Reviewer's report
Title:Expression, localization and polymorphisms of the nuclear receptor PXR in
Barrett's esophagus and esophageal adenocarcinoma
Version: 10Date:5 May 2011
Reviewer: Barry Forman
Reviewer's report:
This manuscript was reviewed jointly by Dr. Lily Lai (llai@coh.org) and Dr. Barry
Forman (bmforman@earthlink.net), City of Hope, Duarte, CA, USA.
Esophageal carcinoma (EAC) remains one of the most lethal solid cancers with
an overall 5 yr survival of less than 15%. In the last twenty years,
adenocarcinoma of the esophagus has become epidemic in incidence. Factors
increasing the risk of EAC includes obesity, white race, male, reflux disease and
Barrett’s esophagus (BE). While the association with BE with EAC is well
established, understanding the molecular changes involved with progression to
disease remains limited. In addition, there is currently no ability to identify specific
populations who are at highest risk for the development of EAC amongst those
with reflux and BE.
In this paper, Dr. Van de Winkel et.al. argue that a nuclear receptor, the
xenobiotic receptor PXR increases its level of expression from normal
esophageal squamous epithelium to BE to EAC; that ligands for PXR induce
nuclear translocation of the receptor in an EAC cell line; and analyze
polymorphisms in the PXR gene that could be predictive for BE. These findings
provide important evidence suggesting a possbile link between PXR and the
conversion of normal esophageal epithelium to BE, and suggest PXR as a
potential treatment target for EAC. Future studies will be required to extend and
confirm these conclusions.
Major Compulsory Revisions
1. Expression of PXR in BE patients
The RNA expression data in figure 1A are all normalized to Sq=1 for all patients.
This reporting scheme is appropriate to make the point of relative differences in
expression, but masks any significant interindividual differences in PXR
expression that may exist among the population. It would be useful if the authors
included this data as a supplemental figure.
The methods do not clearly explain how protein expression patterns were
analyzed in the human samples. Was the positive staining graded on presence
or absence of protein, on intensity of stain, on percentage of cells? Was there a
separate scoring for cytosolic staining, nuclear staining, and overall staining?
Were the readers blinded to disease and to patient? In the BE patients, what was
the relative staining pattern of the squamous epithelium v. the columnar
epithelium? The authors note that there is a greater percentage of nuclear
staining in the EAC specimens than in the BE specimens. However, there is
cytosolic and nuclear staining as well as differences in intensity of staining in the
BE and the EAC slides (as shown in Figure 2C and 2D) and without a more
refined scoring scale and method of determining cytosolic v. nuclear expression,
it is difficult to conclude as the authors did that “. . .PXR protein expression was
observed predominantly in the cytoplasm of Barrett’s epithelial cells and not in
the nucleus” (page 11/28).
2. PXR ligands, LCA, tauro-LCA and rifampicin, induce nuclear translocation in
esophageal carcinoma cell line
The authors report these experiments in two cell lines. HET1A is representative
of squamous cell epithelial cells and the other OE19 is derived from esophageal
adenocarcinoma cells. There is a difference in expression of PXR between the
two cell lines, confirming a difference of expression of PXR in squamous cells v.
columnar cells as shown in human tissues. However, PXR expression is not
induced in either cell line (Figure 3A). The authors only show the lack of PXR
induction with TLCA. This is of particular concern as tauro-conjugated bile acids
such as TLCA cannot enter cells without specific transporters and its not clear
that these transporters are expressed in these cells. In addition, LCA is often
toxic at the concentrations used and LCA/TLCA also target other nuclear
receptors and TGR5. There is therefore a potential lack of specificity with these
compounds at the doses used. It would be helpful to repeat these experiments
with other PXR ligands and particularly with rifampicin and hyperforin which have
become “standard” ligands for human PXR in the literature. These additional
PXR ligands should be included as key comparators to the literature and given
the nuclear translocation data in Figure 3B. Although PXR ligand TLCA does not
induce expression, TLCA does induce nuclear translocation. Is this also true for
rifampicin and LCA?
Discretionary Revisions
1. Expression of PXR in BE patients
The gene expression of PXR was evaluated using QPCR of specimens from
patients with reflux esophagitis, BE, and EAC. Figure 1A clearly demonstrates
the difference in PXR expression between the squamous cells and the columnar
cells of patients with BE. However, the authors do not demonstrate the
expression of PXR in normal epithelium of healthy patients (Figure 2B). What is
the mRNA expression of PXR in normal squamous cell epithelium in healthy
patients (n = 5, as stated in the methods but not shown in the figure)? In addition,
much as the authors have paired specimens in the BE patients (Sq and CE), the
relative PXR expression changes between normal epithelium and the disease
states (RE, BE, and EAC) in each individual patient would greatly enhance the
conclusion of a progressive change of PXR as disease progresses from normal
to cancer.
2. PXR polymorphism is associated with BE
Although there were clinical characteristics presented (Table 2), no multivariate
analysis was shown for whether 7635AG is an independent predictor of risk of
developing BE. In addition, in a previous publication on this cohort, EAC patients
were included in the evaluation of another SNP. No data is shown on the EAC
patients in this paper. Data from the EAC cohort with respect to the PXR SNPs
will add greatly to this paper with regard to progression of disease and relevance
of the SNP from normal to BE to EAC.
The above data will not only add to the authors’ conclusion but potentially could
change treatment recommendations. Currently, while the 7635 AG SNP may
help to identify patients at risk for development of BE, no information is available
to assist in the determination of who is at risk for EAC in patients with BE.
Without that information, patients with BE are left with serial upper endoscopies
until dyplasia is identified. At that time, a highly morbid esophagectomy may be
offered to prevent EAC. If the 7635AG SNP is predictive of EAC, it may save
patients the procedure if they don’t carry the genetic polymorphism.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
This manuscript was reviewed jointly by Dr. Lily Lai (llai@coh.org) and Dr. Barry
Forman (bmforman@earthlink.net), City of Hope, Duarte, CA, USA. The
reviewers do
not have competing interests.
Reviewer's report
Title:Expression, localization and polymorphisms of the nuclear receptor PXR in
Barrett's esophagus and esophageal adenocarcinoma
Version: 10Date:5 May 2011
Reviewer: Barry Forman
Reviewer's report:
This manuscript was reviewed jointly by Dr. Lily Lai (llai@coh.org) and Dr. Barry
Forman (bmforman@earthlink.net), City of Hope, Duarte, CA, USA.
Esophageal carcinoma (EAC) remains one of the most lethal solid cancers with
an overall 5 yr survival of less than 15%. In the last twenty years,
adenocarcinoma of the esophagus has become epidemic in incidence. Factors
increasing the risk of EAC includes obesity, white race, male, reflux disease and
Barrett’s esophagus (BE). While the association with BE with EAC is well
established, understanding the molecular changes involved with progression to
disease remains limited. In addition, there is currently no ability to identify specific
populations who are at highest risk for the development of EAC amongst those
with reflux and BE.
In this paper, Dr. Van de Winkel et.al. argue that a nuclear receptor, the
xenobiotic receptor PXR increases its level of expression from normal
esophageal squamous epithelium to BE to EAC; that ligands for PXR induce
nuclear translocation of the receptor in an EAC cell line; and analyze
polymorphisms in the PXR gene that could be predictive for BE. These findings
provide important evidence suggesting a possbile link between PXR and the
conversion of normal esophageal epithelium to BE, and suggest PXR as a
potential treatment target for EAC. Future studies will be required to extend and
confirm these conclusions.
Major Compulsory Revisions
1. Expression of PXR in BE patients
The RNA expression data in figure 1A are all normalized to Sq=1 for all patients.
This reporting scheme is appropriate to make the point of relative differences in
expression, but masks any significant interindividual differences in PXR
expression that may exist among the population. It would be useful if the authors
included this data as a supplemental figure.
The methods do not clearly explain how protein expression patterns were
analyzed in the human samples. Was the positive staining graded on presence
or absence of protein, on intensity of stain, on percentage of cells? Was there a
separate scoring for cytosolic staining, nuclear staining, and overall staining?
Were the readers blinded to disease and to patient? In the BE patients, what was
the relative staining pattern of the squamous epithelium v. the columnar
epithelium? The authors note that there is a greater percentage of nuclear
staining in the EAC specimens than in the BE specimens. However, there is
cytosolic and nuclear staining as well as differences in intensity of staining in the
BE and the EAC slides (as shown in Figure 2C and 2D) and without a more
refined scoring scale and method of determining cytosolic v. nuclear expression,
it is difficult to conclude as the authors did that “. . .PXR protein expression was
observed predominantly in the cytoplasm of Barrett’s epithelial cells and not in
the nucleus” (page 11/28).
2. PXR ligands, LCA, tauro-LCA and rifampicin, induce nuclear translocation in
esophageal carcinoma cell line
The authors report these experiments in two cell lines. HET1A is representative
of squamous cell epithelial cells and the other OE19 is derived from esophageal
adenocarcinoma cells. There is a difference in expression of PXR between the
two cell lines, confirming a difference of expression of PXR in squamous cells v.
columnar cells as shown in human tissues. However, PXR expression is not
induced in either cell line (Figure 3A). The authors only show the lack of PXR
induction with TLCA. This is of particular concern as tauro-conjugated bile acids
such as TLCA cannot enter cells without specific transporters and its not clear
that these transporters are expressed in these cells. In addition, LCA is often
toxic at the concentrations used and LCA/TLCA also target other nuclear
receptors and TGR5. There is therefore a potential lack of specificity with these
compounds at the doses used. It would be helpful to repeat these experiments
with other PXR ligands and particularly with rifampicin and hyperforin which have
become “standard” ligands for human PXR in the literature. These additional
PXR ligands should be included as key comparators to the literature and given
the nuclear translocation data in Figure 3B. Although PXR ligand TLCA does not
induce expression, TLCA does induce nuclear translocation. Is this also true for
rifampicin and LCA?
Discretionary Revisions
1. Expression of PXR in BE patients
The gene expression of PXR was evaluated using QPCR of specimens from
patients with reflux esophagitis, BE, and EAC. Figure 1A clearly demonstrates
the difference in PXR expression between the squamous cells and the columnar
cells of patients with BE. However, the authors do not demonstrate the
expression of PXR in normal epithelium of healthy patients (Figure 2B). What is
the mRNA expression of PXR in normal squamous cell epithelium in healthy
patients (n = 5, as stated in the methods but not shown in the figure)? In addition,
much as the authors have paired specimens in the BE patients (Sq and CE), the
relative PXR expression changes between normal epithelium and the disease
states (RE, BE, and EAC) in each individual patient would greatly enhance the
conclusion of a progressive change of PXR as disease progresses from normal
to cancer.
2. PXR polymorphism is associated with BE
Although there were clinical characteristics presented (Table 2), no multivariate
analysis was shown for whether 7635AG is an independent predictor of risk of
developing BE. In addition, in a previous publication on this cohort, EAC patients
were included in the evaluation of another SNP. No data is shown on the EAC
patients in this paper. Data from the EAC cohort with respect to the PXR SNPs
will add greatly to this paper with regard to progression of disease and relevance
of the SNP from normal to BE to EAC.
The above data will not only add to the authors’ conclusion but potentially could
change treatment recommendations. Currently, while the 7635 AG SNP may
help to identify patients at risk for development of BE, no information is available
to assist in the determination of who is at risk for EAC in patients with BE.
Without that information, patients with BE are left with serial upper endoscopies
until dyplasia is identified. At that time, a highly morbid esophagectomy may be
offered to prevent EAC. If the 7635AG SNP is predictive of EAC, it may save
patients the procedure if they don’t carry the genetic polymorphism.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
This manuscript was reviewed jointly by Dr. Lily Lai (llai@coh.org) and Dr. Barry
Forman (bmforman@earthlink.net), City of Hope, Duarte, CA, USA. The
reviewers do
not have competing interests.
Please review this paper.
user
Reviewer's report
Title:Expression, localization and polymorphisms of the nuclear receptor PXR in
Barrett's esophagus and esophageal adenocarcinoma
Version: 10Date:5 May 2011
Reviewer: Barry Forman
Reviewer's report:
This manuscript was reviewed jointly by Dr. Lily Lai (llai@coh.org) and Dr. Barry
Forman (bmforman@earthlink.net), City of Hope, Duarte, CA, USA.
Esophageal carcinoma (EAC) remains one of the most lethal solid cancers with
an overall 5 yr survival of less than 15%. In the last twenty years,
adenocarcinoma of the esophagus has become epidemic in incidence. Factors
increasing the risk of EAC includes obesity, white race, male, reflux disease and
Barrett’s esophagus (BE). While the association with BE with EAC is well
established, understanding the molecular changes involved with progression to
disease remains limited. In addition, there is currently no ability to identify specific
populations who are at highest risk for the development of EAC amongst those
with reflux and BE.
In this paper, Dr. Van de Winkel et.al. argue that a nuclear receptor, the
xenobiotic receptor PXR increases its level of expression from normal
esophageal squamous epithelium to BE to EAC; that ligands for PXR induce
nuclear translocation of the receptor in an EAC cell line; and analyze
polymorphisms in the PXR gene that could be predictive for BE. These findings
provide important evidence suggesting a possbile link between PXR and the
conversion of normal esophageal epithelium to BE, and suggest PXR as a
potential treatment target for EAC. Future studies will be required to extend and
confirm these conclusions.
Major Compulsory Revisions
1. Expression of PXR in BE patients
The RNA expression data in figure 1A are all normalized to Sq=1 for all patients.
This reporting scheme is appropriate to make the point of relative differences in
expression, but masks any significant interindividual differences in PXR
expression that may exist among the population. It would be useful if the authors
included this data as a supplemental figure.
The methods do not clearly explain how protein expression patterns were
analyzed in the human samples. Was the positive staining graded on presence
or absence of protein, on intensity of stain, on percentage of cells? Was there a
separate scoring for cytosolic staining, nuclear staining, and overall staining?
Were the readers blinded to disease and to patient? In the BE patients, what was
the relative staining pattern of the squamous epithelium v. the columnar
epithelium? The authors note that there is a greater percentage of nuclear
staining in the EAC specimens than in the BE specimens. However, there is
cytosolic and nuclear staining as well as differences in intensity of staining in the
BE and the EAC slides (as shown in Figure 2C and 2D) and without a more
refined scoring scale and method of determining cytosolic v. nuclear expression,
it is difficult to conclude as the authors did that “. . .PXR protein expression was
observed predominantly in the cytoplasm of Barrett’s epithelial cells and not in
the nucleus” (page 11/28).
2. PXR ligands, LCA, tauro-LCA and rifampicin, induce nuclear translocation in
esophageal carcinoma cell line
The authors report these experiments in two cell lines. HET1A is representative
of squamous cell epithelial cells and the other OE19 is derived from esophageal
adenocarcinoma cells. There is a difference in expression of PXR between the
two cell lines, confirming a difference of expression of PXR in squamous cells v.
columnar cells as shown in human tissues. However, PXR expression is not
induced in either cell line (Figure 3A). The authors only show the lack of PXR
induction with TLCA. This is of particular concern as tauro-conjugated bile acids
such as TLCA cannot enter cells without specific transporters and its not clear
that these transporters are expressed in these cells. In addition, LCA is often
toxic at the concentrations used and LCA/TLCA also target other nuclear
receptors and TGR5. There is therefore a potential lack of specificity with these
compounds at the doses used. It would be helpful to repeat these experiments
with other PXR ligands and particularly with rifampicin and hyperforin which have
become “standard” ligands for human PXR in the literature. These additional
PXR ligands should be included as key comparators to the literature and given
the nuclear translocation data in Figure 3B. Although PXR ligand TLCA does not
induce expression, TLCA does induce nuclear translocation. Is this also true for
rifampicin and LCA?
Discretionary Revisions
1. Expression of PXR in BE patients
The gene expression of PXR was evaluated using QPCR of specimens from
patients with reflux esophagitis, BE, and EAC. Figure 1A clearly demonstrates
the difference in PXR expression between the squamous cells and the columnar
cells of patients with BE. However, the authors do not demonstrate the
expression of PXR in normal epithelium of healthy patients (Figure 2B). What is
the mRNA expression of PXR in normal squamous cell epithelium in healthy
patients (n = 5, as stated in the methods but not shown in the figure)? In addition,
much as the authors have paired specimens in the BE patients (Sq and CE), the
relative PXR expression changes between normal epithelium and the disease
states (RE, BE, and EAC) in each individual patient would greatly enhance the
conclusion of a progressive change of PXR as disease progresses from normal
to cancer.
2. PXR polymorphism is associated with BE
Although there were clinical characteristics presented (Table 2), no multivariate
analysis was shown for whether 7635AG is an independent predictor of risk of
developing BE. In addition, in a previous publication on this cohort, EAC patients
were included in the evaluation of another SNP. No data is shown on the EAC
patients in this paper. Data from the EAC cohort with respect to the PXR SNPs
will add greatly to this paper with regard to progression of disease and relevance
of the SNP from normal to BE to EAC.
The above data will not only add to the authors’ conclusion but potentially could
change treatment recommendations. Currently, while the 7635 AG SNP may
help to identify patients at risk for development of BE, no information is available
to assist in the determination of who is at risk for EAC in patients with BE.
Without that information, patients with BE are left with serial upper endoscopies
until dyplasia is identified. At that time, a highly morbid esophagectomy may be
offered to prevent EAC. If the 7635AG SNP is predictive of EAC, it may save
patients the procedure if they don’t carry the genetic polymorphism.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
This manuscript was reviewed jointly by Dr. Lily Lai (llai@coh.org) and Dr. Barry
Forman (bmforman@earthlink.net), City of Hope, Duarte, CA, USA. The
reviewers do
not have competing interests.
assistant