Title: CLDN18.2 Expression and Its Impact on Prognosis and the Immune Microenvironment
in Gastric Cancer
Version: 1 Date: 14 Jul 2023
Reviewer's report:
Using immunohistochemistry on TMA analysis authors evaluated expression of Claudin 18.2 in
series of 451 therapy-naive gastric cancers (GC) and performed analysis of clinicopathological
features and survival data.  The methods seem appropriate and the results are described in detail.
However, I have some important points that the authors should consider:
1) In the introduction section, a hypothesis could be added. What was the hypothesis at the
beginning of the study?
2) In the “Immunohistochemical evalutation” section it is shortly mentioned that representative
samples of GC tissue, normal tissue and lymph nodes are selected for preparation of TMAs. How
many TMAs were used per case? In particular, how many tissue cores per case were taken from
the cancer area? I think this information is very important, as previous studies have described
the heterogenous expression of Claudin 18.2 in the cancer tissue.
3) How did you assess the percentage of stained cells? Did you counted only the cancer cells /
the epithelial cells in the normal adjacent tissue? Or was the stromal compartment also included
in the assessment? From what I see in the representative images (Figure 2A), this could be the
case. If yes, this should be noted in Materials & Methods.
4) Why did you use the cutoff H-Score = 6? Does it differ from previous studies and the cut-
offs being used for eligibility in Zolbetuximab studies?
5) Were there any cases that showed different expression levels in cancer area and in matched
lymph node metastase? Do cases with high CLDN18.2 expression in the metastasis were
associated with poor prognosis when compared to CLDN18.2-negative cases?
6) The paragraph “Relationship between expression of the CLDN18.2 protein and
clinicopathological features” can lead to confusion, as the p values in the text/Figure 2 does not
fit to the data in the Table 2. If 2 different statistical methods were used, this could be shortly
indicated in the text. That would make the results more clear.
7) Different from previous studies, a positive correlation between CLDN18.2 and PD-L1 is
demonstrated in this study. How would you explain this association and the discrepancies with
previous data?
8) The strengths and limitations of the study should be added at the end of the discussion section.
What are the strengths and limitations of this study?
9) In my opinion, the Figure 1 (plots from databases) could be transfered to the supplement.
You could add larger representative histological images here, because Figure 2A is too small to
appreciate the CLDN18.2 expression.
10) If possible, professional language editing should be carried out. For example, there is no
such “Lauran” type, it is Lauren or Laurén. Perhaps better use the term “adjacent tissue” or
“peritumoral tissue” instead of paraneoplastic tissue.
Title: CLDN18.2 Expression and Its Impact on Prognosis and the Immune Microenvironment
in Gastric Cancer
Version: 1 Date: 14 Jul 2023
Reviewer's report:
Using immunohistochemistry on TMA analysis authors evaluated expression of Claudin 18.2 in
series of 451 therapy-naive gastric cancers (GC) and performed analysis of clinicopathological
features and survival data.  The methods seem appropriate and the results are described in detail.
However, I have some important points that the authors should consider:
1) In the introduction section, a hypothesis could be added. What was the hypothesis at the
beginning of the study?
2) In the “Immunohistochemical evalutation” section it is shortly mentioned that representative
samples of GC tissue, normal tissue and lymph nodes are selected for preparation of TMAs. How
many TMAs were used per case? In particular, how many tissue cores per case were taken from
the cancer area? I think this information is very important, as previous studies have described
the heterogenous expression of Claudin 18.2 in the cancer tissue.
3) How did you assess the percentage of stained cells? Did you counted only the cancer cells /
the epithelial cells in the normal adjacent tissue? Or was the stromal compartment also included
in the assessment? From what I see in the representative images (Figure 2A), this could be the
case. If yes, this should be noted in Materials & Methods.
4) Why did you use the cutoff H-Score = 6? Does it differ from previous studies and the cut-
offs being used for eligibility in Zolbetuximab studies?
5) Were there any cases that showed different expression levels in cancer area and in matched
lymph node metastase? Do cases with high CLDN18.2 expression in the metastasis were
associated with poor prognosis when compared to CLDN18.2-negative cases?
6) The paragraph “Relationship between expression of the CLDN18.2 protein and
clinicopathological features” can lead to confusion, as the p values in the text/Figure 2 does not
fit to the data in the Table 2. If 2 different statistical methods were used, this could be shortly
indicated in the text. That would make the results more clear.
7) Different from previous studies, a positive correlation between CLDN18.2 and PD-L1 is
demonstrated in this study. How would you explain this association and the discrepancies with
previous data?
8) The strengths and limitations of the study should be added at the end of the discussion section.
What are the strengths and limitations of this study?
9) In my opinion, the Figure 1 (plots from databases) could be transfered to the supplement.
You could add larger representative histological images here, because Figure 2A is too small to
appreciate the CLDN18.2 expression.
10) If possible, professional language editing should be carried out. For example, there is no
such “Lauran” type, it is Lauren or Laurén. Perhaps better use the term “adjacent tissue” or
“peritumoral tissue” instead of paraneoplastic tissue.
Please review this paper.
user
Title: CLDN18.2 Expression and Its Impact on Prognosis and the Immune Microenvironment
in Gastric Cancer
Version: 1 Date: 14 Jul 2023
Reviewer's report:
Using immunohistochemistry on TMA analysis authors evaluated expression of Claudin 18.2 in
series of 451 therapy-naive gastric cancers (GC) and performed analysis of clinicopathological
features and survival data.  The methods seem appropriate and the results are described in detail.
However, I have some important points that the authors should consider:
1) In the introduction section, a hypothesis could be added. What was the hypothesis at the
beginning of the study?
2) In the “Immunohistochemical evalutation” section it is shortly mentioned that representative
samples of GC tissue, normal tissue and lymph nodes are selected for preparation of TMAs. How
many TMAs were used per case? In particular, how many tissue cores per case were taken from
the cancer area? I think this information is very important, as previous studies have described
the heterogenous expression of Claudin 18.2 in the cancer tissue.
3) How did you assess the percentage of stained cells? Did you counted only the cancer cells /
the epithelial cells in the normal adjacent tissue? Or was the stromal compartment also included
in the assessment? From what I see in the representative images (Figure 2A), this could be the
case. If yes, this should be noted in Materials & Methods.
4) Why did you use the cutoff H-Score = 6? Does it differ from previous studies and the cut-
offs being used for eligibility in Zolbetuximab studies?
5) Were there any cases that showed different expression levels in cancer area and in matched
lymph node metastase? Do cases with high CLDN18.2 expression in the metastasis were
associated with poor prognosis when compared to CLDN18.2-negative cases?
6) The paragraph “Relationship between expression of the CLDN18.2 protein and
clinicopathological features” can lead to confusion, as the p values in the text/Figure 2 does not
fit to the data in the Table 2. If 2 different statistical methods were used, this could be shortly
indicated in the text. That would make the results more clear.
7) Different from previous studies, a positive correlation between CLDN18.2 and PD-L1 is
demonstrated in this study. How would you explain this association and the discrepancies with
previous data?
8) The strengths and limitations of the study should be added at the end of the discussion section.
What are the strengths and limitations of this study?
9) In my opinion, the Figure 1 (plots from databases) could be transfered to the supplement.
You could add larger representative histological images here, because Figure 2A is too small to
appreciate the CLDN18.2 expression.
10) If possible, professional language editing should be carried out. For example, there is no
such “Lauran” type, it is Lauren or Laurén. Perhaps better use the term “adjacent tissue” or
“peritumoral tissue” instead of paraneoplastic tissue.
assistant