Title: Spontaneous Feline Mammary Intraepithelial Lesions as a Model for Human Estrogen Receptor- and Progesterone Receptor-Negative Breast Lesions

Transcription

1 Author's response to reviews Title: Spontaneous Feline Mammary Intraepithelial Lesions as a Model for Human Estrogen Receptor- and Progesterone Receptor-Negative Breast Lesions Authors: Giovanni P Burrai (vanniburrai@tiscali.it) Sulma I Mohammed (mohammes@purdue.edu) Margaret A Miller (mille188@purdue.edu) Vincenzo Marras (marrasv@gmail.com) Salvatore Pirino (pirino@uniss.it) Maria F Addis (addis@portocontericerche.it) Sergio Uzzau (uzzau@portocontericerche.it) Elisabetta Antuofermo (eantuofermo@uniss.it) Version: 2 Date: 9 February 2010 Author's response to reviews: see over

2 Dear BioMed Central Editorial Team We have revised our manuscript and to avoid confusion we ve change the title: Spontaneous Feline Mammary Intraepithelial Lesions as a Model for Human Estrogen- and Progesterone-Receptors Negative Breast Lesions as follow: Spontaneous Feline Mammary Intraepithelial Lesions as a Model for Human Estrogen Receptor- and Progesterone Receptor-Negative Breast Lesions. We appreciate the reviewers careful critiques and comments, and have revised the manuscript accordingly. We would like to confirm that the manuscript contains 4 color plates. Below is our response in italics to the reviewers. Editorial points: Please clarify where the human samples in the Figures were obtained from and clarify whether ethical approval we needed for the study. To clarify the source of the human specimens, we added this sentence in Methods- Histology section: Human samples were obtained from the Institute of Anatomy and Histopathology, Sassari University School of Medicine. The study protocol was approved by the Ethical Committee at the University of Sassari. Acknowledgements. We strongly encourage you to include an Acknowledgements section between the Authors contributions section and Reference list. We included an Acknowledgements section: This study was supported by the Ministero dell Istruzione, dell Università e della Ricerca (MIUR), FAR grant We are grateful to Munazzah Rahman for valuable consultation, to Dott. Antonello Floris for assistance with statistical analysis and to Dott. Antonica Mura for technical support. Comments from our Associate Editor: The authors should address all reviewers questions/concerns and particularly the issue regarding how well the model fits progression of human ER negative tumors (reviewer #2). Strengths and weaknesses should be discussed. Reponses to the reviewers Reviewer 1: Linda Munson The manuscript investigates the morphological, selected immunohistochemical, and prognostic features of mammary intraepithelial lesions in cats with the goal of establishing domestic cats as a model for breast cancer in women. The manuscript is clearly written, well illustrated, and provides convincing data in support of this model. Overall, this is a significant contribution to knowledge of early morphological features associated with cancer risk in cats, and the similarities to human breast lesions support the hypothesis of a common pathway of carcinogenesis in these species. Publication is strongly recommended if the following minor essential revisions are addressed:

3 In the Methods on pg 8 it should be specified that the control tissues were feline. The 2 nd sentence in the Methods section was changed to: Histologic sections (5 µm thick) from formalin-fixed, paraffin-embedded feline mammary tissue with IELs and without IELs (control tissue) were mounted on positively charged Superfrost slides (Fisher Scientific). The number of cells/nuclei or microscopic fields assessed to determine % positive staining should be included. Sentence inserted in the Methods section - Immunohistochemistry- page 8 : Nuclear immunostaining for ER, PR and Ki-67 was evaluated counting a total of 1000 cells in 10 representative fields at high magnification (400x) whereas for smaller lesions, the entire lesion was considered. The number of immunopositive cells was expressed as a percentage (mean, median, minimum and maximum values ). Because myoepithelial components to tumors determined categorization of lesions, the authors should include how myoepithelial components were confirmed. IELs were categorized according to criteria established for classification of the human breast IELs [refs 26 and 27], in which myoepithelial cells are identified by their shape and cytologic features in HE-stained sections. The authors have applied the same criteria to classify mammary IELs in dogs [ref 47]. The use of the term relative risk is not clear in the manuscript (text and Table 1). Traditionally, this is a quantitative term, not categorical, so possibly a different term would be more appropriate. The authors also should include definitions for low, intermediate, and high The word relative is omitted, and the P-values were rounded up. Table 2. Cystpapillary, not cystopapillary is the traditional spelling. Corrected as tubulopapillary carcinoma as reported by W.H.O Histological Classification of Mammary Tumors of the Dog and Cat. In WHO Histological Classification of Mammary Tumors of the Dog and Cat, the papillary-cystic type is considered a variant of tubulopapillary carcinoma, so we have simply included this variant with the other tubulopapillary carcinomas and modified Table 2 accordingly. The overexpression of HER2/neu was considered unusual for normal tissues, yet the authors did not include information on how it was determined that those tissues were normal. Were frozen sections examined morphologically to assure no tumors were present? Tissue from normal mammary gland was evaluated histologically before protein extraction for western blot analysis. However, positive HER-2 expression in normal mammary tissues is not unusual in woman as reported by [ ref - Natali PG.: Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues. Int J Cancer Mar 15;45(3):457-61] and by [ ref - Press MF: Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene Jul;5(7):953-62].

4 The legend for Fig 4 states five tissues, yet nine tissues are represented. The Fig 4 legend has been changed to indicate that 4 mammary tumors were evaluated. Non neoplastic mammary tissue (4 specimens) and liver (1 specimen) were used for comparison. Reviewer 2: Ander Urruticoechea The present work has a clear design and is technically correct. The material and methods and the results section are clear and easy to read. Nevertheless, a major weakness of this work lies in the conclusion drawn by the authors stating that feline IEL lesions may be a good model for the study of the progression from IEL to invasive cancer in human ER negative tumours. This conclusion is the base of the interest of this paper. To reach this conclusion a number of non explained findings should be addressed by the authors: The fact that ovariectomized cats reduced in around 90% the risk of developing tumours while this paper seems to reveal the lack of importance of ER driven biology in the pathophysiology of feline breast cancer. Ovariohysterectomy reportedly reduces the risk of mammary cancer in cats by about 90% if ovariectomy is performed before 6 months or 1 year of age. However, later ovariectomy, between 13 and 24 months of age, has an insignificant protective effect (11%) and had a negative effect if done after two years of age, actually increasing the risk of feline mammary carcinoma development [13]. In our study, the median age of cats was 10 years, and most had been spayed after 1 year of age or were still intact. Thus, highlight the importance of ER driven biology in the pathophysiology of feline mammary tumor. Moreover also in women ER-negative invasive breast cancers are considered to be the result of tumor progression from ER-positive premalignant lesions or ER-positive breast cancers by genetic alteration (ref -: Ferguson et al. 1995) or may be directly ER-negative via a hormone-independent pathway (ref -: Korach 1994, Hewitt et al. 2002) The apparent discrepancy between the published Her-2 positivity rate in feline cancer and the findings of the authors. We are aware that some authors have shown overexpression of HER-2 in mammary feline carcinoma (39% De Maria [23], 40% Ordas [24], 59,6% Millanta [25]). This discrepancy could reflect a difference in preanalytical factors, such as tissue fixation, or in analytical factors, such as choice of Her-2 antibodies. On the other hand, this is the first study to address HER-2 expression in feline mammary IELs. The unexplained incidence of Her-2 positivity in non-neoplastic tissue. It is not uncommon for normal human breast tissue to express HER-2. [ref - : Natali PG.: Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues. Int J Cancer Mar 15;45(3):457-6]. In our study, the Her-2 positivity in normal feline mammary tissue could be the result of an antibody cross-reaction with another epidermal growth factor receptor, physiologically expressed in feline mammary gland. However, further investigations, such as Fluorescence in Situ Hybridization (FISH), are required to detect HER- 2/neu gene amplification, clarifying the exact nature of this unexpected reactivity.

5 The fact that 50% of usual hyperplasia is related to malignant tumours. In this study, the specimens were mastectomy or lumpectomy specimens from cats with mammary tumors, most of which were malignant. So, the presence of UH in the vicinity of the tumor is not surprising. It will be interesting to see what percentage of pure UH in feline mammary glands without palpable tumors will progress to cancer. This will be evaluated in future studies to compare feline IELs in glands without palpable tumors to those in the human breast in order to elucidate the pathogenesis of breast cancer. The apparent lost of ER expression in the gradient between IEL and cancer. All this facts highlight essential differences between feline and human IEL to cancer progression. Hence it is difficult to support the major conclusion of authors. We recognize that most human breast DCIS are ER-positive, especially well and moderately differentiated tumors and that ER expression usually increases with progression from normal to DCIS to invasive carcinoma. However, a large fraction of high-grade DCIS and the associated invasive carcinoma lack ER expression. [ref - : Diab SG: Concordance between estrogen receptor status of ductal carcinoma-in-situ and invasive disease: Analysis of the Surveillance, Epidemiology, and End Results program. Breast Cancer Res Treat 82:S62, 2003]. Thus, in cats, the loss of ER expression in the gradient from IELs to cancer is comparable to that reported in woman for Estrogen-Receptor-Negative breast lesions. Reviewer 3: Fernanda Seixas Travassos This paper describes a series of spontaneous intraepithelial lesions on feline mammary gland samples. This is the first description of such lesions in this species, which provides this manuscript of major significance. However, there are several points which need to be addressed to improve the comprehensibility of this paper. Major Compulsory Revisions In the Material and Methods section, page 9, 1st paragraph (Immunohistochemistry evaluation of ER, PR and Ki-67) it is not clear if the percentage of positive cells was evaluated using a quantitative method (continuous variable) or a semi-quantitative method (categorical variable). Authors should clarify this point, and data should be presented in a table format. This will help the analysis, because in pages 11 and 12 (Result sections) the interpretation is very confusing since reported percentages have a large variation. For example, regarding the extent of ER positivity it was observed in 60-70% of the nonneoplastic mammary cells, in 1-90% of UH cells, in less than 5% of ADH and in 2-10% of the mammary carcinomas cells. If the analysis was quantitative, the mean, the median, the minimum and the maximum values should be shown in the manuscript. If the evaluation was semi-quantitative, the cut-off value facilitates the analysis. In the Methods section - Immunohistochemistry - page 8, last paragraph the sentence: nuclear immunoreactivity for ER, PR, and Ki67 in IELs and tumors was analyzed and reported as the percentage of labeled epithelial cells was reworded as follow: Nuclear immunostaining for ER, PR and Ki-67 was evaluated counting a total of 1000 cells in 10 representative fields at high magnification (400x) whereas for smaller lesions, the entire lesion was considered. The number of immunopositive cells was expressed as a percentage (mean, median, minimum and maximum values ).

6 In the Result section - Immunohistochemistry - pages 11 and 12 we clarified this point showing several data. In the Results section Histology (page 11, 1st paragraph), the growth patterns observed in DCIS are not described, although in the material and methods sections, page 7, authors report that different patterns were observed (cribriform, papillary, micropapillary, solid and solid with comedo-type necrosis). As this is a descriptive paper of IELs we think it is important to inform readers about this issue and if the growth pattern of DCIS was in accordance with the histotype of invasive lesions or if no relationship was found between in situ and invasive carcinomas growth patterns. In the Results section Histology -, we add the following sentence to clarify the different growth patterns in DCIS: Different morphological patterns were observed in DCIS: 4 were cribriform (1 low grade, 3 intermediate grade), 9 papillary (2 low grade, 7 intermediate grade), 4 micropapillary (2 low grade, 1 low grade, 1 high grade), 6 solid (1 low grade, 1 intermediate grade, 4 high grade) and 5 were comedo type DCIS (5 high grade). Concordance between DCIS histotype and associated tumors was found. However, because invasive carcinomas were classified according to the predominant pattern (>50%), the relationship between growth patterns of invasive and in situ carcinomas was difficult to measure. In the Results section Immunohistochemistry - (Expression of KI67 in IELs, page 12) a statistic analysis comparing IEL proliferation was performed but data was not inserted in the manuscript. Additional information must be introduced to increase the comprehensibility of the article. A table with Ki67 results in the various IELs is suitable. In the Results section Immunohistochemistry -, page 12, authors inserted statistical data about immunohistochemistry expression of Ki67 in IELs In Table 1, the lesions classification according to their relative risk is speculative and should be removed from the table. In fact, in this moment there are no studies to support that feline IELs have the same biological behavior and prognosis as woman breast IELs. Probably the behavior is similar but we need further histological, biological, and genetic studies, to validate this theory. If authors intend to maintain the relative risk classification, they must inform (in the legend or as a footnote) that this classification is adapted from woman breast and requests confirmation. The table has been modified to clarify that the IELs are from cats and that the risk categories are based on human epidemiologic studies. In Table 2, authors classified 8 carcinomas as cystopapillary carcinomas. Nevertheless, in the Material and method sections, page 7, authors mentioned that they used the WHO Histological Classification of Mammary Tumors of the Dogs and Cats, and in this classification this histotype is not included. Authors should correct the classification. In WHO Histological Classification of Mammary Tumors of the Dog and Cat, the papillary-cystic type is considered a variant of tubulopapillary carcinoma, so we have simply included this variant with the other tubulopapillary carcinomas and modified Table 2 accordingly.

7 In Figure 3, we are not convinced that D5 is an intermediate-grade DCIS. The cells show high nuclear pleomorphism and frequent mitosis. This figure should be replaced by a more illustrative one. The figures D5 and D6 have been changed with two more representative figures. In the Background section, page 5, 1st paragraph, in the sentence Mammary neoplasia has been reported to occur in cats from 9 months to 23 years of age (mean, 10 to12 years) a space is missing. The phrase should be replaced by Mammary neoplasia? years of age (mean, 10 to 12 years) A space was inserted to correct to 10 to 12 years. In the Results section - Histology, page 10, 1st paragraph, the number of cases with UH, ADH and DCIS should be insert in the manuscript (n, %). The number of lesions in each category has been added. Table 2: In the legend Elston and Hallis should be replaced by Elston and Ellis. Thank you for catching this error; it has been corrected. In the Results section Histology page 11, 1st paragraph, additionally to the percentage value, the number of cases with UH, ADH and DCIS should be inserted in the manuscript. The number of UH, ADH, and DCIS lesions was inserted in response #7, preceding paragraph. In the Results section, Immunohistochemistry (Expression of PR in IELs - pages 11-12) the number of intermediate-grade DCIS and high- grade DCIS positive cases is missing. When authors state Expression of PR was not detected in the examined mammary tumours authors mean invasive carcinomas or intermediate and high grade DCIS? This item must be clarified. The sentence is changed: Expression of PR was not detected in the intermediate or high-grade DCIS lesions or in examined invasive mammary tumors. In the Results section, Immunohistochemistry (Expression of KI67 in IELs? page 12), the sentence This regression model is significant with a p-value of should be replaced by This regression model is significant with a p-value < Corrected as suggested. In the Discussion section, page 13, last paragraph, the sentence For example, the benign mixed tumor...never develops in cats or women. [38] should be replaced by For example, the benign mixed tumor...never develops in cats or women [38]. Thank you. Corrected as suggested. In the Discussion section, page 14, 1st paragraph, the cited references ([39-40]) should be removed because they are not relevant. We agree that no citation is necessary at this point. We deleted them and renumbered references and citations accordingly.

8 In the Discussion section, page 14, 3rd paragraph, authors state In this study, ER was expressed in 62.5% of UH and in 2% of ADH, however, in the result sections page 11, 3rdparagraph, authors report a 7% of ADH positive cases. Authors must elucidate this issue. This sentence has been reworded as follow: In this study, ER was expressed in 62.5 % of UH and in 7% of ADH. In the Discussion section, page 15, 1st paragraph, the authors state that the ER and PR expression decreased with increasing lesion or tumour grade, however, in the result section there is no information regarding the immunoexpression of these markers in the 3 histological grades of invasive carcinomas. We changed the sentence as follow: As for ER, PR expression decreased with increasing grade of IEL. Discretionary revision Authors describe and sub-classify DCIS. Please state if the noninvasive phenotype was confirmed using myoepithelial markers, because in practice, we many times see invasive carcinomas that mimic in situ lesions. Reviewer #1 also made this valuable suggestion. IELs were categorized according to criteria established for classification of the human breast IELs (refs 26 and 27), for which myoepithelial cells are identified by their shape and cytologic features in HE-stained sections. The authors have applied the same criteria to classify mammary IELs in dogs [ref 47]. In the Discussion section, page 14, 4th paragraph, what do authors mean with nonlesional mammary gland We changed the sentence as follow: non-lesional mammary gland