Author's response to reviews Title: Synuclein Gamma Predicts Poor Clinical Outcome in Colon Cancer with Normal Levels of Carcinoembryonic Antigen Authors: Caiyun Liu (liucaiyun23@yahoo.com.cn) Bin Dong (dongbin2001@163.com) Aiping Lu (aiping_lu@sina.com) Xiaofang Xing (bubblefangfang@gmail.com) Like Qu (qulike@bjcancer.org) Lin Meng (menglin3330@sina.com) Jian Jian Wu (wujian997@bjcancer.org) Eric Shi (ericshi99@yahoo.com) Chengchao Shou (scc@bjcancer.org) Version: 3 Date: 18 December 2009 Author's response to reviews: see over
Dear members of BioMed Central Editorial Team: We appreciate your kind consideration of our manuscript (No: 8262050442961259) and the valuable comments from the reviewers. We have revised the manuscript according to the reviewer s concerns point-to-point as below (Responses to the comments from the reviewers). For your convenience, both the original comments and questions are kept in this letter below. All the changes in the manuscript have also been highlighted by track-change function. We understand the significance for us to provide clarification and additional analysis for our data. We have done with our best to fulfill this goal. Please let us know if there are any additional questions and comments. Yours sincerely, Chengchao Shou, Ph.D, MD Professor and Director Department of Biochem & Mol Biol Peking University School of Oncology Beijing Cancer Hospital & Institute 52# Fu-Cheng road, Haidian district Beijing, 100142, PRC Tel. 86-10-88196766 Fax: 86-10-88122437 e-mail: scc@bjcancer.org
Responses to the comments from the reviewers Reviewer 1: 1. Introduction: In colon cancer, CEA is mainly used as a marker for serial follow-up to detect tumor recurrence and progression rather than as a prognostic/predictive marker. The rationale to study synuclein as a prognostic marker in conjunction with serum CEA levels is therefore not clear. Thanks for what the reviewer concerned. The data from different labs about the utility of preoperative CEA in CRC remains controversial as regards to its application in the diagnosis, prognosis, and management and follow up of CRC patients. The preoperative CEA in CRC patients identifies subsets with favorable, indolent and uneven biological behavior (The cut-off for a positive CEA ranges from 3 to 15 ng/ml between the various studies) (Tan E, et al. Surg Oncol. 2009;18:15-24). Moreover, the addition of preoperative CEA level to conventional staging forms a strong prognostic tool and supplies adopted practice guideline initiative for follow up and therapy in CRC (El-Awady S,et al. Hepatogastroenterology 2009;56:361-366). The serum CEA has high specificity but insufficient sensitivity for detecting CRC recurrence (Tan E, et al. Surg Oncol. 2009;18:15-24) and approximate 30% of all CRC recurrences have normal CEA serum levels (Safi F, et al. Dis Colon Rectum 1993; 36:636 643). Since any single marker is not sufficiently predictive, combination of different markers representing different aspects of tumor biology will have a better prognostic evaluation (Zlobec I, et al.cancer 2008, 112:495-502, Uen YH, et al. Ann Surg 2007, 246:1040-1046). In the present study we found SNCG overexpression correlates with poor outcome and is an independent prognostic indicator. As SNCG level was not associated with preoperative serum CEA level, we were interested in investigating whether a combination of SNCG and CEA could improve prognostic evaluation in CRC. 2. Materials and Methods: Overall survival should be calculated from the date of surgery to the date of recorded death. Data of patients with lost follow-up should be censored. Same applies for disease free survival.
According to the reviewer s comments, we have corrected Overall survival time was calculated from the date of surgery to the date of death due to any cause or last follow-up visit. Disease-free survival (DFS) time was calculated for patients from the date of surgery to the date of disease progression (local recurrence or distant metastasis) or last follow-up visit. to Overall survival time was calculated from the date of surgery to the date of death due to any cause. Disease-free survival (DFS) time was calculated for patients from the date of surgery to the date of disease progression (local recurrence or distant metastasis).. 3. In the results section, where the correlation between SNCG expression and survival is presented, the second half of the first paragraph A significant difference in survival rate was observed is redundant to the first half of this paragraph. Additionally, in the first half a p-value of <0.0001 is presented for survival association, while in the second half a p-value of 0.001 is presented. Which one is correct? Please clarify and remove repetitions. According to the reviewer s comments, we have deleted the repetitions A significant difference in survival rate was observed in 5-year follow up. While there were 58.0% for DFS and 58.7% for OS in patients without SNCG expression, SNCG-positive patients had a 32.9% DFS and 32.5% OS. from the paragraph. The P<0.0001 in the first half represented the difference of mean time for survival between SNCG negative group and SNCG positive group (P<0.0001),while in the second half the P=0.001 showed the hazard ratio of death according to SNCG level was 2.601 (95%CI, 1.471-4.601, P=0.001). 4. Results: The conclusion that SNCG is a stronger predictive factor than intravascular embolus or weaker then depth of tumor invasion cannot be drawn from the data presented. This conclusion could only be drawn if the 95% confidence intervals of the hazard ratios for both markers were not overlapping. According to the reviewer s comments, we have corrected Multivariate analysis revealed that the
influence of SNCG on DFS and OS (P=0.039, 0.048) of the 229 patients was significantly stronger than intravascular embolus (P=0.049, 0.050) but weaker than TNM stage (P=0.008, 0.006) and depth of tumor invasion (P=0.025, 0.028). Based on these analyses, we concluded that tumor SNCG level can be considered as an independent prognostic factor for DFS and OS of the patients with colon adenocarcinoma. to Multivariate analysis revealed that SNCG was an independent prognostic factor for DFS (P=0.039) and OS(P= 0.048) of the patients with colon adenocarcinoma. 5. The same applies for the comparison of hazard ratios. If the 95% confidence intervals overlap (which is the case for all data presented in the next paragraph regarding CEA/SNCG combo versus CEA and SNCG alone), no statement regarding which one of the investigated markers is better than the other can be made. Instead they are then assumed to be of NO significant difference in this study population. Therefore all the statements about the suggested superiority of the marker combination compared to the single marker analyses may be wrong. According to the reviewer s comments, we have corrected Very importantly, the hazard ratio of combined CEA and SNCG for DFS and OS were 3.517 and 3.645, significantly higher than those of CEA (2.440 and 2.639), and SNCG (2.213 and 2.141), respectively. These data suggest that combination of CEA and SNCG indicates a worsen survival outcome than either SNCG or CEA alone. to Very importantly, the hazard ratio of combined CEA and SNCG for DFS and OS were 3.517 and 3.645, 2.440 and 2.639 for CEA, and 2.213 and 2.141 for SNCG, respectively. These data suggested that the combination of CEA and SNCG was a strong prognostic indicator.. 6. The same mistake is unfortunately carried on in the next (last) paragraph of the results as well: You really cannot compare pure hazard ratios. For all the markers (SNCG, Tumor size, Tumor differentiation) the 95% confidence intervals are largely overlapping (Table 4). There is NO significant difference in the presented study population for these markers.
We have corrected The hazard ratio of recurrence according to combined SNCG with CEA was 4.056 (95%CI, 1.679-9.800, P=0.001), higher than SNCG (2.958, 95% CI, 1.452-6.028, P = 0.002) and CEA (3.401, 95%CI, 1.648-7.023, P=0.001). Combination of CEA with SNCG improves prognostic evaluation for patients with colon adenocarcinoma. to The hazard ratio of recurrence according to combined SNCG with CEA was 4.056 (95%CI, 1.679-9.800, P=0.001), 2.958 for SNCG (95% CI, 1.452-6.028, P = 0.002) and 3.401 for CEA (95%CI, 1.648-7.023, P=0.001). Combination of CEA with SNCG might improve prognostic evaluation for patients with colon adenocarcinoma.. 7. What is the rationale to look at the subgroup of CEA negative patients in specific? The sample size becomes pretty small and CEA is not a strong predictive marker for colon cancer anyway, rather than a serial follow-up marker. As the information gathered from this subgroup does not add any useful data to the observations among the whole study population, I would suggest to remove this sub-evaluation. Approximate 30% of all CRC recurrences is negative of CEA serum levels and few studies have investigated the surveillance of those sub-patients. Our results showed SNCG was overexpressed in 35% (34/97) of colon cancer tissues from patients with negative CEA level and SNCG level still was an independent prognostic factor for DFS and OS for colon cancer patients with negative serum CEA level. 8. Discussion: The discussion is mainly based on the assumed higher prognostic value of SNCG or the CEA/SNCG combo which is unfortunately statistically wrong as it cannot be assessed within the available data. It should therefore be rewritten accordingly. We have corrected We investigated the impact of SNCG level on the clinical outcome of patients with normal preoperative serum CEA levels. Our results demonstrated that SNCG remained an independent prognostic variable for these patients and affected patients survival more than TNM stage, lymph node metastasis, depth of invasion, tumor size and differentiation grade into We investigated the impact of SNCG level on the clinical outcome of patients with normal
preoperative serum CEA levels and our results demonstrated that SNCG remained an independent prognostic variable for these patients and affected patients survival, but the clinicopathologic factors such as TNM stage, lymph node metastasis, and depth of invasion,, all didn t influence the patients survival. The sentence We also demonstrated that combination of CEA and SNCG has a significant additive value and provides a higher prognostic value in colon cancer than each of them considered separately for patients with stages I-II and III-IV. Tumor SNCG and preoperative CEA can provide mutual complementary prognostic value and combined analyses of SNCG with CEA provide a better prognosis on survival outcome for patients compared with either marker was modified to We also demonstrated that combination of CEA and SNCG has a significant additive value and provides a high prognostic value in colon cancer. Tumor SNCG and preoperative CEA may provide mutual complementary prognostic value and combined analyses of SNCG with CEA provide a strong prognosis on survival outcome for patients with colon cancer. 9. Mat/Met: Was the analysis carried out on whole tissue sections, or were the cases arrayed in a tissue-micro-array? Please specify. The analysis carried out on whole tissue sections, which was specified in the Method section. 10. I don t understand the scoring system for synuclein expression. Specifically a total score for area adding grade of 3 remains unclear. Finally the authors only derive a positive and negative category for their statistical evaluation. What is the reason to use the complex scoring system? Please clarify. This is a classical immunohistochemical category method that is called four-value classification scale (Lee JC et al. Eur J Cancer.2000; 36:748-753).
We have changed A semiquantitative scoring system was performed according to the following scoring system:.. into Immunoreactivity for SNCG in tumour cells was graded as either negative or positive according to a four-value classification scale as follows: 11. Results: What exactly was the heterogeneous expression pattern of synuclein? Was there a predominance of staining e.g. at the tumor margins? Or was the staining randomly dispersed among the tumor cells? It is well known that the cells in cancer tissue are heterogeneous in terms of morphology and differentiation status, even if the cancer tissue consists of progeny developed from a single neoplastic cell. (Nowell, PC. Science 1976, 194:23-28). Heterogeneous expression between cells has only been fully realized with the staining of sections of tumors with monoclonal antibodies. ( Edwards PAW. Br J Cancer 1985; 51:149-160.) 12. If 4 cases showed staining of normal mucosa, synuclein is not specifically expressed in tumors and is not limited to adenocarcinomas. Please rephrase. We changed SNCG specifically expressed in colon adenocarcinoma cells and SNCG level was associated with intravascular embolus into SNCG is overexpressed in colon adenocarcinoma cells and is associated with intravascular embolus and deleted the phrase These data indicate that expression of SNCG in colon epithelium is limited to colon adenocarcinomas. Reviewer 2: 1. The authors should specifically address the frequency, rate of DFS, and OS of SNCG negative patients that are CEA positive. This will give insight to the readers as to how useful SNCG expression is to improving current diagnostic methods (CEA positive versus negative) versus improving diagnostic tests for patients who have normal CEA levels.
Thanks for the suggestions. We have added There were 42% (45/108) of patients with elevated CEA levels in the SNCG negative group, rates of DFS and OS of these patients were 43% and 39% at 5 year after surgery in section 4 of results. 2. para. 1 of Background section - "Traditionally, TNM stage is the..." The word system should be removed from this sentence. According to the reviewer s suggestions, we have removed system from this sentence. 3. para. 1 of Background section "...but accurate classification of intermediate-stage cases is imprecise and prognosis varies greatly in CRC patients even in the same stage." It is unclear why the authors are making this point since this paragraph is motivating the This sentence want to tell readers why it is necessary for discovery new biomarkers for predicting prognosis of CRC patients. To make it is more clearly, we have changed but accurate classification of intermediate-stage cases is imprecise and prognosis varies greatly in CRC patients even in the same stage into but current classification of CRC cann t predict prognosis precisely even for the patients in the same clinical stage." 4. para. 1 of Background section - Correct the grammar. "Approximately 30% of all CRC recurrences..." We have corrected Approximately 30% of all CRC recurrences... into Approximate 30% of all CRC recurrences. 5. para. 3 of Background section - Correct the grammar. "However, the prognostic significance of SNCG in other cancers remains unknown.." We have corrected However, prognostic significance of SNCG in other cancers has remained unknown. to However, the prognostic significance of SNCG in other cancers remains
unknown. 6. para. 3 of Background section - The authors need to be more specific as to how SNCG can be used prognostically. In particular, the authors are comparing SNCG levels as assessed by immunohistochemistry of tumor sections with serum CEA levels for their prognostic value. The current manner in which these points are described may be confusing to the reader. For example, "In the current study, we investigated whether SNCG expression levels as assessed by immunohistochemical analysis of excised tumor sections could be a useful prognostic marker for colon cancer patients and particularly for those with normal preoperative serum CEA level." According to the reviewer s suggestions, we have changed However, prognostic significance of SNCG in other cancers has remained unknown. In the current study, we investigated whether SNCG level could be a useful prognostic marker for colon cancer patients and particularly for those with normal preoperative serum CEA level. We also evaluated whether combination of SNCG with CEA could improve prognostic value. into However, the prognostic significance of SNCG in other cancers remains unknown. In the current study, SNCG level as assessed by immunohistochemistry of tumor sections is an independent prognostic factor of a shorter DFS and OS for colon cancer patients. Importantly, SNCG remains a prognostic determinant of DFS and OS for colon cancer patients with normal preoperative serum CEA level.. 7. para. 3 of Background section - The last sentence of this paragraph needs to be clarified. According to the reviewer s suggestions, we have changed In the current study, we investigated whether SNCG level could be a useful prognostic marker for colon cancer patients and particularly for those with normal preoperative serum CEA level. We also evaluated whether combination of SNCG with CEA could improve prognostic value. into In the current study, SNCG level as assessed by immunohistochemistry of tumor sections is an independent prognostic factor of a shorter DFS and OS for colon cancer patients. Importantly, SNCG remains a prognostic determinant of DFS and OS for colon cancer patients with normal preoperative serum CEA
level.. 8. The authors should comment on what is considered positive versus negative CEA levels and how that compares with the cut off they used in their analysis. We have changed The serum levels of CEA were considered positive when they were equal to or higher than 5.0 ng/ml according to the manufacture s instructions. into The serum levels of CEA were considered positive when they were equal to or higher than 5.0 ng/ml (cutoff value) and negative when lower than that according to the manufacture s instructions.. 9. Section title 1 of Results section - grammar should be improved. We have corrected SNCG specifically expressed in colon adenocarcinoma cells and SNCG level was associated with intravascular embolus into SNCG is overexpressed in colon adenocarcinoma cells and is associated with intravascular embolus 10. para. 2, section 1, results - Correct the grammar. "Interestingly, in contrast to previous observations of an association between SNCG expression and tumor stage..." We have corrected Interestingly, in contrast to previous observation of association between SNCG expression and tumor stages in many different cancers [11,15], into Interestingly, in contrast to previous observations of an association between SNCG expression and tumor stage in many different cancers [11,15],. 11. para. 2, section 1, results - Correct the grammar. "Expression of SNCG in primary tumors (p=0.023) was..."
We have corrected Expression of SNCG in primary tumor (P=0.023) also into Expression of SNCG in primary tumors (P=0.023) was also. 12. para. 2, section 1, results - Correct the grammar. "There were 61 patients that developed tumor recurrence during the follow-up period." We have corrected There were 61 patients developed recurrence during the follow-up. into There were 61 patients developed tumor recurrence during the follow-up period.. 13. para. 2, section 1, results - Correct the grammar. "While 37% patients with SNCG positive primary tumor developed recurrence, only 22% patients with SNCG negative tumors developed recurrence (P = 0.02)." We have corrected While 37% patients with SNCG positive primary tumor developed recurrence, only 22% patients with SNCG negative tumors developed recurrence (P = 0.02). into While 37% of patients with SNCG positive primary tumor developed tumor recurrence, only 22% of patients with SNCG negative tumors developed tumor recurrence (P = 0.02).. 14. Section title 2 of Results section - grammar should be improved. We have changed SNCG overexpression correlated with poor outcome and was an independent prognostic indicator into SNCG overexpression correlates with poor outcome and is an independent prognostic indicator. 15. Throughout, "in SNCG negative group" should be written as "in the SNCG negative group" and likewise for the SNCG positive group.
We have changed in SNCG negative/positive group into in the SNCG negative/positive group throughout the manuscript. 16. para. 1 section 3, results - Correct the grammar. "whether a combination of SNCG and CEA." We have corrected whether combination of SNCG and CEA into whether a combination of SNCG and CEA. 17. para. 1, section 3, results - The last sentence of this paragraph needs to be corrected to be grammatically correct. We have corrected These data suggest that combination of CEA and SNCG indicates a worsen survival outcome than either SNCG or CEA alone. into These data suggested that the combination of CEA and SNCG was a strong prognostic indicator.. 18. para. 2, section 3, results - Correct the grammar. "Figure 2 illustrates..." We have corrected Figure 2 illustrated into Figure 2 illustrates. 19. para. 2, section 3, results - The grammar of this paragraph needs to be improved. We have corrected Combination of CEA with SNCG improves prognostic evaluation for patients with colon adenocarcinoma. Into Combination of CEA with SNCG might improve prognostic evaluation for patients with colon adenocarcinoma.. 20. The grammar of the title of Table 2 needs to be corrected.
We have corrected SNCG Specifically Expressed on Colon Adenocarcinoma. SNCG level in total 460 clinical colon specimens was investigated by immunohistochemistry into SNCG Expressing Profile in Colon Adenocarcinoma. according to the reviewer s suggestions. Reviewer 3: 1. For the most part the introduction and discussion lack description of the potential biological function of synuclein gamma. More detailed introduction and discussion of the biological role of this protein should be provided. Thanks very much for the suggestions. We have added the potential biological function of synuclein gamma in the Introduction section. 2. The quality of Figure 1 panel A should be improved. Particularly the normal crypts are difficult to visualize as the image is too bright. We have improved the quality of Figure 1 panel A according to the reviewer s comments.