Author's response to reviews

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1 Author's response to reviews Title:Prognostic value of the microrna regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and mir-126 predicts poor survival Authors: Kenneth Lønvik Sveinung W Sørbye (sveinung.sorbye@unn.no) Marit N Nilsen (marit.n.nilsen@uit.no) Ruth H Paulssen (ruth.h.paulssen@uit.no) Version:3Date:4 September 2014 Author's response to reviews: see over

2 Kenneth Lønvik, MSc Department of Clinical Pathology, University Hospital of Northern Norway N-9038, Tromsø, Norway Tel , Fax , Tromsø, Dear Madam/Sir We hereby resubmit a revised version of our manuscript (MS ) entitled: Prognostic value of the microrna regulators Dicer and Drosha in Non-Small Cell Lung Cancer. We have adressed each point brougth up by the reviewers. We like to take the opportunity to thank you for the valuable and important comments and questions and believe that the manuscript has substantially been improved. We provide a marked-up copy of the changes made from the previous article file as a Manuscript with Tracked Changes. We are looking forward to your answer. With kind regards, on behalf of all co-authors, Sincerely Yours, Kenneth Lønvik, MSc UNN HF Besøksadresse: Kontonr: Org nr: Telefon: * Internett: 9038 Tromsø Sykehusveien MVA NO Telefaks: Tromsø

3 Reviewer's report Title:Prognostic value of the microrna regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and mir-126 predicts poor survival Version:2Date:7 May 2014 Reviewer:Mandi Murph Reviewer's report: Overall, the manuscript is an interesting read for those of us in the field of mirna research and it brings in the very important aspect patient specimens. Thus, it is worthy of publication. However, it needs condensation in some areas and further explanation in others. There is significant brevity within the writing style of the introduction and the results, but the discussion is overly wordy and speculative. I would suggest re-writing the discussion based on some of my comments below. The discussion has been re-written based on the comments. Minor essential revisions 1. Citation 15 on Line 87: The citation may be inappropriate for what the authors are trying to establish. My interpretation of this reference is a little different as much of the focus is on mir-155 and let-7a-2. A line in the discussion reads, Indeed, approximately 30% of the 43 mirnas that showed different expression in lung cancer tissue versus noncancerous lung tissue are located within exons or introns of known protein-coding genes, such as TMEM49 (transmembrane protein 49) for hsa-mir-21, EGFL7 (EGF-like-domain, multiple 7) for hsa-mir-126# and hsa-mir-126. In Figure 1 of this reference, the authors are validating the detection of mir-126* in cancer versus noncancerous lung tissue, but this is not the same as evaluating a prognostic biomarker. Thus, the authors may want to reconsider using this citation. We omitted the citation and replaced it with a more appropriate reference: Ebrahimi F, Gopalan V, Smith RA, Lam, A K-Y: mir-126 in human cancers: Clinical roles and current perspectives. Exp Mol Pathol 2014, 96: The authors use DSS without sufficient explanation in this manuscript. The National Cancer Institute defines this as the disease-specific survival rate, which = (% of people)/(defined period of time). This does not appear to be the usage it receives here. What is the defined period of time? In this regards, I am asking if it is 120 or 250 months, since both are used here interchangeably. In addition, it is unusual to see this abbreviated; more common convention uses OS, PFS or DFS. Are these more appropriate here?

4 The DSS rate was defined in the background section, line 76. The period of time has been changed to 120 months throughout all figures. In addition, we have included a figure regarding the overall survival (OS), Figure 1B, 1D, and 4B. 3. The ECOG description in lines fails to mention that this is the (ECOG) [performance status] grade and the whole sentence is not clear. They should remove the words strong and for survival. The sentence has been completely removed and replaced by text shown in pt: 4 (see below). 4. Further description is needed to explain the difference between ECOG performance status grade 0 versus 2, which includes those patients who are unable to work. Otherwise, the novice reader may be left with confusion about this measurement. In addition, the whole description of ECOG may be better served in the method section (lines ) where the rest of it appears. A further description of ECOG performance status has been now included in the background section and reads as follows: The Eastern Cooperative Oncology Group (ECOG) performance status [33] provides scales and criteria in order to assess how a patient s disease is progressing and helps to determine appropriate treatment options and prognosis. Many studies include patients with an ECOG performance status grade of 0 and 1 only. Patients in these groups are either, fully active and able to carry on all pre-disease performance withour restrictions or are restricted in physically strenous activity and able to carry out work of a light or dedentary nature. In this study we have also included NSCLC patients with an ECOG grade of 0 2 and included also patients that are capable of all selfcare but unable to carry out any work activities and have more than 50% of waking hours. Discretionary Revisions 1. Background: The authors first describe mir-126 and then Dicer/Drosha. However, in their study, mir-126 has a minor focus. Thus, why not highlight the others first and mir-126 last? In our study, mir-126 has a minor focus. However, following the previously published the results on mir-126 (ref. #19) we wished to put our attention to Dicer/Drosha with respect to mir-126. That s the reason for the order of information given in the background section. 2. Perhaps the authors may want to describe the adjustments for other to repeat the study (Methods, line 179)? Minor adjustments on hybridization time have been made due to different batches of labelled probes. This is now mentioned in the Materials & Methods section. However, if the cited procedure is used, it should be no problem to reproduce the experiments by a different laboratory. Minor adjustments need to be done by any laboratory for optimal performance.

5 3. Background (line 88): controlling may be too strong of a verb here. controlling has been replaced by influencing. 4. For results described in lines , could the authors write data not shown? (data not shown) has been now included. 5. The best piece of data begins with Figure 4B, perhaps the authors might consider starting with this? We have changed the order of figures and results depicted in former figure 4B are now shown in Figure 2F. Figure 2 now summarizes data obtained for Dicer. 6. The Inter-Observer variability (lines ) could be moved to the methods section, where appropriate. The Inter-Observer variability has been moved to the methods section. 7. Apparently line 287 isn t the word choice that is appropriate here. The word apparently has been removed from the text. 8. Citation needed in line 349 after angiogenesis. Citation #48 as indicated in the reference list has been included: Kuehlbacher A, Ubrich C, Zeiher AM, Dimmler S: Role of Dicer and Drosha for endothelial microrna expression and angiogenesis. Circ Res 2011, 101: Major Compulsory revisions 1. Background (lines 76-78): The authors describe their desire to investigate possible prognostic factors as a means to increase survival in malignancy. However, I don t quite believe this association is appropriate. Perhaps they could consider a softer tone and describe the study as an investigation of biomarkers that would allow a better understanding of malignancy or a biomarker to triage patients into different treatments (i.e. clinical trials for NSCLC)? Changes to the text have been made according to this comment as follows: Therefore, it is important to investigate possible prognostic factors among the survivors in order to gain a better undestanding of NSCLC malignancy and to develop treatments options for different NSCLC patient groups 2. Background (lines 89-91): The authors are describing a study, but without the important fact that mir-126 targets VEGF-A by binding to the 3 untranslated

6 region in the mrna. This is important in light of their findings. A reference regarding this important fact has been now included in the manuscript (now reference #18 in the text): Zhu X, Li L, Hui L, Chen H, Wang X, et al: mir-126 enhances the sensitivity of non-small cell lung cancer cells to anticancer agents by targeting vascular endothelial growth factor A. Acta Biochim Biophys Sin 2012, 44: Figures 1 and 2 add little, if anything, to this study. My recommendation is to place these into supplementary figures 1 and 2 and pull them out of the main figure section. This would allow them to serve as a reference guiding the staining for others seeking to repeat the study or validate these findings. Otherwise, more description is desperately needed. For example, what percentage of cells had staining? Features need to be quantified and interpreted further. Figures 1 and 2 have been moved to the supplemental section (now Supplemental Figures S2 and S3). 4. I fail to see any correlation in figure 3 and the r value suggests there is none. Perhaps the significance (p) correlation is measuring the degree of randomness? Am I missing something? Figure 3 shows a weak, but significant correlation. We have moved this figure to the supplemental section indicated as Supplemental Figure S4. 5. Figure 4 nice data for Dicer in SCC. Why isn t the same performed for Drosha? It was performed, but the data for Drosha were not significant. Anyway, we have now included the results for Drosha into a figure (Figure 3). 6. Why does the time (months) vary from figure to figure? The time varied from figure to figure for no obvious reasons. The time has been now set to 120 months throughout all figures in the manuscript in order to aviod confusion. 7. Figure 5 is not a strong, stand-alone figure. Perhaps the authors could combine figures 4 and 5 together so that 5 could serve as a control to bolster the findings in 4. We have combined figures and results regarding Dicer and Drosha that are now depicted as figure 2 and figure The same for figures 6 and 7 could these be combined? The only issue is that the time (months) varies significantly between these why?

7 We have combined figures and results regarding Dicer and Drosha that are now depicted as figure 2 and figure 3, and adjusted the time to 120 months (see also comment in pt. 6). 9. The fact that Drosha + mir-126 shows a significant impact on DSS, but not Dicer + mir-126 indicates that something is wrong with the overall analyses. Without being privy to the data I cannot comment or speculate on what this might be. We have included a figure depicting overall survival (OS) of Drosha/miR-126 in addition the disease-specific survival (DSS), now figure 4A and 4B. We have repeated the statistical analyses and the results remain the same. mir-126 alone has a strong effect on DSS. This result has been published before [ref#19]. Drosha alone has no significant effect on DSS. However, the patients with combined high Drosha/high mir-126 have an especially poor survival. The patients with high Dicer/high mir-216 show no significant difference regarding the survival compared to patients with high mir-126 alone. 10. The discussion is too speculative on angiogenesis. Show more in vitro data if you are going in this direction. The discussion on angiogenesis has been toned down since this was not the major focus for this study. Level of interest:an article of outstanding merit and interest in its field Quality of written English:Acceptable Statistical review:yes, but I do not feel adequately qualified to assess the statistics. Declaration of competing interests: No competing interest

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10 Reviewer's report Title:Prognostic value of the microrna regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and mir-126 predicts poor survival Version:2Date:19 May 2014 Reviewer:Fabio Klamt Reviewer's report: The manuscript entitled Prognostic Value of the MicroRNA Regulators Dicer and Drosha in Non-Small-Cell Lung Cancer: Co-expression of Drosha and mir-126 Predicts Poor Survival by Lønvik et al., submitted to BMC Clinical Pathology investigated the prognostic impact of Dicer and Drosha and their correlation with mir-126 expression in a large cohort of non-small cell lung cancer (NSCLC) patients. Major Compulsory Revisions: Regarding the immunohistochemical analysis, the authors state on page 189 line 7: "In case of disagreement (variance score> 1), the slides were re-examined and an agreement was reached by the observers." In case of discrepancy, it might be more correct to include a third observer. The re-analysis and discussion may constitute bias. We have included four cores from each patient. The cores are scored by two independent observers. This gives in total eight scores per patient, resulting in an accurate score (mean of 8 scores). It is easy to detect outliers, and usually it is obvious when the scores need correction. It is unusual that observers disagree after reanalysis. The total score is an average of all single scores as described in the methods section. On page 9 line 223 the authors states that: "A strong and significant correlation between Dicer expression in cytoplasm and Drosha expression in the nucleus of neoplastic tumor cells (r = 0.473, p < 0.001) was observed (see Figure 3)". This data eventually becomes questionable, since most authors show the correlation between the expression of Dicer and survival of patients (High expression = better prognosis) but it does not reproduce with Drosha, then it is understood that the terms do not "accompanied. Figure 3 shows a weak, but significant correlation. We have made changes in the text accordingly ( strong replaced by weak ) and moved this figure to the supplemental section now indicated as Figure S3. -After the paragraph that begins at line 233 and ends at line 239 the authors state that The expression of Dicer had no significant impact on survival in Patients with squamous cell carcinoma (see Figure 4A) or in other histological

11 subtypes (data not shown). But then the authors say in EGOG 0 patients with SCC with higher dicer had longer survival. For this reason, I believe that the authors should show the data of the other histological types (even if only for patients in ECOG 0)... in addition, the S1 table it shows that different clinico-pathological variables have influence on survival, including the histological type. These suggested data have now been included and are depicted in figures 2A-2F. In addition we have replaced table S1 with a new table showing data of the other histological types. Later in the discussion, in line 314, the authors argue: "In general, patients with reduced ECOG have a more advanced disease and poor prognosis, independent of the tumor's biological properties." In this case, to avoid misinterpretations, authors must determine whether the reduced ECOG refers to the scale or characteristics of each degree (since lower ECOG value is better PS) We have reformulated the sentence to : In general, patients with reduced performance status (ECOG = 1 4) have a more advanced disease and poor prognosis, independent of the tumor s biological properties in order to avoid misinterpretation. linhas : "Our results clearly show that low Dicer expression is a significant negative prognostic marker in patients with normal performance status ECOG = 0 (see Figure 7A). This patient group might therefore have an advantage in receiving adjunctive treatment". From the description of the cohort, all samples are from patients who underwent surgery (stage III-IIIA and ECOG 0-2). The prognostic power of his marker only was effective in patients with ECOG 0 which according to the link pasted above, are "Fully active, able to carry on all pre-disease performance without restriction". That is, as a rule, everyone should receive adjuvant therapy. Adjuvant therapy would be a preventive therapy to avoid the feeling micrometastases after tumor removal... everyone wins and it is what is already being done, according to this table ( from NCI. So, would not this be the great advantage of the marker?...the authors should further explore the prognostic issue. In our study none of the patients received adjuvant therapy. Nowadays a large porportion of lung cancer patients receive adjuvant therapy. However, we have identified a subpopular highrisk group that might benefit from an even more aggressive treatment. This has been now mentioned in the discussion. In line 325 the authors begin to explore the relationship of Dicer and angiogenesis, but the major support of this hypothesis comes from data from previous studies (in vitro) (line 346). Furthermore, silencing of Dicer and Drosha decreases angiogenesis, but the

12 study most striking result of this study is the correlation between the increased expression of Dicer with better prognosis... paradoxically... the authors did not comment on the data. The discussion has been rewritten and the paradox finding is now commented. Level of interest:an article whose findings are important to those with closely related research interests Quality of written English:Acceptable Statistical review:yes, but I do not feel adequately qualified to assess the statistics. Declaration of competing interests: I have no competing interest in relation to the paper.

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14 Reviewer's report Title:Prognostic value of the microrna regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and mir-126 predicts poor survival Version:2Date:4 July 2014 Reviewer:Giuliana Cortese Reviewer's report: Major Compulsory Revisions 1) In the section 'Statistical Methods', the first sentence about statistical methods being described in other previous publications is nor really necessary here. We have omitted the first sentence in the section Statistical methods citing the previous publications about statistical methods. 2) I could not see the definition of DSS in the manuscript. Please define the first time. In the abstract it should also be written as disease-specific survival instead of DSS. DSS was defined in the background section (line 76). DSS has been now written as disease-specific survival (DSS) rate in the abstract. In addition, we have included a figure regarding the overall survival (OS), Figure 1B, 1D, and 4B. 3) It is important to specify when the microrna Dicer and Drosha were obtained, that is when the timor cells were selected from the patients. Was it done at the beginning of the follow-up or later on? In the second case, the results from the analysis might be biased because some assumptions related to the statistical models would be violated. All tumor tissues were selected in primary surgery of previous non-treated lung cancer patients. The follow-up started directly after surgery. This has been now mentioned in the method section. 4) In the section 'Results', I would not define an r=0.47 as a 'strong' correlation. The word strong is now removed from the text and has been replaced by weak. 5) Some clinicopathological variables are significantly correlated with Drosha and some angiogenic markers are significantly correlated with both Drosha and Dicer. These results are not shown here and they are important in order to assess the combined effects on the survival. It would be interesting to see also these results. We have now included histological data in the Cox regression analysis (Table 1 and Supplemental Tables S3-S7). 6) It seems as if there are more factors that, when combined, have an effect on survival, but their independent effect is not shown. For instance, survival is better for high expression of Dicer, only in patients with ECOG=0 and squamous cell carcinoma. This fact suggest that there is an interaction between expression of Dicer and ECOG, or between expression of Dicer and histology. In the addition to performance status we have now included histological data in the Cox regression analysis (Table 1 and Supplemental Tables S3-S7).

15 Here the appropriate analysis is to consider a regression model for the mortality rate including both Dicer expression and Drosha expression as covariates on a continuos scale rather than categorical covariates with High/low levels. The results might strongly depend on the cut-off points chosen to create the high/low groups. Moreover, based on the comments given above, it is naturally of interest to study directly into the regression model the statistical interaction between each of the two expressions (as continuous variables) and some of the clinicopathological variables. We have included a table with a Cox regression analysis using Dicer and Drosha as covariates on a continuous scale (Supplemental Table S3). Differences in expression of Dicer, Drosha, and mir-126 as continuous variables by histology are analyzed using ANOVA. The results are depicted in Supplemental Table S2. 6a) If a Cox model is used, the assumptions under the model, especially proportionality of the hazards, should be carefully tested. Cox regression analysis of Dicer and Drosha on a continuous scale showed no significant differences in expression. Therefore, we believe that it is not necessary to test the proportionality of the hazards. 7) lines Given that the classification high/low might be arbitrary (this information is not reported by the authors), the authors should look at the association between the expression of Dicer with the timor subgroups, keeping the expression on a continuos scale (continuous variable). That is, here the interest is on investigating if the expression of Dicer id significantly different between subgroups. As mentioned in the methods section Scoring of IHC, high expression of both Dicer and Drosha in neoplastic tumor cells was defined as a mean score 2. For mir-126 we used the same cut-off value and the same scoring system as previously described in detail [19,38] % of patients had low scores and % had high scores for each of the markers. We have replaced table S2 with a new table showing expression of Dicer, Drosha and mir-126 grouped by histology keeping the expression on a continuos scale (continuous variable). We have made changes in the manuscript where appropriate, according to the comments above. 7) If some interactions are significative, the authors could use these variables for showing differences in the survival curves between subgroups. Moreover, I think it is a good practice to show also the estimated curves for ECOg=1-2 and other histology, although not significative (better if they are given in the same figure 4B and 4A). We have now included figures for ECOG=1-2 and other histology, both for Dicer and Drosha (now depicted as figure 2 and figure 3). 7) What is the percentage of patients under study who died from other causes? Please report this information, if possible. The percentage of patients who died from othe causes are now reported in the results section. 8) It could be useful to see Cox-model-based survival predictions for different values of the expressions (low or high).

16 This has been done, and the results are depicted in Supplemental Tables S4- S7. Minor Essential Revisions 8) line 311: the word correlation is not really appropriate here when used for survival (better association or influence on..) Correlation has been replaced by association. Level of interest:an article of importance in its field Quality of written English:Acceptable Statistical review:yes, and I have assessed the statistics in my report. Declaration of competing interests: I declare that I have no competing interests.

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