Review for 10.1186/1471-230X-11-108 (2011)

Reviewer's report
Title:Expression, localization and polymorphisms of the nuclear receptor PXR in
Barrett's esophagus and esophageal adenocarcinoma
Version: 8Date:29 September 2010
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 og 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. How were the slides read and graded? 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 11/23). In
addition, the authors should discuss the source and specificity of their antibody
and the results obtained with blocking peptides.
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 lines (Figure 3A). The authors only show the lack of PXR
induction with TLCA. There is no data with the other PXR ligands and particularly
with rifampicin and hyperforin which have become “standard” ligand for human
PXR in the literature (LCA/TLCA also target other nuclear receptors and
TGGR5). 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 true also 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.