Title:COX-2 overexpression in resected pancreatic head adenocarcinomas correlates with favourable prognosis
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1 Author's response to reviews Title:COX-2 overexpression in resected pancreatic head adenocarcinomas correlates with favourable prognosis Authors: Ewa Pomianowska Aasa R Schjølberg (aassch@ous-hf.no) Ole Petter F Clausen (oclausen@ous-hf.no) Ivar P Gladhaug (igladhau@ous-hf.no) Version:2Date:6 March 2014 Author's response to reviews: see over
2 Editor, BMC Cancer BioMed Central Editorial Office Institute of Clinical Medicine PO Box 1171 Blindern 0318 Oslo Norway Date: March 5, 2014 Your ref.: Our ref.: Telephone: (+47) Telefax: (+47) Re-submission MS COX-2 overexpression in resected pancreatic head adenocarcinomas correlates with favourable prognosis Dear Editor, Thank you for your review and valuable comments on our manuscript COX-2 overexpression in resected pancreatic head adenocarcinomas correlates with favourable prognosis. We have now revised the manuscript according to the reviewers comments; see our detailed point-by-point response to each comment below. Reviewer #1: Answers to comments 1. Title of the manuscript tumour types included in the study cohort Answer: The term pancreatic head adenocarcinomas in the title refers to the terminology currently used, taking into account that adenocarcinomas may originate from three different anatomical sites in the head of the pancreas, i.e. from the ampulla, the distal bile duct, and the ductular structures of the pancreatic parenchyma. In our opinion this is a useful terminology to denote the cancers which derive from the different anatomical compartments of this region (for a review, see for example Verbeke & Gladhaug, Br J Surg, 2012; 99: ). A particular feature of these tumours is that they can be characterised by their type of histopathological differentiation, i.e pancreatobiliary or intestinal type (see for example Westgaard et al, Ann Surg Oncol, 2013; 20:430-39). In the present paper we present novel data on the pattern of COX-2 expression in these subtypes related to the prognosis of the three tumour types, which is one main issue of the paper as also pointed out by reviewer #2. Description of COX-2 expression in tumours of all three anatomical sites in a single series of consecutive pancreatoduodenectomies adds to the literature on this field, hence we do not find it appropriate to leave out the ampullary and the bile duct tumours although more data is presented on the bigger cohort of pancreatic tumours. Dokumentet er elektronisk produsert og godkjent ved UiO i tråd med UiOs reglement for elektronisk godkjenning.
3 UNIVERSITY OF OSLO Page 2 of 4 2. Table 1. There is one case missing under Lymph Node Ratio (LNR), the total number is 91. Answer: LNR is defined as the ratio of the number of metastatic lymph nodes to the total number of lymph nodes evaluated. Since in one case there were no lymph nodes retrieved in the specimen, LNR evaluation could not be performed in this patient, hence one patient was not included in the cohort, making the total number 91 in this table, although the total number of assessed patients with pancreatic cancer was 92. In the revised manuscript this has been added as footnote c in Table Figure 1. Better if the authors used pictures show the expression of COX-2 (NEGATIVE AND POSITIVE) in pancreatic cancer only. No need for the pictures showed SMA, it may mixed the information. Answer: We aimed at determining the exact localization of COX-2 immunoreactivity within the tumour, in particular to see if COX-2 immunostaining was restricted to the cancer cells proper, or whether COX-2 was also expressed in the pancreatic stellate cells of the tumour stroma. We therefore employed double immunostaining with COX-2 and α-smooth muscle actin (αsma) antibodies as shown in Figure 1 a-d. αsma staining is only present in activated pancreatic stellate cells of the tumour stroma and not in the cancer cells. There was no evidence of cytoplasmic COX-2 staining in the αsma positive cells. Thus we were able to conclude that COX-2 staining was only present in the cancer cells and not in the stromal cells. No need for the picture showing Western plot, it may mix the information Answer: To test the validity of the results obtained with the Thermo antibody used for the study cohort we performed additional immunostaining with a different monoclonal COX-2 mouse antibody, Invitrogen (Camarillo, CA, USA). To test the specificity of this antibody, western blotting analysis was performed, showing a highly specific band for COX-2. The Thermo antibody was not suitable for western blotting (producer recommendation). The immunostaining data were essentially similar for the two antibodies. Taken together these data substantiate the specificity of the two antibodies used in the study. Since demonstration of antibody specificity is essential for the interpretation of the results, we have included the western blotting of Invitrogen in Figure 1 f. 4. The majority of the references are quite old, better to use the more recent one. Answer: To comply with the reviewer s suggestion, three recent references ( ref 14,15 and 16) have been included in the revised manuscript.
4 UNIVERSITY OF OSLO Page 3 of 4 Reviewer #2: Answers to comments 1. «Some more detail about morphological parameters to define these two data group (histological subtypes) could be useful. Answer: We agree. In the Patients and methods section (p. 6) of the revised manuscript, we have therefore added the following sentence: In brief, pancreatobiliary tumours typically have simple or branching glands and small solid nests of cells surrounded by a desmoplastic stroma, and have cuboideal to low columnar epithelium arranged in a single layer and the nuclei are rounded but with marked variation in size and shape from one cell to the next. Intestinal tumours typically resembled colon cancer, have tall and often pseudostratified columnar epithelium with oval nuclei located in the more basal aspect of the cytoplasm, and there may also often be presence of mucin. 2. It is intriguing the absence of significant correlation between COX-2 expression and intestinal type of differentiation. Answer: The comment refers to the pancreatic adenocarcinomas. Only about 10% of pancreatic adenocarcinomas exhibit an intestinal type of differentiation (Albores-Saavedra et al, Ann Diag Pathol, 2007; 11:3-9; Westgaard et al, Ann Surg Oncol, 2013; 20:430-39). In our cohort, all intestinally differentiated pancreatic adenocarcinomas (n=8) were COX-2 positive, whereas about two thirds of the tumours of panreaticobiliary differentiation were COX-2 positive. From our data, one would expect that there is a correlation between COX-2 positivity and intestinal differentiation. We believe there is such a correlation, but the present dataset is not large enough to prove the statistical significance of this correlation. To comply with the reviewer s comment, we have therefore added the following sentence in the Results section (p. 9) of the revised manuscript: COX-2 positive tumours were more likely associated with high degree of differentiation (p=0.002) and with intestinal type of differentiation, although, the latter did not reach significance (p= 0.099) (Table 1) probably due to the low number of tumours of the intestinal differentiation type. 3. COX-2 as a marker of good differentiation without a real role from the prognostic point of view? Answer: The question as to whether or not COX-2 expression in fully developed pancreatic cancer plays a causative role in the clinical course of these tumours, and thus has prognostic significance from a causative point of view, has at the present time not been answered. We agree with the reviewer that it might be conceivable that COX-2 expression is associated with a favourable phenotype and thus is a marker of good prognosis rather than a cause of good prognosis. Answering this question is considered to be beyond the scope of the present work.
5 UNIVERSITY OF OSLO Page 4 of 4 4. Intermediate group with respect to survival Answer: Our data are consistent with the idea that there is a gradient of tumour phenotypes with respect to prognosis from COX-2 pos/well differentiated tumours, to COX-2 neg/poorly differentiated tumours, the latter having the worst prognosis. That the mixed phenotypes COX-2 pos/poor differentiation and COX-2 neg/high differentiation fall as an intermediate group with similar survival fits this general pattern. The present data does not allow any conclusion as to which factor, ie. COX-2 expression or tumour grade, is the more important. Probably these two factors are closely linked, making it difficult to resolve this question without studying a much larger patient cohort. 5. Typing errors and small inaccuracies Answer: The typing errors and small inaccuracies pointed out by reviewer have been corrected in the revised version of the manuscript. Editor s comment The statement on ethical approval has been moved to the Patients and methods section in the revised manuscript. Concluding remarks As stated previously, we have no competing interests to declare. We hope that the manuscript will now be found suitable for publication in BMC Cancer. Yours sincerely, Ewa Pomianowska, MD.
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