Response to reviewer comments on “The VEGF -634G>C promoter polymorphism is associated
with risk of gastric cancer” submitted to BMC (MS: 5494242782750212) for publication
Reviewer 1: Maria Gazouli
This reviewer does not have any comments.
Response: none.
Reviewer 2: Xinru Wang
Reviewer's report:
The authors studied correlation between genetic polymorphisms of TGF-ß1 and
VEGF genes and gastric cancer in a case-control study consisting of 171
patients with gastric cancer and 353 cancer-free controls. They found that
VEGF-634G>C SNP may contribute to the risk of developing gastric cancer.
General comments:
The experiment seems well performed and data are clearly exposed. The results
are of interest, although the sample sizes are rather small.
Response: We agree. This study is rather small. This is our initial effort in building up gastric
cancer research at MD Anderson.
However, several points deserve great attention.
Comment 1. In table 3, the reason why the authors have chosen the age 57 years as the cut-off
value is not clear and should be explained.
Response: This is a cutoff value according to the median of the controls. We now expalin this
in the revision as follows: “Age was also dichotomized according to the median age (57 years)
of the controls” (page 6).
Comment 2. The data show that the genotype frequencies of the VEGF -634CG heterozygous
was significantly higher in cases (42.1%) than the controls (28.0%), and the authors got the
conclusion that VEGF-634G>C SNP may be associated with susceptibility to gastric cancer.
Intuitively one would think that the CC genotype may have a stronger association with the risk of
gastric cancer. However, from the table 2, it shows that VEGF -634CC genotype was less
frequent in the cases (17.5%) than in the controls (21.0%). This issue should be discussed.
Response: This was because the common genotype was more frequent in controls (51.0%)
than in cases (40.4%). The final OR estimates were the cross-products of these numbers.
Comment 3. The interesting aspect of the alcohol use effects should be discussed more
intensively, and explain how the genetic effects was more profound in the never drinkers.
Response: This result appears to be common in genotype association studies, in which the risk
associated with adverse genotypes appears to be higher in non-exposed or less exposed
subjects, suggesting the related susceptibility is high in such subgroups. Alternatively, there
may be some unknown risk factors that were not identified in these subgroups. We have
explained this clearly in the revision (page 10).
Reviewer 3: Jing Shen
Reviewer's report:
A hospital-based case-control study was conducted to examine the association between TGFB1
and VEGF genetic polymorphisms and gastric cancer risk. Although the sample size is
moderate (171 patients and 353 controls), one positive result was observed that VEGF 634CG
genotype and the combined CG+CC genotype were associated with a significantly elevated risk
of gastric cancer. The study design is well, and the statistical methods are suitable. The
results were described correctly, and no over-interpretation. These results are valuable for
understanding the susceptibility of gastric cancer in Caucasian although several studies have
been done in Asian population.
Minor Essential Revisions
Comment 1) It is unclear how many gastric cancer cases are histological diagnosed intestinal
or diffuse types in the current study. It is important because different subtypes may have
different susceptibility.
Response: We have added the tumor type information in the revision (page 6 and Table 1)
Comment 2) Providing the information about the false-positive report probability (FPRP) for
the positive findings is necessary.
Response: In response to this comment, we performed the FPRP analysis and found that
under the assumption of a prior probability of 0.1, the FPRP of VEGF -634 CG vs. GG yielded
values of 0.13, suggesting that the VEGF -634 CG vs. GG findings was noteworthy. However,
the FPRPs of VEGF -634 CG+CC vs. GG for all subjects, non-whites, ever smokers and never
drinkers yielded values of 0.31, 0.54, 0.49 and 0.38, respectively, which were likely to be false
positive findings. We have added this information and discussed accordingly (pages 6 and 8).
Comment 3) More discussions are also needed to evaluate the potential influence of the
SNP that is not in agreement with the Hardy–Weinberg equilibrium. Whether it may bias the
result?
Response: We agree that departure from the Hardy–Weinberg equilibrium may cause bias.
However, we had checked the assays results carefully and did not see any irregularities in the
results. We believe at lease the results may not be biased by the genotyping assay. It may be
possible that the study population was selected due to the nature of a hospital-based study.
Therefore, we had called for validation or replication by larger and better designed studies in
then future.
Comment 4) What is the advantage of the current study or the feature that are different
from other previous studies in gastric cancer?
Response: As we have discussed in the introduction, this is a unique US study population, in
which the gastric cancer is relatively uncommon, compared with that in Asian coutries.
Comment 5) As mentioned by the authors that an obvious limitation of the current study
was the lack of information on the H. pylori infection status of the subjects, more
evidence that the observed positive results may be unlikely to be impacted by the
relatively lower H. pylori infectious rare in US population will strengthen the
discussion.
Response: Unfortunately, as we mentioned in the manuscript, the H. pylori infection was not
routinely tested at MD Anderson. This study, if published, will call upon such a need in the clinic
for future studies.
Reviewer 4: Annamaria Ruzzo
Reviewer's report:
The manuscript by Guan et al reports that exist an association between gastric cancer and
VEGF -634 G/C polymorphism.
There are some important points unclear:
Comment 1. Gastric cancer consists of two pathological variants, intestinal and diffuse: how
many patients in this study are intestinal? how many diffuse?
Response: we have added this information in Table of the revision. There were about 12%
patients who had diffuse type of tumors (page 6 and Table 1).
Comment 2. The association of Helicobacter Pylori(HP) and gastric cancer is well established
and Authors underlay this statement under “introduction”, but at the same time they don’t
mention if samples are or not infected by HP. HP is more diffuse in Asians than Caucasians, but
this is not a good reason for not reporting this information among variables.
Response: Unfortunately, as we mentioned in the manuscript, the H. pylori infection was not
routinely tested at MD Anderson. This study, if published, will call upon such a need in the clinic
for future studies.
Comment 3. What the author mean with Non-Hispanic white? The term Caucasian race (also
Caucasoid or Europid) has been used to denote the general physical type of some or all of the
indigenous populations of Europe, North Africa, the Horn of Africa, West Asia, Central Asia and
South Asia (The Races of Europe by Carlton Stevens Coon. From Chapter XI: ).
Response: Because Houston area has many Hispanic or Mexican Americans. To distinguish
the difference between Hispanic and European decendence, “Non-Hispanic white” was
recommended for use in our previous manuscripts.
Comment 4. Authors report under “TGFB1 and VEGF genotypes and risk of gastric cancer” that
the SNPs were all in agreement with H-W equilibrium except for TGFB1 -509 C/T and VEGF 634 G/C. The VEGF -634 G/C is the only SNP associated with gastric cancer. In my opinion the
H-W disequilibrium for VEGF -634 can influence the results reported and address researchers
to wrong conclusions.
Response: We agree that there was such a possibility. However, we had checked the assays
results carefully and did not see any irregularities in the results. We believe at lease the results
may not be biased by the genotyping assay. It may be possible that the study population was
selected due to the nature of a hospital-based study. Therefore, we had called for validation or
replication by larger and better designed studies in then future.
Reviewer 5: Stefania Boccia
Reviewer's report:
The paper of Guan et al. reports the results of a hospital-based case- control study conducted in
US aimed to investigate the association between selected SNP in TGFB1 and VEGF genes and
risk of gastric cancer. Results suggest that subjects heterozygous for the -634 CG genotype of
VEGF gene have an increased risk for gastric cancer. Haplotype analysis and stratified
analyses did not show significant associations.
Even though the study is methodologically well conducted, authors acknowledge the fact that
the genotype data of VEGF-634 among controls do not respect the Hardy-Weinberg equilibrium.
This observation strongly affects their results, as the p-value for the chi-square test (not
reported in the results) is 5 x10 -13, which is particularly high. Selection bias of the control is not
likely to explain such a large deviation of HWE, as authors claim. The most common reason for
the violation of the equilibrium is a genotyping error, and even though authors acknowledge that
for 10% of samples the assay was repeated with 100% concordance, this cannot exclude that a
genotyping error might affect the validity of the results of the entire study. Additionally, if they
exclude that a laboratory error happened, they should have stratify the VEGF -634 CG results
according to ethnicity (e.g., by excluding non-white), as the second most probable reason for
violation of the HWE is population stratification. As such, I find hard to accept the results of the
manuscript in view of the aforementioned observations.
Response: We agree that there was such a possibility. However, we had checked the assays
results carefully and did not see any irregularities in the results. We believe at lease the results
may not be biased by the genotyping assay. It may be possible that the study population was
selected due to the nature of a hospital-based study. Therefore, we had called for validation or
replication by larger and better designed studies in then future.
As a matter of fact, we did stratify the data by ethnicity in Table 3, and only non-whites had high
risk associated with the advers genotypes.