Synchrotron radiation therapy for the treatment

of cancer

Lloyd Mark Lee Smyth

ORCID ID: 0000-0002-6675-5807

Doctor of Philosophy

September 2018

Department of Obstetrics and Gynaecology

Faculty of Medicine, Dentistry and Health Sciences

University of Melbourne

Submitted in total fulfilment of the requirements of the degree Abstract

Despite advances in radiation oncology there remain numerous clinical scenarios where radiation therapy, in conjunction with other cancer therapies, is unable to significantly improve disease prognosis. Advanced lung cancer, pancreatic cancer, and aggressive paediatric brain tumours such as Diffuse Intrinsic Pontine Glioma are examples of incurable diseases with an extremely poor prognosis.

Synchrotron-based radiation therapy modalities challenge classical radiobiology paradigms and could address these unmet clinical needs. The two synchrotron radiation therapy modalities investigated in this thesis are microbeam radiation therapy (MRT) and high dose-rate synchrotron broad-beam radiation therapy (SBBR). The brilliance and minimal divergence of synchrotron-generated radiation gives MRT and SBBR physical properties that are distinctly different to conventional radiation therapy (CRT).

Pre-clinical animal studies demonstrate the potential of these unique physical characteristics to both control tumours and reduce radiation-induced damage to healthy tissue.

The purpose of this thesis was to present radiobiological data that would inform future veterinary and clinical trials of MRT and/or SBBR. Specific objectives were to: 1) produce systematic toxicity data for organs of the head, thorax and abdomen, 2) characterise dose-equivalence between MRT, SBBR and CRT based on both in vitro and in vivo techniques, 3) describe the differential effects of MRT and broad-beam radiation therapy on healthy tissue at a molecular level, and, 4) identify optimal clinical scenarios were MRT could be applied, considering the limitations imposed by normal tissue toxicity.

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Based on total and partial body dose-escalation studies in a murine model, MRT peak doses of approximately 120 Gy and 260 Gy were equivalent to approximately 7 Gy and

12.5 Gy, respectively. The MRT valley dose was a better predictor of normal tissue toxicity than the MRT peak dose, and for SBBR, a normal tissue sparing effect (ie. a

‘FLASH’ effect) could not be detected at a dose-rate of 35-40 Gy/s. Based on a treatment planning study using clinical datasets, small recurrent glioblastomas and head and neck tumours demonstrated the most favourable MRT dosimetry.

This thesis includes the first in vivo dose-equivalence data for synchrotron radiation therapy compared to conventional radiation therapy and the first MRT toxicity data for total body, abdominal and thoracic irradiation. These data are essential for designing safe treatment regimens for future veterinary trials and ultimately, the first human clinical trial of MRT and/or SBBR.

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Declaration

This thesis contains only original work towards the degree of Doctor of Philosophy. No material in this thesis has been accepted for the award of any other degree at any university or other institution. To the best of my knowledge, this thesis contains no material published previously or written by another person, except where reference is made in the text.

This thesis is fewer than the maximum word limit in length, exclusive of tables, bibliographies and appendices.

All animal studies referred to in this thesis were subject to review and approved by the

University of Melbourne Office for Research Ethics and Integrity and performed in accordance with all relevant guidelines and regulations. All human studies were approved by the relevant institutional human ethics committee and conducted in accordance with all relevant guidelines and regulations.

Signed:

Lloyd Smyth

12th September 2018

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List of publications and presentations arising from thesis

Publications

Smyth, LML, Day, LR, Woodford, K, Rogers, PAW, Crosbie, JC & Senthi, S. ‘Identifying optimal clinical scenarios for synchrotron microbeam radiation therapy: a treatment planning study’. Submitted to Radiotherapy and Oncology August 2018.

Smyth, LML, Donoghue, JF, Ventura, JA, Livingstone, J, Bailey, T, Day, LRJ, Crosbie, JC & Rogers, PAW 2018. ‘Comparative toxicity of synchrotron and conventional radiation therapy based on total and partial body irradiation in a murine model’. Scientific Reports, vol. 8, no. 1, pp. e12044.

Smyth, LM, Rogers, PAW, Crosbie, JC & Donoghue, JF 2018. ‘Characterisation of diffuse intrinsic pontine glioma radiosensitivity using synchrotron microbeam radiotherapy and conventional radiation therapy in vitro’. Radiation Research, vol. 189, no. 2, pp. 146-155.

Smyth, LM, Senthi, S, Crosbie, JC & Rogers, PA 2016. ‘The normal tissue effects of microbeam radiotherapy: what do we know, and what do we need to know to plan a human clinical trial?’ International Journal of Radiation Biology, vol. 92, no. 6, pp. 302-311.

Presentations

Oral

Smyth, LML, Ventura, JA, Donoghue, JF, Crosbie, JC & Rogers, PAW Nov 2017. ‘Biological dose-equivalence between synchrotron and conventional radiation therapy based on normal tissue toxicity’. Engineering and Physical Sciences in Medicine Annual Meeting, Hobart, Australia.

Smyth, LML, Ventura, JA, Day, LR, Donoghue, JF, Crosbie, JC & Rogers, PAW Nov 2017. ‘Biological dose-equivalence between synchrotron and conventional radiation therapy based on normal tissue toxicity’. Royal Women’s Hospital Research Week, Melbourne, Australia.

Smyth, LML, Ventura, JA, Crosbie, JC, Donoghue, JD, Senthi, S & Rogers, PAW May 2017. ‘Normal tissue and dose-equivalence of synchrotron radiotherapy

iv modalities’. European Society for Radiotherapy and Oncology 36th Annual Congress, Vienna, Austria.

Smyth, LML, Donoghue, JD, Crosbie, JC, Senthi, S & Rogers, PAW Mar 2017. ‘Towards a human clinical trial of synchrotron radiotherapy’. 12th Annual Scientific Meeting of Medical Imaging and Radiation Therapy, Perth, Australia.

Smyth, LM, Rogers, PA, Crosbie JC & Donoghue, JD Nov 2016. ‘Determining the dose equivalence of synchrotron microbeam radiotherapy and conventional radiotherapy using diffuse intrinsic pontine glioma cell lines’. Engineering and Physical Sciences in Medicine Annual Meeting, Sydney, Australia.

Smyth, LM, Donoghue, JD, Crosbie, JC & Rogers, PAW Nov 2016. ‘Evaluating the response of aggressive glioma cell lines to synchrotron microbeam radiotherapy compared to conventional broad-beam irradiation’. Australian Synchrotron Annual User Meeting, Melbourne, Australia.

Smyth, LML, Senthi, S, Crosbie, JC & Rogers, PAW Mar 2016. ‘The normal tissue effects of synchrotron radiotherapy: towards the first human clinical trial’. 14th International Workshop on Radiation Damage to DNA, Melbourne, Australia.

Poster

Smyth, LML, Crosbie, JC, Rogers, PAW & Donoghue, JD, May 2017. ‘In vitro response of diffuse intrinsic pontine glioma cell lines to microbeam versus conventional radiotherapy’. European Society for Radiotherapy and Oncology 36th Annual Congress, Vienna, Austria.

Smyth, LML, Crosbie, JC & Rogers, PAW May 2015. ‘The normal tissue effects of synchrotron microbeam radiotherapy: what do we know, and what do we need to know to conduct a human clinical trial?’ Epworth HealthCare Research Week, Melbourne, Australia.

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Preface

Publication status, co-author contributions and funding sources of papers included in thesis

Chapter 3: published by Radiation Research on November 30, 2017.

- Full citation: Smyth, LM, Rogers, PAW, Crosbie, JC & Donoghue, JF 2018. Characterization of diffuse intrinsic pontine glioma radiosensitivity using synchrotron microbeam radiotherapy and conventional radiation therapy in vitro. Radiation Research, vol. 189, no. 2, pp. 146-155. - Author contributions: All authors conceived the idea for this project. J Donoghue supplied cell lines for irradiation. L Smyth prepared cell lines for irradiation, performed the irradiations, clonogenic assays and dose-equivalence calculations. L Smyth and J Donoghue performed the flow cytometry assays. J Crosbie oversaw the synchrotron radiation therapy dosimetry. L Smyth and J Donoghue interpreted the results and wrote the manuscript. All authors commented on the manuscript. - Funding sources: L Smyth was supported by a Research Training Program (RTP) scholarship from the Australian Government. J Crosbie was supported by an Early Career Researcher (ECR) fellowship from the National Health and Medical Research Council (NHMRC). This work was supported by a Project grant (1061772) from the NHMRC of Australia and grants from the Robert Connor Dawes Foundation and the Isabella and Marcus Foundation.

Chapter 4: published by Scientific Reports on August 13, 2018.

- Full citation: Smyth, LML, Donoghue, JF, Ventura, JA, Livingstone, J, Bailey, T, Day, LRJ, Crosbie, JC & Rogers, PAW 2018. Comparative toxicity of synchrotron and conventional radiation therapy based on total and partial body irradiation in a murine model. Scientific Reports, vol. 8, no. 1, pp. e12044. - P Rogers, J Crosbie and L Smyth designed the experiments; all authors participated in scientific discussions; L Smyth, J Ventura, J Donoghue, J Crosbie, J Livingstone, T Bailey, L Day and P Rogers performed the irradiations; L Smyth, J Donoghue and J Ventura carried out post-irradiation animal monitoring; L Smyth, J Donoghue and P Rogers performed the histological analysis; L Day and T Bailey performed Monte

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Carlo simulations relating to dosimetry; L Smyth wrote the manuscript with input from all authors. - Funding sources: L Smyth was supported by a Research Training Program (RTP) scholarship from the Australian Government. This work was supported by a Project grant (1061772) from the NHMRC of Australia.

Chapter 5: submitted for publication to Radiotherapy and Oncology on August 21, 2018

- Author contributions: L Smyth, J Crosbie, P Rogers and S Senthi conceived the idea for this project. L Smyth, J Crosbie and S Senthi designed the study. L Day implemented and optimised the dose calculation algorithm use for treatment planning. L Smyth created and evaluated the MRT plans. K Woodford evaluated the clinical treatment plans. All authors participated in scientific discussions. L Smyth drafted the manuscript, with input from all co-authors. - Funding sources: L Smyth was supported by a Research Training Program scholarship from the Australian Government. This project was supported by a grant from the Australian National Health and Medical Research Council [Project Grant 1061772].

Appendix A: published by the Interntional Journal of Radiation Biology on March 16, 2016

- Full citation: Smyth, LM, Senthi, S, Crosbie, JC & Rogers, PA 2016. The normal tissue effects of microbeam radiotherapy: What do we know, and what do we need to know to plan a human clinical trial? International Journal of Radiation Biology, vol. 92, no. 6, pp. 302-311. - Author contributions: L Smyth, J Crosbie and P Rogers conceived the idea for literature review. L Smyth performed the literature search, screened articles for relevance and reviewed relevant articles. L Smyth wrote the article with input from all authors. - Funding sources: L Smyth was supported by a RTP scholarship from the Australian Government. S Senthi and J Crosbie were supported by ECR fellowships from the NHMRC of Australia. This work was supported by a Project Grant (1061772) from the NHMRC and a Grant-in-aid from the Cancer Council Victoria.

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Acknowledgements

The role-change from radiation therapist to researcher – leaping from linear-accelerator to laboratory – was not without risk and not without a retinue of supporters and advisers who made the transition not only possible, but also rewarding.

Firstly, I am indebted to Prof. Peter Rogers and A. Prof. Jeffrey Crosbie for coaxing me away from the comfort of the clinic and into the exciting potential – and daunting unknown – of research. Peter and Jeff introduced me to the synchrotron radiation therapy project and were my first impression, and example, of translational research.

Both formally and informally, Peter has shared with me wisdom and experience from his many fruitful years of research. From how to develop hypotheses and plan experiments, to strategies for grant writing, publishing papers and networking (and not to mention several lessons in navigating bureaucracy and administration), Peter has taught me invaluable skills that will serve me long after my PhD candidature has ended.

Jeff has been, and still is, a mentor who in many ways has gone before me on the path from clinic to laboratory, from bedside to bench. He has provided me with insight into this career pathway, shown me how to leverage my clinical knowledge for research gain and facilitated many collaborative connections, all of which will undoubtedly prove pivotal in continuing a career in full-time research.

I am grateful also to Dr. Jacqueline Donoghue, who as a supervisor alongside Jeff and

Peter, has invested numerous hours of training, planning and constructive feedback into my development as a researcher. I am especially thankful for Jacqui’s patience and calm presence; she was the first to introduce and orientate me to the (sometimes fickle) art of cell-culture.

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There are many others who offered support, mentorship and camaraderie throughout my

PhD candidature and are deserving of thanks. Dr. Yasmin Jayasinghe, Dr. Harry

Georgiou and Dr. Jane Girling provided sterling oversight as members of my advisory committee, contributing encouragement and affirmation, asking pertinent and constructive questions and providing timely advice to ensure I reached each milestone of my candidature. The many members of the GRC research group – Dr. Sarah

Holdsworth-Carson, Dr. Premila Paiva, Ms. Leonie Cann, Dr. Megan Cock, Ms. Jessica

Ventura – on countless times and in various ways provided technical support, scientific

(and non-scientific) discussion, listening ears and counsel. Externally, the staff at the

Imaging and Medical Beam-line, particularly Dr. Andrew Stevenson and Dr. Jayde

Livingstone, and at the Alfred Hospital, particularly Dr. Sashendra Senthi, lent their expertise to studies conducted during my candidature.

I am also grateful to my managers and colleagues at Epworth Radiation Oncology (now

Icon Cancer Centre) for supporting my decision to pursue research and providing flexibility to continue working one day per week in the clinic.

Finally, I give praise to Jesus Christ, the author and perfector of my faith, for the gift of

His endless grace and mercy, and for the ability to ponder the wonders of creation. I honour my family, who are my greatest treasure in this world. To my daughters, Adelie

Reuelle and Eleanor Lina, you have brought me indescribable joy. You are treasured and loved simply for being who you are. To my wife, Emily, your sacrificial love is unrivalled and humbles me completely. You have encouraged and released me to pursue the passions in my heart. These are not my accomplishments; they are equally yours.

Lloyd Smyth, 21st August 2018

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Table of Contents

Abstract ...... i

Declaration ...... iii

List of publications and presentations arising from thesis ...... iv

Preface ...... vi

Publication status, co-author contributions and funding sources of papers included in thesis ...... vi

Acknowledgements ...... viii

Table of Contents ...... x

List of tables/figures ...... xii

List of 3rd party copyright material ...... xv

1. General Introduction ...... 1

1.1 Problem statement ...... 1

1.2 Thesis objectives ...... 2

1.3 Overview of thesis chapters ...... 4

2. Literature review ...... 6

2.1 Preface ...... 6

2.2 Introduction to clinical radiation therapy ...... 6

2.3 External beam radiation therapy: physics principles ...... 10

2.4 Principles of radiobiology ...... 14

2.5 Technical developments in modern external beam radiation therapy ...... 23

2.6 Introduction to synchrotron radiation therapy ...... 28

2.7 Normal tissue toxicity ...... 36

2.8 Biological dose-equivalence ...... 52

2.9 Medical physics developments in synchrotron radiation therapy ...... 55

2.10 Conclusion ...... 60

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3. In vitro characterisation of diffuse intrinsic pontine glioma radiosensitivity using microbeam and broad-beam radiotherapy ...... 62

3.1 Preface ...... 62

3.2 Published manuscript ...... 63

3.3 Extended methods for flow-cytometry assays ...... 96

4. In vivo dose-equivalence between synchrotron and conventional radiation therapy based on murine normal tissue toxicity ...... 100

4.1 Preface ...... 100

4.2 Published manuscript ...... 101

4.3 Supplementary information ...... 128

5. Identification of optimal clinical scenarios for MRT ...... 140

5.1 Preface ...... 140

5.2 Submitted manuscript ...... 141

6. Discussion, conclusions and future directions ...... 173

6.1 Summary of key findings ...... 173

6.2 Discussion ...... 175

6.3 Work in progress ...... 187

6.4 Future directions ...... 190

6.5 Conclusion ...... 193

Bibliography ...... 195

Appendix ...... 212

Appendix A: MRT normal tissue toxicity review ...... 212

Appendix B: Final published version of in vivo dose-equivalence manuscript ...... 241

Appendix C: Methods for mouse brainstem comparative gene expression study .... 253

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List of tables/figures

Figure no. and description Location of figure in thesis

Figure 2.1 X-ray energy spectra following the interaction of Chpt 2.3, p. 12 electrons with a tungsten target at various excitation/acceleration potentials

Figure 2.2. Schematic diagram of a linear accelerator in x-ray Chpt 2.3, p. 14 therapy mode and electron therapy mode

Figure 2.3. The direct and indirect actions of radiation on DNA Chpt 2.4, p. 17

Figure 2.4. The linear-quadratic model for cell survival following Chpt 2.4, p. 20 irradiation

Figure 2.5. Conventional 3D CRT versus IMRT for prostate cancer Chpt 2.5, p. 24 treatment

Figure 2.6. A lateral dose profile through a field of microbeams Chpt 2.6, p. 30 with 50 µm width and 400 µm centre-to-centre spacing

Figure 2.7. Cerebellar histology approximately 15 months Chpt 2.7, p. 38 following MRT irradiation of weanling piglets. Clear stripes of cell loss, corresponding to microbeam paths, are evident

Table 2.1. MRT-induced histological damage to normal brain tissue Chpt 2.7, p. 41- 42 Table 2.2. Functional outcomes after brain irradiation with MRT Chpt 2.7, p. 43 Table 2.3. Effects of MRT on normal brain microvasculature Chpt 2.7, p. 44- 45 Table 2.4. MRT-induced toxicity in various normal tissue types Chpt 2.7, p. 46- 47 Figure 2.8. A robotic patient position system used at the Heidelberg Chpt 2.9, p. 59 Ion Beam therapy centre in Germany

Table 3.1. Interpolated equivalent CRT doses for increasing MRT Chpt 3.2, p. 73 doses Table 3.2. Percentage of apoptotic cells and fold change in Chpt 3.2, p. 74

xii irradiated compared to control groups Figure 3.1. Clonogenic survival of DIPG cells following MRT and Chpt 3.2, p. 89 CRT

Figure 3.2. Dose-dependent increase in apoptotic DIPG cells Chpt 3.2, p. 90 following MRT and CRT

Figure 3.3. Differential cell cycle response between JHH and Chpt 3.2, p. 91 SF7761 cell lines following MRT and CRT

Figure 3.4. Differential development of polyploidy following Chpt 3.2, p. 92 irradiation

Figure 3.5. Proposed mechanism of radio-resistance development in Chpt 3.2, p. 94 DIPG cells

Sup. Figure 3.1. PVDR versus depth based on Monte Carlo Chpt 3.2, p. 95 simulations of a 140 mm x 30 mm field of microbeams with a width of 50 μm and a centre-to-centre spacing of 400 μm.

Table 4.1. Equivalent (TD50) doses for MRT, SBBR and CRT (Gy) Chpt 4.2, p. 107

Table 4.2. Dose groups, field sizes and dose-rates for total and Chpt 4.2, p. 115- partial body irradiations 116

Figure 4.1. Dose response curves following conventional radiation Chpt 4.2, p. 122 therapy (CRT), microbeam radiation therapy (MRT) and high dose- rate synchrotron broad-beam radiation therapy (SBBR)

Figure 4.2. Post-irradiation weight gain for surviving mice Chpt 4.2, p. 123

Figure 4.3. Intestinal histopathology following abdominal partial Chpt 4.2, p. 124 body irradiation

Figure 4.4. Histopathological changes to the cerebellum following Chpt. 4.2, p. 125 microbeam radiation therapy (MRT)

Figure 4.5. Pulmonary damage and fibrosis 170 to 180 days Chpt. 4.2, p. 126 following thoracic partial body irradiation

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Figure 4.6. Mouse positioning for irradiation Chpt. 4.2, p. 127

Supplementary Figure 4.1 Monte Carlo (GEANT4) generated Chpt. 4.3, p. 132 percentage depth dose (PDD) curves for keV x-ray beams incident on a water phantom

Supplementary Figure 4.2. Geometry and composition of the Chpt 4.3, p. 133 theoretical phantom used to model the mouse and plastic (poly- methyl-methacrylate; PMMA) holder. Table 5.1. Summary of clinical plans used for MRT planning Chpt 5.2, p. 148- 149 Table 5.2. Single fraction tolerance doses for organs at risk Chpt 5.2, p. 150 Table 5.3. Dosimetry achieved in MRT plans Chpt 5.2, p. 153- 154 Figure 5.1. Dose-volume histograms for single fraction MRT valley Chpt 5.2, p. 167- dose plans. 168 Figure 5.2. Plots of mean PVDR for the each PTV versus the Chpt 5.2, p. 169 volume and central depth of the PTV. Figure 5.3. PVDR depth profiles for the glioblastoma, head and Chpt 5.2. p. 170- neck, locoregionally recurrent breast cancer and sacral schwannoma 172 target volumes. Figure 6.1. Equivalent CRT doses for 112 Gy MRT based on three Chpt 6.2, p. 176 different microbeam collimator geometries Figure 6.2. A recurrent glioblastoma case selected for MRT Chpt 6.2, p. 184 treatment planning. Figure 6.3. Principal component analysis plots of gene expression Chpt 6.3, p. 190 following MRT and SBBR irradiations of mouse brainstem.

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List of 3rd party copyright material

Citation Information for Copyright Material Location Permission of Item in Granted Thesis (Y/N) Figure 3.11 from Khan, MF & Gibbons, JP 2014, Khan’s Chpt 2.3, Yes the physics of radiation therapy, 5th edn, Lippincott p. 12 Williams & Wilkins, Philadelphia, PA.

Figure 4.9 from Khan, MF & Gibbons, JP 2014, Khan’s the Chpt 2.3, Yes physics of radiation therapy, 5th edn, Lippincott Williams & p. 14 Wilkins, Philadelphia, PA.

Figure 1.8 from Hall, EJ & Giaccia, AJ 2012, Radiobiology Chpt 2.4, Yes for the radiologist, 7th edn, Lippincott Williams & Wilkins, p. 17 Philadelphia, PA.

Figure 3.3 from Hall, EJ & Giaccia, AJ 2012, Radiobiology Chpt 2.4, Yes for the radiologist, 7th edn, Lippincott Williams & Wilkins, p. 20 Philadelphia, PA. Figure 1 from Zelefsky, MJ, Fuks, Z & Leibel, SA 2002. Chpt 2.5, Yes 'Intensity-modulated radiation therapy for prostate cancer', p. 24 Seminars in Radiation Oncology, vol. 12, no. 3, pp. 229- 237. Figure 6 from Siegbahn, EA, Bräuer-Krisch, E, Stepanek, J, Chpt 2.6, Yes Blattmann, H, Laissue, JA & Bravin, A 2005. 'Dosimetric p. 30 studies of microbeam radiation therapy (MRT) with monte carlo simulations', Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, vol. 548, no. 1–2, pp. 54-58. Figure 2 from Laissue, JA, Blattmann, H, Wagner, HP, Chpt 2.7, Yes Grotzer, MA & Slatkin, DN 2007. 'Prospects for microbeam p. 38 radiation therapy of brain tumours in children to reduce neurological sequelae', Development Medicine & Child Neurology, vol. 49, no. 8, pp. 577-581.

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Table 1 and 2 (adapted) from Smyth, LM, Senthi, S, Chpt 2.7, Yes Crosbie, JC & Rogers, PA 2016. 'The normal tissue effects p. 41-47, of microbeam radiotherapy: What do we know, and what do we need to know to plan a human clinical trial?', International Journal of Radiation Biology, vol. 92, no. 6, pp. 302-311. Figure 1 from Nairz, O, Winter, M, Heeg, P & Jakel, O Chpt 2.9, Yes 2013. 'Accuracy of robotic patient positioners used in ion p. 59 beam therapy', Radiation Oncology, vol. 8, pp. e124. Accepted Manuscript of an article published by the Chpt 3.2, Yes Radiation Research Society © in the Radiation Research p. 63-95 th journal on 30 November 2017, available online: Smyth, LM, Rogers, PaW, Crosbie, JC & Donoghue, JF 2018a.

'Characterization of diffuse intrinsic pontine glioma radiosensitivity using synchrotron microbeam radiotherapy and conventional radiation therapy in vitro', Radiation Research, vol. 189, no. 2, pp. 146-155, https://doi.org/10.1667/RR4633.1 Accepted Manuscript of an article published by Springer Chpt 4.2, Yes Nature Limited in the journal Scientific Reports on August p. 101-139 13, 2018. Available online: Smyth, LML, Donoghue, JF, Ventura, JA, Livingstone, J, Bailey, T, Day, LRJ, Crosbie, JC & Rogers, PaW 2018b. 'Comparative toxicity of synchrotron and conventional radiation therapy based on total and partial body irradiation in a murine model', Scientific Reports, vol. 8, no. 1, pp. e12044, https://doi.org/10.1038/s41598-018-30543-1 Accepted manuscript of an article published by Taylor and Appendix Yes Francis in the International Journal of Radiation Biology on A, p. 213- 16th March 2016, available online: Smyth, LM, Senthi, S, 240 Crosbie, JC & Rogers, PA 2016. The normal tissue effects of microbeam radiotherapy: What do we know, and what do we need to know to plan a human clinical trial?

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International Journal of Radiation Biology, vol. 92, no. 6, pp. 302-11, https://www.tandfonline.com/doi/abs/10.3109/09553002.20 16.1154217

Published version of manuscript, available online: Smyth, Appendix Yes (Open LML, Donoghue, JF, Ventura, JA, Livingstone, J, Bailey, T, B, p. 242- Access) Day, LRJ, Crosbie, JC & Rogers, PAW 2018. Comparative 252 toxicity of synchrotron and conventional radiation therapy based on total and partial body irradiation in a murine model. Scientific Reports, vol. 8, no. 1, pp. e12044, https://doi.org/10.1038/s41598-018-30543-1

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CHAPTER 1 General Introduction

1.1 Problem statement

Despite advances in modern healthcare, cancer remains a pervasive, insidious disease that is estimated to account for approximately 30% of all deaths registered in

Australia (Australian Institute of Health and Welfare 2017). It is estimated that one in every two Australians will be diagnosed with cancer before their 85th birthday

(Australian Institute of Health and Welfare 2017).

Radiation therapy is a vital tool in the oncological armamentarium, curing or palliating many forms of cancer either as a stand-alone treatment or as an adjuvant therapy alongside chemotherapy, surgery and more recently, immunotherapy.

Australian and European research estimates that approximately 50% of all cancer patients should receive radiation therapy as part of an optimal treatment course for their disease (Borras et al. 2015, Barton et al. 2014). However, despite the utility of radiation therapy, there are a number of clinical scenarios where conventional radiation therapy is contraindicated, ineffective in curing cancer, or unable to provide a significant improvement in survival or prognosis. Examples of these scenarios include advanced lung cancer, pancreatic cancer, and aggressive brain tumours such as diffuse intrinsic pontine glioma.

In light of these clinical needs, novel synchrotron-based radiation therapy modalities have been proposed as an alternative to conventional radiation therapy

(CRT). At the Australian Synchrotron (Clayton, Victoria), two radiation therapy

1 modalities are being developed for future clinical use; microbeam radiation therapy

(MRT) and synchrotron broad-beam radiation therapy (SBBR). The physical characteristics of the radiation utilised by these modalities – most notably, ultra-high dose-rates (SBBR and MRT) and highly non-uniform dose-distributions (MRT) – are a radical departure from the paradigm of CRT. Pre-clinical animal studies have demonstrated the therapeutic effect of both modalities and the potential to improve the balance between tumour control and normal tissue toxicity compared to CRT. The radiobiological mechanisms which underpin the therapeutic effect of SBBR and MRT remain to be conclusively determined.

Given that the principle of non-maleficence – ‘primum non nocere’ (first, do no harm) – is a cornerstone of medical ethics, questions relating to normal tissue toxicity remain at the core of the quest to translate synchrotron radiation therapy to clinical use.

Simply put, the safety and toxicity profile of synchrotron radiation therapy, as well as dose-equivalence with CRT, must be systematically characterised in order to progress towards a human clinical trial. However, there is a distinct absence of systematic normal tissue data in animal models to provide a basis for determining appropriate, and most importantly, safe dose regimens for clinical trials of synchrotron radiation therapy.

Furthermore, the optimal clinical scenarios where MRT might be deployed safely have yet to be determined. This thesis aims to address these unanswered questions.

1.2 Thesis objectives

The aim of this thesis is to address three fundamental issues in the progression of MRT and/or SBBR to a clinical trial; 1) the lack of systematic normal tissue toxicity data, 2) a poor understanding of dosimetric equivalence between MRT, SBBR and

2

CRT, and 3) the need to identify safe and optimal clinical scenarios for the future deployment of MRT. There are four specific aims of this thesis, with corresponding hypotheses:

• Aim 1: To determine equivalent doses of MRT, SBBR and CRT using in vitro

and in vivo methods.

• Aim 2: To systematically characterise the normal tissue toxicity related to MRT

and SBBR for tissues of the head, thorax and abdomen using systematic dose-

escalation studies.

• Aim 3: To determine the differential effects of MRT, SBBR and CRT on healthy

and tumour tissue at a cellular and molecular level.

• Aim 4: To identify optimal treatment sites for MRT using a treatment planning

system.

• Hypothesis 1: For MRT, the dose delivered to the valley region is the most

important determinant of normal tissue toxicity; valley doses for MRT will most

closely reflect CRT tolerance doses.

• Hypothesis 2: A tissue-sparing effect related to the use of an ultra-high dose-rate

exists for SBBR; for the same dose of radiation, normal tissue will be more

tolerant to SBBR compared to CRT.

• Hypothesis 3: There will be differences, at a cellular and molecular level, in the

response of normal tissue and tumours to MRT, SBBR and CRT.

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• Hypothesis 4: Optimal treatment sites for MRT will be determined by

characteristics intrinsic to the tumour target volume; small and relatively

superficial target volumes will yield the most favourable dosimetry.

1.3 Overview of thesis chapters

Chapter 1 is a general introduction, containing a problem statement that underpins the objectives of the thesis.

Chapter 2 provides a scientific introduction and literature review of concepts relating to synchrotron radiation therapy, including relevant radiobiological and medical physics principles. This chapter includes a review of the available literature reporting on dose-equivalence and normal tissue toxicity in pre-clinical synchrotron radiation therapy experiments, as well as developments in treatment and planning technologies.

Chapter 3 compares the radio-sensitivity of two glioma cell lines to MRT and

CRT. This chapter addresses Aim 1 and Aim 3 of this thesis, discussing dose- equivalence and the differential biological effects of MRT and CRT. This chapter is written in the style of a scientific paper and takes the form of a paper published in the journal Radiation Research.

Chapter 4 draws together Aims 1 and 2, discussing dose-equivalence between synchrotron radiation therapy modalities and CRT using an in vivo model, as well as systematically characterising organ specific normal tissue toxicities acutely following irradiation. Chapter 4 is written in the style of a scientific paper and takes the form of a paper published in the journal Scientific Reports.

Chapter 5 relates specifically to Aim 4 and is a treatment planning study for

MRT using a commercially available treatment planning system that is used in clinical

4 radiation oncology. This chapter shows the feasibility of MRT for different clinical scenarios and identifies optimal treatment sites for future clinical use. Chapter 5 is written in the style of a scientific paper and takes the form of a manuscript submitted to the journal Radiotherapy and Oncology for publication

Chapter 6 contains a discussion and presents overall conclusions drawn from the findings of the individual chapters of the thesis. This chapter also discusses work in progress and plans for future experiments which will build on the findings of this thesis.

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CHAPTER 2 Literature review

2.1 Preface

This chapter introduces the scientific concepts underpinning this thesis. It also provides a review of pre-clinical radiobiological findings and medical physics developments pertinent to MRT and SBBR. Section 2.7 of this chapter, which reviews normal tissue toxicity data in MRT, includes tables adapted from a review article published in the International Journal of Radiation Biology, of which I am the primary author. These sections have been cited appropriately and the peer-reviewed manuscript, accepted for publication, has been included in Appendix A.

2.2 Introduction to clinical radiation therapy

Cancer epidemiology and radiation therapy utilisation

In 2014, cancer accounted for 29% of all deaths in Australia (Australian Institute of Health and Welfare 2017); the second most common cause of death after heart disease. One in every three males and one in every four females are estimated to be diagnosed with cancer by the age of 75 (Australian Institute of Health and Welfare

2017). Prostate cancer and breast cancer are the most common diagnoses for men and women respectively, with colorectal cancer, lung cancer and melanoma the next most common types of cancer for both men and women (Australian Institute of Health and

Welfare 2017).

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A range of tumour and patient related factors determine the optimal treatment course for individual cancer cases. Tumour factors include the organ of origin, tumour stage and size, histological grade, the presence of absence of metastatic (distant) cancer spread away from the original tumour site and the operability of the tumour (Symonds et al. 2012). Patient factors such as age, performance status and personal preference also play a key role in determining whether a radical (curative) or palliative treatment strategy is adopted (Symonds et al. 2012).

Radiation therapy is used as a stand-alone treatment or as part of a multi-modal approach alongside surgery and/or chemotherapy to cure or palliate many forms of cancer. A significant proportion of all patients diagnosed with cancer in Australia

(48.3%) will require radiation therapy at least once during the course of their disease

(Barton et al. 2014). This estimate corresponds closely with the optimal utilisation proportion of radiation therapy for cancer patients across Europe (Borras et al. 2015).

Projected increases in population growth and aging will significantly increase the need for radiation therapy in absolute terms over the next decade (Borras et al. 2016).

A short history of radiation therapy

Radiation has been used in medicine ever since the serendipitous discovery of x- rays by the German-Dutch physicist Wilhelm Roentgen in 1895 (Reed 2011). In 1896,

Emile Grubbe (United States of America), Leopold Freund (Austria) and Victor

Despeignes (France) were amongst the first to use radiation to treat cancerous lesions.

Radiation was shown to slow tumour growth, reduce pain and in some cases, completely cure superficial skin lesions in the decades that followed (Holsti 1995).

Research and treatment in the field of radiation therapy was propelled into the twentieth century by the discovery of naturally occurring radioactive elements by Henri Becquerel

7 and Marie and Pierre Curie, for which a Nobel Prize was awarded in 1903 (Einhorn

1995).

The first radiation therapy treatments were delivered either using early versions of the x-ray tube or radium in various forms. Large, single doses of radiation were typically used until the early 1920s when Claude Regaud (France) demonstrated that delivering multiple, smaller doses over consecutive days was less toxic (Holsti 1995).

Regaud and his compatriot Henri Coutard were the first use this technique, called

‘simple fractionation’, to treat head and neck cancers, which was accepted and commonly practiced by the wider radiation therapy community by the 1930s. The concept of simple, or temporal, fractionation, as demonstrated by Regaud and Coutard, remains a cornerstone of modern radiation oncology.

Early versions of the x-ray tube produced radiation with a relatively low energy, typically in the kilovoltage (50 to 150 kilo-electron-volts (keV)) and orthovoltage (150 to 500 keV) range. Given the limited ability of low energy x-rays to penetrate tissue, early radiation therapy was limited to superficial tumours and skin lesions and was largely used in a palliative rather than curative setting. In 1937, St. Bartholomew’s

Hospital in London installed the first x-ray tube capable of delivering megavoltage radiation therapy treatments (Thwaites & Tuohy 2006), which paved the way for the development of the linear accelerator and the capacity to treat deep-seated tumours.

The first dedicated medical linear accelerator was commissioned and used to treat cancer patients in 1953 at Hammersmith Hospital in London and was capable of producing x-rays with an energy of 8 mega-electron-volts (MeV). By 1962, there were

15 clinical linear accelerators in operation globally, including the United States of

America, Australia, Russia and Japan (Thwaites & Tuohy 2006). The linear accelerator remains the workhorse of modern radiation therapy clinics around the world today.

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Modern radiation therapy modalities

Modern radiation therapy is predominantly delivered using three different modalities: 1) external beam radiation therapy, 2) brachytherapy and 3) radionuclide therapy.

External beam radiation therapy (EBRT), also known as teletherapy, is the most widely used form of radiation therapy. In this mode, radiation is delivered using an external radiation source, such as a linear accelerator, from outside the patient’s body.

Modern linear accelerators have a gantry that can rotate 360 degrees around the patient, allowing for radiation to be delivered to the target from multiple angles. This creates a highly conformal dose distribution around the tumour target region whilst minimising the radiation dose delivered to healthy tissue.

In contrast to EBRT, brachytherapy employs sealed radioactive sources to deliver radiation over very short distances. These sources may be placed inside a target organ like the uterus (intra-cavitary), implanted into the target tissue such as the breast or prostate (interstitial), or placed in direct contact with the body surface. Historically,

Radium-226 was the most commonly used source for brachytherapy, however, artificial radionuclides such as Cesium-137, Iridium-192, Palladium-103 and Iodine-125 have gained popularity due to their more favourable physical properties such as their radiation energy, half-life and size (Khan & Gibbons 2014). The key advantage of brachytherapy compared to EBRT in certain clinical situations is the ability to place a source in very close proximity to, or even within, the target. This allows for very rapid dose fall-off beyond the target region and therefore provides excellent normal tissue sparing.

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While EBRT and brachytherapy deliver radiation in a focal manner, radionuclide therapy utilises unsealed radiation sources that are administered systemically, usually orally or via injection. These sources are subsequently directed towards the cancerous or anaplastic tissues at a cellular level using anti-bodies or tissue- specific metabolites which are chemically attached to the radionuclide (Wessels &

Meares 2000, McDougall 2000). Thyroid cancer, bone metastases, neuroendocrine tumours and lymphoma are commonly treated using radionuclide therapy, which can reduce the radiation dose delivered to healthy tissue through the close-range emission of radiation at the target tissue.

2.3 External beam radiation therapy: physics principles

X-ray production

X-rays are a form of high-energy electromagnetic radiation with a frequency greater than approximately 3 x 1016 hertz (Hz). Given the high frequency of x-rays, their behaviour and interaction with matter is best explained using their particle nature (Khan

& Gibbons 2014). There are two main mechanisms of x-ray generation in EBRT which relate to two different modes of interaction between high-speed electrons and an atom.

Bremsstrahlung x-rays are produced when an electron propagating through space is deflected by the attractive action of Coulomb forces as it passes closely to the positively charged nucleus of an atom (Khan & Gibbons 2014). During this deflection and change in velocity, the electron can lose some or all of its energy, which is converted to bremsstrahlung radiation. This gives rise to a spectrum of x-rays with a maximum energy equal to the initial kinetic energy of the electron. As the initial energy

10 of the electrons increases, the direction of x-ray emission approaches the same direction as the incident electron beam (Khan & Gibbons 2014).

The second type of x-ray production occurs when a high speed electron ionises an atom by ejecting one of its orbital electrons. When this occurs, an electron from a higher energy, outer orbit falls down to fill this vacancy. This orbital transition results in the release of electromagnetic radiation with an energy that is discrete and characteristic of the atomic number and difference in energy between the two electron orbits (Khan &

Gibbons 2014).

The overall radiation energy spectrum produced by the atomic interactions of high-speed electrons includes both bremsstrahlung x-rays and characteristic x-rays

(Figure 2.1). This spectrum, and the efficiency of energy production, can be manipulated by changing the atomic number of the target and the incident energy of the electrons.

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Figure 2.1. X-ray energy spectra following the interaction of electrons with a tungsten target at various excitation/acceleration potentials. Figure adapted from Khan, MF &

Gibbons, JP 2014, Khan’s the physics of radiation therapy, 5th edn, Lippincott Williams

& Wilkins, Philadelphia, PA, with permission.

Linear accelerators collide electrons with a metallic target, typically composed of tungsten, to produce therapeutic radiation (Figure 2.2A). Electrons are accelerated from a low energy of approximately 50 keV to energies up to 20 to 25 MeV by high- frequency electromagnetic waves (Khan & Gibbons 2014, Thwaites & Tuohy 2006). As electrons bombard the target, a divergent x-ray beam is produced predominantly through bremsstrahlung interactions. This x-ray beam consists of a spectrum of energies, with a maximum beam energy equal to the incident energy of the electrons and mean energy that is approximately one third the maximum (Khan & Gibbons 2014).

As shown in Figure 2.2A, the diverging x-ray beam then passes through a primary beam-shaping collimator and a flattening filter, which ensures a uniform intensity

12 across the entire field (Symonds et al. 2012). This beam can then be shaped further using a secondary collimator and a multi-leaf collimator in order to produce field shapes specific to the patient’s target volume. In addition to producing x-rays, high-energy linear accelerators can operate in electron beam mode (Figure 2.2B) in order to treat superficial tumours. In this mode, the metallic target is moved out of the electron pencil-beam path and replaced by a thin metallic scattering foil which spreads the pencil beam out into a broader treatment field (Khan & Gibbons 2014).

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Figure 2.2. Schematic diagram of a linear accelerator in x-ray therapy mode (A) and electron therapy mode (B). From Khan, MF & Gibbons, JP 2014, Khan’s the physics of radiation therapy, 5th edn, Lippincott Williams & Wilkins, Philadelphia, PA, with permission.

2.4 Principles of radiobiology

Interactions of x-rays with matter

X-rays, electrons and other charged particles utilised in medicine are classified as ionising radiation and cause biological damage by breaking chemical bonds in biomolecules. While larger charged particles cause damage through direct ionisation, x- rays are more likely to be indirectly ionising, exerting a biological effect via the production of fast-moving electrons and ionised molecular species as the x-rays are absorbed in the body (Hall & Giaccia 2012, Baskar et al. 2014).

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X-rays produce fast-moving electrons predominantly through three different mechanisms, with the dominant mechanism largely dependent on the incident beam energy (Khan & Gibbons 2014). In the body, the photoelectric effect is dominant when incident x-rays have an energy less than 30 keV (Symonds et al. 2012). During this process, photons transfer all of their energy to an inner orbital electron. Part of this energy is used to eject the electron from its orbit and the remainder is transferred to the ejected electron as kinetic energy (Hall & Giaccia 2012). At energies above 100 keV and up to 20 MeV, typical of conventional radiation therapy, the Compton Effect is dominant. In contrast to the photoelectric effect, Compton interactions take place with outer shell electrons and the incident photon only loses some of its energy, which is transferred to the ejected electron as kinetic energy. The photon is deflected and then continues with a lower energy, interacting with further atoms and producing more fast- moving electrons (Hall & Giaccia 2012). Finally, x-rays may interact with the nuclear

Coulomb field, with all energy from a photon being converted to the rest mass and kinetic energy of an electron-positron pair (Symonds et al. 2012). This process, also known as pair-production, becomes possible at x-ray energies higher than 1.02 MeV and significant at energies greater than 10 MeV.

X-rays and DNA damage

In biological systems, DNA is considered to be the key target for radiation- induced damage, with biological consequences ranging from cell death within days of irradiation, to genetic mutations that may be expressed after a number of years (Maier et al. 2016, Hall & Giaccia 2012). The fast-moving electrons generated as x-rays are absorbed in tissue and cause DNA damage either directly, or indirectly via a cascade of ionisation events that culminate in the production of free radical species (Figure 2.3).

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Although free radicals have a biological lifetime in the range of a nanosecond, this is sufficient time to diffuse towards DNA and break chemical bonds within the molecule.

Given that the body is largely composed of water, the hydroxyl free radical is estimated to account for the majority of DNA damage caused in mammalian cells due to radiation

(Hall & Giaccia 2012). Inducing the death of tumour cells is the primary aim of radiation therapy, however, this cannot be separated from the risk of inducing damage or mutations in the DNA of normal tissue; this can lead to tissue damage or in the long- term, secondary cancer.

Absorbed radiation dose is quantified as the energy imparted by the ionising radiation per unit mass and is given the SI unit gray (Gy). This unit is defined as 1 Gy =

1 Joule/kilogram (Khan & Gibbons 2014).

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Figure 2.3. The direct and indirect actions of radiation on DNA. The indirect action of radiation to cause DNA damage is predominantly mediated by the hydroxyl free radical, which is formed via hydrolysis. From Hall, EJ & Giaccia, AJ 2012, Radiobiology for the radiologist, 7th edn, Lippincott Williams & Wilkins, Philadelphia, PA, with permission.

Mechanisms of radiation induced cell death

DNA double strand breaks (DSBs) are the most important form of biological damage caused by ionising radiation in terms of cell death (Hall & Giaccia 2012, Maier et al. 2016). DSBs can be defined as breaks in both strands of DNA that are less than a few base pairs apart. A small proportion (approximately less than 5%) of all DSBs caused by x-rays cannot be repaired during the cell-cycle, either during G1 phase via non-homologous end-joining or at S/G2 phase by homologous recombination (Maier et al. 2016). Unrepaired DSBs may give rise to chromosomal abnormalities such as

17 dicentric or ring aberrations that can cause cell death, senescence, mutations or genomic instability (Hall & Giaccia 2012). Mechanisms of cell death include apoptosis, necroptosis and mitotic catastrophe.

Mitotic catastrophe is the predominant form of cell death following ionising radiation and is characterised by premature, uncontrolled mitosis and the over- duplication of chromosomes (Maier et al. 2016). As a result of the aberrant duplication and segregation of chromosomes, cells with multiple nuclei, micronuclei, or abnormally high DNA content may form, leading to death during mitosis or interphase of the first or second cell-cycle after irradiation (Maier et al. 2016).

Apoptosis and necroptosis are both forms of programmed cell death. Apoptosis is regulated by tumour-suppressor genes such as p53 and involves the activation of the caspase family of proteins (Matt & Hofmann 2016), which cleave intra-cellular contents for subsequent packaging and controlled release from the dying cell. In contrast to this, necroptosis operates independently of the caspase family. Necroptosis is a regulated form of necrosis, characterised by cellular swelling, controlled rupturing of the plasma membrane and the expulsion of cellular contents directly into the extracellular matrix

(Matt & Hofmann 2016).

An alternative p53 mediated signalling pathway involving the activation of p21 may lead irradiated cells to become senescent rather than undergoing apoptosis

(Roninson 2003, Roninson, Broude & Chang 2001). Senescent cells are generally arrested in the G1 phase however they remain viable and active, able to influence the tumour microenvironment through the secretion of signalling factors. For normal tissue, cellular senescence can be used as a protective mechanism against the deleterious effects of ionising radiation, however for tumour cells, this mechanism can be adopted as a means of radio-resistance (Maier et al. 2016).

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Radio-sensitivity and clonogenic cell survival

The sensitivity of different tissue and tumour types to radiation vary significantly and this is largely dependent on the level of proliferative activity within that tissue. A meaningful endpoint of cell survival or death is clonogenic potential; the ability of a given cell to continue to divide and reproduce itself indefinitely (Hall &

Giaccia 2012). The clonogenic assay was first pioneered by Theodore Puck and Philip

Marcus in 1956 (Puck & Marcus 1956) and is the gold standard for measuring radio- sensitivity in vitro. This kind of assay measures the ability of a single cells to reproduce indefinitely, or to grow into a colony, following some form of cytotoxic insult such as irradiation (Franken et al. 2006). Survival curves generated from clonogenic assays plot radiation dose on the horizontal axis with clonogenic survival on the vertical axis

(Figure 2.4). Survival is defined as the number of colonies generated relative to the number of cells initially seeded, with the survival of treated cells then normalised against the ability to non-treated cells to form colonies (Franken et al. 2006).

The linear quadratic model describes the mathematical relationship between dose and clonogenic survival and provides a quantitative method of comparing the shape of cell survival curves for different tissue or tumour types. According to this model, the fraction of cells surviving after irradiation is expressed as: S = e-αD-βD^2, where D is dose (Gy) and α and β are constants that represent the linear and quadratic components of cell killing, respectively (Hall & Giaccia 2012).

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Figure 2.4. The linear-quadratic model for cell survival following irradiation. Linear and quadratic components of cell killing are shown. Adapted from Hall, EJ & Giaccia,

AJ 2012, Radiobiology for the radiologist, 7th edn, Lippincott Williams & Wilkins,

Philadelphia, PA, with permission.

The α and β coefficients describe the initial and subsequent slopes of the dose- response survival curves, which relate to the linear and quadratic portions of cell killing, respectively (Hall & Giaccia 2012). The α/β ratio equals the dose at which linear and quadratic components of cell killing contribute equally and describes the curvature of the cell survival curve (Figure 2.4). Tissues with a low α/β ratio (less than approximately four) such as the bladder, rectum, kidney, brain and lung are typically classed as late responding tissues that are more resistant to radiation, having a slower rate of proliferation and lower levels of radiation-induced apoptosis (Hall & Giaccia

2012, Fowler 2005). The converse is true for tissues with a high α/β ratio of ten or greater; these tissues such as the skin, mucosa, testis and gut are early-responding, relatively radio-sensitive and highly proliferative (Fowler 2005, Hall & Giaccia 2012).

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Most tumours have a high α/β ratio, with the most notable exception being prostate cancer, which has a α/β ratio of approximately one to two (Fowler 2005).

Fractionation and the ‘Five Rs of Radiobiology’

The primary aim of radiation therapy is to maximise damage to the tumour whilst maximally sparing surrounding normal tissue. Fractionating a single large radiation dose into several smaller sequential doses of radiation, a technique first adopted by Regaud and Coutard in the 1920s (Holsti 1995), is fundamental to exploiting the radiobiological differences between tumours and normal tissue and optimising the therapeutic ratio of conventional radiation therapy (CRT). Typically, curative treatment regimens for common diseases such a breast and prostate cancer consist of daily 2 Gy treatments for five to seven weeks, for a total dose of 50 to 80 Gy. Temporal fractionation draws on five key radiobiological principles; repair, repopulation, redistribution, re-oxygenation and radio-sensitivity (Hall & Giaccia 2012, Rodney

Withers 1992, Steel, McMillan & Peacock 1989).

Normal tissue with a low α/β ratio, which typically proliferates slowly, has a greater ability to repair sub-lethal DNA damage compared to most tumour types.

Therefore, temporal fractionation allows greater recovery in normal tissue compared to malignant tissue when treatments are separated by at least six hours (Rodney Withers

1992). Similarly, the time between treatments allows proliferative normal tissue, such as mucosa and skin, which have a high α/β ratio, to repopulate the damaged tissue with functional cells (Hall & Giaccia 2012).

Temporal fractionation also has implications for proliferative malignant tissue, with the synchronisation and redistribution of surviving radio-resistant cells into the

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G2/M phase, which is most radio-sensitive phase of the cell cycle, prior to the next treatment (Rodney Withers 1992). Furthermore, tumour tissue is re-oxygenated between treatments, increasing the radio-sensitivity of cells that may have previously been deprived of oxygen due to the size of the tumour (Rodney Withers 1992).

Given these radiobiological principles, the efficacy of radiation therapy for specific cancers can be improved by selecting an appropriate dose-fractionation scheme.

Conventionally fractionated treatment typically consists of daily treatments of 1.8 to 2

Gy over several weeks and is appropriate for many cancer types, including breast, prostate, lung and bowel cancer (Symonds et al. 2012). Hyper-fractionation involves giving multiple smaller fractions per day, approximately 1 Gy each, to deliver at least the same total dose over the same length of time as conventional treatment. This has been shown to be more effective for some highly proliferative tumours, such as head and neck squamous cell carcinomas, leading to better survival and loco-regional disease control (Bourhis et al. 2006).

Conversely, hypo-fractionated treatment regimens use relatively large dose per fractions (greater than 2 Gy), delivering a higher biologically equivalent dose over a shorter period of time than conventionally fractionated treatment. Theoretically, this may provide an advantage for radio-resistant cancers (Benjamin, Tree & Dearnaley

2017). However for prostate cancer, which has a low α/β ratio, clinical trials demonstrate only mild improvements in survival and disease control (Cao et al. 2017) with no significant evidence that hypo-fractionation is superior (Incrocci et al. 2016).

Nevertheless, hypo-fractionation can provide the benefit of a shorter overall treatment time and reduced costs to the patient. For example, hypo-fractionated radiation therapy for early stage breast cancer can be delivered in three weeks and provides non-inferior

22 cosmetic outcomes, disease control, and overall survival compared to conventionally fractionated treatment which is delivered over five weeks (Whelan et al. 2010).

2.5 Technical developments in modern external beam radiation therapy

Optimising geometric dose conformity

Improvements in radiation therapy can be broadly characterised as either, 1) fundamental changes to the dose-fractionation regimens used for specific cancers, or, 2) technological advancements which improve our ability to control the geometry of physical dose-distributions. These technical advancements have to a large extent made it possible to deliver either larger total doses (dose-escalation) or larger doses per fraction

(hypo-fractionation) by improving the geometric conformity of dose to the tumour, thereby reducing the volume of normal tissue collaterally irradiated during treatment.

While the idea of conforming a dose of radiation to a limited volume had been articulated and applied using simple techniques since the 1930s, the era of modern three-dimensional conformal radiation therapy (3DCRT) began early in the 1970s when the Computed Tomography (CT) scanner was first introduced (Fraass 1995). The concurrent development of computer controlled dynamic multi-leaf collimators (MLCs) during this time period further improved the capacity to deliver highly conformal radiation therapy treatments and paved the way for the next significant development in radiation therapy; intensity modulated radiation therapy (IMRT) (Fraass 1995, Boyer &

Yu 1999). IMRT, using dynamic MLCs, allows for more complex field profiles to be produced, even allowing for high levels of dose conformity to irregularly shaped target volumes. A comparison of IMRT versus 3DCRT plans for prostate cancer radiation therapy clearly illustrates this point. IMRT is capable of creating a dose-distribution that

23 can be sculpted to the concave prostate target volume, while the 3DCRT leads to increased irradiation of the rectum, which is the dose-limiting organ at risk (Figure 2.5).

Figure 2.5. Conventional 3DCRT (left) versus IMRT (right) for prostate cancer treatment. For IMRT, the highest dose is sculpted to the concave prostate volume

(yellow contour) while sparing the rectum (red contour). Figure from Zelefsky, Fuks and Leibel (2002), with permission.

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Normal tissue sparing can be further achieved by reducing positional uncertainties in the target volume. Image guided radiation therapy (IGRT) techniques provide a suite of corrective options that can be applied to account for random and systematic errors in target positioning during and between treatments (van Herk 2007).

IGRT involves taking images of patient positioning, typically using x-rays, before, during or after treatment in order to understand the deviations in anatomical positioning between the planned and delivered treatment. Additionally, organ motion during treatment can be managed via real-time tumour tracking using electromagnetic beacons

(Mantz 2014, Lin et al. 2013) or implanted fiducial markers (Nguyen et al. 2017), and respiratory control using various breath-hold techniques and biofeedback (Zagar et al.

2017, Boda-Heggemann et al. 2016, Pollock et al. 2015).

Anatomical and physiological changes in a patient, such as tumour regression, morphological changes in healthy organs, weight loss and variable organ distension, can significantly impact the treatment of cancers of the head and neck (Hamming-Vrieze et al. 2012), lung (Booth et al. 2016), liver (Leinders et al. 2013), bladder (Foroudi et al.

2014), prostate (Keall et al. 2017) and cervix (Seppenwoolde et al. 2016). Adaptive radiation therapy (ART) strategies are designed to evaluate anatomical and physiological changes during a treatment course and to modify the original treatment plan accordingly (Yan 2010). The feedback loop provided by ART ensures that the actual dose delivered to the patient does not deviate significantly from the planned dosimetry, preventing under-dosing of the target volume and excessive exposure of healthy tissue to radiation. The integration of imaging modalities such as Magnetic

Resonance Imaging (MRI) with radiation therapy treatment systems (Kupelian & Sonke

2014) and developments in deformable image registration and dose-accumulation algorithms (Jaffray et al. 2010, Al-Mayah et al. 2015, Cazoulat et al. 2016) continue to

25 improve the efficacy and scope of ART and will allow for an increasingly personalised approach to radiation therapy.

Charged particle therapy

There has been significant interest and investment over the past two decades in utilising charged particles such as protons and carbon ions, rather than photons, to improve the therapeutic effect of EBRT. Charged particles offer improved dose conformality to the target by depositing maximum dose, known as a ‘Bragg Peak’, at a well-defined depth with a steep reduction in dose beyond this region (Schulz-Ertner,

Jäkel & Schlegel 2006, Khan & Gibbons 2014). These characteristics can be exploited to reduce the total dose delivered to the patient, thereby reducing the risk of radiation- induced secondary malignancies, and also to better protect organs that lie in close proximity to the tumour target region. While the dosimetric advantages of proton therapy for a number of cancer types are well documented (Harrabi et al. 2016, Ding et al. 2014, Holliday et al. 2016, Stokkevåg et al. 2014), robust evidence of this translating into meaningful, long-term clinical improvements compared to photon-based therapy is still required (Romesser et al. 2016, Remick et al. 2017, Henderson et al. 2017, Yock et al. 2016, Leroy et al. 2016).

Combined radio-immunotherapy

The developments in EBRT and clinical radiation oncology discussed above ultimately focus on improving the conformity of the radiation dose to the target volume, facilitating improved normal tissue sparing and dose-escalation to the tumour. However, there is evidence that radiation therapy can be harnessed to modulate and enhance

26 immunological activity across a wide range of doses, both locally at the tumour site and also systemically (Demaria & Formenti 2012, Kang, Demaria & Formenti 2016). These observations have propelled research into the combination of radiation therapy with immunotherapy in order to exploit their potential synergy. Radiation therapy can increase anti-tumour immune effects by increasing the expression of surface receptors on immune cells and tumour antigen presentation (Reits et al. 2006, Garnett et al. 2004) and by reducing the expression of receptors on tumours that would otherwise down- regulate immune cell activation (Demaria et al. 2005) or programmed cell death (Hamid et al. 2013). Combined radio-immunotherapy is under investigation in a growing number of clinical trials for a broad range of cancer types (Kang, Demaria & Formenti

2016, Weichselbaum et al. 2017), with significant interest in the response of patients with metastatic disease and advanced disease. The results of these trials will shed light on the optimal way to combine radiation therapy and immunotherapy in terms of sequence, timing, radiation therapy dose regimens and types of immunomodulatory targets (Demaria & Formenti 2012, Demaria et al. 2014).

Limitations of modern radiation oncology

Despite the technical advances of modern radiation therapy and the increased use of novel dose-fractionation schemes, there are many types of cancer that have not seen significant improvements in prognosis.

For diffuse intrinsic pontine glioma, a devastating paediatric brain tumour, survival has not improved significantly over the past decade; more than 90% of children die within two years of diagnosis (Hargrave, Bartels & Bouffet 2006). Locally advanced pancreatic cancer has a five year overall survival rate of approximately 7% (Siegel,

Miller & Jemal 2015); radiation therapy plays a central role in this scenario, especially

27 when the disease is inoperable (Myrehaug et al. 2016). In these scenarios, the intrinsic characteristics of the disease and the exquisite sensitivity of surrounding organs to radiation, hinders any opportunity for durable disease control.

Given the apparent limitations of current radiation therapy strategies, a radical departure from conventional medical physics and radiobiological principles may be required to improve the prognosis, and potentially provide the possibility of cure, for types of cancer such as these.

2.6 Introduction to synchrotron radiation therapy

Synchrotron physics

Synchrotron radiation is emitted by a charged particle as it approaches the speed of light while moving in an arc (Hofmann 2004). At synchrotron light sources such as the Australian Synchrotron, electrons are accelerated to relativistic speeds and stored in a ring for the emission of synchrotron radiation, which can then be utilised for research, medical, or industrial purposes. At the Australian Synchrotron, electrons are produced by thermionic emission from an electron gun, accelerated to an initial energy of approximately 100 MeV by a linear accelerator and subsequently to an energy of 3 giga- electron-volts (GeV) by a series of steering and focussing electromagnets before being transitioned to a storage ring (Australian Synchrotron 2008). The Imaging and Medical

Beamline (IMBL) at the Australian Synchrotron, which opened in 2007, captures synchrotron radiation emitted from electrons circulating the storage ring for applications such as phase-contrast imaging, tomography and more recently, experimental radiation therapy (Stevenson et al. 2017).

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The three physical characteristics of synchrotron-generated radiation that are distinct from conventional therapeutic radiation from a linear accelerator are the extremely high dose-rate, minimal beam divergence and keV energy spectrum. These unique features facilitate two novel forms of radiation therapy; microbeam radiation therapy (MRT) and high dose rate synchrotron broad-beam radiation therapy (SBBR).

Microbeam radiation therapy

The minimal divergence and the kilovoltage energy of radiation generated by a synchrotron allows for the collimation of a broad radiation field into virtually parallel micro-planar beams. In MRT, radiation fields typically consist of 25 to 100 µm wide quasi-parallel micro-planar beams that have a centre-to-centre spacing of 100 to 400 µm

(Brauer-Krisch et al. 2010). As seen in Figure 2.6, this kind of array creates a periodically alternating dose profile of ‘peaks’ and ‘valleys’, where the in-beam doses

(peaks) can potentially be up to 100 times higher than the dose deposited between the beams (valleys) due to scatter (Blattmann et al. 2005). A keV beam energy is essential to maintaining this peak-valley geometry by reducing the range of secondary electrons scattered into the valley region (Slatkin et al. 1992).

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Figure 2.6. A lateral dose profile through a field of microbeams with 50 µm width and

400 µm centre-to-centre spacing. A comparison of a Monte Carlo (MC) simulated dose distribution to measured dose distribution using radiochromic film is shown. Figure from Siegbahn et al. (2005), with permission.

Peak doses used in pre-clinical MRT experiments typically lie in the range of

100 to 1000 Gy (Ibahim et al. 2014, Laissue et al. 2013, Schultke et al. 2008, Serduc et al. 2008). These extremely high peak doses, which are at least an order of magnitude greater than typical doses delivered using conventional radiation therapy (CRT), can be delivered within a clinically feasible time-frame given the extremely high dose-rate of synchrotron radiation. At the IMBL, an in-beam dose-rate of 300 to 400 Gy/s can be achieved (Livingstone et al. 2017). At the European Synchrotron Research Facility in

Grenoble, France, in-beam dose-rates in the order of several thousand Gy/s are achievable (Renier et al. 2008). In comparison, typical CRT treatments are delivered at a dose-rate in the order of 0.05 Gy/s.

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The tolerance of normal tissue to radiation increases as the irradiated volume of that specific tissue is reduced (Emami et al. 1991, Marks et al. 2010, Hopewell, Morris

& Dixon-Brown 1987). This dose-volume premise underpins therapies such as MRT, which is intrinsically focussed on spatial dose-fractionation. A German radiologist named Albert Köhler first conceived the idea of spatial fractionation in 1909. Köhler showed that skin toxicity could be reduced using ‘grid therapy’, where a 3mm-square grid of woven iron wire was pressed closely to the skin of patients during kilovoltage irradiation (Laissue, Blattmann & Slatkin 2012). Today, grid therapy using megavoltage x-rays from a linear accelerator can be used to de-bulk large and advanced tumours prior to CRT (Zhang et al. 2008) and has therapeutic potential for skin cancers such as melanoma (Zhang et al. 2014).

Synchrotron based MRT was first explored by Daniel Slatkin and colleagues at the Brookhaven National Laboratory in New York in the early 1990s (Slatkin et al.

1992, Slatkin et al. 1995) and has been in pre-clinical development at a small number of synchrotrons across the world ever since, including the European Synchrotron Research

Facility (ESRF) (Grenoble, France), SPring-8 (Hyogo Prefecture, Japan) and the

Australian Synchrotron (Melbourne, Australia). In addition to the remarkable tolerance of normal tissue to peak MRT doses (Laissue et al. 2013, Laissue et al. 2007, Serduc et al. 2008, van der Sanden et al. 2010), pre-clinical studies show that MRT can slow tumour growth and even facilitate tumour control despite not irradiating the entire tumour with a uniform field (Miura et al. 2006, Laissue et al. 1998, Bouchet et al.

2016).

There are a number of mechanisms that have been proposed for the therapeutic effect of MRT. Firstly, normal tissue retains its cellular architecture and the ability to launch a coordinated repair response following MRT while tumour tissue demonstrates

31 marked cellular migration and reduced proliferative capacity (Crosbie et al. 2010). The intermixing of maximally irradiated tumour cells in the peak region with minimally irradiated cells in the valley region could enhance the propagation of cytotoxic bystander effects such as the induction of DNA double strand breaks, which has been demonstrated in glioma cell lines following MRT (Kashino et al. 2009).

Secondly, MRT exerts differential transcriptomic effects on tumour and normal tissue, with differences in key pathways relating to immunity and inflammation

(Bouchet et al. 2015, Bouchet et al. 2013b). The regulation of inflammation and immune response is also different when comparing tissue irradiated using MRT versus

CRT (Ibahim et al. 2016, Yang et al. 2014, Sprung et al. 2012, Bouchet et al. 2016), which further highlights the potential importance of these pathways to the therapeutic effect of MRT. Exploiting the immunomodulatory properties of radiation is an exciting frontier in modern radiation oncology (Kang, Demaria & Formenti 2016,

Weichselbaum et al. 2017). MRT could be utilised to further explore the synergy between radiation therapy and immunotherapy, however, the topic of immunomodulation is beyond the scope of this thesis.

Lastly, tumour micro-vasculature has a greater radio-sensitivity to MRT compared to normal brain micro-vasculature (Bouchet et al. 2013a, Bouchet et al. 2010) which has implications for vascular permeability and the delivery of micro-nutrients and cellular mediators of damage repair. As further evidence of this, MRT has been shown to selectively reduce blood flow and cause an acute inflammatory response in immature microvasculature of zebra-fish (Brönnimann et al. 2016), which could be used as a model for the pro-angiogenic environment characteristic of many tumours. In contrast, mature microvasculature, typical of healthy tissue, remains unaffected by MRT

(Brönnimann et al. 2016). The dependence of MRT-induced tissue damage on the stage

32 of microvasculature maturation has also been shown in a chick chorioallantoic membrane model (Sabatasso et al. 2011). An impaired blood supply deprives tissue of nutrients and other molecules necessary for repair, and in this way, MRT might preferentially damage tumour tissue. Importantly, the differential effect of MRT on immature versus mature microvasculature is lost when the beam width is increased to greater than 400 µm (Brönnimann et al. 2016). Broad-beam irradiation also has the capacity to increase vascular permeability in tumours. However, the increase in permeability induced by MRT occurs earlier, is greater in magnitude and is longer in duration compared to broad-beam irradiation (Bouchet et al. 2017).

Mini-beam radiation therapy

A variant of MRT is mini-beam radiation therapy (MBRT), which borrows the same concept of a periodically alternating, heterogeneous dose-distribution but uses arrays of relatively thick microbeams as wide as 700 µm and spaced by at least 1 mm

(Dilmanian et al. 2006, Prezado et al. 2015). The mechanical demands of collimation and target positioning are lower for MBRT given the use of thicker microbeams, which makes the interlacing of two or more minibeam arrays to produce a homogenous dose at the target region more easily achievable (Dilmanian et al. 2006). MBRT also allows for the use of higher energy beams, which have greater tissue penetration compared to

MRT (Prezado et al. 2015). The clinical potential of MBRT has been demonstrated in pre-clinical studies on glioma-bearing rats, with an increase in lifespan for rats irradiated with two orthogonally cross-fired minibeam arrays interlacing at the target region compared to non-treated controls (Prezado et al. 2012, Deman et al. 2012).

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Synchrotron broad-beam radiation therapy

SBBR is characterised by uniform broad-beam fields similar to CRT, but with dose-rates that are thousands of times greater than those used in the clinic today. In principle, the dose-rate of SBBR at the IMBL could be in the range of 300 Gy/s

(Livingstone et al. 2017); however, dose-rates are currently limited to approximately 40

Gy/s for pre-clincial studies to ensure accurate dosimetry.

Recently, ultra-high dose-rate radiation therapy has risen to prominence within the radiation oncology community due to the observation of reduced toxicity compared to conventional dose-rates (Favaudon et al. 2014, Montay-Gruel et al. 2017). For this phenomenon - known as a ‘FLASH’ effect - to occur, dose-rates must exceed 30 to 60

Gy/s. SBBR at the IMBL is capable of these dose-rates and therefore has the potential to elicit a FLASH effect.

Favaudon et al. first demonstrated the FLASH effect by irradiating the whole thorax of mice using ultra-high versus conventional dose-rates, showing markedly reduced long-term lung fibrosis in the ultra-high dose-rate group (Favaudon et al. 2014).

The tissue-sparing benefits of ultra-high dose-rate radiation therapy have since been confirmed in a veterinary dose-escalation study performed on cats with spontaneously growing nasal tumours (Vozenin et al. 2018). Alongside the potential normal tissue sparing advantages, ultra-high dose-rate radiation therapy could significantly reduce overall treatment times, which has clinical and economic value. Faster treatment delivery would reduce the impact of patient movement during treatment, perhaps leading to smaller treatment margins, as well as enhancing patient comfort and increasing patient throughput.

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Current pre-clinical data for FLASH radiation therapy has been based on high dose-rate electron beams from experimental linear accelerators (Favaudon et al. 2014,

Montay-Gruel et al. 2017), and more recently, x-rays from synchrotron sources

(Montay-Gruel et al. 2018). Additionally, there is interest in modifying clinical linear accelerators to enable the delivery of electron beams at the ultra-high dose-rates required to research the FLASH effect (Schüler et al. 2017). In contrast to these methods, synchrotrons generate ultra-high dose-rate x-ray beams. This would control for radiation quality and allow for a direct comparison to x-ray based CRT, making

SBBR an attractive technique to quantify and understand the FLASH effect.

Future applications and directions

Alongside applications in radiation oncology, MRT has the potential to alleviate symptoms of neurological conditions. In this context, MRT could be used to deliberately transect key neuronal pathways, or ablate highly localised regions of the brain, in order to modulate or suppress the networks responsible for abnormal movement (Romanelli & Bravin 2011, Serduc et al. 2010b). Proof of principle data supporting this neurosurgical application of MRT exists in pre-clinical models of spinal cord injury (Dilmanian et al. 2013) and epilepsy related to the somatosensory cortex

(Pouyatos et al. 2013). Epilepsy induced by mesial temporal sclerosis may also benefit from this application of MRT (Fardone et al. 2018).

For cancer, harnessing the unique physical characteristics of synchrotron generated x-rays in MRT or SBBR could provide the paradigm shift required to address currently unmet clinical meeds in clinical radiation oncology. While there is promising data to suggest that MRT and SBBR could be used therapeutically, three key issues

35 must be addressed for synchrotron radiation therapy to progress towards a human clinical trial: 1) safety and normal tissue toxicity, 2) dose-equivalence with conventional radiation therapy (CRT) and 3) the development of beam-line infrastructure, such as treatment planning systems and image-guidance, to support human use.

2.7 Normal tissue toxicity

Although remarkable normal tissue sparing is an apparent hallmark of MRT and ultra-high dose-rate radiation therapy, a robust characterisation of the toxicity induced by synchrotron radiation therapy is still required to safely conduct a human clinical trial.

While MRT has accumulated a substantial body of toxicity data since its inception in the early 1990s, ultra-high dose-rate modalities are relatively new. Normal tissue toxicity data for both MRT and ultra-high dose-rate radiation therapy are reviewed in this section.

MRT

The first MRT studies reported neuropathological effects in rodent brain tumour models (Laissue et al. 1998, Slatkin et al. 1995) with the brain continuing to be a focus of pre-clinical studies in the following decades. A body of literature reports brain toxicity in animal models, with the majority of data reported at a histological level

(Table 1) and a smaller number of studies reporting functional outcomes, such as survival, memory and cognitive function (Table 2). A further subset of studies specifically focuses on the effects of MRT on brain microvasculature (Table 3). Table 1 to 3 summarise brain normal tissue toxicity data presented across the MRT literature,

36 highlighting animal models, MRT array parameters, irradiation geometry and peak and valley dose in relation to toxicity outcomes.

Slatkin et al. (1995) first demonstrated the tolerance of healthy rat brain tissue to radiation doses of up several thousand Gy when delivered as microplanar beams 20 µm wide and spaced by 200 µm; brain tissue retained its structural integrity and histological damage was confined to the path of the microbeams. While early pre-clinical MRT studies continued to show the effects of MRT on the brain structure at a histological level, long-term functional and developmental outcomes were not investigated for a further decade.

In a landmark study on weanling piglets, used as a model for the immature brain tissue of human paediatric patients, Laissue et al. (2007) showed that peak MRT entrance doses up to 263 Gy at the depth of the cerebellum could be tolerated without any impact on neurological or behavioural development. Cerebellar architecture was preserved, however, clear stripes of long-term cell loss, corresponding to the microbeam paths, were observed over a year following irradiation (Figure 2.7). Schultke et al.

(2008) followed this study by evaluating the impact of MRT on the long-term cognitive function of rats and found no significant differences in memory function between control rats and those irradiated with two cross-fired microbeam arrays each delivering a peak dose of 350 Gy.

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Figure 2.7. Cerebellar histology approximately 15 months following MRT irradiation of weanling piglets. Clear stripes of cell loss, corresponding to microbeam paths, are evident. Figure from Laissue et al. (2007), with permission.

In parallel with pre-clinical studies characterising normal tissue toxicity, several studies also demonstrated the ability of MRT to slow the growth of tumours and prolong the survival of tumour bearing animals. Tumour models investigated in these studies include murine mammary carcinoma (Dilmanian et al. 2003), squamous cell carcinoma (Miura et al. 2006), and most frequently, 9L Gliosarcoma (Dilmanian et al.

2002, Regnard et al. 2008b, Schultke et al. 2008, Serduc et al. 2009a, Serduc et al.

2009b), which is a widely used rodent model for high-grade brain tumours. With the weight of evidence for the therapeutic potential of MRT increasing, interest grew in the mechanisms that might underpin the differential effect of MRT on tumour and normal tissue.

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The regenerative ability of endothelial cells was first suspected to play a key role in the resistance of normal tissue to MRT by Slatkin et al. (1995) and a preferential tumouricidal effect by Laissue et al. (1998) and Dilmanian et al. (2002). Histological data from subsequent experiments on avian embryonic brain (Dilmanian et al. 2001), rat skin (Zhong et al. 2003) and rat brain (Dilmanian et al. 2005) supported this hypothesis.

Further experiments verified these histological results with functional outcomes, demonstrating that changes to the normal brain, in terms of blood volume (Serduc et al.

2006) and cerebral oedema (Serduc et al. 2008) following peak MRT doses of up to

1000 Gy were only transient and minor. MRI-based studies demonstrated evidence of preferential damage to tumour microvasculature in regard to oedema and blood vessel morphology (Bouchet et al. 2010) and the induction of hypoxia in tumour but not normal brain tissue (Bouchet et al. 2013a).

While brain toxicity has been explored in significant depth, a disproportionately small number of studies report normal tissue toxicity in other organs (Table 4), including the skin (Zhong et al. 2003, Dilmanian et al. 2003, Priyadarshika et al. 2011,

Potez et al. 2018), spinal cord (Laissue et al. 2013), eyes (Sandmeyer et al. 2008) and major blood vessels (van der Sanden et al. 2010). Nevertheless, these studies similarly demonstrate the remarkable tolerance of healthy tissue to peak doses of MRT that are at least an order of magnitude greater than the doses that would be tolerated using conventional broad-beam radiation therapy (Smyth et al. 2016).

For example, Dilmanian et al. (2003) and Zhong et al. (2003) demonstrated the ability of mouse skin to withstand peak MRT doses in the order of 1000 Gy without undergoing moist desquamation. For comparison, the median effective dose (ED50) for this effect in pigs, as a model for human skin, using CRT is approximately 30 to 40 Gy

(van den Aardweg, Arnold & Hopewell 1990, van den Aardweg et al. 1989). Similarly

39 for the spinal cord, Laissue et al. (2013) showed that the ED50 for foreleg paresis in rats following MRT was 373 Gy (peak dose) delivered in a single dose, while the ED50 for synchrotron based broad-beam radiation therapy was 130 Gy. In clinical radiation oncology, the maximum dose to the spinal cord is typically kept to less than 13 Gy in a single fraction in order to minimise the risk of myelopathy (Kirkpatrick, van der Kogel

& Schultheiss 2010).

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Table 2.1. MRT-induced histological damage to normal brain tissue

Beam Irradiation Field Size MB Animal Anatomical Time post- energy geometry in normal (Width x Width/Spacing Structural damage to normal Study Model Target MRT (keV) tissue Height) (mm) (µm/µm) PED (Gy) VD (Gy) brain tissue

Mukumoto et C57BL/6 Whole brain Up to 90 90 (peak) Unidirectional (AP) 10 x 12 25/200 480 ~13 (entrance) Neuronal cell loss in MB al. (2017) Mice days paths; no demyelination; preserved cortical architecture

Serduc et al. Healthy Rats Upper cerebral Two 107 Unidirectional (AP) 4 x 4 25/400 500 3.8 (entrance) No necrosis or other (2014) hemispheres, months (median) observable damage corpus callosum 50/400 280 3 (entrance) No necrosis or other observable damage

50/400 500 5.3 (entrance) Microstructural damage to 100/400 150 – 500 2.6 – 8.6 white matter; no necrosis (entrance)

Laissue et al. Healthy Rat Entire Approx. NR Unidirectional 10 x 10 28/105 150 9.5 (at centre of Significant cerebellar (2007); Hanson, Pups (12-14 cerebellum, one year (LR) hindbrain) hypoplasia; Slatkin and days old) dorsal pons, microcalcifications in 90% of Laissue (2013) dorsal medulla, animals posterior aspect of occipital lobes 28/105 50 3.1 (at centre of Moderate cerebellar (immature hindbrain) hypoplasia; hindbrain tissue) 28/210 150 2.7 (at centre of microcalcifications in 4-10% hindbrain) of animals

28/210 50 0.9 (at centre of Slight cerebellar hypoplasia; hindbrain) no microcalcifications observed

Laissue et al. Healthy Cerebellum Approx. NR Unidirectional 15 x 15 28/210 150 – 600 3.2 – 12.6 (at No necrosis; no abnormalities (2007) Weanling one year (lateral) (66 – 263 at depth of compared to unirradiated Piglets (42 depth of cerebellum) controls except loss of and 48 days cerebellum) astroglial and neuronal nuclei old) confined to MB path

Dilmanian et al. Fischer Rats Cerebrum and Up to 16 120 (half Unidirectional (AP) 10 x 10 27/200 800 (560 at NR Loss of neurons in MB path, (2005) cerebellum days power depth of intact capillaries energy) cerebellum)

Dilmanian et al. 9LGS Anterior part of Four 66 or 74 Unidirectional (RL) 8-10 x 11.4 27/100 500 19 (at centre of No white matter necrosis, (2002) bearing Rats the cerebrum months (median) (359 at centre brain) vascularisation or cerebral (frontal lobe) and at of brain) oedema observed; significant death microcalcifications

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(variable) 27/75 500 33 (at centre of Moderate white matter (359 at centre brain) necrosis, significant of brain) microcalcifications; slight cerebral oedema; significant vascularisation;

27/75 250 17 (at centre of No white matter necrosis or (179 at centre brain) vascularisation; slight cerebral of brain) oedema; significant microcalcifications

Laissue et al. 9LGS Right Striatum At death 49.3 Bidirectional 10 x 12 25/100 2 x 312 NR Highest scores of brain (1998) bearing Rats (variable) (median) (AP and RL) 2 x 625 damage; loss of tissue structure

Unidirectional 25/100 312, 625 NR Median brain damage score of (AP) 0; no loss of tissue structure

Slatkin et al. Healthy Rats Right cerebral 31 days 48.5 Unidirectional (AP) 4 x 4 (20 MBs) 20/200 312, 625 NR No observable changes in peak (1995) and cerebellar (median) or valley hemispheres 20/200 2500, 5000 NR Loss of astrocytic and neuronal nuclei confined to MB path; no necrosis

14 days 42/200 2500 NR Loss of astrocytic and neuronal nuclei confined to MB path; no necrosis

42/200 10000 NR Tissue necrosis

31 days 3 MBs 20/200 2500, 5000, NR Loss of astrocytic and 10000 neuronal nuclei confined to MB path; no necrosis

MB, microbeam; PED, Peak entrance dose; VD, Valley dose; 9LGS, 9L Gliosarcoma; NR, not reported; NA, not applicable; AP, anterior-to-posterior; RL, right-to-left; LR, left-to-right

Table 2.1 is adapted from data presented by Smyth et al. (2016). The full publication is available in the appendix.

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Table 2.2. Functional outcomes after brain irradiation with MRT

Beam Irradiation Field Size MB Animal Anatomical Time post- energy geometry in normal (Width x Width/Spacing Study Model Target MRT (keV) tissue Height) (mm) (µm/µm) PED (Gy) VD (Gy) Toxicity Results

Mukumoto C57BL/6 Whole brain Up to 90 90 (peak) Unidirectional (AP) 10 x 12 25/200 360 ~10 (entrance) 100% survival et al. (2017) mice days 600 ~16 (entrance) Predicted LD50 720 ~19 (entrance) 100% mortality

25/100 120 ~4 (entrance) 100% survival 240 ~7 (entrance) 100% mortality

25/300 600 ~16 (entrance) 100% survival 840 ~22 (entrance) 100% mortality

Schultke et Wistar and Right cerebral One year 83 (peak) Bidirectional (AP 10 x 14 25/210 2 x 350 NR No decrease in memory al. (2008) Fischer Rats hemisphere and RL) function/object recognition compared to non-irradiated controls

Laissue et al. Healthy Cerebellum Approx. NR Unidirectional 15 x 15 28/210 600 (263 at 12.6 (at depth No changes in behaviour or (2007), Weanling one year (lateral) depth of of cerebellum) neurological development Hanson, Piglets (42 or cerebellum) Slatkin and 48 days old) Laissue (2013)

Healthy Rat Entire Approx. NR Unidirectional 10 x 10 28/105 150 9.5 (at centre of Sub-normal body weight and Pups (12-14 cerebellum, one year (LR) hindbrain) strength, ataxia, asthenia, days old) dorsal pons, hyperactivity dorsal medulla, posterior aspect 28/105 50 3.1 (at centre of No differences compared to of occipital lobes hindbrain) controls (immature hindbrain tissue) 28/210 150 2.7 (at centre of No differences compared to hindbrain) controls

MB, microbeam; PED, Peak entrance dose; VD, Valley dose; 9LGS, 9L Gliosarcoma; NR, not reported; AP, anterior-to-posterior; RL, right-to-left; LD50, predicted lethal dose for 50% of animals

The template for data summarised in Table 2.2 is adapted from Smyth et al. (2016). The full publication is available in the appendix.

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Table 2.3. Effects of MRT on normal brain microvasculature

Beam Irradiation Field Size MB Animal Anatomical Time post- energy geometry in normal (Width x Width/Spacing Physical and functional Study Model Target MRT (keV) tissue Height) (mm) (µm/µm) PED (Gy) VD (Gy) changes to microvasculature

Bouchet et F98 glioma Anterior right Up to 14 105 Unidirectional 8 x 10 50/200 241 10.5 (at 9mm No changes to blood brain al. (2017) bearing rats cerebral days (median) (contralateral depth in brain) barrier hemisphere hemisphere)

Bidirectional (brain 8 x 10 50/200 2 x 241 21 (at 9mm Significant, transient increase tissue at overlap of depth in brain) in vascular permeability one AP and lateral week post-irradiation cross-fried arrays))

Bouchet et 9LGS Arrays centred at Up to 55 90 Unidirectional 8 x 10 50/200 400 12.5 (at brain No change in blood oxygen al. (2013a) bearing Rats tumour located at days (median) (lateral, anterior and (350 at brain depth) saturation; slight; minor right caudate posterior brain depth) changes to vessel morphology nucleus tissue proximal to and decrease in vessel density target)

Bouchet et 9LGS Arrays centred at Up to 45 90 Unidirectional 8 x 10 50/200 400 12.5 (at brain No changes to blood vessel al. (2010) bearing Rats the anterior part days (median) (lateral, anterior and (350 at brain depth) size, permeability or of right cerebral posterior brain depth) morphology; no change to hemisphere tissue proximal to blood volume fraction target)

Bidirectional (brain 50/200 2 x 400 25 (at brain No changes to blood vessel tissue at overlap of (700 at brain depth) permeability and blood volume AP and lateral depth) fraction; increase in vessel cross-fried arrays) size; some changes in vessel morphology

Serduc et al. Female Swiss Upper part of left Up to one 90 Unidirectional 3.6 x 4 25/211 312 NR No observable cerebral (2008) nude mice cerebral month (mean) (AP) oedema hemisphere 25/211 1000 10.5 (at 1cm Mild, transient oedema which depth) resolves a week after irradiation

Serduc et al. Swiss nude Upper part of left Up to three 90 Unidirectional 4 x 4 25/211 312 NR No observable changes to (2006) mice cerebral months (mean) (AP) blood volume, no hemisphere haemorrhage, and no significant morphological changes

25/211 1000 NR Transient increase in blood brain barrier permeability, down localised to MB path up

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to 12 days post-irradiation; no other observable changes

MB, microbeam; PED, Peak entrance dose; VD, Valley dose; 9LGS, 9L Gliosarcoma; NR, not reported; AP, anterior-to-posterior

Table 2.3 is adapted from data presented by Smyth et al. (2016). The full publication is available in the appendix.

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Table 2.4. MRT-induced toxicity in various normal tissue types Beam Irradiation Field Size MB Animal Anatomical Time energy geometry in (width x height) Width/Spacing Study Model Target post-MRT (keV) normal tissue (mm) (µm/µm) PED (Gy) VED (Gy) Toxicity

Eye

Sandmeyer et Rats Right 12 months NR Unidirectional 10 x 14 25/211 350 ~5 Retinal lesions in 8/16 left eyes, no cataracts al. (2008) bearing C6 Cerebral (proximal to observed in left eyes Glioma Hemisphere target region)

Bidirectional 2 x 350 ~14 Retinal lesions in 16/16 right eyes; cataracts (tissue at in 12/37 right eyes overlap of AP and RL cross- fried arrays) Spinal Cord

Laissue et al. Healthy Cervical Up to 383 100 Unidirectional 11 x 21 35/210 253 (at cord 9 (at cord Paresis free at 383 days post irradiation (2013) Rats spinal cord days (mean) (RL) depth) depth) (11mm length) 357 (at cord 12.7 (at cord Spinal cord tissue appears histologically depth) depth) normal

373.3 (at 13.2 (at cord Dose calculated to cause paralysis in 50% of cord depth) depth) rats

Skin

Potez et al. Healthy Ear pinnae Up to 240 105 Unidirectional 5.5 x 14 50/200 200 3.2 No early or late manifestations of epidermal (2018) mice days (mean) 400 6.5 damage. Increase in ventral dermis thickness (C57BL/6) 240 days post-irradiation.

800 12.9 Increase in ventral and dorsal dermal thickness 240 days post-irradiation. Transient oedema, increase in ventral and dorsal epidermal thickness, ear thickness and sebaceous gland density 15-30 days post- irradiation

Priyadarshika Healthy Dorsal skin 5 days 125 Unidirectional 6 x 6 25/200 200 ~2 – 4 Epidermal and adnexal cell death, epidermal et al. (2011) mice flap (mean) nuclear enlargement, focal spongiosis, leukocyte infiltration in <10% of dermis

400 ~4 – 8 Epidermal and adnexal cell death, epidermal nuclear enlargement, focal spongiosis, leukocyte infiltration in <10% of dermis

800 ~8 – 16 Epidermal and adnexal cell death, epidermal nuclear enlargement, focal spongiosis sometimes involving lower half of epidermis, leukocyte infiltration in 10 – 70% of dermis

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Dilmanian et Mice Right hind 7 months 100 Unidirectional 20 x 20 90/300 970 19 No moist desquamation or complete epilation al. (2003) bearing leg (median) EMT6 or 118 No moist desquamation; complete epilation murine (median) 1900 38 in 7/10 rats; failure to full regrow hair in 4/5 mammary rats carioma

Zhong et al. Healthy Right hind Up to 30 100 Unidirectional 35 x 15 90/300 1005 25 No moist desquamation; minor hair clumping (2003) mice leg days (median) or 120 1335 33 Moist desquamation evident; severe hair (median) clumping

Vasculature (major vessels) van der Healthy Saphenous Up to 12 107 Unidirectional 10 x 10 50/400 312 2.7 Stable thinning of tunica media in peak Sanden et al. mice artery months (median) (ventro-dorsal) regions, hypertrophy of tunica media in (2010) valley regions ; no vascular occlusions

2000 17.6 Complete loss of vascular smooth muscle cells and tunica media in peak regions, hypertrophy of tunica media in valley regions; no vascular occlusions

MB, microbeam; PED, Peak entrance dose; VED, Valley entrance dose; AP, anterior-to-posterior; RL, right-to-left; BB, broadbeam; NR, not reported

Table 2.4 is adapted from data presented by Smyth et al. (2016). The full publication is available in the appendix.

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While there is a wide body of literature reporting the toxic effects of MRT, it is difficult to synthesise these data due to differences in animal models, and most pertinently, microbeam array parameters (width and spacing) between studies. In some cases, the deleterious effects of the tumours being irradiated also confound the tissue toxicity data. The toxicity studies on healthy animals are therefore of the greatest value.

Furthermore, only a limited number of studies report clinical outcomes and even fewer studies report long term toxicity at least one year following irradiation. Of these studies, only Mukumoto et al. (2017) and Laissue et al. (2013) provide robust dose-response curves based on systematic dose-escalation. These studies provide an important template for future studies designed to characterise the toxicity of organs outside the central nervous system, such as organs of the thorax, abdomen and pelvis.

Nevertheless, a number of key observations can be made from the existing MRT toxicity literature. Firstly, maintaining a unidirectional peak-valley spatial dose- distribution is essential to preserving the normal tissue sparing effects of MRT. At a histological level, normal tissue that falls in the overlap area between two or more cross-fired MRT arrays shows higher levels of damage compared to tissue irradiated from one direction only (Bouchet et al. 2017, Bouchet et al. 2010, Laissue et al. 1998).

Therefore, cross-firing two or more MRT arrays to maximise the peak dose delivered to the tumour, either through an overlapping (Laissue et al. 1998, Bouchet et al. 2012,

Bouchet et al. 2010, Serduc et al. 2009b, Schultke et al. 2008) or seamlessly interlaced approach (Brauer-Krisch et al. 2005, Dilmanian et al. 2006, Serduc et al. 2010b), comes with significant risk which can only be partly mitigated through conformal radiation therapy techniques. The development of systems to facilitate conformal synchrotron radiation therapy is discussed in section 2.9.

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Secondly, there is a clear relationship between microbeam centre-to-centre spacing and normal tissue toxicity. When keeping microbeam width constant, increasing the microbeam spacing leads to improved histological (Laissue et al. 2007) and functional (Mukumoto et al. 2017) outcomes following the irradiation of tissue in healthy animals. It is therefore essential that MRT tolerance doses are reported with a corresponding microbeam width and spacing. Microbeam spacing is an essential parameter in striking the balance between normal tissue sparing and tumour control. In this regard, Regnard et al. (2008b) compare a microbeam centre-to-centre spacing of

100 µm and 200 µm for the irradiation of 9L gliosarcoma in rats and advocate for a larger spacing of 200 µm in order to provide more clinically acceptable long-term functional outcomes in surviving mice.

Mini-beam radiation therapy

There are a number of mini-beam radiation therapy (MBRT) animal experiments reporting normal tissue damage in the brain. In a recent dose escalation study on healthy rats, Prezado et al. (2015) show the entire rat brain can tolerate in-beam doses up to 150

Gy, with no clinical signs of toxicity up to 560 days post-irradiation, when using an array of mini-beams 600 µm wide and spaced by 1200 µm. Doses of 200 Gy resulted in abnormal behavioural signs such as apathy and an inability to feed within a few days of irradiation (Prezado et al. 2015). The results of this study reaffirmed findings of a previous experiment performed on healthy rats which demonstrated no observable changes in the structure or function of brain microvasculature 298 days after exposure four mini-beams, 620 µm wide and spaced by 1220µm, delivering 123 Gy to the right cerebral hemisphere (Deman et al. 2012).

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In contrast to the normal tissue sparing demonstrated following exposure to a single MBRT array, focal brain lesions are produced at the overlapping region of two orthogonally cross-fired interlacing arrays (Deman et al. 2012, Dilmanian et al. 2006).

Contralateral regions of the brain exposed to only one mini-beam array remained undamaged up to six months following a dose of 120 Gy (Dilmanian et al. 2006).

Similarly to MRT, these observations reaffirm the assertion that maintaining a peak- valley dose-distribution is essential for the normal tissue sparing benefits of MBRT and suggest that using a unidirectional approach would provide the highest level of safety in a future clinical trial.

Dilmanian et al. (2006) report the effects of MBRT on the thoracic spinal cord of rats. An in-beam dose of 400 Gy with a single array of four mini-beams spaced by 4 mm could be delivered with no paralysis or leg weakness within an observation period of 7 months when the mini-beam width was restricted to 500 µm (Dilmanian et al.

2006). However, when the mini-beam width was increased to 680 µm, a 400 Gy peak dose caused loss of leg strength relative to untreated controls and signs of paralysis 10 to 20 days following irradiation (Dilmanian et al. 2006). By comparison, Laissue et al.

(2013) report no paresis following an MRT dose of 253 Gy following spinal cord irradiation and paresis in 50% of rats following a dose of 373 Gy using microbeams 35

µm wide and spaced by 210 µm.

Ultra-high dose-rate radiation therapy

To date, only a small number of published studies report normal tissue toxicity outcomes following ultra-high dose-rate radiation therapy compared to CRT. The

FLASH effect was first reported by Favaudon et al. (2014) in mice following bilateral

50 thorax irradiation using electrons. In this study, mice exposed to 17 Gy using CRT

(conventional dose-rate of 0.03 Gy/s) showed pulmonary fibrosis beginning 8 weeks following thorax irradiation in addition to severe cutaneous lesions, while no mice developed fibrosis or any other complication up to 36 weeks post-irradiation up to doses of 20 Gy delivered at an ultra-high dose-rate of 60 Gy/s (Favaudon et al. 2014). In the ultra-high dose-rate group, doses higher than 23 Gy resulted in cachexia within 32 weeks of irradiation, and a dose of 30 Gy led to pulmonary oedema, inflammation and fibrosis after 24 weeks. Interestingly, Favaudon et al. (2014) demonstrated that radiation delivered at an ultra-high dose-rate was just as effective as CRT in regards to tumour growth suppression, which suggests that ultra-high dose-rates could improve the therapeutic ratio for lung cancer treatments.

Montay-Gruel et al. (2017) subsequently demonstrated that the FLASH effect is dose-rate dependent. Based on whole-brain irradiations in mice using electrons, 10 Gy delivered at a dose-rate of 60 Gy/s or lower led to significantly poorer memory outcomes compared to non-irradiated control mice while mice receiving this dose at a dose-rate of 100 Gy/s or higher performed just as well as controls (Montay-Gruel et al.

2017). Nevertheless, mice in the groups irradiated using dose-rates of 30 Gy/s and 60

Gy/s performed significantly better in object recognition tests compared to mice receiving 10 Gy at a conventional dose-rate, demonstrating that a FLASH normal tissue sparing effect is still evident at dose-rates as low as 30 Gy/s (Montay-Gruel et al. 2017).

A number of pre-clinical MRT studies report on toxicity following SBBR (x- rays), but without a comparison to CRT. In particular, Mukumoto et al. (2017) report survival outcomes after whole brain irradiation of healthy mice (120 Gy/s) and Laissue et al. (2013) report foreleg paresis following cervical spinal cord irradiation in rats.

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In conclusion, ultra-high dose-rate radiation therapy has just begun to emerge as a novel radiation therapy modality and toxicity data are limited. Current data exists in a predominantly proof-of-concept format, with systematic dose-escalation studies that provide a direct comparison to CRT needed to characterise the safety profile of ultra- high dose-rate modalities such as SBBR. In the same way that MRT toxicity data should be reported alongside the corresponding MRT parameters (width and spacing), SBBR toxicity data should be reported alongside dose-rate, which has been shown to influence normal tissue sparing outcomes (Montay-Gruel et al. 2017). Further pre-clinical studies of both SBBR and MRT, which report acute and long-term toxicity data for clinically relevant, functional outcomes, are still required for the planning of future clinical trials.

2.8 Biological dose-equivalence

Drawing sensible comparisons between conventional and synchrotron radiation therapy modalities is not straightforward given their contrasting physical and radiobiological characteristics. For MRT specifically, dose-equivalence is an even more complex issue given its intrinsically inhomogeneous dose-distribution compared to the uniform fields employed in CRT. Furthermore, the extremely high dose gradients between the peak and valley regions of an MRT field significantly challenge the accuracy and utility of conventional, physical dosimetric techniques (Crosbie et al.

2008).

Despite these difficulties, defining dose-equivalence remains a crucial task.

Clinicians must understand how to prescribe doses of synchrotron radiation therapy and how these prescriptions would compare to CRT in regard to both tumour control and normal tissue toxicity. Assessing dose-equivalence based on biological endpoints is

52 therefore both practical and clinically relevant, and provides an attractive alternative to physical dose-equivalence. In vitro and in vivo biological endpoints, used as surrogates for dose-equivalence, are discussed in the literature.

In vitro techniques

Ibahim et al. (2014) calculated equivalent MRT and broad-beam doses based on the in vitro response of tumour and normal tissue cell lines using two biological endpoints: clonogenic cell survival, which is a widely used technique for characterising radio-sensitivity in vitro (Franken et al. 2006), and cell impedance (RT-Cis), which provides a real-time integrated measure of cell number, morphology and metabolism

(Roa et al. 2011). Cell lines investigated in this study included EMT6.5 and 4T1.2 mouse mammary tumours, SaOS-2 osteosarcoma, and NMuMG, representing normal mouse epithelium. These data validate in vitro methods of determining dose- equivalence between MRT and CRT, however, discrepancies in the calculated equivalent doses between the cell-lines and biological endpoint assays suggest that these techniques may not be robust. Nevertheless, these data demonstrate the dependence of dose-equivalence on microbeam parameters. The MRT array with 50 µm microbeam width and 200 µm centre to centre spacing (50/200) had higher equivalent broad-beam doses, on average, compared to the array with 25 µm beam width and 200 µm microbeam spacing (25/200) (Ibahim et al. 2014). A larger microbeam width at a constant centre to centre spacing will lead to greater energy deposition by the array, and therefore, a higher equivalent broad-beam dose.

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In vivo techniques

Several pre-clinical rodent studies evaluate dose-equivalence based on the response of normal tissue to irradiation, comparing synchrotron MRT to SBBR delivered at an ultra-high dose rate.

Based on a histological scoring system for acute dermatological toxicity in mice,

Priyadarshika et al. (2011) found that MRT peak doses of 200 Gy and 400 Gy (25 µm microbeam width and 200 µm centre to centre spacing) elicited an equivalent response to low doses of SBBR ranging from 11 Gy to 44 Gy. The broad-beam dose-rate used in this study was 100 Gy/s. No comparison to CRT was made; therefore, no conclusions can be drawn regarding the presence or absence of a FLASH effect in this model.

Systematic dose-escalation studies provide a more specific measure of dose- equivalence between MRT and SBBR. Laissue et al. (2013) investigated the response of rat spinal cord to increasing doses of both radiation modalities, using foreleg paresis as a biological endpoint to calculate dose-equivalence. Based on the ED50, a mean (± standard error) microbeam peak and valley dose of 373.3 (± 90.8) Gy and 13.3 (± 3.2)

Gy, respectively, was equivalent to 130 (± 31) Gy delivered using SBBR (Laissue et al.

2013). Microbeams in this study were 35 µm wide and spaced by 210 µm. Similarly,

Mukumoto et al. (2017) performed a dose-escalation study comparing the toxic effects of whole brain irradiation in C57BLJ/6 mice using MRT and synchrotron broad-beam radiation therapy. Based on the lethal dose to 50% of test animals (LD50), a MRT peak dose of 600 Gy (microbeam width and spacing of 25 µm and 200 µm, respectively) was equivalence to 80 Gy using SBBR.

Similarly to Priyadarshika et al. (2011), Laissue et al. (2013) and Mukumoto et al. (2017) did not deliver broad-beam radiation therapy at a clinically comparable dose-

54 rate. Given the possibility of eliciting a FLASH effect, the equivalent broad-beam doses calculated in these studies may have been significantly overestimated due to using

SBBR dose-rates in the order of 100 Gy/s. There remains a need for systematic dose- escalation studies that compare synchrotron radiation therapy modalities to a CRT control series (dose-rate of approximately 0.05 Gy/s). Nevertheless, these studies provide a useful template for the design of future dose-equivalence studies, with ED50 or LD50 values for each modality providing a clinically relevant endpoint for both biological dose-equivalence and normal tissue toxicity.

2.9 Medical physics developments in synchrotron radiation therapy

The ability to deliver radiation accurately and in a way that conforms to the tumour relies on robust patient positioning, image-guidance, and treatment planning systems. Treatment planning systems furthermore allow for clinicians to model the planned dose distribution in three dimensions and in the context of a patient’s anatomy, which is essential for predicting normal tissue toxicity and evaluating radiation therapy treatment plans. In order to prepare synchrotrons for clinical use, each of these three domains must be developed.

Treatment planning system

Two key components of a treatment planning system for synchrotron radiation therapy, and in particular, MRT, are 1) a dose-calculation algorithm that can model the delivered dose, and 2) a graphical user interface that allows clinicians to view, manipulate and evaluate the calculated dose in an anatomical context. These kinds of treatment planning systems are routinely used in the clinic for CRT today.

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For MRT, the physical characteristics of the dose distribution are unique from

CRT – in particular the low keV energy spectrum and the peak-valley geometry on a microscopic scale – and therefore, conventional dose calculation algorithms are not adequate. Monte Carlo simulations with various codes such as EGS4 (Company &

Allen 1998, Orion et al. 2000, De Felici et al. 2005), PENELOPE (Siegbahn et al. 2005,

Siegbahn et al. 2006, Martinez-Rovira et al. 2010) and GEANT4 (Spiga et al. 2007,

Cornelius et al. 2014) offer a robust approach to modelling MRT dose distributions in tissue compared to measured experimental dosimetry (Martinez-Rovira, Sempau &

Prezado 2012a) but at the expense of long calculation times. In the first Monte Carlo based treatment system for MRT implemented at the ESRF for veterinary trials, almost one day was required to calculate a plan to treat the head of a dog (Martinez-Rovira,

Sempau & Prezado 2012b).

In the clinic however, constraints on time and computational resources render arbitrarily high levels of dose calculation accuracy unfeasible. For CRT, it is expected and shown that even the most complex plans, such as those involving IMRT or treatment sites such as the lung, be calculated in a matter of minutes (Kroon, Hol &

Essers 2013, Margarida et al. 2010). Researchers at RMIT University (Melbourne,

Australia) developed a fast and simple dose-calculation algorithm, based on percentage depth-dose data for microbeams in water, which was integrated into the commercially available EclipseTM (Varian Medical Systems Inc., Palo Alto, CA, USA) treatment planning system (Poole et al. 2017). While this algorithm is fast and integrated into a user interface that is clinically recognisable, its use is significantly limited by its inability to account for tissue inhomogeneity (Poole et al. 2017), which is essential for accurate dose calculations in human patients.

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Convolution based algorithms (Bartzsch & Oelfke 2013, Debus, Oelfke &

Bartzsch 2017), which do not rely purely on time-consuming Monte Carlo simulations, are an alternative and accurate method of MRT dose-calculation and have been used in pre-clinical and veterinary trials at the ESRF. Most recently, a ‘hybrid’ approach that combines Monte Carlo and convolution-based methods of dose calculations has been developed for use at the ESRF (Donzelli et al. 2018). This hybrid algorithm has also been adopted by the IMBL and has been implemented in a research version of the

Eclipse™ treatment planning system. This hybrid approach offers dose-calculation accuracy comparable to pure Monte Carlo methods, but with clinically realistic computation times (Donzelli et al. 2018).

Image-guidance and patient positioning

While an efficient treatment planning system for MRT is essential for future clinical trials, robust patient positioning and image-guidance technology must also be developed in parallel in order to translate these plans into accurately delivered treatments. Clinically available patient positioning and image-guidance systems for radiation therapy allow for complex CRT treatments to be delivered with sub-millimetre accuracy. This level of accuracy is necessary to ensure radiation is delivered to the tumour with minimal irradiation of nearby organs at risk. For example, when treating tumours in the brain, critical structures such as the brainstem, optic nerves, or hippocampus, may abut the tumour or treatment volume. For MRT, which delivers peak doses of radiation that are at least an order of magnitude higher than CRT doses, treatment accuracy is of utmost importance.

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Patient positioning is a particular challenge for synchrotron radiation therapy given the fixed horizontal beam-line within the irradiation enclosure. In comparison to this, the gantry of a clinical linear accelerator is able to rotate around the patient by a full 360 degrees, enabling treatment from a complete range of angles. At the synchrotron, the patient must be moved with respect to the beam in order to facilitate a range of treatment angles. The current patient positioning solution adopted by the ESRF for brain treatments is a ‘medical chair’ which can rotate, tilt and move in three translational planes, and fixes the head of a sitting patient in position using a stereotactic frame (Renier et al. 2008). Another solution, which could be applied for non-cranial treatments, is the use of industrial robotic positioning tables with six axes of movement

(Figure 2.8). These are employed at other medical radiation facilities that have a fixed horizontal beam-line, such as the Heidelberg Ion-Beam Therapy Centre in Germany, and have been shown to have comparable levels of positioning accuracy to current clinical positioning systems (Nairz et al. 2013).

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Figure 2.8. A robotic patient position system used at the Heidelberg Ion Beam therapy centre in Germany. The treatment couch can be moved with six degrees of freedom in front of a fixed horizontal beam line. Figure from Nairz et al. (2013), with permission.

In the clinic, image-guidance systems work seamlessly with patient positioning technology to maintain treatment accuracy, providing the anatomical imaging data that informs the necessary changes in patient position. Both the ESRF and IMBL (Australian

Synchrotron) describe image guidance protocols that use synchrotron radiation for imaging in pre-clinical animal trials for MRT (Pelliccia et al. 2016, Serduc et al. 2010a).

Similarly to the clinic, at the ESRF it is possible to position samples with 1mm accuracy based on the co-registration of pre-treatment images with radiographs generated from a treatment plan (Donzelli et al. 2016). Importantly, the imaging dose using the synchrotron beam is comparable to imaging doses delivered clinically (Donzelli et al.

2016).

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There are significant drawbacks to using the synchrotron beam for both imaging and treatment. Firstly, the beams produced at the ESRF and IMBL are limited to 40mm

(Donzelli et al. 2016) and 30mm (Pelliccia et al. 2016) in width, respectively. This means that for human trials, several images must be taken in order to capture enough anatomical data for accurate positioning. Secondly, the optimal beam energy for image quality is different from the energy spectrum used for therapy. In the protocol described by Pelliccia et al. (2016), a low (50 keV) monochromatic beam is selected for image- guidance at the IMBL and therefore a switch between imaging and therapy modes is required. Both of these limitations increase the overall time required for image-guided treatment, which would increase the risk of patient movement between corrections in patient position and the delivery of treatment. The implementation of an external x-ray at the IMBL, which operates independently of the synchrotron beam, allows for a larger imaging field size and increases the efficiency of the image guidance process. This kind of approach is analogous to the on-board imaging systems of modern linear accelerators, where the planes for imaging and therapy are perpendicular to one another while sharing a common centre of rotation.

2.10 Conclusion

While the therapeutic potential of synchrotron-based MRT and SBBR is exciting, the development of appropriate treatment planning, patient positioning and image-guidance systems, are required in order to facilitate human clinical use. These medical physics developments must be done in addition to collecting robust normal tissue toxicity data, which is fundamental to understanding the safety profile of synchrotron-based radiation therapy, and how the radiobiological effects of MRT and

SBBR can be compared to CRT. Identifying optimal anatomical treatment sites for

60 implementing synchrotron radiation therapy will provide further focus in the quest to conduct a human clinical trial of MRT or SBBR. These are key themes of this thesis.

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CHAPTER 3 In vitro characterisation of diffuse intrinsic pontine glioma radiosensitivity using microbeam and broad-beam radiotherapy

3.1 Preface

This purpose of this chapter is to address the issue of biological-dose equivalence between MRT and CRT (Aim 1) and to investigate the differential effects of each modality (Aim 3) at a cellular level. This chapter reproduces a paper published in the Radiation Research journal (Smyth et al. 2018a), with permission from the

Radiation Research Society ©. The final published version is available online at: https://doi.org/10.1667/RR4633.1. This chapter contains its own bibliography and reference style which is separate from the rest of the thesis. The figures appear at the end of the chapter and have been renumbered for the purpose of this thesis. An extended methods section, not published with the manuscript, has also been included. I am the primary author of this publication, involved in planning and responsible for carrying out the experimental work. I was responsible for analysing and interpreting the data and for writing the manuscript, with input and oversight from the co-authors.

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3.2 Published manuscript

Characterisation of Diffuse Intrinsic Pontine Glioma

Radiosensitivity using Synchrotron Microbeam Radiotherapy

and Conventional Radiation Therapy In Vitro

L. M. Smytha,b, P. A. W. Rogersa, J. C. Crosbiec,d and J. F. Donoghuea,c,e, aUniversity of Melbourne, Department of Obstetrics and Gynaecology, Royal Women’s

Hospital, Parkville, Victoria 3052, Australia; bEpworth Radiation Oncology, Epworth

HealthCare, Richmond, Victoria 3121, Australia; cSchool of Science, RMIT University,

Melbourne, Victoria 3001, Australia; dWilliam Buckland Radiotherapy Centre, Alfred

Hospital, Melbourne, Victoria 3004, Australia; eHudson Institute of Medical Research,

Monash University, Victoria 3168, Australia

Running Title: Radiosensitivity of paediatric glioma cell lines

Final published version: Smyth LM, Rogers PAW, Crosbie JC, Donoghue, JF.

‘Characterisation of Diffuse Intrinsic Pontine Glioma Radiosensitivity using

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Synchrotron Microbeam Radiotherapy and Conventional Radiation Therapy In Vitro’

2018 Radiat Res; 189: 146-55. © 2018 Radiation Research Society.

Abstract

Synchrotron microbeam radiation therapy (MRT) is a promising pre-clinical radiotherapy modality that has been proposed as an alternative to conventional radiotherapy (CRT) for diseases such as Diffuse Intrinsic Pontine Glioma (DIPG), a devastating paediatric tumour of the brainstem. The primary aim of this study was to characterise and compare the radio-sensitivity of two DIPG cell lines (SF7761 and

JHH-DIPG-1) to MRT and CRT. We hypothesised that these DIPG cell lines would exhibit differential responses to MRT and CRT. Single cell suspensions were exposed to MRT (112, 250, 560, 1180 Gy peak dose) or CRT (2, 4, 6, and 8 Gy) to produce clonogenic cell-survival curves. Apoptosis induction and the cell cycle were also analysed five days following irradiation using flow cytometry. JHH-DIPG-1 cells displayed greater radio-resistance than SF7761 to both MRT and CRT, with higher colony formation and increased accumulation of G2/M phase cells. Apoptosis was significantly increased in SF7761 cells compared to JHH-DIPG-1 following MRT exposure demonstrating cell-line specific differential radio-sensitivity to MRT.

Additionally, biologically equivalent doses of MRT and CRT were calculated based on clonogenic survival, furthering our understanding of the response of cancer cells to

MRT compared to CRT.

Introduction

Approximately 20% of all paediatric cancers are brain tumours and of these none is more deadly than Diffuse Intrinsic Pontine Glioma (DIPG) (1, 2). DIPG is

64 typically diagnosed in children between 5 and 10 years of age after a relatively short latency period where clinical signs such as ataxia, motor deficits and cranial nerve palsies begin to manifest (3). Radiotherapy remains the only treatment capable of prolonging survival in children diagnosed with DIPG (2, 4) and despite efforts over the past 30 years to improve survival with concurrent chemotherapy, length of survival after diagnosis has remained static at 8-14 months (1-4). Trials of hyper-fractionated (5) and hypo-fractionated (6, 7) radiotherapy regimens have been similarly inconsequential to improving overall survival. These results highlight the need to radically change the paradigm of conventional radiotherapy (CRT) in order to improve the prognosis of patients diagnosed with DIPG.

Microbeam radiotherapy (MRT) is a pre-clinical radiotherapy modality that has been proposed as an alternative treatment for DIPG (8) and other paediatric brain tumours (9) based on its extraordinary ability to spare normal brain tissues (10-12). The normal tissue sparing effects of MRT are likely to be related to its periodically alternating spatial dose distribution where in-beam ‘peak’ regions 25-50 microns (μm) wide are spaced by ‘valley’ regions 200-400 μm wide (13). This arrangement allows peak regions to receive a dose in the order of 100–1000 Gy whilst the valley regions receive a dose in the order of 10 Gy, depending on the field size and tissue depth (13).

At present, only a synchrotron source is capable of generating x-rays with the high flux and minimal divergence required to maintain the field characteristics of MRT.

Pre-clinical studies in rodents with intracranial rat syngeneic glioma xenografts or subcutaneous human glioma xenografts have yielded promising results, showing the ability of MRT to slow the progression of tumours (14-17), and in some cases, ablate them completely (18). Remarkably, brain tissue has been shown to tolerate peak doses up to hundreds of Gy with no evidence of long-term motor or cognitive dysfunction in

65 mice (12), memory impairment in rats (17) or developmental abnormalities in weanling piglets used as a human paediatric model (9). Taken together, these pre-clinical data indicate the potential for MRT to improve the therapeutic interventions for DIPG and the need for continued development of DIPG tumour models for future pre-clinical experiments (19). Similarly to pre-clinical MRT studies using rodent glioma models

(14, 20), future experiments should compare the efficacy of MRT in regard to both

DIPG tumour control and normal tissue sparing.

The primary aim of this work was to characterise the radio-sensitivities of two different DIPG cell lines of human paediatric origin (21, 22). Secondarily, we aimed to compare the effect of MRT and CRT on radiation induced apoptosis and the cell cycle.

We hypothesised that the two DIPG cells lines would have different intrinsic radio- sensitivities and would respond to MRT and CRT differently. Dose equivalence was determined by clonogenic assays, which are widely used for assessing the intrinsic radio-sensitivity of cells (23) and have been previously used to evaluate dose equivalence between MRT and CRT (24). Alpha/beta (α/β) ratios were calculated from the CRT clonogenic survival curves to provide a further indication of radio-sensitivity.

Most tumours have a α/β ratio higher than 10, reflecting a high proliferation rate and a greater acute sensitivity to radiation compared to late-responding and less proliferative normal tissues, such as the brain, which have a α/β ratio or 1.5 to 5 (25, 26). Given the impact of radiation induced damage on proliferation and cell survival (27), we also analysed cell cycle responses and apoptosis induction between the cell lines and radiation modalities using flow cytometry.

Materials and methods

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Cell culture

Two human DIPG cell lines were used in this study. JHH-DIPG-1 (referred herein as JHH) originated from a six year old male who had previously undergone combined chemotherapy and radiotherapy and harboured a p53C277F missense mutation

(21). The SF7761 DIPG cell line was isolated after surgical biopsy from a previously untreated six year old female and was p53 wild type (22). The cell lines were propagated as neurospheres in KnockOut Dulbecco’s Modified Eagle Media: Nutrient mixture F12 (Gibco, Life Technologies, Mulgrave, Australia) supplemented with

StemPro neural supplement (Gibco, Life Technologies, Mulgrave, Australia), 20 μg/mL basic fibroblast growth factor, 20 μg/mL human epidermal growth factor, 200 mM

GlutaMAX (Gibco, Life Technologies, Mulgrave, Australia) and 1% penicillin and streptomycin.

Cell preparation for irradiation

Single cell suspensions were produced one to two hours prior to irradiation by incubating neurospheres with 2-5 mL of StemPro Accutase dissociation reagent (Gibco,

Life Technologies, Mulgrave, Australia) in a water-bath at 37°C for 20 minutes. Single cells were then washed in Dulbecco’s Phosphate Buffered Saline (DPBS) (Gibco, Life

Technologies, Mulgrave, Australia), counted using the Countess automated cell counter

(Invitrogen, Life Technologies, Mulgrave, Australia) and resuspended in complete culture medium as described above. Single cells in suspension were then transferred into 2mL eppendorf microtubes in triplicate per dose/modality. Samples were kept on ice during transportation to radiation facilities and following irradiation to minimise temperature interference with DNA repair and cell cycle processing.

MRT irradiations

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Cells were exposed to synchrotron MRT at the Imaging and Medical Beamline

(IMBL) of the Australian Synchrotron (Clayton, Australia). A superconducting multi- pole wiggler with a peak magnetic field strength of 3 Tesla produced the raw (or white) synchrotron x-ray beam, which was filtered through in vacuo graphite and copper absorbers to produce a polychromatic x-ray beam with a mean energy of 94 keV (28,

29). A tungsten-carbide multi-slit collimator was used to segment the beam into an array of vertical microbeams with a width of 50 μm and a centre-to-centre spacing of

400 μm. Tubes containing the suspended cells were positioned on a perspex jig fixed to a sample stage that was driven vertically through the array of microbeams. The in-beam

MRT doses used were 112 Gy, 250 Gy, 560 Gy and 1180 Gy at the centre of the eppendorf tubes (5mm depth) with the dose rate approximately 380 Gy/s. GEANT4

Monte Carlo simulations were performed to estimate the peak to valley dose ratio

(PVDR) and the corresponding valley doses. Gafchromic HD-V2 radiochromic film

(Ashland, Covington, Kentucky, USA) was attached to the perspex jig distally to the tubes and used to verify the delivered MRT doses.

A spatially alternating peak-valley dose distribution was maintained at all MRT doses. The PVDR was estimated to be 18 at the centre of the 2mL centrifuge tubes

(Supplementary Figure 3.1), meaning that peak doses of 112 Gy, 250 Gy, 560 Gy and

1180 Gy had corresponding valley doses of 6.2 Gy, 13.9 Gy, 31.1 Gy and 65.6 Gy, respectively. While the peak dose was attenuated by 12.5% at 10mm depth, the valley dose increased by 56% due to forward and lateral scattering dose build-up. In MRT, the peak dose attenuates in an exponential-like fashion, much like a broad beam of kilovoltage x-rays. However, the valley dose builds up over a distance of approximately 10-15 mm before attenuating, as previously described (30, 31). A

68 kilovoltage energy spectrum is necessary to maintain the unique peak-valley geometry that is characteristic of MRT.

CRT irradiations

CRT was performed at the Walter and Eliza Hall Institute (Melbourne,

Australia) using a Co60 teletherapy unit with a dose rate of 0.03 Gy/s. Tubes containing the suspended cells were mounted on a perspex jig at a distance of 50 cm from the radiation source and received a dose of 2 Gy, 4 Gy, 6 Gy or 8 Gy for the clonogenic assay and 3 Gy, 6 Gy and 9 Gy for the flow cytometry based assays.

Clonogenic assays

DIPG cells grow as spheres in suspension rather than colonies as traditionally seen in clonogenic assays. Therefore, a modified clonogenic assay (23, 32) was performed for each cell line after both CRT and MRT with sphere formation used as a substitute for clonal survival. Between one and two hours following irradiation, single cells were transferred from the irradiated tubes to a six-well plate at a seeding density of

1 x 104 cells per well (non-irradiated controls, 2 and 4 Gy CRT, 112 and 250 Gy MRT) or 3 x 104 cells per well (560 and 1180 Gy MRT, 6 and 8 Gy CRT). Seeding densities were increased at the highest CRT and MRT doses to account for higher levels of cell death following irradiation. Complete culture medium as described above was added to the 6-well plates and cells were incubated at 37°C and 5% CO2. Fourteen days after irradiation, neurospheres in each well were counted manually using an inverted microscope and normalised against the number of spheres in non-irradiated control wells. Only spheres with a diameter greater than 50 μm were counted, as determined using a DP 200TM digital camera with InSight software (DeltaPix, Smorum, Denmark).

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The number of neurospheres was used to generate semi-log form clonogenic survival curves of survival versus dose. GraphPad Prism (version 7.0) software

(GraphPad Software Inc, San Diego, California, USA) was used to fit the linear- quadratic model (S = e-(αD+βD2)) which describes the surviving fraction (S) with increasing dose (D) (33), to the CRT data using non-linear regression. The linear quadratic model was fit only to the CRT data, for the purpose of interpolating equivalent CRT doses for each MRT dose and comparing the radio-sensitivities of the two cell lines. The alpha (α) and beta (β) coefficients, which represent the initial slope and subsequent curvature of the cell-survival curve, respectively, were used to calculate the alpha/beta (α/β) ratio for each cell line.

Apoptosis and cell cycle assays

Following irradiation, single cells were transferred into T25 flasks and cultured in complete media. As the doubling time of the SF7761 cell lines is approximately 48 hours (22), we selected a time point of five days post irradiation for analysis to allow at least two full cell cycles to occur. At this time, neurospheres were dissociated into single cells using StemPro Accutase and washed twice with DPBS before being split into two fractions. A number of limitations are associated with performing assays using single cell suspensions (34), including the need to disrupt normal cell function by the necessary dissociation of cells. In our study, this limitation was minimised by the short period of time between cell dissociation and fixation.

The first fraction was incubated with Annexin V (AnV) and Propidium Iodide

(PI) (Molecular ProbesTM, Life Technologies, Mulgrave, Australia) according to the manufacturer’s instructions and analysed for apoptosis induction by flow cytometry using the BD Canto Flowcytometer (BD Biosciences, San Jose, CA). Cells were

70 classified as necrotic (PI positive), apoptotic (PI and AnV positive) or non-apoptotic (PI and AnV negative). The fold change in total apoptotic cells between irradiated and non- irradiated controls groups was analysed. The necrotic population was not analysed.

The second fraction was fixed and permeabilised in 70% cold ethanol and stored at -20°C for a minimum of 24 hours. Cells were then washed in DPBS and incubated in

500 µL of Propidium Iodide/RNAse solution (Molecular ProbesTM, Life Technologies,

Mulgrave, Australia) for 30 minutes. Samples were analysed using the BD Canto

Flowcytometer to characterise the DNA content of cells. Cells were characterised as

Sub G0, G0/G1 or G2/M. The fold change for each cell cycle phase in irradiated compared to non-irradiated control groups was analysed for each cell line and irradiation modality.

Statistics

All data are presented as mean ± SEM, unless otherwise stated. Statistical analysis was performed using an independent Student’s t-test or Mann Whitney U test, with p < 0.05 considered statistically significant. All data interpolations and statistical analyses were performed using GraphPad Prism version 7.0 (GraphPad Software Inc,

San Diego, California, USA). Assays were performed in triplicate and repeated in at least three independent experiments.

Results

Clonogenic assays

Fourteen days following MRT, DIPG sphere formation was lower at all doses for SF7761 compared to JHH, with a significantly lower surviving fraction at 112 Gy (P

= 0.02) (Figure 3.1a). Following CRT, the surviving fraction was also lower for SF7761

71 than JHH at all experimental doses, with significance at 2 Gy (P = 0.03) and 4 Gy (P =

0.02) (Figure 3.1b). The linear quadratic model was fitted to the CRT data (Figure 3.1b) for SF7761 (R2 = 0.95) and

JHH (R2 = 0.95). The best-fit values for the α and β coefficients, with 95% confidence intervals, were 0.36 [0.22 - 0.51] and 0.013 [0.0075 - 0.0034] for SF7761 compared to

0.16 [0.084 - 0.24] and 0.019 [0.0075 - 0.031] for JHH. The α/β ratio for the SF7761 and JHH cell lines were 27.5 Gy and 8.3 Gy, respectively. Based on these survival curves and α/β ratios, the SF7761 cell line was intrinsically more radio-sensitive to both

MRT and CRT compared to the JHH cell line.

The interpolated equivalent CRT dose for each MRT dose, as predicted by the linear-quadratic model based on clonogenic survival 14 days after irradiation, is summarised in Table 3.1. The equivalent MRT and CRT doses for each cell line were not significantly different. For both cell lines, the surviving fraction at 1180 Gy was too low to interpolate equivalent CRT doses from the experimental data.

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Table 3.1. Interpolated equivalent CRT doses for increasing MRT doses

Equivalent CRT doses (Gy)a

MRT Peak

Dose SF7761 JHH P Value

112 Gy 2.5 ± 0.4 2.1 ± 0.4 0.5

250 Gy 4.7 ± 0.7 5.1 ± 0.7 0.7

560 Gy 8.0 ± 0.2 8.8 ± 0.5 0.2

a Interpolated from linear quadratic dose-response curves fitted to the clonogenic survival data. Data are presented as mean ± SEM, n = 3 (JHH), n = 4 (SF7761).

Apoptosis

MRT

There was a dose-dependent increase in apoptotic cells for both SF7761 and

JHH in response to MRT (Table 3.2). Five days following irradiation with a peak dose of 560 Gy, apoptotic cells increased 5.4 ± 0.7 fold for SF7761 and 2.9 ± 0.5 fold for

JHH when compared to non-irradiated controls, demonstrating that both cell lines were sensitive to MRT. The increase in apoptosis following irradiation was significantly greater for SF7761 than JHH cells at all three MRT doses (Figure 3.2a) indicating that

SF7761 cells were more sensitive to MRT than JHH, which was in agreement with the day fourteen clonogenic assay data.

CRT

Both cell lines exhibited increased apoptosis in response to CRT in a dose- dependent manner (Table 3.2). Following 9 Gy CRT, apoptotic cells increased 4.2 ± 0.9

73 fold for SF7761 and 3.3 ± 0.8 fold for JHH. Similar to the MRT data, there was a greater fold increase in apoptotic cells for SF7761 compared to JHH at all CRT doses, however, these differences were not significant (Figure 3.2b).

Table 3.2. Percentage of apoptotic cells and fold change in irradiated compared to control groups SF7761 JHH Apoptotic Fold P Value Apoptotic Fold P Value Cells (%) Change Cells (%) Change MRT

Control 12.0 ± 1.3 - - 13.5 ± 2.5 - -

112 Gy 37.2 ± 4.9 3.2 ± 0.4 0.0005* 18.6 ± 3.7 1.4 ± 0.06 0.3

250 Gy 53.6 ± 5.7 4.6 ± 0.6 0.0003* 26.1 ± 4.1 2.0 ± 0.3 0.04*

560 Gy 63.3 ± 6.5 5.4 ± 0.7 0.0002* 38.3 ± 7.4 2.9 ± 0.5 0.02*

CRT

Control 12.4 ± 0.4 - - 11.7 ± 1.0 - -

3 Gy 31.0 ± 5.9 2.5 ± 0.5 0.03* 13.9 ± 2.2 1.2 ± 0.1 0.4

6 Gy 41.1 ± 6.8 3.3 ± 0.6 0.01* 24.3 ± 4.7 2.1 ± 0.5 0.06

9 Gy 51.9 ± 9.8 4.2 ± 0.9 0.02* 37.6 ± 7.6 3.3 ± 0.8 0.03*

Data are presented as mean ± SEM, n = 4 (MRT), n = 3 (CRT)

*Statistically significant changes in irradiated compared to control groups

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Cell cycle distribution

MRT

MRT induced a dose-dependent increase in Sub G0 phase, decrease in G0/G1 and decrease in G2/M in SF7761 cells (Figure 3.3a-c). At 560 Gy, the Sub G0 population increased 6.6 ± 0.7 fold and the G0/G1 and G2/M populations decreased by a factor of 0.6 ± 0.1 and 0.3 ± 0.1, respectively, compared to non-irradiated controls.

For JHH, there was a dose-dependent increase in Sub G0 cells and decrease in G0/G1 cells which was associated with an increase in G2/M phase accumulation (Figure 3.3a- c). At 560 Gy, the Sub G0 and G2/M populations increased 4.2 ± 0.6 fold and 1.4 ± 0.2 fold, respectively, while G0/G1 decreased by a factor of 0.4 ± 0.03 compared to non- irradiated controls.

CRT

Following 9 Gy irradiation of the SF7761 cell line, the Sub G0 population increased 5.3 ± 1.1 fold while the G0/G1 and G2/M populations decreased by a factor of

0.8 ± 0.1 and 0.3 ± 0.04, respectively, compared to the non-irradiated controls (Figure

3.3d-f). For JHH, there was a dose-dependent increase in both the Sub G0 and G2/M population of cells, while the G0/G1 populations decreased relative to the non-irradiated controls. 9 Gy CRT increased the Sub G0 and G2/M populations 4.8 ± 1.7 fold and 1.2

± 0.2 fold, respectively, while the G0/G1 population decreased by a factor of 0.4 ± 0.1

(Figure 3.3d-f).

SF7761 versus JHH

The cellular responses of the two DIPG cell lines to irradiation were distinctly different. The induction of Sub G0 cells was greater in SF7761 compared to JHH following MRT and CRT (Figure 3.3a and 3.3d) which supported the apoptosis data

75

(Figure 3.2a and 3.2b). Furthermore, changes in the G2/M population were significantly different following all MRT and CRT doses (Figure 3.3c and 3.3f), with an increase for

JHH but a marked decreased for the SF7761 cell line. Following both MRT and CRT, polyploidy was evident for JHH, but not SF7761 (Figure 3.4a and 3.4b), suggesting that while both radiation modalities were acutely detrimental to the viability of JHH cells

(increased Sub G0), a sub-set of cells were still able to continue through the cell cycle and replicate their DNA but unable to divide, resulting in an accumulation of cells in the

G2/M phase. Polyploidy was not observed in SF7761 cells, which instead showed a reduction in G0/G1 and G2/M populations following both MRT and CRT. Together these findings indicate that each cell line has a different response to radiation, with

SF7761 more radio-sensitive than JHH regardless of the radiation modality. There were no significant differences in the biological effect of increasing doses of MRT compared to CRT for either of the two cell lines.

Discussion

This is the first study to compare the radio-sensitivity of different DIPG cell lines using both MRT and CRT. Assays for apoptosis and clonogenic cell survival showed that the JHH cell line was more resistant to both MRT and CRT when compared to the SF7761 cell line. Cell cycle analysis also demonstrated increased G2/M phase accumulation for JHH following irradiation which has previously been linked with radio-resistance in meningioma and glioma cell lines (35, 36). Importantly, we found that the JHH cell line, but not SF7761, formed polyploid cells following both

MRT and CRT.

76

MRT and CRT have been previously shown to regulate genes and molecular pathways differently (37, 38). In a study on the response of the EMT6.5 mouse mammary tumour cell line to MRT and CRT, only 19% of up-regulated pathways and

52% of down-regulated pathways were common between MRT and CRT within 24 hours of treatment (38). These differentially regulated pathways included biological processes such as apoptosis, which were differentially up-regulated by MRT, and mitotic cell checkpoints, which were differentially down-regulated by CRT (38).

However, we found that DIPG cell lines responded similarly to both MRT and

CRT, with no significant differences in the change of apoptotic cells or cell cycle distribution following increasing doses of both modalities. The lack of differential response to MRT versus CRT in this study could be accounted for by the intrinsic differences between DIPG and mammary tumour cell lines. Also, we assessed apoptosis and the cell cycle five days following irradiation (due to the slow doubling time of the

DIPG cells), compared to the mammary tumour gene arrays which were performed four to 48 hours post-irradiation (37, 38).

Previously, Ibahim and colleagues (24) calculated equivalent MRT and CRT doses based upon clonogenic survival and real-time cell impedance, which provides an integrated measure of cell attachment, proliferation and metabolism. However, the equivalent CRT doses for 112 to 560 Gy MRT calculated by Ibahim and colleagues were higher on average compared to this study by approximately 2 Gy to 4 Gy (24).

These discrepancies could be accounted for by differences in the MRT collimator configuration, which determines microbeam width and centre-to-centre spacing. The centre-to-centre spacing used in this work was 400 μm, while Ibahim and colleagues used 200 μm spacing. Reducing the microbeam spacing by half effectively doubles the number of microbeams within a given field. The resultant doubling in energy deposition

77 could account for the higher equivalent CRT doses calculated by Ibahim and colleagues

(24). Differences in MRT collimator configurations could be clinically significant, with pre-clinical animal models demonstrating that microbeam spacing of at least 200 μm, leads to better normal tissue sparing in animal models but at the expense of tumour control (14, 15). Accordingly, new models for normal tissue toxicity and tumour control for MRT should also consider different collimator configurations, particularly in regard to microbeam spacing (13).

While the DIPG cell lines showed a similar response to both MRT and CRT, there was a distinct differential response between the JHH and SF7761 cell lines. It is likely that these are due to genetic heterogeneity between the two cell lines. Cell cycle arrest is normally mediated at the G1 phase by p53 and p21 (CDKN1A) in order to repair DNA damage caused by genotoxic insults such as radiation. The SF7761 cell line expresses wild type p53 and p21 (22), and following irradiation exhibited a significant arrest in the G1 phase and the induction of apoptosis. The SF7761 cells were significantly radio-sensitive, and this may have been partially due to p53 and p21 mediated checkpoint activity within the G1 phase. Further evaluation of the checkpoint regulators during each phase of the cell cycle following irradiation will be required in the future.

In contrast to this, the JHH cells were previously exposed to several genotoxic agents including temozolomide, irinotecan, bevacizumab and CRT and exhibited a somatic missense mutation within p53 (C277F) (21). It is unclear whether the JHH cell line endogenously expressed this mutation or acquired it through previous genotoxic exposure. The p53C277F mutation reduces the tumour suppressive activity of p53 by inhibiting sequence specific DNA binding to response elements such as GADD45 through steric hindrance and reduced p53 contact with DNA (39), and as a consequence,

78 has been shown to increase radio-resistance in SaO-2 osteosarcoma cells (40). From our data, irradiated JHH cells appeared to move through the G1 phase and were arrested in the G2/M phase, as demonstrated by an increase in the G2/M population following both MRT and CRT. This may have then resulted in prolonged mitosis and/or mitotic slippage where DNA is re-duplicated and accumulated (41). Continued mitotic activity in the absence of cellular division may explain why polyploidy was observed, as has been reported in the literature (42, 43).

Although polyploid cells have previously been characterised as reproductively dead, there is evidence that a small fraction of these cells can escape death and retain clonogenic potential (42-44). Cellular senescence is also a potential product of DNA damage and has been shown to be reversible (44, 45). Puig and colleagues report the slow depolyploidisation and recovery of clonogenicity in a small fraction (10-5 to 10-4) of cells from a colon carcinoma cell line after chemotherapeutic treatment with cisplatin

(42). A similar process may explain why clonogenic survival at 14 days was higher for

JHH compared SF7761 at all MRT and CRT doses (Figure 3.5) and will require further investigations for confirmation. Both reversible senescence and reversible polyploidy have been proposed as potential mechanisms of therapeutic resistance and the progression or recurrence of tumours after therapy (42, 43, 45). Further to this, polyploidy may enable swift tumour evolution and the acquisition of mutations that further enhance resistance to other anti-cancer therapies (46).

It is unlikely that the JHH cells were undergoing delayed apoptosis following mitotic catastrophe, given that the clonogenic assays were performed 14 days following irradiation and that delayed apoptotic pathways are usually activated by mitotic catastrophe within six days of irradiation (47).

79

While the formation of polyploid cells could be linked to radio-resistance in the

JHH cell line, the abnormally high amount of genetic material and the associated increase in metabolic activity could be exploited as an alternative therapeutic target

(46). Despite the typically low proportion of polyploidy cells in a given tumour population, progress toward making polyploidy a therapeutic target has been made.

Strategies include the inhibition of Chk1 (48), targeting mitotic kinesin Eg5 (49), the induction of cell-specific autophagy (50) and targeting elevated levels of reactive oxygen species with antioxidants (51). Profiling DIPG cell lines for the ability to produce polyploid cells could be useful in determining an appropriate course of treatment, especially given the increased use of re-irradiation with CRT for local tumour recurrences (52). Genomic profiling has now characterised distinct molecular subgroups of DIPG (53), whilst new screening methods that quantify the prevalence of polyploid cells are in development (46). For cells that have not developed radio-resistance, such as

SF7761, MRT could provide an alternative radio-therapeutic treatment that is more damaging to tumour cells and less damaging to normal tissue.

In summary, this is the first study to characterise the biological response of

DIPG cell lines to MRT and CRT. The JHH cell line, was intrinsically more radio- resistant to both irradiation modalities compared to SF7761 and demonstrated the ability to develop polyploid cells in response to irradiation. Whilst patients with radio- sensitive tumours may benefit from MRT, the inclusion of adjuvant therapies targeting altered gene signatures and cell functions such as polyploidy may be required to improve the efficacy of radiotherapy in patients with a radio-resistant tumour in the future.

80

Acknowledgements

LMLS is supported by an Australian Government Research Training Project

Scholarship. JCC was previously supported by an Early Career Research Fellowship from the National Health and Medical Research Council (NHMRC) of Australia

[1036174]. This project was supported by grants from the Robert Connor Dawes

Foundation, the Isabella and Marcus Foundation and the NHMRC [Project Grant

1061772]. The authors wish to thank Jayde Livingstone, Andrew Stevenson and the team at the IMBL for their assistance in irradiations and dosimetry at the Australian

Synchrotron, Liam Day for performing Monte Carlo simulations of MRT dosimetry,

Premila Paiva for assistance with dose equivalence calculations and Sandy Fung at the

Monash Institute of Pharmaceutical Sciences Flow Cytometry Facility.

81

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FIGURES

AB

Figure 3.1. Clonogenic survival of DIPG cells following MRT and CRT. The total number of spheres formed from single cells following irradiation was used as a surrogate to assess clonal survival. SF7761 cells were more radio-sensitive than JHH cells to both MRT (A) and CRT (B) with significantly less sphere formation at 112 Gy

MRT and 2 Gy and 4 Gy CRT. The linear quadratic model was fitted to the CRT data using non-linear regression (B), with R2 values of 0.95 for both SF7761 and JHH cell lines indicating that the model was adequately fitted to the data. Data are presented as mean ± SEM, n = 3, *P < 0.05.

89

A B

Figure 3.2. Dose-dependent increase in apoptotic DIPG cells following MRT and CRT.

For SF7761, the fold change in apoptotic cells was higher compared to JHH at all MRT

(C) and CRT (D) doses, with significant differences following MRT. Data are shown as

mean ± SEM, n = 4 (MRT), n = 3 (CRT), *P < 0.05, **P < 0.01.

90

A B C

D E F

Figure 3.3. Differential cell cycle response between JHH and SF7761 cell lines

following MRT and CRT. SF7761 and JHH cells exhibited an increase in Sub G0

(apoptotic) cells following irradiation, with a significantly greater fold change for

SF7761 compared to JHH following MRT (A) but not CRT (D). There was a dose-

dependent decrease in G0/G1 phase cells for both cell lines following MRT (B) and

CRT (E) with no significant difference in the fold change between JHH and SF7761

cells. There was also a dose-dependent decrease in G2/M phase cells for SF7761

following MRT (C) and CRT (F), while JHH demonstrated a dose-dependent increase

in G2/M phase cells. Data are shown as mean ± SEM, n = 3, *P < 0.05, **P < 0.01,

***P < 0.001.

91

A

B

Figure 3.4. Differential development of polyploidy following irradiation. Flow cytometry plots (A) show a tail beyond the 100K region for JHH which represents cells

92 with greater than the normal diploid DNA content. This polyploidy population was only evident in JHH cells following MRT and CRT. This was confirmed morphologically

(B). Cells were stained with the cell surface marker Phalloidin, DNA damage marker gH2AX and the nuclear marker DAPI, with JHH showing multi-nucleated single cells

(white arrows) while SF7761 showed an aggregate of cells with single nuclei.

93

Figure 3.5. Proposed mechanism of radio-resistance development in DIPG cells. The differential response of SF7761 and JHH cells to radiation-induced damage is proposed to occur as a result of impaired p53 function and the development of polyploidy cells following G2/M arrest in JHH cells. Further work will be required to confirm the functional role of the p53C277F mutation in the development of radio-resistance and polyploidy.

94

SUPPLEMENTARY FIGURES

Supplementary Figure 3.1. PVDR versus depth based on Monte Carlo simulations of a

140 mm x 30 mm field of microbeams with a width of 50 μm and a centre-to-centre spacing of 400 μm.

95

3.3 Extended methods for flow-cytometry assays

Preparation of irradiated cells

1. Five days post-irradiation, cells were taken out of culture and transferred to 15 mL

tubes for centrifugation at 1100 revolutions per minute (rpm) for 5 minutes.

2. The supernatant was decanted and discarded. The remaining pellet was washed and

resuspended in 2mL of warm Dulbecco’s Phosphate Buffered Saline (DPBS)

(Gibco, Life Technologies, Mulgrave, Australia) for centrifugation at 1100 rpm for

5 minutes.

3. The supernatant was decanted and discarded and the cell pellet was resuspended in 1

mL of warm (37°C) StemPro Accutase dissociation reagent (Gibco, Life

Technologies, Mulgrave, Australia). The cell suspension was left to incubate at

37°C for 15 to 20 minutes, or until single cells were formed. An additional 1 mL of

Accutase was added if clumps of cells were still present following the initial

incubation period.

4. The single cell suspension was centrifuged at 1100 rpm for 5 minutes. The

supernatant was decanted and discarded.

5. Single cells were washed and resuspended in 2 mL of warm DPBS and split into

two 1 mL fractions.

Cell-cycle assay – fixed cells

1. Single cells underwent centrifugation at 1100 rpm for 5 minutes. The supernatant

was decanted and discarded.

2. The remaining pellet of single cells was disrupted and resuspended in the residual

DPBS left in the tube.

96

3. 1 mL of cold 70% ethanol was added to fix the single cells by slowing pipetting the

ethanol down the sides of the tube. The tube was gently shaken to mix the single cell

suspension with the ethanol.

4. Cells were placed in a freezer at -20°C for fixation for at least 24 hours.

5. Cells were thawed and underwent centrifugation at 1100 rpm for 5 minutes.

6. The supernatant was decanted and discarded. The pellet was washed and

resuspended in 5 mL of cold DPBS for centrifugation at 1100 rpm for 5 minutes.

7. The supernatant was decanted and discarded.

8. 0.5 mL of FxCycleTM Propidium Iodide (PI)/RNase staining solution (Molecular

ProbesTM, Life Technologies, Mulgrave, Australia) was added to each cell pellet and

mixed well via pipetting.

9. Samples were incubated for at least 30 minutes at room temperature and kept

protected from light.

10. Samples were analysed with a BD Canto Flowcytometer (BD Biosciences, San Jose,

CA) using a 488 nm blue laser. Maximum excitation and emission wavelengths

were 535 nm and 617 nm, respectively. Emission was collected using a 670 nm

long-pass filter. A total of 3000 to 10000 events were recorded per sample.

11. The fluorescence profile of PI/Rnase indicated the DNA content of cells, and

allowed cells to be grouped by cell-phase; Sub G0, G0/G1, or G2/M.

Apoptosis assay – live cells

1. Three working solutions were prepared from the Fluorescein (FITC) Annexin

V/Dead cell Apoptosis Kit (Molecular ProbesTM, Life Technologies, Mulgrave,

Australia):

97

a. 1X Annexin binding buffer: 5X Annexin binding buffer from the kit was

diluted in four volumes of deionised water.

b. Propidium iodide (PI) working solution: 1 mg/mL PI stock solution was

diluted in 9 volumes of the 1X Annexin binding buffer from 1a), for a total

PI concentration of 100 µg/mL

c. Cell stain solution: one volume of PI working solution (1b) added to 5

volumes of FITC Annexin V stain and 100 volumes of 1X Annexin V

binding buffer (1a)

2. Three non-irradiated control samples were prepared:

a. Annexin V only: 5 µL of FITC Annexin V stain

b. PI only: 1.5 µL of PI working solution (1b)

c. Cells only: 350 µL of 1X Annexin binding buffer (1a)

3. Samples were stained with 100 µL of the cell stain solution (c), with the exception

of the cells-only control, and incubated at room temperature and away from light for

at least 15 minutes.

4. 350 µL of the 1X Annexin V binding buffer (1a) was added to all the samples and

controls (with the exception of the cells only control).

5. Samples were placed on ice and analysed with a BD Canto Flowcytometer (BD

Biosciences, San Jose, CA) using the 488 nm blue laser. The maximum excitation

and emission wavelengths for FITC-Annexin V conjugates were 490 nm and 525

nm, respectively, and emission was collected using a 530/30 nm band-pass filter.

Emission for PI was collected using a 670 nm long-pass filter, as per the cell cycle

assay. A total of 5000 to 10000 events were recorded per sample. Based on the level

of fluorescence, cells were grouped as necrotic (PI positive), apoptotic (PI and

Annexin V positive) or non-apoptotic (PI and Annexin V negative).

98

99

CHAPTER 4 In vivo dose-equivalence between synchrotron and conventional radiation therapy based on murine normal tissue toxicity

4.1 Preface

This chapter evaluates dose-equivalence between MRT, SBBR and CRT (Aim

1) based on a systematic characterisation of normal tissue toxicity across of a range of organs (Aim 2). Here, a paper published in the journal Scientific Reports (Smyth et al.

2018b) is reproduced, with permission from Springer Nature Limited. This chapter contains its own bibliography and reference style which is separate from the rest of the thesis. The figures appear at the end of the chapter and have been renumbered for the purpose of this thesis. A supplementary methods and data section, which is available online with the published manuscript, is also included in this chapter. The open-access, final published version of this article is available online at https://doi.org/10.1038/s41598-018-30543-1 and is attached as Appendix B. I am the primary author of this paper and was involved in planning the experiments and carrying out all aspects of the experimental work. I wrote the approved animal ethics protocol and was responsible for the post-irradiation animal monitoring. I analysed and interpreted the data, and drafted the manuscript, with input from all co-authors.

100

4.2 Published manuscript

Comparative toxicity of synchrotron and conventional

radiation therapy based on total and partial body irradiation

in a murine model

Lloyd M. L. Smyth1, Jacqueline F. Donoghue1,2, Jessica A. Ventura1, Jayde Livingstone3, Tracy Bailey4, Liam R. J. Day2, Jeffrey C. Crosbie2,5 and Peter A. W. Rogers1

1Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women’s Hospital, Parkville, Victoria 3052, Australia

2School of Science, RMIT University, Melbourne, Victoria 3001, Australia;

3Imaging and Medical Beamline, Australian Synchrotron, Clayton, Victoria 3168, Australia

4Australian Radiation Protection and Nuclear Safety Agency, Yallambie, Victoria 3085, Australia

5William Buckland Radiotherapy Centre, Alfred Hospital, Melbourne, Victoria 3004, Australia.

Jeffrey C. Crosbie and Peter A. W. Rogers contributed equally to this work.

Correspondence and requests for material should be addressed to P.R. (email: [email protected])

101

Abstract

Synchrotron radiation can facilitate novel radiation therapy modalities such as microbeam radiation therapy (MRT) and high dose-rate synchrotron broad-beam radiation therapy (SBBR). Both of these modalities have unique physical properties that could be exploited for an improved therapeutic effect. While pre-clinical studies report promising normal tissue sparing phenomena, systematic toxicity data are still required.

Our objective was to characterise the toxicity of SBBR and MRT and to calculate equivalent doses of conventional radiation therapy (CRT). A dose-escalation study was performed on C57BLJ/6 mice using total body and partial body irradiations. Dose- response curves and TD50 values were subsequently calculated using PROBIT analysis.

For SBBR at dose-rates of 37 to 41 Gy/s, we found no evidence of a normal tissue sparing effect relative to CRT. Our findings also show that the MRT valley dose, rather than the peak dose, best correlates with CRT doses for acute toxicity. Importantly, longer-term weight tracking of irradiated animals revealed more pronounced growth impairment following MRT compared to both SBBR and CRT. Overall, this study provides the first in vivo dose-equivalence data between MRT, SBBR and CRT and presents systematic toxicity data for a range of organs that can be used as a reference point for future pre-clinical work.

Introduction

Advances in clinical radiation oncology over the past few decades have revolved around improving the conformity of dose-distributions to the tumour or modifying fractionation regimens to maximise the therapeutic ratio. More recently, the use of experimental radiation sources has led to novel radiobiological findings with potential

102 clinical applications. Examples of this include remarkable normal-tissue sparing of the lung1 and brain2 following ultra-high dose-rate radiation therapy, known as a ‘FLASH’ effect, and the spinal cord3 and brain4,5 when using micron scale spatially fractionated fields (microbeam radiation therapy; MRT). Given the demonstrated tumouricidal potential of these modalities1,6,7, their novel radiobiology could potentially be exploited for therapeutic benefit.

Radiation generated by a synchrotron source has the physical characteristics necessary to facilitate MRT and the potential to produce a FLASH normal tissue sparing effect using high dose-rate synchrotron broad-beam radiation therapy (SBBR). Both

MRT and SBBR are being developed for future clinical use at the Imaging and Medical

Beamline (IMBL) of the Australian Synchrotron. MRT is characterised by arrays of quasi-parallel micro-planar beams with a width of 25 to 100 µm that are typically spaced by 100 to 400 µm8. This arrangement allows for the delivery of in-beam (peak) doses that are at least an order of magnitude greater than the doses delivered between the beams (valley) due to scatter. In-beam dose-rates can exceed several hundred Gy/s.

MRT exerts a differential effect on normal versus tumour tissue in regard to gene pathway modulation9, post-irradiation tissue repair10 and vascular architecture11. The mechanism behind the FLASH normal tissue sparing effect is yet to be determined, however, hypotheses include the differential activation of DNA damage pathways1 and the induction of transient hypoxia1,12. The oxygen depletion hypothesis is supported by in vitro data13 and in vivo experiments where transient radio-resistance was induced in mouse tails at high dose-rates14.

While there are a significant number of pre-clinical animal studies reporting on tissue responses to MRT, there is a lack of systematic dose-escalation data across a broad range of organs15. Currently, there is no published toxicity data for total body,

103 abdominal or thoracic irradiation using MRT. Robust toxicity data for SBBR is similarly lacking. These are significant issues that must be addressed prior to a clinical trial.

Through a systematic dose-escalation study of conventional radiation therapy

(CRT) versus MRT and SBBR using C56BLJ/6 mice, we provide the first in vivo report of dose-equivalence between these modalities. Dose-response curves for normal tissue toxicity were generated for each radiation modality following total body irradiation

(TBI) and partial body irradiation (PBI) of the whole abdomen, head and thorax. The aim of this current study was to calculate TD50 values for each modality based on acute clinical endpoints related to weight and overall wellbeing, as a means of assessing dose- equivalence. We hypothesised that compared to CRT, there would be a normal tissue sparing effect using SBBR and that for synchrotron MRT, the valley dose would be the most important determinant of toxicity.

Biological methods of determining dose-equivalence between CRT and MRT are particularly insightful given the unique challenges of physically comparing a spatially homogenous field to an intrinsically inhomogeneous one. Endpoints such as clonogenic survival16, skin histopathology17, and normal tissue toxicity (current study), provide a measure of the gross effect of an entire array of microbeams compared to a homogenous field. Toxicity endpoints were chosen in this study given the fundamental need to determine the safety profile of both MRT and SBBR compared to CRT.

Additionally, by reporting TD50 outcomes of TBI and PBI, we can compare MRT and

SBBR to decades of classical radiobiology literature. Normal tissue toxicity data is essential to the planning of future pre-clinical animal studies and ultimately, for the selection of safe dose regimens in future clinical trials using MRT and/or SBBR15.

Results

104

Total body irradiation

Mice in the two highest SBBR and CRT dose groups and the highest MRT dose group displayed signs of acute radiation syndrome, losing at least 15 to 20% body- weight within five to nine days following TBI. Several other animals in these groups showed delayed weight loss (at least 15 to 20%) and moribund behaviour, including lack of grooming, hunched posture and low activity levels between eleven and eighteen days following irradiation. The TD50 doses (Fig. 4.1A, Table 4.1) for these toxicities in the SBBR and CRT groups were 6.7 Gy (3.9 – 8.4 Gy) and 6.9 Gy, respectively. The

MRT valley and peak TD50 doses were 3.8 (2.7 – 10.8) and 120 (84.3 – 343), respectively.

Surviving mice from all three irradiation modalities had sub-normal weights compared to control mice (Fig. 4.2A). All groups returned to their pre-experimental weight within 60 days of treatment, except the 133 Gy MRT group. These mice had a mean change in body weight of -5.6 ± 1.5% compared to their pre-experimental

(baseline) weight (Fig. 4.2A).

Abdominal PBI

Mice receiving the two highest MRT and SBBR doses and the highest CRT dose displayed acute signs of gastro-intestinal syndrome, losing at least 15 to 20% body- weight and showing signs of severe diarrhoea, within five to six days of irradiation. The

MRT peak and valley TD50 doses were 257 Gy and 7.7 Gy, and the SBBR and CRT

TD50 doses were 11.3 Gy (7.6 – 14.5 Gy) and 12.7 Gy (8.7 – 17.3 Gy), respectively

(Fig. 4.1B, Table 4.1). At necropsy, affected mice showed severe signs of dehydration and intestinal water retention, reflected histologically by the destruction of normal crypt-villus architecture and denudation of villi (Fig. 4.3B). No mice in the 166 Gy

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MRT group, 7.4 Gy CRT group or 8.3 and 5.5 Gy SBBR groups showed signs of acute gastrointestinal syndrome.

Surviving mice had sub-normal weights compared to control mice, regardless of the radiation modality used (Fig. 4.2B). All groups returned to their pre-experimental weight within 60 days of treatment except the 249 Gy MRT group, which had a mean change in body weight of -0.9 ± 0.9%. The gross crypt-villus architecture of surviving mice following irradiation was relatively normal, except for mice in the 249 Gy MRT group. The villi of these mice showed lifting of epithelial cells from the lamina propria layer and significant extension of the sub-epithelial space (Fig. 4.3A). The disruption of the epithelial-capillary interface in villi is consistent with abnormal mucosal absorption18 and could explain the subsequent growth impairment in the MRT groups.

Head PBI

Mice in the two highest SBBR and CRT dose-groups, as well as the 255 Gy, 317

Gy and 377 Gy MRT groups, showed a decline in activity levels, grooming, appetite and water intake and/or 15 to 20% weight loss between seven to twelve days following irradiation. The TD50 values for these toxicities were 12.3 Gy (8.0 – 16.4 Gy) and 13.1

Gy (9.2 – 17.2 Gy) for CRT and SBBR, respectively. The MRT TD50 doses were 268

Gy (232 – 313 Gy) and 7.2 Gy (6.2 – 8.4 Gy) for the peak and valley, respectively (Fig.

4.1C, Table 4.1).

In addition to weight loss and a moribund state, MRT caused neurological toxicities including ataxia, loss of balance and fitting within two to four hours at 455 Gy

(5/5 mice) and 377 Gy (2/5 mice). These symptoms were not evident in the SBBR and

CRT groups at the doses used in this study. Distinct and evenly spaced bands of cerebellar granular cell loss, corresponding with microbeam paths, were observed in

106 mice that experienced acute neurological toxicities following MRT (Fig. 4.4). Cerebral and brainstem histology was unremarkable in these mice and these cerebellar changes were not observed in surviving mice from any other dose/modality. However, narrower bands of cerebellar scarring were observed in some long-term MRT survivors (Fig. 4.4), consistent with previous findings19.

Surviving mice in all irradiated groups had sub-normal weight gain compared to non-irradiated controls 37 days following irradiation, with the weight of mice in the 211

Gy MRT group 3.7 ± 1.9% below baseline (Fig. 4.2C). No signs of neurological toxicity or significant weight loss were observed in the 9.8 Gy CRT group or 7.6 and 11.3 Gy

SBBR groups.

Table 4.1. Equivalent (TD50) doses for MRT, SBBR and CRT (Gy) MRT-peak MRT-valley SBBR CRT TBI 120 (84.3–343) 3.8 (2.7–10.8) 6.7 (3.9–8.4) 6.9

Abdominal 257 7.7 11.3 (7.6–14.5) 12.7 (8.7–17.3) PBI

Head PBI 268 (232–313 ) 7.2 (6.2–8.4) 13.1 (9.2–17.2) 12.3 (8.0–16.4)

26 TD50 doses calculated using PROBIT analysis . 95% confidence intervals are stated in parentheses.

Thoracic PBI

Several mice in the 587 Gy (4/5), 515 Gy (1/5) and 459 Gy (2/5) MRT groups experienced severe neurological toxicities within 2 to 4 hours of irradiation, including ataxia, loss of balance and fitting. These toxicities were unexpected and might be explained by collateral irradiation of upper spinal column during the thoracic irradiations. Further mice from the 587 Gy (1/5), 515 Gy (2/5) and 459 Gy (2/5) MRT

107 groups presented with severe clinical symptoms including hunched posture, lack of grooming and poor body condition and were sacrificed due to 20% weight loss within six weeks of irradiation. Several mice from the 27.9 Gy SBBR (3/5), 24.2 Gy CRT (1/5) and 21.2 Gy CRT (2/5) groups presented with severe in-field cutaneous lesions within five weeks of irradiation and were sacrificed.

Remaining mice steadily gained weight following irradiation, albeit having sub- normal weight gain compared to non-irradiated control mice (Fig. 4.2D). Weight gain was significantly impaired in the 515 Gy MRT group relative to controls (p < 0.05)

(Fig. 4.2D). Between 140 and 180 days after irradiation, n = 6 mice from CRT dose- groups and n = 1 mouse from the 23.3 Gy SBBR group began to show signs of cachexia. These mice were sacrificed following the loss of 20% body weight over the course of two weeks, with some mice presenting with low activity levels, laboured respiration, poor grooming and hunched posture.

The lungs of surviving mice from each radiation modality showed signs of inflammation and long-term pulmonary destruction 170 to 180 days following irradiation with the most severe damage evident at the highest doses of each modality.

For every dose and modality, histopathology revealed alveolar destruction, airspace enlargement and the thickening of alveolar walls (Fig. 4.5A). Masson’s Trichrome stained sections confirmed the presence of fibrosis, with collagen deposition in sub- pleural and intra-parenchymal regions (Fig. 4.5B). PROBIT analysis was not performed on the thoracic PBI series due to the premature sacrifice of mice from all three irradiation modalities.

Discussion

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To our knowledge, this is the first in vivo study reporting dose-equivalence between MRT, SBBR and CRT. Tissue specific TD50 values were calculated for each modality. There was no gender bias specific to the toxicities observed across the TBI and PBI experiments. Key findings of this study include: 1) no clear evidence of a normal tissue sparing effect using a SBBR dose-rate of 37 to 41 Gy/s, 2) the valley

MRT dose as the most relevant parameter for acute toxicity, and, 3) long-term detrimental effects of MRT on growth, despite the acute tolerance of irradiation.

Importantly, the TD50 values for TBI and abdominal PBI calculated for CRT in

20 our study were comparable to LD50 values previously reported by Booth et al. . These data were based upon similar levels of toxicity in the same mouse model and at a comparable dose rate of 0.01 Gy/s. Furthermore, Favaudon et al.1 report pulmonary fibrosis at doses higher than 15 Gy when using a conventional dose-rate of 0.03 Gy/s, consistent with our findings. There is no comparable data for head PBI previously reported in the literature to validate the CRT group in our study.

Our TD50 data provides no clear evidence for a normal tissue-sparing effect using SBBR, and at the doses used for thoracic PBI, SBBR was not protective against destructive pulmonary changes relative to CRT. The protective effect of FLASH radiation therapy has previously been shown to be dose-rate dependent2. Following whole brain irradiation, the memory of C57BLJ/6 mice was preserved when 10 Gy was delivered at 100 Gy/s but was significantly impaired relative to non-irradiated controls with a dose-rate of 60 Gy/s or less2. A significant decline in memory function was also observed when the dose-rate was reduced from 60 Gy/s to 30 Gy/s2. Furthermore, lung- sparing benefits of FLASH radiation therapy are reported at dose-rates of 60 Gy/s1. It is therefore possible that the SBBR dose-rates of approximately 37 to 41 Gy/s used in our study were too low to elicit a definitive normal tissue sparing effect compared to CRT.

109

Although Favaudon et al.1 and Montay-Gruel et al.2 delivered FLASH radiation therapy using electron radiation rather than photons, there was no difference in lung fibrogenesis when comparing electrons versus gamma-ray photons at a conventional dose rate1. Furthermore, given that x-rays, gamma-rays and electrons are all examples of sparsely ionising radiation and have a comparable relative biological effectiveness21, it is unlikely that radiation quality influenced the SBBR toxicity outcomes presented in our study or those reported previously1,2.

The TD50 values for CRT correlated more closely with the MRT valley dose that the MRT peak dose across the TBI and PBI experiments. This supported our hypothesis that the valley dose was the most useful parameter for comparing MRT and CRT with regards to acute normal tissue toxicity. However, the TD50 values for the MRT valley were still substantially lower than the CRT TD50 doses, suggesting that the peak and intermediate doses delivered by MRT still influence acute toxicity. Further to this, it is apparent from our head PBI series that acute neurological toxicity is possible within hours of MRT irradiation. This is most likely due to high peak doses, rather than the valley doses, given that the corresponding valley doses in the affected MRT groups were markedly lower than the SBBR and CRT doses that were tolerated without acute neurotoxicity.

Histological analysis demonstrated a marked loss of granular cells in the cerebellum of MRT-irradiated mice that experienced acute neurological toxicities. This loss of cells directly correlated with the microbeam paths. The cerebellum has been previously shown to tolerate peak MRT doses in the order of 200 Gy without significant signs of long-term neurological or developmental difficulties19, which is much lower than the highest MRT doses used in this study. To our knowledge, our results provide the first report of acute neurological symptoms within hours of MRT and suggest that

110 the cerebellum should be considered separately to the cerebrum as an organ at risk in

22 future studies. Mukumoto et al. reported an LD50 of 600 Gy for MRT (peak dose) following whole brain irradiation of C57BLJ/6 mice, compared to a TD50 of 268 Gy in this study. Aside from assessing lethality rather than toxicity, the significantly higher

22 LD50 reported by Mukumoto et al. could be explained by a number of factors including the use of narrower microbeams (25 µm versus 50 µm) and a smaller field size.

Finally, while spatial fractionation in MRT enables the delivery and acute tolerance of high doses of radiation, the long-term effects on growth could be profound.

We consistently observed greater growth impairment following MRT for TBI and PBI compared to SBBR and CRT. For abdominal PBI, this was associated with late morphological changes to the intestinal mucosa. Increasing the dose delivered per fraction is classically considered to increase the risk of late irradiation damage23. As such, hypofractionated treatment regimens (greater than 2 Gy per fraction) are used in the clinic with a high degree of selectivity. Our data suggests that similar caution should be taken when using high peak MRT doses and that the radiobiology of tumours and surrounding normal tissue should be considered when determining possible treatment sites for the future use of MRT. However, it is important to acknowledge that while the

TBI and PBI irradiations used in this study provide fundamental toxicity data, they are not representative of the conformal CRT treatments typically delivered in the clinic today which minimise the exposure of organs at risk to radiation. This limitation is important given the relationship between irradiation volume and normal tissue toxicity24,25.

There are limitations of our experimental protocol that should be noted. The dose-rates of the MRT groups were in the order of 300 Gy/s. However, it still took

111 several seconds to move the mouse vertically through the synchrotron x-ray beam for

TBI and PBI. Thus, it is possible there will be effects of both organ motion (e.g. cardiac pulsation) and gross changes in animal position during these irradiations. These movements may lead to some blurring or smearing of the peak-valley MRT dose distribution, in effect, exaggerating the physical microbeam width. In addition, the lead shields placed on the plastic mouse holder contributed to some scattered x-ray dose.

The effects of animal motion might be evident in the 455 Gy PBI head group

(Fig. 4.4), where the observed band of cerebellar granular cell damage was closer to 100

μm in width rather than 50 μm. Additionally, there is a lateral penumbra directly on either side of each microbeam, which represents regions of dose intermediate between the peak and valley dose. The penumbra could also explain why biological bands of microbeam damage appear wider than the physical microbeams.

Undoubtedly, refinements could be made to our experimental protocol in future studies, but these should not detract from the important data we report on organ toxicity, particularly for the gut and lung. Finally, a notable limitation of our TD50 data is the relatively small sample size per group for each radiation modality and dose. This restriction on group size was necessary to satisfy animal ethics requirements, given the expectation and manifestation of severe radiation-induced toxicities.

To further investigate the radiobiological differences between SBBR, MRT and

CRT at a molecular level, and to histologically quantify radiation-induced damage, future in vivo studies using the median toxicity doses calculated in this study are planned. Furthermore, these toxicity-based dose-equivalence data will be used in pre- clinical studies of tumour-bearing mice to investigate whether an improved therapeutic ratio can be achieved using MRT or SBBR compared to CRT.

112

To conclude, the observations made in this current dose-equivalence study provide an important step towards understanding the relative toxicity of SBBR and

MRT compared to CRT. We did not observe a FLASH normal tissue sparing effect at the SBBR dose-rates used in this study. We report pulmonary and gastrointestinal toxicity data for MRT for the first time and demonstrate that the MRT valley dose is a better predictor of acute toxicity than the peak dose. Importantly, we also report long- term growth impairment following MRT. The dose-response curves and toxicity data generated in this study will provide a reference point for future in vivo studies. These studies should aim to identify scenarios where the potential radiobiological advantages of SBBR and MRT can be best exploited for an enhanced therapeutic effect.

Methods

Irradiation sources

MRT was performed at the IMBL at the Australian Synchrotron using an array of vertically orientated quasi-parallel microbeams (Fig. 4.6A) with a width of 50 µm and centre-to-centre spacing of 400 µm. The in-beam dose-rate was between 276 and

319 Gy/s and the mean photon energy was 95 keV. SBBR was also performed at the

IMBL at a dose-rate between 37 and 41 Gy/s with a mean photon energy of 124 keV.

CRT was delivered using a Comet MXR-320/26 x-ray tube with Gulmay GX320 generator at the Australian Radiation Protection and Nuclear Safety Agency using a dose-rate of 0.05 to 0.06 Gy/s. The effective CRT photon energy was 93 keV.

Monte Carlo generated percentage depth-dose curves for MRT, SBBR and CRT are presented in Supplementary Fig. 4.1. The relative change in dose-deposition with depth is almost identical for each of the three modalities within the first 30 mm,

113 therefore making any dosimetric differences negligible in the context of the size of a mouse.

Dosimetry and field placement

The dose, dose-rate and field size for each type of irradiation is specified in

Table 4.2. Mice were positioned in a plastic holder, held vertically in the radiation path

(Fig. 4.6B) and irradiated in an anterior to posterior direction. For abdominal PBI, the radiation field encompassed the entire abdomen and pelvis with the superior field border at level of the xyphoid process of the sternum (Fig. 4.6B). Head PBI included the entire brain, brainstem and superior part of the cervical spinal cord (Fig. 4.6B). During thoracic PBI, both lungs and the heart were irradiated, with the inferior field border at the xyphoid process of the sternum to avoid collateral irradiation of the abdomen (Fig.

4.6B). Lead shields were used to achieve the required field borders.

All doses were prescribed to a depth of 5 mm in water. A full description of our dosimetry protocol for all three irradiation modalities is provided in a Supplementary

Methods section. In short, SBBR and CRT dosimetry is based on full-scatter reference conditions as previously described26,27,28 with Monte Carlo simulations used to adjust for the loss of backscatter due to the plastic mouse holder. The theoretical phantom used to mimic the scatter conditions of a mouse in the plastic mouse holder is shown in

Supplementary Fig. 4.2. MRT peak doses were derived from the SBBR dosimetry29 and

Monte Carlo simulations of the peak-to-valley dose ratio (PVDR) used to calculate the valley doses. The PVDR at 5 mm depth for the MRT irradiations was between 31.8

(TBI) and 41.3 (Thoracic PBI). The total uncertainty (k=1) for the irradiations are as follows; CRT – 6.1%, MRT peak – 5.1%, MRT valley – 8.6%, SBBR – 4.8%. A detailed uncertainty budget is available as Supplementary Data.

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Table 4.2. Dose groups, field sizes and dose-rates for total and partial body irradiations TBI MRT Peak (Valley) (Gy) SBBR (Gy) CRT (Gy) 30 mm x 100 mm 30 mm x 100 mm 100 mm diameter circle Dose-rate: 291 Gy/s Dose-rate: 39.1 Gy/s Dose-rate: 0.05 Gy/s 44.4 (1.4) 3.6 5.1 59.1 (1.9) 5.4 7.6 88.9 (2.8) 7.2 10.1 133 (4.2) 9.0

Abdomen PBI MRT Peak (Valley) (Gy) SBBR (Gy) CRT (Gy) 30 mm x 60 mm 30 mm x 60 mm 100 mm x 60 mm Dose-rate: 288 Gy/s Dose-rate: 38.3 Gy/s Dose-rate: 0.06 Gy/s 166 (5.0) 5.5 7.4 249 (7.5) 8.3 11.1 329 (9.9) 11.1 14.8 412 (12.4) 13.8

Head PBI MRT Peak (Valley) (Gy) SBBR (Gy) CRT (Gy) 30 mm x 30 mm 30 mm x 30 mm 100 mm x 30 mm Dose-rate: 280 Gy/s Dose-rate: 41.3 Gy/s Dose-rate: 0.06 Gy/s 147 (3.9) 7.6 9.8 178 (4.8) 11.3 14.7 211 (5.7) 15.1 19.6 255 (6.8) 18.9

Dose-rate: 319 Gy/s 317 (8.5) 377 (10.1) 455 (12.2)

Thoracic PBI MRT Peak (Valley) (Gy) SBBR (Gy) CRT (Gy)

115

30 mm x 20 mm 30 mm x 20 mm 100 mm x 20 mm Dose-rate: 276 Gy/s Dose-rate: 36.8 Gy/s Dose-rate: 0.06 Gy/s 391 (9.5) 13.9 18.1 459 (11.1) 18.6 21.2 515 (12.5) 23.3 24.2 587 (14.2) 27.9

Mice and ethics statement

A total of 235 male and female C57BLJ/6 mice aged 8 to 10 weeks old at irradiation were purchased from the Monash Animal Research Platform and housed at the animal facilities of the Australian Synchrotron and Royal Melbourne Hospital.

Animal procedures were approved by the University of Melbourne Office for Research

Ethics and Integrity (ethics identification no. 1613833) and performed in accordance with relevant guidelines and regulations.

Mice in the TBI, abdominal PBI and head PBI groups were monitored at least once per day post-irradiation and euthanized according to strict intervention criteria.

Mice receiving thoracic PBI were monitored twice per week following irradiation.

Specific toxicity endpoints were severe (20%) weight loss compared to pre- experimental weight, 15% weight loss and signs of poor well-being (severe diarrhoea, moribund behaviour, hunched posture, lack of grooming) and abnormal neurological signs (seizures, fitting, balance disorders). Growth following irradiation was assessed by calculating the percentage change in weight of each mouse at a given time-point compared to baseline, which was defined as the weight immediately prior to irradiation.

Histopathology

Mice were humanely euthanized and tissue was collected for each mouse once reaching one of the aforementioned toxicity endpoints or at the end of the experimental

116 post-irradiation follow-up period (60 to 68 days for TBI and abdominal PBI, 38 days for head PBI and 170 to 180 days for thoracic PBI). The intestinal tract and brain were harvested and fixed in formalin for 24 hours. Lungs were gently inflation-fixed using formalin and post-fixed in formalin for 24 hours. After fixation, tissue was embedded in paraffin and 4 µm thick sections were stained with hematoxylin-eosin reagent for analysis. Lung sections were additionally stained with Masson’s Trichrome. Slides were viewed using an Olympus IX83 wide-field microscope (Olympus Corp., Tokyo, Japan) and images were taken using an Olympus DP22 colour camera (Olympus Corp., Tokyo,

Japan).

Statistics

N = 5 mice were irradiated per dose group for each modality. PROBIT analysis30 was performed using the IBM SPSS Statistics suite (version 24) (IBM Corp.,

Armonk, NY, USA) to model the probability of toxicity as a function of radiation dose.

The TD50 dose, which was the dose associated with a 50% incidence of a specified toxicity, was calculated for each radiation modality with a 95% confidence interval stated in parentheses where possible. Weight data was analysed using a one-way analysis of variance (ANOVA) in GraphPad Prism version 7.0 (GraphPad Software Inc,

San Diego, CA, USA) with a significance level of 0.05. These data are presented as a mean percentage change compared to baseline (pre-experimental weight) ± s.e.m; *p <

0.05; **p < 0.01; ***p<0.001; ****p < 0.0001; ns, not statistically significant.

Data Availability

All datasets generated and analysed during this study are available from the corresponding author upon reasonable request.

References

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1. Favaudon, V. et al. Ultrahigh dose-rate FLASH irradiation increases the differential response between normal and tumor tissue in mice. Sci. Transl. Med. 6, 245ra93 (2014). 2. Montay-Gruel, P. et al. Irradiation in a flash: unique sparing of memory in mice after whole brain irradiation with dose rates above 100Gy/s. Radiother. Oncol. 124(3), 365-396 (2017). 3. Laissue, J. A. et al. Response of the rat spinal cord to x-ray microbeams. Radiother. Oncol. 106(1), 106-111 (2013). 4. Schultke, E. et al. Pencilbeam irradiation technique for whole brain radiotherapy: technical and biological challenges in a small animal model. PloS One 8, e54960 (2013). 5. Bouchet, A. et al. Synchrotron microbeam radiation therapy induces hypoxia in intracerebral gliosarcoma but not in the normal brain. Radiother. Oncol. 108(1), 143-148 (2013). 6. Laissue, J. A. et al. Neuropathology of ablation of rat gliosarcomas and contiguous brain tissues using a microplanar beam of synchrotron-wiggler-generated X rays. Int. J. Cancer. 78, 654-660 (1998). 7. Dilmanian, F. A. et al. Response of rat intracranial 9L gliosarcoma to microbeam radiation therapy. J. Neurooncol. 4, 26-38 (2002). 8. Brauer-Krisch, E. et al. Effects of pulsed, spatially fractionated, microscopic synchrotron x-ray beams on normal and tumoral brain tissue. Mutat. Res. 704(1-3), 160-166 (2010). 9. Bouchet, A. et al. Identification of AREG and PLK1 pathway modulation as a potential key of the response of intracranial 9L tumor to microbeam radiation therapy. Int. J. Cancer. 136(11), 2705-2716, (2015). 10. Crosbie, J. C. et al. Tumor cell response to synchrotron microbeam radiation therapy differs markedly from cells in normal tissues. Int. J. Radiat. Oncol. Biol. Phys. 77(3), 886-894 (2010). 11. Bouchet, A. et al. Preferential effect of synchrotron microbeam radiation therapy on intracerebral 9L gliosarcoma vascular networks. Int. J. Radiat. Oncol. Biol. Phys. 78(5), 1503-1512 (2010). 12. Inada, T., Nishio, H., Amino, S., Abe, K. & Saito, K. High Dose-rate Dependence of Early Skin Reaction in Mouse. Int.J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 38, 139-145 (1980).

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13. Weiss, H., Epp, E. R., Heslin, J. M., Ling, C. C. & Santomasso, A. Oxygen Depletion in Cells Irradiated at Ultra-high Dose-rates and at Conventional Dose- rates. Int.J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 26, 17-29 (1974). 14. Hendry, J. H., Moore, J. V., Hodgson, B. W. & Keene, J. P. The constant low oxygen concentration in all the target cells for mouse tail radionecrosis. Radiat. Res. 92, 172-181 (1982). 15. Smyth, L. M., Senthi, S., Crosbie, J. C. & Rogers, P. A. The normal tissue effects of microbeam radiotherapy: what do we know, and what do we need to know to plan a human clinical trial? Int. J. Radiat. Biol. 92(6), 302-311 (2016). 16. Ibahim, M. J. et al. An evaluation of dose equivalence between synchrotron microbeam radiation therapy and conventional broadbeam radiation using clonogenic and cell impedance assays. PloS One 9, e100547 (2014). 17. Priyadarshika, R. C., Crosbie, J. C., Kumar, B. & Rogers, P. A. Biodosimetric quantification of short-term synchrotron microbeam versus broad-beam radiation damage to mouse skin using a dermatopathological scoring system. Br. J. Radiol. 84(1005), 833-842 (2011). 18. Chiu, C., McArdle, A. H., Brown, R., Scott, H. J. & Gurd, F. N. Intestinal mucosal lesion in low-flow states: a morphological, hemodynamic, and metabolic reappraisal. Archives of Surgery 101(4), 478-483 (1970). 19. Laissue, J. A., Blattmann, H., Wagner, H. P., Grotzer, M. A. & Slatkin, D. N. Prospects for microbeam radiation therapy of brain tumours in children to reduce neurological sequelae. Dev. Med. Child Neurol. 49(8), 577-581 (2007). 20. Booth, C., Tudor, G., Tudor, J., Katz, B. P. & MacVittie, T. The acute gastrointestinal syndrome in high-dose irradiated mice. Health Phys. 103(4), 383- 399 (2012). 21. Hall, E. J. & Giaccia, A. J.. Radiobiology for the Radiologist. 7th edn, (Lippincott Williams & Wilkins, 2012). 22. Mukumoto, N. et al. Sparing of tissue by using micro-slit-beam radiation therapy reduces neurotoxicity compared with broad-beam radiation therapy. J. Radiat. Res. 58(1), 17-23 (2017). 23. Fowler, J. F. The linear-quadratic formula and progress in fractionated radiotherapy. Br. J. Radiol. 62(740), 679-694 (1989). 24. Rodney Withers, H., Taylor, J. M. G. & Maciejewski, B. Treatment volume and tissue tolerance. Int. J. Radiat. Oncol. Biol. Phys. 14(4), 751-759 (1988).

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25. Hopewell, J. W. The volume effect in radiotherapy--its biological significance. Br. J. Radiol. 70(Special Issue 1), S32-40 (1997). 26. Lye, J. E. et al. Absolute dosimetry on a dynamically scanned sample for synchrotron radiotherapy using graphite calorimetry and ionization chambers. Phys. Med. Biol. 61(11), 4201-4222 (2016). 27. Livingstone, J. et al. Preclinical radiotherapy at the Australian Synchrotron's Imaging and Medical Beamline: instrumentation, dosimetry and a small-animal feasibility study. J Synchrotron Radiat. 24(4), 854-865 (2017). 28. British Institute of Radiology, Central axis depth dose data for use in radiotherapy departments. Suppl. 25 (British Institute of Radiology, London 1996). 29. Poole, C. M., Day, L. R., Rogers, P. A. & Crosbie, J. C. Synchrotron microbeam radiotherapy in a commercially available treatment planning system. Biomed. Phys. Eng. Express 3, 025001 (2017). 30. Finney, D. J. Probit analysis (Cambridge University Press, London 1964).

Acknowledgements

The authors wish to acknowledge beam-time access from The Australian

Synchrotron. We are grateful to Duncan Butler and the team at the Australian Radiation

Protection and Nuclear Safety Agency for assistance with the CRT irradiations and dosimetry, Andrew Stevenson, Dr. Mitzi Klein, Helen Forrester and Claire Scott for support during SBBR and MRT irradiations at the IMBL, Leonie Cann for assistance in preparing tissue samples for histological analysis, Shenna Langenbach for interpretation of the lung histology and Andrew Naughton and the animal welfare team at the

University of Melbourne. We are grateful to Trent Smith from The Australian

Synchrotron for constructing the plastic mouse holders to position the mice. L.S. is supported by a Research Training Program scholarship from the Australian

Government. This project was supported by a grant from the NHMRC [Project Grant

1061772].

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Author contributions

P.R., J. C. and L. S. designed the experiments; L.S., J.C., J.D., J.V., J.L., T.B., L.D. and

P.R participated in scientific discussions; L.S., J.V., J.D., J.C., J.L., T.B., L.D. and P.R. performed the irradiations; L.S., J.D. and J.V. carried out post-irradiation animal monitoring; L.S., J.D. and P.R. performed the histological analysis; L.D. and T.B. performed Monte Carlo simulations relating to dosimetry; L.S., J.C. and P.R. wrote the manuscript.

Competing interests

The authors declare no competing interests.

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Figures

Figure 4.1. Dose response curves following conventional radiation therapy (CRT), microbeam radiation therapy (MRT) and high dose-rate synchrotron broad-beam radiation therapy (SBBR) for (a) total body irradiation (TBI), (b) abdominal partial body irradiation (PBI) and (c) head

PBI. The horizontal TD50 line indicates the dose predicted to cause toxicity (>15-20% weight loss, severe diarrhoea, moribund behaviour) in 50% of the animals. For all three irradiation sites, there was no significant difference in TD50 values between CRT and

SBBR. The peak MRT TD50 dose was an order of magnitude higher for each irradiation site. Dose response curves were generated using PROBIT analysis with N = 3-4 doses per modality and N = 4-5 mice per dose.

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Figure 4.2. Post-irradiation weight gain for surviving mice. Weight gain is measured by a percentage change in weight compared to pre-experimental weight following (a) total body irradiation (TBI) 60 days post-irradiation, (b) abdominal partial body irradiation (PBI) 60 days post-irradiation, (c) head PBI 37 days post-irradiation and, (d) thoracic PBI 140 days following irradiation. Mice had subnormal weights compared to controls following irradiation regardless of the modality used or irradiation site. Mice in the high dose-rate synchrotron broad-beam radiation therapy (SBBR) and microbeam radiation therapy (MRT) groups had the most significant growth impairment. There was no statistically significant difference in weight gain between SBBR and conventional radiation therapy (CRT) at near equal doses, except for following TBI, with the 5.4 Gy SBBR group having significantly less weight gain compared to 5.1 Gy CRT. Differences between groups were analysed using ANOVA with N = 2-5 surviving mice per dose/modality; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Figure 4.3. Intestinal histopathology following abdominal partial body irradiation. (a) Hematoxylin and eosin (HE) stained sections of small intestine from surviving mice showed relatively normal crypt villus architecture 60 days following irradiation with the exception of the 249 Gy microbeam radiation therapy (MRT) group. Short arrows point to regions where the sub-epithelial space has been extended and long arrows show where the epithelial cells of the villus have completely lifted off from the underlying lamina propria. For the MRT groups, valley doses are indicated in parentheses. (b) HE stained sections of small intestine from mice that were euthanized due to acute gastrointestinal syndrome showed a loss of normal crypt-villus architecture.

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Figure 4.4. Histopathological changes to the cerebellum following microbeam radiation therapy (MRT). Top panels depict hematoxylin and eosin stained cerebellar sections and the lower panels depict magnified images corresponding to regions of interest (boxed). Wide, evenly spaced bands of cerebellar granular cell loss (short arrows), corresponding with microbeam paths, were evident following 455 Gy MRT and was associated with neurotoxicity within two to four hours of irradiation. Narrow bands of granular layer scarring (long arrows) were evident at 38 days following 377 Gy MRT in surviving mice.

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Figure 4.5. Pulmonary damage and fibrosis 170 to 180 days following thoracic partial body irradiation. (a) Hematoxylin and eosin stained lung sections showed severe pulmonary damage, including alveolar destruction, airspace enlargement (asterisks) and the thickening of alveolar walls (short arrows), in all dose groups for each modality. For the microbeam radiation therapy groups, valley doses are indicated in parentheses. (b) Masson’s Trichrome staining revealed the deposition of collagen fibres (blue) in sub- pleural (short arrows) and intra-parenchymal (asterisks) regions.

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Figure 4.6. Mouse positioning for irradiation. (a) For microbeam radiation therapy at the Imaging and Medical Beamline, microbeams were orientated vertically and therefore in parallel with respect to the superior-inferior plane of mice. Microbeam width and spacing are not to scale in this beams-eye-view diagram. (b) Radiographic imaging shows mice positioned vertically in the path of radiation. Mice were gently strapped to a plastic holder, with support provided by small positioning pegs. Superior and inferior field borders for the partial body irradiations are denoted by overlayed dotted lines.

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4.3 Supplementary information

Further explanation of dosimetry method, traceability and uncertainty

In all three delivery modes (SBBR, MRT and CRT), the absorbed dose to water was determined at a point on the beam axis at a depth of 5 mm in a theoretical phantom designed to mimic the scatter conditions of a mouse in the custom plastic (poly-methyl- methacrylate; PMMA) holder used during the irradiations (Supplementary Fig. 4.2). In this phantom the mouse is modelled as a rectangular water slab (2 cm wide x 10 cm high x 1.5 cm thick) with a 0.5 cm air gap, and PMMA holder of 7 cm wide x 16 long and 2.4 cm thick. While this model does not take into account any details of the mouse itself, it does include the overall scatter conditions, and therefore is a more accurate choice than using the dose in a full-scatter water phantom, or the incident air kerma.

Initially the phantom calculations were performed to estimate the uncertainty in using the full-scatter water dose as a surrogate for the mouse dose. However, when the magnitude of the difference between full-scatter water and the mouse phantom became apparent, it became clear that this was a better surrogate for the actual mouse dose.

Importantly, the mouse plastic phantom (with a lack of backscatter) has a pronounced effect on MRT valley dose compared to the full-scatter water phantom.

SBBR

Dosimetry for SBBR was performed using a PTW (Freiburg, Germany) model

31014 pinpoint ionization chamber in a virtual water phantom according to the protocols previously described by Lye et al.1 and Livingstone et al2. The chamber was calibrated by the National Measurement Institute of Germany, Physikalisch-Technische

Bundesanstalt (PTB), for absorbed dose to water in conventional X-ray beams, and the calibration coefficient was interpolated in HVL to the synchrotron beam spectrum to

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9 obtain ND,w = 2.70 x 10 Gy/C with an uncertainty of 2.2% (k=1). We estimate an additional 0.5% uncertainty arises from the difference in the field size, depth and spectrum of the synchrotron beam compared to the PTB calibration beams. Charge from the Pinpoint chamber was integrated as it and the phantom were scanned through the 1 mm high x 30 mm wide synchrotron beam to deliver a uniform rectangular field. After correction for recombination, the dose at the measurement depth (20 mm) was multiplied by a measured depth-dose relationship (PDD) to obtain the dose at 5 mm depth. Dosimetry was performed for the four field sizes (30 mm x 20 mm, 30 mm x 30 mm, 30 mm x 60 mm, 30 mm x 100 mm) which produce slightly different doses due to increased backscatter in the larger fields. In this dynamic delivery mode, the incident air kerma and beam size is constant. The dose is controlled by the velocity of the sample as it is scanned through the beam. Uncertainties in all of these factors give rise to a combined uncertainty in the delivered dose at 5 mm depth in a water phantom of 2.4%

(k=1). An additional uncertainty due to air around the mouse and loss of backscatter in the mouse phantom was calculated from Monte Carlo simulations to be in the range of

0.94-0.90 leading to combined uncertainties of 4.8 % for SBBR.

MRT

Dosimetry for MRT was determined relative to the measurements made for

SBBR. Previous work by Poole et al.3 used GEANT4 Monte Carlo models to establish the output factor (OF) for the peak dose and the peak to valley dose ratio (PVDR). The

OF is the ratio of the dose in the peaks to the dose in the broad beam when the MRT collimator is removed (i.e. the SBBR case). The PVDR is the ratio of the dose in the peaks to the dose in the valleys. In the current study, the PVDR varied from 31.8 (TBI) to 41.3 (Thoracic PBI). The uncertainty in the OF is estimated to be 1.7% and in the

PVDR is 6.9% (k=1). These uncertainties are added in quadrature with the SBBR

129 uncertainty to obtain a combined uncertainty of 5.1% for the peak doses and 8.6% for the valley doses in MRT.

CRT

Dosimetry for CRT was performed with an IBA (Schwarzenbruck, Germany)

FC65-G ionization chamber in air without its build-up cap. The chamber was calibrated by the Australian Radiation Protection and Nuclear Safety Agency (ARPANSA) for air kerma, and absorbed dose to water at the surface of a full-scatter water phantom was calculated following the American Association of Physicists in Medicine Task Group

61 protocol4, for the largest field size (approximately 10 cm diameter). A PDD from

Supplement 25 of the British Journal of Radiology5 was applied to calculate dose at 5 mm depth, and Monte Carlo calculations were performed to account for the loss of backscatter during the mouse irradiations. These calculations included the beam size for each case, replaced the full-scatter water phantom with a model of the PMMA mouse holder and, for the PBI cases, the Pb shields. The mouse itself was modelled as a rectangular slab of water with a 5 mm air gap surrounding it (Supplementary Fig. 2).

The combined DW uncertainty at 5mm depth was calculated to be 6.1% for CRT. The full uncertainty budget for CRT, SBBR and MRT is included as a Supplementary

Dataset.

References

1. Lye, J. E. et al. Absolute dosimetry on a dynamically scanned sample for

synchrotron radiotherapy using graphite calorimetry and ionization chambers.

Physics in Medicine & Biology 61, 4201 (2016).

2. Livingstone, J. et al. Preclinical radiotherapy at the Australian Synchrotron's

Imaging and Medical Beamline: instrumentation, dosimetry and a small-animal

feasibility study. Journal of synchrotron radiation 24, 854-865 (2017).

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3. Poole, C. M., Day, L. R., Rogers, P. A. & Crosbie, J. C. Synchrotron microbeam

radiotherapy in a commercially available treatment planning system. Biomed. Phys.

Eng. Express 3, 025001 (2017)

4. Ma, C. M. et al. AAPM protocol for 40-300 kV x-ray beam dosimetry in

radiotherapy and radiobiology. Med. Phys. 28(6), 868-893 (2001).

5. British Institute of Radiology, Central axis depth dose data for use in radiotherapy

departments. Suppl. 25 (British Institute of Radiology, London 1996).

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Supplementary figures

Percentage depth dose curves – all modalities

Supplementary Figure 4.1. Monte Carlo (GEANT4) generated percentage depth dose

(PDD) curves for keV x-ray beams incident on a water phantom. Curves are for mono- energetic x-ray beams representing microbeam radiation therapy (MRT; 95 keV), ultra- high dose rate radiation therapy (SBBR; 124 keV) and conventional radiation therapy

(CRT; 93 keV). The Mo-Mo curve represents a beam with a spectrum of x-ray energies and a mean energy of 124 keV (SBBR). These PDD plots show that the relative change in dose deposition with depth is almost identical for the first 30 mm (> thickness of a mouse) for all three modalities.

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Plastic mouse-holder geometry

Supplementary Figure 4.2. Geometry and composition of the theoretical phantom used to model the mouse and plastic (poly-methyl-methacrylate; PMMA) holder. This phantom was used in the Monte Carlo modelling of the scatter conditions of all three irradiation modalities. While this model does not take into account the exact size or composition of the mouse itself, it does include the overall scatter conditions, and is therefore a more accurate choice than using the dose in a full-scatter water phantom, or the incident air kerma. The geometry of this ‘mouse and plastic’ phantom has a pronounced effect on MRT valley dose owing to the lack of back-scatter compared to a full-scatter water phantom.

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Uncertainty calculation for mouse irradiation dosimetry - CRT

u (%) Air kerma rate Value Type A Type B

7 FC65-G calibration coefficient NK 4.38 x 10 Gy/C 0.70 [1] chamber-source distance for PBI (TBI) 300 mm (343 mm) 0.50 [2] Ionization current 1126 pA (833 pA) for 16.5 mA tube current 0.05 0.05 [3] irradiation non-uniformity PBI (TBI) 1 (0.94) 4.00 [4]

temperature/pressure correction (kTP) 0.30 [5] X-ray stability (no monitor used) 0.30 [6] possible spectral differences at short distance 0.29 [7] Quadratic sum 0.05 4.12 Combined air kerma rate uncertainty 4.12

Monte Carlo statistical uncertainty 0.30 [8] differences in geometry/backscatter 4.00 [9] difference in modelled/actual field size/shape 1.20 [10] Quadratic sum 0.30 4.18 Combined Monte Carlo uncertainty 4.19

Dw uncertainty Air kerma rate 0.05 4.12 Shutter timing (very small, long exposures) 0.00 PDD correction to 5 mm depth 0.9966 0.20 Monte Carlo (field size, mouse holder) 0.30 4.18 Mass energy attenuation coefficients 1.086 1.5 [11]

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Quadratic sum 0.30 6.06 Combined D_(w,z=0.5) uncertainty (k=1) 6.07

!",$%&.( " 2/220122 !",$%&.( = *+, -. 4 8 - 8 3 !",567 567 567 9:

[1] Inherent calibration uncertainty, from ARPANSA calibratkion for air kerma at this beam quality (NXE250) [2] Approximate 1/r^2 uncertainty in positioning accuracy of 0.5-1mm in 300mm. Also note that SSD was d - 5 mm [3] Type A is ESDM in typical current measurement, Type B is calculated in worksheet ‘IC current type B’, U8 spreadsheet [4] Beam non-uniformity for the largest exposed site (measured with film) is a 26% correction with significant asymmetry at one end (down to 40% for TBI). Estimate uncertainty is 100% of largest correction, which is 6% (DB - I've kept this at 6% but I think its defintion should include more geometry of the mouse) [5] See U26 - different lab and detector but the estimate is still valid and dominated by possible difference between temperatures at chamber and thermistor [6] Based on therapy QC history [7] Guess, chamber NK changes 0.5% with HVL change of ~30%, unlikely HVL will change more than that with distance, treated as rectangular distribution [8] from Monte Carlo calculation [9] Monte Carlo calculation based on jig irradiated at centre (actually moved depending on exposed site) and no lead shielding included in model which affects backscatter Rough calculation based on ratios of irradiated field sizes and TG-61 backscatter factors (which assumes full scatter conditions) gives up to 4% correction for smallest exposed sites (head and thorax, see 'backscatter with lead' tab), estimate uncertainty is 100% of this correction DB: Even when including the full MC calculation of the dose, there is about a 4% uncertainty due to the air around the mouse. I think this cannot be got rid of. [10] Estimated difference between FWHM of 9.5 cm and 10.3 cm (approximate measured value and value used in model) [11] From TG-61

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Uncertainty calculation for mouse irradiation dosimetry - SBBR

u (%) Absorbed dose to water Value Type A Type B

9 Pinpoint chamber calibration coefficient ND,w 2.70 x 10 Gy/C 2.20 [1] chamber depth in phantom 0.50 [2] Ionization current 0.05 0.05 [3] temperature/pressure correction (kTP) 0.50 [4] Beam stability (no monitor used) 0.13 [5] Interpolation to synchrotron spectrum 0.50 [6] Field size and distance differences 0.50 [7] Correction to 5 mm depth 0.00 [8] Quadratic sum 0.05 2.42 Combined dose to water uncertainty 2.42

Monte Carlo statistical uncertainty 0.30 differences in geometry/backscatter 0.944 for 30x30 4.00 [9] Cu, Cu BSF correction applied to Mo,Mo 1.00 [10] difference in modelled/actual field size/shape 0.50 [11] Quadratic sum 0.30 4.15 Combined Monte Carlo uncertainty 4.16

Dw uncertainty Dw rate 0.05 2.42 Monte Carlo (field size, mouse holder) 0.30 4.15

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Quadratic sum 0.30 4.81 Combined D_(w,z=0.5) uncertainty (k=1) 4.82

!",$%&.( = ;!!<.&&,&.&(*+=,"

[1] Inherent calibration uncertainty, from PTB Certificate [2] ApproximatePDD uncertainty in positioning accuracy of 0.5-1mm in water PDD [3] Type A is ESDM in typical current measurement, Type B is calculated in worksheet ‘IC current type B’, U8 spreadsheet [4] TP variation mostly due to possible temperatre and pressure lags. Larger in the tron than ARPANSA. [5] Tron output variation in top up mode: max 0.5 mA in 200 mA - so 0.25% max (divide by 2) [6] Estimate of interpolating to Mo/Mo spectrum - kQ from PTB is a slowly varying function with energy [7] Possible variation due to different fields sizes etc. But this is an ion chamber - insensitive to such things [8] Ion chamber measurements performed at 5mm depth in soid water. Positional uncertainty accounted for in [2]. [9] A correction is applied to account for the difference between fullscatter conditions and the simplified mouse model. The uncertainty is 4% due to variable field size due to Pb strips during exp. [10] An additional 1% since the backscatter correction factor is applicable to the Cu,Cu spectrum only. [11] The beam is almost parallel when the BDA defines the beam's static height. Small beam divergence accounted for in simulation. Field size variation conservatively estimated to be +- 4mm Corresponds to a 0.5% change in dose

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Uncertainty calculation for mouse irradiation dosimetry -MRT

u (%) Type Type Absorbed dose to water - peak dose A B SBBR absorbed dose at 5 mm depth 0.30 4.81 [1] Output factor OF 0.40 1.69 [2] Quadratic sum 0.50 5.10 Combined dose to water uncertainty 5.12

Type Type Absorbed dose to water - valley dose A B Absorbed Peak dose at 5 mm depth 0.50 5.10 PVDR 0.80 6.85 [3] Quadratic sum 0.94 8.54 Combined dose to water uncertainty 8.59

Peak dose !",$%&.( = OF x !",$%&.(

Valley dose !",$%&.( = PVDR x OF x !",$%&.(

[1] Measured for SBBR, including corrections for water cube to mouse model. The interpolation to Mo,Mo uncertainty from SBBR [6] is not included since it is measured in Cu,Cu.

Also absent is the additional 1% Mo,Mo uncertainty in the SBBR MC backscatter mouse model correction [9].

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[2] The output factor is calculated from MC. The positional uncertainty is estimated to be 1.64% due to a +- 1mm shift with depth. The uncertainty due to variation in field size is 0.38% based on a +- 4mm vertical shift. The uncertainty estimates are combined in quad. [3] PVDR also calculated using MC. The PVDR is heavily dependent on the depth and field size. The positional uncertainty is estimated to be 5.9% due to a +- 1mm shift with depth. The uncertainty due to variation in field size is 3.6% based on a +- 4mm vertical shift The uncertainty estimates are combined in quad

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CHAPTER 5 Identification of optimal clinical scenarios for MRT

5.1 Preface

The purpose of this chapter is to determine which human disease settings best facilitate the unique dosimetry of MRT (Aim 4). This chapter reproduces a manuscript submitted to the journal Radiotherapy and Oncology for publication. This paper contains its own bibliography and reference style which is separate from the rest of the thesis. The figures appear at the end of the chapter, and have been renumbered for the purpose of this thesis. I am the primary author of this manuscript, involved in planning the study and responsible for carrying out the treatment planning work. I was responsible for analysing and interpreting the data and writing the manuscript, with input from all co-authors.

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5.2 Submitted manuscript

Identifying optimal clinical scenarios for synchrotron microbeam radiation therapy: a treatment planning study

Lloyd M. L. Smytha,*, Liam. R. Dayb, Katrina Woodfordc,d, Peter. A. W. Rogersa, Jeffrey C. Crosbieb,c & Sashendra Senthic,d

aDepartment of Obstetrics & Gynaecology, University of Melbourne, Royal Women’s Hospital, Parkville, Victoria 3052, Australia; bSchool of Science, RMIT University, Melbourne, Victoria 3001, Australia; cAlfred Health Radiation Oncology, Alfred Health, Melbourne, Victoria 3004, Australia; dDepartment of Surgery, Central Clinical School, Monash University, Melbourne, Victoria 3004, Australia.

*Corresponding author. Address: Level 7, Royal Women’s Hospital, Corner Grattan Street and Flemington Road, Parkville, Victoria 3052, Australia; email: [email protected]

Funding sources:

L.S. is supported by a Research Training Program scholarship from the Australian Government. This project was supported by a grant from the Australian National Health and Medical Research Council [Project Grant 1061772].

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Abstract

Background and purpose: Synchrotron Microbeam Radiation Therapy (MRT) is a pre-clinical modality characterised by a spatially alternating peak-valley dose- distribution. Dosimetry studies using clinical datasets have not yet been conducted. Our aim was to investigate MRT dosimetry in scenarios refractory to conventional treatment and to identify optimal settings for a future Phase I trial.

Materials and methods: MRT plans were generated for seven clinical scenarios where re-irradiation was performed clinically. A hybrid algorithm, combining Monte

Carlo and convolution-based methods, was used for dose-calculation. The valley dose to organs at risk (OARs) had to respect the single fraction tolerance doses achieved in the corresponding re-irradiation plans. The resultant peak dose and the peak-to-valley dose ratio (PVDR) at the tumour target volume were assessed.

Results: Peak doses greater than 80 Gy in a single fraction, and PVDRs greater than 10, could be achieved for plans with small (<35 cm3) or shallow volumes, particularly recurrent glioblastoma, head and neck tumours, and select loco-regionally recurrent breast cancer sites. Treatment volume was a more important factor than treatment depth in determining the PVDR. The mean PVDR correlated strongly with the size of the target volume (rs = -0.70, p = 0.01). The PVDRs achieved in these clinical scenarios are considerably lower than those reported in previous pre-clinical studies.

Conclusion: Our findings suggest that intra-cranial and head and neck sites will be optimal scenarios for a future Phase I trial of MRT.

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Introduction

For many recurrent, metastatic or treatment-resistant cancers, dismal prognosis persists despite the use of modern radiation oncology techniques. Synchrotron microbeam radiation therapy (MRT) is a pre-clinical modality that could improve outcomes through physical parameters and phenomena that are a departure from current radiobiological paradigms [1, 2]. MRT is delivered as an array of quasi-parallel micro- planar beams tens of microns wide and spaced by hundreds of microns which generates an alternating peak-valley dose-distribution in tissue [3, 4]. In pre-clinical models, peak

(in-beam) doses are reported to be in the order of 50 times greater than the corresponding valley doses [5, 6]. Several characteristics of synchrotron radiation are essential to maintaining a high-resolution peak-valley dose-distribution in tissue [7]; 1) a keV x-ray energy to minimise the range of secondary electrons, 2) an ultra-high dose- rate (several hundred Gray per second) to mitigate the effects of physiological tissue motion, and 3) minimal beam divergence.

In animal models, normal tissue tolerates single fraction MRT doses up to several hundred Gray [4, 5, 8-10] while tumour control can be achieved despite only a fraction of the tumour receiving the peak dose [11, 12]. These observations challenge a central dogma of conventional radiation oncology; a dose-response and therapeutic index that is dependent on highly conformal doses delivered to the entire target volume, while avoiding adjacent organs at risk. For MRT, the therapeutic effect is thought to rely on optimising the interplay between the peak and valley doses [2, 7], making the peak-to-valley dose ratio (PVDR) one key metric for assessing the effectiveness of clinical plans.

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Previous studies showing the possible PVDRs and peak doses achievable by

MRT use simple, homogenous phantoms [7, 13-17]. A smaller number of studies use more complex inhomogeneous phantoms of the human lung [18], human head [19-21] or rat spinal cord [5]. Importantly, many of these studies used field sizes suitable for either pre-clinical rodent irradiations or for small human tumours. Such results are generally not applicable to the re-irradiation setting in humans, particularly given the influence of field size on the PVDR [3].

Although cranial malignancies have been proposed as an ideal future target for

MRT [1, 2], no studies have demonstrated the dosimetry of MRT in different clinical contexts. The purpose of this treatment planning study is to identify scenarios that would be optimal for a future Phase I trial of MRT in humans. Here, we selected a variety of intra-cranial and extra-cranial targets with a broad range of target volumes.

Specifically, we aim to inform clinicians about the PVDRs achievable, the peak doses reasonably deliverable and clinical features that may be a contraindication for MRT. We hypothesise that the depth and volume of tumours will correlate with PVDR. To our knowledge, this is the first MRT dosimetry study to use clinical datasets.

Materials and methods

Clinical datasets

Seven clinical computed tomography (CT) datasets and corresponding external beam radiation therapy plans were supplied by Alfred Health Radiation Oncology following approval by the institutional Human Ethics Review Committee. Datasets were de-identified, anonymised and imported into a research version of the Eclipse™ (Varian

Medical Systems, Palo Alto, CA) treatment planning system (TPS). We performed

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MRT planning using datasets for intra-cranial, head and neck, chest and spine sites.

Details for the datasets, including demographic and clinical planning data, are summarised in Table 5.1. Patients included in this planning study had all developed predominantly local, symptomatic recurrence following radical radiotherapy treatment and were treated with re-irradiation using conventional fractionated treatment having exhausted systemic treatment options. In each case there were organ at risk (OAR) constraints limiting the total dose that could be delivered.

Treatment planning system characteristics

A hybrid MRT dose-calculation algorithm, previously described by Donzelli et al. [22], was implemented in the Eclipse™ TPS environment. In short, the hybrid algorithm calculates the dose delivered due to photon and electron mediated energy transport separately; Monte Carlo simulations calculate photon transport while secondary electron transport is calculated with a convolution-based algorithm. The resultant accuracy of the hybrid algorithm is comparable to pure Monte Carlo approaches but with substantially shorter, and clinically feasible, calculation times [22].

The algorithm was adapted to model the parameters of the Imaging and Medical

Beamline (IMBL) at the Australian Synchrotron [3]. MRT at the IMBL is delivered using a horizontally propagating array of vertically orientated microbeams 50 µm wide with a centre-to-centre spacing of 400 µm. The microbeam energy spectrum is between

50 and 200 keV (weighted mean energy of 94 keV) and the in-beam dose rate is approximately 300 Gy/s [3].The TPS limits the field width and length to 30 mm and

140 mm, respectively, and uses a fixed horizontal beamline which reflects the physical limitations of the IMBL. The TPS generates separate, corresponding plans for the peak and valley doses

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Planning method and evaluation

The OAR constraints used for re-irradiation were used to generate acceptable

MRT valley plans. Once an acceptable MRT valley plan was achieved, a corresponding

MRT peak plan was generated to determine the peak doses that could be delivered throughout the target volumes. Dose-volume histograms and dose statistics from the

Eclipse™ TPS were used to evaluate and compare the MRT and clinical treatment plans. A plan showing the PVDR was derived from the peak and valley plans for each dataset. PVDR depth profiles were generated based on a line parallel to the field direction and passing through the geometric centre of the target volume. Because MRT is delivered as a single fraction treatment [1, 23], the tolerance doses for OARs achieved in the fractionated clinical plans were scaled to a single fraction equivalent. Scaling was based on the linear quadratic (LQ) model, as described previously [24], with a/b ratios sourced predominantly from QUANTEC studies. This is summarised in Table 5.2. Skin doses, which were not considered in the clinical plans but are relevant to MRT due to the use of a keV beam energy, were restricted to V26 Gy < 0.035 cm3 and V23 Gy < 10 cm3 [25].

A uni-directional field approach, required to preserve the peak-valley geometry optimal for normal tissue sparing [4], was used for all plans. Field directions for individual plans were chosen based on a balance between avoiding critical OARs and minimising the target volume depth relative to the field direction. Where the size of a target volume exceeded the field width or height restrictions at the IMBL, a series of fields were stitched together to achieve coverage. For the head and neck sites, plans assumed a supine position and direct lateral fields. For the remaining sites, plans assumed a seated position, with couch rotations used to change the field entrance angle.

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Statistics

Spearman’s rank correlation (rs) was used to analyse the relationship between target volume characteristics and PVDR. Statistical analyses were performed using

GraphPad Prism version 7.0 (GraphPad Software Inc, San Diego, California, USA),

95% confidence intervals are reported in square brackets and p < 0.05 was considered to be statistically significant.

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Table 5.1. Summary of clinical plans used for MRT planning Patient Gender Age at re- Diagnosis Primary Site(s) of Re-irradiation PTV Depth at Re-irradiation technique ID irradiation irradiation dose recurrence dose Volume centre of (Years) (Gy)/Number of (Gy)/Number of (cm3) PTV (cm) fractions fractions GBM1 F 52 Glioblastoma 60/30 Left temporal 30/5 11.1 4.9 3 non-coplanar DCAs lobe GBM2 M 58 Glioblastoma 60/30 Right lateral 30/5 31.3 4.9 4 non-coplanar DCAs ventricle HN1 M 70 Sino-nasal 66/33 Left cavernous 50/20 13.2 6.3 3 DCAs + 4 IMRT fields at 3 undifferentiated sinus non-coplanar couch angles carcinoma HN2 F 64 Adenoid cystic 60/30 Right base of 50/20 20.2 5.4 4 non-coplanar DCAs carcinoma of the skull submandibular gland Chest1 F 41 Multifocal 50.4/28 Right IMC 50.4/28 223.2 3.0 5 static fields invasive ductal Right Neck 44.1 2.5 carcinoma Right 45/28 55.3 2.5 SCF/ICF Chest2 F 45 Infiltrating 50.4/28 Right IMC 50.4/28 84.1 2.4 5 IMRT fields ductal Left IMC 77.0 2.3 carcinoma Right ICF 35.4 2.8 Right Axilla 49.0 3.4 Right SCF 30.1 2.4 Sac1 F 42 Schwannoma Surgery Sacrum 54/30 237.2 4.0 5 IMRT fields

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PTV; planning target volume, DCA; dynamical conformal arc, IMRT; intensity modulated radiation therapy, SCF; supra-clavicular fossa, ICF; infra-clavicular fossa, IMC; internal mammary chain

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Table 5.2. Single fraction tolerance doses for organs at risk Plan Number of Organ at risk Endpoint a/b ratio Fractionated dose Single Fraction Fractions (Gy) equivalent dose (Gy)

GBM1 5 Brainstem Cranial Neuropathy 2.1 [26] Dmax = 8.8 Dmax = 4.9

Optic apparatus Optic Neuropathy 1.6 [27] Dmax = 1.9 Dmax = 1.3

GBM2 5 Brainstem Cranial Neuropathy 2.1 Dmax = 24.6 Dmax = 12.1

Optic apparatus Optic Neuropathy 1.6 Dmax = 5.9 Dmax = 3.3

HN1 20 Brainstem Cranial Neuropathy 2.1 Dmax = 20.7 Dmax = 7.1

Optic apparatus Optic Neuropathy 1.6 Dmax = 29.2 Dmax = 8.7

HN2 20 Brainstem Cranial Neuropathy 2.1 Dmax = 30 Dmax = 9.4

Optic apparatus Optic Neuropathy 1.6 Dmax = 30 Dmax = 8.9

Chest1 28 Spinal cord Myelopathy 0.87 [28] Dmax = 45 Dmax = 10.1 Lungs Pneumonitis 4 [29] V20 Gy = 27.1% V7.9 Gy = 27.1%

Chest2 28 Spinal cord Myelopathy 0.87 Dmax = 45 Dmax = 10.1 Lungs Pneumonitis 4 V20 Gy = 20.0% V7.9 Gy < 20.0%

Sac1 30 Spinal nerve roots Neurological toxicity 3 [31] Dmax = 56.6 Dmax = 15.2

Rectum RTOG Grade ³ 2 late 3 [30] Dmax = 54.8 Dmax = 14.8 toxicity [30]

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Results

Valley dose to organs at risk

Table 5.3 summarises the dosimetry achieved in the plans. Single fraction valley doses for each plan met the dose constraints achieved clinically and are displayed in dose-volume histograms (Fig. 5.1). For the recurrent glioblastoma and head and neck cases, the optic apparatus and the brain were the dose-limiting organs. Maximum valley doses to the brainstem were equal to or less than 6.0 Gy and clearly within tolerance.

Very small volumes of the brain, limited to regions bordering the skull, received high doses, up to a maximum point dose of 24.1 Gy. However, the volume of brain receiving

12 Gy was less than 5 cm3 in all cases [32]. Given its proximity to the bony spinal canal, the spinal cord was the most dose-limiting organ for the recurrent breast cancer plans. For the sacrum plan, the rectum was the dose-limiting normal tissue structure.

The highest V26 Gy and V23 Gy values for the skin recorded across all the plans were

0.003 and 0.02 cm3, respectively, both for a recurrent glioblastoma plan (GBM1). The maximum point dose to bone ranged from 20.5 for the sacral schwannoma (Sac1) to

46.7 Gy for the recurrent left cavernous sinus tumour (HN1).

Peak dose to target volumes

For acceptable doses to the OARs, the mean, single fraction peak doses ranged from 47.7 Gy for the sacral schwannoma (Sac1) to 131.3 Gy for the right supra- clavicular breast cancer recurrence (Chest2). The highest mean peak doses were achieved for plans with either small or superficial volumes. One of the recurrent breast cancer plans (Chest2) achieved mean peak doses greater than 100 Gy for all target volumes. The glioblastoma and head and neck plans still achieved mean peak doses of

80 to 100 Gy despite having the greatest treatment depths of all plans. The interquartile

151 range for peak doses was largest for PTVs encompassing high or low-density regions, with bony regions responsible for very high maximum peak doses. The recurrent glioblastoma plans showed the least variation in peak dose across their respective PTVs, reflecting their location at depth within the brain and distance away from relatively low or high-density structures.

Peak to valley dose ratio

Mean PVDRs for the PTVs of each scenario ranged from 7.5 for the recurrent sacral schwannoma (Sac1) to 16.0 for a recurrent glioblastoma plan (GBM1). Even at depths greater than 5 to 6 cm, glioblastoma and head and neck plans could achieve

PVDRs greater than 10. The mean PVDR correlated strongly with the size (i.e. volume) of the PTV (rs = -0.70 (-0.90, -0.22), n = 13, p = 0.01) but no significant correlation was found between mean PVDR and central PTV depth (Fig. 5.2).

Figure 5.3 illustrates the change in PVDR as a function of depth and tissue density. In soft tissue, PVDR was at a maximum at the field entrance and decreased sharply within the first 2 cm depth in tissue. The rate of decrease in PVDR reduced with depth, with PVDR approaching a plateau for most plans before increasing again slightly as the field exited the body. PVDR decreased sharply when moving from regions of soft tissue to bone and increased again sharply when moving back into soft tissue. Smaller target volumes, with smaller corresponding field sizes, had the highest entrance PVDR values and the highest PVDR plateaus. The PVDR approached zero in air gaps or cavities (Fig. 5.3A and 3D).

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Table 5.3. Dosimetry achieved in MRT plans

Patient ID/PTV Valley dose for OARs Mean PVDR Mean Peak dose Median Peak dose

for PTV to PTV (Gy) (IQR) to PTV (Gy)

GBM1 Brainstem: Dmax = 3.1 Gy 16.0 96.7 95.8 (87.1 – 105.3)

Optic apparatus: Dmax = 1.3 Gy

Brain: V12 Gy = 0.1 cm3

GBM2 Brainstem Dmax = 5.5 Gy 10.2 87.6 86.8 (77.9 – 96.0)

Optic apparatus: Dmax = 2.5 Gy

Brain: V12 Gy = 4.9 cm3

HN1 Brainstem Dmax = 4.2 Gy 11.7 102.9 101.6 (91.0 – 112.6)

Optic apparatus Dmax = 8.2 Gy

Brain: V12 Gy = 4.9 cm3

HN2 Brainstem Dmax = 6.0 Gy 9.4 81.2 75.2 (64.3 – 92.9)

Optic apparatus Dmax = 8.9 Gy

Brain: V12 Gy = 0.3 cm3

Chest1 Spinal Canal Dmax = 10.1 Gy

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R IMC Lungs V7.9 Gy = 0.1 % 8.7 62.4 57.6 (47.2 – 76.8)

R Neck 11.1 67.0 65.1 (55.1 – 78.2)

R SCF/ICF 9.0 65.6 63.7 (56.7 – 71.9)

Chest2 Spinal Canal Dmax = 10.0 Gy

R IMC Lungs V7.9 Gy = 7.9 % 9.5 100.0 101.2 (77.8 – 119.4)

L IMC 11.1 124.4 105.6 (85.2 – 199.6)

R IC 10.6 103.3 121.5 (102.1 – 143.0)

R Ax 11.4 103.0 98.1 (84.3 – 120.1)

R SCF 13.1 131.3 128.9 (113.8 – 149.5)

Sac1 Spinal nerve roots Dmax = 13.5 7.5 47.7 45.7 (37.4 – 55.5)

Rectum: Dmax = 14.8 Gy

IQR; Inter-quartile range, SCF; supra-clavicular fossa, ICF; infra-clavicular fossa, IMC; internal mammary chain

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Discussion

The purpose of this treatment planning study was to inform clinicians about the dosimetry possible with MRT, and in doing so, to identify optimal scenarios for its clinical testing. For an acceptable valley dose to OARs, shallow PTVs, or those with a volume less than 35 cm3, achieved the highest PVDRs and peak doses. These scenarios included intra-cranial tumours, head and neck tumours, and select recurrent breast cancer sites. Treatment volume was a more critical factor than treatment depth in determining the PVDRs.

The PVDRs presented in our study are considerably lower than those reported in pre-clinical rodent studies, which are typically between 20 and 50 [5, 6, 33-35]. This observation is important given the centrality of peak-valley dosimetry to the novel radiobiology of MRT. Furthermore, previous MRT dosimetry studies typically use field sizes that would be too small to cover the target volumes selected in our study [3, 19,

21]. Consequently, clinically achievable PVDRs, in scenarios with a justification for a

Phase I test, have previously been overestimated. For instance, Martinez-Rovira et al.

[19] assume a field size of 2 cm2 for a hypothetical intra-cranial tumour, yielding a substantially higher PVDR (~21) and lower valley doses compared to the intra-cranial tumour recurrences presented in our study.

Although small tumours are the most dosimetrically favourable for MRT, Phase

I testing will not be justifiable in scenarios where combinations of surgery and stereotactic radiotherapy are routinely used [36-38]. However, a trial of MRT could be justified in settings where a standard, second line therapy for local recurrence has not been established and especially for patients with repeated local recurrences and poor prognosis. Datasets used in our study were selected from patients who had been offered

155 potentially toxic re-irradiation in the clinic, in the context of no further surgical or systemic treatment options.

Glioblastoma Multiforme (GBM) is an example of an insidious disease which continues to have a dismal survival rate and limited therapeutic options for local recurrences despite significant research investment [39-41]. In our study, both GBM plans demonstrated features that might be favourable for MRT; relatively high PVDRs and mean peak doses to the PTV as well as a relatively low variation in peak dose across the target. Given the high incidence of GBM compared to other primary brain tumours, it should be possible to recruit a sub-set of patients with a treatment volume favourable for MRT.

Loco-regionally recurrent breast cancer is another clinical scenario where a trial of MRT could be justified, and in our study, showed potential for high peak doses to the target volume. The relatively shallow depth of the lymph nodes targeted in these plans favours the keV energy of MRT, with maximum peak doses and PVDRs occurring close to the skin surface. The difference in dosimetry between the two recurrent breast cancer scenarios presented in our study can be explained by differences in the total field size and the proximity of their respective PTVs to the dose-limiting spinal cord. The proximity of target volumes to the spinal canal could be a contraindication for MRT given the local increase in valley dose in bone which would increase the risk of spinal cord toxicity. To mitigate this risk, an angled, rather than direct anterior field approach, could be taken to completely avoid the MRT field exiting through or close to the spinal canal. However, this approach would also substantially increase the total field size required and the depth of the target volumes relative to the MRT field.

Given the dosimetry observed in the loco-regionally recurrent breast cancer plans, superficially recurrent cancers or metastatic cutaneous lesions would also be

156 dosimetrically favourable targets for MRT. Breast tumours which metastasize to the skin [42] and in-transit cutaneous or sub-cutaneous melanoma recurrences [43] can be unresponsive to standard local and systemic therapies and remain a therapeutic challenge [44, 45]. These scenarios would be good candidates for MRT and future dosimetric investigation is warranted. Recurrent melanoma confined to the limbs would be a particularly attractive target for a Phase I test of MRT, given the lack of closely situated vital organs.

Pragmatically speaking, the treatment of recurrent intra-cranial or head and neck lesions would currently be more feasible than breast cancer recurrences to the chest wall and regional lymph nodes. Firstly, patient-positioning solutions to overcome the limitations of the fixed horizontal beamline are more readily available for head and neck targets. A medical treatment chair has been implemented at the European Synchrotron

Research Facility [46] and provides sufficient accuracy for stereotactic intra-cranial irradiations. This would be a feasible positioning solution for treatments of the head or neck at the IMBL. Positioning for extra-cranial treatment sites would be more difficult, however, a robotic patient positioning couch recently installed at the IMBL might be suitable for clinical purposes following further testing and development. Secondly, the accuracy of the hybrid algorithm in lung tissue remains a point of contention due to the complex pulmonary microstructure and the presence of air. This may preclude its use for thoracic treatment fields, where the use of more time-consuming pure Monte Carlo algorithms would be required.

A potential disadvantage of treating intra-cranial tumours with MRT is the need for fields to traverse the skull prior reaching the target volume. Martinez-Rovira et al.

[19] estimate that a maximum peak dose of 75 to 90 Gy would be deliverable to an intra-cranial tumour at a depth of 4.5 to 5.5 cm, if desiring to restrict the probability of

157 long-term bone-related complications to less than 5% in 5 years [47]. The higher atomic number of bone is associated with an increased probability of Compton-scattered photons being absorbed in the valley region via the photo-electric effect, leading to a sharp increase in valley dose in the skull [21]. We chose to accept higher maximum valley doses to bone given that: 1) osteoradionecrosis is a late-effect of radiation and that for a Phase I test of MRT, acute toxicity would be the most relevant outcome for patients with an otherwise short life-expectancy (< 6 months), 2) bone is highly resistant to radiation, with single fraction doses of at least 50 Gy routinely used in the extracorporeal irradiation of malignant bone tumours [48, 49], and, 3) if patients survive long enough to experience long-term osteoradionecrosis of the skull, cranioplasty is a common and well-established neurosurgical procedure to preserve cosmesis and brain protection [50].

A key assumption in our study, and of previous MRT dosimetry investigations

[19], is that the MRT valley dose is the best parameter for the evaluation of normal tissue complication probability. A recently published in vivo study investigating the equivalence of MRT and conventional broad-beam radiation therapy in a murine model showed that median toxic valley doses were lower than the median toxic conventional radiation therapy dose by a factor of 1.6 to 1.8 [51].This finding suggests that peak doses cannot be ignored in the context of MRT toxicity and that caution is required when assuming the equivalence of MRT valley dose with broad-beam dose. Further in vivo work is required to determine more precise and toxicity-specific scaling factors that would bring MRT valley doses to within OAR tolerance.

We have used the LQ model to scale total fractionated OAR doses to a single fraction equivalent. While the applicability of the LQ model at high doses per fraction is controversial [52], there is robust data to suggest that the model is valid up to 10 Gy per

158 fraction and sufficient data to justify its use in the design of clinical trials using 15 to 18

Gy per fraction [53-55]. The majority of the single fraction tolerance doses we utilized as dose constraints were less than 10 Gy. Additionally, our tolerance doses were at least as conservative as the single fraction tolerance doses recommended by QUANTEC [56] or used in recent radiosurgery trials [25, 57].

In conclusion, our findings affirm that intra-cranial and head and neck sites will be the most optimal scenarios for a future Phase I trial of MRT, especially when also considering patient positioning solutions and the physical limitations of the IMBL.

Superficially recurrent or cutaneous metastatic lesions should also be investigated further. While the PVDRs presented in this study are relatively low, peak doses that are a factor of ten larger that the tissue tolerance dose are still possible. This could profoundly improve the therapeutic effect of radiation therapy and is worth exploring in clinical scenarios where alternative options have been exhausted or where standard therapeutic strategies have not been established.

Conflict of interest

The authors declare there are no conflicts of interest

Acknowledgements

The authors thank Frank Gagliardi and Vanessa Panetierri at Alfred Health

Radiation Oncology for assistance in anonymising and exporting the clinical radiation therapy datasets plans for MRT planning use.

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L.S. is supported by a Research Training Program scholarship from the

Australian Government. This project was supported by a grant from the Australian

National Health and Medical Research Council [Project Grant 1061772].

References

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[37] Lo SS, Moffatt-Bruce SD, Dawson LA, Schwarz RE, Teh BS, Mayr NA, et al. The role of local therapy in the management of lung and liver oligometastases. Nat Rev Clin Oncol 2011;8:e405. https://doi.org/10.1038/nrclinonc.2011.75. [38] Tree AC, Khoo VS, Eeles RA, Ahmed M, Dearnaley DP, Hawkins MA, et al. Stereotactic body radiotherapy for oligometastases. Lancet Oncol 2013;14:28- 37. https://doi.org/10.1016/S1470-2045(12)70510-7. [39] Hanif F, Muzaffar K, Perveen k, Malhi SM, Simjee SU. Glioblastoma multiforme: a review of its epidemiology and pathogenesis through clinical presentation and treatment. Asian Pac J Cancer Prev 2017;18:3-9. https://doi.org/10.22034/APJCP.2017.18.1.3. [40] Young RM, Jamshidi A, Davis G, Sherman JH. Current trends in the surgical management and treatment of adult glioblastoma. Ann Transl Med 2015;3:e121. https://doi.org/10.3978/j.issn.2305-5839.2015.05.10. [41] Gallego O. Nonsurgical treatment of recurrent glioblastoma. Curr Oncol 2015;22:273-81. http://dx.doi.org/10.3747/co.22.2436. [42] Krathen RA, Orengo IF, Rosen T. Cutaneous metastasis: a meta-analysis of data. South Med J 2003;96:164-7. https://doi/org/10.1097/01.SMJ.0000053676.73249.E5. [43] Balch CM, Buzaid AC, Soong S-J, Atkins MB, Cascinelli N, Coit DG, et al. Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma. J Clin Oncol 2001;19:3635-48. https://doi.org/10.1200/JCO.2001.19.16.3635. [44] Testori A, Faries MB, Thompson JF, Pennacchioli E, Deroose JP, van Geel A, N., et al. Local and intralesional therapy of in-transit melanoma metastases. J Surg Oncol 2011;104:391-6. https://doi.org/10.1002/jso.22029. [45] Adams S, Kozhaya L, Martiniuk F, Meng T-C, Chiriboga L, Liebes L, et al. Topical TLR7 agonist imiquimod can induce immune-mediated rejection of skin metastases in patients with breast cancer. Clin Cancer Res 2012;18:6748-57. https://doi.org/10.1158/1078-0432.CCR-12-1149. [46] Renier M, Brochard T, Nemoz C, Requardt H, Brauer E, Esteve F, et al. The radiotherapy clinical trials projects at the ESRF: technical aspects. Eur J Radiol 2008;68:S147-50. https://doi.org/10.1016/j.ejrad.2008.04.057.

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[47] Emami B, Lyman J, Brown A, Coia L, Goitein M, Munzenrider JE, et al. Tolerance of normal tissue to therapeutic irradiation. Int J Radiat Oncol Biol Phys 1991;21:109-22. https://doi.org/10.1016/0360-3016(91)90171-Y. [48] Hong AM, Millington S, Ahern V, McCowage G, Boyle R, Tattersall M, et al. Limb preservation surgery with extracorporeal irradiation in the management of malignant bone tumor: the oncological outcomes of 101 patients. Ann Oncol 2013;24:2676-80. https://doi.org/10.1093/annonc/mdt252. [49] Hayashi K, Araki N, Koizumi M, Suzuki O, Seo Y, Naka N, et al. Long-term results of intraoperative extracorporeal irradiation of autogenous bone grafts on primary bone and soft tissue malignancies. Acta Oncol 2015;54:138-41. https://doi.org/10.3109/0284186X.2014.930172. [50] Piazza M, Grady MS. Cranioplasty. Neurosurg Clin N Am 2017;28:257-65. https://doi.org/10.1016/j.nec.2016.11.008. [51] Smyth LML, Donoghue JF, Ventura JA, Livingstone J, Bailey T, Day LRJ, et al. Comparative toxicity of synchrotron and conventional radiation therapy based on total and partial body irradiation in a murine model. Sci Rep 2018;8:e12044. https://doi.org/10.1038/s41598-018-30543-1. [52] Kirkpatrick JP, Meyer JJ, Marks LB. The linear-quadratic model is inappropriate to model high dose per fraction effects in radiosurgery. Sem Radiat Oncol 2008;18:240-3. https://doi.org/10.1016/j.semradonc.2008.04.005. [53] Brenner DJ. The linear-quadratic model is an appropriate methodology for determining isoeffective doses at large doses per fraction. Sem Radiat Oncol 2008;18:234-9. https://doi.org/10.1016/j.semradonc.2008.04.004. [54] van der Kogel AJ. Chronic effects of neutrons and charged particles on spinal cord, lung, and rectum. Radiat Res 1985;104:S208-16. [55] Garcia LM, Leblanc J, Wilkins D, Raaphorst GP. Fitting the linear–quadratic model to detailed data sets for different dose ranges. Phys Med Biol 2006;51:2813. https://doi.org/10.1088/0031-9155/51/11/009. [56] Marks LB, Yorke ED, Jackson A, Ten Haken RK, Constine LS, Eisbruch A, et al. Use of normal tissue complication probability models in the clinic. Int J Radiat Oncol Biol Phys 2010;76:S10-9. https://doi.org/10.1016/j.ijrobp.2009.07.1754. [57] Ryu S, Pugh SL, Gerszten PC, Yin F-F, Timmerman RD, Hitchcock YJ, et al. RTOG 0631 phase II/III study of image-guided stereotactic radiosurgery for

165 localized (1-3) spine metastases: phase II results. Pract Radiat Oncol 2014;4:76- 81. https://doi.org/10.1016/j.prro.2013.05.001.

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Figures

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Figure 5.1. Dose-volume histograms for single fraction MRT valley dose plans. The optic apparatus and brain were the most dose-limiting OARs for the glioblastoma (Panel A and B) and the head and neck (Panel C and D) recurrences. The spinal canal and rectum were the dose-limiting OARs for the loco-regionally recurrent breast cancer (Panel E and F) and sacral schwannoma (Panel G) plans, respectively.

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Figure 5.2. Plots of mean PVDR for the each PTV versus the volume (Panel A) and central depth (Panel B) of the PTV. While the size of the PTV correlated strongly and negatively with PVDR (rs = -0.70 [-0.90, -0.22], p = 0.01), central PTV depth did not (rs = 0.007 [-0.56, 0.57], p = 0.98). The minimum central PTV depth for the targets selected for MRT planning was 2.3 cm. Correlation was analysed using Spearman’s rank correlation (n = 13) with p < 0.05 considered statistically significant.

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Figure 5.3. PVDR depth profiles for the glioblastoma (Panel A and B), head and neck (Panel C and D), locoregionally recurrent breast cancer (Panel E to L) and sacral schwannoma (Panel M) target volumes. Regions of relatively high electron density, such as bone, are shaded in dark grey, while regions of relatively low density, such as lung, are white. PVDR increased sharply in regions of bone and approached zero in air (Panel A and D). In most cases, the PVDR increased marginally and gradually as the field passed through lung tissue, with sharper changes evident at tissue interfaces.

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CHAPTER 6 Discussion, conclusions and future directions

6.1 Summary of key findings

The overall aim of this thesis was to gather and present radiobiological data which will contribute to the translation of synchrotron radiation therapy to clinical use.

Key themes included dose-equivalence with conventional radiation therapy (CRT)

(chapters 3 and 4), the differential effects of MRT and CRT (chapters 3), acute and long-term normal tissue toxicity (chapter 4) and the identification of disease settings most suitable for microbeam radiation therapy (MRT) (chapter 5). Key findings of this thesis were:

• For Diffuse Intrinsic Pontine Glioma (DIPG) tumour cell-lines, differences in

cellular response following irradiation were related to the heterogeneity between cell

lines rather than the choice of radiation modality.

• MRT peak doses of 112 Gy, 250 Gy and 560 Gy were equivalent to 2 to 2.5 Gy, 4.5

to 5 Gy and 8 to 9 Gy, respectively, based on clonogenic assays (in vitro) using

DIPG tumour cell-lines.

• MRT peak doses of approximately 120 Gy and 260 Gy were equivalent to

approximately 7 Gy and 12.5 Gy, respectively, based on murine normal tissue

toxicity (in vivo).

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• The MRT valley dose was a better predictor of normal tissue toxicity than the MRT

peak dose, based on a comparison of median toxic doses with CRT.

• Long-term growth, as measured by weight-gain, was significantly impaired

following MRT despite the acute tolerance of radiation.

• High dose-rate synchrotron broad-beam radiation therapy (SBBR) at 35-40 Gy/s did

not provide a normal tissue sparing effect (ie. a ‘FLASH’ effect), supporting the

notion that the ‘FLASH’ phenomenon is dose-rate dependent.

• Clinically achievable peak to valley dose ratios (PVDRs) and peak doses are

substantially lower than those used in pre-clinical animal studies.

• In clinical scenarios, target volume correlates most strongly (negatively) with PVDR

and is a parameter which could be useful for the screening of patients for a future

clinical trial of MRT.

• From a range of clinical scenarios where a trial of MRT could be justified, small

recurrent intra-cranial and head and neck tumours show the greatest potential for

high PVDRs and peak doses at the target and the least variation in peak dose

delivered to the total target volume.

• Large but superficial target volumes, such as cutaneous metastases or recurrences of

breast cancer, could also yield high PVDRs and peak doses and warrant further

investigation.

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6.2 Discussion

Dose-equivalence between MRT and CRT

To plan a clinical trial for MRT, it will be vital to communicate to clinicians how a dose delivered by a spatially heterogeneous field at the synchrotron compares to a broad-beam dose delivered by a linear accelerator in the clinic. In essence, we must be able to tell a clinician that ‘X’ Gy delivered at the synchrotron will be equivalent to ‘Y’

Gy delivered in the clinic, in terms of biological endpoints such as tumour control or specific normal tissue complications. In chapter 3, clonogenic survival using DIPG tumour cells was used as an endpoint to calculate dose-equivalence. This kind of approach to evaluating MRT-CRT dose-equivalence has been established previously using a range of other tumour and normal tissue cell-lines (Ibahim et al. 2014).

A comparison of the data published by Ibahim et al. (2014) and the data presented in chapter 3 reveals an important principle of MRT: microbeam width and spacing are intrinsically linked to biological effectiveness and will influence equivalence with CRT. Figure 6.1 compares equivalent CRT and MRT doses presented in chapter 3 (Smyth et al. 2018a) with those calculated by Ibahim et al. (2014). Three different microbeam collimator geometries are represented; 25 µm beam width with 200

µm centre-to-centre spacing (25/200), 50 µm beam width with 200 µm spacing (50/200) and 50 µm beam width with 400 µm spacing (50/400). These data show that as microbeam width is increased while spacing is kept constant, the equivalent CRT dose also increases. Conversely, as spacing is increased for a constant microbeam width, biological effectiveness decreases.

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9 8 7 6 5 4 3 2 1 Equivalent CRT Dose (Gy) 0 25/200 50/200 50/400 MRT collimator geometry (width/spacing) (µm)

Figure 6.1. Equivalent CRT doses for 112 Gy MRT based on three different microbeam collimator geometries. Data for the 25/200 (n = 6) and 50/200 (n = 3) geometries are from Ibahim et al. (2014) and the 50/400 (n = 2) geometry from Smyth et al. (2018a).

These dose-equivalence data are based on the in vitro clonogenic survival of a range of different cell lines and are presented as the mean equivalent CRT dose ± standard error of the mean.

Selecting a microbeam collimator geometry that strikes an optimal balance between tumour control and healthy tissue sparing will be paramount to the success of

MRT as a therapeutic tool. To this end, several studies have investigated the impact of varying microbeam width or microbeam spacing on the therapeutic effect of MRT in vivo. Regnard et al. (2008b) determined that at a constant microbeam width of 25 µm, a centre-to-centre spacing of 100 µm was excessively neurotoxic despite providing a greater extension of lifespan in mice treated for intra-cranial gliosarcomas. As such a

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200 µm spacing was deemed more appropriate to balance tumour control and normal tissue toxicity (Regnard et al. 2008b). Serduc et al. (2009a) found that if microbeam spacing and the valley dose are kept constant at 211 µm and 18 Gy, respectively, a microbeam width of 50 µm provides better brain tissue preservation than a 75 µm wide beams and better tumour control than a 25 µm wide beams. A microbeam collimator geometry of 50 µm beam width and 400 µm spacing is currently in use at the Imaging and Medical Beamline (IMBL) of the Australian Synchrotron. A spacing of 400 µm is conservative and gives pre-eminence to normal tissue preservation, even compared to a

200 µm spacing (Schultke et al. 2013). The experiments presented in this thesis could not explore the effect of modifying the microbeam width or spacing on biological effectiveness given that the collimator in use at the IMBL has a fixed geometry.

An important observation from the data presented in chapters 3 and 4 is that in vivo versus in vitro assays can lead to substantial differences in dose-equivalence calculations. For a 50/400 MRT collimator, peak doses of 112 Gy, 250 Gy and 560 Gy had relatively low equivalent CRT doses of 2 to 2.5 Gy, 4.5 to 5 Gy and 8 to 9 Gy, respectively, based on the clonogenic assay data presented in chapter 3 using DIPG tumour cell-lines. The same collimator geometry was then used for the normal tissue toxicity study presented in chapter 4. The in vivo equivalent doses, however, were notably higher – at least twice as high – than those calculated in vitro using clonogenic assays; peak doses of approximately 120 Gy and 260 Gy were equivalent to CRT doses of approximately 7 Gy and 12.5 Gy, respectively.

An explanation for the large disparity between the in vitro and in vivo data could be the difference in scatter conditions between the two sets of experiments. For the cell irradiations, cells were suspended in culture media and irradiated in small centrifuge tubes surrounded by air. The effective field size, that is, the area of cells subject to the

177 radiation field, was approximately 1 cm x 3 cm. For the mouse irradiations in chapter 4, the effective field size covering the body of mice during total and partial body irradiations was at least 2 cm x 3 cm (head and thorax) and up to 2 cm x 10 cm (total body irradiation). The amount of scatter produced in the mouse irradiations would therefore be substantially higher than the cell irradiations, therefore increasing the biological effect of the microbeam array and the calculated equivalent doses. It should be noted that the valley doses presented in chapter 3 for the DIPG cell irradiations were based on Monte Carlo simulations which assumed full scatter conditions and a field size of 30 mm x 140 mm. In contrast, the valley doses for the mouse irradiations were based on Monte Carlo simulations that factored in the true scatter conditions, including the plastic mouse holder and possible air gaps. In order to compare the valley doses presented in chapter 3 and 4, simulation parameters for the in vitro irradiation conditions could be adjusted to reflect the geometry of the tubes and culture media containing the DIPG cells. The true valley doses for the in vitro irradiations in chapter 3 will be substantially lower than the full-scatter valley doses currently presented.

The discrepancy between in vitro and in vivo findings demonstrates the potential weakness of both kinds of assays to evaluate the novel radiobiology of MRT, especially if inferences will be drawn for the human application of MRT. Care should be taken when planning future pre-clinical experiments to ensure that 1) scatter conditions, including field size and treatment depth, could be reasonably comparable to clinical scatter conditions, and 2) that these conditions are simulated as rigorously as possible in order to provide the most accurate estimate of both valley and peak doses. This will allow pre-clinical dose-equivalence data, as well as other radiobiological findings, to be reliably translated to future clinical practice.

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Normal Tissue Toxicity

Chapter 4 presented novel normal tissue toxicity data for synchrotron MRT and high dose-rate synchrotron broad-beam radiation therapy (SBBR). To the candidate’s knowledge, these are the first reported toxicity outcomes for MRT and SBBR to the lungs and abdomen. These are also the first TBI experiments performed using synchrotron radiation therapy modalities.

Two important hypotheses were tested by the dose-escalation experiments presented in chapter 4; firstly, that the valley dose would be the most important parameter for normal tissue toxicity during MRT, and secondly, that SBBR would convey a normal tissue sparing effect relative to CRT, also known as a ‘FLASH’ effect

(Favaudon et al. 2014).

The first hypothesis was confirmed; the median toxic valley doses for MRT were not substantially different to the median toxic CRT doses for each type of irradiation while the median toxic peak doses were at least an order of magnitude greater. An important caveat here, however, is that while the valley dose was most closely associated with toxicity, the median toxic valley doses were consistently lower than the median toxic CRT doses by a factor of 1.6 to 1.8. This observation communicates a key point; peak doses should not be ignored when considering MRT toxicity. The peak and valley regions make a combined contribution to the toxicity associated with MRT. Previous treatment planning studies of MRT, including the treatment planning study outlined in chapter 5, make the assumption that the valley dose to healthy tissue should respect conventional tolerance dose limits (Martinez-Rovira et al. 2010). The data presented in chapter 4 suggests that this assumption may not be

179 appropriate. To be more conservative with respect to toxicity and safety in a future

Phase I trial of MRT, it may be necessary to apply a scaling factor to ensure that the valley dose to organs at risk (OARs) is lower than the conventional broad-beam tolerance dose. However, until organ and toxicity-specific dose-equivalence data is available for MRT, it will be difficult to select an appropriate scaling factor for MRT treatment planning purposes.

The second hypothesis was not proven; there was no normal tissue sparing advantage for SBBR relative to CRT. Given the dose-rate dependence of the ‘FLASH’ normal tissue sparing phenomenon (Montay-Gruel et al. 2017), it is likely that the dose- rate was not high enough for significant differences in the median toxic dose to be observed between the SBBR and CRT groups in any of the TBI or PBI experiments.

Montay-Gruel et al. (2018) recently proved that SBBR, using x-rays delivered at an average dose rate of 37 Gy/s, was capable of triggering a FLASH effect in a rodent whole brain irradiation model. While the instantaneous SBBR dose-rate in chapter 4 ranged from approximately 35 to 40 Gy/s, the average dose-rate for the entire total or partial body irradiation was substantially lower. This is because it took several seconds to vertically scan mice through the synchrotron beam. It is also possible that the endpoint chosen for chapter 4 (severe acute toxicity) was not sensitive enough to detect the presence of a FLASH effect. Future in vivo studies exploring the FLASH phenomenon should use smaller irradiation volumes and focus on organ-specific, quantifiable endpoints related to function or tissue structure.

The SBBR dose-rate is limited at the IMBL due to the mechanical tolerances of the motorised stage used to vertically scan mice through the horizontally propagating synchrotron beam. If delivering relatively small doses (up to 10 to 20 Gy) at instantaneous dose-rates greater than 40 Gy/s, the speed that the sample stage must

180 move at would substantially – and unacceptably – sacrifice dosimetric accuracy. In order to maximally exploit SBBR for a ‘FLASH’ effect by accessing dose-rates close to

100 Gy/s (Montay-Gruel et al. 2017), the stage set-up at the IMBL will need to be optimised. One solution is to move the sample stage further away from the source to increase the overall field size, which will negate the need for vertical sample scanning through the synchrotron x-ray beam. However, the dose-rate would need to be recovered to compensate for the increase in distance away from the x-ray source. Work is underway at the IMBL to prepare Hutch 3 – which is over 100m further away from the x-ray source than Hutch 2B (where the experiments presented in chapter 4 were performed) – for future veterinary trials of MRT. These trials will be an important step in the translational of MRT and/or SBBR to human use.

A significant limit to the clinical applicability of the in vivo data presented in chapter 4 is the use of large irradiation volumes in proportion to the overall size of the mouse model. Therapeutic applications of MRT or SBBR are unlikely to involve TBI or even PBI to entire regions of the body. In alignment with current clinical practice, MRT treatment fields are likely to conform to the shape of the tumour target volume, with fields encompassing as little normal tissue as reasonably achievable. The choice to perform total and partial body experiments in chapter 4 was both conservative and pragmatic. PBI and TBI data is conservative because if large volumes can tolerate the doses investigated, it is reasonable to assume that these doses could also be tolerated if delivered to a smaller volume. This dose-volume effect in conventional radiation therapy is well documented (Hopewell, Morris & Dixon-Brown 1987, Emami et al.

1991).

The choice to perform TBI and PBI was also pragmatic. While it would have been ideal to prospectively generate organ and complication-specific toxicity data –

181 similar to the clinical data retrospectively summarised by the QUANTEC group (Marks et al. 2010) and Emami et al. (1991) – this would have required an enormous amount of time and resources. Such a line of experimentation would also be difficult to justify from an animal ethics perspective, given the need to respect the principles of

‘refinement’ and ‘reduction’. The TBI and PBI experiments performed in chapter 4 also allowed for the irradiation of multiple organs at a time. While the toxicity endpoints were not specific to a particular organ (aside from the lung fibrosis data), critical doses that would acutely endanger well-being were determined. At the very least, the non- toxic doses observed in these experiments provide a conservative starting point for the planning of dose-regimens for future veterinary trials of MRT.

Optimal sites for MRT

The fifth chapter of this thesis demonstrated MRT dosimetry in human subjects, particularly in clinical scenarios where first-line therapeutic strategies failed. These patients, diagnosed with recurrent tumours and lesions resistant to conventional radiation therapy, could be justifiable candidates for a future Phase I trial. While dosimetry studies using various anthropomorphic phantoms have been performed

(Martinez-Rovira et al. 2010), studies using human clinical computed tomography (CT) datasets do not currently exist. Exploration of MRT dosimetry beyond the intra-cranial setting is limited. Subsequently, the need to identify optimal settings for MRT remains unaddressed.

A future Phase I trial of MRT will be focussed on safety and toxicity. Therefore, dose prescriptions are likely to be conservative and limited by the valley dose, given that the toxic effects of MRT are related most closely to the valley region (Smyth et al.

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2016). Assuming that it is desirable to maximise the peak dose delivered to a tumour, scenarios with a high PVDR would be optimal for MRT. For the clinical scenarios presented in chapter 5, the primary objective of the treatment plans was therefore conservative; to ensure that the MRT valley dose respected the clinically accepted dose constraints for OARs. As previously discussed, the valley dose is not a perfect surrogate for broad-beam toxicity, however, this assumption was necessary in the absence of endpoint-specific toxicity data for individual organs.

For an acceptable valley dose to OARs, recurrences of intra-cranial, head and neck and breast cancers could receive single fraction peak doses of at least 80 to 100

Gy. Small (less than 35 cm3) or superficial target volumes showed the greatest potential for high peak doses and peak-to-valley dose ratios (PVDRs). PVDRs greater than 10 were possible, with the highest PVDR (16.0) achieved for a recurrent glioblastoma plan.

An example of the kind of intra-cranial tumour that might be treated using MRT, a recurrent glioblastoma, is shown in Figure 7.2. Based on achieving the least variation in peak dose across the planning target volume, as well as the challenges in patient- positioning at the fixed horizontal IMBL, the glioblastomas and head and neck tumour recurrences would provide the most optimal target for a future Phase I test of MRT.

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Figure 6.2. A recurrent glioblastoma case selected for MRT treatment planning. The valley dose distribution is displayed for an axial slice of the clinical computed tomography dataset, through the centre of the planning target volume (cyan).

While maximising the PVDR is optimal for normal tissue sparing, lowering the

PVDR in the tumour volume by increasing the valley dose could enhance the tumour control probability (Prezado et al. 2009). Dose-enhancement studies using high atomic- number particles have been conducted to artificially increase the valley dose in the tumour (Engels et al. 2016, Martinez-Rovira & Prezado 2011) with promising survival improvements reported in glioma-bearing rats (Regnard et al. 2008a). The use of these nanoparticles requires a cautious approach, however, the potential synergy between

MRT and dose-enhancing particles would be of benefit to intra-cranial lesions where the

184 skull might otherwise limit the dose delivered to the tumour – if bone is deemed to be a critical OAR in a future Phase I trial.

A notable finding of chapter 6 was that the PVDRs achieved in the clinical plans were substantially lower than the PVDRs reported in previous pre-clinical studies. In rodent models (Mukumoto et al. 2017, Laissue et al. 2013, Schültke et al. 2018), as well as phantoms (Livingstone et al. 2017, Martinez-Rovira et al. 2010), PVDRs with a value of at least 20 – and up to 70 – are reported. These studies used field sizes that would be too small for the tumour recurrences scenarios presented in chapter 5. The valley dose is particularly sensitive to field size, with increases in the size of the microbeam array leading to an increase in the number of scattered photons (Siegbahn et al. 2006,

Livingstone et al. 2017). Therefore, underestimating the field size has resulted in overestimating the PVDR that might be achievable in clinical scenarios. Given the importance of PVDR to the therapeutic effect and novel radiobiology of MRT, the validity of scaling radiobiological responses between animal studies, particularly on rodents, and humans should be questioned. Veterinary studies on spontaneous canine tumours – which will be conducted at the IMBL in the near future – will provide a more robust in vivo model of MRT dosimetry, comparable to what can be expected for clinical applications.

While it would have been ideal to investigate MRT dosimetry in a wider range of treatment sites, the study presented in chapter 5 was limited by the availability of clinical datasets. This MRT dosimetry study could be expanded in future to include other disease scenarios, such as cutaneous metastases, chest-wall recurrences of breast cancer, advanced pancreatic cancer, recurrent melanoma and advanced lung cancer.

These are settings where a clinical trial of MRT could be justified.

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A consideration for future dosimetry studies, as well as veterinary or human trials using MRT, are the substantial uncertainties in dose-calculation for treatments that pass through the lung. Imaging the complex microstructure and air-tissue composition of the lung, especially the alveoli, is beyond the capability and spatial resolution of typical computed tomography technology. This means that modelling the MRT dose- distribution in lung tissue is problematic regardless of the calculation algorithm used

(Donzelli et al. 2018). Treatment sites that require collateral irradiation of the lung, such as the recurrent breast cancer scenarios presented in chapter 5, are therefore not ideal candidates for MRT.

Superficial lesions, such as those involving cutaneous or sub-cutaneous metastases or recurrences, are the most likely alternative scenario to intra-cranial or head and neck tumours for the first trial of MRT. Firstly, treatments for these types of lesions will not require microbeams to pass through structures such as bone or lung prior to reaching the tumour target volume. Bone degrades the peak-valley geometry and substantially lowers the PVDR, as seen in chapter 5. Secondly, treating superficial lesions favours the intrinsic properties of MRT, particularly the keV beam energy which is essential to a microscopic peak-valley dose-distribution but leads to limited tissue penetration. Lastly, superficial lesions will be simpler to treat from a patient-positioning and treatment planning perspective. Broad-beam keV radiation has been used for decades to treat skin cancers and these treatments are simple to plan and deliver

(Symonds et al. 2012). Computed tomography planning scans and fixed patient- positioning are typically not required, which could overcome the challenge of having a fixed horizontal beam-line at the Australian Synchrotron.

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6.3 Work in progress

Differential regulation of gene expression in the brainstem following MRT versus SBBR

The equivalent median toxic doses presented in chapter 4 provide a newfound basis for studying the differential radiobiological effects of MRT versus broad-beam radiation. Nominating doses to compare the properties of MRT and broad-beam radiation therapy has previously been difficult, given the distinctly different physical dose-distributions of the two modalities. Many studies have simply utilised a broad- beam dose which matches the physical valley dose of the corresponding MRT array at a given depth in tissue (Yang et al. 2014, Sprung et al. 2012, Bouchet et al. 2017). Ibahim et al. (2016) was the first to use a biological endpoint, in vitro clonogenic survival

(Ibahim et al. 2014), to select broad-beam doses for comparison to MRT.

A study investigating the regulation of genes and molecular pathways in the brainstem following MRT and SBBR is currently in progress. In this work, biologically equivalent doses of MRT and SBBR, as calculated in chapter 4, were used. These were the doses predicted to cause severe acute toxicity within two weeks of whole head irradiation in 50% of irradiated mice (Smyth et al. 2018b).

Adult and paediatric gliomas, including those of the brainstem, are possible oncological targets for MRT (Schultke et al. 2017, Grotzer et al. 2015). Furthermore, the in vitro sensitivity of two genetic variants of DIPG (a brainstem glioma) to MRT was shown in chapter 3 (Smyth et al. 2018a) and intra-cranial or head and neck tumour targets were found to be optimal dosimetric settings for a trial of MRT in chapter 5.

Brainstem tumours are notoriously difficult to resect and are often not amenable to surgery (Green & Kieran 2015). For high-grade paediatric brainstem gliomas such as

DIPG, radiation therapy is the only effective treatment strategy, however, almost all

187 children still die within two years of diagnosis (Hargrave, Bartels & Bouffet 2006,

Grimm & Chamberlain 2013). Adult DIPG has a more favourable prognosis but it still aggressive, often lethal and continues to have limited therapeutic options (Eisele &

Reardon 2016). For low-grade, focal brainstem gliomas in both adults and children, remission is often induced with combinations of chemotherapy and radiation therapy.

However, the long-term toxicities of irradiation can be severe, especially for those treated as children (Green & Kieran 2015). MRT therefore has the potential to improve tumour control for high-grade brainstem gliomas, and to improve neurocognitive outcomes for long-term survivors of lower grade brainstem gliomas.

Despite the potential to use MRT as a novel therapy for brainstem gliomas, studies focussed specifically on the radiobiological response of the brainstem to MRT are notably absent from the literature. Gene expression following MRT has previously been investigated through a rodent model of high-grade glioma, with the response of normal brain tissue, but not the brainstem, also being characterised (Bouchet et al. 2015,

Bouchet et al. 2013b). No comparison with broad-beam radiation therapy was made in these studies. The aim of the current study is to characterise the regulation of gene expression in mouse brainstem acutely following MRT and to compare this to SBBR.

The materials and methods used for this work is outlined in Appendix C.

Previous studies on breast cancer models indicate that MRT and broad-beam irradiation differentially regulate the expression of genes and molecular pathways within 4 to 48 hours of treatment (Yang et al. 2014, Sprung et al. 2012). In these studies, differentially regulated pathways included those related to transcription and translation, macro-molecule metabolism, oxidoreductase activity and signalling transduction (Yang et al. 2014). Genes related to immune function were also regulated differently (Sprung et al. 2012). The immunomodulatory effect of MRT is a potentially

188 exciting avenue of further research which could have important implications for brainstem tumours, as Bouchet et al. (2013b) showed that the early response of glioma- bearing rats to MRT is chiefly based on immune and inflammatory pathways.

Gene expression data from the mouse brainstem study is currently being analysed. Even though no differences in cellular response were observed in DIPG

(brainstem tumour) cell lines following broad-beam and microbeam irradiation five days following treatment (chapter 3), we expect that gene expression profiling at a more immediate time point following irradiation (4 and 48 hours) will be a more sensitive and informative endpoint for radiobiological differences between the two modalities.

Preliminary findings, based on principal component analysis (Figure 6.3), are suggestive of differential gene expression in the brainstem between MRT and SBBR groups, particularly at four hours post-irradiation.

189

Figure 6.3. Principal component analysis of gene expression following the irradiation of mouse brainstem with equivalent doses of MRT and SBBR.

6.4 Future directions

Tumour control studies in animal models

Previous studies testing the hypothesis that MRT is more efficacious than broad- beam radiation therapy for tumour control have selected broad-beam and MRT doses either arbitrarily or based on valley dose (Bouchet et al. 2016, Miura et al. 2006,

Regnard et al. 2008b). It is difficult to infer from these studies whether MRT improves tumour control because of intrinsic radiobiological advantages over broad-beam radiation therapy, or if a higher and more ablative dose of MRT was given compared to the broad-beam groups.

190

Similarly to the mouse brainstem study currently in progress, we plan to use toxicity-based dose-equivalence data to choose MRT, SBBR and CRT doses in future studies on tumour-bearing mice. The aim of these studies will be to determine if MRT and/or SBBR improves tumour control compared to CRT at doses predicted to cause the same level of normal tissue toxicity (ie. the TD50 or TD10) or at the maximally tolerated doses. Using equivalent toxic doses for each modality will provide a more robust test of the hypothesis that MRT provides a higher therapeutic index compared to CRT.

More specifically, we will use mouse three tumour cell-lines that align with the types of partial body irradiations performed in chapter 4; B104-1-1 (brain), LL/2 (lung) and CT26-CL25 (colon). B104-1-1 is a mouse fibroblast cell-line transfected with the neu oncogene, which is actively expressed in neuro- and glioblastomas (Shih et al.

1981). LL/2 is a mouse derived lung carcinoma cell line (Dus, Budzynski &

Radzikowski 1985) and CT26-CL25 is a mouse derived colon adenocarinoma (Wang et al. 1995). Tumours will be imaged via Computed Tomography (CT) prior to irradiation to visualise tumour growth and to enable a treatment plan to be generated. At the IMBL, radiographic imaging will be used to precisely position mice in the radiation beam.

Mice will either receive the maximally tolerated dose, TD50 or TD10, as inferred from the data generated in chapter 4, relevant to the specific tumour location.

Following treatment, mice will be imaged via small animal Positron Emission

Tomography (PET/CT) to monitor tumour progression. Non-irradiated control mice are expected to become moribund (15% weight loss, lethargic, ataxia), due to tumour growth within 4-6 weeks. Tumour volume measurements will be calculated using

PET/CT data and Kaplan-Meier survival charts will be developed as mice are euthanised. At this time, tissue will be collected (brain, lung, intestine). Tissue will then

191 be divided into tumour and normal tissue and stored in RNA later for molecular studies and fixed in formalin for immunohistochemical analysis.

In summary, these tumour studies in mice will build on the dose-equivalence and normal tissue toxicity data presented in this thesis and also test various other aspects of the synchrotron radiation therapy work-flow, including treatment planning, image-guidance and patient-positioning systems. These studies will mimic the workflow of a clinical radiation therapy department and will be crucial preparation for veterinary studies, and eventually, a human trial at the IMBL.

MRT and immunomodulation

Radiation therapy has known immunomodulatory properties, having the potential to both stimulate and suppress the immune system (Weichselbaum et al.

2017). Optimal parameters to enhance an anti-tumour immune response, such as the choice of radiation fractionation regimen and ideal combinations of immune-directed therapies, remain to be defined (Kang, Demaria & Formenti 2016). Choosing an optimal total radiation dose and fractionation regimen could be particularly critical to the efficacy of a combined radio-immunotherapy strategy (Demaria & Formenti 2012).

Immunomodulation has been shown to be a central early effect of MRT that contributes to its therapeutic efficacy (Bouchet et al. 2013b). However, despite its importance, understanding the synergy between MRT and the immune system has remained a relatively unexplored frontier (Smilowitz et al. 2006). MRT could be a powerful tool to explore the immunomodulatory effects of radiation therapy in future studies. Firstly, MRT has the potential to deliver exceedingly high peak doses which could trigger a significant antigenic response. As shown in chapter 5, single fraction

192 doses in the order of 100 Gy are realistic. These doses are at least four to five times higher than the single fraction doses delivered in current radio-immunotherapy clinical trials (Kang, Demaria & Formenti 2016). Secondly, MRT provides a novel platform to explore the effect of spatial fractionation, rather than temporal fractionation, on host immune response. The choice of fractionation, not just total dose, is an equally important consideration for combined radio-immunotherapy (Demaria & Formenti

2012).

If MRT can be optimised to elicit a therapeutic immune response – either as a stand-alone therapy or as part of a combined radio-immunotherapy strategy – it may not be necessary to treat the entire tumour volume with a conformal high dose. This would be a significant paradigm shift in radiation oncology and make deep-seated tumours possible candidates for MRT, despite the limited penetration of keV microbeams.

6.5 Conclusion

This thesis makes an important contribution to the pre-clinical field of synchrotron radiation therapy, exploring radiobiological themes of dose-equivalence with CRT, normal tissue toxicity and clinically achievable dosimetry. The data presented has value for both pre-clinical researchers and clinicians who are involved in the translational pathway of MRT and SBBR. For pre-clinical researchers, the dose- equivalence data could inform the dose regimens of future studies comparing MRT or

SBBR versus CRT in terms of tumour control or differences in cellular or molecular regulation. For clinicians, the toxicity data and observations from the MRT treatment planning study may contribute to guidelines for a future Phase I trial, including optimal sites and contraindications for MRT, and safe dose regimens. Ultimately, the impact of

193 this thesis will be an improved platform for exploring the novel radiobiological properties of synchrotron radiation and intensified research focus towards disease settings where these properties might be best exploited for therapeutic use.

194

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Appendix Appendix A: MRT normal tissue toxicity review

Appendix A is the published manuscript of a review published by Taylor and

Francis in the International Journal of Radiation Biology on 16th March 2016, available online: https://www.tandfonline.com/doi/abs/10.3109/09553002.2016.1154217. Tables and content from this article were adapted for use in Chapter 2.7 and cited appropriately. I am the first author of this publication and contributed to 70% of the work. I was involved in the planning of the review article and was responsible for performing the literature search, screening articles, synthesising the data and writing the review, with input from the co-authors. This accepted manuscript is reproduced with permission from Taylor and Francis.

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The normal tissue effects of microbeam radiotherapy: what

do we know, and what do we need to know to plan a human

clinical trial?

Lloyd M.L. Smyth1,2, Sashendra Senthi3, Jeffrey C. Crosbie3,4 & Peter A.W. Rogers1

1Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne,

Victoria, Australia

2Epworth Radiation Oncology, Epworth HealthCare, Melbourne, Victoria, Australia

3William Buckland Radiotherapy Centre, Alfred Hospital, Melbourne, Victoria,

Australia

4School of Applied Sciences, RMIT University, Melbourne, Victoria, Australia

Running Title: Normal Tissue Effects of Microbeam Radiotherapy

Keywords: Microbeams, Radiotherapy, Radiosurgery, Dose-response curve

Corresponding Author: Lloyd Mark Lee Smyth, Department of Obstetrics and

Gynaecology, Level 7, Royal Women’s Hospital, Parkville, VIC 3052, Australia, Ph:

+61 3 8345 3724, Fax: +61 3 8345 3702, Email: [email protected]

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Abstract

Purpose: Microbeam Radiotherapy (MRT) is a promising pre-clinical cancer therapy which represents a radical departure from the radiobiological principles of conventional radiotherapy (CRT). In order to translate MRT to human clinical trials, robust normal tissue toxicity data are required. This review summarises the normal tissue effects reported by pre-clinical MRT animal studies and compares these data to clinical recommendations in CRT.

Conclusion: Few pre-clinical studies are specifically designed to evaluate the dose- response of normal tissue to MRT. However, it remains clear that a range of normal tissues can tolerate peak MRT doses at least an order of magnitude higher than CRT.

Furthermore, the dose deposited in the valley regions, predominantly determined by microbeam spacing, has a greater influence on the normal tissue response to MRT compared to the peak regions. The development of a new normal tissue complication probability model for MRT, in conjunction with a treatment planning system, will be pivotal in the collection of robust normal tissue toxicity data and the translation of MRT to clinical use.

Keywords: Microbeams, Radiotherapy, Radiosurgery, Dose-response curve

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Introduction

Clinical radiotherapy has evolved over decades to optimize the therapeutic ratio between tumour control and normal tissue toxicity. Temporal fractionation was one of the first major advances, exploiting the ability of normal tissue to repair sub-lethal damage between treatments. More recently, technological advances such as intensity modulation and image guidance have reduced the dose that is delivered to organs adjacent to tumours. This has culminated in stereotactic ablative radiotherapy, allowing dose escalation to the tumour without an increased risk of toxicity. Despite these advances, further improvements are needed, with high-grade gliomas, locally advanced lung cancer and pancreatic cancer being specific examples where modern delivery techniques such as stereotactic ablative radiotherapy, particle-beam therapies and Boron

Neutron Capture Therapy, have had limited impact or simply cannot be used.

Microbeam Radiotherapy (MRT) from a synchrotron source may represent the paradigm shift needed to address these unmet clinical needs.

MRT is characterised by an array of high flux, quasi-parallel beams, up to 100 microns

(µm) wide, which generate ‘peak’ entrance doses up to 10000Gy. These beams are spaced by 100-400µm, creating ‘valley’ doses that can be up to 100 times lower

(Blattmann et al. 2005). This periodically alternating spatial dose distribution produced by MRT is a radical departure from the dosimetry of conventional radiotherapy (CRT), which aims to deliver a nearly homogenous high dose to ablate or control the tumour.

However, despite only a fraction of tissue receiving peak doses in MRT, tumour control can be achieved (Laissue et al. 1998, Dilmanian et al. 2003b, Miura et al. 2006). In contrast, normal tissue retains its structural integrity (Laissue et al. 2007, Crosbie et al.

2010) and remains able to mount a coordinated repair response to MRT-induced damage (Crosbie et al. 2010), maintaining the therapeutic index (Laissue et al. 1998,

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Dilmanian et al. 2003b, Miura et al. 2006). Hypotheses for the efficacy of MRT due to the periodically alternating dose distribution are based on observations including: 1) preferential damage to tumour microvasculature compared to normal brain microvasculature in vivo (Bouchet et al. 2010, 2013a), 2) in-field bystander effects related to cellular migration in vitro and in vivo (Kashino et al. 2009, Crosbie et al.

2010) and the communication of stress factors in vitro (Tomita et al. 2010,

Autsavapromporn et al. 2013) between peak and valley regions, 3) the huge increase in surface area between heavily (peaks) and lightly (valleys) irradiated thin tissue slices when a broadbeam is converted into an array of microbeams (Laissue et al. 2013) and,

4) the differential modulation of immune and inflammatory gene pathways in tumour compared to normal tissue in vivo (Bouchet et al. 2013b, 2015).

A challenge to the clinical feasibility of MRT is the need to use low energy x-ray beams, typically with a median energy less than 200keV, which limits their ability to penetrate tissue (Dilmanian et al. 2006). The half value layer in water for these orthovoltage beams is approximately 5cm (Siegbahn et al. 2006). Furthermore, the peak to valley dose ratio of MRT arrays decreases with increasing target tissue depth and treatment field size (Anderson et al. 2012, Siegbahn et al. 2006). Current pre-clinical rodent experiments which have demonstrated the ability of MRT to control tumours have not been significantly impacted by these effects due to the small target volumes and anatomical dimensions of mice and rats. However, to maintain the delivery of a tumour-controlling dose at depth in the brain of a larger animal or human, the dose delivered to normal tissue proximal to the target could result in significantly increased damage. In an attempt to overcome this issue, an alternative irradiation technique using thick microbeams (also known as ‘minibeams’) as wide as 680µm has been proposed

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(Dilmanian et al. 2006), however an assessment of the therapeutic efficacy and normal tissue effects of minibeam radiotherapy is beyond the scope of this review.

Given the clinical potential of MRT, robust normal tissue toxicity data, especially pre- clinical depth-dose data, must be collected in order to successfully translate these therapies to human clinical trials. This review summarises available normal tissue toxicity data from MRT animal studies and considers how these relate to current normal tissue toxicity data and clinical dose constraints using CRT.

It is important to note that throughout the published MRT literature, doses are often reported as entrance doses only, and not as a dose at depth in the tissue of interest.

Where possible, this review quotes the dose at depth, otherwise, the entrance dose is specified. Knowledge of the entrance doses alone is not enough to draw conclusions about the dose-dependence of toxicity in tissues at depth. The following syntax is used throughout the remainder of this paper to describe microbeam arrays. Geometrical parameters are designated in parentheses as follows; (beam width/beam centre-to-centre spacing; field width x field height). Peak and mid-valley doses are quoted as either an entrance dose or a dose at depth in the following form; peak-dose(Gy)/mid-valley- dose(Gy). ‘NR’ designates an experimental parameter not reported or not possible to infer from the literature.

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The tolerance of normal tissue to MRT

The majority of MRT related normal tissue toxicity data are for the brain tissue of healthy and tumour-inoculated mice and rats. Limited data exist for tissue types such as the spinal cord, skin, and optic structures. No toxicity data are currently available for organs of the respiratory, gastrointestinal, or genitourinary systems. The following section summarises the findings of studies presenting organ specific toxicity data from

MRT experiments on both healthy and tumour-inoculated animals. Table I summarises the macroscopic and microscopic effects of MRT on normal brain tissue and Table II summarises the normal tissue effects of MRT in a range of other tissues.

Brain

The brain of several animal species, including rats, mice and pigs, is remarkably resistant to gross, macroscopic damage caused by MRT. There is no evidence of tissue necrosis one month after an entrance dose of 5000Gy/NR from a single array of sequentially fired microbeams (20µm/200µm: 4mm x 4mm) delivered anteroposteriorly to the right cerebral and cerebellar hemispheres (Slatkin et al. 1995). The dimensions of the array (the field size), and hence the volume of brain tissue in the field, is an important determinant of necrosis, since three microbeams (20µm/200µm; ~400 µm x

4mm) with an entrance dose of 10000Gy/NR cause only in-beam cellular loss, whereas

20 microbeams (42µm/200µm; ~4mm x 4mm) at the same entrance dose cause acute tissue necrosis within 14 days of irradiation (Slatkin et al. 1995). The doses used in this study and other pre-clinical rodent studies of MRT significantly exceed the doses at which radiation necrosis is predicted to occur with CRT (Lawrence et al. 2010).

However, the concern in CRT is the occurrence of late radiation necrosis one to two years following treatment (Lawrence et al. 2010) which is mechanistically different to the acute brain necrosis reported in MRT animal studies. Therefore, there is a need for

218 longer term follow up in pre-clinical animal experiments in order to more adequately assess the resistance of brain tissue to late brain necrosis following MRT.

The microvasculature of normal brain tissue also demonstrates a tolerance to MRT when compared with tumour microvasculature (Bouchet et al. 2010, 2013a). An initial study of healthy mice demonstrated that a single anteroposterior array of microbeams

(25µm/211µm; 3.6mm x 4mm) delivering an entrance dose of 1000Gy/NR to the upper part of the left cerebral hemisphere, causes only minor, transient oedema that resolves approximately one week after irradiation (Serduc et al. 2008). More recently, studies on tumour-bearing rats showed that two orthogonally cross-fired microbeam arrays

(50µm/400µm; 8mm x 10mm), centred on the right caudate nucleus and delivering a dose of 350Gy/12.5Gy per array to the brain, cause preferential damage to tumour microvasculature in comparison to normal brain microvasculature in regard to vessel morphology, size and permeability (Bouchet et al. 2010) and oxygen perfusion

(Bouchet et al. 2013a). With two orthogonally fired microbeam arrays, bi-directionally irradiated areas of normal brain tissue experience greater damage compared to unidirectionally irradiated tissue in regard to overall structure (Laissue et al. 1998) and microvasculature (Bouchet et al. 2010), which highlights the importance of maintaining the unidirectional, periodically alternating peak-valley dose distribution to the normal tissue to uphold the benefits of MRT.

In regard to memory function, healthy rats show no deficits compared to normal untreated rats, at one month and one year after MRT delivered by two orthogonally cross-fired microbeam arrays (25µm/211µm; 10mm x 14mm) to the right cerebral hemisphere with entrance doses of 350Gy/NR (Schültke et al. 2008). Furthermore, the memory performance of healthy mice is equivalent to non-irradiated controls six months after receiving a dose of 2298Gy/5.7Gy at 3mm depth from a single right to left

219 lateral array of pencilbeams (50µm x 50µm square pencilbeams, spaced on centre by

400 µm; 18mm x 8mm) targeting the whole brain (Schültke et al. 2013). In contrast, when the spacing of pencilbeams is reduced to 200µm, a dose of 431Gy/4.3Gy or higher at 3mm depth in the brain causes a reduction in memory performance six months post-irradiation compared to non-irradiated controls (Schültke et al. 2013). Using the same array of square pencilbeams with a spacing of 400µm, mice tolerate doses up to

1767Gy/4.4Gy with no significant decline in motor performance compared to non- irradiated controls, however at 200µm spacing, motor performance is worse at six months for a dose of only 431Gy/4.3Gy (Schültke et al. 2013). These results demonstrate that microbeam spacing is more important than valley dose alone in determining brain dysfunction after MRT.

The ability of the brain to tolerate a large range of peak entrance doses without undergoing necrosis (Slatkin et al. 1995) or brain dysfunction (Laissue et al. 2001,

Schültke et al. 2008, Schültke et al. 2013) is remarkable given that at a microscopic level, a single microbeam array creates discrete bands where neuronal and astroglial nuclei are lost, corresponding to the path of microbeams through the cerebrum

(Dilmanian et al. 2005, Serduc et al. 2006, Laissue et al. 2007, Schültke et al. 2013) and cerebellum (Dilmanian et al. 2005, Laissue et al. 2007). This demonstrates that surviving cells in the valley region are able to maintain tissue integrity and in some measure compensate for the loss of functional cells in the peak region. These observations support the hypothesis that for typical microbeam arrays, gross brain toxicity is more dependent on valley region parameters rather than just the peak dose.

Neurological Development

In 42 and 48 day old weanling piglets, used as a human paediatric model, there are no significant changes in behavioural or neurological development at one year post-

220 irradiation when the cerebellum is irradiated to 263Gy/12.6Gy at depth by a single lateral array of microbeams (28µm/211µm; 15mm x 15mm) (Laissue et al. 2007).

However, at a dose of 150Gy/9.5Gy delivered by a single left to right lateral array

(28µm/105µm; 15mm x 15mm) to the centre of the hindbrain, 12 to 14 day old rat pups with structurally immature hindbrain tissue exhibit ataxia, asthenia and subnormal body weights, as well as marked cerebellar hypoplasia over one year post-irradiation (Laissue et al. 2007, Hanson et al. 2013). Evidence of developmental abnormalities in these rat pups has led to conservative suggestions that maximum valley doses in the hindbrain should not exceed 5Gy during human paediatric MRT treatments (Hanson et al. 2013).

It should be noted that the brain of human infants are much more developed at birth when compared to young rodents (Clancy et al. 2007) and therefore the extrapolation of neurological comparisons between human infants and these rat pups is difficult.

(Table I)

Optic Nerve and Eye

The optic nerve and radiosensitive structures of the eye lie in close anatomical proximity to intra-cranial MRT targets and have been incidentally exposed to microbeam arrays in most pre-clinical animal studies to date. No deliberate effort has yet been made to restrict the collateral dose delivered to these structures. Light microscopy and electroretinography confirm that two orthogonally fired microbeam arrays (25µm/211µm; 10mm x 14mm), centred at the right cerebral hemisphere, with entrance doses of 350Gy/NR cause long-term retinal degeneration in rats one year post- irradiation (Sandmeyer et al. 2008). Importantly, this effect is spatially-dependent, with retinal lesions in 8 of 16 left eyes partially exposed to unidirectional lateral irradiation, versus 16 of 16 right eyes subject to the added anteroposterior irradiation (Sandmeyer et al. 2008). In this study, optic nerves of the left eyes were exposed to a valley dose of

221 approximately 6Gy and the left retinae possibly subject to a valley dose of 5Gy, while the posterior structures of the right eye, including the optic nerve, right central artery, and possibly parts of the posterior eyeball, were subject to a radiotoxic valley dose of approximately 14Gy (Laissue et al. 2012). Optic neuropathy is typically considered to develop over the course of months to years and no long-term data are available for

MRT-irradiated animals. For CRT, the incidence of optic neuropathy is extremely rare when the maximum dose delivered to any part of the optic nerve in a single fraction is kept to less than 8Gy but increases to greater than 10% in the range of 12 to 15Gy

(Mayo et al. 2010).

Spinal Cord

Literature regarding the structural and functional response of the spinal cord to MRT is limited, however the available data suggest it can withstand high peak doses of MRT without a decline in motor function. No histological changes are observed up to doses of

357Gy/12.7Gy at depth in the cervical spinal cord using a single right to left lateral array of microbeams (35µm/210µm; 11mm x 21mm) to irradiate the entire circumference of an 11mm long segment of spinal cord, however focal white and grey matter necrosis, fibrinoid necrosis of microvasculature and leukocytic infiltration are evident at 507Gy/18Gy (Laissue et al. 2013). In regard to motor function, rats receiving doses of 253Gy/9Gy at depth in the spinal cord show no signs of foreleg paresis up to

383 days post-irradiation, whilst the MRT peak/valley dose which causes foreleg paresis in 50% of rats is approximately 373.3Gy/13.2Gy (Laissue et al. 2013).

Importantly, for foreleg paresis the dose-effect relationship is steeper with the valley dose than with the peak dose (Laissue et al. 2013), further emphasising the importance of the valley region in normal tissue sparing. In single fraction radiosurgery using CRT there is a less than 1% rate of myelopathy when the maximum point dose to the spinal

222 cord is kept to less than 13Gy (Kirkpatrick et al. 2010). The volume of irradiated tissue is of particular importance in the spinal cord with the tolerance dose increasing when the irradiated volume is decreased (Hopewell and Trott 2000). As such, the contrasting rates of myelopathy between MRT and CRT may correlate given that the entire volume of spinal cord within the 11mm long MRT field received at least 13Gy whilst the CRT rate is in reference to a maximum point dose.

Skin

In comparison to megavoltage radiotherapy, the maximum dose of an orthovoltage microbeam array lies at a depth of 1mm to the skin surface (Crosbie et al. 2008).

Therefore in MRT, the skin must tolerate high entrance doses in order to allow a tumour-ablating dose to be delivered at depth. Based on a non-mechanistic arbitrary histological scoring system, a single microbeam array (25µm/200µm; 6mm x 6mm) delivering a dose of 400Gy/4-8Gy to the dorsal skin flap of mice has a similar dermatopathological effect 120 hours following irradiation to low dose synchrotron generated broadbeam radiotherapy (11Gy, 22Gy and 44Gy in a single fraction) 84 hours following irradiation (Priyadarshika et al. 2011). In contrast, an MRT dose of

800Gy/~8-16Gy causes higher levels of leukocytic infiltration, epidermal spongiosis and nuclear enlargement compared to both 400Gy/~4-8Gy MRT and a single 44Gy broadbeam dose (Priyadarshika et al. 2011). However, the acute erythematous reaction associated with radiation-induced changes to basal cell density is expected to occur over a number of weeks following broadbeam doses ranging from 11 to 44Gy (Hopewell

1990). Therefore, the histological endpoints and the short follow-up time of this study are not sufficient to conclude that high dose MRT produces an equivalent acute skin reaction to low dose CRT.

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Earlier MRT studies report the gross skin toxicity associated with the treatment of mammary and squamous cell carcinomas implanted in the hind legs of mice (Dilmanian et al. 2003b, Miura et al. 2006). With a single array of microbeams (90µm/300µm;

20mm x 20mm) delivered in successive 1.5mm high tiers, mice tolerate doses up to

970Gy/19Gy at tumour depth without acute moist desquamation or epilation (Dilmanian et al. 2003b). With the same array parameters, 1740Gy/35Gy causes acute epilation and the failure to fully regrow hair by 7 months post irradiation (Dilmanian et al. 2003b) and 1335Gy/33Gy causes transient moist desquamation (Zhong et al. 2003). In comparison, mice receiving broad-beam in the study conducted by Dilmanian et al.

(2003b), experienced complete epilation and failure to fully regrow hair at 30Gy, and moist desquamation at 45Gy, which is close to the MRT valley doses which caused similar skin toxicity. In comparison, late changes such as dermal atrophy and telangiectasia occur in humans a year or more after CRT at single doses of approximately 10.5Gy and 12.5Gy, respectively (Hopewell 1990). However, pig skin is a model that is better suited to approximate human radiotoxicity data when compared to murine models (Hopewell 1990).

Vasculature (major vessels)

The tolerance of vasculature to MRT was first demonstrated in the rat carotid artery, with no histological damage observed at peak/valley doses of 150Gy/4.5Gy using a single array of microbeams (27µm/200µm; 15mm x 7.6mm) delivered as two 3.8mm high vertical tiers (Dilmanian et al. 2003a). With a single microbeam array

(50µm/400µm; 10mm x 10mm) targeting the left hind leg of mice, there is a stable thinning of the tunica media of the saphenous artery in the microbeam path by 25% at 6 and 12 months after an entrance dose of 312Gy/2.7Gy, and complete disappearance of the vascular smooth muscle cells and the tunica media in the microbeam path after

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2000Gy/17.6Gy (van der Sanden et al. 2010). Conversely, the tunica media in the valley regions undergoes hypertrophy which might compensate for a loss of blood pressure along the artery due to damage in the peak regions (van der Sanden et al. 2010). No vascular occlusions were observed in the one-year period following MRT at entrance doses of 312Gy/2.7Gy or 2000Gy/17.6Gy (van der Sanden et al. 2010). For context, stenosis has been reported at doses as low as 10Gy in the middle cerebral artery of human patients receiving a single fraction of radiosurgery using CRT (Yamamoto et al.

1997).

(Table II)

Limitations of current data and future directions

Developing a new normal tissue complication probability model

The mechanisms underlying the efficacy of the periodic spatial alternation of peak/valley dose distributions used in MRT are not well understood and are beyond the scope of this review. However, it can be assumed that a radiation dose of hundreds of

Gray causes saturated cell killing in the peak regions based on the observed loss of nuclei directly in the path of microbeams (Slatkin et al. 1995, Dilmanian et al. 2005,

Serduc et al. 2006, Laissue et al. 2007, Laissue et al. 2013, Schültke et al. 2013).

Therefore, the tolerance of normal tissue to MRT and its ability to exert a self-reparative effect is likely to rely upon the maintenance of a sufficient population of sub-lethally damaged normal progenitor and functional cells in the valley regions.

Consequently, for typical microbeams 25-50µm wide, microbeam spacing is the most critical factor in normal tissue sparing since spacing influences both the magnitude of the valley dose and width of the valley region. Current normal tissue toxicity data for

MRT support this hypothesis, demonstrating that valley dose, rather than peak dose,

225 correlates with existing normal tissue constraints for single fraction radiosurgery using

CRT. However, given that the valley dose does not sufficiently predict the biological equivalence of MRT and CRT (Ibahim et al. 2014) and evidence that near equal valley doses do not equate to similar rates of brain toxicity (Schültke et al. 2013), the proportion of normal tissue receiving the valley dose, or less than tissue-specific threshold dose (Blattmann et al. 2005), could be the most important determinant of gross normal tissue toxicity. Factors related to this parameter include the shape of the valley region (Blattmann et al. 2005), the quantitative relationship between microbeam width and spacing, and the surface area of the interface between peak and valley tissue slices. With the use of highly heterogeneous dose distributions in MRT compared to near homogenous dose distributions in CRT, new normal tissue complication probability and tumour control probability models must be developed and should take into account differing microbeam configurations, particularly microbeam spacing and the shape of the valley.

Few pre-clinical studies are specifically designed to characterise the dose-response of normal tissue to MRT. The diversity of array configurations – including beam spacing – used throughout the existing body of pre-clinical MRT literature makes drawing conclusions about the dose-dependence of normal tissue toxicity difficult. Future studies should characterise organ-specific dose-response relationships based on microbeam spacing, and these data could be seminal in the development of a new normal tissue complication probability model for MRT.

Treatment planning system

A further difficulty with current MRT toxicity data is the lack of depth-dose data at the tissue of interest and the absence of dose-volume data in an anatomical context. Some pre-clinical MRT studies report peak and valley doses at depth in the tissue of interest

226 based on Monte Carlo simulations, but only as a single dose at a specified depth rather than a volume-defined dose. In the absence of three-dimensional treatment planning capabilities, retrospective Monte Carlo simulations based on the varying MRT parameters used in previous studies will allow for the computation of volume-defined doses at depth and a better evaluation of the dose-response relationship of specific normal tissues. The development of a treatment planning system will be fundamental in the translation of MRT to a human trial, facilitating the collection and modelling of dose-volume data and implementation of volumetric dose constraints which are standard in the clinical radiation oncology paradigm (Grotzer et al. 2015). Accurate dose-calculation algorithms suitable for MRT pencil beams (Bartzsch and Oelfke 2013) and planar microbeams (Martínez-Rovira et al. 2012) are currently in development.

Further to this, the development of a treatment planning system, in conjunction with image-guidance capabilities, will allow for MRT to be delivered to clinically realistic proportions of normal tissue in comparison to tumour/target volume. Pre-clinical MRT studies to date have delivered treatment to excessive volumes of normal tissue. As such, the volume of normal tissue exposed to MRT fields in proportion to tumour volumes are vastly exaggerated compared to current CRT treatments. Estimates of the proportion of tumour volume to normal brain in pre-clinical MRT studies are as low as 0.06 (Laissue et al. 1998). Considering the correlation between the size of the target volume and the incidence of adverse events in cranial radiosurgery using CRT (Lawrence et al. 2010) and what is known about the volume effect and the importance of anatomical and physiological factors in normal tissue toxicity (Hopewell 1997, Hopewell and Trott

2000), data should be collected using image-guided MRT to simulate the irradiation of realistic proportions of the brain, and other types of normal tissue, comparable to clinical scenarios. When image-guided MRT, based on pre-determined three-

227 dimensional treatment plans, is implemented, the normal tissue damage seen outside of target tumour volumes in pre-clinical MRT studies could be reduced or even avoided altogether.

Long-term toxicity studies

Long-term toxicity data for MRT a year or more post-irradiation are limited. Only a handful of studies present long-term toxicity outcomes a year or more after MRT

(Laissue et al. 2007, Schültke et al. 2008, van der Sanden et al. 2010, Laissue et al.

2013). Given that MRT delivers large doses in a single fraction, the late effects could be profound and must be evaluated if MRT is shown to be effective and result in prolonged survival.

228

Conclusion

Despite the difficulties in defining dose-response relationships for normal tissue toxicity, it is still clear that a range of normal tissues are remarkably tolerant to MRT, and can tolerate peak doses at least an order of magnitude greater than CRT. Table I and

II summarise the effect of MRT on normal tissue, as a function of peak/valley dose and microbeam parameters.

The proportion of tissue receiving the valley dose, or below a tissue-specific threshold dose, is likely to be the most important metric for predicting normal tissue toxicity in

MRT. New normal tissue complication probability models and organ specific dose- response relationships should therefore take into account microbeam spacing, since spacing determines the magnitude of the valley dose and width of the valley region.

Finally, developing a treatment planning system with the capability of modelling three- dimensional dose distributions for MRT at depth in tissue is a crucial step towards collecting robust normal tissue toxicity data and translating MRT to clinical use.

229

Acknowledgements

None

Declaration of interest

JCC and SS are supported by Early Career Researcher Fellowships from the National

Health & Medical Research Council (NH&MRC) of Australia. LMLS is the recipient of an Australian Postgraduate Award scholarship. The authors acknowledge funding from the NH&MRC and Cancer Council Victoria.

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References

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Table I. MRT-induced macroscopic and microscopic damage to normal brain tissue

Beam Irradiation Field Size MB Animal Anatomical Time post- energy geometry in (Width x Width/Spacing Structural damage to normal Study Model Target MRT (keV) normal tissue Height) (mm) (µm/µm) PED (Gy) VD (Gy) brain tissue

Serduc et al. Healthy Rats Upper cerebral 2 months 107 Unidirectional 4 x 4 25/400 500 3.8 (entrance) No necrosis or other 2014 hemispheres, (median) (AP) observable damage corpus callosum 50/400 280 3 (entrance) No necrosis or other observable damage

50/400 500 5.3 (entrance) Microstructural damage to 100/400 150 – 500 2.6 – 8.6 white matter resulting in the (entrance) increased diffusivity of water; no necrosis

Bouchet et 9LGS Arrays centred at Up to 55 90 Unidirectional 8 x 10 50/200 400 12.5 (at brain No change in blood oxygen al. 2013 bearing Rats tumour located at days (median) (lateral, anterior (350 at brain depth) saturation; slight; minor right caudate and posterior brain depth) changes to vessel morphology nucleus tissue proximal to and decrease in vessel density target)

Bouchet et 9LGS Arrays centred at Up to 45 90 Unidirectional 8 x 10 50/200 400 12.5 (at brain No changes to blood vessel al. 2010 bearing Rats the anterior part days (median) (lateral, anterior (350 at brain depth) size, permeability or of right cerebral and posterior brain depth) morphology; no change to hemisphere tissue proximal to blood volume fraction target)

Bidirectional 50/200 2 x 400 25 (at brain No changes to blood vessel (brain tissue at (700 at brain depth) permeability and blood overlap of AP and depth) volume fraction; increase in lateral cross-fried vessel size; some changes in arrays) vessel morphology

Serduc et al. Genetic Arrays centred at Up to 30 107 Unidirectional 2 x 2 52/200 200 NR No detectable damage 2010 Absence right caudate days (mean) Epilepsy nucleus Multidirectional - 52/200 4 x 200 (700 NR No detectable cerebral lesion; Rats Four arrays at 1cm depth no change in blood brain

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barrier permeability 4 x 200 (700 Multidirectional - 52/200 at 1cm NA(a) Induction of cerebral lesion; Four Interlaced depth) increased blood brain arrays (Seamless) permeability; changes to vessel morphology;

Serduc et al. Healthy Upper part of left Up to 1 90 Unidirectional 3.6 x 4 25/211 312 NR No observable cerebral 2008 Female cerebral month (mean) (AP) oedema Swiss nude hemisphere mice 25/211 1000 10.5 (at 1cm Mild, transient oedema which depth) resolves a week after irradiation

Laissue et Healthy Rat Entire Approx. 1 NR Unidirectional 10 x 10 28/105 150 9.5 (at centre Significant cerebellar al. 2007; Pups (12-14 cerebellum, year (LR) of hindbrain) hypoplasia; Hanson et days old) dorsal pons, microcalcifications in 90% of al. 2013 dorsal medulla, animals posterior aspect of occipital lobes 28/105 50 3.1 (at centre Moderate cerebellar (immature of hindbrain) hypoplasia; hindbrain tissue) 28/210 150 2.7 (at centre microcalcifications in 4-10% of hindbrain) of animals

28/210 50 0.9 (at centre Slight cerebellar hypoplasia; of hindbrain) no microcalcifications observed

Laissue et Healthy Cerebellum Approx. 1 NR Unidirectional 15 x 15 28/210 150 – 600 3.2 – 12.6 (at No necrosis; no abnormalities al. 2007 Weanling year (lateral) (66 – 263 at depth of compared to unirradiated Piglets (42 depth of cerebellum) controls except loss of and 48 days cerebellum) astroglial and neuronal nuclei old) confined to MB path

Dilmanian 9LGS Anterior part of 4 months 66 or 74 Unidirectional 8-10 x 11.4(b) 27/100 500 19 (at centre of No white matter necrosis, et al. 2002 bearing Rats the cerebrum and at (median) (RL) (359 at brain) vascularisation or cerebral (frontal lobe) death centre of oedema observed; significant (variable) brain) microcalcifications

Moderate white matter 27/75 500 33 (at centre of necrosis, significant

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(359 at brain) microcalcifications; slight centre of cerebral oedema; significant brain) vascularisation;

No white matter necrosis or 27/75 250 17 (at centre of vascularisation; slight (179 at brain) cerebral oedema; significant centre of microcalcifications brain)

Laissue et 9LGS Right Striatum At death 49.3 Bidirectional 10 x 12 25/100 2 x 312 NR Highest scores of brain al. 1998 bearing Rats (variable) (median) (AP and RL) 2 x 625 damage; loss of tissue structure

Unidirectional 312, 625 NR Median brain damage score (AP) of 0; no loss of tissue structure

Slatkin et al. Healthy Rats Right cerebral 31 days 48.5 Unidirectional 4 x 4 (20 20/200 312, 625 NR No observable changes in 1995 and cerebellar (median) (AP) Microbeams) peak or valley hemispheres 20/200 2500, 5000 NR Loss of astrocytic and neuronal nuclei confined to MB path; no necrosis

14 days 42/200 2500 NR Loss of astrocytic and neuronal nuclei confined to MB path; no necrosis

42/200 10000 NR Tissue necrosis

31 days 3 Microbeams 20/200 2500, 5000, NR Loss of astrocytic and 10000 neuronal nuclei confined to MB path; no necrosis

MB, microbeam; PED, Peak entrance dose; VD, Valley dose; 9LGS, 9L Gliosarcoma; NR, not reported; NA, not applicable; AP, anterior-to-posterior; RL, right-to-left; LR, left-to-right (a) Four interlacing arrays were used to produce a quasi-seamless 700Gy dose at depth in the target region (b) Field was administered sequentially in three 3.8mm high tiers of microbeams

237

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Table II. MRT-induced toxicity in various normal tissue types with comparison to recommended dose constraints in human CRT

Time Beam Irradiation Field Size MB Animal Anatomical post- energy geometry in (width x height) Width/Spacing Study Model Target MRT (keV) normal tissue (mm) (µm/µm) PED (Gy) VD (Gy) Comments

Eye Human CRT: recommended maximum single fraction doses: lens – 3Gy, retina – 5Gy (Grimm et al. 2011); >10% chance of optic neuropathy with single dose of 12- 15Gy (Mayo et al. 2010)

Sandmeyer et Rats Right 12 NR Unidirectional 10 x 14 25/211 350 ~5 (Laissue et Retinal lesions in 8/16 left eyes, no al. 2008 bearing Cerebral months (proximal to al. 2012) cataracts observed in left eyes C6 Hemisphere target region) Glioma Bidirectional 2 x 350 ~14 (Laissue Retinal lesions in 16/16 right eyes; (tissue at et al. 2012) cataracts in 12/37 right eyes overlap of AP and RL cross- fried arrays) Spinal Cord Human CRT: Max. point dose of 13Gy causes spinal cord myelopathy in less than 1% of cases (Kirkpatrick et al. 2010)

Laissue et al. Healthy Cervical Up to 383 Mean ~ Unidirectional 11 x 21 35/210 253 (at cord 9 (at cord Paresis free at 383 days post irradiation 2013 Rats spinal cord days 100 (RL) depth) depth) (11mm length) 357 (at cord 12.7 (at cord Spinal cord tissue appears histologically depth) depth) normal

373.3 (at 13.2 (at cord Dose calculated to cause paralysis in 50% cord depth) depth) of rats

Skin Human CRT: Threshold single fraction dose to cause: skin atrophy – 10.5Gy, telangiectasia – 12.5Gy (Hopewell 1990)

Dilmanian et Mice Right hind 7 months Median Unidirectional 20 x 20(a) 90/300 970 19 (entrance) No moist desquamation or complete al. 2003b bearing leg of 100 epilation EMT6 or 118 murine 1900 38 (entrance) No moist desquamation; complete epilation

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mammary in 7/10 rats; failure to full regrow hair in carioma 4/5 rats

Zhong et al. Healthy Right hind Up to 30 Median Unidirectional 35 x 15(a) 90/300 1005 25 (entrance) No moist desquamation; minor hair 2003 mice leg days of 100 clumping or 120 1335 33 (entrance) Moist desquamation evidence; severe hair clumping

Priyadarshika Healthy Dorsal skin 5 days Mean Unidirectional 6 x 6 25/200 400 ~4 – 8 Low levels of dermopathological damage, et al. 2011 mice flap of (entrance) comparable to 11, 22 and 44Gy BB 125keV Significantly higher scores of 800 ~8 – 16 dermopathological damage compared to (entrance) 44Gy BB and 400Gy peak MRT

Vasculature Human CRT: Vascular stenosis observed at (major doses as low as 10Gy in a single fraction vessels) (Yamamoto et al. 1997) van der Healthy Left hind Up to 12 Median Unidirectional 10 x 10 50/400 312 2.7 (entrance) Stable thinning of tunica media in peak Sanden et al. mice leg, months of 107 (ventro-dorsal) regions, hypertrophy of tunica media in 2010 including valley regions ; no vascular occlusions saphenous 17.6 artery 2000 (entrance) Complete loss of vascular smooth muscle cells and tunica media in peak regions, hypertrophy of tunica media in valley regions ; no vascular occlusions

MB, microbeam; PED, Peak entrance dose; VD, Valley dose; AP, anterior-to-posterior; RL, right-to-left; BB, broadbeam; NR, not reported (a) Field was administered sequentially in 1.5mm high tiers of microbeams

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Appendix B: Final published version of in vivo dose-equivalence manuscript

Appendix B is the electronic, final published version of the accepted manuscript reproduced in Chapter 4.2. This manuscript was published under an open access agreement in the journal Scientific Reports (Smyth et al. 2018b) by Springer Nature

Limited and is available online at https://doi.org/10.1038/s41598-018-30543-1. I am the primary author of this manuscript and contributed to 60% of the final manuscript. I was involved in planning the experiments and carrying out all aspects of the experimental work, with input from co-authors. I wrote the approved animal ethics protocol and was responsible for the post-irradiation animal monitoring. I analysed and interpreted the data, and drafted the manuscript, with input from all co-authors.

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Appendix C: Methods for mouse brainstem comparative gene expression study

Appendix C contains the methods and materials used for the brainstem irradiations, RNA extractions and next generation RNA sequencing performed for a project currently in progress, as discussed in chapter 6.3. This study intends to compare the response of the brainstem to MRT versus SBBR, based on the acute regulation of gene expression following irradiation. This section is written in the style of a journal article and will be submitted as part of future manuscript. The references for this section are found in the thesis bibliography.

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Methods and materials

Ethics, study design and animal procedures

Twenty-four C57BL/6 mice (male and female, 8 to 10 weeks old) were purchased from the Monash Animal Research Platform and housed at the Australian

Synchrotron animal holding facility at least 48 hours prior to irradiation and for the duration of the experiments. Animal procedures were approved by the University of

Melbourne Office for Research Ethics and Integrity and performed according to relevant guidelines and regulations.

Mice were allocated across three experimental arms. The first two experimental arms received equivalent, median toxic doses (TD50) of MRT (n = 8) and SBBR (n = 8).

These were the TD50 values predicted to cause greater than 15 to 20% weight loss and/or moribund behaviour within two weeks of whole head irradiation (Smyth et al.

2018b). The MRT peak (valley) TD50 was 241 Gy (6.4 Gy) and SBBR TD50 was 13.2

Gy. The third experimental arm received a substantially higher MRT dose of 455 Gy (n

= 4). A further n = 4 mice were allocated to a non-irradiated control group.

Mice were anaesthetised with an intraperitoneal injection of ketamine (80mg/kg) and xylazine (10 mg/kg) and secured gently to a plastic mouse holder. Mice were positioned vertically and irradiated in an anterior to posterior direction. The radiation field for both MRT and SBBR had a total size of 30 x 30 mm which covered the whole brain, brainstem and upper cervical spinal cord. Radiochromic film was placed over the intended target to verify the positional accuracy of the irradiation field.

Mice in the MRT and SBBR TD50 groups were humanely euthanised 4 or 48 hours following irradiation (n = 4 mice per time point and modality). Mice in the 455

Gy MRT group were monitored closely for severe, acute neurological toxicities

254 following irradiation, and were euthanised immediately if ataxia, fitting or loss of balance was observed. Whole brains, including the brainstem, were harvested from the mice and divided into left and right hemispheres. The left hemispheres were stored in

RNAlater RNA stabilisation reagent (QIAGEN®, Hilden, Germany) and kept at -80°C.

The right hemispheres were fixed in formalin for 24 hours before being washed in

Dulbecco’s Phosphate Buffered Saline (DPBS) (Gibco, Life Technologies, Mulgrave,

Australia) and stored at 4°C.

MRT and SBBR irradiation parameters

All irradiations were performed at the Imaging and Medical Beamline (IMBL) of the Australian Synchrotron (Clayton, Victoria). Parameters for beam production at the IMBL, including filtration, magnetic field strength and other radiation therapy components, are described previously by Livingstone et al. (2017). In brief, MRT was delivered as an array of horizontally propagating, vertically orientated microbeams with width of 50 μm and a centre-to-centre spacing of 400 μm. The weighted mean energy of the MRT spectrum was 94 keV and the dose-rate was 319 Gy/s. SBBR consisted of a homogenous field of radiation with mean photon energy of 124 keV, delivered at a dose-rate of 41.3 Gy/s. All doses were prescribed to 5 mm depth in water, assuming scatter conditions and dosimetry previously described in chapter 5 (Smyth et al. 2018b).

The estimated uncertainty (k=1) for the doses delivered for MRT was 5.1% for the peak and 8.6% for the valley. For SBBR the uncertainty (k=1) was 4.8%.

RNA isolation

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The medulla and pons regions of the brainstem were dissected away from the left brain hemispheres under an optical microscope and RNA was isolated from the brainstem using the RNeasy Minikit (QIAGEN®), according to the manufacturer’s instructions. In brief, 30 to 50 μg of brainstem tissue was homogenised with lysis buffer and zirconium oxide ceramic beads (2.8 mm) and then centrifuged for 3 minutes. The aqueous portion of the lysate was collected, combined with an equal volume of 100% ethanol and added to a RNeasy spin column. DNase I (QIAGEN®) incubation was performed on-column to remove genomic DNA contamination from the samples.

Following washing, RNase free water was added to the spin-column membrane and

RNA eluted via centrifugation. The concentration and purity of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermofisher Scientific, Waltham, MA).

RNA sequencing

RNA sequencing (RNA-seq) (Wang, Gerstein & Snyder 2009) was performed at the Australian Genome Research Facility (AGRF, Parkville, Australia). Prior to sample library preparation and sequencing, RNA sample integrity was assessed via electrophoresis using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa

Clara, CA) on the 2100 Bioanalyzer system (Agilent Technologies). The integrity of the brainstem samples was high, with a minimum RIN of 7.6.

A total of 1 μg of RNA per sample was used for library preparation. Samples were processed using the Illumina TruSeq® Stranded mRNA Library Prep kit

(Illumina, San Diego, CA) according to the manufacturer’s protocol. In short, mRNA was purified with Oligo-dT magnetic beads and fragmented via exposure to divalent cations and heat. First strand cDNA was then synthesised via reverse transcription using

256 random primers and second strand cDNA, marked with dUTP in place of dTTP, was synthesised to replace the RNA template and form double stranded (ds) cDNA. The 3’ ends were then adenylated and adapters ligated to the ends of the ds cDNA fragments.

Finally, the non-uracil containing first strand cDNA fragments were selectively enriched by use of a uracil-sensitive polymerase and cDNA amplification was performed.

Library preparations were sequenced on the Novaseq 6000 Sequencing System

(Illumina) with 100 base-pair single end reads generated. There were at least 25 million single end reads per sample. Sequencing reads were in the reverse orientation. Four biological replicates were sequenced per experimental group. Image analysis was performed in real time by the NovaSeq Control Software v1.3.0 and Real Time Analysis v3.3.3 (Illumina). The Illumina bclfastq 2.20.0.422 pipeline was used to generate the sequence data.

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Smyth, Lloyd

Title: Synchrotron radiation therapy for the treatment of cancer

Date: 2019

Persistent Link: http://hdl.handle.net/11343/220148

File Description: Synchrotron radiation therapy for the treatment of cancer

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