2,466
Views
20
CrossRef citations to date
0
Altmetric
Review

Immunotherapy as sensitizer for local radiotherapy

ORCID Icon, , ORCID Icon, , , , & show all
Article: 1832760 | Received 27 May 2020, Accepted 02 Oct 2020, Published online: 30 Oct 2020

References

  • Cooper JS, Guo MD, Herskovic A, Macdonald JS, Martenson, Jr JA, Al-Sarraf M, Byhardt R, Russell AH, Beitler JJ, Spencer S, et al. Chemoradiotherapy of locally advanced esophageal cancer: long-term follow-up of a prospective randomized trial (RTOG 85-01). Radiation therapy oncology group. JAMA. 1999;281(17):1623. doi:10.1001/jama.281.17.1623.
  • Curran WJ Jr, Paulus R, Langer CJ, Komaki R, Lee JS, Hauser S, Movsas B, Wasserman T, Rosenthal SA, Gore E, et al. Sequential vs. concurrent chemoradiation for stage III non-small cell lung cancer: randomized phase III trial RTOG 9410. J Natl Cancer Inst. 2011;103(19):1452. doi:10.1093/jnci/djr325.
  • Bosset JF, Collette L, Calais G, Mineur L, Maingon P, Radosevic-Jelic L, Daban A, Bardet E, Beny A, Ollier JC. EORTC radiotherapy group trial 22921. chemotherapy with preoperative radiotherapy in rectal cancer. N Engl J Med. 2006;355(11):1114. doi:10.1056/NEJMoa060829.
  • Pignon JP, le Maître A, Maillard E, Bourhis J. MACH-NC collaborative group. Meta-analysis of chemotherapy in head and neck cancer (MACH-NC): an update on 93 randomised trials and 17,346 patients. Radiother Oncol. 2009;92(1):4. doi:10.1016/j.radonc.2009.04.014.
  • Bartelink H, Roelofsen F, Eschwege F, Rougier P, Bosset JF, Gonzalez DG, Peiffert D, van Glabbeke M, Pierart M. Concomitant radiotherapy and chemotherapy is superior to radiotherapy alone in the treatment of locally advanced anal cancer: results of a phase III randomized trial of the European organization for research and treatment of cancer radiotherapy and gastrointestinal cooperative groups. J Clin Oncol. 1997;15:2040.
  • Dueñas-González A, Zarbá JJ, Patel F, Alcedo JC, Beslija S, Casanova L, Pattaranutaporn P, Hameed S, Blair JM, Barraclough H, et al. Phase III, open-label, randomized study comparing concurrent gemcitabine plus cisplatin and radiation followed by adjuvant gemcitabine and cisplatin versus concurrent cisplatin and radiation in patients with stage IIB to IVA carcinoma of the cervix. J Clin Oncol. 2011;29(13):1678. doi:10.1200/JCO.2009.25.9663.
  • Mak RH, Hunt D, Shipley WU, Efstathiou JA, Tester WJ, Hagan MP, Kaufman DS, Heney NM, Zietman A. Long-term outcomes in patients with muscle-invasive bladder cancer after selective bladder-preserving combined-modality therapy: a pooled analysis of radiation therapy oncology group protocols 8802, 8903, 9506, 9706, 9906, and 0233. J Clin Oncol. 2014;32(34):3801. doi:10.1200/JCO.2014.57.5548.
  • Van Limbergen EJ, De Ruysscher DK, Olivo Pimentel V, Marcus D, Berbee M, Hoeben A, Rekers N, Theys J, Yaromina A, Dubois LJ, et al. Combining radiotherapy with Immunotherapy: the past, the present and the future. Br J Radiol. 2017 Aug;90(1076):20170157. doi:10.1259/bjr.20170157.
  • Demaria S, Coleman CN, Formenti SC. Radiotherapy: changing the game in immunotherapy. Trends Cancer. 2016;2(6):286–11. doi:10.1016/j.trecan.2016.05.002.
  • Demaria S, Golden EB. Formenti SC role of local radiation therapy in cancer immunotherapy. JAMA Oncol. 2015;1(9):1325–1332. doi:10.1001/jamaoncol.2015.2756.
  • De Ruysscher D, Niedermann G, Burnet NG, Siva S, Lee AWM, Hegi-Johnson F. Radiotherapy toxicity. Nat Rev Dis Primers. 2019;5:14.
  • Reynders K, Illidge T, Siva S, Chang JY, De Ruysscher D. The abscopal effect of local radiotherapy: using immunotherapy to make a rare event clinically relevant. Cancer Treat Rev. 2015;41:503–510. doi:10.1016/j.ctrv.2015.03.011.
  • Baumann M, Petersen C, Schulz P, Baisch H. Impact of overall treatment time on local control of slow growing human GL squamous cell carcinoma in nude mice treated by fractionated irradiation. Radiother Oncol. 1999 Jan;50(1):107–111. doi:10.1016/S0167-8140(98)00112-1.
  • Plautz GE, Inoue M, Shu S. Defining the synergistic effects of irradiation and T-cell immunotherapy for murine intracranial tumors. Cell Immunol. 1996 Aug 1;171(2):277–284. doi:10.1006/cimm.1996.0204.
  • Everse LA, Renes IB, Jürgenliemk-Schulz IM, Rutgers DH, Bernsen MR, Dullens HF, Den Otter W, Battermann JJ. Local low-dose interleukin-2 induces systemic immunity when combined with radiotherapy of cancer. A pre-clinical study. Int J Cancer. 1997 Sep 17;72(6):1003–1007. doi:10.1002/(SICI)1097-0215(19970917)72:6<1003::AID-IJC14>3.0.CO;2-5.
  • Jürgenliemk-Schulz IM, Renes IB, Rutgers DH, Everse LA, Bernsen MR, Den Otter W, Battermann JJ. Anti-tumor effects of local irradiation in combination with peritumoral administration of low doses of recombinant interleukin-2 (rIL-2). Radiat Oncol Investig. 1997;5(2):54–61. doi:10.1002/(SICI)1520-6823(1997)5:2<54::AID-ROI3>3.0.CO;2-I.
  • Meng Y, Carpentier AF, Chen L, Boisserie G, Simon JM, Mazeron JJ, Delattre JY. Successful combination of local CpG-ODN and radiotherapy in malignant glioma. Int J Cancer. 2005 Oct 10;116(6):992–997. doi:10.1002/ijc.21131.
  • Mason KA, Neal R, Hunter N, Ariga H, Ang K, Milas L. CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors. Radiother Oncol. 2006 Aug;80(2):192–198. doi:10.1016/j.radonc.2006.07.024.
  • Zegers CM, Rekers NH, Quaden DH, Lieuwes NG, Yaromina A, Germeraad WT, Wieten L, Biessen EA, Boon L, Neri D, et al. Radiotherapy combined with the immunocytokine L19-IL2 provides long-lasting antitumor effects. Clin Cancer Res. 2015 Mar 1;21(5):1151–1160. doi:10.1158/1078-0432.CCR-14-2676.
  • van den Heuvel MM, Verheij M, Boshuizen R, Belderbos J, Dingemans AM, De Ruysscher D, Laurent J, Tighe R, Haanen J, Quaratino S. NHS-IL2 combined with radiotherapy: preclinical rationale and phase Ib trial results in metastatic non-small cell lung cancer following first-line chemotherapy. J Transl Med. 2015 Jan 27;13:32. doi:10.1186/s12967-015-0397-0.
  • Schölch S, Rauber C, Tietz A, Rahbari NN, Bork U, Schmidt T, Kahlert C, Haberkorn U, Tomai MA, Lipson KE, et al. Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors. Oncotarget. 2015 Mar 10;6(7):4663–4676. doi:10.18632/oncotarget.3081.
  • Connolly KA, Belt BA, Figueroa NM, Murthy A, Patel A, Kim M, Lord EM, Linehan DC, Gerber SA. Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes. Oncotarget. 2016 Dec 27;7(52):86522–86535. doi:10.18632/oncotarget.13287.
  • Wu CJ, Tsai YT, Lee IJ, Wu PY, Lu LS, Tsao WS, Huang YJ, Chang CC, Ka SM, Tao MH. Combination of radiation and interleukin 12 eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment. Oncoimmunology. 2018 Jul 23;7(9):e1477459. doi:10.1080/2162402X.2018.1477459.
  • Zhuang Y, Li S, Wang H, Pi J, Xing Y, Li G. PD-1 blockade enhances radio-immunotherapy efficacy in murine tumor models. J Cancer Res Clin Oncol. 2018 Oct;144(10):1909–1920. doi:10.1007/s00432-018-2723-4.
  • Buchegger F, Rojas A, Delaloye AB, Vogel CA, Mirimanoff RO, Coucke P, Sun LQ, Raimondi S, Denekamp J, Pèlgrin A, et al. Combined radioimmunotherapy and radiotherapy of human colon carcinoma grafted in nude mice. Cancer Res. 1995 Jan 1;55(1):83–89.
  • Chiang CS, Hong JH, Wu YC, McBride WH, Dougherty GJ Combining radiation therapy with interleukin-3 gene immunotherapy. Cancer Gene Ther. 2000 Aug;7(8):1172–1178. doi:10.1038/sj.cgt.7700217.
  • Lohr F, Hu K, Haroon Z, Samulski TV, Huang Q, Beaty J, Dewhirst MW, Li CY. Combination treatment of murine tumors by adenovirus-mediated local B7/IL12 immunotherapy and radiotherapy. Mol Ther. 2000 Sep;2(3):195–203. doi:10.1006/mthe.2000.0114.
  • Teitz-Tennenbaum S, Li Q, Rynkiewicz S, Ito F, Davis MA, McGinn CJ, Chang AE. Radiotherapy potentiates the therapeutic efficacy of intratumoral dendritic cell administration. Cancer Res. 2003 Dec 1;63(23):8466–8475.
  • Huang J, Wang Y, Guo J, Lu H, Lin X, Ma L, Teitz-Tennenbaum S, Chang AE, Li Q Radiation-induced apoptosis along with local and systemic cytokine elaboration is associated with DC plus radiotherapy-mediated renal cell tumor regression. Clin Immunol. 2007 Jun;123(3):298–310. doi:10.1016/j.clim.2007.02.005
  • Meng Y, Efimova EV, Hamzeh KW, Darga TE, Mauceri HJ, Fu YX, Kron SJ, Weichselbaum RR. Radiation-inducible immunotherapy for cancer: senescent tumor cells as a cancer vaccine. Mol Ther. 2012 May;20(5):1046–1055. doi:10.1038/mt.2012.19.
  • Wang YS, Liu SJ, Huang SC, Chang CC, Huang YC, Fong WL, Chi MS, Chi KH. Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy. Front Oncol. 2012 Oct 29;2:149. doi:10.3389/fonc.2012.00149.
  • Wei S, Egenti MU, Teitz-Tennenbaum S, Zou W, Chang AE. Effects of tumor irradiation on host T-regulatory cells and systemic immunity in the context of adoptive T-cell therapy in mice. J Immunother. 2013 Feb;36(2):124–132. doi:10.1097/CJI.0b013e31828298e6.
  • Dovedi SJ, Adlard AL, Lipowska-Bhalla G, McKenna C, Jones S, Cheadle EJ, Stratford IJ, Poon E, Morrow M, Stewart R, et al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014 Oct 1;74(19):5458–5468. doi:10.1158/0008-5472.CAN-14-1258.
  • Lim JY, Brockstedt DG, Lord EM, Gerber SA. Radiation therapy combined with Listeria monocytogenes-based cancer vaccine synergize to enhance tumor control in the B16 melanoma model. Oncoimmunology. 2014 Jun 3;3:e29028. doi:10.4161/onci.29028.
  • Rekers NH, Zegers CM, Yaromina A, Lieuwes NG, Biemans R, Senden-Gijsbers BL, Losen M, Van Limbergen EJ, Germeraad WT, Neri D, et al. Combination of radiotherapy with the immunocytokine L19-IL2: additive effect in NK cell dependent tumour model. Radiother Oncol. 2015 Sep;116(3):438–442. doi:10.1016/j.radonc.2015.06.019.
  • Blanchard M, Shim KG, Grams MP, Rajani K, Diaz RM, Furutani KM, Thompson J, Olivier KR, Park SS, Markovic SN, et al. Definitive management of oligometastatic melanoma in a murine model using combined ablative radiation therapy and viral immunotherapy. Int J Radiat Oncol Biol Phys. 2015 Nov 1;93(3):577–587. doi:10.1016/j.ijrobp.2015.07.2274.
  • Mondini M, Nizard M, Tran T, Mauge L, Loi M, Clémenson C, Dugue D, Maroun P, Louvet E, Adam J, et al. Synergy of radiotherapy and a cancer vaccine for the treatment of HPV-associated head and neck cancer. Mol Cancer Ther. 2015 Jun;14(6):1336–1345. doi:10.1158/1535-7163.MCT-14-1015.
  • Sharabi AB, Nirschl CJ, Kochel CM, Nirschl TR, Francica BJ, Velarde E, Deweese TL, Drake CG. Stereotactic radiation therapy augments antigen-specific PD-1-mediated antitumor immune responses via cross-presentation of tumor antigen. Cancer Immunol Res. 2015 Apr;3(4):345–355. doi:10.1158/2326-6066.CIR-14-0196.
  • Monjazeb AM, Kent MS, Grossenbacher SK, Mall C, Zamora AE, Mirsoian A, Chen M, Kol A, Shiao SL, Reddy A, et al. Blocking Indolamine-2,3-dioxygenase rebound immune suppression boosts antitumor effects of radio-immunotherapy in murine models and spontaneous canine malignancies. Clin Cancer Res. 2016 Sep 1;22(17):4328–4340. doi:10.1158/1078-0432.CCR-15-3026.
  • Young KH, Baird JR, Savage T, Cottam B, Friedman D, Bambina S, Messenheimer DJ, Fox B, Newell P, Bahjat KS, et al. Optimizing timing of immunotherapy improves control of tumors by hypofractionated radiation therapy. PLoS One. 2016 Jun 9;11(6):e0157164. doi:10.1371/journal.pone.0157164.
  • Zheng W, Skowron KB, Namm JP, Burnette B, Fernandez C, Arina A, Liang H, Spiotto MT, Posner MC, Fu YX. Weichselbaum RR combination of radiotherapy and vaccination overcomes checkpoint blockade resistance. Oncotarget. 2016 Jul 12;7(28):43039–43051. doi:10.18632/oncotarget.9915.
  • Oweida A, Lennon S, Calame D, Korpela S, Bhatia S, Sharma J, Graham C, Binder D, Serkova N, Raben D, Karam SD, et al. Ionizing radiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopic murine head and neck squamous cell carcinoma. Oncoimmunology. 2017 Aug 3;6(10):e1356153. doi:10.1080/2162402X.2017.1356153.
  • Weiss T, Weller M, Guckenberger M, Sentman CL, Roth P. NKG2D-Based CAR T cells and radiotherapy exert synergistic efficacy in Glioblastoma. Cancer Res. 2018 Feb 15;78(4):1031–1043. doi:10.1158/0008-5472.CAN-17-1788.
  • Choi CW, Jeong MH, Park YS, Son CH, Lee HR, Koh EK. Combination treatment of stereotactic body radiation therapy and immature dendritic cell vaccination for augmentation of local and systemic effects. Cancer Res Treat. 2019 Apr;51(2):464–473. doi:10.4143/crt.2018.186.
  • Wang H, Lin X, Luo Y, Sun S, Tian X, Sun Y, Zhang S, Chen J, Zhang J, Liu X, et al. α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8+ T-cell infiltration. Immunotherapy. 2019 Feb;11(2):101–118. doi:10.2217/imt-2018-0049.
  • Brinkmann OA, Bruns F, Gosheger G, Micke O, Hertle L. Treatment of bone metastases and local recurrence from renal cell carcinoma with immunochemotherapy and radiation. World J Urol. 2005 Jul;23(3):185–190. doi:10.1007/s00345-004-0479-8.
  • Jacobs JJ, Hordijk GJ, Jürgenliemk-Schulz IM, Terhaard CH, Koten JW, Battermann JJ, Den Otter W. Treatment of stage III-IV nasopharyngeal carcinomas by external beam irradiation and local low doses of IL-2. Cancer Immunol Immunother. 2005 Aug;54(8):792–798. doi:10.1007/s00262-004-0641-6.
  • Seung SK, Curti BD, Crittenden M, Walker E, Coffey T, Siebert JC, Miller W, Payne R, Glenn L, Bageac A, et al. Phase 1 study of stereotactic body radiotherapy and interleukin-2–tumor and immunological responses … . Sci Transl Med. 2012 Jun 6;4(137):137ra74. doi:10.1126/scitranslmed.3003649.
  • Barker CA, Postow MA, Khan SA, Beal K, Parhar PK, Yamada Y, Lee NY, Wolchok JD. Concurrent radiotherapy and ipilimumab immunotherapy for patients with melanoma. Cancer Immunol Res. 2013 Aug;1(2):92–98. doi:10.1158/2326-6066.CIR-13-0082.
  • Abei M, Okumura T, Fukuda K, Hashimoto T, Araki M, Ishige K, Hyodo I, Kanemoto A, Numajiri H, Mizumoto M, et al. A phase I study on combined therapy with proton-beam radiotherapy and in situ tumor vaccination for locally advanced recurrent hepatocellular carcinoma. Radiat Oncol. 2013 Oct;16(8):239. doi:10.1186/1748-717X-8-239.
  • Kiess AP, Wolchok JD, Barker CA, Postow MA, Tabar V, Huse JT, Chan TA, Yamada Y, Beal K. Stereotactic radiosurgery for melanoma brain metastases in patients receiving ipilimumab: safety profile and efficacy of combined treatment. Int J Radiat Oncol Biol Phys. 2015 Jun 1;92(2):368–375. doi:10.1016/j.ijrobp.2015.01.004.
  • Twyman-Saint Victor C, Rech AJ, Maity A, Rengan R, Pauken KE, Stelekati E, Benci JL, Xu B, Dada H, Odorizzi PM, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature. 2015 Apr 16;520(7547):373–377. doi:10.1038/nature14292.
  • Nardin C, Mateus C, Texier M, Lanoy E, Hibat-Allah S, Ammari S, Robert C, Dhermain F. Tolerance and outcomes of stereotactic radiosurgery combined with anti- programmed cell death-1 (pembrolizumab) for melanoma brain metastases. Melanoma Res. 2018 Apr;28(2):111–119. doi:10.1097/CMR.0000000000000413.
  • Bourhis J, Sire C, Graff P, Grégoire V, Maingon P, Calais G, Gery B, Martin L, Alfonsi M, Desprez P, et al. Concomitant chemoradiotherapy versus acceleration of radiotherapy with or without concomitant chemotherapy in locally advanced head and neck carcinoma (GORTEC 99-02): an open-label phase 3 randomised trial. Lancet Oncol. 2012;13(2):145. doi:10.1016/S1470-2045(11)70346-1.
  • Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, Jones CU, Sur R, Raben D, Jassem J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck.N. Engl J Med. 2006 Feb 9;354(6):567–578. doi:10.1056/NEJMoa053422.
  • James ND, Hussain SA, Hall E, Jenkins P, Tremlett J, Rawlings C, Crundwell M, Sizer B, Sreenivasan T, Hendron C, et al. BC2001 investigators. Radiotherapy with or without chemotherapy in muscle-invasive bladder cancer. N Engl J Med. 2012 Apr 19;366(16):1477–1488. doi:10.1056/NEJMoa1106106.
  • Northover J, Glynne-Jones R, Sebag-Montefiore D, James R, Meadows H, Wan S, Jitlal M. Ledermann JChemoradiation for the treatment of epidermoid anal cancer: 13-year follow-up of the first randomised UKCCCR Anal Cancer Trial (ACT I). Br J Cancer. 2010 Mar 30;102(7):1123–1128. doi:10.1038/sj.bjc.6605605.
  • Rudd CE. CTLA-4 co-receptor impacts on the function of Treg and CD8+ T-cell subsets. Eur J Immunol. 2009 Mar;39(3):687–690. doi:10.1002/eji.200939261.
  • Kachikwu EL, Iwamoto KS, Liao YP, DeMarco JJ, Agazaryan N, Economou JS, McBride WH, Schaue D. Radiation enhances regulatory T cell representation. Int J Radiat Oncol Biol Phys. 2011;81(4):1128–1135. doi:10.1016/j.ijrobp.2010.09.034.
  • Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Lao CD, Cowey CL, Schadendorf D, Wagstaff J, Dummer R, et al. Five-year survival with combined nivolumab and ipilimumab in advanced melanoma. N Engl J Med. 2019 Oct 17;381(16):1535–1546. doi:10.1056/NEJMoa1910836.
  • Serre R, Barlesi F, Muracciole X, Barbolosi D. Immunologically effective dose: a practical model for immuno-radiotherapy. Oncotarget. 2018 Aug 7;9(61):31812–31819. doi:10.18632/oncotarget.25746.
  • Yovino S, Kleinberg L, Grossman SA, Narayanan M, Ford E. The etiology of treatment-related lymphopenia in patients with malignant gliomas: modeling radiation dose to circulating lymphocytes explains clinical observations and suggests methods of modifying the impact of radiation on immune cells. Cancer Invest. 2013;31:140–144. doi:10.3109/07357907.2012.762780.
  • Merrick A, Errington F, Milward K, O’Donnell D, Harrington K, Bateman A, Pandha H, Vile R, Morrison E, Selby P, et al. Immunosuppressive effects of radiation on human dendritic cells: reduced IL-12 production on activation and impairment of naive T-cell priming. Br J Cancer. 2005;92:1450. doi:10.1038/sj.bjc.6602518.
  • Filatenkov A, Baker J, Mueller AM, Kenkel J, Ahn GO, Dutt S, Zhang N, Kohrt H, Jensen K, Dejbakhsh-Jones S, et al. Ablative tumor radiation can change the tumorimmune cell microenvironment to induce durable complete remissions. Clin Cancer Res. 2015;21:3727–3739. doi:10.1158/1078-0432.CCR-14-2824.
  • Honeychurch J, Illidge TM. The influence of radiation in the context of developing combination immunotherapies in cancer. Ther Adv Vaccines Immunother. 2017 Dec;5(6):115–122. doi:10.1177/2051013617750561.
  • Park HJ, Griffin RJ, Hui S, Levitt SH, Song CW. Radiation-induced vascular damage in tumors: implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS). Radiat Res. 2012;177(3):311–327. doi:10.1667/RR2773.1.
  • Koontz BF, Verhaegen F, De Ruysscher D. Tumour and normal tissue radiobiology in mouse models: how close are mice to mini-humans? Br J Radiol. 2017;90:20160441. doi:10.1259/bjr.20160441.
  • Schwartz LH, Litière S, de Vries E, Ford R, Gwyther S, Mandrekar S, Shankar L, Bogaerts J, Chen A, Dancey J, et al. 1.1-Update and clarification: from the RECIST committee. Eur J Cancer. 2016 Jul;62:132–137. doi:10.1016/j.ejca.2016.03.081.
  • Wolchok JD, Hoos A, O’Day S, Weber JS, Hamid O, Lebbé C, Maio M, Binder M, Bohnsack O, Nichol G, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immunearelated response criteria. Clin Cancer Res. 2009;15(23):7412. doi:10.1158/1078-0432.CCR-09-1624.
  • Seymour L, Bogaerts J, Perrone A, Ford R, Schwartz LH, Mandrekar S, Lin NU, Litière S, Dancey J, Chen A, et al. RECIST working group. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017;18(3):e143. doi:10.1016/S1470-2045(17)30074-8.