The Effect of Lung Resection for NSCLC on Circulating Immune Cells: A Pilot Study

Round 1

Reviewer 1 Report

In the study “The Effect of Lung Resection for NSCLC on Circulating Immune Cells” Phillips et al analyzed the effects of surgical resection on the profile of circulating immune cells at different time points.

This is an indeed interesting manuscript. However, some issues should be addressed before publication can be recommended from my side. Please find below my suggestions

1)      Number of analyzed patients should be stated in the abstract

2)      Study design should be stated in the title e.g. The Effect of Lung Resection for NSCLC on Circulating Immune Cell – a pilot study

3)      At the end of the introduction you state that you performed analyses in four additional patients but it is unclear to me how many total patients have been analyzed until the end of the introduction

4)      Regarding pathologic staging, do you mean benign=carcinoid? This should not be the case since carcinoids should be staged according to the 8^th TNM classification, just like NSCLC and you should think about excluding the carcinoid, since it is not fitting well to the rest of the study population, same for Granuloma (maybe this is the benign case? Than you should clarify that it serves as control) and Carcinosarcoma

5)      During the whole manuscript you should state how many samples/patients have been represented in the corresponding results

6)      Since I am no expert in clustering analyses, I recommend to get the manuscript reviewed by an expert in this field and a biostatistician. From my impression there might be a severe issue regarding multiple testing and testing different kind of clusters

7)      In general, the reasons why these multiple analyses have been performed is hard to understand from the clinical point of view. You should declare in your manuscript and also in your abstract the main findings. Sometimes, the manuscript is hard to follow and overloaded with different kind of clusters that – in the end – showed all the more or less same result that you summarize in line 344. Please focus on telling a key message

8)      At the end, the Tregs did not change the way one might expect if I got it right. If this is the finding, it is okay and you should also state this result in your abstract and results so that the reader knows the key finding

9)      The most important findings would be the changes at time of recurrence. You should at least discuss this issue (also I don´t expect –based on your results - such changes since the surgical removal did not change the Tregs and the other way around is thus also unrealistic)

10)   In general, this is an interesting topic and we need further research regarding changes of immune cell population during cancer therapy. If the Tregs have not changed you still should publish this results BUT you have to discuss the study limitations (small number of patients, heterogeneous study population, histology etc) and think of verification/falsification possibilities of your results since you performed a pilot study which indicates that a bigger study should follow…

none

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript by Phillips J and colleagues the authors set out to investigate the compositions and status of circulating immune cells, specifically regulatory T cells (Treg) post lung resection in NSCLC patients. Samples from a total of 19 patients were used for this study. Immunophenotyping was done using standard flow cytometry, as well as high-resolution spectral flow cytometry, single cell (sc) RNAseq and scTCR sequencing. Analysis was performed using pre-operative samples and compared with samples from post-operation period 2 weeks for short-term assessment and post operation times 6, 12 and 18 months for long-term assessment. Long-term assessment was done in a subset of patients. The authors mentioned that while previous studies have reported increased frequency of Tregs post resection, which specific subset of Treg increases and the functional consequence of this increase is unknown. Considering the emerging importance of Treg in cancer prognosis, recurrence and response to immune therapy, the significance of this study is undoubtedly very high.

Unfortunately, the study is clearly negative as the standard gating or the use of more cutting-edge technologies failed to detect any meaningful difference in Treg subsets post-surgery. In fact, they did not detect much change in any immune cell types 2-weeks post-surgery or at longer time points. There is no doubt that a nicely designed study could be negative, but the knowledge gained from the negative study could be valuable for the scientific community. The negative outcome of this study does not bother this reviewer as much its design.

The major problem with this manuscript is its complexity and the lack of proper descriptions of the samples used for various analyses and their justifications. The title of this manuscript describes this study as the one about circulating immune cells. However, both lung tissues and lymph nodes  were also analyzed. Which analysis used what kind of sample and the rationale behind this decision is severely lacking throughout the manuscript. For example, many times authors have used the term ‘paired samples”; are those paired within patients, across times or between various analyses types? Overall, I had a difficult time following this manuscript to make any conclusion.

Author Response

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Reviewer 3 Report

Phillips et al. described the levels of immune cells, particularly regulatory T cell (Treg) subsets, before and after lung resection for non-small cell lung cancer with the aim of assessing the correlation between peripheral blood changes in Treg populations and those present in the tumor and local lymph node microenvironments. This is an interesting topic; however, the study has some concerns.

1. The 'Introduction' and ‘Methods’ sections need to be improved and clarified. For instance, it is not clear to the reader the role of circulating Treg in tumor microenvironment and their link to immune checkpoint (line 49-50).

2. The number of enrolled subjects must be explicit, even in the abstract, as it appears only in Table 1.

3. Furthermore, it would be useful to have more information on the pharmacological treatment of the enrolled subjects and their comorbidities, such as COPD. Indeed, as reported by several authors, alterations in immune homeostasis caused by changes in the number or function of Treg cells or immune checkpoints, like PD-1/PD-L1 axis, occur frequently in COPD patients (PMID:32411140 and 36575294). Given the frequency of lung cancer in COPD subjects, it will be helpful to acknowledge this in the manuscript.

 

As a minor consideration, bibliography notes should be taken before the dot of the statement that they refer to.

Moderate editing of English language

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have adequately addressed all my issues. Regarding biostatistics, I would suggest to get the manuscript checked by an expert on this field.

Author Response

Thank you for your positive review. Dr. Christensen is an expert in biostatistics and bioinformatics and a co-author of the manuscript. 

Reviewer 2 Report

I have no more concerns.

Author Response

Thank you for your positive review.

Reviewer 3 Report

I appreciate the efforts of the authors to address all comments with additional data and explanations. However, there are still some points that need clarification, and I suggest that the authors further revise the manuscript.

1. As for the subjects enrolled, it is difficult to know whether there are twenty-five or twenty-one or nineteen in the abstract. It may be useful to schematize the analyses performed and the number of subjects for each analysis. If two patients were excluded due to technical problems, they should not be considered in the total number of patients enrolled, as there are no data on them.

2. I appreciate the effort to emphasize the link between t-reg and immune checkpoints in lung cancer and COPD as well as the presentation of demographic data. It would be appropriate to include a reference to the table (PMID:36575294).

Minor editing of English language required

Author Response

Thank you for your positive review. We have undertaken additional revisions as suggested as noted below. 

Point 1: As for the subjects enrolled, it is difficult to know whether there are twenty-five or twenty-one or nineteen in the abstract. It may be useful to schematize the analyses performed and the number of subjects for each analysis. If two patients were excluded due to technical problems, they should not be considered in the total number of patients enrolled, as there are no data on them.

Response 1: Thank you for your positive review and this comment. We apologize for the continued confusion. Per institutional policy, any patient that has samples collected would be considered “enrolled” in a study by our institutional definition. The 2 patients that had samples collected that were unusable due to technical issues would still be considered to be “enrolled” in the study by our institutional definition. We appreciate that this definition may not be universal. In accordance with reviewer suggestions, we have attempted to be consistent with language throughout the manuscript and have changed “enrolled” to “consented” where appropriate.  We have revised the abstract to further clarify the number of patients consented, which is 25. Twenty-one of these patients were consented for circulating immune cell studies, with 4 additional patients included for single-cell RNA and single-cell TCR sequencing analysis of blood, tumor and lymph node tissue. In addition, one of the original 21 patients for circulating immune cell studies had blood, tumor and lymph node collected for single-cell analyses, for a total of 5 patients. We have changed the heading of Table 1 to “Clinical characteristics of consented patients”. Additional clarification has been made in the Materials and Methods section 2.2 and a schematic has been added as Supplemental Figure 1 to clarify patients consented, enrolled and analyzed. Additional clarification has also been added to the Results section 3.1. Changes to the manuscript are highlighted in red with tracked changes.

 

Point 2: I appreciate the effort to emphasize the link between t-reg and immune checkpoints in lung cancer and COPD as well as the presentation of demographic data. It would be appropriate to include a reference to the table (PMID:36575294).

Response 2: Thank you for this comment. The above noted reference has been added to the manuscript as reference 20, line 60-61.