Differential Immune Infiltration Profiles in Colitis-Associated Colorectal Cancer versus Sporadic Colorectal Cancer

Round 1

Reviewer 1 Report

The study underscores the differences in immune infiltration between CAC and sCRC. While high CD3+ and CD20+ infiltrates are associated with improved survival in CAC, this correlation is absent in sCRC. The study also highlights the distinct roles of TIGIT and TOX expression in these cancer types, suggesting that T-cell exhaustion might play a minor role in CAC development.

Understanding the immune infiltration profiles in different types of CRC can provide insights into disease progression and potential therapeutic targets. This study sheds light on the differential immune responses in CAC and sCRC, which can have implications for treatment strategies and patient prognosis.

The research provides valuable insights into the immune landscape of CAC and sCRC, emphasizing the need to consider the unique immune profiles when devising therapeutic strategies. The study is well-structured, with clear objectives and robust methodologies. Future research could delve deeper into the mechanisms underlying these differential immune responses and explore potential therapeutic interventions targeting these pathways.

In my opinion this study is ready to be submitted, just reviewed some points.

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Author Response

Thank you for your positive feedback. The introduction has been revised in response to various comments.

Reviewer 2 Report

Simple summary must be added. In the present version guidelines from journal site (not text) are presented

Citations of references in the text are not in agreement with journal guidelines. guidelines  because In the text, reference numbers should be placed in square brackets

In the introduction previous studies on immune infiltration profiles during Crohn's disease and ulcerative colitis as well as during colorectal cancer should be described in more detail.

The novelty of investigation and eventually clinical significance should be underlined at the end of the introduction.

The information that investigation has been performed in agreement with Declaration of Helsinki should be added.

Specifications of antibodies used in the study (with catalogue number, working dilutions, name of company, city and country (in the case of the USA also the abbreviation of the state) should be presented in table. Table will be more readable.

In specification of all reagents and equipment the name of city should be added. For example in line instead “Zeiss, Germany” should be “Zeiss, the name of city, where the company is located, Germany”

Microphotographs in the figure 1 are not readable. Scale bares should be added on photographs.

What is the clinical relevance of the research? This issue should be thoroughly discussed in the discussion.

Conclusion is rather enigmatic.  I strongly suggest redrafting this part of the manuscript

Author Response

Thank you very much for these comprehensive comments. We have adjusted the manuscript accordingly and believe that it has thereby gained in quality.

Point by point response: 

1. Simple summary must be added. In the present version guidelines from journal site (not text) are presented

We added a simple summary.

[SIMPLE SUMMARY: Chronic inflammation plays a significant role in colorectal cancer (CRC) development, particularly in colitis-associated CRC (CAC). This study examined immune infiltration patterns in CAC patients compared to sporadic CRC (sCRC) patients and their impact on prognosis. Twenty CAC and twenty sCRC patients, matched by tumor characteristics, were analyzed. Immunohistochemistry targeted various immune markers, including T-cell and B-cell markers, in tumor and adjacent mucosal tissues. The results revealed differences between CAC and sCRC in T-cell exhaustion markers (TOX, TIGIT) and immune cell infiltration. High CD3+ and CD20+ cell levels correlated with improved survival in CAC but not in sCRC. This study highlighted distinct immune profiles in CAC and sCRC, suggesting that T-cell exhaustion might play a different role in CAC development than in sCRC. Understanding these immune differences could impact treatment strategies and prognosis for CAC patients.]

2. Citations of references in the text are not in agreement with journal guidelines. guidelines  because In the text, reference numbers should be placed in square brackets

We adapted the references according to the journal guidelines.

3. The novelty of investigation and eventually clinical significance should be underlined at the end of the introduction.

We added the following sentence to further emphasize the clinical significance of the performed investigation to the introduction: 

[To minimize confounders, we conducted the first study on CAC and sCRC with meticulously matching tumor location, TNM stages, and grading.]

4. The information that investigation has been performed in agreement with Declaration of Helsinki should be added.

The investigation has been performed in agreement with Declaration of Helsinki. This statement was added to the text into the Methods section.

5. Specifications of antibodies used in the study (with catalogue number, working dilutions, name of company, city and country (in the case of the USA also the abbreviation of the state) should be presented in table. Table will be more readable. In specification of all reagents and equipment the name of city should be added.

We have organized the information regarding the antibodies utilized in this study within a table.

 6. For example in line instead “Zeiss, Germany” should be “Zeiss, the name of city, where the company is located, Germany”

 We added this information to the companies mentioned in the text.

7. Microphotographs in the figure 1 are not readable. Scale bares should be added on photographs.

 We have included scale bars in the photographs in figure 1.

We will also upload the figure separately for higher resolution.

8. What is the clinical relevance of the research? This issue should be thoroughly discussed in the discussion.

We added this sentence:

[At present, the clinical significance is of course constrained. Our objective has been to obtain initial indications of which immune factors might contribute to a notably aggressive course of CAC. Specifically, easily measurable parameters detectable through immunohistochemistry could be of great utility in this regard. In fact, heightened infiltration of TIGIT+ and FOXP3+ cells could potentially result in an immunosuppressive phenotype. This, in turn, could streamline the consideration of initiating a more aggressive adjuvant therapy at earlier UICC stages.]

9. Conclusion is rather enigmatic. I strongly suggest redrafting this part of the manuscript.

We redrafted the conclusion:

[The study revealed a significant divergence in T-cell exhaustion markers TIGIT and TOX, shedding light on the distinct nature of CAC. Due to the relatively small number of patients, further investigation is needed to gain deeper insights of the role of immune infiltrates in CAC.]

Reviewer 3 Report

An interesting topic that opens up new study opportunities. I hope you will continue the research you have started.

Author Response

Thank you for your valuable positive feedback. We are planning an expansion of the study.

Reviewer 4 Report

The present paper deals with some differences of lymphoid cell populations and neutrophils in sporadic colorectal cancer (sCRC) versus colitis-associated cancer (CAC).  The samples of inflamed and tumor tissues were taken in representative groups of well-verified CAC and sCRC patients excluding hereditary cancers. Immunochemistry was targeted for the basic T- and B-cell cell surface markers, as well as neutrophil-specific antigen and T-cell exhaustion markers. In general, the study revealed somewhat different B- and T cell patterns for sporadic and colitis-associated cancer cases.  E.g., the TOX+ cell infiltration proved to be lower in CAC than in sCRC. TIGIT+ infiltration was lower at higher sCRC stages. In CAC, high CD3+ and CD20+ infiltrates relate to improved survival, being absent in sCRC. In general, these multiple differences do not provide a systematic view on biological significance of these findings. 

Remarks:

Line 97: one may briefly refer to the literature sources or guidelines on hereditary factors of CRC and CAC which served as exclusion criteria in the study cohort. 

Lines 125-130: The panel of Mabs for immunostaining is well chosen. However, the authors did not evaluate the populations of macrophages/dendritic cells which are also an important component of any tumor microenvironment. The data on resident macrophages in the CRC samples (not found in Results) would be quite valuable for subsequent data interpretation, or the literature data may be, at least, mentioned under Discussion . 

Line245 ….. (Prognostic analysis) The survival curves are based on relatively small number of cases, with nearly-marginal p values. Therefore, these data could be interpretetd quite cautiously as a preliminary result (this should be stressed in Conclusions).

 Minor remarks:

The editorial instruction concerning simple summary should be deleted. In general, copy editing is not required.

Author Response

Thank you very much for this productive review. We have adjusted the manuscript accordingly and believe that it has thereby further gained in quality.

Point by point response

• In general, these multiple differences do not provide a systematic view on biological significance of these findings.

Unfortunately, the data thus far have not revealed a conclusive correlation between infiltration and survival. The study design is an observational study aimed at analyzing the existing immune infiltrates in tumors and mucosa in CAC and sporadic CRC. Further analyses, including prospective investigations, are required to fully elucidate this matter.

1. Remarks: Line 97: one may briefly refer to the literature sources or guidelines on hereditary factors of CRC and CAC which served as exclusion criteria in the study cohort.

We specified the inclusion criteria in the Methods section:

[The exclusion criterion was a known familial predisposition, such as variants associated with non-polyposis colorectal cancer (HNPCC), polyposis variants like Familial Adenomatous Polyposis (FAP), or MUTYH-associated syndromes]

2. Lines 125-130: The panel of Mabs for immunostaining is well chosen. However, the authors did not evaluate the populations of macrophages/dendritic cells which are also an important component of any tumor microenvironment.

The role of macrophages was not specifically addressed in this study due to conflicting data regarding their prognostic impacts in the literature. The primary focus of this study centers on lymphocyte infiltration. Indeed, conducting a comprehensive investigation that includes macrophages and dendritic cells could be quite intriguing.

3. The data on resident macrophages in the CRC samples (not found in Results) would be quite valuable for subsequent data interpretation, or the literature data may be, at least, mentioned under Discussion .

A very intriguing point made by the reviewer; a more detailed examination of macrophages would be highly interesting here. For the sake of completeness, a brief paragraph discussing the potential role of macrophages in CAC development has been added to the discussion section:

[In this study, we did not address dendritic cells such as macrophages, Tumor-Associated Macrophages (TAMs) are a diverse population of immune cells that play a complex role in CRC and probably in CAC prognosis [44]. Macrophages can polarize into M1 and M2 types, with distinct roles. M1 promotes inflammation and has anti-tumor effects, while M2 has anti-inflammatory properties and supports cancer development [45]. The type and location of TAMs can have different prognostic significance: M2 TAMs have been associated with poor CRC prognosis, high M1 macrophage and CD68+TAMs infiltration is correlated with a better prognostic situation in CRC patients [45]. The transition from UC to CAC involves two key processes: inflammation and tumorigenesis, both of which involve macrophages [44]. For example, in the UC inflammatory phase, excessive macrophage infiltration could worsen the inflammation by inducing proinflammatory cytokine production and promoting CAC; Then, before tumor formation, M1 primarily drives inflammation and tumorigenesis. In established CAC, M1 inhibits progression via anti-tumor immunity, while M2 promotes tumor growth and metastasis [44]. Notably, some studies suggest that M2 not only advances tumors but also raises CAC risk, possibly due to their early-stage tumor-promoting role [46]. ]

4. Line245 ….. (Prognostic analysis) The survival curves are based on relatively small number of cases, with nearly-marginal p values. Therefore, these data could be interpretetd quite cautiously as a preliminary result (this should be stressed in Conclusions).

We have reiterated this fact in both the limitations and the conclusion:

Second, a relatively small number of CAC patients recruited in this study may impose limitations. [Hence, survival analyses should be interpreted with caution, especially in this context.]

AND

[Due to the relatively small number of patients], further investigation is needed to gain deeper insights of the role of immune infiltrates in CAC.

• Minor remarks: The editorial instruction concerning simple summary should be deleted. In general, copy editing is not required.

We followed the journal's instructions to provide a simple summary.