Author's response to reviews

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1 Author's response to reviews Title: Characteristic mtor activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease - a combined tissue microarray, in vitro and in vivo study Authors: Agnes Mark (markagi@korb1.sote.hu) Melinda Hajdu (melindahajdu@gmail.com) Zsofia Varadi (v.zsofka@fre .hu) Tamas B Sticz (tsticz@korb1.sote.hu) Noemi Nagy (n.noncsi@fre .hu) Judit Csomor (jmj.csomor@gmail.com) Lajos Berczi (berczi33@gmail.com) Viktoria Varga (vivarga@gmail.com) Monika Csoka (csokam@t-online.hu) Laszlo Kopper (kopper@korb1.sote.hu) Anna Sebestyen (anna@korb1.sote.hu) Version: 2 Date: 21 January 2013 Author's response to reviews: see over

2 Author's response to reviews Title: Characteristic mtor activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease a combined tissue microarray, in vitro and in vivo study Authors: Ágnes Márk (markagi@korb1.sote.hu), Melinda Hajdu (melindahajdu@gmail.com), Zsófia Váradi (v.zsofka@fre .hu), Tamás Béla Sticz (tsticz@korb1.sote.hu), Noémi Nagy (n.noncsi@fre .hu), Judit Csomor (jmj.csomor@gmail.com), Lajos Berczi (berczi33@gmail.com), Viktória Varga (vivarga@gmail.com), Monika Csóka (csokam@tonline.hu), László Kopper (kopper@korb1.sote.hu), Anna Sebestyén (anna@korb1.sote.hu) Version: 2 Date: 18 January 2013 Author's response to reviews: see over 1

3 Answer for the revision of the manuscript MS: entitled Characteristic mtor activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease a combined tissue microarray, in vitro and in vivo study sent to Journal of BMC Cancer Dear Editor and Reviewers, I would like to thank you for the evaluation of our manuscript and for your helpful comments. All changes are highlighted in purple in the manuscript. Answers for Reviewer 1 (Miguel A Piris) - a more clear description of the results should be given, mean and standard errors for both tumoral and control samples Legends and results have been completed with mean and standard errors of in vitro and in vivo experiment data on Figure 5b-c, and on Figure 6. Significant correlations have been indicated in the figures and in the legends. - IHC results should be confirmed using Western blot in at least some of the experiments The specificity of ps6 antibody used for IHC was confirmed with Western-blotting in human Hodgkin lymphoma and Burkitt-lymphoma cell lines. Xenograft tumors from the same cell lines were also shown by IHC to display mtor activity. The other IHC antibodies are not recommended for other applications, but the data we obtained with antibody clones applicable for Western blotting support our IHC results. Please see the figure attached here for these WB and IHC results. The reliability of ps6 as a marker for mtor activity has been described in more detail in the results section, according to the reviewer s request (page 9) HT58 KMH2 HT58 KMH2 HT58 KMH2 HT58 xenograft control, p-s6 HT58 xenograft Rapamune, p-s6 ps6 32 kda pmtor (289kDa) mtor (289 kda) KMH2 xenograft control, p-s6 KMH2 xenograft Rapamune, p-s6 2

4 Figure: Evaluation of ps6 antibody specifity - it is unclear whether the results for HL justify some of the conclusions on the prognostic value of mtor expression in HL. The authors should provide more data on this, validation series, Kaplan-Meier analysis. The size of the series is probably insufficient for reaching valid conclusions According to the suggestions of both reviewers, figure 3 has been updated with Kaplan-Meier plots and log-rank test has been performed (Figure 3b). The methods, results and discussion sections have been completed with a specification of 72 cases and the connection of mtor activity (page 7-8, 12, 14). - experiments on a single cell lines should be confirmed using different cell lines Combination treatments were performed on KMH2, DEV and L1236 HL cell lines. Rapamycin significantly increased the apoptotic effect of doxorubicin, vincristine and etoposide in KMH2 and DEV cell lines. Results have been completed with these results, along with a clarified description of in vitro experiments (page 13). - rates on lymphoma incidence in Europe should have a citation The Background section has been completed with a citation about lymphoma incidence in Europe (page 4). The text has been modified accordingly. Modifications are highlighted in purple in the manuscript. Answer for Reviewer 2 (JUAN F GARCIA): 1. The series of patients is heterogeneous and include cases of both, early and advanced HL, and a relevant number of pediatric cases. Thus, limitations and potential bias must be comment in the discussion. As presented, correlations between mtor activation and clinical data are unclear. It could be useful the inclusion of relevant clinical data (ages, stages, histopathological Hodgkin subtypes ), and follow-up for the 72 cases analyzed in a table, showing the distribution and comparisons (Student s t-test) between cases with high versus low mtor activity. All clinical data, which was investigated for correlation with mtor activity have been clarified in the results and discussion, according to reviewer suggestions (page 7-8 and page 12 and 14). As the clinical data and mtor activity showed no significant correlations, we attach the table with all clinical characteristics of HL patients for the reviewer. We completed the results and the discussion section indicating which clinical data were examined for correlation with mtor activity, as the reviewer suggested; as we found no significant correlation, the table with detailed data is presented here for the reviewer only (see attached table). 3

5 We also included a sentence in the discussion about the difficulty of drawing statistically significant consequenses in this heterogeneous patient group, as the reviewer suggested (page 14-15). Characteristics Total number of cases High mtor activity Low mtor activity p All HL cases 83 77/83 (93%) 6/83 (7%) Age, y (n=83) <18 27 (33%) 24/27 (89%) 3/ (49%) 39/41 (95%) 2/41 >45 15 (18%) 14/15 (93%) 1/ # Gender (n=83) Male 40 (48%) 36/40 (90%) 4/40 Female 43 (52%) 41/43 (95%) 2/ φ Ann Arbor stage (n=83) I-II 53 (64%) 49/53 (96%) 4/53 III-IV 30 (36%) 28/30 (97%) 2/30 1 φ B symptoms (n=83) Yes 25 (30%) 25/25 (100%) 0/25 No 58 (70%) 52/58 (95%) 6/ φ Histological type (n=83) NS 47 (57%) 44/47 (94%) 3/47 MC 18 (22%) 17/18 (94%) 1/18 LR 8 (10%) 8/8 (100%) 0/8 LD 3 (4%) 3/3 (100%) 0/3 NLPHL 7 (8%) 5/7 (71%) 2/ # All clinical data avalaible (n=72) Complete remission (disease free) 44/72 (61%) 40/44 (91%) 4/44 Relapsed patients (n=25) 25/72 (35%) 23/25 (92%) 2/25 CR after relapse 15/25 (60%) 13/15 (87%) 2/15 Transplanted 13/25 (52%) 12/13 (92%) 1*/13 Died 11/72 (15%) 11/11 (100%) 0/ # *:This case with low mtor activity is in complete remission (more than 5 years) after transplantation. Statistical analysis was done with Fisher s exact test (φ) and with chi square test (#). Table. No correlation was found between mtor activity and the examined clinical parameters. The table includes the summary of all data with p values. 2. Also, the association between mtor activity and outcome could be better presented using Kaplan-Meier plots and log-rank test. The authors have the follow-up and survival data for the 72 cases and this could be easily included in material and methods and explained in the results section (page 11). According to the suggestions of both reviewers, figure 3 has been updated with Kaplan-Meier plots and log-rank test has been performed (Figure 3b). The methods, results and discussion 4

6 sections have been completed with a specification of 72 cases and the connection of mtor activity (page 7-8, 12, 14). 3. Figure 1, in panel 1F, positive cells seems to be mainly infiltrating macrophages, similarly to normal reactive germinal centers (panel 1L), but not real Burkitt lymphoma tumor cells; this need to be clarified; if not, it could be better to replace the microphotograph for panel 1F. According to reviewer s suggestion, panel 1F in figure1 has been changed for another Burkitt lymphoma case, in which there are no apoptotic forms and other disturbing effects which would complicate the interpretation of the figure. (The photo previously used was taken from a treated patient, showing a lot of apoptotic/necrotic forms as well as reactive macrophages. However, the specificity of the immunostaining can be demonstrated here by the negativity of the apoptotic/necrotic cells.) 4. Figure 2 only show results of high mtor activity for KMH2 cell line (WB and ELISA), whereas in page 11 the authors state that this high activity is found in all cell lines. This must be clarified. The activity of mtor has been determined using more methods in HL cell lines, and the text has been clarified accordingly in the results section (page 11). HL cell lines KMH2, UH-01, L428, L1236, HDLM2 and DEV showed high mtor activity by ICC (Figure 2b). ICC results were confirmed by both Western-blotting and ELISA in KMH2 cells, and either Western-blotting or ELISA was performed in the other cell lines as well (Figure 2). 5. In page 12, the authors indicate that mtorc2 expression is detected only in one HL case. A representative microphotograph can be included in Figure 4a for comparison, since only cases with very low expression are illustrated. Figure 4a has been modified, and the new version includes one photograph for Rictor overexpression as well. 6. In figure 4b, nuclear expression of p50 seems to be associated with high mtor activity; if this is the case, this could be further described and clarified in the legend and in the text (page 12). We described and clarified in the legend and in the text (page 12, 15) that Bcl-xL and NFkBp50 expression and mtor activity may correlate, but it was not significant. 7. In Figure 5, panel b do not show significant increase in apoptosis after 72 h of treatment, but in panel c the authors describe the combinatorial effects after 72 h. These discrepancies must be clarified. Also, figure 5c includes combinations of rapamycin and vincristine, but in the manuscript (page 12) the authors only mention the combinations with doxorubicin and etoposide. Do the asterisks (*) mean statistical significant increase?, this must be included in figure legend. The legends have been updated and the significant effects of rapamycin have been indicated in Figure 5b. Figure 5b shows that rapamycin monotherapy induced apoptosis at 96, 120 és 144h, which was significant (p<0.05). Figure 5c shows that rapamycin is able to enhance the effect of doxorubicin, vincristine and etoposide already after 24h. Asterisks (*) were included in Figure 5b to indicate statistical significance. 5

7 We would like to thank you for your constructive suggestions. We hope that the modifications and the comments will satisfy you and our revised manuscript can be considered for publication in BMC Cancer. We are submitting the revised version with the modifications highlighted. Figure 1, Figure 3, Figure 4a, Figure 5b-c and Figure 6 have been updated. Yours Sincerely, Anna Sebestyén, PhD corresponding author 6