A Multiomics Assessment of Preoperative Exercise in Pancreatic Cancer Survivors Receiving Neoadjuvant Therapy: A Case Series

Round 1

Reviewer 1 Report

The paper is clear and well written. Usually, all numbers are considered results. Additionally, the integration of the Table 1-2 inside the methods make not fluid the read. Did the patient have a tailored diet or any particular facilitation, such as specialistic visit, during or before the study? Was assessed the nutritional status? Are the patients familiar with the exercise?

Author Response

Reviewer 1

Reviewer Comment: The paper is clear and well written. Usually, all numbers are considered results. Additionally, the integration of the Table 1-2 inside the methods make not fluid the read. Did the patient have a tailored diet or any particular facilitation, such as specialistic visit, during or before the study? Was assessed the nutritional status? Are the patients familiar with the exercise?

Authors’ Response: Thank you for your kind comments. We have moved Table 1 and 2 to the results section. Regarding diet and exercise familiarization, the following has been added to Section 2.1: ‘Participants 1 and 3 reported regular exercise prior to diagnosis and Participant 2 reported regular but declining physical activity over the past several years. All participants received standard of care interventions in addition to the exercise intervention, which included appointments with a registered dietician.’

Reviewer 2 Report

The manuscript titled “A Multiomics Assessment of Preoperative Exercise in Pancreatic Cancer Survivors Receiving Neoadjuvant Therapy: A Case Series” aims to investigate the effects of exercise intervention prior to tumor resection to alter circulating metabolites, proteins, lipids, and skeletal muscle mass in patients with pancreatic cancer. The authors utilize a multifaceted approach incorporating metabolomics, proteomics, lipidomics, and body composition (DXA) to identify correlations between circulating molecular markers and changes to skeletal muscle mass in patients after exercise therapy prior to surgery. The authors identify multiple, significantly changed molecules involved in inflammation and immune response, mitochondrial metabolism, and proteostasis that correlate with improved functional outcomes (ie., 400 MWT, sit to stand, etc…) in patients. This study reports interesting results and provides an exercise therapy to improve the quality of care of patients with pancreatic cancer. However, minor revisions to the manuscript would enhance its impact, my suggestions are below.

1.     If possible, the addition of relevant metabolomic, proteomic, or lipidomic data from patients in other studies investigating similar changes with neoadjuvant therapy in pancreatic cancer or other cancers would enhance the study. Even though patients with similar age is unlikely, the addition of data reporting changes in circulating metabolites, proteins, lipids with exercise could also be used if available. This would give some detail as to which changes are influenced by exercise and which changes are influenced by the neoadjuvant therapy or tumor resection. Including these data as a panel figure, supplement, or a short dialogue in the discussion would improve the manuscript.

2.     The authors provide data for correlations between exercise intervention and skeletal muscle mass. Are there any correlations between exercise intervention and changes in fat mass? If so, these results should be included in the manuscript.

3.     Were there any significant or notable differences in metabolites, proteins, lipids between patient one and patient two from baseline to pre-surgery time points? It is interesting that patient one gained approximately 8 kg of total mass and patient two lost 2 kg prior to surgery, while both had physiological and functional improvements with exercise intervention. Inclusion or discussion of these data, if possible, would improve the study.

4.     Figure 1B, there is a misspelling of appendicular. Figure 1C, there is a misspelling of metabolomics. 

The paper is well written. There are minor spelling errors that are identified in my comments.

Author Response

Reviewer 2

The manuscript titled “A Multiomics Assessment of Preoperative Exercise in Pancreatic Cancer Survivors Receiving Neoadjuvant Therapy: A Case Series” aims to investigate the effects of exercise intervention prior to tumor resection to alter circulating metabolites, proteins, lipids, and skeletal muscle mass in patients with pancreatic cancer. The authors utilize a multifaceted approach incorporating metabolomics, proteomics, lipidomics, and body composition (DXA) to identify correlations between circulating molecular markers and changes to skeletal muscle mass in patients after exercise therapy prior to surgery. The authors identify multiple, significantly changed molecules involved in inflammation and immune response, mitochondrial metabolism, and proteostasis that correlate with improved functional outcomes (ie., 400 MWT, sit to stand, etc…) in patients. This study reports interesting results and provides an exercise therapy to improve the quality of care of patients with pancreatic cancer. However, minor revisions to the manuscript would enhance its impact, my suggestions are below.

Reviewer Comment: If possible, the addition of relevant metabolomic, proteomic, or lipidomic data from patients in other studies investigating similar changes with neoadjuvant therapy in pancreatic cancer or other cancers would enhance the study. Even though patients with similar age is unlikely, the addition of data reporting changes in circulating metabolites, proteins, lipids with exercise could also be used if available. This would give some detail as to which changes are influenced by exercise and which changes are influenced by the neoadjuvant therapy or tumor resection. Including these data as a panel figure, supplement, or a short dialogue in the discussion would improve the manuscript.

Authors’ Response: Thank you for this salient point. We are actively and prospectively collecting samples now in other patient populations to expand this research, though these findings would not be able to be included in this manuscript. To the best of our ability, we have been unable to find any additional omics studies looking at cancer survivors undergoing exercise therapy that we can include in the discussion either. We hope that this case study serves as a foundation to motivate future research in this area.

Reviewer Comment: The authors provide data for correlations between exercise intervention and skeletal muscle mass. Are there any correlations between exercise intervention and changes in fat mass? If so, these results should be included in the manuscript.

Authors’ Response: Thank you for the suggestion. We focus on correlations with lean mass in this study as this may be more clinically relevant in this population, which is at risk for cancer-related cachexia (seen in two participants). Our focus on associations with measures of fitness and lean mass is supported by research demonstrating an association between these constructs and surgical outcomes (references 6 and 7 in the manuscript). We did analyze these data for correlation with fat mass as well now, which provided supportive data for the approach by reproducing published findings. We have added these data to a new Figure 3, expanded the results section, and provided additional discussion in lines 380-383.

Reviewer Comment: Were there any significant or notable differences in metabolites, proteins, lipids between patient one and patient two from baseline to pre-surgery time points? It is interesting that patient one gained approximately 8 kg of total mass and patient two lost 2 kg prior to surgery, while both had physiological and functional improvements with exercise intervention. Inclusion or discussion of these data, if possible, would improve the study.

Authors’ Response: Thank you for the observation. Patient 2 did not present with cachexia related weight loss at baseline unlike Patient 1. Changes for both patients were beneficial for their given fitness status: a recovery of recent lost weight in Patient 1 and a decrease in fat mass and increase in lean mass in Patient 2. As such, these represent two potential “templates” of patients: cachectic vs normal body composition. However, more elaborate analyses such as correlation analyses are difficult to perform with such a limited sample set.

Reviewer Comment: Figure 1B, there is a misspelling of appendicular. Figure 1C, there is a misspelling of metabolomics. 

Authors’ Response: Thank you for catching these. Fig 1B spelling has been corrected. Fig 1C was referring to the measurement of metals, referred to as “Metalomics”. However, to avoid confusion, we specified that these measurements were ICP-MS based and therefore separate from metabolomics.

Reviewer 3 Report

Travis Nemkov and colleagues examined the influence of preoperative exercise in pancreatic cancer patients treated with neoadjuvant therapy. Although the effects of exercise during neoadjuvant treatment are quite important, there are some flaws that should be corrected before publication.

The authors stated in the title that the patients were cancer survivors. How did the authors define that? Since one patient was not resected and received palliative treatment, it is hard to believe that this patient survived for a long time. Therefore, the authors should remove cancer survivors from the title and replace it with e.g. patients.

On page 4, line 163, the authors state that the data were analysed using "an in-house R pipeline". Please specify this, otherwise it is difficult to understand the process of data analysis.

Since only subjects 1 and 2 underwent resection, subject 3 should be excluded from the analysis, as this patient most likely already had metastatic disease at the time of enrollment. This is because micrometastases are often not detected during staging with CT or MRI imaging.

The authors highlighted the anti-inflammatory role of exercise. Were the authors able to validate the results of the GO enrichment analysis with changes in blood cell populations?

In addition, the ECOG status of the patients would be very interesting as this is a limitation for exercise in many cases. The authors should also report the ECOG status after resection.

The number of cases is very limited, it would be useful to compare the results of the study with other studies. Otherwise it remains unclear whether the results can be transferred to the clinical situation.

Furthermore, the authors should discuss the significance of the observed changes in more detail to highlight how the observed changes could contribute to better patient outcomes or be used for new therapeutic approaches.

Since the part of the discussion on page 12, line 378 and following, does not discuss relevant aspects of the research presented in the paper, and also consists mostly of self-citations, this part should be removed.

please check the report

Author Response

Reviewer #3

Travis Nemkov and colleagues examined the influence of preoperative exercise in pancreatic cancer patients treated with neoadjuvant therapy. Although the effects of exercise during neoadjuvant treatment are quite important, there are some flaws that should be corrected before publication.

Reviewer Comment: The authors stated in the title that the patients were cancer survivors. How did the authors define that? Since one patient was not resected and received palliative treatment, it is hard to believe that this patient survived for a long time. Therefore, the authors should remove cancer survivors from the title and replace it with e.g. patients.

Authors’ Response: We utilized the widely accepted NCI definition of cancer survivor (Source: https://www.cancer.gov/about-cancer/coping/survivorship#:~:text=An%20individual%20is%20considered%20a,States%20who%20are%20cancer%20survivors) and have qualified this definition starting in line 36 of the revised text.

Reviewer Comment: On page 4, line 163, the authors state that the data were analysed using "an in-house R pipeline". Please specify this, otherwise it is difficult to understand the process of data analysis.

Authors’ Response: Thank you for pointing this out, as it was mistakenly included in the submitted draft. This statement was ultimately not relevant to this manuscript. R was only used to generate enrichment plots using the ggplot package. This has been clarified by removing the statement on page 4 and adding clarity in Section 2.9.

Reviewer Comment: Since only subjects 1 and 2 underwent resection, subject 3 should be excluded from the analysis, as this patient most likely already had metastatic disease at the time of enrollment. This is because micrometastases are often not detected during staging with CT or MRI imaging.

Authors’ Response: The Reviewer’s point is well taken, even in consideration that all samples were collected in close proximity of multidisciplinary reviews (we have added this detail to section 2.1). The goal of this manuscript is to demonstrate the potential of this approach, highlight the type of data that can be acquired, and the subsequent hypotheses that can be generated. We acknowledge the small number of subjects recruited for this pilot study, and while we cannot rule out the presence of micro metastases, do not feel that a re-analysis of all datasets would provide substantial changes to a case study with limited number of subjects. To help compensate, we have clearly labeled the graphs indicating individual participant data points. In addition, we have added additional statements in lines 372-375 to highlight this is as a proof of principal rather than a comprehensive study into molecular mechanisms of exercise response in PDAC survivors.

Reviewer Comment: The authors highlighted the anti-inflammatory role of exercise. Were the authors able to validate the results of the GO enrichment analysis with changes in blood cell populations?

Authors’ Response: This is an excellent point by the Reviewer, and we wished for the same. The current study, however, was only able to utilize the banked samples linked with a de-identified dataset. As blood cell outcomes were not collected in the original protocol, they are not accessible for this investigation. We have included text in lines 398-403 to forecast this research.

Reviewer Comment: In addition, the ECOG status of the patients would be very interesting as this is a limitation for exercise in many cases. The authors should also report the ECOG status after resection.

Authors’ Response: The medically documented ECOG was not recorded in the initial protocol and is not available in the current dataset. However, detailed descriptions of recruitment and patient functional status (including objective and patient-reported outcomes) are presented in detail in the original report, cited in this investigation. Retrospectively, the senior author is a physical therapist and would estimate that all participants had an ECOG status of 0-1 at all reported timepoints, as they were able to complete the study physical assessments. This point has been emphasized in the text (p.2 line 81): ‘All participants were functionally able to complete all fitness and functional assessments along with a personalized exercise intervention.’

Reviewer Comment: The number of cases is very limited, it would be useful to compare the results of the study with other studies. Otherwise it remains unclear whether the results can be transferred to the clinical situation.

Authors’ Response: We acknowledge the Reviewer’s point and kindly recognize the limitation of presenting data from a case series. To compensate, we have tried to focus on the proof-of-concept nature of the current investigation. We have added citations in the first paragraph of the discussion describing a recent clinical trial in this population demonstrating similar fitness and functional outcomes as were observed here, and the one other known investigation of exercise-associated mechanisms in this population, observing possible tumor vasculature remodeling.

Reviewer Comment: Furthermore, the authors should discuss the significance of the observed changes in more detail to highlight how the observed changes could contribute to better patient outcomes or be used for new therapeutic approaches. Since the part of the discussion on page 12, line 378 and following, does not discuss relevant aspects of the research presented in the paper, and also consists mostly of self-citations, this part should be removed.

Authors’ Response: The final paragraph of the discussion has been rewritten to better address how the presented data could inform novel therapeutic approaches and increase relevance to the rest of the paper. We have expanded the discussion to provide better context for the focus of this paragraph, which is to describe how future advancements in the fields of remote sampling combined with mass spectrometry can expand adaptability of the case study presented herein. We have cited additional publications for use of dried blood sampling for omics analysis, as well as included our manuscripts that are some of the few which have used this technique for monitoring exercise responses.

Round 2

Reviewer 3 Report

The revised version of the study shows significant improvements. As previously emphasized, the definition of cancer survivor used is inappropriate for patients with ductal adenocarcinoma of the pancreas, as there are virtually no long-term survivors who can be considered true cancer survivors. Cancer survivor should therefore be removed from the title and the study as it is misleading, especially as one participant in the study has already developed metastatic disease.

As mentioned previously, the authors should exclude the metastatic patient from the analyses, as there is considerable doubt that the results of the analyses limited to the two patients who underwent the full study protocol will reach the same conclusion as the current analyses, which include the data of the metastatic patients. The fact that this is a pilot study should not lead to false or misleading results being published due to lack of precision in data analysis.

Author Response

Reviewer #3

Reviewer Comment: The revised version of the study shows significant improvements. As previously emphasized, the definition of cancer survivor used is inappropriate for patients with ductal adenocarcinoma of the pancreas, as there are virtually no long-term survivors who can be considered true cancer survivors. Cancer survivor should therefore be removed from the title and the study as it is misleading, especially as one participant in the study has already developed metastatic disease.

Authors’ Response: We appreciate the reviewer’s concern about the term ‘cancer survivor’ and acknowledge the unfortunately very low long term survival rate for patients diagnosed with PDAC. However, we are hesitant to change this term as we are using it as defined by the National Cancer Institute, which we state in the introduction (https://cancercontrol.cancer.gov/ocs/definitions). This definition is also supported by the American Society of Clinical Oncology (https://www.cancer.net/survivorship/what-cancer-survivorship) and the American Cancer Society (https://www.cancer.org/cancer/survivorship.html). Per the NCI “There is no one face or kind of cancer survivor. Some have been treated for their cancer and remain cancer-free, while others continue to live with cancer.” This indicates that the term ‘survivor’ should not be used solely in reference to an individual who is cancer-free or has lived with their disease for an extended period of time. Our title and manuscript clearly indicates the phase of survivorship the current participants are experiencing by stating that they are receiving neoadjuvant treatment, with more details on each individual in the Methods section.”

Reviewer Comment: As mentioned previously, the authors should exclude the metastatic patient from the analyses, as there is considerable doubt that the results of the analyses limited to the two patients who underwent the full study protocol will reach the same conclusion as the current analyses, which include the data of the metastatic patients. The fact that this is a pilot study should not lead to false or misleading results being published due to lack of precision in data analysis.

Authors’ Response: We would like to kindly emphasize that the value of this case study is to establish the utility of a multiomics platform in molecularly characterizing blood in response to exercise of all cancer patients, not just patients who respond positively. And while we cannot definitely know whether this participant was metastatic when the blood sample was taken, to accommodate this critique, we have performed an analysis of ASMI and include the GO enrichment analysis below as an example. The removal of Participant 3 from the analysis has only a minimal effect on the results. For example, while acute phase response is the top hit followed by opsonization in the original analysis, their order is flipped in the analysis that excludes Participant 3 though the p-values remain the same (please see the attached figure, with the original GO Enrichment results on the right, and the new results on the left generated with proteomics data only from Participants 1 and 2).

In that light, we believe it is important to include participant 3, as this reveals important information. For example, Patient 3 had the highest baseline albumin and lowest LPI(18:1) and PI (16:0/22:6). The former might indicate a higher inflammatory status at the inception of the exercise program, which is supported by higher levels of CRP, SAA1, and SAA4 (among other inflammatory proteins) in this participant. We’ve amended Figure 4 and have added the following to the discussion:

“The primary limitation of this proof-of-concept study was low patient recruitment. Due to the limited number of participants in this study, systems analysis were performed on all five samples (including before and after samples for Participants 1 and 2, and only before for Participant 3). Subsequent analysis of ASMI protein correlates when including only samples from Participants 1 and 2 revealed highly similar GO enrichment (Supple-mental Figure 1). Moreover, inclusion of all samples from participants in the data analysis highlighted that Participant 3 had higher inflammatory protein levels at the outset, including albumin, SAA1/2/4, and CRP. As a case study with only 3 subjects, it is not possible to make broad conclusions regarding inflammatory status. However, these data do highlight the potential in monitoring patient status and forecasting exercise or treatment response, as it was discovered prior to surgery that Participant 3’s cancer had metastasized and she was transitioned to palliative care.”

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Thank you for the detailed response.

The use of the term 'cancer survivors' is not appropriate, even if it is based on the NCI guidelines mentioned. This is because the term is misleading, as emphasized by ASCO.

Furthermore, the term primarily refers to studies in large populations and is not suitable in the context of the pilot study with only three participants.

To compound the issue, the authors have not provided any details regarding the patients' follow-up, potentially leading readers to believe that they are long-term survivors. This is particularly inappropriate for patient three, given their metastatic stage.

After demonstrating that the analysis of two patients yields comparable results to the analysis including metastasized patients, the authors should limit the overall analyses to the two patients.

Including metastatic patients based on particularly favorable preliminary values for the analysis is unscientific cherry-picking and should be avoided. This is especially true since the character of a pilot study should not allow the publication of incorrect data. Regardless of the pilot study, statements are made that have potential clinical implications.

Author Response

Thank you for your detailed review and insightful feedback. We appreciate your concerns regarding the use of the term "cancer survivors" and our study's scope and methodology. While we acknowledge the dismal survival rates for patients with pancreatic cancer, we have sought to align our terminology with broad consensus within the oncology field and have referenced guidelines that advocate for this term's use across various cancer stages to emphasize its relevance and inclusivity. Our study's pilot nature aimed to explore preliminary effects of exercise interventions in pancreatic cancer patients as read out by multi-omics, understanding the inherent limitations of our small sample size.

Including a participant with metastatic cancer was a deliberate choice, aimed at capturing a broad spectrum of responses to exercise interventions, which we believe enriches our preliminary findings and contributes to a more comprehensive understanding that can guide future research. This individual was included in our prior study, which is referenced throughout, and thus we assure you that our study design and participant inclusion were driven by predefined scientific criteria, thus ensuring transparency and adherence to rigorous standards.