Reviewer's report 
 Humoral immune response to MUC5AC in patients with colorectal polyps and colorectal Title: 
carcinoma 
 2  8 September 2005 Version: Date: 
 Celso A Reis Reviewer: 
Reviewer's report: 
General 
This paper describes the use of a ELISA assay for the evaluation of the incidence of naturally 
occurring MUC5AC antibodies in the sera of normal individuals, patients with colonic polyps and 
patients with advanced colorectal carcinoma. This paper also addresses the relationship of naturally 
occurring MUC5AC antibodies with the prognosis of colorectal carcinoma. 
The free circulating MUC5AC antibodies of the patients in the sera were measured 
using an enzyme- linked immunosorbant assay with a synthetic peptide corresponding to an 8 aa. 
segment of MUC5AC tandem repeat region. 
The results obtained showed that natural MUC5AC antibodies were detected in 6 of 22 (27.3%) 
healthy subjects, 9 of 20 (45%) polyp patients, 18 of 30 (60%) patients with colorectal cancer. The 
presence of circulating free MUC5AC antibody levels was significantly correlated with expression of 
MUC5AC in polyp sections. MUC5AC antibody in the sera of colorectal carcinoma patients was not 
significantly correlated with clinicopathologic parameters, but disease free survival and overall 
survival were shorter in MUC5AC antibody positive patients. 
The authors concluded that the decreased survival in patients with MUC5AC antibody positivity may 
be due to a decreased in the MUC5AC expression in tumor tissues of surviving carcinoma patients. 
The authors discuss properly the limited number of individuals included in this study. Overall the 
results of this work are promising and supports further studies for testing that serum MUC5AC antibody may be used as a prognostic factor in colorectal carcinoma. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
There are two issues in this study that may be addressed in the discussion section: 
1) The peptide used in the assay contains only one repeat sequence of 8 aa. It is not clear whether 
the use of a longer peptide will provide epitopes for better recognition of antibodies present in the 
sera of the patients and eventually improve this ELISA, and consequently improving the capabilities of the assay. It is a suggestion that the author may test in the future an ELISA which uses other MUC5AC tandem repeat peptides, containing at least 2 repeats of 8 aa. 
2) Another concern is that the peptide used in this assay is based on the tandem repeat sequence of 
MUC5AC. This sequence is highly O-glycosylated in the MUC5AC mucin. The capability of 
antibodies present of in the sera of the patients may stem from the variation of O-glycosylation in the 
tandem repeat of MUC5AC. It has been shown in the literature that MUC5AC glycopeptides can be 
efficiently produced by chemoenzymatic synthesis (Reis et al., Int. J Cancer 1997, 74:112-121). The 
hypothesis of using an ELISA assay with MUC5AC glycopeptides (Different MUC5AC glycoforms) 
may be tested in the future in order to evaluate whether it can provide a better discrimination 
between different groups of patients and their clinical features. The author may discuss the possibility of testing different MUC5AC glycopeptides in this ELISA. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Accept after minor essential revisions What next?: 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
This Reviewer declares that has monoclonal antibodies available to the MUC5AC mucin. 
Reviewer's report 
 Humoral immune response to MUC5AC in patients with colorectal polyps and colorectal Title: 
carcinoma 
 2  8 September 2005 Version: Date: 
 Celso A Reis Reviewer: 
Reviewer's report: 
General 
This paper describes the use of a ELISA assay for the evaluation of the incidence of naturally 
occurring MUC5AC antibodies in the sera of normal individuals, patients with colonic polyps and 
patients with advanced colorectal carcinoma. This paper also addresses the relationship of naturally 
occurring MUC5AC antibodies with the prognosis of colorectal carcinoma. 
The free circulating MUC5AC antibodies of the patients in the sera were measured 
using an enzyme- linked immunosorbant assay with a synthetic peptide corresponding to an 8 aa. 
segment of MUC5AC tandem repeat region. 
The results obtained showed that natural MUC5AC antibodies were detected in 6 of 22 (27.3%) 
healthy subjects, 9 of 20 (45%) polyp patients, 18 of 30 (60%) patients with colorectal cancer. The 
presence of circulating free MUC5AC antibody levels was significantly correlated with expression of 
MUC5AC in polyp sections. MUC5AC antibody in the sera of colorectal carcinoma patients was not 
significantly correlated with clinicopathologic parameters, but disease free survival and overall 
survival were shorter in MUC5AC antibody positive patients. 
The authors concluded that the decreased survival in patients with MUC5AC antibody positivity may 
be due to a decreased in the MUC5AC expression in tumor tissues of surviving carcinoma patients. 
The authors discuss properly the limited number of individuals included in this study. Overall the 
results of this work are promising and supports further studies for testing that serum MUC5AC antibody may be used as a prognostic factor in colorectal carcinoma. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
There are two issues in this study that may be addressed in the discussion section: 
1) The peptide used in the assay contains only one repeat sequence of 8 aa. It is not clear whether 
the use of a longer peptide will provide epitopes for better recognition of antibodies present in the 
sera of the patients and eventually improve this ELISA, and consequently improving the capabilities of the assay. It is a suggestion that the author may test in the future an ELISA which uses other MUC5AC tandem repeat peptides, containing at least 2 repeats of 8 aa. 
2) Another concern is that the peptide used in this assay is based on the tandem repeat sequence of 
MUC5AC. This sequence is highly O-glycosylated in the MUC5AC mucin. The capability of 
antibodies present of in the sera of the patients may stem from the variation of O-glycosylation in the 
tandem repeat of MUC5AC. It has been shown in the literature that MUC5AC glycopeptides can be 
efficiently produced by chemoenzymatic synthesis (Reis et al., Int. J Cancer 1997, 74:112-121). The 
hypothesis of using an ELISA assay with MUC5AC glycopeptides (Different MUC5AC glycoforms) 
may be tested in the future in order to evaluate whether it can provide a better discrimination 
between different groups of patients and their clinical features. The author may discuss the possibility of testing different MUC5AC glycopeptides in this ELISA. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Accept after minor essential revisions What next?: 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
This Reviewer declares that has monoclonal antibodies available to the MUC5AC mucin. 
Please review this paper.
user
Reviewer's report 
 Humoral immune response to MUC5AC in patients with colorectal polyps and colorectal Title: 
carcinoma 
 2  8 September 2005 Version: Date: 
 Celso A Reis Reviewer: 
Reviewer's report: 
General 
This paper describes the use of a ELISA assay for the evaluation of the incidence of naturally 
occurring MUC5AC antibodies in the sera of normal individuals, patients with colonic polyps and 
patients with advanced colorectal carcinoma. This paper also addresses the relationship of naturally 
occurring MUC5AC antibodies with the prognosis of colorectal carcinoma. 
The free circulating MUC5AC antibodies of the patients in the sera were measured 
using an enzyme- linked immunosorbant assay with a synthetic peptide corresponding to an 8 aa. 
segment of MUC5AC tandem repeat region. 
The results obtained showed that natural MUC5AC antibodies were detected in 6 of 22 (27.3%) 
healthy subjects, 9 of 20 (45%) polyp patients, 18 of 30 (60%) patients with colorectal cancer. The 
presence of circulating free MUC5AC antibody levels was significantly correlated with expression of 
MUC5AC in polyp sections. MUC5AC antibody in the sera of colorectal carcinoma patients was not 
significantly correlated with clinicopathologic parameters, but disease free survival and overall 
survival were shorter in MUC5AC antibody positive patients. 
The authors concluded that the decreased survival in patients with MUC5AC antibody positivity may 
be due to a decreased in the MUC5AC expression in tumor tissues of surviving carcinoma patients. 
The authors discuss properly the limited number of individuals included in this study. Overall the 
results of this work are promising and supports further studies for testing that serum MUC5AC antibody may be used as a prognostic factor in colorectal carcinoma. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
There are two issues in this study that may be addressed in the discussion section: 
1) The peptide used in the assay contains only one repeat sequence of 8 aa. It is not clear whether 
the use of a longer peptide will provide epitopes for better recognition of antibodies present in the 
sera of the patients and eventually improve this ELISA, and consequently improving the capabilities of the assay. It is a suggestion that the author may test in the future an ELISA which uses other MUC5AC tandem repeat peptides, containing at least 2 repeats of 8 aa. 
2) Another concern is that the peptide used in this assay is based on the tandem repeat sequence of 
MUC5AC. This sequence is highly O-glycosylated in the MUC5AC mucin. The capability of 
antibodies present of in the sera of the patients may stem from the variation of O-glycosylation in the 
tandem repeat of MUC5AC. It has been shown in the literature that MUC5AC glycopeptides can be 
efficiently produced by chemoenzymatic synthesis (Reis et al., Int. J Cancer 1997, 74:112-121). The 
hypothesis of using an ELISA assay with MUC5AC glycopeptides (Different MUC5AC glycoforms) 
may be tested in the future in order to evaluate whether it can provide a better discrimination 
between different groups of patients and their clinical features. The author may discuss the possibility of testing different MUC5AC glycopeptides in this ELISA. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Accept after minor essential revisions What next?: 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
This Reviewer declares that has monoclonal antibodies available to the MUC5AC mucin. 
assistant