PONE-D-22-21974CELA3B immunostaining is a highly specific marker for acinar cell carcinoma of the pancreasPLOS ONE

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Additional Editor Comments :

Congratulations on the good work! Only minor revision is recommeded!

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an extensive IHC analysis of an impressive number of human tumors and normal tissues studying the expression of chymotrypsin-like elastase family member 3B (CELA3B), a pancreatic enzyme with digestive function in the intestine. Since previous RNA analyses had suggest that CELA3B expression is limited to the pancreas, the authors wanted to explore the diagnostic utility of CELA3B IHC for the distinction of pancreatic from extrapancreatic cancers and for the distinction of the rare acinar cell carcinomas from ductal adenocarcinomas.

Using TMAs, CELA3B expression was successfully analyzed by IHC in 13,223 tumor samples from 132 tumor types/subtypes and 76 different normal tissues. In principle, CELA3B staining was limited to acinar cells and to a fraction of ductal cells of the pancreas. In tumors, CELA3B was only expressed in 12 of 16 (75%) pancreatic acinar cell carcinomas, including 6 cases with strong staining as well as in 5 of 13,207 other tumors (0.04%). The latter included a few cases of salivary gland tumors. The authors conclude that the CELA3B IHC data shows good sensitivity (75%) and high specificity (99.9%) for diagnosing acinar cell carcinoma of the pancreas.

This is a solid and well-written paper exploring the possible use of CELA3B IHC for the diagnosis of a rare and difficult to diagnose subset of pancreatic cancers. The results are clear and convincing and the study is the first to use IHC to study the expression of CELA3B in tumors. The authors have long and well-documented experience in producing and analyzing TMAs. I only have a few minor comments.

To resolve the issue of CELA3B staining in non-acinar pancreatic cells, I suggest the authors use RNA in situ hybridization to look for mRNA expression in these cells.

The authors should discuss the observation of CELA3B staining in the few salivary gland tumors.

The quality of the immunostainings in Fig 3 (“non-pancreatic” tumors with positive CELA3B staining) could be improved.

Reviewer #2: Immunohistochemistry in a tissue microarray format (TMA) was used to analyze the CELA3B expression in 13,223 tumor samples from 132 different tumor types and subtypes as well as 8 samples each of 76 different normal tissue types. The authors have found that the CELA3B immunostaining was seen in 12 of 16 (75%) acinar cell carcinoma of the pancreas including 6 cases with strong staining (37.5%) as well as in 5 of 13,207 other tumors (0.04%). This study is of important significance for the clinical pathological diagnosis. However, evidence for good sensitivity (75%) of CELA3B immunohistochemistry for diagnosing acinar cell carcinoma of the pancreas is not sufficient. Here are some questions before this article was published:

1. Most importantly, the number of positive cases of acinar cell carcinoma of the pancreas, which was only 16, was low not only in the overall database (13,223 cases) but also in comparison. More cases of acinar cell carcinoma of the pancreas are needed in order to draw a conclusion.

2. How do the authors draw conclusions of high sensitivity (99.9%) of CELA3B immunohistochemistry for diagnosing acinar cell carcinoma of the pancreas?

3. Although CELA3B immunostaining was seen in 12 of 16 (75%) acinar cell carcinoma of the pancreas，but was essentially negative in the remaining 4 cases (20%)，suggesting that the absence of CELA3B immunostaining is a good predictor of acinar cell carcinoma of the pancreas.

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Reviewer #1: No

Reviewer #2: No

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CELA3B immunostaining is a highly specific marker for acinar cell carcinoma of the pancreas

PONE-D-22-21974R1

Dear Dr. Simon,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Shuai Ren

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The final version is ready for publication now.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 2) The authors should discuss the observation of CELA3B staining in the few salivary gland tumors.

Authors reply: We have discussed similarities between salivary glands and the pancreatic gland that might explain occasional CELA3B staining in salivary gland tumors on page 8, lines 187-195.

As far as I can see there is no discussion or mentioning of salivary gland tumors on page 8, lines 187-195. This part only concerns colorectal cancers. Please add the discussion about CELA3B staining in salivary gland tumors.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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