Cancer Cell
Previews Radiotherapy Complements Immune Checkpoint Blockade Shin Foong Ngiow,1,2 Grant A. McArthur,3,4 and Mark J. Smyth1,2,* 1Immunology
in Cancer and Infection Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia of Medicine, University of Queensland, Herston, QLD 4006, Australia 3Cancer Therapeutics Programs, Trescowthick Laboratories, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, VIC 3002, Australia 4Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2015.03.015 2School
Adaptive immune resistance ablates effective anti-tumor immune responses. In a recent issue of Nature, Victor and colleagues describe that anti-PD-L1 combats adaptive immune resistance upon localized radiation plus anti-CTLA-4 therapy. The superior activity of radiation and dual immune checkpoint blockade is mediated by non-redundant immune mechanisms in cancer.
Combinations of two or more immunotherapies with additive or synergistic benefit in preclinical models (Uno et al., 2006) have set the framework for combination successes in the clinic (Wolchok et al., 2013). With the recent success of immune checkpoint inhibitors and other immunomodulators, there has been renewed interest in evaluating the combination of such agents with radiation therapy (RT) in clinical trials (Verbrugge et al., 2012; Formenti and Demaria, 2013). The biologic premise behind such a strategy is that the tumor-antigen release and change in the tumor microenvironment achieved by localized RT will promote specific tumor targeting by the adaptive immune system, which can be augmented further by systemic immunestimulating agents (Tang et al., 2014). In this manner, clinicians hope to induce a phenomenon known as the abscopal effect, whereby localized RT results in immune-mediated tumor regression in disease sites well outside of the radiation field. RT also induces DNA damage and tumor cell death by promoting tumor-cell expression of Fas and MHC class I. Dying tumor cells release not only tumor antigens but also ATP and danger signals such as HMGB1 and calreticulin. RT also has some potentially deleterious effects by increasing the tumor cell expression of PD-L1, secretion of TGFb, and induction of Treg. A small pilot phase I/II study in prostate cancer (Slovin et al., 2013) and case reports in melanoma (Postow et al., 2012; Hiniker et al., 2012; Golden et al., 2013) combining RT with ipilimu-
mab show the possible clinical activity of this approach. Now, Victor et al. (2015) report a phase I clinical trial of 22 patients with advanced melanoma treated with RT and antiCTLA-4. In the trial, a single lesion was irradiated with hypofractionated stereotactic body radiation (6–8 Gy delivered over two or three fractions), followed by four cycles of ipilimumab beginning 3– 5 days after the last fraction of RT. Assessment of unirradiated lesions using RECIST criteria demonstrated 18% patients had a partial response as the best response, 18% had stable disease, and 64% had progressive disease. So, although partial responses were observed, like monotherapy with anti-CTLA-4, the majority of the patients did not respond. The authors then describe the use of the B16-F10 melanoma mouse model, where RT plus anti-CTLA-4 was more effective than either treatment alone, promoting the regression of both irradiated and unirradiated tumors (Victor et al., 2015). RT given before, or concurrently with, anti-CTLA-4 yielded similar results. Complete responses in these combination-treated mice were CD8+ T celldependent, and memory to rechallenge was demonstrated. However, not all mice responded, and melanoma cell lines from relapsing mice were derived. In these cell lines, resistance to the combination was confirmed, but the lesions were not RT resistant. Random forest machine learning analysis for sub-types of TIL isolated from the resistant melanomas demonstrated that the top predictor for
resistance was the CD8+CD44+ to Treg ratio, which failed to increase after RT plus anti-CTLA-4 (as it did in sensitive tumors). Transcriptomic profiling revealed that PD-L1 was among the top 0.2% of upregulated genes in melanoma (RT plus anti-CTLA-4 resistance signature). Genetic elimination of PD-L1 on the therapy-resistant Res499 melanoma cells by CRISPR dramatically restored response to RT plus anti-CTLA-4 (0% to 60% survival), suggesting upregulation of PD-L1 was one major mechanism, but not the only mechanism of resistance to RT plus anti-CTLA-4. In the original parental B16-F10, RT plus anti-CTLA-4 increased the proportion of PD-1+Eomes+ CD8 T cells and the proportion that were Ki67+ and granzyme (Grz) B+. But in the resistant sublines, the Ki67+GrzB+ CD8 T cells were not increased. The frequency of PD-1+CD8+ T cells that were Eomes+ was a striking modifier of the likelihood of a complete response (CR), because nearly all the CRs occurred when the frequency of Ki67+GrzB+ in PD-1+CD8+ was high, but the relative size of the PD1+Eomes+-exhausted population was small. Critically, adding anti-PD-L1 improved responses to the naive B16F10 and resistant sublines after RT plus anti-CTLA-4. Similar results were obtained in the mouse TSA mammary tumor and a mouse pancreatic tumor model. Random forest modeling showed that anti-CTLA-4 predominantly caused a decrease in Treg cells, anti-PD-L1 strongly increased CD8 TIL frequency,
Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 437
Cancer Cell
Previews
common, and most patients eventually die from progressive metastatic disease. A considerable amount of work remains to be done to create successful combinations of immunotherapeutics and radiation, which includes identifying the optimal radiation dose, fractionation, and sequence for use in combination with immune checkpoint inhibitors. ACKNOWLEDGMENTS We wish to acknowledge funding support from the National Health and Medical Research Council of Australia, The Susan Komen for the Cure, and the Cancer Council of Queensland.
REFERENCES
Figure 1. Schematic Model for Non-redundant Mechanisms of RT/Anti-CTLA-4/Anti-PD-L1 Therapy to Combat Immune Resistance in Melanoma Adapted from Victor et al. (2015) Extended Data Figure 6.
and blockade of both increased the CD8/ Treg ratio. RT only caused a modest increase in CD8 TILs; however, TCR sequencing revealed that there was an increased diversity of TCR clonotypes, which was also observed with RT plus anti-CTLA-4. This effect was also seen in the spleen and peripheral blood. Some clones reached 20% post-treatment in the blood with triple treatment. By contrast, peripheral T cell expansion was modest with RT and anti-CTLA-4 alone. So the concept gleaned from the mouse studies was that favorable immune changes in TILs after immune checkpoint blockade promoted their peripheral clonal expansion. When combined with increased TCR repertoire diversity afforded by RT, selection and oligoclonal peripheral expansion of clones with distinct TCR traits were favored (Figure 1). From the clinical trial of RT + ipilimumab, 12 patients had pre-treatment tumor biopsies, and pre- and post-treatment blood was available on 10 patients. From these low numbers, it was revealed that PD-L1lo intensity on melanoma cells was associated with re-invigoration of PD-1+Eomes+ CD8+ T cells after RT plus anti-CTLA-4, while PD-L1hi status was associated with persistent exhaustion of the CD8+T cells. PD-L1hi on melanoma biopsies collected from therapy-resistant patients is potentially reflecting the
failure of CTL anti-tumor cytotoxicity, consistent with the lower frequencies of Ki67+GrzB+PD1+CD8+ T cells. As discussed above, it is also worth emphasizing that a proportion of the mice with PD-L1 knockout Res499 cells still succumbed to disease after RT plus antiCTLA-4 treatment, suggesting a role for non-tumor PD-L1 in promoting resistance to RT/anti-CTLA-4. Intriguingly, despite other recent reports (Herbst et al., 2014) PD-L1 status on the macrophages was neither associated with reinvigoration nor independently predictive of progression-free survival. Although there may be activation of other T cell checkpoint pathways, the hierarchy of PD-L1 expression on tumor cells and non-tumor cells in driving T cell exhaustion warrants further study in larger patient cohorts. Nonetheless, the mechanism of radiation to diversify TCR clonotypes remains to be discerned. Radiotherapy is a major component of cancer treatment, with over 50% of cancer patients receiving radiation during the course of their disease. Radiation is used for both palliation of local symptoms and curative therapy in some malignancies, like lung, prostate, or head and neck cancer. However, although radiation offers clear benefits in terms of both local control and survival, disease recurrence outside the radiation field remains all too
438 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.
Formenti, S.C., and Demaria, S. (2013). J. Natl. Cancer Inst. 105, 256–265. Golden, E.B., Demaria, S., Schiff, P.B., Chachoua, A., and Formenti, S.C. (2013). Cancer Immunol Res 1, 365–372. Herbst, R.S., Soria, J.C., Kowanetz, M., Fine, G.D., Hamid, O., Gordon, M.S., Sosman, J.A., McDermott, D.F., Powderly, J.D., Gettinger, S.N., et al. (2014). Nature 515, 563–567. Hiniker, S.M., Chen, D.S., Reddy, S., Chang, D.T., Jones, J.C., Mollick, J.A., Swetter, S.M., and Knox, S.J. (2012). Transl Oncol 5, 404–407. Postow, M.A., Callahan, M.K., Barker, C.A., Yamada, Y., Yuan, J., Kitano, S., Mu, Z., Rasalan, T., Adamow, M., Ritter, E., et al. (2012). N. Engl. J. Med. 366, 925–931. Slovin, S.F., Higano, C.S., Hamid, O., Tejwani, S., Harzstark, A., Alumkal, J.J., Scher, H.I., Chin, K., Gagnier, P., McHenry, M.B., and Beer, T.M. (2013). Ann. Oncol. 24, 1813–1821. Tang, C., Wang, X., Soh, H., Seyedin, S., Cortez, M.A., Krishnan, S., Massarelli, E., Hong, D., Naing, A., Diab, A., et al. (2014). Cancer Immunol Res 2, 831–838. Uno, T., Takeda, K., Kojima, Y., Yoshizawa, H., Akiba, H., Mittler, R.S., Gejyo, F., Okumura, K., Yagita, H., and Smyth, M.J. (2006). Nat. Med. 12, 693–698. Verbrugge, I., Hagekyriakou, J., Sharp, L.L., Galli, M., West, A., McLaughlin, N.M., Duret, H., Yagita, H., Johnstone, R.W., Smyth, M.J., and Haynes, N.M. (2012). Cancer Res. 72, 3163–3174. Victor, C.T., Rech, A.J., Maity, A., Rengan, R., Pauken, K.E., Stelekati, E., Benci, J.L., Xu, B., Dada, H., Odorizzi, P.M., et al. (2015). Nature Published online March 9, 2015. http://dx.doi.org/10. 1038/nature14292. Wolchok, J.D., Kluger, H., Callahan, M.K., Postow, M.A., Rizvi, N.A., Lesokhin, A.M., Segal, N.H., Ariyan, C.E., Gordon, R.A., Reed, K., et al. (2013). N. Engl. J. Med. 369, 122–133.
Previews Radiotherapy Complements Immune Checkpoint Blockade Shin Foong Ngiow,1,2 Grant A. McArthur,3,4 and Mark J. Smyth1,2,* 1Immunology
in Cancer and Infection Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia of Medicine, University of Queensland, Herston, QLD 4006, Australia 3Cancer Therapeutics Programs, Trescowthick Laboratories, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, VIC 3002, Australia 4Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2015.03.015 2School
Adaptive immune resistance ablates effective anti-tumor immune responses. In a recent issue of Nature, Victor and colleagues describe that anti-PD-L1 combats adaptive immune resistance upon localized radiation plus anti-CTLA-4 therapy. The superior activity of radiation and dual immune checkpoint blockade is mediated by non-redundant immune mechanisms in cancer.
Combinations of two or more immunotherapies with additive or synergistic benefit in preclinical models (Uno et al., 2006) have set the framework for combination successes in the clinic (Wolchok et al., 2013). With the recent success of immune checkpoint inhibitors and other immunomodulators, there has been renewed interest in evaluating the combination of such agents with radiation therapy (RT) in clinical trials (Verbrugge et al., 2012; Formenti and Demaria, 2013). The biologic premise behind such a strategy is that the tumor-antigen release and change in the tumor microenvironment achieved by localized RT will promote specific tumor targeting by the adaptive immune system, which can be augmented further by systemic immunestimulating agents (Tang et al., 2014). In this manner, clinicians hope to induce a phenomenon known as the abscopal effect, whereby localized RT results in immune-mediated tumor regression in disease sites well outside of the radiation field. RT also induces DNA damage and tumor cell death by promoting tumor-cell expression of Fas and MHC class I. Dying tumor cells release not only tumor antigens but also ATP and danger signals such as HMGB1 and calreticulin. RT also has some potentially deleterious effects by increasing the tumor cell expression of PD-L1, secretion of TGFb, and induction of Treg. A small pilot phase I/II study in prostate cancer (Slovin et al., 2013) and case reports in melanoma (Postow et al., 2012; Hiniker et al., 2012; Golden et al., 2013) combining RT with ipilimu-
mab show the possible clinical activity of this approach. Now, Victor et al. (2015) report a phase I clinical trial of 22 patients with advanced melanoma treated with RT and antiCTLA-4. In the trial, a single lesion was irradiated with hypofractionated stereotactic body radiation (6–8 Gy delivered over two or three fractions), followed by four cycles of ipilimumab beginning 3– 5 days after the last fraction of RT. Assessment of unirradiated lesions using RECIST criteria demonstrated 18% patients had a partial response as the best response, 18% had stable disease, and 64% had progressive disease. So, although partial responses were observed, like monotherapy with anti-CTLA-4, the majority of the patients did not respond. The authors then describe the use of the B16-F10 melanoma mouse model, where RT plus anti-CTLA-4 was more effective than either treatment alone, promoting the regression of both irradiated and unirradiated tumors (Victor et al., 2015). RT given before, or concurrently with, anti-CTLA-4 yielded similar results. Complete responses in these combination-treated mice were CD8+ T celldependent, and memory to rechallenge was demonstrated. However, not all mice responded, and melanoma cell lines from relapsing mice were derived. In these cell lines, resistance to the combination was confirmed, but the lesions were not RT resistant. Random forest machine learning analysis for sub-types of TIL isolated from the resistant melanomas demonstrated that the top predictor for
resistance was the CD8+CD44+ to Treg ratio, which failed to increase after RT plus anti-CTLA-4 (as it did in sensitive tumors). Transcriptomic profiling revealed that PD-L1 was among the top 0.2% of upregulated genes in melanoma (RT plus anti-CTLA-4 resistance signature). Genetic elimination of PD-L1 on the therapy-resistant Res499 melanoma cells by CRISPR dramatically restored response to RT plus anti-CTLA-4 (0% to 60% survival), suggesting upregulation of PD-L1 was one major mechanism, but not the only mechanism of resistance to RT plus anti-CTLA-4. In the original parental B16-F10, RT plus anti-CTLA-4 increased the proportion of PD-1+Eomes+ CD8 T cells and the proportion that were Ki67+ and granzyme (Grz) B+. But in the resistant sublines, the Ki67+GrzB+ CD8 T cells were not increased. The frequency of PD-1+CD8+ T cells that were Eomes+ was a striking modifier of the likelihood of a complete response (CR), because nearly all the CRs occurred when the frequency of Ki67+GrzB+ in PD-1+CD8+ was high, but the relative size of the PD1+Eomes+-exhausted population was small. Critically, adding anti-PD-L1 improved responses to the naive B16F10 and resistant sublines after RT plus anti-CTLA-4. Similar results were obtained in the mouse TSA mammary tumor and a mouse pancreatic tumor model. Random forest modeling showed that anti-CTLA-4 predominantly caused a decrease in Treg cells, anti-PD-L1 strongly increased CD8 TIL frequency,
Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 437
Cancer Cell
Previews
common, and most patients eventually die from progressive metastatic disease. A considerable amount of work remains to be done to create successful combinations of immunotherapeutics and radiation, which includes identifying the optimal radiation dose, fractionation, and sequence for use in combination with immune checkpoint inhibitors. ACKNOWLEDGMENTS We wish to acknowledge funding support from the National Health and Medical Research Council of Australia, The Susan Komen for the Cure, and the Cancer Council of Queensland.
REFERENCES
Figure 1. Schematic Model for Non-redundant Mechanisms of RT/Anti-CTLA-4/Anti-PD-L1 Therapy to Combat Immune Resistance in Melanoma Adapted from Victor et al. (2015) Extended Data Figure 6.
and blockade of both increased the CD8/ Treg ratio. RT only caused a modest increase in CD8 TILs; however, TCR sequencing revealed that there was an increased diversity of TCR clonotypes, which was also observed with RT plus anti-CTLA-4. This effect was also seen in the spleen and peripheral blood. Some clones reached 20% post-treatment in the blood with triple treatment. By contrast, peripheral T cell expansion was modest with RT and anti-CTLA-4 alone. So the concept gleaned from the mouse studies was that favorable immune changes in TILs after immune checkpoint blockade promoted their peripheral clonal expansion. When combined with increased TCR repertoire diversity afforded by RT, selection and oligoclonal peripheral expansion of clones with distinct TCR traits were favored (Figure 1). From the clinical trial of RT + ipilimumab, 12 patients had pre-treatment tumor biopsies, and pre- and post-treatment blood was available on 10 patients. From these low numbers, it was revealed that PD-L1lo intensity on melanoma cells was associated with re-invigoration of PD-1+Eomes+ CD8+ T cells after RT plus anti-CTLA-4, while PD-L1hi status was associated with persistent exhaustion of the CD8+T cells. PD-L1hi on melanoma biopsies collected from therapy-resistant patients is potentially reflecting the
failure of CTL anti-tumor cytotoxicity, consistent with the lower frequencies of Ki67+GrzB+PD1+CD8+ T cells. As discussed above, it is also worth emphasizing that a proportion of the mice with PD-L1 knockout Res499 cells still succumbed to disease after RT plus antiCTLA-4 treatment, suggesting a role for non-tumor PD-L1 in promoting resistance to RT/anti-CTLA-4. Intriguingly, despite other recent reports (Herbst et al., 2014) PD-L1 status on the macrophages was neither associated with reinvigoration nor independently predictive of progression-free survival. Although there may be activation of other T cell checkpoint pathways, the hierarchy of PD-L1 expression on tumor cells and non-tumor cells in driving T cell exhaustion warrants further study in larger patient cohorts. Nonetheless, the mechanism of radiation to diversify TCR clonotypes remains to be discerned. Radiotherapy is a major component of cancer treatment, with over 50% of cancer patients receiving radiation during the course of their disease. Radiation is used for both palliation of local symptoms and curative therapy in some malignancies, like lung, prostate, or head and neck cancer. However, although radiation offers clear benefits in terms of both local control and survival, disease recurrence outside the radiation field remains all too
438 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.
Formenti, S.C., and Demaria, S. (2013). J. Natl. Cancer Inst. 105, 256–265. Golden, E.B., Demaria, S., Schiff, P.B., Chachoua, A., and Formenti, S.C. (2013). Cancer Immunol Res 1, 365–372. Herbst, R.S., Soria, J.C., Kowanetz, M., Fine, G.D., Hamid, O., Gordon, M.S., Sosman, J.A., McDermott, D.F., Powderly, J.D., Gettinger, S.N., et al. (2014). Nature 515, 563–567. Hiniker, S.M., Chen, D.S., Reddy, S., Chang, D.T., Jones, J.C., Mollick, J.A., Swetter, S.M., and Knox, S.J. (2012). Transl Oncol 5, 404–407. Postow, M.A., Callahan, M.K., Barker, C.A., Yamada, Y., Yuan, J., Kitano, S., Mu, Z., Rasalan, T., Adamow, M., Ritter, E., et al. (2012). N. Engl. J. Med. 366, 925–931. Slovin, S.F., Higano, C.S., Hamid, O., Tejwani, S., Harzstark, A., Alumkal, J.J., Scher, H.I., Chin, K., Gagnier, P., McHenry, M.B., and Beer, T.M. (2013). Ann. Oncol. 24, 1813–1821. Tang, C., Wang, X., Soh, H., Seyedin, S., Cortez, M.A., Krishnan, S., Massarelli, E., Hong, D., Naing, A., Diab, A., et al. (2014). Cancer Immunol Res 2, 831–838. Uno, T., Takeda, K., Kojima, Y., Yoshizawa, H., Akiba, H., Mittler, R.S., Gejyo, F., Okumura, K., Yagita, H., and Smyth, M.J. (2006). Nat. Med. 12, 693–698. Verbrugge, I., Hagekyriakou, J., Sharp, L.L., Galli, M., West, A., McLaughlin, N.M., Duret, H., Yagita, H., Johnstone, R.W., Smyth, M.J., and Haynes, N.M. (2012). Cancer Res. 72, 3163–3174. Victor, C.T., Rech, A.J., Maity, A., Rengan, R., Pauken, K.E., Stelekati, E., Benci, J.L., Xu, B., Dada, H., Odorizzi, P.M., et al. (2015). Nature Published online March 9, 2015. http://dx.doi.org/10. 1038/nature14292. Wolchok, J.D., Kluger, H., Callahan, M.K., Postow, M.A., Rizvi, N.A., Lesokhin, A.M., Segal, N.H., Ariyan, C.E., Gordon, R.A., Reed, K., et al. (2013). N. Engl. J. Med. 369, 122–133.
