PONE-D-21-12611

Incidence of malignant transformation in the oviductal fimbria in laying hens, a preclinical model of spontaneous ovarian cancer

PLOS ONE

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Academic Editor

PLOS ONE

Additional Editor Comments:

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2. Please provide the product number and any lot numbers of the antibodies purchased for your study.

3.  At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(1) Please state the source of hens used in the study

(2) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment), and any steps taken to minimize animal suffering and distress

(3) Please include the method of euthanasia

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

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 manuscript communicates the very interesting occurrence of tumors located on the fimbriae of the laying hen. This is significant in that high grade serous cancer in women is thought to arise from a primary lesion in the fimbria, characterized by a p53 signature in the secretory cells. Other groups have identified commonalities in gene expression between hen ovarian tumors and normal oviduct (see Trevino, LS, Giles, JR Wang, W, Urick ME Johnson PA. Gene Expression Profiling Reveals Differentially Expressed Genes in Ovarian Cancer of the Hen: Support for Oviductal Origin? Hormones and Cancer. August 2010; 1(4):177-86 DOI: 10.1007/s12672-010-0024-8) and mutations in p53 have been identified by sequencing cDNA from ovarian tumors (reference 16, Hakim, et al). These observations are relevant to this manuscript but have been glossed over in the introduction (lines 94-96).

Until the authors are able to demonstrate that the p53 gene is mutated, they should refrain from referring to it as mutant and instead describe it as abnormal staining. The assumption that human p53 staining in tumors is the result of a mutation has been backed up innumerable times by sequencing the gene or transcript. Furthermore, the PAX family (2, 5 and 8) has been shown to inhibit p53 transcription, so the increase in p53 transcript may simply reflect loss of PAX2 protein expression (ref. Stuart ET, Haffner R, Oren M, Gruss P. Loss of p53 function through PAX-mediated transcriptional repression. EMBO J. 1995 Nov 15; 14(22):5638-45. PMID: 8521821; PMCID: PMC394679.)

Additionally, the Pax 8 gene is not present in avian species including chickens (ref Paixao-Cortes VR, Salzano FM, Bortolini MC (2013) Evolutionary History of Chordate PAX Genes: Dynamics of Change in a Complex Gene Family. PLoS ONE 8(9): e73560. doi:10.1371/journal.pone.0073560). Therefore it is recommended that the authors remove of all mention of PAX8.

The data on PAX8 protein raises the question of how all of the antibodies used were validated for use in the chicken. For example, the western blot of WT-1 in figure 3E shows the molecular weight to be 65 kDa whereas other references show chicken embryo WT-1 to have a molecular weight of 42 kDa (Cell Tissue Res (2001) 303:173–186 DOI 10.1007/s004410000307 R. Carmona · M. González-Iriarte J.M. Pérez-Pomares · R. Muñoz-Chápuli Localization of the Wilms’ tumour protein WT1 in avian embryos).

Additionally, the antibodies that were used in this manuscript should be identified by catalog number or clone.

The intensity of Ki67 expression in figure S2 seems relatively comparable between normal fimbria and the tumor cells, although normal fimbriae are described to have “few” Ki67 positive cells (page 18, line382). In fact, it is difficult to determine if any of the normal fimbrial epithelial cells are negative for Ki67. It would be more accurate to report the number of positive cells relative to the total number of epithelial cells in several fields. It is misleading to use actin as a normalization protein in the western blot as there are varying amounts of non-Ki67 positive tissues (actin-containing stroma) in the samples. An epithelial specific marker would more accurately reflect the Ki67 quantity in the epithelial compartments.

A table should be added that shows the number of hens in each category as described on page 10 in Gross Presentation. Hens categorized in group 4 had masses in ovaries and/or fimbriae (line 226 page 11) these should be separated into ovary, fimbria and both (three subcategories of group 4). It is not clear if all hens in group 2 had ovarian tumors, or also oviductal tumors. The use of and/or (page 10 line 211 and page 11 line 226) renders these categorizations very murky

If the study was undertaken to examine the incidence, (page 5, line 119 and page 18 line 394), what is the incidence of malignant transformation in the fimbriae? The answer might be more obvious by the inclusion of the table, if not explicitly stated.

Were patient specimens from BRCA1 mutated fimbrial tissue used in this manuscript? If not, should they be included in the description of clinical specimens (page 6 line 138)?

Figure 1 C is described as a tumor in the ovary involving the fimbria. An arrow pointing to this involvement would be useful. A suggestion for future photographs would be to remove the tumors and reproductive tract from the animal for clearer viewing.

Page 5 line 111. Should read Tumor protein p53, not tissue.

Reviewer #2: The aim of this study was to examine if the oviductal fimbria in hens is a site of origin of Ovarian high grade serous carcinoma (HGSC) and whether it expresses several putative markers expressed in ovarian HGSC in patients. Authors showed that, compared with normal fimbriae, intensities of expression of WT-1, mutant p53, CA-125, PAX8, PAX2 and Ki67 were higher in fimbrial tumors, however, the expression of PAX2 decreased gradually as the tumor progressed to late stages. The patterns of expression of these markers were similar to those in ovarian HGSC patients. Thus, authors suggest that tumors of the oviductal fimbria in hens may offer a preclinical model to study different aspects of spontaneous ovarian HGSC in women.

This study is designed with unique idea to use a chicken model that enables to investigate the factors responsible for the ovarian and infundibular cancer. The results show interesting and novel findings that expression profiles of specific markers were similar to those in ovarian HGSC patients, and thus tumors of the oviductal fimbria in hens may offer a preclinical model to study the spontaneous ovarian HGSC.

Major comments

1. Authors describe the examined tissue only as the infundibulum. However, the hen infundibulum has a unique structure; namely, at the edge of the fimbria (approximately 2 mm), mucosal surface on the inner side extends even on the outer side before the epithelium transit to serosa. Thus, both inner and outer surface of this part of the fimbria are surrounded serosa by mucosal epithelium. Furthermore, lymohocyst-like structures may appear occasionally in that outer mucosal epithelium. Authors show the micrographs for the immunohistochemistry; they should make it clear which parts of the infundibulum were examined, cephalic, middle, caudal, inner side or outer side.

The following paper may help the authors to understand the comments of this reviewer.

Fujii S et al. (!981) The regional morphology of the infundibulum of the hen's oviduct with special reference to the mechanism of the engulfing of the ovulated ovum. J. Fac. Appl. Biol. Sci. Hiroshima Univ. 20: 87-98. ( https://ir.lib.hiroshima-u.ac.jp/files/public/2/23426/20141016143413572892/24-2126.pdf )

2. In the Results section, Figure legends are presented. It makes difficult to read the manuscript, and thus the figure legends should be put on separate pages.

3. Quality of Fig. 2A is not acceptable. Authors should prepare a new photograph to replace with the current one.

Specific comments

L134: Give the name of the institute that approved the animal experiment.

L154: What is D2O? Add full spelling at the place where the abbreviation appears first.

L155: What is neutralization? You may mean the endogenous peroxidase was quenched.

L159-160: Add the name of animals used to raise the antibodies.

L172: Add how to confirm the specificity of the immunostaining. Although western blotting was performed, IHC specificity is also required.

L174-183: Is the word of “intensity of the staining” correct? This reviewer assumes it may the positive area in 20,000 μm2.

L193: Procedure for the total RNA extraction, revers transcription for CDNA synthesis, and qRT-PCR condition should be added.

L202: Actin should be β-actin.

L241: Delete cloaca, cloaca is not a part of oviduct.

L260: Crypts in the funnel part may not be the secretory glands, although the chalaza is secreted in the tubular region of the infundibulum.

L265: Specify the pleomorphic nuclei containing mitotic bodies by arrows on the micrographs.

L284: Specify the intense staining for WT-1 by arrows on the micrographs.

L344: Although authors describe the expression is significant, P=0.075 does not show significance.

L355: Specify the intense staining for PAX8 by arrows on the micrographs.

L459-450: This sentence is difficult to follow.

Figures: Magnification should be shown by scale bars. “X40” is not a correct magnification.

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Reviewer #1: No

Reviewer #2: No

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Incidence of malignant transformation in the oviductal fimbria in laying hens, a preclinical model of spontaneous ovarian cancer

PONE-D-21-12611R1

Dear Dr. Barua,

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Yihong Wang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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