with the combination of RT and PD-1 or sion on CD8+ T-cells, decreased numbers of CD4 PD-L1 blockade compared to single modality and con- +/FOXP3+ regulatory T-cells (Tregs) and myeloid-derived trol arms identified elevations in tumor cell PD-L1 ex- suppressor cells (MDSCs), upregulation of PD-L1 on pression that were CD8+ T-cell and IFNγ-dependent dendritic cells and tumor cells in irradiated tumors, following irradiation (10 Gy over 5 daily fractions) com- RT-induced upregulation of major histocompatibility pared to non-irradiated mice with peak levels occurring complex (MHC) class I tumor-associated antigen 72 h after last dose of RT [86]. RT-induced increases in complexes, and enhanced antigen cross-presentation in the CD8+/Treg ratio and PD-L1 expression occurred draining lymph nodes compared to single modality 24–96 h post-RT in a separate mouse model [81]. In arms [71, 72, 74, 76–79]. colon carcinoma tumors, the addition of PD-L1 Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 5 of 17 blockade on day 1 of RT (schedule A), day 5 of RT cell-intrinsic activation of the type I IFN pathway as medi- (schedule B), or 7 days after RT (schedule C) showed ated by cyclic GMP-AMP (cGAMP) synthase (cGAS) and that there was no significant difference in overall sur- stimulator of interferon genes (STING) signaling [89]. vival (OS) between schedule A and B (p > 0.05) though RT-induced abscopal responses with PD-1 blockade were sequential therapy (schedule C) was ineffective in enhan- additionally shown to be regulated by Trex1 where induc- cing OS compared to RT alone (median OS 30 days vs. tion of Trex1 expression in cancer cells resulted in loss of 35 days, p > 0.05) [86]. Notably, PD-1 expression was sig- abscopal responses in mice treated with the combination. nificantly decreased on CD8+ T-cells 7 days after RT compared to time-matched controls (p < 0.05). Combined modality therapy reverses T-cell exhaustion Abscopal effects and systemic immunity and resistance to RT and anti-PD-1 therapy On subcutaneous tumor flank rechallenge of Murine tumor xenografts have shown that increasing treatment-naïve mice and mice cured by combination levels of PD-1 and TIM-3 co-expression in CD4+ RT and checkpoint blockade, immunologic memory was T-cells, CD8+ T-cells, and Tregs over time contribute to established in cured mice but not in treatment-naïve an exhausted or impaired T-cell phenotype [90]. mice suggesting that the immune system in cured mice Furthermore, resistance to anti-PD-1 therapy in retained the ability to recognize tumor-associated RT-refractory tumors has been characterized by signifi- antigens and mount an immune response of greater cant elevations in expression of genes associated with magnitude and speed upon rechallenge, i.e., systemic im- T-cell exhaustion, increased levels of checkpoints includ- munity [71, 72]. Abscopal effects have been shown to be ing LAG-3, TIM3, and CTLA-4 on CD4+ T-cells, and mediated, in part, by PD-1 as administration of a single decreased number of CD11c + tumor-associated macro- fraction of 15 Gy by stereotactic ablative radiotherapy phages (TAMs) [81]. The addition of immune check- (SABR) to the primary tumor in a melanoma subcutane- point inhibitors to RT has been shown to enhance ous mouse model resulted in significant reduction in tumor response compared to controls across several tumor volumes of secondary nonirradiated tumors in mouse tumor models through reinvigoration of PD-1-knockout mice compared to PD-1-wild-type (WT) exhausted CD8+ TILs characterized by increased Ki67+ mice [87]. Addition of a PD-1 inhibitor to SABR resulted GzmB+ T-cells within the exhausted PD-1+ Eomes+ in synergistic antitumor activity on the primary tumor T-cell pool, increased CD8+ CD44+ TILs, and increased compared to PD-1 inhibitor or SABR alone and recapit- CD8+/Treg ratio [61, 77, 85]. ulated abscopal effects on secondary nonirradiated tu- Moreover, an anti-PD-1-resistant murine lung cancer mors in PD-1-WT mice when treatment alone with model established through sequential in vivo passage of anti-PD-1 or SABR did not reduce secondary tumor nonresponsive tumors to ongoing anti-PD-1 therapy was growth. Furthermore, following RT, higher levels of characterized by significant downregulation of MHC high PD-1+ CD11a CD8+ T-cells were seen in primary tu- class I and II genes including β2-microglobulin and mors compared to secondary tumors and higher levels reduction in CD4+/CD8+ TILs and IFN-γ production in in irradiated compared to nonirradiated tumors; this resistant tumors compared to parental tumors [91]. population of cells appeared to comprise the principal Addition of RT induced IFN-γ production and MHC tumor-specific reactive phenotype. This latter finding class I expression and ultimately restored response to has been confirmed in another study where RT in- PD-1 blockade in resistant tumors. Addition of a PD-L1 creased T-cell receptor (TCR) repertoire clonality and inhibitor has been shown to reverse RT-induced tumor diversity of the TCR repertoire in irradiated tumors equilibrium in favor of tumor regression in mice sub- compared to controls, however, the addition of PD-1 in- cutaneously injected with melanoma and breast tumors hibition to RT increased TCR diversity both in irradiated demonstrating RT-induced stable disease (SD, defined as and out-of-field sites [88]. Further analysis revealed that ≥3 weeks) characterized by a transient rise and fall in most of these TCR clones arose from progenitor clones levels of tumor-infiltrating CD8+ T-cells and IFNγ [92]. that were established in tumors prior to therapy, and it Extrinsic RT resistance has been recently shown to be is the influx of tumor-infiltrating lymphocytes (TILs) contributed by RT-induced host STING activation from outside the tumor along with resident-tumor resulting in immunosuppressive MDSC recruitment that infiltrating T-cells that contribute to the enhanced is mediated by chemokine receptor type 2 (CCR2) in a tumor responses seen with combination therapy. syngeneic mouse model of colon carcinoma [93]. Treat- Recently, durable regression of irradiated tumors and ment with anti-CCR2 antibodies could potentially serve abscopal responses observed in mammary tumor-bearing a role in reversing RT resistance by attenuating host mouse models treated with combination RT and check- STING-mediated immunosuppression and complement point blockade were shown to be dependent on cancer RT and checkpoint blockade combinations. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 6 of 17 A growing body of preclinical evidence supports the or after PD-1 blockade produced 6- and 12-month combination of other immunotherapeutic agents with median OS rates of 85 and 78%, respectively [115]. One RT or radiofrequency ablation (RFA), immune check- retrospective study investigated 53 patients with meta- point blockade, and/or chemotherapy to enhance static melanoma treated with RT sequential or concur- tumor growth control (and often systemic control)in rent to anti-PD-1 therapy or as salvage therapy in the preclinical mouse models; synergistic antitumor activ- setting of progression on anti-PD-1 therapy (35 patients ity with multimodality therapy was characterized by received extracranial RT or intracranial SRS and 21 pa- tumor cell PD-L1 expression in a JAK/Stat1-depen- tients received whole brain radiotherapy (WBRT)) and dent manner and reduced numbers of CD11b + Gr1+ showed that median OS and ORR were not significantly cells (MDSCs) [90, 94–99]. different between concurrent and sequential RT/SRS cohorts (Table 2)[116]. Toxicities A single-institute retrospective trial analyzed the effi- Several preclinical studies have investigated the toxicity cacy of concurrent SRS and anti-PD-1 or anti-CTLA-4 of combined RT and checkpoint blockade. Notably, one therapy (defined as SRS within 4 weeks of administration investigation of lung-irradiated (20 Gy) C57bl/6-WT of checkpoint inhibitors) in 75 patients with melanoma mice receiving anti-PD-1 antibody (10 mg/kg intraperi- brain metastases and identified significantly improved toneal twice per week for 5 doses) showed more findings median percent reduction in lesion volume with concur- of abnormal alveoli, inflammatory changes, and exudates rent compared to nonconcurrent arms and with in the alveolar septa associated with a 2.1-fold increase anti-PD-1 compared to anti-CTLA-4 arms at 3 months in CD8+ T-cells in the irradiated lung tissues of mice in and 6 months [117]. However, when both anti-PD-1 and the RT and PD-1 blockade arm though post-RT mortal- anti-CTLA-4 therapies were combined there was no sig- ity up to 120 days was not significantly different in the nificant difference in median OS between nonconcurrent RT alone vs. RT and PD-1 blockade arm (p = 0.657) (9.0 months, range 2.1–61.8) and concurrent arms [100]. A separate study, however, using a similar dose of (19.1 months, range 2.7–64.2, p = 0.0691). In solely 20 Gy of thoracic RT (designed to induce mortality) to metastatic NSCLC patients (n = 21), combined RT to oli- C57bl/6 mice identified worse survival with RT and goprogressive sites along with PD-1/PD-L1 blockade or PD-1 blockade (36% survived) than RT alone (70% other immune therapies resulted in excellent local con- survived, p = 0.0169) at 21 days post-RT and increased trol, median time to systemic progression of 2.3 months T-cell infiltrates in lung and cardiac tissues (both in- (95% confidence interval (CI) 1.0–4.5), and median OS and out-of-field) of mice treated with RT and PD-1 of 7.2 months (95% CI 4.2–11.1) [118]. Among 25 pa- blockade compared to RT alone putatively due to tients with unresectable melanoma, abscopal responses enhanced healthy tissue damage by T-cell activation with (CR or PR) were observed in 56% of patients with the the addition of PD-1 blockade to thoracic RT [101]. addition of late RT (> 3 months of insufficient response Incorporating PD-1 blockade to cardiac RT in mice to anti-PD-1 monotherapy) [119]. has also shown to decrease survival and exacerbate A group of 137 patients with metastatic melanoma, cardiac dysfunction and myocarditis that are CD8+ NSCLC, and RCC treated with WBRT, SRS, or extracra- T-cell-mediated [102]. nial RT before or after initiation of PD-1 blockade expe- rienced a median OS 249 days (8 months; interquartile Clinical studies range (IQR) 90–689) following the start of anti-PD-1 Retrospective studies therapy though OS was 25.7 months in the cohort re- Numerous case reports and case series have documented ceiving brain RT as first form of palliative RT [120]. On clinically significant, and often durable, tumor responses multivariate analysis, melanoma patients fared best as to the combination of RT and PD-1/PD-L1 blockade in the hazard ratio (HR) for death was 3.1 (95% CI 1.7–5.9) advanced or metastatic melanoma, NSCLC, Hodgkin for NSCLC and HR of 3.2 (95% CI 1.2–7.9) for RCC lymphoma, RCC, and cervical cancer [103–112]. Initial compared to melanoma (p = 0.0008) possibly due to im- retrospective series of patients with melanoma brain proved responses to checkpoint inhibitors in melanoma metastases treated with SRS or fractionated RT within with the incorporation of both PD-1 and CTLA-4 inhib- 3–6 months of receiving anti-PD-1 therapy produced itors into standard care. promising 1-year OS rates and significantly improved 6- A secondary analysis of the phase I KEYNOTE-001 and 12-month distant brain metastasis control and OS trial of 98 patients with locally advanced or metastatic rates in those treated with SRS and anti-PD-1 therapy NSCLC treated with pembrolizumab showed signifi- vs. SRS and chemotherapy (Table 2)[113, 114]. In 24 cantly improved median OS of 10.7 months (95% CI patients with brain metastases from melanoma (54%) 6.5–18.9) vs. 5.3 months (95% CI 2.7–7.7, HR 0.58, 95% and NSCLC (46%), treatment with SRS before, during, CI 0.36–0.94, p = 0.026) in those who ever did and did Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 7 of 17 Table 2 Retrospective clinical studies with available results on the antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade Study n Design Outcomes Toxicities Ref. RS 26 Melanoma BMs treated with SRS or Median OS 11.8 mo (range 0.5–33.9) 1 grade 2 headache relieved with [114] FSRT (16–30 Gy X 1–5 fractions) and 1-year OS 55% in unresected steroids within 6 mo of nivolumab (1, 3, or BMs; median OS not reached and 10 mg/kg every 2 weeks for 12 1-year OS 100% in resected BMs doses then every 12 weeks for 8 doses) RS 96 Melanoma BMs treated with SRS 6- and 12-mo distant BM control rate For anti-PD-1 therapy: 1 grade 2 [113] (majority 24 Gy X 1 fraction) within 3 61%/38% anti-PD-1, 26%/21% headache managed with steroids mo of nivolumab 3 mg/kg every anti-CTLA-4, 53%/20% BRAF/MEK 2 weeks, pembrolizumab 2 mg/kg inhibitor, 15%/5% chemotherapy every 3 weeks, or other systemic (p = 0.008); 6- and 12-mo OS 81%/ therapies 66% anti-PD-1, 84%/50% anti-CTLA-4, 83%/75% BRAF/MEK inhibitor, 70%/15% chemotherapy (p = 0.004) RS 24 Melanoma and NSCLC BMs treated 6- and 12-mo OS 85 and 78%; 2 patients grade ≥ 3 CNS toxicity: 1 [115] with SRS (median 20 Gy/fraction, IQR median OS not reached; 6- and seizure and 1 symptomatic 16–21) within median 19 weeks 12-mo distant brain progression rate radionecrosis requiring surgery (range 0–107) of nivolumab or 37 and 65% pembrolizumab (median 5 cycles, IQR 3–6) RS 53 Metastatic melanoma treated with Medians OS 6.4 vs. 8.6 mo For RT arm: 3 patients grade ≥ 3 [116] extracranial RT/intracranial SRS (8– (p = 0.7672) for concurrent vs. rash, 1 grade ≥ 3 diarrhea, 2 grade ≥ 30 Gy X 1–10 fractions) or WBRT sequential RT/SRS; ORR 31% vs. 36% 3 radiation dermatitis, 1 grade ≥ 3 (median 30 Gy X10 fractions) and (p = 1) for concurrent vs. sequential radionecrosis; for WBRT arm: 1 pembrolizumab 2 mg/kg every RT/SRS; lesional response rate 45% grade ≥ 3 nausea, 1 grade ≥ 3 3 weeks or nivolumab 3 mg/kg for 30 progressing lesions treated cognitive changes, 2 grade ≥ 3 rash every 2 weeks as concurrent, with salvage RT/SRS sequential, or salvage (following progression on anti-PD-1 therapy) therapy RS 75 Melanoma BMs treated with SRS Median % lesion volume reduction NR [117] (median 20 Gy, range 12–24 Gy) at 3 mo (− 83.0% vs. -52.8%, within ±4 weeks (concurrent) of p < 0.0001) and 6 mo (− 94.9% vs. pembrolizumab 2 or 10 mg/kg every -66.2%, p < 0.0001) for concurrent vs. 2–3 weeks or nivolumab 3 mg/kg noncurrent; median % lesion volume every 2–3 weeks or ipilimumab reduction at 3 mo (− 89.3% vs. -66.2%, p < 0.0001) and 6 mo (− 95.1% vs. -75.9%, p = 0.0004) for anti-PD-1 vs. anti-CTLA-4 RS 21 Metastatic NSCLC treated with RT 6- and 12-mo local control rates 91.7 1 grade 4 cerebral edema (WBRT) [118] (8–30 Gy X 1–10 fractions) while and 85.2%; median time to systemic and 1 grade 3 pneumonitis receiving anti-PD-1, anti-PD-L1, progression 2.3 mo (95% CI 1.0–4.5); and/or anti-CTLA-4, or other immune median OS 7.2 mo (95% CI 4.2–11.1) therapy RS 25 Unresectable melanoma treated with CR, PR, SD, and PD rates for radiated No unusual AEs reported [119] hypofractionated RT (1 weekly sites 24, 8, 44, and 28% and for fraction over 4–5 weeks (84%) or 1 nonirradiated sites 29, 19, 19, and gammaknife RT for BMs (16%)) 33%, respectively; abscopal within 3 mo of anti-PD-1 (early) or > responses (CR or PR) in 56% for 3 mo after anti-PD-1 therapy (late) addition of late RT RS 15 Metastatic melanoma, RCC, NSCLC Safety analysis All-grade immune-related AEs in 3 [123] treated with palliative RT (total patients (20%) and 1 RT-related AE 8–36 Gy via 3–8 Gy fractions) (7%) of moderate mucositis; no cases within ±75 days of PD-1 inhibitor of pneumonitis RS 84 Metastatic melanoma, NSCLC, and No significant differences in toxicity For all-grade AEs: 6 patients with [124] other solid tumors treated with rates between PD-1/PD-L1 and pneumonitis (7.2%, 1 grade ≥ 3); for thoracic RT (median total dose CTLA-4 inhibitors or concurrent and grade ≥ 2 AEs: 14 fatigue, 9 rash, 10 3000 cGy (range 600–7400 X 10 sequential treatment GI toxicities, 12 infections, 8 thyroid fractions) within 1 month dysfunction, 7 renal injury, and 9 (concurrent) or up 6 months other (sequential) of PD-1/PD-L1 and/or CTLA-4 blockade Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 8 of 17 Table 2 Retrospective clinical studies with available results on the antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade (Continued) Study n Design Outcomes Toxicities Ref. RS 29 Metastatic NSCLC treated with Median PFS and OS of 3.8 mo (95% Possible treatment-related AEs: 1 [125] thoracic RT (10–70 Gy X 1–35 CI 1.9–8) and 9.2 mo (95% CI 5.1-not grade 5 pneumonitis and 2 grade 3 fractions) within 6 mo of PD-1/PD-L1 reached) pneumonitis and/or CTLA-4 blockade RS 133 Metastatic NSCLC, melanoma, and No significant difference in immune- All-grade immune-related AEs: 20% [127] RCC treated with palliative RT related AEs between those receiving dermatitis, 8% colitis, 5% transaminitis; (8–37.5 Gy X 1–15 fractions) within RT during/after checkpoint inhibitors grade ≥ 3 immune-related AEs: 4% 180 days of PD-1 or CTLA-4 inhibitor and before checkpoint inhibitors colitis, 2% transaminitis, 2% (p = 0.053), receiving RT within hypophysitis 14 days or outside 14 days of checkpoint blockade (p = 0.06), and of site of irradiation RS 137 Metastatic NSCLC, melanoma, and Median OS 249 days (IQR 90–689) No grade 4–5 immune-related AEs [120] RCC treated with WBRT (12–39 Gy), following PD-1 blockade; on SRS (15–30 Gy), or extracranial RT multivariate analysis HR for death 3.1 (8–66 Gy) within a median 85 days (95% CI 1.7–5.9) for NSCLC and HR (IQR 34–181) of anti-PD-1 therapy 3.2 (95% CI 1.2–7.9) for RCC vs. melanoma (p = 0.0008) RS 17 NSCLC BMs treated with SRS or FSRT Distant brain control rate 57% No neurologic/ cutaneous AEs with [128] (18–25 Gy X 1–5 fractions) within ±6 (RT during or before PD-1/PD-L1 SRS and anti-PD-1/PD-L1 therapy mo of nivolumab or durvalumab blockade) vs. 0% (RT after, p = 0.05); (41% received prophylactic median OS for SRS during/before dexamethasone before SRS); 1 PD-1/PD-L1 blockade vs. SRS after patient each discontinued (HR 3.6, 95% CI 0.74–26.9, p = 0.11) PD-1/PD-L1 inhibitor due to colitis on multivariate analysis and pneumonitis RS 137 Melanoma BMs treated with SRS or Median OS 16.9 mo; for See outcomes [129] WBRT (median 20 Gy, range 12–30) radionecrosis: 37 patients (27%); within 1 year of PD-1 or CTLA-4 no difference in risk between blockade ipilimumab and pembrolizumab (p = 0.549) or CTLA-4 and PD-1 (p = 0.86); 1-year OS 78.4% vs. 55.06% (without radionecrosis, p = 0.341) RS 98 Advanced NSCLC treated with Any previous RT vs. no previous RT: All-grade treatment-related pulmon- [121] palliative RT any time point before median PFS 4.4 mo (95% CI 2.1–8.6) ary toxicity in 3 patients (13%, with (median 9.5 mo, range 1–106) first vs. 2.1 mo (95% CI 1.6–2.3, HR 0.56, RT) vs. 1 (1% without RT, p = 0.046); cycle of pembrolizumab 2 or 95% CI 0.34–0.91, p = 0.019); median grade ≥ 3 treatment-related 10 mg/kg every 2–3 weeks OS 10.7 mo (95% CI 6.5–18.9) vs. 5.3 pulmonary toxicity similar in both mo (95% CI 2.7–7.7, HR 0.58, 95% CI arms (1 each, p = 0.44) 0.36–0.94, p = 0.026) RS 108 Melanoma BMs treated with SRS In combination with RT: median OS 2 radiation necrosis (SRS + anti-PD-1) [122] and/or WBRT (dose NR) within 7.5 mo with CTLA-4 (95% CI 4.4– treated with surgery, steroids, and ±6 weeks of various systemic 15.6), 20.4 mo PD-1 (95% CI 8.8-NA), bevacizumab therapies and 17.8 mo BRAF ± MEK inhibitor (95% CI 11.8-NA) RS retrospective study, BMs brain metastases, SRS stereotactic radiosurgery, FSRT fractionated stereotactic RT, Gy Gray, OS overall survival, NSCLC non-small cell lung cancer, IQR interquartile range, CNS central nervous system, RT radiotherapy, WBRT whole brain radiation therapy, ORR overall response rate, NR not reported, CI confidence interval, CR complete response, PR partial response, SD stable disease, PD progressive disease, AEs adverse events, RCC renal cell carcinoma, GI gastrointestinal, HR hazard ratio, PFS progression-free survival, NA not applicable not receive RT, respectively [121]. In spite of these inter- Safety analyses esting clinical results, no data are provided on the type, Retrospective safety analyses in patients with ad- dose, schedule of radiotherapy or the tumor burden of vanced solid tumors receiving RT and PD-1/PD-L1 patients receiving therapy making the results hard to in- and/or CTLA-4 blockade have generally not demon- terpret. Interestingly, one retrospective series of 108 pa- strated increased risk of toxicity with the combination tients with melanoma brain metastases treated with SRS beyond those expected with each modality independ- and/or WBRT concurrently with various contemporary ently [123, 124]. There were no significant differences systemic therapies highlighted that RT in combination in toxicity rates between choice of PD-1/PD-L1 and with anti-PD-1 therapy produced among the best OS in CTLA-4 inhibitor or concurrent and sequential treat- the cohort without clinically significant increases in ment with RT [124]. However, another series of 29 neurotoxicity [122]. metastatic NSCLC patients given thoracic RT and Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 9 of 17 PD-1/PD-L1 and/or CTLA-4 inhibitors identified 1 assessment at all irradiated sites and the best ORR was case of possibly treatment-related grade 5 pneumonitis in 44% (4 patients with partial responses (PRs)) by World a patient who received 20 Gy over 5 fractions of thoracic Health Organization (WHO) criteria (Table 3). A phase RT initiated 1 month after the last dose of anti-PD-1 ther- I/II study investigated the safety and efficacy of concur- apy [125]. Interestingly, case reports have documented the rent local palliative RT and durvalumab (PD-L1 inhibi- existence of PD-1 inhibitor-induced radiation recall pneu- tor) in 10 patients with unresectable or metastatic monitis even after 2 years of RT [126]. advanced solid tumors [136]. When RT (to 15 localized A multicenter safety analysis demonstrated no signifi- lesions) was given a median of 8.5 days (range 1–35) cant differences in immune-related AEs regardless of site from the last dose of durvalumab, the combination was of irradiation, between those receiving RT during/after generally tolerated with no grade ≥ 3 RT-related AEs checkpoint inhibitors and before checkpoint inhibitors (Table 3). The 1-year OS and progression-free survival (p = 0.053), and between those receiving RT within 14 days (PFS) rates were 44% (95% CI 12–77) and 30% (95% CI or outside 14 days of checkpoint blockade (p =0.06) [127]. 2–58), respectively. One retrospective series demonstrated that brain RT and Preliminary results from a phase I dose-finding study PD-1/PD-L1 blockade was relatively well-tolerated in pa- of stereotactic body RT (SBRT; 8 Gy X 1 or 5 Gy X 5) tients with NSCLC brain metastases as toxicity rates were and durvalumab or the CTLA-4 inhibitor tremelimumab consistent with those seen with checkpoint inhibitors (or combination of all 3) was administered as alone [128]. Interestingly, the distant brain control (out-- second-line therapy to 24 metastatic pancreatic adeno- of-field) rate for RT during/before PD-1/PD-L1 blockade carcinoma patients. No DLTs have been observed so far was 57% compared to 0% (RT after, p = 0.05). Another [137]. The best response was SD in 5 patients (21%) with retrospective series of 137 patients with melanoma brain rapid progression within 4 weeks in an additional 5 pa- metastases identified 37 patients (27%) who developed tients. A phase II trial involving locally advanced NSCLC radionecrosis following SRS or WBRT and anti-CTLA-4 patients recently reported preliminary results from part I or anti-PD-1 therapy with a median time of onset of of the study [138]. Out of 10 enrolled patients, 7 have 6months (range1.3–31.4 months), which is comparable received atezolizumab added to consolidation carbopla- to rates seen in other series though prospective studies are tin and paclitaxel following weekly carboplatin/paclitaxel limited [129–132]. Notably, 1-year OS did not signifi- and RT and 2 patients have demonstrated PD after 6 and cantly differ between those that developed radionecro- 8 doses of the PD-L1 inhibitor. Given the safety and sis vs. those without (Table 2). However, risk of tolerability of patients in part I, criteria were met for ad- radionecrosis was significantly associated with concur- vancement to part II of the study where atezolizumab rent use of chemotherapy within 6 months of SRS will be added to the chemoradiation portion followed by (HR 2.20, 95% CI 1.22–3.97, p = 0.009) and increased consolidation atezolizumab, carboplatin, and paclitaxel. number of lesions treated (HR 1.09, 95% CI 1.03– Recently, the PD-L1 inhibitor durvalumab was granted 1.15, p = 0.002). The lack of significant difference in FDA approval based on superior PFS but similar safety OS between presence and absence of radionecrosis compared to placebo following platinum-based chemo- conflicts with the results of other studies though the radiation in locally advanced, unresectable NSCLC in number of patients treated with brain RT and PD-1 the phase III PACIFIC trial [139]. Patients who did not blockade were likely much smaller [130, 133]. demonstrate PD after ≥2 cycles of platinum-based chemotherapy concurrent with definitive RT were ad- Prospective studies ministered durvalumab or placebo within 1–42 days for A combined preclinical and phase I study was among the up to 1 year (Table 3). Improved outcomes were ob- first to provide preliminary results for the efficacy of com- served in the experimental arm irrespective of PD-L1 bined RT and checkpoint blockade in the prospective set- status or histology. ting [134]. In the phase I dose-finding cohort of 5 patients given local RT for mixed response or asymptomatic pro- Discussion gression to atezolizumab, dual RT and anti-PD-L1 therapy Elucidated mechanisms underlying the immune stimula- was well-tolerated without any dose-limiting toxicities tory properties of RT are growing in complexity (Fig. 1). (DLTs) or severe immune-mediated AEs and all 5 patients The CD8+ T-cell remains a crucial component in the experienced at least SD (Table 3). ability of RT to elicit an antitumor immune response In another phase I trial, 9 patients with advanced mel- within and beyond the radiation field [140]. In addition, anoma received RT during induction, between induction evidence is mounting to support that RT specifically and maintenance, or during maintenance therapy with upregulates MHC tumor-associated antigen complexes, ipilimumab and/or nivolumab [135]. Combined RT and enhances tumor antigen cross-presentation in draining checkpoint inhibition resulted in SD or response by first lymph nodes, and increases T-cell infiltration into Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 10 of 17 Table 3 Prospective clinical studies with available results on the antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade Study n Design Outcomes Toxicities Ref. Phase I 4 solid tumors, 1 Atezolizumab 0.01–20 mg/kg Stabilization of systemic Transient grade 1–2 [134] hematologic every 3 weeks (dose-finding progression in all 5 patients inflammatory AEs (fevers, malignancy cohort) + local fractionated RT (PR at systemic site in 1 flu-like symptoms) observed (dose NR) for mixed responses or patient) but no DLTs or serious asymptomatic PD immune-related AEs Phase I 9 advanced Nivolumab 0.3–10 mg/kg every ORR 44% (4 PRs) as best 5 patients with non- [135] melanoma 3 weeks X 21 weeks (induction) response by WHO criteria; laboratory grade ≥ 3 AEs, 2 then every 12 weeks X 84 weeks median OS 27 mo; 1- and RT-related grade ≥ 3 AEs (maintenance) ± ipilimumab 3 or 2-year OS rates of 89 and (intracranial hemorrhage, 10 mg/kg every 3 weeks X 78%, respectively diarrhea) 9 weeks (induction) then every 12 weeks X 84 weeks (maintenance) or combined nivolumab 1 mg/kg and ipilimumab 3 mg/kg every 3 weeks X 12 weeks then nivolumab 3 mg/kg every 2 weeks up to 96 weeks + RT (median 30 Gy X 5 fractions, range 21–37.5 Gy X 1–15 fractions) during induction or maintenance Phase I/II 10 unresectable or Durvalumab 10 mg/kg every In-field ORR 60% (2/10 CRs, 5 cases of (50%) RT-related [136] metastatic solid 2 weeks + local RT (median 20 Gy, 4/10 PRs); median OS 11.5 grade 2 AEs (3 mucositis, tumors (≥5% PD-L1 range 6–33 X median 5 fractions, mo (95% CI 8.8–13.7); median 1 diarrhea, 1 vomiting) expression) range 1–10) given a median of PSF 6.2 months (95% CI 8.5days (range1–35) of last dose 4.5–12.4); out-of-field 10/14 of durvalumab SD, no responses or abscopal effects were seen Phase I 24 metastatic SBRT (8 Gy X 1 fraction or 25 Gy SD as best ORR in 5 patients No DLTs observed; most [137] pancreatic X 25 fractions) + durvalumab (21%) common AE was grade 1–2 adenocarcinoma (dose NR) every 2 weeks or fatigue at dose level 2 tremelimumab (dose NR) every 4 weeks X 6 doses then every 12 weeks for 3 doses or triple therapy Phase II 10 locally advanced Weekly carboplatin (AUC 2) and Out of 7 patients receiving 3 patients with potential [138] NSCLC weekly paclitaxel 50 mg/m +RT atezolizumab, 2 patients immune-related AEs (1 grade 5 days/week for 6–7 weeks developed PD after 6 and 8 3 arthralgia, 1 grade 2 (60–66 Gy over 30–33 fractions) doses of atezolizumab pneumonitis resolved with followed by atezolizumab steroids, 1 grade 3 dyspnea) 1200 mg every 3 weeks + consolidation carboplatin (AUC 6) and paclitaxel 200 mg/m on days 1 and 22 for 2 cycles then atezolizumab alone up to 1 year Phase III 709 stage III, locally 2 or more cycles of platinum- Median PFS 16.8 months Grade 3–4 AEs 29.9% vs. [139] advanced, based chemotherapy (defined by (95% CI 13.0–18.1) vs. 26.1% (placebo); most unresectable NSCLC local practice) + concurrent 5.6 months (95% CI 4.6–7.8) common grade 3–4 AEs definitive RT (54–66 Gy with with placebo (HR 0.52, 95% CI pneumonia (4.4% vs. 3.8%), mean dose to the lung < 20 Gy 0.42–0.65, p <0.001); median pneumonitis (3.4% vs. 2.6%), or volume of lung parenchyma TTD or distant metastasis and anemia (2.9% vs. 3.4%) in receiving ≥20 Gy < 35%) 23.2 months (95% CI 23.2-NE) durvalumab vs. placebo arms followed by (within 1–42 days) vs. 14.6 months (95% CI durvalumab 10 mg/kg every 10.6–18.6) with placebo 2 weeks up to 1 year or placebo (HR 0.52, 95% CI 0.39–0.69, if no PD during chemoradiation p < 0.001); ORR 28.4% vs. 16.0% with placebo (p <0.001) RT radiation therapy, NR not reported, PD progressive disease, PR partial response, DLT dose-limiting toxicity, AEs adverse events, Gy Gray, ORR overall response rate, PR partial response, WHO World Health Organization, CI confidence interval, SD stable disease, SBRT stereotactic body radiation therapy, NSCLC non-small cell lung cancer, AUC area under curve, CR complete response, PFS progression-free survival, HR hazard ratio, TTD time to death, NE not estimable or reached Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 11 of 17 tumors [79, 141]. Local RT appears necessary in eliciting Several points of consideration remain that could po- abscopal effects, but RT alone remains insufficient in tentially impact the rational combination of RT and complete eradication of local and distant tumors likely, PD-1/PD-L1 inhibitors and their efficacy. Firstly, im- in part, due to activation of negative T-cell regulatory munogenic cell death has been shown to be induced by pathways including the PD-1/PD-L1 axis and immune RT in a dose-dependent manner in vitro [68]. In other checkpoints such as CTLA-4 [76, 86, 87]. However, RT preclinical studies, increasing radiation doses (single has been shown to upregulate expression of PD-1 and fractions above 7.5 Gy but not 5 Gy) were immunosti- PD-L1 on immune and tumor cells rendering it an at- mulatory, associated with elevated IFN-γ production, tractive modality to combine with PD-1/PD-L1 blockade and prevented increases in Tregs [143]. At higher doses [71, 76, 78, 79, 86, 97]. Activation of cGAS-STING sig- (single fractions ≥15 Gy), dose-dependent increases in naling has also been recognized to mediate systemic Tregs were observed and associated with no improve- tumor rejection by combined RT and checkpoint block- ment in antitumor immune responses. Fractionation of ade given that knockdown of cGAS and STING in can- the 15 Gy generally resulted in superior immune re- cer cells abrogated priming of CD8+ T-cells in sponses compared to single-fraction 15 Gy. In a seminal tumor-draining sites and infiltration of abscopal tumors study of 2 preclinical mouse carcinoma models, evalu- by CD8+ T-cells [89]. ation of RT (20 Gy X 1, 8 Gy X 3, or 6 Gy X 5 fractions In efforts to characterize the synergistic antitumor ac- over consecutive days) in combination with an tivity of combined RT and PD-1/PD-L1 blockade, nu- anti-CTLA-4 antibody determined that fractionated RT merous studies have identified significant elevations in but not single-dose RT achieved significantly enhanced CD8+ IFNγ+ TNFα+ T-cells but decreases in CD4+ tumor responses both within and outside the radiation FOXP3+ Tregs leading to an increased CD8+/Treg ratio, field (abscopal effects) when combined with CTLA-4 increases in tumor-antigen specific CD8+ TILs with a blockade [55]. It has been further corroborated that frac- CD44+ effector memory phenotype, decreases in im- tionated RT (8 Gy X 3) with checkpoint blockade was munosuppressive MDSCs, reinvigoration of CD8+ TILs able to elicit abscopal effects whereas checkpoint block- with an exhausted phenotype, and increases in TCR rep- ade with RT doses ≥20 Gy single dose were character- ertoire clonality and diversity of the TCR repertoire in ir- ized by complete loss of abscopal responses through radiated and out-of-field sites as a consequence of induction of Trex1 and downregulation of type I IFN combination radioimmunotherapy [61, 72, 76, 79, 88]. signaling [89]. Furthermore, addition of anti-PD-L1 therapy to tumors The timing of RT in relation to administration of that are nonresponsive to RT has shown the ability to checkpoint inhibitors represents another issue of discus- reverse RT-induced tumor equilibrium in favor of tumor sion. Preclinical data support that RT-associated in- regression [92]. Resistance to RT also appears to be creases in the CD8+ T-cell/Treg ratio, CD8+ T-cell regulated by host STING activation via CCR2; add- PD-1 expression, and tumor cell PD-L1 expression often itional targeting of the CCR2 pathway may therefore occur early with peak levels occurring within 24–96 h aid in reversing RT resistance in the context of post-RT [81, 86]. In an elegant study exploring combined checkpoint blockade [93]. Conversely, integration of anti-PD-L1 therapy and fractionated RT (10 Gy in 5 daily RT to anti-PD-1-resistant tumors restores response to fractions), the addition of PD-L1 blockade on day 1 of RT PD-1 blockade highlighted by RT-induced IFN-γ pro- (concurrent regimen starting at the beginning of RT), day duction and MHC class I expression [91]. 5 of RT (concurrent regimen starting at the end of RT), or Immune modulation from immune checkpoint in- 7 days after RT (sequential therapy) showed that there hibitors and RT through nonredundant pathways that was no significant difference in OS between either concur- altogether contribute to synergistic antitumor activity rent therapy schedules [86]. However, sequential therapy now represents an emerging theme from ongoing in- was ineffective in enhancing OS compared to RT alone vestigations in combination RT and immunotherapy (median OS 30 days vs. 35 days, p > 0.05). Interestingly, [61, 77, 85, 88, 90, 142]. For example, anti-CTLA-4 the rise in PD-1 expression on CD8+ T-cells was evident therapy has been shown to predominantly inhibit Tregs, up to 7 days after the last dose of RT, after which PD-1 increase the CD8+ T-cell/Treg ratio, and promote T-cell levels significantly decreased compared to time-matched expansion. Radiation enhances the diversity of the TCR controls. In the clinical setting, retrospective series have repertoire, shapes the TCR repertoire of expanded periph- documented a wider range of schedules in combining eral T-cell clones in an antigen-driven selection manner, radioimmunotherapy ranging from RT at any point prior and promotes tumor infiltration by antigen-specific CD8+ to immune checkpoint therapy, within 1 month of admin- T-cells. Addition of PD-1/PD-L1 blockade reverses T-cell istration of checkpoint inhibitors, or up to 1 year of check- exhaustion to offset decreases in the CD8+ T-cell/Treg ra- point blockade [117, 121, 124, 129]. Moreover, results tio and further enhances oligoclonal T-cell proliferation. have been largely mixed on the impact of scheduling of Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 12 of 17 RT and checkpoint blockade on survival as several retro- the limited number of larger prospective trials, PD-1 and spective studies have identified that there is no significant PD-L1 blockade have often been incorporated into stand- difference in OS between concurrent and nonconcurrrent ard dosing regimens of SBRT and chemoradiation rou- radioimmunotherapy while another study demonstrated a tinely used in the treatment of locally advanced pancreatic significant improvement in PFS and OS in patients having cancer and NSCLC, for example (Table 3). ever received RT prior to PD-1 blockade compared to It is worthwhile to mention that the Phase III PACIFIC those with no prior RT [116, 117, 121]. It is worthwhile to trial demonstrated the superiority of chemoradiation mention that these retrospective studies were likely lim- followed by durvalumab when the latter was included ited by variability in RT modality, tumor histology, patient within 1–42 days of chemoradiation over chemoradia- characteristics, and cohort size. Notably, abscopal effects tion followed by placebo in locally advanced NSCLC have been observed in 56% of patients with the addition of [139]. On review of the study protocol and Supplementary late RT to PD-1 blockade as well (> 3 months of insuffi- Appendix, the investigators emphasized the initiation of cient response to anti-PD-1 monotherapy) [119]. durvalumab as close as possible to chemoradiation when Another point of consideration in clinical trial design is antigen release and PD-L1 expression is likely to be at its the issue of toxicity with combined RT and PD-1/PD-L1 greatest. An analysis of benefit in those receiving durvalu- blockade. Several preclinical studies demonstrated more mab closer to chemoradiation compared to those treated findings of abnormal alveoli, inflammatory changes, exu- later relative to chemoradiation was not provided; an ana- dates in the alveolar septa, and cardiac toxicity in mice re- lysis of this nature may provide further insight on the pro- ceiving thoracic RT and anti-PD-1 therapy, when posed synergism offered by this combination. For reasons compared to controls, though effects on survival have which are unclear, the median PFS of the placebo arm been mixed [100–102]. Retrospective analyses have gener- (5.6 months) appears worse than historical standards ally shown no increased risk of toxicity with the combin- [145]. It is also unclear whether the benefit derived from ation of RT and checkpoint blockade beyond those the combination arm is due to the efficacy of immuno- expected with either modality alone [121, 124, 127]. For therapy in settings of smaller disease volume as seen pre- brain RT, a study of 137 patients treated with SRS or viously [146]. All of these are potential factors that may WBRT in combination with PD-1 or CTLA-4 blockade contribute to the difference seen in efficacy between ex- identified radionecrosis in 27% though 1-year OS did not perimental and control arms. significantly differ between those that developed radione- Despite the promising results and feasibility of the PA- crosis and those that did not [129]. Reassuringly, retro- CIFIC trial, clinical studies on an upper threshold RT spective series of > 200 patients receiving combined RT dose with checkpoint inhibition by which no further im- and immunotherapy have demonstrated that there are no provement in antitumor immunity is offered (as foresha- significant differences in toxicities regardless of site of ir- dowed by preclinical evidence discussed previously) are radiation, choice of checkpoint inhibitor, or treatment virtually nonexistent, yet duly warranted. Dedicated schedule (concurrent vs. sequential) [124, 127]. dose-escalation studies on combination PD-1/PD-L1 in- Taking together the preclinical evidence on the kinet- hibitors and RT are also needed in other tumor types to ics of PD-1 and PD-L1 expression in relation to RT and determine safety and tolerability. Early phase studies of the clinical data on the safety and tolerability of radioim- this nature are emerging and have demonstrated the munotherapy, there is growing evidence to support that feasibility of this combination while recognizing the im- PD-1/PD-L1 blockade is optimal when synchronized portance of timing of checkpoint blockade with respect with the administration of fractionated RT to prevent to RT administration [147]. Extrapolation of RT dose the development of immunological anergy [144]. Indeed, effects from animal to human studies is not straightfor- the concept of administering PD-1/PD-L1 inhibitors ward and great caution is needed in applying dosing concurrently or immediately following fractionated RT schemes and regimens involving combination RT and has already been employed in clinical trials with evi- PD-1/PD-L1 blockade in human patients [148]. Further dence that the combination is generally well-tolerated understanding of the mechanistic and dynamic immunos- (Table 3). However, despite our increased understanding, timulatory properties of RT and PD-1/PD-L1 blockade are preclinical and clinical data have yet to offer a consensus undoubtedly warranted with validation in (ideally) pro- on optimal dosing and modality sequencing to date [68]. spective cohorts prior to maximizing tumor responses The majority of retrospective and prospective studies on with the combination. The ability to optimize immune combination RT and checkpoint blockade have predomin- responses in the future with radioimmunotherapy may antly used fractionated dosing schemes (Tables 2 and 3). potentially depend on the immunotherapeutic strategy However, depending on the tumor type, target site, and used, tumor histology, balance between proimmuno- modality employed, total RT doses from retrospective genic and immunosuppressive effects of either modal- series have ranged widely from 8 to 74 Gy (Table 2). Of ity, and other host factors [50, 148]. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 13 of 17 Lastly, phase I trials of RT and anti-PD-1 therapy have Received: 26 December 2017 Accepted: 16 May 2018 already provided glimpses into potential mechanisms of failure even with the combination as 1 patient with metastatic RCC who rapidly progressed on combined References 1. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. 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Radiation therapy and PD-1/PD-L1 blockade: the clinical development of an evolving anticancer combination

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Abstract

Several inhibitors of programmed cell death-1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved as a form of immunotherapy for multiple cancers. Ionizing radiation therapy (RT) has been shown to enhance the priming and effector phases of the antitumor T-cell response rendering it an attractive therapy to combine with PD-1/PD-L1 inhibitors. Preclinical data support the rational combination of the 2 modalities and has paved way for the clinical development of the combination across a spectrum of cancers. In this review, we highlight the preclinical and clinical development of combined RT and PD-1/PD-L1 blockade to date. In addition to a comprehensive evaluation of available safety and efficacy data, we discuss important points of consideration in clinical trial design for this promising combination. Keywords: Radiation therapy, PD-1, PD-L1, Clinical trials, Preclinical, Antitumor, Immune response, Checkpoint inhibitor Background tumor response with associated regression of untreated Early preclinical evidence demonstrated that activation metastases outside of the radiation field has been of the programmed cell death 1 (PD-1) and programmed reported and was first described as the abscopal effect death ligand 1 (PD-L1) axis suppressed the activation [44]. Increasing evidence supports that the abscopal ef- and proliferation of tumor antigen-specific T-cells and fect is likely immune-mediated – largely, in a T-cell promoted tumorigenesis [1, 2]. These processes were dependent manner with a complex interplay between reversed with PD-1/PD-L1 blockade and supported proimmunogenic and proinflammatory factors [45–53]. the concept of PD-1/PD-L1 blockade as a potential Over time, recognition of the immunomodulatory prop- form of anti-cancer immunotherapy. The first agents erties of radiation has led to the integration of RT with in the family of PD-1/PD-L1 inhibitors to be ap- immune-modulating agents including immune check- proved by the Food and Drug Administration (FDA) point inhibitors to potentially develop a combination were the humanized monoclonal IgG4 antibodies, therapy with enhanced or synergistic anticancer activity pembrolizumab and nivolumab, that targeted PD-1 in (Fig. 1). unresectable or advanced melanoma [3–10]. There are Indeed, an initial preclinical study showed that com- currently 5 PD-1/PD-L1 inhibitors approved by the bining RT (1–2 fractions of 12 Gray (Gy) to the primary FDA for the treatment of a number of solid tumors tumor) with an anti-cytotoxic T lymphocyte-associated and hematologic malignancies [11–43]. antigen-4 (CTLA-4) monoclonal antibody resulted in Ionizing radiation therapy (RT) is widely used in the synergistic antitumor activity in a poorly immunogenic definitive and metastatic setting for local tumor control; metastatic mammary carcinoma mouse model when however, the ability of radiation to elicit a systemic CTLA-4 blockade by itself was ineffective [54]. Enhanced antitumor responses have also been observed across several preclinical animal models treated with * Correspondence: richard.tuli@cshs.org combined RT and CTLA-4 blockade [55–58]. Since the Departments of Radiation Oncology and Biomedical Sciences, Samuel first preclinical studies that highlighted the synergistic Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, AC 1023, Los Angeles, CA 90048, USA antitumor activity of combination RT and CTLA-4 Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 2 of 17 Fig. 1 Proposed mechanisms of synergy between RT and PD-1/PD-L1 inhibitors. Emerging evidence demonstrates that immune modulation from PD-1/PD-L1 inhibitors and RT through nonredundant pathways contributes to synergistic antitumor activity, thereby forming the basis for the rationale combination of the two modalities. RT, radiation therapy; PD-1, programmed cell death 1 receptor; PD-L1, programmed death ligand 1; IFN-γ, interferon-γ; cGAS, cyclic GMP-AMP (cGAMP) synthase; STING, stimulator of interferon genes; MHC, major histocompatibility complex; TCR, T-cell receptor; TILs, tumor-infiltrating lymphocytes, Tregs; regulatory T cells; MDSCs, myeloid-derived suppressor cells blockade, several prospective clinical trials have re- Preclinical studies ported on the activity of RT and ipilimumab in ad- The efficacy of combination RT and checkpoint blockade vanced solid tumors [59–66]. Similarly, there are is associated with modulation of immune parameters numerous ongoing clinical trials investigating the within the tumor microenvironment combination of RT and CTLA-4 blockade that have Early investigations in mouse models of solid and been extensively reviewed and are beyond the scope hematologic malignancies showed enhanced antitumor of this manuscript [67, 68]. Herein, we review in effects when treated with PD-1 or PD-L1 blockade in detail the preclinical and clinical development of the combination with in-field RT, sublethal total body combination of RT and PD-1/PD-L1 inhibitors in can- irradiation (TBI), or stereotactic radiosurgery (SRS) cer therapy. compared to single modality treatment (Table 1)[69–85]. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 3 of 17 Table 1 Preclinical studies demonstrating antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade Cell line Experimental model RT dose PD-1/PD-L1 dose Ref. B16-D5 (melanoma) Mice subcutaneous TBI 600 cGy PD-L1 mAb 20 mg/kg IP starting on [69] (1 fraction) day 4 then every 3–4 days +1X10 gp100 or OVA pulsed dendritic 257–264 cell vaccine SC on day 4 and 11 ± 1X10 pmel T-cells (adoptive transfer) IV on day 4 after inoculation AT.3 (triple-negative Mice xenograft 12 Gy (1 fraction) or PD-1 mAb 100 μg + CD137 mAb [70, 71] mammary) 4–5 Gy (4 fractions) 100 μg IP on days 0, 4, 8, and 12 of RT GL261 (glioma) Mice xenograft 10 Gy (1 fraction) PD-1 mAb 10 mg/kg IP on days 10, [72] 12, and 14 of RT B16-SIY (melanoma) TUBO Mice subcutaneous 25 Gy (2 fractions) PD-L1 mAb 200 μg IP every 3 days [92] (mammary) 15 Gy (1 fraction) for 4 doses starting 3 weeks after RT 5 T33 (myeloma) Mice intravenous TBI 500 cGy PD-L1 mAb 200 μg IP on days 12, 14, [75] A20 (B-cell lymphoma) (1 fraction) 17, 19, 21, 26, and 28 after C1498 (leukemia) inoculation 5 T33 (myeloma) Mice intravenous TBI 1100 cGy HSCT on day 0 + PD-L1 mAb 200 μg [73] (1 fraction) IP on days 3, 5, 10, 12, 17, and 19 after HSCT ± vaccine (irradiated 5 T33 cells or 5 T33 cells transfected with empty vectors) on days 3, 10, and 17 after HSCT 5 T33 (myeloma) Mice intravenous TBI 500 cGy PD-L1 mAb 200 μg IP on days 12, 14, [74] (1 fraction) 17, 19, 21, 26, and 28 after inoculation ± LAG-3, TIM-3, or CTLA- 4 mAbs 200 μg IP on same days V600E CT26 (colon 4434 (BRAF - Mice subcutaneous 10 Gy (5 fractions) PD-1 or PD-L1 mAb 10 mg/kg IP 3 [86] mutant melanoma) times weekly up to 3 weeks starting 4 T1 (triple-negative on day 1 of RT mammary) TUBO (mammary) Mice subcutaneous 12 Gy (1 fraction) PD-L1 mAb 200 μg IP every 3 days [76] MC38 (colon for 4 doses starting on day 0 or 1 of RT TSA (mammary) Mice subcutaneous 24 Gy (3 fractions) PD-1 mAb (dose NR) starting on day [77] 15 after inoculation and every 4 days thereafter B16-OVA (melanoma) Mice subcutaneous 15 Gy (1 fraction) PD-1 mAb 10 mg/kg ± CTLA-4 mAb [87] RENCA (renal) 10 mg/kg IP on days 7, 9, 11, 14, and 16 following tumor cell inoculation B16-OVA (melanoma) Mice subcutaneous 12 Gy (1 fraction) PD-1 mAb 200 μg IP every 3 days for [79] 4 T1-HA (mammary) 3 doses starting 1 day prior to RT PyMT (mammary) Mice subcutaneous 12 Gy (1 fraction) PD-1 mAb dose NR + single dose of [78] CTLA-4 mAb (dose NR) 3 days prior to PD-1 and RT B16-F10 (melanoma) Mice subcutaneous 20 Gy (1 fraction) PD-L1 mAb 200 μg + CTLA-4 mAb [61] 200 μg IP every 3 days for 3 doses starting 5 or 9 days after inoculation Meer (head and neck Mice subcutaneous 1, 6, 10 Gy fractions PD-L1 antibody dose NR [82] squamous) Adeno-Cre viral vector (lung) GEMM intrathoracic 8.5 Gy twice daily PD-1 mAb 200 μg IP 3 times weekly [81] injection over 2 days starting 6 h after second RT dose MB49 (bladder) Mice xenograft 12 Gy (1 fraction) PD-L1 mAb 250 μg IP twice weekly [84] for 4 doses starting 1 day prior to RT MC38 (colon Mice subcutaneous 24 Gy (3 fractions) PD-1 mAb ± CD137 mAb 5–10 mg/ [83] 4 T1 (mammary) B16F10-OVA kg IP on days 13, 15, and 17 after (melanoma) inoculation Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 4 of 17 Table 1 Preclinical studies demonstrating antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade (Continued) Cell line Experimental model RT dose PD-1/PD-L1 dose Ref. 4-hydroxytamoxifen GEMM topical 14 Gy (1 fraction) PD-1 + CD137 or PD-1 + CTLA-4 mAb [99] induction (BRAF-mutant, induction 100 μg IP twice weekly for 4 doses PTEN-deficient melanoma) on day 1 of RT 344SQ (lung) Mice subcutaneous 36 Gy (3 fractions) PD-1 mAb 10 mg/kg IP starting on [91] day 1 of RT and continued for additional 3–4 doses ARK (esophageal squamous) Mice subcutaneous 20 Gy (10 fractions) PD-1 mAb (dose NR) starting 2 days [85] before RT and every 3 days thereafter ± carboplatin and paclitaxel IP (dose NR) on day 1 of RT and every 3 fractions GL261 (glioma) Mice xenograft 10 Gy (1 fraction) PD-1 mAb 200 μg IP on days 10, 12, [90] and 14 of RT ± TIM-3 mAb 250 μgIP days 7, 11, and 15 of RT V600E CT26 (colon 4434 (BRAF - Mice subcutaneous 10 Gy (5 fractions) PD-1 or PD-L1 mAb 10 mg/kg IP 3 [88] mutant melanoma) times weekly for 1 week starting on day 1 of RT TSA (mammary) Mice subcutaneous 24 Gy (3 fractions) on PD-1 mAb 200 μg IP on days 12, 15, [89] days 12, 13 and 14 19, 22 and 26 after inoculation after inoculation Hep-55.1c (hepatocellular) Mice orthotopic 30 Gy (3 fractions) PD-1 mAb 250 μg IP on days 7, 14, [96] and 21 after inoculation KPC and Pan02 (pancreatic) Mice subcutaneous 6, 12, or 20 Gy PD-L1 mAb 10 mg/kg IP on days 4, [95] (1 fraction) 7, 10, and 13 after inoculation + 10 Gy (5 fractions) gemcitabine 100 mg/kg IP on days 0 15 Gy (5 fractions) and 3 of inoculation HCa-1 (hepatocellular) Mice intramuscular 10 Gy (1 fraction) PD-L1 mAb 10 mg/kg IP every [97] 3 days for 4 doses starting on day 1 of RT LM8 (osteosarcoma) Mice subcutaneous 10 Gy (1 fraction) PD-L1 mAb 150 μg + CTLA-4 mAb [98] 150 μg IP every 3 days for 3 doses starting on days 9, 12, and 15 after inoculation CT26 (colon Mice intradermal RFA 17-gauge single PD-1 mAb 200 μg IP every 3 days for [94] ablation electrode for 4 doses 3.5–4.5 min at target temperature of 70 degrees C RT radiation therapy, TBI total body irradiation, cGy centigray mAb monoclonal antibody, IP intraperitoneal, SC subcutaneous, IV intravenous, Gy Gray, HSCT hematopoietic stem cell transplantation, LAG-3 lymphocyte-activation gene 3, TIM-3 T-cell immunoglobulin mucin-3, NR not reported, GEMM genetically engineered mouse model, RFA radiofrequency ablation Combined modality therapy was associated with higher Combination modality-induced immune profile changes levels of CD8+/interferon-γ (IFNγ)+/tumor necrosis may be time-dependent factor-α (TNFα) + cytotoxic T-cells, increased PD-1, T-cell Early syngeneic mouse tumor models demonstrating immunoglobulin mucin-3 (TIM-3), lymphocyte-activation significant improvements in survival and tumor volume gene 3 (LAG-3), and 2B4 (immune checkpoints) expres- reduction with the combination of RT and PD-1 or sion on CD8+ T-cells, decreased numbers of CD4 PD-L1 blockade compared to single modality and con- +/FOXP3+ regulatory T-cells (Tregs) and myeloid-derived trol arms identified elevations in tumor cell PD-L1 ex- suppressor cells (MDSCs), upregulation of PD-L1 on pression that were CD8+ T-cell and IFNγ-dependent dendritic cells and tumor cells in irradiated tumors, following irradiation (10 Gy over 5 daily fractions) com- RT-induced upregulation of major histocompatibility pared to non-irradiated mice with peak levels occurring complex (MHC) class I tumor-associated antigen 72 h after last dose of RT [86]. RT-induced increases in complexes, and enhanced antigen cross-presentation in the CD8+/Treg ratio and PD-L1 expression occurred draining lymph nodes compared to single modality 24–96 h post-RT in a separate mouse model [81]. In arms [71, 72, 74, 76–79]. colon carcinoma tumors, the addition of PD-L1 Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 5 of 17 blockade on day 1 of RT (schedule A), day 5 of RT cell-intrinsic activation of the type I IFN pathway as medi- (schedule B), or 7 days after RT (schedule C) showed ated by cyclic GMP-AMP (cGAMP) synthase (cGAS) and that there was no significant difference in overall sur- stimulator of interferon genes (STING) signaling [89]. vival (OS) between schedule A and B (p > 0.05) though RT-induced abscopal responses with PD-1 blockade were sequential therapy (schedule C) was ineffective in enhan- additionally shown to be regulated by Trex1 where induc- cing OS compared to RT alone (median OS 30 days vs. tion of Trex1 expression in cancer cells resulted in loss of 35 days, p > 0.05) [86]. Notably, PD-1 expression was sig- abscopal responses in mice treated with the combination. nificantly decreased on CD8+ T-cells 7 days after RT compared to time-matched controls (p < 0.05). Combined modality therapy reverses T-cell exhaustion Abscopal effects and systemic immunity and resistance to RT and anti-PD-1 therapy On subcutaneous tumor flank rechallenge of Murine tumor xenografts have shown that increasing treatment-naïve mice and mice cured by combination levels of PD-1 and TIM-3 co-expression in CD4+ RT and checkpoint blockade, immunologic memory was T-cells, CD8+ T-cells, and Tregs over time contribute to established in cured mice but not in treatment-naïve an exhausted or impaired T-cell phenotype [90]. mice suggesting that the immune system in cured mice Furthermore, resistance to anti-PD-1 therapy in retained the ability to recognize tumor-associated RT-refractory tumors has been characterized by signifi- antigens and mount an immune response of greater cant elevations in expression of genes associated with magnitude and speed upon rechallenge, i.e., systemic im- T-cell exhaustion, increased levels of checkpoints includ- munity [71, 72]. Abscopal effects have been shown to be ing LAG-3, TIM3, and CTLA-4 on CD4+ T-cells, and mediated, in part, by PD-1 as administration of a single decreased number of CD11c + tumor-associated macro- fraction of 15 Gy by stereotactic ablative radiotherapy phages (TAMs) [81]. The addition of immune check- (SABR) to the primary tumor in a melanoma subcutane- point inhibitors to RT has been shown to enhance ous mouse model resulted in significant reduction in tumor response compared to controls across several tumor volumes of secondary nonirradiated tumors in mouse tumor models through reinvigoration of PD-1-knockout mice compared to PD-1-wild-type (WT) exhausted CD8+ TILs characterized by increased Ki67+ mice [87]. Addition of a PD-1 inhibitor to SABR resulted GzmB+ T-cells within the exhausted PD-1+ Eomes+ in synergistic antitumor activity on the primary tumor T-cell pool, increased CD8+ CD44+ TILs, and increased compared to PD-1 inhibitor or SABR alone and recapit- CD8+/Treg ratio [61, 77, 85]. ulated abscopal effects on secondary nonirradiated tu- Moreover, an anti-PD-1-resistant murine lung cancer mors in PD-1-WT mice when treatment alone with model established through sequential in vivo passage of anti-PD-1 or SABR did not reduce secondary tumor nonresponsive tumors to ongoing anti-PD-1 therapy was growth. Furthermore, following RT, higher levels of characterized by significant downregulation of MHC high PD-1+ CD11a CD8+ T-cells were seen in primary tu- class I and II genes including β2-microglobulin and mors compared to secondary tumors and higher levels reduction in CD4+/CD8+ TILs and IFN-γ production in in irradiated compared to nonirradiated tumors; this resistant tumors compared to parental tumors [91]. population of cells appeared to comprise the principal Addition of RT induced IFN-γ production and MHC tumor-specific reactive phenotype. This latter finding class I expression and ultimately restored response to has been confirmed in another study where RT in- PD-1 blockade in resistant tumors. Addition of a PD-L1 creased T-cell receptor (TCR) repertoire clonality and inhibitor has been shown to reverse RT-induced tumor diversity of the TCR repertoire in irradiated tumors equilibrium in favor of tumor regression in mice sub- compared to controls, however, the addition of PD-1 in- cutaneously injected with melanoma and breast tumors hibition to RT increased TCR diversity both in irradiated demonstrating RT-induced stable disease (SD, defined as and out-of-field sites [88]. Further analysis revealed that ≥3 weeks) characterized by a transient rise and fall in most of these TCR clones arose from progenitor clones levels of tumor-infiltrating CD8+ T-cells and IFNγ [92]. that were established in tumors prior to therapy, and it Extrinsic RT resistance has been recently shown to be is the influx of tumor-infiltrating lymphocytes (TILs) contributed by RT-induced host STING activation from outside the tumor along with resident-tumor resulting in immunosuppressive MDSC recruitment that infiltrating T-cells that contribute to the enhanced is mediated by chemokine receptor type 2 (CCR2) in a tumor responses seen with combination therapy. syngeneic mouse model of colon carcinoma [93]. Treat- Recently, durable regression of irradiated tumors and ment with anti-CCR2 antibodies could potentially serve abscopal responses observed in mammary tumor-bearing a role in reversing RT resistance by attenuating host mouse models treated with combination RT and check- STING-mediated immunosuppression and complement point blockade were shown to be dependent on cancer RT and checkpoint blockade combinations. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 6 of 17 A growing body of preclinical evidence supports the or after PD-1 blockade produced 6- and 12-month combination of other immunotherapeutic agents with median OS rates of 85 and 78%, respectively [115]. One RT or radiofrequency ablation (RFA), immune check- retrospective study investigated 53 patients with meta- point blockade, and/or chemotherapy to enhance static melanoma treated with RT sequential or concur- tumor growth control (and often systemic control)in rent to anti-PD-1 therapy or as salvage therapy in the preclinical mouse models; synergistic antitumor activ- setting of progression on anti-PD-1 therapy (35 patients ity with multimodality therapy was characterized by received extracranial RT or intracranial SRS and 21 pa- tumor cell PD-L1 expression in a JAK/Stat1-depen- tients received whole brain radiotherapy (WBRT)) and dent manner and reduced numbers of CD11b + Gr1+ showed that median OS and ORR were not significantly cells (MDSCs) [90, 94–99]. different between concurrent and sequential RT/SRS cohorts (Table 2)[116]. Toxicities A single-institute retrospective trial analyzed the effi- Several preclinical studies have investigated the toxicity cacy of concurrent SRS and anti-PD-1 or anti-CTLA-4 of combined RT and checkpoint blockade. Notably, one therapy (defined as SRS within 4 weeks of administration investigation of lung-irradiated (20 Gy) C57bl/6-WT of checkpoint inhibitors) in 75 patients with melanoma mice receiving anti-PD-1 antibody (10 mg/kg intraperi- brain metastases and identified significantly improved toneal twice per week for 5 doses) showed more findings median percent reduction in lesion volume with concur- of abnormal alveoli, inflammatory changes, and exudates rent compared to nonconcurrent arms and with in the alveolar septa associated with a 2.1-fold increase anti-PD-1 compared to anti-CTLA-4 arms at 3 months in CD8+ T-cells in the irradiated lung tissues of mice in and 6 months [117]. However, when both anti-PD-1 and the RT and PD-1 blockade arm though post-RT mortal- anti-CTLA-4 therapies were combined there was no sig- ity up to 120 days was not significantly different in the nificant difference in median OS between nonconcurrent RT alone vs. RT and PD-1 blockade arm (p = 0.657) (9.0 months, range 2.1–61.8) and concurrent arms [100]. A separate study, however, using a similar dose of (19.1 months, range 2.7–64.2, p = 0.0691). In solely 20 Gy of thoracic RT (designed to induce mortality) to metastatic NSCLC patients (n = 21), combined RT to oli- C57bl/6 mice identified worse survival with RT and goprogressive sites along with PD-1/PD-L1 blockade or PD-1 blockade (36% survived) than RT alone (70% other immune therapies resulted in excellent local con- survived, p = 0.0169) at 21 days post-RT and increased trol, median time to systemic progression of 2.3 months T-cell infiltrates in lung and cardiac tissues (both in- (95% confidence interval (CI) 1.0–4.5), and median OS and out-of-field) of mice treated with RT and PD-1 of 7.2 months (95% CI 4.2–11.1) [118]. Among 25 pa- blockade compared to RT alone putatively due to tients with unresectable melanoma, abscopal responses enhanced healthy tissue damage by T-cell activation with (CR or PR) were observed in 56% of patients with the the addition of PD-1 blockade to thoracic RT [101]. addition of late RT (> 3 months of insufficient response Incorporating PD-1 blockade to cardiac RT in mice to anti-PD-1 monotherapy) [119]. has also shown to decrease survival and exacerbate A group of 137 patients with metastatic melanoma, cardiac dysfunction and myocarditis that are CD8+ NSCLC, and RCC treated with WBRT, SRS, or extracra- T-cell-mediated [102]. nial RT before or after initiation of PD-1 blockade expe- rienced a median OS 249 days (8 months; interquartile Clinical studies range (IQR) 90–689) following the start of anti-PD-1 Retrospective studies therapy though OS was 25.7 months in the cohort re- Numerous case reports and case series have documented ceiving brain RT as first form of palliative RT [120]. On clinically significant, and often durable, tumor responses multivariate analysis, melanoma patients fared best as to the combination of RT and PD-1/PD-L1 blockade in the hazard ratio (HR) for death was 3.1 (95% CI 1.7–5.9) advanced or metastatic melanoma, NSCLC, Hodgkin for NSCLC and HR of 3.2 (95% CI 1.2–7.9) for RCC lymphoma, RCC, and cervical cancer [103–112]. Initial compared to melanoma (p = 0.0008) possibly due to im- retrospective series of patients with melanoma brain proved responses to checkpoint inhibitors in melanoma metastases treated with SRS or fractionated RT within with the incorporation of both PD-1 and CTLA-4 inhib- 3–6 months of receiving anti-PD-1 therapy produced itors into standard care. promising 1-year OS rates and significantly improved 6- A secondary analysis of the phase I KEYNOTE-001 and 12-month distant brain metastasis control and OS trial of 98 patients with locally advanced or metastatic rates in those treated with SRS and anti-PD-1 therapy NSCLC treated with pembrolizumab showed signifi- vs. SRS and chemotherapy (Table 2)[113, 114]. In 24 cantly improved median OS of 10.7 months (95% CI patients with brain metastases from melanoma (54%) 6.5–18.9) vs. 5.3 months (95% CI 2.7–7.7, HR 0.58, 95% and NSCLC (46%), treatment with SRS before, during, CI 0.36–0.94, p = 0.026) in those who ever did and did Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 7 of 17 Table 2 Retrospective clinical studies with available results on the antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade Study n Design Outcomes Toxicities Ref. RS 26 Melanoma BMs treated with SRS or Median OS 11.8 mo (range 0.5–33.9) 1 grade 2 headache relieved with [114] FSRT (16–30 Gy X 1–5 fractions) and 1-year OS 55% in unresected steroids within 6 mo of nivolumab (1, 3, or BMs; median OS not reached and 10 mg/kg every 2 weeks for 12 1-year OS 100% in resected BMs doses then every 12 weeks for 8 doses) RS 96 Melanoma BMs treated with SRS 6- and 12-mo distant BM control rate For anti-PD-1 therapy: 1 grade 2 [113] (majority 24 Gy X 1 fraction) within 3 61%/38% anti-PD-1, 26%/21% headache managed with steroids mo of nivolumab 3 mg/kg every anti-CTLA-4, 53%/20% BRAF/MEK 2 weeks, pembrolizumab 2 mg/kg inhibitor, 15%/5% chemotherapy every 3 weeks, or other systemic (p = 0.008); 6- and 12-mo OS 81%/ therapies 66% anti-PD-1, 84%/50% anti-CTLA-4, 83%/75% BRAF/MEK inhibitor, 70%/15% chemotherapy (p = 0.004) RS 24 Melanoma and NSCLC BMs treated 6- and 12-mo OS 85 and 78%; 2 patients grade ≥ 3 CNS toxicity: 1 [115] with SRS (median 20 Gy/fraction, IQR median OS not reached; 6- and seizure and 1 symptomatic 16–21) within median 19 weeks 12-mo distant brain progression rate radionecrosis requiring surgery (range 0–107) of nivolumab or 37 and 65% pembrolizumab (median 5 cycles, IQR 3–6) RS 53 Metastatic melanoma treated with Medians OS 6.4 vs. 8.6 mo For RT arm: 3 patients grade ≥ 3 [116] extracranial RT/intracranial SRS (8– (p = 0.7672) for concurrent vs. rash, 1 grade ≥ 3 diarrhea, 2 grade ≥ 30 Gy X 1–10 fractions) or WBRT sequential RT/SRS; ORR 31% vs. 36% 3 radiation dermatitis, 1 grade ≥ 3 (median 30 Gy X10 fractions) and (p = 1) for concurrent vs. sequential radionecrosis; for WBRT arm: 1 pembrolizumab 2 mg/kg every RT/SRS; lesional response rate 45% grade ≥ 3 nausea, 1 grade ≥ 3 3 weeks or nivolumab 3 mg/kg for 30 progressing lesions treated cognitive changes, 2 grade ≥ 3 rash every 2 weeks as concurrent, with salvage RT/SRS sequential, or salvage (following progression on anti-PD-1 therapy) therapy RS 75 Melanoma BMs treated with SRS Median % lesion volume reduction NR [117] (median 20 Gy, range 12–24 Gy) at 3 mo (− 83.0% vs. -52.8%, within ±4 weeks (concurrent) of p < 0.0001) and 6 mo (− 94.9% vs. pembrolizumab 2 or 10 mg/kg every -66.2%, p < 0.0001) for concurrent vs. 2–3 weeks or nivolumab 3 mg/kg noncurrent; median % lesion volume every 2–3 weeks or ipilimumab reduction at 3 mo (− 89.3% vs. -66.2%, p < 0.0001) and 6 mo (− 95.1% vs. -75.9%, p = 0.0004) for anti-PD-1 vs. anti-CTLA-4 RS 21 Metastatic NSCLC treated with RT 6- and 12-mo local control rates 91.7 1 grade 4 cerebral edema (WBRT) [118] (8–30 Gy X 1–10 fractions) while and 85.2%; median time to systemic and 1 grade 3 pneumonitis receiving anti-PD-1, anti-PD-L1, progression 2.3 mo (95% CI 1.0–4.5); and/or anti-CTLA-4, or other immune median OS 7.2 mo (95% CI 4.2–11.1) therapy RS 25 Unresectable melanoma treated with CR, PR, SD, and PD rates for radiated No unusual AEs reported [119] hypofractionated RT (1 weekly sites 24, 8, 44, and 28% and for fraction over 4–5 weeks (84%) or 1 nonirradiated sites 29, 19, 19, and gammaknife RT for BMs (16%)) 33%, respectively; abscopal within 3 mo of anti-PD-1 (early) or > responses (CR or PR) in 56% for 3 mo after anti-PD-1 therapy (late) addition of late RT RS 15 Metastatic melanoma, RCC, NSCLC Safety analysis All-grade immune-related AEs in 3 [123] treated with palliative RT (total patients (20%) and 1 RT-related AE 8–36 Gy via 3–8 Gy fractions) (7%) of moderate mucositis; no cases within ±75 days of PD-1 inhibitor of pneumonitis RS 84 Metastatic melanoma, NSCLC, and No significant differences in toxicity For all-grade AEs: 6 patients with [124] other solid tumors treated with rates between PD-1/PD-L1 and pneumonitis (7.2%, 1 grade ≥ 3); for thoracic RT (median total dose CTLA-4 inhibitors or concurrent and grade ≥ 2 AEs: 14 fatigue, 9 rash, 10 3000 cGy (range 600–7400 X 10 sequential treatment GI toxicities, 12 infections, 8 thyroid fractions) within 1 month dysfunction, 7 renal injury, and 9 (concurrent) or up 6 months other (sequential) of PD-1/PD-L1 and/or CTLA-4 blockade Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 8 of 17 Table 2 Retrospective clinical studies with available results on the antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade (Continued) Study n Design Outcomes Toxicities Ref. RS 29 Metastatic NSCLC treated with Median PFS and OS of 3.8 mo (95% Possible treatment-related AEs: 1 [125] thoracic RT (10–70 Gy X 1–35 CI 1.9–8) and 9.2 mo (95% CI 5.1-not grade 5 pneumonitis and 2 grade 3 fractions) within 6 mo of PD-1/PD-L1 reached) pneumonitis and/or CTLA-4 blockade RS 133 Metastatic NSCLC, melanoma, and No significant difference in immune- All-grade immune-related AEs: 20% [127] RCC treated with palliative RT related AEs between those receiving dermatitis, 8% colitis, 5% transaminitis; (8–37.5 Gy X 1–15 fractions) within RT during/after checkpoint inhibitors grade ≥ 3 immune-related AEs: 4% 180 days of PD-1 or CTLA-4 inhibitor and before checkpoint inhibitors colitis, 2% transaminitis, 2% (p = 0.053), receiving RT within hypophysitis 14 days or outside 14 days of checkpoint blockade (p = 0.06), and of site of irradiation RS 137 Metastatic NSCLC, melanoma, and Median OS 249 days (IQR 90–689) No grade 4–5 immune-related AEs [120] RCC treated with WBRT (12–39 Gy), following PD-1 blockade; on SRS (15–30 Gy), or extracranial RT multivariate analysis HR for death 3.1 (8–66 Gy) within a median 85 days (95% CI 1.7–5.9) for NSCLC and HR (IQR 34–181) of anti-PD-1 therapy 3.2 (95% CI 1.2–7.9) for RCC vs. melanoma (p = 0.0008) RS 17 NSCLC BMs treated with SRS or FSRT Distant brain control rate 57% No neurologic/ cutaneous AEs with [128] (18–25 Gy X 1–5 fractions) within ±6 (RT during or before PD-1/PD-L1 SRS and anti-PD-1/PD-L1 therapy mo of nivolumab or durvalumab blockade) vs. 0% (RT after, p = 0.05); (41% received prophylactic median OS for SRS during/before dexamethasone before SRS); 1 PD-1/PD-L1 blockade vs. SRS after patient each discontinued (HR 3.6, 95% CI 0.74–26.9, p = 0.11) PD-1/PD-L1 inhibitor due to colitis on multivariate analysis and pneumonitis RS 137 Melanoma BMs treated with SRS or Median OS 16.9 mo; for See outcomes [129] WBRT (median 20 Gy, range 12–30) radionecrosis: 37 patients (27%); within 1 year of PD-1 or CTLA-4 no difference in risk between blockade ipilimumab and pembrolizumab (p = 0.549) or CTLA-4 and PD-1 (p = 0.86); 1-year OS 78.4% vs. 55.06% (without radionecrosis, p = 0.341) RS 98 Advanced NSCLC treated with Any previous RT vs. no previous RT: All-grade treatment-related pulmon- [121] palliative RT any time point before median PFS 4.4 mo (95% CI 2.1–8.6) ary toxicity in 3 patients (13%, with (median 9.5 mo, range 1–106) first vs. 2.1 mo (95% CI 1.6–2.3, HR 0.56, RT) vs. 1 (1% without RT, p = 0.046); cycle of pembrolizumab 2 or 95% CI 0.34–0.91, p = 0.019); median grade ≥ 3 treatment-related 10 mg/kg every 2–3 weeks OS 10.7 mo (95% CI 6.5–18.9) vs. 5.3 pulmonary toxicity similar in both mo (95% CI 2.7–7.7, HR 0.58, 95% CI arms (1 each, p = 0.44) 0.36–0.94, p = 0.026) RS 108 Melanoma BMs treated with SRS In combination with RT: median OS 2 radiation necrosis (SRS + anti-PD-1) [122] and/or WBRT (dose NR) within 7.5 mo with CTLA-4 (95% CI 4.4– treated with surgery, steroids, and ±6 weeks of various systemic 15.6), 20.4 mo PD-1 (95% CI 8.8-NA), bevacizumab therapies and 17.8 mo BRAF ± MEK inhibitor (95% CI 11.8-NA) RS retrospective study, BMs brain metastases, SRS stereotactic radiosurgery, FSRT fractionated stereotactic RT, Gy Gray, OS overall survival, NSCLC non-small cell lung cancer, IQR interquartile range, CNS central nervous system, RT radiotherapy, WBRT whole brain radiation therapy, ORR overall response rate, NR not reported, CI confidence interval, CR complete response, PR partial response, SD stable disease, PD progressive disease, AEs adverse events, RCC renal cell carcinoma, GI gastrointestinal, HR hazard ratio, PFS progression-free survival, NA not applicable not receive RT, respectively [121]. In spite of these inter- Safety analyses esting clinical results, no data are provided on the type, Retrospective safety analyses in patients with ad- dose, schedule of radiotherapy or the tumor burden of vanced solid tumors receiving RT and PD-1/PD-L1 patients receiving therapy making the results hard to in- and/or CTLA-4 blockade have generally not demon- terpret. Interestingly, one retrospective series of 108 pa- strated increased risk of toxicity with the combination tients with melanoma brain metastases treated with SRS beyond those expected with each modality independ- and/or WBRT concurrently with various contemporary ently [123, 124]. There were no significant differences systemic therapies highlighted that RT in combination in toxicity rates between choice of PD-1/PD-L1 and with anti-PD-1 therapy produced among the best OS in CTLA-4 inhibitor or concurrent and sequential treat- the cohort without clinically significant increases in ment with RT [124]. However, another series of 29 neurotoxicity [122]. metastatic NSCLC patients given thoracic RT and Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 9 of 17 PD-1/PD-L1 and/or CTLA-4 inhibitors identified 1 assessment at all irradiated sites and the best ORR was case of possibly treatment-related grade 5 pneumonitis in 44% (4 patients with partial responses (PRs)) by World a patient who received 20 Gy over 5 fractions of thoracic Health Organization (WHO) criteria (Table 3). A phase RT initiated 1 month after the last dose of anti-PD-1 ther- I/II study investigated the safety and efficacy of concur- apy [125]. Interestingly, case reports have documented the rent local palliative RT and durvalumab (PD-L1 inhibi- existence of PD-1 inhibitor-induced radiation recall pneu- tor) in 10 patients with unresectable or metastatic monitis even after 2 years of RT [126]. advanced solid tumors [136]. When RT (to 15 localized A multicenter safety analysis demonstrated no signifi- lesions) was given a median of 8.5 days (range 1–35) cant differences in immune-related AEs regardless of site from the last dose of durvalumab, the combination was of irradiation, between those receiving RT during/after generally tolerated with no grade ≥ 3 RT-related AEs checkpoint inhibitors and before checkpoint inhibitors (Table 3). The 1-year OS and progression-free survival (p = 0.053), and between those receiving RT within 14 days (PFS) rates were 44% (95% CI 12–77) and 30% (95% CI or outside 14 days of checkpoint blockade (p =0.06) [127]. 2–58), respectively. One retrospective series demonstrated that brain RT and Preliminary results from a phase I dose-finding study PD-1/PD-L1 blockade was relatively well-tolerated in pa- of stereotactic body RT (SBRT; 8 Gy X 1 or 5 Gy X 5) tients with NSCLC brain metastases as toxicity rates were and durvalumab or the CTLA-4 inhibitor tremelimumab consistent with those seen with checkpoint inhibitors (or combination of all 3) was administered as alone [128]. Interestingly, the distant brain control (out-- second-line therapy to 24 metastatic pancreatic adeno- of-field) rate for RT during/before PD-1/PD-L1 blockade carcinoma patients. No DLTs have been observed so far was 57% compared to 0% (RT after, p = 0.05). Another [137]. The best response was SD in 5 patients (21%) with retrospective series of 137 patients with melanoma brain rapid progression within 4 weeks in an additional 5 pa- metastases identified 37 patients (27%) who developed tients. A phase II trial involving locally advanced NSCLC radionecrosis following SRS or WBRT and anti-CTLA-4 patients recently reported preliminary results from part I or anti-PD-1 therapy with a median time of onset of of the study [138]. Out of 10 enrolled patients, 7 have 6months (range1.3–31.4 months), which is comparable received atezolizumab added to consolidation carbopla- to rates seen in other series though prospective studies are tin and paclitaxel following weekly carboplatin/paclitaxel limited [129–132]. Notably, 1-year OS did not signifi- and RT and 2 patients have demonstrated PD after 6 and cantly differ between those that developed radionecro- 8 doses of the PD-L1 inhibitor. Given the safety and sis vs. those without (Table 2). However, risk of tolerability of patients in part I, criteria were met for ad- radionecrosis was significantly associated with concur- vancement to part II of the study where atezolizumab rent use of chemotherapy within 6 months of SRS will be added to the chemoradiation portion followed by (HR 2.20, 95% CI 1.22–3.97, p = 0.009) and increased consolidation atezolizumab, carboplatin, and paclitaxel. number of lesions treated (HR 1.09, 95% CI 1.03– Recently, the PD-L1 inhibitor durvalumab was granted 1.15, p = 0.002). The lack of significant difference in FDA approval based on superior PFS but similar safety OS between presence and absence of radionecrosis compared to placebo following platinum-based chemo- conflicts with the results of other studies though the radiation in locally advanced, unresectable NSCLC in number of patients treated with brain RT and PD-1 the phase III PACIFIC trial [139]. Patients who did not blockade were likely much smaller [130, 133]. demonstrate PD after ≥2 cycles of platinum-based chemotherapy concurrent with definitive RT were ad- Prospective studies ministered durvalumab or placebo within 1–42 days for A combined preclinical and phase I study was among the up to 1 year (Table 3). Improved outcomes were ob- first to provide preliminary results for the efficacy of com- served in the experimental arm irrespective of PD-L1 bined RT and checkpoint blockade in the prospective set- status or histology. ting [134]. In the phase I dose-finding cohort of 5 patients given local RT for mixed response or asymptomatic pro- Discussion gression to atezolizumab, dual RT and anti-PD-L1 therapy Elucidated mechanisms underlying the immune stimula- was well-tolerated without any dose-limiting toxicities tory properties of RT are growing in complexity (Fig. 1). (DLTs) or severe immune-mediated AEs and all 5 patients The CD8+ T-cell remains a crucial component in the experienced at least SD (Table 3). ability of RT to elicit an antitumor immune response In another phase I trial, 9 patients with advanced mel- within and beyond the radiation field [140]. In addition, anoma received RT during induction, between induction evidence is mounting to support that RT specifically and maintenance, or during maintenance therapy with upregulates MHC tumor-associated antigen complexes, ipilimumab and/or nivolumab [135]. Combined RT and enhances tumor antigen cross-presentation in draining checkpoint inhibition resulted in SD or response by first lymph nodes, and increases T-cell infiltration into Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 10 of 17 Table 3 Prospective clinical studies with available results on the antitumor activity of combined radiation therapy and PD-1/PD-L1 blockade Study n Design Outcomes Toxicities Ref. Phase I 4 solid tumors, 1 Atezolizumab 0.01–20 mg/kg Stabilization of systemic Transient grade 1–2 [134] hematologic every 3 weeks (dose-finding progression in all 5 patients inflammatory AEs (fevers, malignancy cohort) + local fractionated RT (PR at systemic site in 1 flu-like symptoms) observed (dose NR) for mixed responses or patient) but no DLTs or serious asymptomatic PD immune-related AEs Phase I 9 advanced Nivolumab 0.3–10 mg/kg every ORR 44% (4 PRs) as best 5 patients with non- [135] melanoma 3 weeks X 21 weeks (induction) response by WHO criteria; laboratory grade ≥ 3 AEs, 2 then every 12 weeks X 84 weeks median OS 27 mo; 1- and RT-related grade ≥ 3 AEs (maintenance) ± ipilimumab 3 or 2-year OS rates of 89 and (intracranial hemorrhage, 10 mg/kg every 3 weeks X 78%, respectively diarrhea) 9 weeks (induction) then every 12 weeks X 84 weeks (maintenance) or combined nivolumab 1 mg/kg and ipilimumab 3 mg/kg every 3 weeks X 12 weeks then nivolumab 3 mg/kg every 2 weeks up to 96 weeks + RT (median 30 Gy X 5 fractions, range 21–37.5 Gy X 1–15 fractions) during induction or maintenance Phase I/II 10 unresectable or Durvalumab 10 mg/kg every In-field ORR 60% (2/10 CRs, 5 cases of (50%) RT-related [136] metastatic solid 2 weeks + local RT (median 20 Gy, 4/10 PRs); median OS 11.5 grade 2 AEs (3 mucositis, tumors (≥5% PD-L1 range 6–33 X median 5 fractions, mo (95% CI 8.8–13.7); median 1 diarrhea, 1 vomiting) expression) range 1–10) given a median of PSF 6.2 months (95% CI 8.5days (range1–35) of last dose 4.5–12.4); out-of-field 10/14 of durvalumab SD, no responses or abscopal effects were seen Phase I 24 metastatic SBRT (8 Gy X 1 fraction or 25 Gy SD as best ORR in 5 patients No DLTs observed; most [137] pancreatic X 25 fractions) + durvalumab (21%) common AE was grade 1–2 adenocarcinoma (dose NR) every 2 weeks or fatigue at dose level 2 tremelimumab (dose NR) every 4 weeks X 6 doses then every 12 weeks for 3 doses or triple therapy Phase II 10 locally advanced Weekly carboplatin (AUC 2) and Out of 7 patients receiving 3 patients with potential [138] NSCLC weekly paclitaxel 50 mg/m +RT atezolizumab, 2 patients immune-related AEs (1 grade 5 days/week for 6–7 weeks developed PD after 6 and 8 3 arthralgia, 1 grade 2 (60–66 Gy over 30–33 fractions) doses of atezolizumab pneumonitis resolved with followed by atezolizumab steroids, 1 grade 3 dyspnea) 1200 mg every 3 weeks + consolidation carboplatin (AUC 6) and paclitaxel 200 mg/m on days 1 and 22 for 2 cycles then atezolizumab alone up to 1 year Phase III 709 stage III, locally 2 or more cycles of platinum- Median PFS 16.8 months Grade 3–4 AEs 29.9% vs. [139] advanced, based chemotherapy (defined by (95% CI 13.0–18.1) vs. 26.1% (placebo); most unresectable NSCLC local practice) + concurrent 5.6 months (95% CI 4.6–7.8) common grade 3–4 AEs definitive RT (54–66 Gy with with placebo (HR 0.52, 95% CI pneumonia (4.4% vs. 3.8%), mean dose to the lung < 20 Gy 0.42–0.65, p <0.001); median pneumonitis (3.4% vs. 2.6%), or volume of lung parenchyma TTD or distant metastasis and anemia (2.9% vs. 3.4%) in receiving ≥20 Gy < 35%) 23.2 months (95% CI 23.2-NE) durvalumab vs. placebo arms followed by (within 1–42 days) vs. 14.6 months (95% CI durvalumab 10 mg/kg every 10.6–18.6) with placebo 2 weeks up to 1 year or placebo (HR 0.52, 95% CI 0.39–0.69, if no PD during chemoradiation p < 0.001); ORR 28.4% vs. 16.0% with placebo (p <0.001) RT radiation therapy, NR not reported, PD progressive disease, PR partial response, DLT dose-limiting toxicity, AEs adverse events, Gy Gray, ORR overall response rate, PR partial response, WHO World Health Organization, CI confidence interval, SD stable disease, SBRT stereotactic body radiation therapy, NSCLC non-small cell lung cancer, AUC area under curve, CR complete response, PFS progression-free survival, HR hazard ratio, TTD time to death, NE not estimable or reached Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 11 of 17 tumors [79, 141]. Local RT appears necessary in eliciting Several points of consideration remain that could po- abscopal effects, but RT alone remains insufficient in tentially impact the rational combination of RT and complete eradication of local and distant tumors likely, PD-1/PD-L1 inhibitors and their efficacy. Firstly, im- in part, due to activation of negative T-cell regulatory munogenic cell death has been shown to be induced by pathways including the PD-1/PD-L1 axis and immune RT in a dose-dependent manner in vitro [68]. In other checkpoints such as CTLA-4 [76, 86, 87]. However, RT preclinical studies, increasing radiation doses (single has been shown to upregulate expression of PD-1 and fractions above 7.5 Gy but not 5 Gy) were immunosti- PD-L1 on immune and tumor cells rendering it an at- mulatory, associated with elevated IFN-γ production, tractive modality to combine with PD-1/PD-L1 blockade and prevented increases in Tregs [143]. At higher doses [71, 76, 78, 79, 86, 97]. Activation of cGAS-STING sig- (single fractions ≥15 Gy), dose-dependent increases in naling has also been recognized to mediate systemic Tregs were observed and associated with no improve- tumor rejection by combined RT and checkpoint block- ment in antitumor immune responses. Fractionation of ade given that knockdown of cGAS and STING in can- the 15 Gy generally resulted in superior immune re- cer cells abrogated priming of CD8+ T-cells in sponses compared to single-fraction 15 Gy. In a seminal tumor-draining sites and infiltration of abscopal tumors study of 2 preclinical mouse carcinoma models, evalu- by CD8+ T-cells [89]. ation of RT (20 Gy X 1, 8 Gy X 3, or 6 Gy X 5 fractions In efforts to characterize the synergistic antitumor ac- over consecutive days) in combination with an tivity of combined RT and PD-1/PD-L1 blockade, nu- anti-CTLA-4 antibody determined that fractionated RT merous studies have identified significant elevations in but not single-dose RT achieved significantly enhanced CD8+ IFNγ+ TNFα+ T-cells but decreases in CD4+ tumor responses both within and outside the radiation FOXP3+ Tregs leading to an increased CD8+/Treg ratio, field (abscopal effects) when combined with CTLA-4 increases in tumor-antigen specific CD8+ TILs with a blockade [55]. It has been further corroborated that frac- CD44+ effector memory phenotype, decreases in im- tionated RT (8 Gy X 3) with checkpoint blockade was munosuppressive MDSCs, reinvigoration of CD8+ TILs able to elicit abscopal effects whereas checkpoint block- with an exhausted phenotype, and increases in TCR rep- ade with RT doses ≥20 Gy single dose were character- ertoire clonality and diversity of the TCR repertoire in ir- ized by complete loss of abscopal responses through radiated and out-of-field sites as a consequence of induction of Trex1 and downregulation of type I IFN combination radioimmunotherapy [61, 72, 76, 79, 88]. signaling [89]. Furthermore, addition of anti-PD-L1 therapy to tumors The timing of RT in relation to administration of that are nonresponsive to RT has shown the ability to checkpoint inhibitors represents another issue of discus- reverse RT-induced tumor equilibrium in favor of tumor sion. Preclinical data support that RT-associated in- regression [92]. Resistance to RT also appears to be creases in the CD8+ T-cell/Treg ratio, CD8+ T-cell regulated by host STING activation via CCR2; add- PD-1 expression, and tumor cell PD-L1 expression often itional targeting of the CCR2 pathway may therefore occur early with peak levels occurring within 24–96 h aid in reversing RT resistance in the context of post-RT [81, 86]. In an elegant study exploring combined checkpoint blockade [93]. Conversely, integration of anti-PD-L1 therapy and fractionated RT (10 Gy in 5 daily RT to anti-PD-1-resistant tumors restores response to fractions), the addition of PD-L1 blockade on day 1 of RT PD-1 blockade highlighted by RT-induced IFN-γ pro- (concurrent regimen starting at the beginning of RT), day duction and MHC class I expression [91]. 5 of RT (concurrent regimen starting at the end of RT), or Immune modulation from immune checkpoint in- 7 days after RT (sequential therapy) showed that there hibitors and RT through nonredundant pathways that was no significant difference in OS between either concur- altogether contribute to synergistic antitumor activity rent therapy schedules [86]. However, sequential therapy now represents an emerging theme from ongoing in- was ineffective in enhancing OS compared to RT alone vestigations in combination RT and immunotherapy (median OS 30 days vs. 35 days, p > 0.05). Interestingly, [61, 77, 85, 88, 90, 142]. For example, anti-CTLA-4 the rise in PD-1 expression on CD8+ T-cells was evident therapy has been shown to predominantly inhibit Tregs, up to 7 days after the last dose of RT, after which PD-1 increase the CD8+ T-cell/Treg ratio, and promote T-cell levels significantly decreased compared to time-matched expansion. Radiation enhances the diversity of the TCR controls. In the clinical setting, retrospective series have repertoire, shapes the TCR repertoire of expanded periph- documented a wider range of schedules in combining eral T-cell clones in an antigen-driven selection manner, radioimmunotherapy ranging from RT at any point prior and promotes tumor infiltration by antigen-specific CD8+ to immune checkpoint therapy, within 1 month of admin- T-cells. Addition of PD-1/PD-L1 blockade reverses T-cell istration of checkpoint inhibitors, or up to 1 year of check- exhaustion to offset decreases in the CD8+ T-cell/Treg ra- point blockade [117, 121, 124, 129]. Moreover, results tio and further enhances oligoclonal T-cell proliferation. have been largely mixed on the impact of scheduling of Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 12 of 17 RT and checkpoint blockade on survival as several retro- the limited number of larger prospective trials, PD-1 and spective studies have identified that there is no significant PD-L1 blockade have often been incorporated into stand- difference in OS between concurrent and nonconcurrrent ard dosing regimens of SBRT and chemoradiation rou- radioimmunotherapy while another study demonstrated a tinely used in the treatment of locally advanced pancreatic significant improvement in PFS and OS in patients having cancer and NSCLC, for example (Table 3). ever received RT prior to PD-1 blockade compared to It is worthwhile to mention that the Phase III PACIFIC those with no prior RT [116, 117, 121]. It is worthwhile to trial demonstrated the superiority of chemoradiation mention that these retrospective studies were likely lim- followed by durvalumab when the latter was included ited by variability in RT modality, tumor histology, patient within 1–42 days of chemoradiation over chemoradia- characteristics, and cohort size. Notably, abscopal effects tion followed by placebo in locally advanced NSCLC have been observed in 56% of patients with the addition of [139]. On review of the study protocol and Supplementary late RT to PD-1 blockade as well (> 3 months of insuffi- Appendix, the investigators emphasized the initiation of cient response to anti-PD-1 monotherapy) [119]. durvalumab as close as possible to chemoradiation when Another point of consideration in clinical trial design is antigen release and PD-L1 expression is likely to be at its the issue of toxicity with combined RT and PD-1/PD-L1 greatest. An analysis of benefit in those receiving durvalu- blockade. Several preclinical studies demonstrated more mab closer to chemoradiation compared to those treated findings of abnormal alveoli, inflammatory changes, exu- later relative to chemoradiation was not provided; an ana- dates in the alveolar septa, and cardiac toxicity in mice re- lysis of this nature may provide further insight on the pro- ceiving thoracic RT and anti-PD-1 therapy, when posed synergism offered by this combination. For reasons compared to controls, though effects on survival have which are unclear, the median PFS of the placebo arm been mixed [100–102]. Retrospective analyses have gener- (5.6 months) appears worse than historical standards ally shown no increased risk of toxicity with the combin- [145]. It is also unclear whether the benefit derived from ation of RT and checkpoint blockade beyond those the combination arm is due to the efficacy of immuno- expected with either modality alone [121, 124, 127]. For therapy in settings of smaller disease volume as seen pre- brain RT, a study of 137 patients treated with SRS or viously [146]. All of these are potential factors that may WBRT in combination with PD-1 or CTLA-4 blockade contribute to the difference seen in efficacy between ex- identified radionecrosis in 27% though 1-year OS did not perimental and control arms. significantly differ between those that developed radione- Despite the promising results and feasibility of the PA- crosis and those that did not [129]. Reassuringly, retro- CIFIC trial, clinical studies on an upper threshold RT spective series of > 200 patients receiving combined RT dose with checkpoint inhibition by which no further im- and immunotherapy have demonstrated that there are no provement in antitumor immunity is offered (as foresha- significant differences in toxicities regardless of site of ir- dowed by preclinical evidence discussed previously) are radiation, choice of checkpoint inhibitor, or treatment virtually nonexistent, yet duly warranted. Dedicated schedule (concurrent vs. sequential) [124, 127]. dose-escalation studies on combination PD-1/PD-L1 in- Taking together the preclinical evidence on the kinet- hibitors and RT are also needed in other tumor types to ics of PD-1 and PD-L1 expression in relation to RT and determine safety and tolerability. Early phase studies of the clinical data on the safety and tolerability of radioim- this nature are emerging and have demonstrated the munotherapy, there is growing evidence to support that feasibility of this combination while recognizing the im- PD-1/PD-L1 blockade is optimal when synchronized portance of timing of checkpoint blockade with respect with the administration of fractionated RT to prevent to RT administration [147]. Extrapolation of RT dose the development of immunological anergy [144]. Indeed, effects from animal to human studies is not straightfor- the concept of administering PD-1/PD-L1 inhibitors ward and great caution is needed in applying dosing concurrently or immediately following fractionated RT schemes and regimens involving combination RT and has already been employed in clinical trials with evi- PD-1/PD-L1 blockade in human patients [148]. Further dence that the combination is generally well-tolerated understanding of the mechanistic and dynamic immunos- (Table 3). However, despite our increased understanding, timulatory properties of RT and PD-1/PD-L1 blockade are preclinical and clinical data have yet to offer a consensus undoubtedly warranted with validation in (ideally) pro- on optimal dosing and modality sequencing to date [68]. spective cohorts prior to maximizing tumor responses The majority of retrospective and prospective studies on with the combination. The ability to optimize immune combination RT and checkpoint blockade have predomin- responses in the future with radioimmunotherapy may antly used fractionated dosing schemes (Tables 2 and 3). potentially depend on the immunotherapeutic strategy However, depending on the tumor type, target site, and used, tumor histology, balance between proimmuno- modality employed, total RT doses from retrospective genic and immunosuppressive effects of either modal- series have ranged widely from 8 to 74 Gy (Table 2). Of ity, and other host factors [50, 148]. Gong et al. Journal for ImmunoTherapy of Cancer (2018) 6:46 Page 13 of 17 Lastly, phase I trials of RT and anti-PD-1 therapy have Received: 26 December 2017 Accepted: 16 May 2018 already provided glimpses into potential mechanisms of failure even with the combination as 1 patient with metastatic RCC who rapidly progressed on combined References 1. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. 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Journal for ImmunoTherapy of CancerSpringer Journals

Published: Jun 4, 2018

References

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