Title: Human chorionic gonadotropin and its relation to grade, stage and patient survival in ovarian cancer

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1 Author's response to reviews Title: Human chorionic gonadotropin and its relation to grade, stage and patient survival in ovarian cancer Authors: Miriam Lenhard Alexandra Tsvilina Lan Schumacher Markus S Kupka (markus.kupka@med.uni-muenchen.de) Nina Ditsch (nina.ditsch@med.uni-muenchen.de) Doris Mayr (doris.mayr@med.uni-muenchen.de) Klaus Friese (klaus.friese@med.uni-muenchen.de) Udo Jeschke (udo.jeschke@med.uni-muenchen.de) Version: 2 Date: 13 December 2011 Author's response to reviews: see over

2 Klinikum der Universität München Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe Innenstadt Direktor: Prof. Dr. med. Klaus Friese Klinikum der Universität München Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe Innenstadt Maistraße 11 D München BMC Cancer Professor Christina Chap Executive Editor Prof. Dr. Udo Jeschke Munich, 12th of December 2011 Phone Fax Revision MS: Dear Professor Chap, thank you for your kind review of our manuscript entitled Human chorionic gonadotropin and its relation to grade, stage and patient survival in ovarian cancer. On behalf of my co-authors, I am now re-submitting the revised version of the manuscript for publication in BMC Cancer. We have thoroughly revised the manuscript, carefully integrating all reviewer s comments into account. Please find our point to point revision note attached below. All authors have agreed to the content in its resubmitted form. I hope the changes will meet your expectations. Please do not hesitate to contact me in case of any questions regarding the manuscript. Sincerely yours, Professor Dr. Udo Jeschke Anschrift: D München Maistraße 11 Telefon (0 89) (Vermittlung) Verkehrsverb.: U1, U2, U3, U6, U7, U8, 16, 17, 18, 27, 31 o. 56 bis Haltestelle Sendlinger Tor Verkehrsverb.: U3, U6 oder 58 bis Haltestelle Goetheplatz

3 Klinikum der Universität München Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe Innenstadt Seite 2 Reviewer's report Title: Human chorionic gonadotropin and its relation to grade, stage and patient survival in ovarian cancer Version: 1 Date: 11 November 2011 Reviewer: Udo Markert Reviewer's report: The paper describes a retrospective study on ßhCG expression in sera and tissues from 156 ovarian cancer patients. The authors describe a positive correlation of ßhCG and tumor grade, but also a positive correlation with survival rate. This point needs to be discussed more deeply. The information of the manuscript is novel and interesting and worthy to be published, but numerous corrections need to be performed. Abstract: Results: Is there a correlation between serum and tissue hcg positiv samples? Unfortunately, correlation analysis for serum and tissue hcg results cannot be performed, since the samples derive from different patient cohorts. Not clear:... We identified significant differences in hcg expression related to tumour grade (p=0.022)... Does this refer to hcg expression in serum or tumor tissue? This refers to tumor tissue results and is now mentioned more preciously in the abstract. The sentence:... but subgroup analysis revealed an increased 5-year survival in LH-R positive and hcg positive tumors (hcg positive 75.0 % vs. hcg negative 50.5%)....does not exactly summarize the results as described in the results section:... there was an increased 5-year survival in patients with hcg positive tumors in the LH-R positive / FSH-R negative subgroup... This is an important remark. For a better understanding of the survival data, we clearly added information on the LH-R, FSH-R and hcg status of the subgroups. Methods: Do the 40 serum samples derive from a subgroup of patients from which also tumor tissue samples were obtained? The serum samples do not derive from a subgroup of patients with tissue samples. Therefore no further correlation analysis from serum and tissue results could be performed. Have they also been collected between 1990 and 2002? The serum samples have been collected from 2003 to 2006, which we now state in the text. How have they been stored? All serum samples have been stored at -80 C. This information in now mentioned. Immunohistochemistry: Have the slides been blinded for the reviewers? Yes, all slides have been blinded for the reviewers. This information is added in the revised manuscript. Results, page 6: ßhCG ELISA: Last sentence: Make clear, if the median calculation has been done by using all samples or after exclusion of negative samples (calculation only on hcg positive samples) The median calculation has been done using all samples. Negative samples were included in calculation. Discussion The surprising result that higher hcg indicates more advanced stage but also higher survival, needs a

4 Klinikum der Universität München Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe Innenstadt Seite 3 more extensive explanation, because it seems to be contradictive. The provided discussion on this point is very short. For a better understanding, this important aspect is now discussed in more detail. Please find our explanation in the revised manuscript. Tables and Figures In several tables and figures:, should be replaced by. This misspelling is now corrected. Table 1: The table should include also the numbers for the 40 serum samples from cancer patients. This information is now included in table 1. Table 2: Table should be reduced and simplified. P-values for non-significant results are not necessary. Two decimal positions instead of 3 are sufficient. Int and IRS data may be combined to reduce redundancies in axis labeling. The suggested corrections have been done. Table 3: Labeling is confusing: Instead of only LH positive etc. LH pos/ FSH neg will be clearer. The term equals is not clear. The correction has been made. Figure 1: It should be stated if presented data derives from all samples or only from the samples with ßhCG levels above (which?) detection limit. What is the meaning of the numbers next to asterisk and circles? The median calculation has been done using all samples. Negative samples were included in calculation. The circle indicate values more than 1.5 box lengths, and the asterisk values more than 3.0 box lengths from the 75 th percentile. Numbers at circle and asterisk indicate sample number. Figure 2b: Please re-check the magnification (upper right and lower left seem to have the same amplification; lower right has a higher amplification) Figure 2b was re-checked. Although size of nuclei differs in pictures, this is due to different tumor material and not size of the picture. Amplification is correct as stated. Figures 3-5: Figures can be combined to Fig 3a-c. For former Fig 5: please provide n for each bar, because this information is not included in Table 1. Figures 3-5 are now combined to figure 3a-c and in former fig 5 are the n provided. Fig 6: Labeling is confusing: Instead of only LH positive etc. LH pos/ FSH neg will be clearer. The term equals is not clear. The correction has been made. Reviewer's report Title: Human chorionic gonadotropin and its relation to grade, stage and patient survival in ovarian cancer Version: 1 Date: 23 November 2011 Reviewer: Jinsong Liu Reviewer's report: Overall, this is a very nice study, well designed, the conclusion is valid. I have only a few questions, which should be addressed: The serous carcinoma, are all high grade or also low grade? What is the definition of benign? Do they include borderline tumor? The serous carcinomas include patients of all grades. Benign tumors include patients with benign cystadenomas, simple ovarian cysts, endometriosis, teratomas or fibromas. Borderline tumors of

5 Klinikum der Universität München Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe Innenstadt Seite 4 the ovary are neither included in the benign nor in the ovarian cancer patient collective. This is now also stated in the material and methods section. The author should also cite a paper published recently in Am J Pathology by Guo et al, "Overexpression of the ß subunit of human chorionic gonadotropin promotes the transformation of human ovarian epithelial cells and ovarian tumorigenesis". This is a recently published important article in the field of hcg and ovarian cancer, which is now discussed and cited in the revised manuscript. Editorial request: Experimental research that is reported in the manuscript must have been performed with the approval of an appropriate ethics committee. Research carried out on humans must be in compliance with the Helsinki Declaration, and any experimental research on animals must follow internationally recognized guidelines. A statement to this effect must appear in the Methods section of the manuscript, including the name of the body which gave approval, with a reference number where appropriate. A statement on ethics is now added in the material and methods section of the article.

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