LC3B, mTOR, AMPK Are Molecular Targets for Neoadjuvant Chemotherapy in Gastric Cancers

Round 1

Reviewer 1 Report

The manuscript has been improved however a few more changes have to take place prior accepting the manuscript for publication.

In the abstract which is still long a change has to be done at least in my opinion: Omit maybe the phrase "The molecular cascades.... studied" and in the phrase "The study aimed... mRNA" add a comment that the specific proteins play a role in autophagy.

Still the epidemiologic data about gastric cancer are not correct. It is obvious that gastric cancer is not the most lethal type of cancer (line 48.)

In Table 1 you give data for more than 25 patients who had received FLOT chemotherapy. I could not find an explanation why in this table number of patients are more than those who had chemotherapy.

Thank you

 

Author Response

Dear, Reviewer,

The Team of the Authors presents the highly effective binding of the Molecular Biology specialists and clinicians. The unique and sometimes unclear facts were obtained that require the further proceeding of the research. The paper is the first experience in this field. We thank the reviewer for the tolerant attitude to the paper. All comment were taken into account.

1. The aim of the study was changed
2. The Abstract of the paper was modified

The introduction and material and methods parts were shortened

Abstract: Autophagy plays a dual role in oncogenesis processes. On the one hand, the autophagy enhances cell resistance to oncogenic factors, and on the other hand, it participates in the tumor progression. The aim of the  to find the associations between the effectiveness of FLOT regimen in resectable gastric cancers (GCs) with the key autophagy-related proteins.

Material and methods. The study included 34 patients with morphologically verified gastric cancer. All patients had FLOT neoadjunvant chemotherapy (NACT) (fluorouracil, leucovorin, oxaliplatin, and docetaxel) followed the gastrectomy. The studied tissue material was the non-transformed and tumor tissues obtained during diagnostic video gastroscopy in patients before the start of combined treatment and after surgical treatment, frozen after collection. The LC3B, mTOR, AMPK expression was determined by real-time PCR. The content of the LC3B protein was determined by the Western Blotting analysis.

3. Gastric cancer remains the urgent problem worldwide and in Russia. The statistic s issues show the global rise in cancers incidence. In Russia gastric cancers incidence-rate takes the first positions in list of cancers. Gastric adenocarcinoma is the fifth most common and the third most lethal cancer worldwide.

The text was changed to the “GC is the one of the most common cancers. It is presents the urgent global problem and belong to the third most lethal tumor worldwide (8.3% of all cancer deaths are attributable to gastric cancer) [2].

4. Table 1 was modified and there is no differences in text explanations.

Dear, Reviewer, you did the second and the next revisions of the paper and wonder what is the reason of the patients number differences. The unexcused reason is trivial. The continuing research leads to the changes in the data. It is difficult to calculate all changes especially in clinical facts and indicators.  Right now all the facts and indicators were carefully revised by the Authors. The updated paper version was changed and found to the best and improved version.

Reviewer 2 Report

In this manuscript, the authors attempt to find the associations of the effectiveness of the FLOT regimen in resectable gastric cancers (GCs) with that of the LC3B, mTOR, and AMPK mRNA. The mRNA level and the content of the LC3B protein are associated with the tumor stage categorization and the presence of signet ring cells. The tumor size and regional lymph node affections were associated with a decrease in mTOR mRNA level. They found the link between the expression of autophagy markers and therapy response. The following points should be clarified.

 

Please use larger fonts in all figures to facilitate a better experience for the reviewers and readers.

Author Response

Reviewer 2

Dear, Reviewer,

The Team of the Researcher revised all the unclear facts and added the expiations according to Your comments.

1. The clarification to the fact of the linckage between the molecular merkers level and cancer spreading and aggressive behavior was added

“Found data show the autophagy involvement in the oncogenesis that followed by the patients prognosis. The highest level of the “self-digesion” reaches in most spreading tumors. Being the biology approved mechanism of cancer protection, autophagy and its marker LC3B determine the inner origin of the ineffective anti-cancer treatment.”

“Aggressive cancers behavior is revealed to be dependent on the onset of the autophagy, and its regulators.”

 

2. The data on the autophagy involvement in to the anti-cancer treatment effect were clarified.

“Even though multiple mechanisms are known to be responsible for the anti-cancer treatment benefits, the autophagy regulation belong to the most powerful processes that could explain the biology of variable response to the therapy in GC patients.”

 

3. The quality of figures was improved

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

Authors present a study on the evaluation of LC3B and AMPK as predictive biomarkers of response to neoadjuvant FLOT in gastric cancer. Overall, the manuscript is difficult to read, a strong revision of english is necessary along with some other minor mistakes (t -800C in the abstract just to provide an example). The abstract should be rewritten and strongly synthesized as it is not focused. Importantly, methods are not adequately described: WB is missing, and all statistical analysis are not properly described, which is a fundamental point in a correlation study. Even more importantly, the same purpose is not clear...why data on flot feasibility? It is outside of the scope and also statistical methodology is not appropriate to this purpose. As this information would not probably add anything new to the field as this regimen is highly consolidated, it is in reviewers opinion that authors should focus on biomarker studies.

Author Response

Authors are pleased for the paper revision.

All marks were taken into account.

1. The linguistic correction was made. Proofreading
2. The abstract was rewritten (all parts)
3. The introduction part of the paper was shortened and rebuilt.
4. The aim of the paper was clarified.
5. The discussion and conclusion of the paper were also rewritten. The main point of the study was rewritten.

Reviewer 2 Report

Interesting study on a timely topic in gastric cancer. The manuscript is quite well written and organized. English could be improved.

I suggest the following changes:

• Introduction section: although the authors correctly included important papers in this setting, I believe some recent papers regarding emerging treatments in gastric cancer should be cited within the introduction ( PMID: 33916206 ; PMID: 33916915 ), only for a matter of consistency. We think it might be useful to introduce the topic of this interesting study.
• Methods and Statistical Analysis: nothing to add.
• Discussion section: Very interesting and timely discussion. Of note, the authors should expand the Discussion section, including a more personal perspective to reflect on. For example, they could answer the following questions – in order to facilitate the understanding of this complex topic for readers: what potential does this study hold? What are the knowledge gaps and how do researchers tackle them? How do you see this area unfolding in the next 5 years? We think it would be extremely interesting for the readers.

However, I think the authors should be acknowledged for their work. In fact, they correctly addressed an important topic in gastric carcinoma, the methods sound good and their discussion is well balanced.

One additional little flaw: the authors could better explain the limitations of their work, in the last part of the Discussion.

I believe this article is suitable for publication in the journal although some revisions are needed. The main strengths of this paper are that it addresses an interesting and very timely question and provides a clear answer, with some limitations.

I suggest a linguistic revision and the addition of some references for a matter of consistency. Moreover, the authors should better clarify some points.

Author Response

Authors are pleased for the paper revision.

All marks were taken into account.

1. Proofreading was made.
2. The abstract was rewritten (all parts)
3. The introduction part of the paper was shortened and rebuilt.
4. The aim of the paper was clarified.
5. The main point of the study was rewritten. The discussion part of the paper was modified

Reviewer 3 Report

Included in the attached file "peer-review-19187747.v2.pdf"

Comments for author File: Comments.pdf

Author Response

Response to the reviewer

1) The abstract was modified

2) Lines 50-52: were rewritten.

3) The number of the patients was corrected

4) Line 25: improved

5) the tables were modified, the number of samples were added

6) Clinical significance is highlighted

7) The conclusions  are rewritten with two paragraphs.

Round 2

Reviewer 1 Report

Authors have merely reduced the length of the manuscript without addressing properly reviewer's comments.

Author Response

Authors are pleased for the paper revision.

All marks were taken into account.

1. The linguistic correction was made. Proofreading
2. The abstract was rewritten (all parts)
3. The introduction part of the paper was shortened and rebuilt.
4. The aim of the paper was clarified.
5. WB is present (point 2.2.)

2.2. Determination of LC3B content.

Electrophoresis SDS-PAGE (Laemmli) was used [29]. The protein was transferred to 0.2-/xm pore-sized PVDF membrane (GE Healthcare, UK), either at 150 mA or 100 V for 1 h by using a Bio-Rad Mini Trans- Blot electrophoresis cell. The membrane was incubated in a 1:2500 dilution of monoclonal mouse anti-human LC3B (Affinity Biosciences, USA) at 4 ºС overnight.

PVDF samples were incubated in Amersham ECL western blotting detection analysis system (Amersham, USA). The results were standardized using the beta-actin expression in a sample and were expressed in percentages to the protein content in non-transformed tissues. The level of protein in normal gastric tissue was indicated as 100%.

6. Statistical analysis methods were modified and clarified.

Statistical analysis was performed using SPSS 19.0 software. Data were expressed as median and ranges. Mann-Whitney test was used for comparing differences in mean values in two independent groups. Nonparametric one-way ANOVA on ranks was carried out to test whether samples originate from the same distribution, which is used to compare two or more independent samples of equal or different sample sizes. Median Test and Kruskal-Wallis test was applied. Nonparametric correlation analysis was performed, and the Spearmen coefficient was calculated.

7. Dear, Reviewer, we were trying to use correlation analysis at the first steps of the study. It can highlight the complex interrelationships in the analysis the link between the mRNA level and protein content.

Reviewer 2 Report

The authors did not address the issues and queries required.

Please revise accordingly. 

Author Response

Authors are pleased for the paper revision.

1. Proofreading was made.
2. PMID: 33916206 ; PMID: 33916915  The references were added to the paper.
3. The abstract was rewritten (all parts)
4. The introduction part of the paper was shortened and rebuilt.
5. The aim of the paper was clarified.
6. The main point of the study was rewritten. The discussion part of the paper was modified

The authors attempted to present, in a small group of patients, the relationship between the variability of tumor response to treatment and the autophagy. The widespread use of the hemotherapeutic and targeted drugs at the present time without their role clarifying in tumor progression becomes meaningless. It is clear that in the next 5 years there will be a turning point in the oncogenesis deeper understanding. Molecular targets capable to influence the anticancer treatment effectiveness will be identified.

7. Authors try to explain the limitation of the work. The adoption of a personalized approach in the cancer patients treatment has been limited so far. At the same time, it becomes clear that the manifestation of the aggressive properties of the tumor during treatment is the result of inadequate treatment.

“Because of biological behavior modification with the growth in tumor aggressiveness is a consequence of the anti-cancer therapeutical agents usage. Chemotherapy interventions need to consider both the early signs of a poor cancer prognosis and molecular-based effect of treatment.”

Round 3

Reviewer 1 Report

Authors have only slightly shortened the manuscript withdrawing a few words. This is not a revised version.

Reviewer 2 Report

The authors addressed the queries we raised.

We recommend Acceptance.