The Radiobiological Effects of Proton Beam Therapy: Impact on DNA Damage and Repair
Round 1
Reviewer 1 Report
The authors have taken into account all the comments from the reviewer and significantly improved the manuscript.
Author Response
The authors have taken into account all the comments from the reviewer and significantly improved the manuscript.
Response: We thank the Reviewer for their comment that our manuscript has been significantly improved, and requested no further changes.
Reviewer 2 Report
The revised manuscript presented by the Authors is structured with a different approach compared to the original version. Specifically, physical and technological aspects of proton therapy are now minimised. The review is now focused on the analysis and discussion of DNA damage and repair aspects of proton therapy. In my opinion, this should be already evident from the title, which appears too general in the current version.
I think that focusing on DNA damage and repair aspects helps targeting the goal of this review. However, significant improvements are still necessary from my point of view. First of all, the discussion of several aspects is too qualitative. For instance, the Authors should try to be more quantitative and accurate in the discussion of RBE-related aspects. It should always be clear what is the specific end-point considered for a specific RBE value, otherwise the information provided might be confounding and sometimes misleading.
The Authors should stress what they exactly mean for CDD, since different definitions have been associated to clustered DNA damage over the years. In the manuscript, CDD essentially refers to a biochemical clustering of different types of DNA lesions. Different approaches should at least be mentioned, as for instance the nanometer-scale clustering of lesions (that partially overlaps with CDD as mentioned in the current paper, e.g. Goodhead 10.1093/rpd/ncl498) and micrometer-scale clustering of DNA DSB (e.g. 10.1667/RR2964.1).
Finally, the paper is not easy to read and in my opinion the use of tables could help summarising the most relevant information provided in the text. Similarly, in the Conclusions the Authors indicate some of the experiments that might help shading light on aspects that currently are not well understood. A bulleted list might help to summarise the key points of the discussion.
Author Response
The revised manuscript presented by the Authors is structured with a different approach compared to the original version. Specifically, physical and technological aspects of proton therapy are now minimised. The review is now focused on the analysis and discussion of DNA damage and repair aspects of proton therapy. In my opinion, this should be already evident from the title, which appears too general in the current version.
Response: We appreciate the Reviewer’s comment and given the specific focus of our review, we have now modified the title accordingly to clarify that this is centred on analysis of DNA damage and repair related to proton therapy.
I think that focusing on DNA damage and repair aspects helps targeting the goal of this review. However, significant improvements are still necessary from my point of view. First of all, the discussion of several aspects is too qualitative. For instance, the Authors should try to be more quantitative and accurate in the discussion of RBE-related aspects. It should always be clear what is the specific end-point considered for a specific RBE value, otherwise the information provided might be confounding and sometimes misleading.
Response: We thank the Reviewer for highlighting that our review is more focussed on DNA damage and repair which helps to achieve its targeted goal. We also appreciate the Reviewer’s comment that our review needs to be more quantitative in several aspects. Therefore we have now gone through all the text in detail, and made significant additions to highlight quantitative changes in DNA damage induction (Section 3.2), generation of ROS and cell cycle progression (Section 3.3), DSB repair pathway choice (Section 3.4) and CDD formation and repair (Sections 3.5 and 3.6). We believe that this has significantly improved the quality, and the precise details, contained within our review.
In relation to RBE, as indicated above and in our response to one of the Reviewer’s comments from the original submission, our intention was to focus our review on the cellular DNA damage response follows protons and the comparison to photon irradiation. Therefore we did not aim to review literature on end-points and determination of RBE values from in vitro studies and the uncertainties regarding the clinical use of an RBE of 1.1, which has been covered in detail elsewhere in other reviews (cited in our paper).
The Authors should stress what they exactly mean for CDD, since different definitions have been associated to clustered DNA damage over the years. In the manuscript, CDD essentially refers to a biochemical clustering of different types of DNA lesions. Different approaches should at least be mentioned, as for instance the nanometer-scale clustering of lesions (that partially overlaps with CDD as mentioned in the current paper, e.g. Goodhead 10.1093/rpd/ncl498) and micrometer-scale clustering of DNA DSB (e.g. 10.1667/RR2964.1).
Response: We thank the Reviewer for their comment and have now added a few statements (Page 3, line 93; Page 3, line 108) regarding our definition of complex DNA damage (CDD), which is two or more DNA lesions within 1-2 helical turns of the DNA. This refers to localised damage over short distances within the DNA. However as we acknowledged later on in our review (Page 8, line 293), CDD is difficult to measure, but also specifically define, using in vivo experiments due to technical limitations.
Finally, the paper is not easy to read and in my opinion the use of tables could help summarising the most relevant information provided in the text. Similarly, in the Conclusions the Authors indicate some of the experiments that might help shading light on aspects that currently are not well understood. A bulleted list might help to summarise the key points of the discussion.
Response: We appreciate the Reviewer’s comment. In the revised review, we have now incorporated two tables within Sections 3.2 and 3.4 which now summarise the key information relating to DNA double strand break induction (Table 1) and repair pathway dependence (Table 2), both of which analysing these in the context of proton versus photon irradiation. As also suggested, the Conclusions and Outlook section now contains a bullet-point list at the end (Page 14, line 448) to summarise the key aspects.
Reviewer 3 Report
General comments
The manuscript entitled “radiobiological effects of proton beam therapy” is a thorough and in depth review that does not only complement existing literature, but really goes beyond literature on the same or similar topic.
I have had the pleasure to review an already revised version of this manuscript, in which major amendments have been performed. There are only a few minor items that need to be addressed, some of them are related to radiation physics aspects that have an impact on radiobiological considerations. Otherwise the review is very complete and detailed. Unfortunately the comments of the reviewer(s) that have resulted in the modification indicated in red were not accessible to me. Thus I cannot assess whether the authors have replied in a satisfactory manner.
Specific comments:
Page 1, Line 11: Sentence “…... can spare irradiation of proximal healthy”. Yes, that is true, but also the low entrance dose contribute to the fact that the low to mid dose volume clinical proton therapy is substantially smaller when compared to radiotherapy with high energy photon beams.
Page 2, line 61: Sentence “… a constant value of 1.1 is established….”. I am wondering whether the word “established” is misleading here. There is a long lasting discussion on whether the clinical practice of using an RBE of 1.1 is scientifically correct. The (over-)simplification of using this constant value is rather related to practical reasons and the absence of a validated and accepted model that accounts for RBE variations as a function of its manifold dependencies.
Page 2, line 67: the RBE does not only decrease with dose, it also depends on the dose per fraction, the LET, the α/β ratio of the tissue/cells considered, and on the endpoint considered.
Page 4, line 129: sentence “….as particles with mass and positive charge, interact…..”. This is basically true, but the intensity of proton beams, their LET and resulting secondary particle spectra depend a big deal on the mode of beam delivery. For example, existing radiobiological data from experiments in passively scattered proton beams are influenced by neutrons generated in the metallic structures that scatter protons. On the other hand, such “contaminating” neutrons are not present in state of the art proton beam delivery systems with scanned pencil beams. This radiation physics aspect needs to be mentioned, but not extensively discussed. However, it clearly indicates that proton therapy research needs a truly interdisciplinary approach.
Author Response
General comments
The manuscript entitled “radiobiological effects of proton beam therapy” is a thorough and in depth review that does not only complement existing literature, but really goes beyond literature on the same or similar topic.
I have had the pleasure to review an already revised version of this manuscript, in which major amendments have been performed. There are only a few minor items that need to be addressed, some of them are related to radiation physics aspects that have an impact on radiobiological considerations. Otherwise the review is very complete and detailed. Unfortunately the comments of the reviewer(s) that have resulted in the modification indicated in red were not accessible to me. Thus I cannot assess whether the authors have replied in a satisfactory manner.
Response: We very much appreciate the Reviewer’s kind comments that our manuscript represents a “a thorough and in depth review” that “really goes beyond the literature on the same or similar topic”.
Specific comments:
Page 1, Line 11: Sentence “…... can spare irradiation of proximal healthy”. Yes, that is true, but also the low entrance dose contribute to the fact that the low to mid dose volume clinical proton therapy is substantially smaller when compared to radiotherapy with high energy photon beams.
Response: We appreciate the Reviewer’s comment and have now modified this sentence for clarity.
Page 2, line 61: Sentence “… a constant value of 1.1 is established….”. I am wondering whether the word “established” is misleading here. There is a long lasting discussion on whether the clinical practice of using an RBE of 1.1 is scientifically correct. The (over-)simplification of using this constant value is rather related to practical reasons and the absence of a validated and accepted model that accounts for RBE variations as a function of its manifold dependencies.
Response: We agree with the Reviewer that the word “established” is too strong a word given the uncertainty surrounding using an RBE of 1.1, so this has been modified to “utilized”
Page 2, line 67: the RBE does not only decrease with dose, it also depends on the dose per fraction, the LET, the α/β ratio of the tissue/cells considered, and on the endpoint considered.
Response: In the previous sentence (Page 2, line 64), we had already acknowledged that “RBE depends on both physical factors such as the proton beam energy, the dose fractionation, and dose rate, and biological factors including the type of the tissue, cell cycle stage, the oxygenation level, but also the position of irradiation along the SOBP”. Therefore we feel that this is sufficient.
Page 4, line 129: sentence “….as particles with mass and positive charge, interact…..”. This is basically true, but the intensity of proton beams, their LET and resulting secondary particle spectra depend a big deal on the mode of beam delivery. For example, existing radiobiological data from experiments in passively scattered proton beams are influenced by neutrons generated in the metallic structures that scatter protons. On the other hand, such “contaminating” neutrons are not present in state of the art proton beam delivery systems with scanned pencil beams. This radiation physics aspect needs to be mentioned, but not extensively discussed. However, it clearly indicates that proton therapy research needs a truly interdisciplinary approach.
Response: We thank the Reviewer for their comment, and whilst the particular section referred to is focussed on DNA damage induction and repair, we have extended this sentence (now Page 4, line 136) as suggested to mention about the physical aspect of protons.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This manuscript reviews recent advances in the physics and biology underpinning our understanding of the use of protons in radiotherapy. In particular the authors focus on some of the physical uncertainties associated with energy deposition along the Bragg curve and also the gaps in our knowledge related to mechanisms of DNA damage and repair involving complex lesions. This is a well written and timely review which will be of interest to many researchers in this area. I have the following comments which may help improve the manuscript.
Main issues
1. Abstract, this needs to highlight that beam delivery methods, sources of dose calculation and delivery uncertainty and current developments with a focus on imaging will be covered.
2. Section 2.1. and Figure 1, This section could be improved by adding a few statements, outlining the interrelationship between the slowing down protons along the Bragg curve and LET as this underpins much of the rest of the paper. For completeness it would useful to show the change in LET with the monoenergetic curve that is presented in Fig 1a.
3. Section 2.4.4, The authors rightly highlight the potential for biological-based treatment planning. Several surrogates have been suggested instead of LET only and these should be included, see for example: McMahon SJ, Paganetti H, Prise KM. LET-weighted doses effectively reduce biological variability in proton radiotherapy planning. Phys Med Biol. 2018 Nov 9;63(22):225009. doi: 10.1088/1361-6560/aae8a5. PubMed PMID: 30412471.
4. Section 3.1., for completeness the authors should briefly mention recent in vivo studies aiming to test whether RBE increases for normal tissue toxicity measured as spinal cord damage (see 1: Saager M, Peschke P, Brons S, Debus J, Karger CP. Determination of the proton RBE in the rat spinal cord: Is there an increase towards the end of thespread-out Bragg peak? Radiother Oncol. 2018 Jul;128(1):115-120. doi:10.1016/j.radonc.2018.03.002. Epub 2018 Mar 21. PubMed PMID: 29573823.)
5. Section 3.2, For completeness the authors should mention the alt-NHEJ pathway in their discussion of repair pathway options in section
Minor
Introduction, line 32, “allowing to irradiate larger” to “allowing the irradiation of larger”
Section 3.2, change “within tumorous cells leading to apoptosis” to “within tumours leading to cell death”
Section 3.2.3, lime 429, change “delay in the rate of repair” to “ reduced DNA repair rate”
Section 3.2.4, line 469, change “extremely lethal” to “lethal”
Line 477, change “We recently described first evidence” to “We recently reported for the first time”
Author Response
This manuscript reviews recent advances in the physics and biology underpinning our understanding of the use of protons in radiotherapy. In particular the authors focus on some of the physical uncertainties associated with energy deposition along the Bragg curve and also the gaps in our knowledge related to mechanisms of DNA damage and repair involving complex lesions. This is a well written and timely review which will be of interest to many researchers in this area. I have the following comments which may help improve the manuscript.
Response: We appreciate the Reviewer’s comment that this “is a well written and timely review which will be of interest to many researchers in this area”. Just to note given the comments from other Reviewers who suggested that the scope of the initial submission was too broad, the revised manuscript is now focussed purely on the radiobiology aspects of proton beam therapy. Therefore there has been substantial restructuring and additions to the text (highlighted in red), which we feel have significantly improved the manuscript.
Main issues
1. Abstract, this needs to highlight that beam delivery methods, sources of dose calculation and delivery uncertainty and current developments with a focus on imaging will be covered.
Response: The changes to the abstract suggested by the Reviewer are no longer required given the focus on proton radiobiology.
2. Section 2.1. and Figure 1, This section could be improved by adding a few statements, outlining the interrelationship between the slowing down protons along the Bragg curve and LET as this underpins much of the rest of the paper. For completeness it would useful to show the change in LET with the monoenergetic curve that is presented in Fig 1a.
Response: Section 2.1 and Figure 1 on physical characteristics has now been removed. However we have included into the Introduction of the revised manuscript, Figure 1 which shows the depth-dose distribution of protons as a pristine beam and a SOBP and the relationship of these to energy and LET which is described fully in the text. As the Reviewer corrected pointed out, this is useful for understanding the biological impact of protons described in the remainder of the review.
3. Section 2.4.4, The authors rightly highlight the potential for biological-based treatment planning. Several surrogates have been suggested instead of LET only and these should be included, see for example: McMahon SJ, Paganetti H, Prise KM. LET-weighted doses effectively reduce biological variability in proton radiotherapy planning. Phys Med Biol. 2018 Nov 9;63(22):225009. doi: 10.1088/1361-6560/aae8a5. PubMed PMID: 30412471.
Response: Section 2.4.4 has been removed from the revised manuscript given the focus on radiobiology.
4. Section 3.1., for completeness the authors should briefly mention recent in vivo studies aiming to test whether RBE increases for normal tissue toxicity measured as spinal cord damage (see 1: Saager M, Peschke P, Brons S, Debus J, Karger CP. Determination of the proton RBE in the rat spinal cord: Is there an increase towards the end of the spread-out Bragg peak? Radiother Oncol. 2018 Jul;128(1):115-120. doi:10.1016/j.radonc.2018.03.002. Epub 2018 Mar 21. PubMed PMID: 29573823.).
Response: We appreciate the Reviewer’s suggestion, and the article determining the RBE of protons from rat cervical spinal cord experiments has now been included into Section 2 (previously Section 3.1).
5. Section 3.2, For completeness the authors should mention the alt-NHEJ pathway in their discussion of repair pathway options in section
Response: We thank the Reviewer for their comment and have now added alternative-NHEJ into Section 3.1 (previously 3.2). Furthermore we have significantly revised the figure accompanying this section (Figure 2), which now incorporates schematics of all the relevant DNA repair pathways (BER, classical-NHEJ, alternative-NHEJ and HR). We feel this aids in complementing the text and particularly in visualising the mechanisms through which the repair pathways proceed.
Minor
Introduction, line 32, “allowing to irradiate larger” to “allowing the irradiation of larger”
Section 3.2, change “within tumorous cells leading to apoptosis” to “within tumours leading to cell death”
Section 3.2.3, lime 429, change “delay in the rate of repair” to “ reduced DNA repair rate”
Section 3.2.4, line 469, change “extremely lethal” to “lethal”
Line 477, change “We recently described first evidence” to “We recently reported for the first time
Response: We thank the Reviewer for identifying these minor changes to the text, and these have now been corrected in the revised manuscript.
Reviewer 2 Report
The paper presented by Eirini-Terpsi Vitti and colleagues is a review paper on advances in the physics and biology of proton therapy. However, despite the promise of the title, the review is not really dedicated to the analysis of recent developments in the field of proton therapy.
The paper rather describes basics aspects of the physics and technology of proton therapy treatments, which can be found in several other reviews, papers and even textbooks. The Biology section, which according to many is the current innovation field also in proton therapy, is mainly restricted to the discussion of DNA double-strand breaks. The information presented is also well-established and not really focused on advancements in the field.
For these reasons, in my opinion the paper is not suitable for publication in Cancers in the current status.
Author Response
The paper presented by Eirini-Terpsi Vitti and colleagues is a review paper on advances in the physics and biology of proton therapy. However, despite the promise of the title, the review is not really dedicated to the analysis of recent developments in the field of proton therapy.
The paper rather describes basics aspects of the physics and technology of proton therapy treatments, which can be found in several other reviews, papers and even textbooks. The Biology section, which according to many is the current innovation field also in proton therapy, is mainly restricted to the discussion of DNA double-strand breaks. The information presented is also well-established and not really focused on advancements in the field.
For these reasons, in my opinion the paper is not suitable for publication in Cancers in the current status.
Response: We are disappointed that the Reviewer felt that our article described only the basic aspects of proton therapy. However in response to this we have now removed the physics aspects, and the revised manuscript is now entirely focussed on the radiobiology of proton beam therapy. As a consequence, there has been substantial restructuring and additions to the text (highlighted in red), to further improve the scope and coverage of the review. In particular, there is:-
· A major modification to Figure 2 which now displays more mechanistic details on the DNA repair pathways.
· Section 3.2 which contains additional studies and details on comparisons of DNA damage and their repair in cells following proton and photon irradiation.
· A new section describing evidence of the differences in reactive oxygen species induction and cell cycle progression in cells in response to protons and photons.
· Additional evidence using biophysical models and experimental evidence to support the generation of complex DNA damage after proton irradiation (section 3.5).
· Extended conclusions and outlook statement (Section 4) to highlight the importance of future studies examining biological factors such as tumour hypoxia and the tumour microenvironment in the cellular response to proton beam therapy.
We would disagree with the Reviewer that Chapter 3 in the original submission was restricted to the discussion of DNA double strand breaks, as in fact there very much a specific focus and consideration of the effects of protons in terms of DNA damage complexity at both the chromatin and cellular level. Nevertheless with the substantial modifications to the revised manuscript, we believe our review is now covering new developments in our understanding of the radiobiological effects of protons, which is timely and of interest to researchers working within the field.
Reviewer 3 Report
There are numerous errors in this manuscript. I haven't commented on all of them. The overall problem is the scope of the manuscript is too broad. Therefore, there are numerous sub-sections which fail to review the advances in any sub-field of research.
At best, these sub-sections refer the reader to more comprehensive reviews, at worse, they over-simplify the subject and mislead the reader.
Overall, it is difficult to see what useful purpose this manuscript serves, as it makes little useful contribution to analysis or review of knowledge beyond that which could be found in introductory textbooks or other more comprehensive review articles (often published in this journal).
There are numerous excellent reviews of proton therapy in the literature, and it is difficult to see where this manuscript contributes to the review literature.
Specific comments below:
Page 3, line 81: There are mainly designs of synchrotoron and it is inaccurate to refer to this accelerator type as producing beams between 70 and 250 MeV. This is not a fundamental limitation of the technology, but a practical choice for PBT. Section 2.2 is too brief to usefully inform the reader. Note, in active beam scanning systems, a SOBP is formed in the energy selection system, not in the "delivery system". The is a confusing usage of beamline and delivery system here.
2.4.3.1. Proton Radiography....
- Not mentioned technical challenges to achieve 330 MeV (plus greater impact on cost of PBT).
- No review of current leaders / technologies for achieving pCT.
- What is the current status of development of pCT - it is inadequate to simply state that it is not ready for clinical implementation.
This section is inadequately referenced. This is an overly simplistic overview of a complex field of research with many technical challenges remaining. The simplicity of the overview given here is misleading.
What has the reader gained from reading this section?
The same criticism can be given to sections 2.4.22, 2.4.3.2, 2.4.3.3.
2.4.2.2. To say that early studies look promising is certainly a misleading overstatement without clarification. MR-proton has not yet been demonstrated at clinically useful energies, nor with a gantry system. For this paragraph to be of any use to the reader, it should review which groups have made which advances and critique the contribution of these efforts regarding the future potential of this development.
Section 2.4.3.3. No reference given to state the current research estimates for prompt-gamma resolution. This is a critical omission for a section reviewing imaging techniques for range verification. What are the encouring results? Just saying that there are encouraging results is of no use to the reader
2.4.4. Biological treatment planning.
This is an extremely hot topic in proton therapy research. To allocate 9 lines, given the importance and relevance to the title of the manuscript, this is inadequate. It is grossly under-referenced, the key reference in this section is from 2013, which overlooks many recent advances. Approaches for biological planning have been extensively reviewed in other publications, which are not cited.
Furthermore, Cancers recently published a far superior review of this topic "Mechanistic Modelling of Radiation Responses
by Stephen J McMahon and Kevin M Prise
Cancers 2019, 11(2), 205; https://doi.org/10.3390/cancers11020205"
Furthermore, it is significantly mis-leading due to teh risks associated with biological planning in that it implies that a move away from RBE = 1.1 and traetment plans based on physical dose is likely. There is still a lack of substantial clinical evidence for RBE, and as yet a move away from RBE = 1.1 looks unlikely in the near future. A critical omission is the discussion of the risk of biological planning.
3.1. A frustratingly short paragraph, that does little other than refer the reader to other reviews. The sentence "In spite of the large fluctuation derived 337 from in vitro data, many researchers propose that the use of a constant RBE of 1.1 is reasonable taking 338 into account in vivo experimental data and the clinical experience of the last 60 years [3, 18, 55, 58]." misleads the reader on a critical point regarding the clinical decision to use RBE = 1.1. This decision was based on a pragmatic choice to avoid under-dosing the CTV given the biological uncertainty. Few would agree that an RBE of 1.1 reflects the data, but the statement as written does not reflect the clinical pragmatic decision to minimise risk given the uncertainty.
Section 3.2
"The therapeutic effect of PBT, similar to conventional radiotherapy techniques, relies on significant DNA damage within tumorous cells leading to apoptosis" - Please comment on the relative proportion of apoptotic cells following proton irradiation. Mitotic catastrophe is the dominent mechanism of cell death.
Sections 3.2 onwards are well written, and well referenced, although Section 3.2.4 seems to mainly review the authors own work. An expansion of section 3 with an emphasis on proton therapy would be useful.
However, there is a superior review of this topic published in Cancers, "Complex DNA Damage: A Route to Radiation-Induced Genomic Instability
and Carcinogenesis", Mavragani et al, 2017.
Author Response
There are numerous errors in this manuscript. I haven't commented on all of them. The overall problem is the scope of the manuscript is too broad. Therefore, there are numerous sub-sections which fail to review the advances in any sub-field of research.
At best, these sub-sections refer the reader to more comprehensive reviews, at worse, they over-simplify the subject and mislead the reader.
Overall, it is difficult to see what useful purpose this manuscript serves, as it makes little useful contribution to analysis or review of knowledge beyond that which could be found in introductory textbooks or other more comprehensive review articles (often published in this journal).
There are numerous excellent reviews of proton therapy in the literature, and it is difficult to see where this manuscript contributes to the review literature.
Response: We understand that the Reviewer thought that the scope of our review was too broad. Therefore we made the decision to remove the physics sections, and to focus entirely on the radiobiology of protons in the context of the impact on DNA damage and how this is repaired through DNA damage response pathways. We’ve made substantial changes and additions to the text (highlighted in red), which we feel have significantly improved the focus of the review and to the overall quality. We believe our particular focus on the impact of protons on DNA damage complexity relevant to distal edge effects and how this is signalled at the level of chromatin and processed in cells, add a different context to what has been reported previously.
Specific comments below:
Page 3, line 81: There are mainly designs of synchrotoron and it is inaccurate to refer to this accelerator type as producing beams between 70 and 250 MeV. This is not a fundamental limitation of the technology, but a practical choice for PBT. Section 2.2 is too brief to usefully inform the reader. Note, in active beam scanning systems, a SOBP is formed in the energy selection system, not in the "delivery system". The is a confusing usage of beamline and delivery system here.
2.4.3.1. Proton Radiography....
- Not mentioned technical challenges to achieve 330 MeV (plus greater impact on cost of PBT).
- No review of current leaders / technologies for achieving pCT.
- What is the current status of development of pCT - it is inadequate to simply state that it is not ready for clinical implementation.
This section is inadequately referenced. This is an overly simplistic overview of a complex field of research with many technical challenges remaining. The simplicity of the overview given here is misleading.
What has the reader gained from reading this section?
The same criticism can be given to sections 2.4.22, 2.4.3.2, 2.4.3.3.
2.4.2.2. To say that early studies look promising is certainly a misleading overstatement without clarification. MR-proton has not yet been demonstrated at clinically useful energies, nor with a gantry system. For this paragraph to be of any use to the reader, it should review which groups have made which advances and critique the contribution of these efforts regarding the future potential of this development.
Section 2.4.3.3. No reference given to state the current research estimates for prompt-gamma resolution. This is a critical omission for a section reviewing imaging techniques for range verification. What are the encouring results? Just saying that there are encouraging results is of no use to the reader.
2.4.4. Biological treatment planning.
This is an extremely hot topic in proton therapy research. To allocate 9 lines, given the importance and relevance to the title of the manuscript, this is inadequate. It is grossly under-referenced, the key reference in this section is from 2013, which overlooks many recent advances. Approaches for biological planning have been extensively reviewed in other publications, which are not cited.
Furthermore, Cancers recently published a far superior review of this topic "Mechanistic Modelling of Radiation Responses
by Stephen J McMahon and Kevin M Prise
Cancers 2019, 11(2), 205; https://doi.org/10.3390/cancers11020205"
Furthermore, it is significantly mis-leading due to teh risks associated with biological planning in that it implies that a move away from RBE = 1.1 and traetment plans based on physical dose is likely. There is still a lack of substantial clinical evidence for RBE, and as yet a move away from RBE = 1.1 looks unlikely in the near future. A critical omission is the discussion of the risk of biological planning.
Response: As mentioned in our response to the general overview, the revised submission has a major focus on the radiobiology of proton beam therapy and therefore all the major comments above do not apply to the current manuscript.
3.1. A frustratingly short paragraph, that does little other than refer the reader to other reviews. The sentence "In spite of the large fluctuation derived 337 from in vitro data, many researchers propose that the use of a constant RBE of 1.1 is reasonable taking 338 into account in vivo experimental data and the clinical experience of the last 60 years [3, 18, 55, 58]." misleads the reader on a critical point regarding the clinical decision to use RBE = 1.1. This decision was based on a pragmatic choice to avoid under-dosing the CTV given the biological uncertainty. Few would agree that an RBE of 1.1 reflects the data, but the statement as written does not reflect the clinical pragmatic decision to minimise risk given the uncertainty.
Response: Our intention in Section 3.1 (now Section 2) was indeed to summarise the clinical use of an RBE of 1.1 and the contribution of LET to RBE determined from in vitro studies, as the uncertainties of this have already been covered in detail in a number of reviews. Indeed we originally cited two recent reviews extensively covering this topic (Willers et al., 2018, Radiother Oncol; Mohan et al., 2017, Acta Oncol). We therefore wanted to focus more on the impact of protons on DNA damage and how this is repaired through the cellular DNA damage response. However we agree with the Reviewer that the statement identified in this section is slightly misleading, and have revised this accordingly. We have also included additional details on observed increases in RBE particularly at the distal fall-off, and cited evidence from a recent in vivo study showing RBE of 1.1-1.3 in rat spinal cords (Saager et al., 2018, Radiother Oncol).
Section 3.2
"The therapeutic effect of PBT, similar to conventional radiotherapy techniques, relies on significant DNA damage within tumorous cells leading to apoptosis" - Please comment on the relative proportion of apoptotic cells following proton irradiation. Mitotic catastrophe is the dominent mechanism of cell death.
Response: We agree with the Reviewer, which was also pointed out by Reviewer 1, and have now changed “apoptosis” to “cell death” (in Section 3.1).
Sections 3.2 onwards are well written, and well referenced, although Section 3.2.4 seems to mainly review the authors own work. An expansion of section 3 with an emphasis on proton therapy would be useful.
However, there is a superior review of this topic published in Cancers, "Complex DNA Damage: A Route to Radiation-Induced Genomic Instability
and Carcinogenesis", Mavragani et al, 2017.
Response: We thank the Reviewer for commenting that Section 3.2 onwards was well written. We would like to point out the significant changes in the revised review which we hope will address the Reviewer’s comment on further expansion of this section. The major changes are as follows:-
· A major modification to Figure 2 which now displays more mechanistic details on the DNA repair pathways.
· Section 3.2 which contains additional studies and details on comparisons of DNA damage and repair in cells following proton and photon irradiation.
· A new section describing evidence of the differences in reactive oxygen species induction and cell cycle progression in cells in response to protons and photons.
· Additional evidence using biophysical models and experimental evidence to support the generation of complex DNA damage after proton irradiation (section 3.5).
· Extended conclusions and outlook statement (Section 4) to highlight the importance of future studies examining biological factors such as tumour hypoxia and the tumour microenvironment in the cellular response to proton beam therapy.
We understand that Section 3.2.4 (now Section 3.6) is mainly focussed on our research although there is little data available in the literature examining the cellular response to protons at the chromatin level, which demonstrates the novelty and importance of our work. We’ve also included citation to our recently published work (Carter et al., 2019, Int. J. Rad. Oncol. Biol. Phys) which has further strengthened our experimental research in this area. Nevertheless, we feel that the numerous additions to other sections of the review provide more coverage of proton radiobiology.
Regarding the indicated review (Mavragani et al., 2017), in fact we had cited this article in our original submission. However this is very much focussed on complex DNA damage and its impact on carcinogenesis, rather than our review which provides current evidence for the generation and impact of complex DNA damage following proton beam therapy. Admittedly the available literature on this topic is quite limited despite this being established as a critical factor in the radiobiology of proton therapy, but we feel it is important to highlight and address the current state of knowledge.
