Helicobacter pylori Status May Differentiate Two Distinct Pathways of Gastric Adenocarcinoma Carcinogenesis

Round 1

Reviewer 1 Report

The manuscript describes the potential use of monoclonal adnab-9   in differentiation of familial and sporadic cases of gaswtric cancer in the context of Helicobacter pylori infection. The metholodology and statistic used are of high quality and in fact, their critical assess,emt is far beyond of my expertise. I havbbe only some general comments:

1. In the introductory section, the statement that intensive treatment of GERD reduce gastric cancer prevention seems to me uncertain.

2. Combined determination nof CEA, CA 19-9 and CA 72-4 can increase the sensitivity and specificity of tumor markers used as a set

3. The startisrtical program used must be specified

4. The authors present their data only centered on sophisticated immunologic methods. Neglecting serologic data seems to me too early (Cag A?).

5. How can you differentiayte gasetriuc from colon cancer by stool assay of adnab-p?

6. And finally: does determination of adnab-9 in gastric cancer cancer  change the therapeutic decisions? (type and extent of surgery, chemotherapy immune checkpoint inhibitors?  

Author Response

Reviewer #1 Changes in text highlighted in yellow.

General Comments: Minor

Query 1. In the introductory section, the statement that intensive treatment of GERD reduce gastric cancer prevention seems to me uncertain.

Response to Query 1:

Gastric cancer of the cardia and fundus appears to be related to GERD as Hp does not have the same impact as it has on the distal stomach. Thus, some have suggested that intensive GERD treatment may be of benefit. However, we do comment that this approach is experimental for most patients at risk. We add the following citation to the conclusion to support this and a supporting statement in the discussion section.

Kim JJ. Upper gastrointestinal cancer and reflux disease. J Gastric Cancer. 2013 Jun;13(2):79-85. doi: 10.5230/jgc.2013.13.2.79. Epub 2013 Jun 25. PMID: 23844321; PMCID: PMC3705136.

2. Combined determination nof CEA, CA 19-9 and CA 72-4 can increase the sensitivity and specificity of tumor markers used as a set.

Response to Query 2:

The above markers have been touted as accurate for diagnosis but these data are outdated. The latest paper looking at these markers singled out CEA as the most effected marker and confirmed that CA72-4 is outmoded. Adnab-9 is one of the consistent front runners according to multiple reviews so we did not do a direct comparison to CEA.

Herrera-Pariente C, Montori S, Llach J, Bofill A, Albeniz E, Moreira L. Biomarkers for Gastric Cancer Screening and Early Diagnosis. Biomedicines. 2021 Oct 12;9(10):1448. doi: 10.3390/biomedicines9101448. PMID: 34680565; PMCID: PMC8533304. Mentions gp87

Cited by Xian X, Tang L, Wu C, Huang L. miR-23b-3p and miR-130a-5p affect cell growth, migration and invasion by targeting CB1R via the Wnt/β-catenin signaling pathway in gastric carcinoma. Onco Targets Ther. 2018 Oct 25;11:7503-7512. doi: 10.2147/OTT.S181706. PMID: 30498363; PMCID: PMC6207250.

Liu HN, Yao C, Wang XF, Zhang NP, Chen YJ, Pan D, Zhao GP, Shen XZ, Wu H, Liu TT. Diagnostic and economic value of carcinoembryonic antigen, carbohydrate antigen 19-9, and carbohydrate antigen 72-4 in gastrointestinal cancers. World J Gastroenterol. 2023 Jan 28;29(4):706-730. doi: 10.3748/wjg.v29.i4.706. PMID: 36742169; PMCID: PMC9896613.

Query 3. The startisrtical program used must be specified

Response to Query 3:

We did provide details of the statistical package we used: MedCalc Online; MedCalc Software Ltd., Ostend, Belgium. Lines 156-157.

Query 4. The authors present their data only centered on sophisticated immunologic methods. Neglecting serologic data seems to me too early (Cag A?).

Response to Query 4:

We agree that serologic markers would have added much insight but as we used historic tissue microscopy, serum was not available.Lines 150-151.

Query 5. How can you differentiayte gasetriuc from colon cancer by stool assay of adnab-p?

Response to Query 5:

This is an excellent question. Adnab-9 binding mirror the activity of the innate immune system as we point out in our latest COVID-19 paper (reference 41). Gastric cancer does not evoke a strong Adnab-9 stool binding as compared to colon cancer for which it was a priori designed, suggesting that it is under the immunologic radar and hence more deadly with fewer treatment options. A lower binding OD might differentiate gastric from colon cancer. Please see the added comment on lines 315-316.

Query 6. And finally: does determination of adnab-9 in gastric cancer change the therapeutic decisions? (type and extent of surgery, chemotherapy immune checkpoint inhibitors? 

Response to Query 6:

This is another insightful question. It does appear that increased Adnab-9 binding in tissue slides confers a better prognosis. This may mirror a better innate immune response. It would be anticipated that response to surgery and advanced chemotherapy with PDL-1 checkpoint inhibitors may be related to Adnab-9 binding but future studies may be needed to confirm this. The below reference was added to the discussion.

Figueroa-Protti L, Soto-Molinari R, Calderón-Osorno M, Mora J, Alpízar-Alpízar W. Gastric Cancer in the Era of Immune Checkpoint Blockade. J Oncol. 2019 Sep 24;2019:1079710. doi: 10.1155/2019/1079710. PMID: 31662748; PMCID: PMC6778883.

Author Response File: Author Response.docx

Reviewer 2 Report

Thank you for the opportunity to review the paper "Helicobacter pylori Status May Differentiate Two Distinct Path-2 ways of Gastric Adenocarcinoma Carcinogenesis". 

The idea of the paper is original and if better discussed and presented, the perspective introduced by the paper may be useful for the clinical practice.

However, before proceeding further,  I recommend the authors to solve the following issues (minor si major).

The introduction, although interesting with some well-pointed remarks, does not have a logical flow of ideas that would help the reader to better follow and understand the problem -> the hypothesis and -> the exact aims of the paper. Please rephrase the last paragraph of the introduction to clearly state the objectives of the study.

The t-student test requires several assumptions to be met (including normal distribution of the data). Did the authors check for normal distributions and the other assumptions before applying t-test?

When comparing groups, did the authors identify and adjust for confounding factors? Nothing is said about confounding factors (regarding each of the comparisons made) or the way to manage them. 

Also, no Clear inclusion and exclusion criteria (for the patients) are provided.

Why data was collected from the time interval 1995 - 2000 but only publish the study now in 2023? Please explain the actuality of the problem identified by you and how old data such as this does not impact the present-day results.

The Limitations section is missing.

In the abstract, not all abbreviations are adequately explained.

The phrases' structure is not typical for english languge and it is hard to follow.

Author Response

Reviewer #2 Changes in text highlighted in orange.

Comments and suggestions for authors:

Thank you for the opportunity to review the paper "Helicobacter pylori Status May Differentiate Two Distinct Path-2 ways of Gastric Adenocarcinoma Carcinogenesis".

The idea of the paper is original and if better discussed and presented, the perspective introduced by the paper may be useful for the clinical practice.

However, before proceeding further,  I recommend the authors to solve the following issues (minor si major).

Query 1: The introduction, although interesting with some well-pointed remarks, does not have a logical flow of ideas that would help the reader to better follow and understand the problem -> the hypothesis and -> the exact aims of the paper. Please rephrase the last paragraph of the introduction to clearly state the objectives of the study.

Response to Query 1:

We have added a statement regarding the aims of the paper as suggested by the reviewer and these changes have been accordingly embodied in the last paragraph of the intoduction, highlighted in purple.

Query 2: The t-student test requires several assumptions to be met (including normal distribution of the data). Did the authors check for normal distributions and the other assumptions before applying t-test?

Response to Query 2:

We undertook an in-depth analysis of our ordinal data to assess for normality of distribution. We looked at data such as age and found the distribution to be normal. However the reviewer was correct in respect of the FERAD data which we found not to be normal in distribution. We summarize this detailed analysis in the table below and have made changes to Figure 6, as a result. We thank the reviewer for their perpicacity in this regard.

. We used the Kolmogorov-Smirnov calculation which can also be accessed online: https://www.socscistatistics.com/tests/kolmogorov/default.aspx

 On all age calculations we obtained the same evaluation: “The data do not differ significantly from that which is normally distributed”. However, the FERAD date are clearly not normal as suspected by the reviewer. This is a sweeping justification not to use a parametric test for the FERAD our paper and believe that the reviewer is justified in his supposition. We accordingly used the non-parametric Chi-square Test and non-parametric Mann-Whitney test to alter our figure accordingly and this has also been added to the statistics section highlighted in purple.

For your interest we post the results in the table below but do not feel that this should be incorporated into the paper as these are merely representative samples from a database . We will accordingly put the relevant results in the paper under Figure 6. Analysis does not reach significance but indicates a strong trend (p=0.08).

Query 3: When comparing groups, did the authors identify and adjust for confounding factors? Nothing is said about confounding factors (regarding each of the comparisons made) or the way to manage them.

Response to Query 3:

We did our best to exclude confounding factors in the kindred by doing exhaustive genetic sequencing and immunohistochemistry in the kindred and the community group. The major challenge was in the FERAD ratio as it has two components which can be altered by medications, food supplements, anemia, and contracting neoplastic disease. There were no diffrences in analysis we performed in terms of the above confounding factors and the extensive follow-up allowed surveillance for these conditions. A statement has been included in the methods section to this effect.

Query 4: Also, no Clear inclusion and exclusion criteria (for the patients) are provided.

Respond to Query 4:

This was an observational study particularly regarding the community database. The only inclusion criteria was the willingness to be part of the data collection and ability to give informed consent. Exclusion criteria was the inability to give consent and inability to provide stool specimens. There was no intervention and the IRB deemed the study to be low-risk.

Query 5: Why data was collected from the time interval 1995 - 2000 but only publish the study now in 2023? Please explain the actuality of the problem identified by you and how old data such as this does not impact the present-day results.

Response to Query 5:

The data from the kindred and from the immunohistochemist was indeed completed by 2000. The study community database was started in 1995 but did not a’ priori focus on patients with gastric cancer but since follow-up continued until the end of the study recently, we were able to record who had develop gastric cancer or related conditions and explore FERAD ratio, the significance of which was only apparent in the last 2 years. There was also an inexplicable reduction in gastric cancer incidence in the years from 2009-2018 which also slowed publications as we have noted in the conclusions section and last reference added.  These are highlighted in purple.

The Limitations section is missing.

Query 6: In the abstract, not all abbreviations are adequately explained.

Response to Query 6:

In scanning the manuscript a[[roximately 10 abbreviations were found and fully explained. We thank the reviewer for pointing this out. They are highlighted in purple.

Query on Comments on the quality of English language:

The phrases' structure is not typical for english languge and it is hard to follow.

Response to Query:

We performed a spell, style and grammar test of the manuscript. The only error found was a capitalization of “M” in the abbreviation for the innate immune system (InImS). This has been corrected and highlighted in purple.

Reviewer 3 Report

Very interesting and comprehensive article regarding the evaluation of the phenotype of sporadic gastric cancer based on helicobacter pylori status and binding of a tumor risk marker monoclonal, Adnab-9. Overall, a well-written and well-depicted original study. Some minor comments:

1) Throught the entire length of the manuscript, the references used are pretty old (before the year 2000 are 17 out of 41 refs, aprox. 45%). I suggest that you use more recent references, later than the year 2010, either change some of the old ones or add some new. Doing so would improve the overall quality of the paper.

2) Figures 1,2,6 are very blurry, and thus very hard to read. I do not think that they are at least 300dpi in .tiff or .jpeg. Please, improve their overall visibility  either by increasing their dpi to more than 300, or by changing their file type.

3) In Materials and Methods section, the 2.3. Statistical Evaluation part needs some minor improvements. Please clearly state which statistical computer software package was utilized apart from Medcalc, for example did you use IBM SPSS or STATA? Moreover, please use the corresponding 95% confidence intervals throught the text, a p-value without the corresponding 95% CI is not accurate enough. Also, clearly state whether you used mean values with % or medians with IQR for demographics, e.g AGE. Overall, the overall quality of the paper will be improved.

Occasional errors in choice of preposition and poor word choice are present in the manuscript. So, there is some benefit to proofreading prior to publication. However, overall, the manuscript is understandable.

Author Response

Reviewer #3 Changes in text highlighted in blue.

Query 1) Throught the entire length of the manuscript, the references used are pretty old (before the year 2000 are 17 out of 41 refs, aprox. 45%). I suggest that you use more recent references, later than the year 2010, either change some of the old ones or add some new. Doing so would improve the overall quality of the paper.

Response to Query 1:

The distribution of publication times reflects the broad interest in immunohistochemistry now superseded by more modern techniques, but still have their place. We agree that this imbalance should be corrected and have added 5 more citations (42-46) published after 2010 highlighted in blue.

Query 2) Figures 1,2,6 are very blurry, and thus very hard to read. I do not think that they are at least 300dpi in .tiff or .jpeg. Please, improve their overall visibility  either by increasing their dpi to more than 300, or by changing their file type.

Response to Query 2:

Anything we can do to make the presentation more attractive will be attempted and we that the reviewer for his contribution to achieve this, we will try suggested corrections on the final version. Please see new renditions of Figures 1,2, and 6.

Query 3) In Materials and Methods section, the 2.3. Statistical Evaluation part needs some minor improvements. Please clearly state which statistical computer software package was utilized apart from Medcalc, for example did you use IBM SPSS or STATA? Moreover, please use the corresponding 95% confidence intervals throught the text, a p-value without the corresponding 95% CI is not accurate enough. Also, clearly state whether you used mean values with % or medians with IQR for demographics, e.g AGE. Overall, the overall quality of the paper will be improved.

Response to Query 3:

As stated in our response to reviewer 1, the online program we used was MedCalc Online; MedCalc Software Ltd., Ostend, Belgium. It is a stand-alone program completely independent from the SPSS and STATA programs which we did not use. We also stated that Any p values of < 0.05 were considered significant which mirror the 95 percentile and we mainly used ordinal data not proportional, where CI are usually given, however we can add CI to Table 2 where applicable lines 233-234). For survival data we used the Log-Rank test which also does not provide CI.

Comments on the Quality of English Language

Occasional errors in choice of preposition and poor word choice are present in the manuscript. So, there is some benefit to proofreading prior to publication. However, overall, the manuscript is understandable.

Response to English Quality Language.

We agree that careful proofreading should improve readability and will review the manuscript accordingly.

Reviewer 4 Report

Abstract:

The abstract is correctly done from a scientific point of view, but it is much too long. As a rule, usually the abstract is limited to 200 words; in this case it is much more than 400 words. I recommend shortening the text and, obviously, condensing the information contained in the abstract.

There are only 3 keywords, correctly chosen and useful, but which do not fully cover the topic of the manuscript. I recommend adding at least 2 keywords; I leave it up to the authors to choose them.

I consider the introduction of a list of abbreviations used in the manuscript necessary, even if they are correctly explained in the text. It remains up to the decision of the authors and the publisher where it would be optimal to place this list.

On a scale of 1 to 10, I will give 6 points for the abstract.

Introduction:

I really liked the introduction; the authors explain very well the context in which their study is placed, using 18 correctly selected bibliographic references.

 On a scale of 1 to 10, I agree 10 points for introduction.

Methodology:

The methodology is correctly described, on approximately one page. I have some reservations, seeing the small number of patients and the diverse way of collecting samples and data, on the possibilities of generalizing the results obtained in these limiting conditions.

On a scale of 1 to 10, I agree 8 points for methodology.

Results:

The results chapter is correct and beautifully written, the 6 figures and 3 tables bringing a welcome graphic illustration. I have no objections to this part of the manuscript.

On a scale of 1 to 10, I agree 10 points for results.

Discussion:

To the discussion chapter should have been allocated a larger space, the topic of the manuscript being interesting and generating controversies. In its current form, I would still say that the discussions are at an acceptable level.

In this situation, on a scale of 1 to 10, I will give 8 points for discussion.

Conclusion:

The conclusions are limited to less than 4 lines, the authors focusing almost exclusively on future research directions. I would like at least a sentence, at the beginning of the conclusions, about their manuscript, then future research directions.

On a scale of 1 to 10, I will give only 6 points for conclusions.

Bibliography/References:

The manuscript has 41 references, current, correctly written and correctly quoted in the text, which is acceptable.

On a scale of 1 to 10, I agree 9 points for the bibliography.

Figures/Tables:

I identified 6 figures and 3 tables, of good quality, with satisfactory resolution, which are necessary and useful for the manuscript. The graphic illustration of this paper does not impress, but rises to an acceptable level.

On a scale of 1 to 10, I agree 8 points for this chapter.

Review Decision:

Accept after minor revision.

The English language used is fine, but fine polish is needed in some places.

Author Response

Reviewer #4 Responses in text highlighted in green.

Query regarding the abstract.

Abstract:

The abstract is correctly done from a scientific point of view, but it is much too long. As a rule, usually the abstract is limited to 200 words; in this case it is much more than 400 words. I recommend shortening the text and, obviously, condensing the information contained in the abstract.

Response to Query regarding the abstract:

We thought that the specifications allowed for a 500-word limit and the current count is 418. We will reduce it to below 400 words at n=398 as can be seen in the abstract sample below and in the text lines 23-34.

There are only 3 keywords, correctly chosen and useful, but which do not fully cover the topic of the manuscript. I recommend adding at least 2 keywords; I leave it up to the authors to choose them.

In addition to the keywords H. pylori; Adnab-9; monoclonal antibody, we will add Familial Gastric Cancer; and FERAD ratio. Line 50.

I consider the introduction of a list of abbreviations used in the manuscript necessary, even if they are correctly explained in the text. It remains up to the decision of the authors and the publisher where it would be optimal to place this list.

We have no objection to this but like the reviewer we will leave the decision to the editor so as not to affect the style requirements.

On a scale of 1 to 10, I will give 6 points for the abstract.

Introduction:

I really liked the introduction; the authors explain very well the context in which their study is placed, using 18 correctly selected bibliographic references.

 On a scale of 1 to 10, I agree 10 points for introduction.

Response to Introduction:

We thank the reviewer for their kind evaluation.

Methodology:

The methodology is correctly described, on approximately one page. I have some reservations, seeing the small number of patients and the diverse way of collecting samples and data, on the possibilities of generalizing the results obtained in these limiting conditions.

On a scale of 1 to 10, I agree 8 points for methodology.

Response to Methodology.

We agree that the number, particularly in the kindred is limited. However, there are no previous pediatric similar cases in our review.

Results:

The results chapter is correct and beautifully written, the 6 figures and 3 tables bringing a welcome graphic illustration. I have no objections to this part of the manuscript.

On a scale of 1 to 10, I agree 10 points for results.

 Response to Results:

We thank the reviewer for their kind evaluation

Discussion:

To the discussion chapter should have been allocated a larger space, the topic of the manuscript being interesting and generating controversies. In its current form, I would still say that the discussions are at an acceptable level.

In this situation, on a scale of 1 to 10, I will give 8 points for discussion.

Response to Discussion

We thank the reviewer for his kind evaluation and enlarged the discussion section (lines 323-331 highlighted in yellow due to overlapping suggestions).

Conclusion:

The conclusions are limited to less than 4 lines, the authors focusing almost exclusively on future research directions. I would like at least a sentence, at the beginning of the conclusions, about their manuscript, then future research directions.

On a scale of 1 to 10, I will give only 6 points for conclusions.

Response to Conclusion

We thank the reviewer for their evaluation and will add the additional sentence.

Bibliography/References:

The manuscript has 41 references, current, correctly written and correctly quoted in the text, which is acceptable.

On a scale of 1 to 10, I agree 9 points for the bibliography.

Response to Bibliography/References:

We thank the reviewer for their kind evaluation and will add latest citations

Figures/Tables:

I identified 6 figures and 3 tables, of good quality, with satisfactory resolution, which are necessary and useful for the manuscript. The graphic illustration of this paper does not impress, but rises to an acceptable level.

On a scale of 1 to 10, I agree 8 points for this chapter.

Response to Figures/Tables:

We thank the reviewer for their kind evaluation and have tried to improve figures 1,2, and 6 as outlined above in green.

Review Decision:

Accept after minor revision.

Comments on the Quality of English Language

The English language used is fine, but fine polish is needed in some places.

Response to English Language:

We thank the reviewer for their kind evaluation and will proofread and make necessary corrections.

Round 2

Reviewer 2 Report

I can only see the reponses given to other reviewer. I can not see the author's responses to my comments.

These were my commnets:

"Thank you for the opportunity to review the paper "Helicobacter pylori Status May Differentiate Two Distinct Path-2 ways of Gastric Adenocarcinoma Carcinogenesis". 

The idea of the paper is original and if better discussed and presented, the perspective introduced by the paper may be useful for the clinical practice.

However, before proceeding further,  I recommend the authors to solve the following issues (minor si major).

The introduction, although interesting with some well-pointed remarks, does not have a logical flow of ideas that would help the reader to better follow and understand the problem -> the hypothesis and -> the exact aims of the paper. Please rephrase the last paragraph of the introduction to clearly state the objectives of the study.

The t-student test requires several assumptions to be met (including normal distribution of the data). Did the authors check for normal distributions and the other assumptions before applying t-test?

When comparing groups, did the authors identify and adjust for confounding factors? Nothing is said about confounding factors (regarding each of the comparisons made) or the way to manage them. 

Also, no Clear inclusion and exclusion criteria (for the patients) are provided.

Why data was collected from the time interval 1995 - 2000 but only publish the study now in 2023? Please explain the actuality of the problem identified by you and how old data such as this does not impact the present-day results.

The Limitations section is missing.

In the abstract, not all abbreviations are adequately explained."

The phrases' structure is not typical for english languge and it is hard to follow.

Author Response

Please see the attachment (the same submission as on 21 June 2023)

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

Dear authors,

Thank you for the careful answers to my comments. 

Generally, I agree with the author's responses. However, when consulting the last version of the uploaded manuscript, none of the modifications, mentioned by the authors as response to my comments, are visible (there is no "text in purple").

Moreover, the comment referring to the fact that the Limitations sections is absent did no receive any response.

I am afraid I cannot agree with the authors' response regarding my comment on the english language of the paper. The authors replied: "We performed a spell, style and grammar test of the manuscript. The only error found was a capitalization of “M” in the abbreviation for the innate immune system (InImS). "

I find this answer very superficial. My comment was about the lack of english language fluency. This is not solved by just correcting a capitalized letter.

Please edit the english language fluency with a specialized service or with a english native.

Author Response

Please see the attachment (new authors' reply).

Author Response File: Author Response.pdf

Round 4

Reviewer 2 Report

- The authors say: "This was an observational study particularly regarding the community database. The only inclusion criteria was the willingness to be part of the data collection and ability to give informed consent. Exclusion criteria was the inability to give consent and inability to provide stool specimens. There was no intervention and the IRB deemed the study to be low-risk."

Why haven't been these inclusion and exclusion criteria (explained to me in the response letter) also included in the manuscript? From my point of view, they should also be integrated in the article.

- The authors say: "The data from the kindred and from the immunohistochemistry was indeed completed by 2000. The study community database was started in 1995 but did not a’ priori focus on patients with gastric cancer but since follow-up continued until the end of the study recently, we were able to record who had develop gastric cancer or related conditions and explore FERAD ratio, the significance of which was only apparent in the last 2 years."

Then... the date until these patients were followed-up must be given/mentioned in the Methodology section.

- The Limitations section is still missing!

- I am afraid I cannot agree with the authors' response regarding my comment on the english language of the paper. The authors replied: "We performed a spell, style and grammar test of the manuscript. The only error found was a capitalization of “M” in the abbreviation for the innate immune system (InImS). " I find this answer very superficial. My comment was about the lack of english language fluency. This is not solved by just correcting a capitalized letter. Please edit the english language fluency with a specialized service or with a english native.

I am afraid I cannot agree with the authors' response regarding my comment on the english language of the paper. The authors replied: "We performed a spell, style and grammar test of the manuscript. The only error found was a capitalization of “M” in the abbreviation for the innate immune system (InImS). " I find this answer very superficial. My comment was about the lack of english language fluency. This is not solved by just correcting a capitalized letter. Please edit the english language fluency with a specialized service or with a english native.

Author Response

Response to final reviewer’s comments.

Query 1: The authors say: "This was an observational study particularly regarding the community database. The only inclusion criteria was the willingness to be part of the data collection and ability to give informed consent. Exclusion criteria was the inability to give consent and inability to provide stool specimens. There was no intervention and the IRB deemed the study to be low-risk."

Why haven't been these inclusion and exclusion criteria (explained to me in the response letter) also included in the manuscript? From my point of view, they should also be integrated in the article.

Answer to Query 1.

In compliance with the reviewer’s comment, we emphasize this is highlighted in red on lines 168-171. It is now clearly stated that the inclusion criteria were the ability to provide informed consent and being physically fit to safely have a procedure. Exclusion criteria were inability or unwillingness to participate.

Query 2:- The authors say: "The data from the kindred and from the immunohistochemistry was indeed completed by 2000. The study community database was started in 1995 but did not a’ priori focus on patients with gastric cancer but since follow-up continued until the end of the study recently, we were able to record who had develop gastric cancer or related conditions and explore FERAD ratio, the significance of which was only apparent in the last 2 years."

Then... the date until these patients were followed-up must be given/mentioned in the Methodology section.

Response to Query 2:

The average time of follow for these patients is 7.65 years and is included in the manuscript, highlighted in red on lines 234-5. The intention to record the follow-time is now noted in the methodology section (line 171) as suggested, but we feel that the actual result is more appropriately recorded in the results section (lines 248-249). However, if the review insists, we can put it into the methodology section.

Query 3:- The Limitations section is still missing!

Response to Query 3:

We did not include a separate limitations section as paragraph subheadings did not allow for it.  We have inserted such a Limitations subsection (2.4) to comply with the reviewer’s suggestion, highlighted in red on lines 185-193.

Query 4: I am afraid I cannot agree with the authors' response regarding my comment on the english language of the paper. The authors replied: "We performed a spell, style and grammar test of the manuscript. The only error found was a capitalization of “M” in the abbreviation for the innate immune system (InImS). " I find this answer very superficial. My comment was about the lack of english language fluency. This is not solved by just correcting a capitalized letter. Please edit the english language fluency with a specialized service or with a english native.

Response to Query 1:

The contributing author is a native English speaker. The attempt to improve the manuscript by scanning was a good faith attempt to improve all aspects of the grammar or readability. Granted that matters of style may affect fluency; we respectfully suggest that the reviewer should therefore provide specific examples of the lack of English Language fluency. We will be happy to correct whatever deficiencies the reviewer suggests.

Round 5

Reviewer 2 Report

The authors replied to all my comments. I have no further observations. Thank you and good luck!

I invite the journal staff to evaluate and adress the english revision for fluency. I have no further scientific comments. After the english revision (as the editors consider), the paper can be accepted for publication.