Microbiota Alterations in Gastrointestinal Cancers

Round 1

Reviewer 1 Report

In the current manuscript Karwowska et al. reviewed the relevant literature of the already discovered changes in the commensal microbiota during cancer progression in several different gastrointestinal tumor type. Following the shift in the stages of tumorigenesis, the authors define the microbiota (and its discrepancies) in healthy, pre-cancerous and cancerous phase in esophageal, gastric, pancreatic, liver, biliary tract, gallbladder, ampullary and colorectal cancer. While the role of the microbiota in tumor initiation, metastasis formation and therapy outcome is a novel, important and rather uncharted territory in cancer research, the present manuscript fall short of providing any new insight for the field.

Being an expert on a given research topic is not required to write and publish a review, however it will give a certain critical point-of-view which creates the proper “soil” for that review article to become more than just a summary of the current knowledge. This manuscript is a perfect factual presentation of what we know about the role of the gastrointestinal microbiota in carcinogenesis, but lacking any kind of critical analysis of this dataset. In the present form this work is a review of reviews which already proved by the fact that out of the 146 references 62 cited articles are reviews. Unfortunately, there are several occasions throughout the manuscript where the original sentence (from a review!) was copy – pasted almost identically and, moreover, even in the source the sentence was a reference to another research article!

For example:                                                        

Karwowska et al. page 12, line 406-408: “In addition, P. gingivalis and another periodontal pathogen related to pancreatic cancer, A. actinomycetemcomitans, were reported to initiate the Toll-like receptor signalling pathways, which have been shown to be critical drivers of pancreatic carcinogenesis in animal models [86].”

Fan et al. (Ref. 86 in the current manuscript): “In addition, P. gingivalis and A. actinomycetemcomitans can initiate the Toll-like receptor (TLR) signaling pathways, and TLR activation is a critical driver of pancreatic carcinogenesis in animal models, as we (Dr. Miller) recently demonstrated [46].”

The authors should cite the ORIGINAL paper from Dr. Miller, read it and then present the findings in the light of other works, connecting the ideas and results and highlighting the significant differences if there are any.

Another problematic aspect is the misrepresentation of some results:

Karwowska et al. page 12, line 404-406: “Those bacteria are believed to promote PDAC development via peptidylarginine deiminase secretion which might lead to p53 and K-ras point mutations by degrading arginine [85].”

In Ref. 85 (Ogrendik, Clin Med Insights Arthritis Musculoskelet Disord., 2017), which is a 2 pages long article, there is no such statement, furthermore, the words PDAC, pancreatic cancer, p53 or Kras is missing completely. I believe Karwowska and her colleagues have a proper reference that could support this sentence, but unfortunately it was not included in the manuscript.

And this in only two example.

Additionally, I would like to suggest to pay extensive attention to the name of the cited authors. For example on page 12, line 372 there is a reference to an article from Pathogens et al, however the real name of the first author is Pötgens.

Together with the lack of critical analysis of the already published literature data, these aforementioned issues make it really hard to recommend this manuscript for publication in the present form. There are also no appropriate figures and tables to help the reader understand the changes in the microbiota in certain stages of tumorigenesis and, in general, there is no added value to the topic. Therefore, I cannot recommend it for publication in the journal Applied Sciences.

Author Response

Dear Reviewer 1,

thank you for valuable suggestions. We have applied changes in our review article as per requested.

Answers to particular issues:

Being an expert on a given research topic is not required to write and publish a review, however it will give a certain critical point-of-view which creates the proper “soil” for that review article to become more than just a summary of the current knowledge. This manuscript is a perfect factual presentation of what we know about the role of the gastrointestinal microbiota in carcinogenesis, but lacking any kind of critical analysis of this dataset. In the present form this work is a review of reviews which already proved by the fact that out of the 146 references 62 cited articles are reviews. Unfortunately, there are several occasions throughout the manuscript where the original sentence (from a review!) was copy – pasted almost identically and, moreover, even in the source the sentence was a reference to another research article!

For example:                                                        

Karwowska et al. page 12, line 406-408: “In addition, P. gingivalis and another periodontal pathogen related to pancreatic cancer, A. actinomycetemcomitans, were reported to initiate the Toll-like receptor signalling pathways, which have been shown to be critical drivers of pancreatic carcinogenesis in animal models [86].”

Fan et al. (Ref. 86 in the current manuscript): “In addition, P. gingivalis and A. actinomycetemcomitans can initiate the Toll-like receptor (TLR) signaling pathways, and TLR activation is a critical driver of pancreatic carcinogenesis in animal models, as we (Dr. Miller) recently demonstrated [46].”

The authors should cite the ORIGINAL paper from Dr. Miller, read it and then present the findings in the light of other works, connecting the ideas and results and highlighting the significant differences if there are any.

Thank you for the comment. Changes were applied as suggested.

Another problematic aspect is the misrepresentation of some results:

Karwowska et al. page 12, line 404-406: “Those bacteria are believed to promote PDAC development via peptidylarginine deiminase secretion which might lead to p53 and K-ras point mutations by degrading arginine [85].”

In Ref. 85 (Ogrendik, Clin Med Insights Arthritis Musculoskelet Disord., 2017), which is a 2 pages long article, there is no such statement, furthermore, the words PDAC, pancreatic cancer, p53 or Kras is missing completely. I believe Karwowska and her colleagues have a proper reference that could support this sentence, but unfortunately it was not included in the manuscript.

Thank you for the comment. Changes were applied as suggested. It was indeed an error in references.

And this in only two example.

Additionally, I would like to suggest to pay extensive attention to the name of the cited authors. For example on page 12, line 372 there is a reference to an article from Pathogens et al, however the real name of the first author is Pötgens.

Thank you for the comment. Changes were applied as suggested.

Together with the lack of critical analysis of the already published literature data, these aforementioned issues make it really hard to recommend this manuscript for publication in the present form. There are also no appropriate figures and tables to help the reader understand the changes in the microbiota in certain stages of tumorigenesis and, in general, there is no added value to the topic. Therefore, I cannot recommend it for publication in the journal Applied Sciences.

Thank you for all valuable suggestions. We have update our references and added clinical trials instead of review articles. Of course it is important to include direct reference in a review article however due to the fact that we have written a really long paper we decided that in few cases we will include appropriate reviews (for example while discussing cancer etiology and incidence. Although this is an important issue, this is not the main subject of our article). Now the ratio clinical trial/ case study reference to review articles in 96 to 55.

Moreover we have added critical analysis of few studies, especially based on methodology. Few research groups that we quoted in this review used culture-based methods, we have emphasized that many members of microbiota community are not cultivable and sequencing techniques are required to perform statistical analyses. Moreover, we have highlighted that results obtained on murine model are not always transferable to human due to significant changes in human and murine GI tract. (All changes added in red)

Reviewer 2 Report

In this Review the Authors describe changes in microbiota in human gastrointestinal malignancies such as esophageal, stomach, liver, biliary tract, pancreas and colon inflammations and cancers. The manuscript is very clearly written, and the reader is easily led to through the main findings on the differences on microbiota in the different inflammatory disorders and cancers of the gastrointestinal tract. The review is well developed, balanced and addresses correctly several potential issues, arising from the summarized literature. Here are a number of points that should be considered in order to improve the work:

A table showing the changes in microbiota in gastric cancer should be included. It would be useful to include in the conclusions a table highlighting the main findings regarding the microbiota associated to the different types of cancer in the gastrointestinal tract, in order to suggest one or more potential microbiota fingerprint(s) per type(s) of tumor.

 

Author Response

Dear Reviewer 2,

thank you for valuable suggestions. We have applied changes as per requested.

 

In this narrative review the authors evaluated the difference, in terms of microbiota profile, among healthy subjects and those with gastrointestinal malignancy.

In the section Introduction when the authors define microbiota, a key step to understand all the review, they should add a reference. I suggest , for example, Derovs et al. Medicina 2019, 55(8), 459; https://doi.org/10.3390/medicina55080459

Thank you for the suggestion. The change has been applied.

Line 44 sentence "Change in gut microbiota can also...", it is better to replace can with "could"

Thank you for the suggestion. The change has been applied.

English language should be revised. For example, line 75 Galiardi et al, it is better to replace report with "reported".

Thank you for the suggestion. The change has been applied.

When the authors discuss "Microbiota in pancreatitis", it should be clarified if there is a difference on the basis of etiology (alcohol, biliary stones or unknown origin). This has a great relevance in terms of potential, future, new therapeutic approaches.

Thank you for the suggestion. The change has been applied. We have added microbiota changes in alcohol driven pancreatitis, however we were unable to find data on alterations in gut microbiome in gallstone driven pancreatitis. (part written in blue)

In case of HCC is not well-discussed if there are data based on some etiology as alcohol-related cirrhosis. Since the relevance of this etiology is increasing, the authors should provide novelties on this issue.

Furthermore, it has been reported that gut microbiota can contribute to persistent non alcoholic steatohepatitis (NASH) (see Kim H-N et al. J. Clin. Med. 2019, 8(8), 1089; https://doi.org/10.3390/jcm8081089). Since NASH will be in the naer future the most important cause of cirrhosis and HCC, this should be discussed.

Thank you for the suggestion. We have added information about alterations in alcohol-related liver cirhossis. Most papers that we quote don’t analyze differences in microbiome between patients with different cirrhosis etiology so we are unable to add this information. (parts written in blue)

Besides, due to the fact that this is a heavy review article we decided not to focus precisely on microbiota changes in different etiologies, even though we keep in mind that the etiology is a very important variable.

In the section discussion the authors should discuss better (not only as question) the possibility that microbiota changes are a consequence and not a cause or concomitant-cause of cancer.  

That is a very good question, it is, in point of fact, the subject of my PhD thesis. We have extended the disscussion part with information about some most common bacteria present in GI cancers. However we are still unable to say what was first: changes in microbiota or cancer and I don’t want to postulate that microbiota alterations are the result of cancer and inflammation. We still need lots of research to understand human microbiota not only on taxonomic level but also to understand its metabolism and transcriptome. Frequently used 16S sequencing techniques are not enough to properly understand microbial community as it is limited by databases content. Shotgun sequencing is very expensive and even available bioinformatic tools are not enough to understand what is present in our microbiome sample as it is also limited to databases (we are now using AI techniques in order to better understand microbiome and Shotgun data).

Line 659, John Cunningham should be reported as John Cunningham virus.

Thank you for the suggestion. The change has been applied.

When the authors discuss CRC it should be stressed that a beneficial treatment of conditions that could predispose to malignancy (for example RCU or Crohn's disease) is associated with changes in microbiota composition (see Ribaldone et al. J Clin Med. 2019 Oct 9;8(10). pii: E1646. doi: 10.3390/jcm8101646.)

Thank you for the suggestion. Even than this is a very interesting topic, due to the fact that our article is already containing lots of information, we decided not to incorporate it.

Some reference should be more appropriate.

For example, the reference number 59 (on extragastric manifestations of H.pylori) should be added to Santambrogio et al. Minerva Gastroenterol Dietol 2019;65:204-13.

Thank you for the suggestion, I am unable however to fin the reference online.

The reference number 88 should be changed with Ruzic M et al. Panminerva Med 2018;60:185-91.

Thank you for the suggestion. The change has been applied.

The reference 140 is old. Recently, Yang et al (Min Medica 2019;110:464-70) have updated the issue.

Thank you for the suggestion, I am unable however to fin the reference online.

In Fig 2 cirhossis should changed with cirrhosis 

Thank you for the suggestion. The change has been applied.

 

Reviewer 3 Report

In this narrative review the authors evaluated the difference, in terms of microbiota profile, among healthy subjects and those with gastrointestinal malignancy.

In the section Introduction when the authors define microbiota, a key step to understand all the review, they should add a reference. I suggest , for example, Derovs et al. Medicina 2019, 55(8), 459; https://doi.org/10.3390/medicina55080459

Line 44 sentence "Change in gut microbiota can also...", it is better to replace can with "could"

English language should be revised. For example, line 75 Galiardi et al, it is better to replace report with "reported".

When the authors discuss "Microbiota in pancreatitis", it should be clarified if there is a difference on the basis of etiology (alcohol, biliary stones or unknown origin). This has a great relevance in terms of potential, future, new therapeutic approaches.

In case of HCC is not well-discussed if there are data based on some etiology as alcohol-related cirrhosis. Since the relevance of this etiology is increasing, the authors should provide novelties on this issue.

Furthermore, it has been reported that gut microbiota can contribute to persistent non alcoholic steatohepatitis (NASH) (see Kim H-N et al.

J. Clin. Med. 2019, 8(8), 1089; https://doi.org/10.3390/jcm8081089). Since NASH will be in the naer future the most important cause of cirrhosis and HCC, this should be discussed.

In the section discussion the authors should discuss better (not only as question) the possibility that microbiota changes are a consequence and not a cause or concomitant-cause of cancer.  

Line 659, John Cunningham should be reported as John Cunningham virus.

When the authors discuss CRC it should be stressed that a beneficial treatment of conditions that could predispose to malignancy (for example RCU or Crohn's disease) is associated with changes in microbiota composition (see Ribaldone et al. J Clin Med. 2019 Oct 9;8(10). pii: E1646. doi: 10.3390/jcm8101646.)

Some reference should be more appropriate.

For example, the reference number 59 (on extragastric manifestations of H.pylori) should be added to Santambrogio et al. Minerva Gastroenterol Dietol 2019;65:204-13.

The reference number 88 should be changed with Ruzic M et al. Panminerva Med 2018;60:185-91.

The reference 140 is old. Recently, Yang et al (Min Medica 2019;110:464-70) have updated the issue.

In Fig 2 cirhossis should changed with cirrhosis 

Author Response

Dear Reviewer 3,

thank you for valuable suggestions. We have applied changes as per requested.

 

In this Review the Authors describe changes in microbiota in human gastrointestinal malignancies such as esophageal, stomach, liver, biliary tract, pancreas and colon inflammations and cancers. The manuscript is very clearly written, and the reader is easily led to through the main findings on the differences on microbiota in the different inflammatory disorders and cancers of the gastrointestinal tract. The review is well developed, balanced and addresses correctly several potential issues, arising from the summarized literature. Here are a number of points that should be considered in order to improve the work:

A table showing the changes in microbiota in gastric cancer should be included.

Thank you for the suggestion. Table was added (Table 2).

It would be useful to include in the conclusions a table highlighting the main findings regarding the microbiota associated to the different types of cancer in the gastrointestinal tract, in order to suggest one or more potential microbiota fingerprint(s) per type(s) of tumor.

Thank you for the suggestion. The change has been applied (Table 7).

Round 2

Reviewer 1 Report

The manuscript improved notably, the newly added notes and remarks to some of the cited studies help substantially the reader to critically approach these works. The English of the manuscript is still high quality, but i've found some typos in the newly included parts. Please correct these.

I still miss some helpful figures, however i understand that the complexity of this topic is making the creation of such figures extremely challenging.

In summary, i think this manuscript is almost ready, and after some spell-check, it should be published in the jorunal of Applied Sciences.