Reviewer's report 
 Alterations of tumor suppressor gene p16INK4A in pancreatic ductal carcinoma Title: 
 1  14 March 2005 Version: Date: 
 Peter Muscarella Reviewer: 
Reviewer's report: 
General 
Pancreatic cancer continues to have a dismal prognosis despite recent advances in surgical 
technique and adjuvant therapy. Molecular characterization of pancreatic adenocarcinoma specimens is crucial to our understanding of these tumors and holds promise to identify potential 
targets for novel therapies. The p16 gene is a component of the Rb/E2F pathway and thus 
contributes to cell cycle regulation at the G1/S phase junction. Inactivating alterations of the p16 tumor suppressor gene have previously been reported to occur frequently in human pancreatic adenocarcinoma cell lines and primary tumors. Known mechanisms of inactivation include 
homozygous deletion, mutation, and promotor hypermethylation. 
The submitted manuscript describes an analysis of p16 inactivation in 25 human pancreatic 
adenocarcinoma primary tumors obtained at the time of surgical resection. Methods of specimen 
collection, tissue processing/DNA isolation, genetic analyses, and immunohistochemical analysis 
are all solidly based on previously published techniques. Appropriate controls are employed, as are statistical analyses of data. The findings of p16 alterations by deletion, mutation and methylation in 12%, 16% and 52% of specimnes, for an overall frequency of 72%, are consistent with previously 
published reports. Analysis of expression is an important aspect of this study and appears to confirm 
the genetic findings. 
The study is well designed and the manuscript is relatively well written, but it does not contribute any 
new findings to our understanding of pancreatic cancer molecular pathogenesis. Instead, the 
findings confirm those of previous investigators and this is acknowledged by the authors. The significance and relevance of the study are of moderate importance at best. Nevertheless, the study does evaluate a previously uncharacterized group of patients (Indian-Asians) whose tumors could 
have potentially arisen through alternative pathogenetic mechanisms. The manuscript appears to be 
suitable for publication in BMC Gastroenterology after the authors have reviewed and considered 
the following comments. 
------------------------------------------------------------------------------ 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
None 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
1) A table summarizing the findings of the genetic analysis and IHC findings for all specimens should 
be included and would make the manuscript easier for the reader to interpret. 
2) In methods - Detection of Homozygous Deletion in p16, please clarify that DNA was isolated from 
frozen tissue. 
3) Background (paragraph 3). Please add "types" to the end of the sentence "....role in the regulation 
of cellular growth in the majority of cell." 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
1) A functional analysis of mutant p16 proteins would be very nice to see. This can be performed by 
cloning mutant cDNAs into expression vectors. Loss of CDK binding and CDK-inhibitory activity 
would support the significance of these mutations and might suggest a stronger role for p16. The 
three specimens that appeared to express p16 by IHC might, in fact, contain nonfunctional or poorly 
functional p16 protein. In addition, some discussion regarding the presence or absence of the 
mutations in the functionally active (CDK-binding domain) region of the p16 protein would be nice. 
2) In the discussion, the authors might want to address the finding that two specimens showed loss 
of p16 expression by IHC, but demonstrated no inactivating alterations by genetic analysis. What 
are the reasons that this might occur? 
 Accept after minor essential revisions What next?: 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Needs some language corrections before being published Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests. 
Reviewer's report 
 Alterations of tumor suppressor gene p16INK4A in pancreatic ductal carcinoma Title: 
 1  14 March 2005 Version: Date: 
 Peter Muscarella Reviewer: 
Reviewer's report: 
General 
Pancreatic cancer continues to have a dismal prognosis despite recent advances in surgical 
technique and adjuvant therapy. Molecular characterization of pancreatic adenocarcinoma specimens is crucial to our understanding of these tumors and holds promise to identify potential 
targets for novel therapies. The p16 gene is a component of the Rb/E2F pathway and thus 
contributes to cell cycle regulation at the G1/S phase junction. Inactivating alterations of the p16 tumor suppressor gene have previously been reported to occur frequently in human pancreatic adenocarcinoma cell lines and primary tumors. Known mechanisms of inactivation include 
homozygous deletion, mutation, and promotor hypermethylation. 
The submitted manuscript describes an analysis of p16 inactivation in 25 human pancreatic 
adenocarcinoma primary tumors obtained at the time of surgical resection. Methods of specimen 
collection, tissue processing/DNA isolation, genetic analyses, and immunohistochemical analysis 
are all solidly based on previously published techniques. Appropriate controls are employed, as are statistical analyses of data. The findings of p16 alterations by deletion, mutation and methylation in 12%, 16% and 52% of specimnes, for an overall frequency of 72%, are consistent with previously 
published reports. Analysis of expression is an important aspect of this study and appears to confirm 
the genetic findings. 
The study is well designed and the manuscript is relatively well written, but it does not contribute any 
new findings to our understanding of pancreatic cancer molecular pathogenesis. Instead, the 
findings confirm those of previous investigators and this is acknowledged by the authors. The significance and relevance of the study are of moderate importance at best. Nevertheless, the study does evaluate a previously uncharacterized group of patients (Indian-Asians) whose tumors could 
have potentially arisen through alternative pathogenetic mechanisms. The manuscript appears to be 
suitable for publication in BMC Gastroenterology after the authors have reviewed and considered 
the following comments. 
------------------------------------------------------------------------------ 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
None 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
1) A table summarizing the findings of the genetic analysis and IHC findings for all specimens should 
be included and would make the manuscript easier for the reader to interpret. 
2) In methods - Detection of Homozygous Deletion in p16, please clarify that DNA was isolated from 
frozen tissue. 
3) Background (paragraph 3). Please add "types" to the end of the sentence "....role in the regulation 
of cellular growth in the majority of cell." 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
1) A functional analysis of mutant p16 proteins would be very nice to see. This can be performed by 
cloning mutant cDNAs into expression vectors. Loss of CDK binding and CDK-inhibitory activity 
would support the significance of these mutations and might suggest a stronger role for p16. The 
three specimens that appeared to express p16 by IHC might, in fact, contain nonfunctional or poorly 
functional p16 protein. In addition, some discussion regarding the presence or absence of the 
mutations in the functionally active (CDK-binding domain) region of the p16 protein would be nice. 
2) In the discussion, the authors might want to address the finding that two specimens showed loss 
of p16 expression by IHC, but demonstrated no inactivating alterations by genetic analysis. What 
are the reasons that this might occur? 
 Accept after minor essential revisions What next?: 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Needs some language corrections before being published Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests. 
Please review this paper.
user
Reviewer's report 
 Alterations of tumor suppressor gene p16INK4A in pancreatic ductal carcinoma Title: 
 1  14 March 2005 Version: Date: 
 Peter Muscarella Reviewer: 
Reviewer's report: 
General 
Pancreatic cancer continues to have a dismal prognosis despite recent advances in surgical 
technique and adjuvant therapy. Molecular characterization of pancreatic adenocarcinoma specimens is crucial to our understanding of these tumors and holds promise to identify potential 
targets for novel therapies. The p16 gene is a component of the Rb/E2F pathway and thus 
contributes to cell cycle regulation at the G1/S phase junction. Inactivating alterations of the p16 tumor suppressor gene have previously been reported to occur frequently in human pancreatic adenocarcinoma cell lines and primary tumors. Known mechanisms of inactivation include 
homozygous deletion, mutation, and promotor hypermethylation. 
The submitted manuscript describes an analysis of p16 inactivation in 25 human pancreatic 
adenocarcinoma primary tumors obtained at the time of surgical resection. Methods of specimen 
collection, tissue processing/DNA isolation, genetic analyses, and immunohistochemical analysis 
are all solidly based on previously published techniques. Appropriate controls are employed, as are statistical analyses of data. The findings of p16 alterations by deletion, mutation and methylation in 12%, 16% and 52% of specimnes, for an overall frequency of 72%, are consistent with previously 
published reports. Analysis of expression is an important aspect of this study and appears to confirm 
the genetic findings. 
The study is well designed and the manuscript is relatively well written, but it does not contribute any 
new findings to our understanding of pancreatic cancer molecular pathogenesis. Instead, the 
findings confirm those of previous investigators and this is acknowledged by the authors. The significance and relevance of the study are of moderate importance at best. Nevertheless, the study does evaluate a previously uncharacterized group of patients (Indian-Asians) whose tumors could 
have potentially arisen through alternative pathogenetic mechanisms. The manuscript appears to be 
suitable for publication in BMC Gastroenterology after the authors have reviewed and considered 
the following comments. 
------------------------------------------------------------------------------ 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
None 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
1) A table summarizing the findings of the genetic analysis and IHC findings for all specimens should 
be included and would make the manuscript easier for the reader to interpret. 
2) In methods - Detection of Homozygous Deletion in p16, please clarify that DNA was isolated from 
frozen tissue. 
3) Background (paragraph 3). Please add "types" to the end of the sentence "....role in the regulation 
of cellular growth in the majority of cell." 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
1) A functional analysis of mutant p16 proteins would be very nice to see. This can be performed by 
cloning mutant cDNAs into expression vectors. Loss of CDK binding and CDK-inhibitory activity 
would support the significance of these mutations and might suggest a stronger role for p16. The 
three specimens that appeared to express p16 by IHC might, in fact, contain nonfunctional or poorly 
functional p16 protein. In addition, some discussion regarding the presence or absence of the 
mutations in the functionally active (CDK-binding domain) region of the p16 protein would be nice. 
2) In the discussion, the authors might want to address the finding that two specimens showed loss 
of p16 expression by IHC, but demonstrated no inactivating alterations by genetic analysis. What 
are the reasons that this might occur? 
 Accept after minor essential revisions What next?: 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Needs some language corrections before being published Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests. 
assistant