Title: Epidermal Growth Factor Receptor (EGFR) and SMAD4 negatively correlated in the
Progression of Gallbladder Adenocarcinoma in Eastern Indian Patients
Version: 4 Date: 15 Aug 2024
Reviewer:  Pramod Kumar Tiwari
Reviewer's report:
In view of the higher incidence of GBC in Eastern Indian population, the authors of the
manuscript have made a detailed analysis on the genetic risk factors, associated with GBC,
including genes, like CCND1, MYC, EGFR, ERBB2/Her2-neu, CDKN2A, and SMAD4,
mutations in KRAS, amplification of ERBB2/Her2-neu gene, significance of certain SNPs of
ERBB2/Her2-neu and CCND1 gene and expression of TGF-beta. They observed EGFR and
SMAD4 expression to be negatively correlated in GBC.  About 30% of the GBC samples showed
overexpression/amplification of ERBB2. The expression of TGF- # was found significantly
downregulated in the studied population. Also, they observed a very low frequency of KRAS
codon 12 mutation in Indian GBC patients. Although the present study has revalidated
certain investigations made in other world populations earlier, apart from some of their new
observations made in the patient population, the study is significant in view of the high incidence
of GBC in this region of India and the reports of variations reported in the genetic regulation of
occurrence of GBC in different geographical and ethnic populations. However, there are certain
comments for the authors, mentioned below, for their careful response.
1. The English language and grammar need careful revision.
2. Methods: The details of setting of all PCR, RT-PCR reactions (as described in the text),
including primers, may be better given in a tabular form to have quick overview by anyone who
wishes to repeat the same. Such technical details in text format are often cumbersome. This can
also reduce the text size to some extent.
3. Sanger sequencing. The methodological details may be given as supplementary file.
4. Results: Page 12, second para, lines 5-10, Page 13, para1 &2. How many GBC samples
showed simultaneous overexpression and downregulation of particular genes, analysed? That is,
how many showed 2, 3 or 4 genes simultaneously over expressed and/with down regulation of
CDKN2A and SMAD4 together or independently and in combination? This is to determine the
relative roles of those genes in GBC. May be presented in a tabular form. It is concerned more
with their expression pattern (might be synergistic or antagonistic or else?) in individual sample,
may be useful in personalized diagnosis/therapy, in future. Only percentage gives an arbitrary
picture. If any correlation appears between expression pattern and gender/sex or tumour stage?
5. Discussion: Some overlapping statements between introduction and discussion may be
omitted to reduce the text length. For example, the second para in discussion section.
Title: Epidermal Growth Factor Receptor (EGFR) and SMAD4 negatively correlated in the
Progression of Gallbladder Adenocarcinoma in Eastern Indian Patients
Version: 4 Date: 15 Aug 2024
Reviewer:  Pramod Kumar Tiwari
Reviewer's report:
In view of the higher incidence of GBC in Eastern Indian population, the authors of the
manuscript have made a detailed analysis on the genetic risk factors, associated with GBC,
including genes, like CCND1, MYC, EGFR, ERBB2/Her2-neu, CDKN2A, and SMAD4,
mutations in KRAS, amplification of ERBB2/Her2-neu gene, significance of certain SNPs of
ERBB2/Her2-neu and CCND1 gene and expression of TGF-beta. They observed EGFR and
SMAD4 expression to be negatively correlated in GBC.  About 30% of the GBC samples showed
overexpression/amplification of ERBB2. The expression of TGF- # was found significantly
downregulated in the studied population. Also, they observed a very low frequency of KRAS
codon 12 mutation in Indian GBC patients. Although the present study has revalidated
certain investigations made in other world populations earlier, apart from some of their new
observations made in the patient population, the study is significant in view of the high incidence
of GBC in this region of India and the reports of variations reported in the genetic regulation of
occurrence of GBC in different geographical and ethnic populations. However, there are certain
comments for the authors, mentioned below, for their careful response.
1. The English language and grammar need careful revision.
2. Methods: The details of setting of all PCR, RT-PCR reactions (as described in the text),
including primers, may be better given in a tabular form to have quick overview by anyone who
wishes to repeat the same. Such technical details in text format are often cumbersome. This can
also reduce the text size to some extent.
3. Sanger sequencing. The methodological details may be given as supplementary file.
4. Results: Page 12, second para, lines 5-10, Page 13, para1 &2. How many GBC samples
showed simultaneous overexpression and downregulation of particular genes, analysed? That is,
how many showed 2, 3 or 4 genes simultaneously over expressed and/with down regulation of
CDKN2A and SMAD4 together or independently and in combination? This is to determine the
relative roles of those genes in GBC. May be presented in a tabular form. It is concerned more
with their expression pattern (might be synergistic or antagonistic or else?) in individual sample,
may be useful in personalized diagnosis/therapy, in future. Only percentage gives an arbitrary
picture. If any correlation appears between expression pattern and gender/sex or tumour stage?
5. Discussion: Some overlapping statements between introduction and discussion may be
omitted to reduce the text length. For example, the second para in discussion section.
Please review this paper.
user
Title: Epidermal Growth Factor Receptor (EGFR) and SMAD4 negatively correlated in the
Progression of Gallbladder Adenocarcinoma in Eastern Indian Patients
Version: 4 Date: 15 Aug 2024
Reviewer:  Pramod Kumar Tiwari
Reviewer's report:
In view of the higher incidence of GBC in Eastern Indian population, the authors of the
manuscript have made a detailed analysis on the genetic risk factors, associated with GBC,
including genes, like CCND1, MYC, EGFR, ERBB2/Her2-neu, CDKN2A, and SMAD4,
mutations in KRAS, amplification of ERBB2/Her2-neu gene, significance of certain SNPs of
ERBB2/Her2-neu and CCND1 gene and expression of TGF-beta. They observed EGFR and
SMAD4 expression to be negatively correlated in GBC.  About 30% of the GBC samples showed
overexpression/amplification of ERBB2. The expression of TGF- # was found significantly
downregulated in the studied population. Also, they observed a very low frequency of KRAS
codon 12 mutation in Indian GBC patients. Although the present study has revalidated
certain investigations made in other world populations earlier, apart from some of their new
observations made in the patient population, the study is significant in view of the high incidence
of GBC in this region of India and the reports of variations reported in the genetic regulation of
occurrence of GBC in different geographical and ethnic populations. However, there are certain
comments for the authors, mentioned below, for their careful response.
1. The English language and grammar need careful revision.
2. Methods: The details of setting of all PCR, RT-PCR reactions (as described in the text),
including primers, may be better given in a tabular form to have quick overview by anyone who
wishes to repeat the same. Such technical details in text format are often cumbersome. This can
also reduce the text size to some extent.
3. Sanger sequencing. The methodological details may be given as supplementary file.
4. Results: Page 12, second para, lines 5-10, Page 13, para1 &2. How many GBC samples
showed simultaneous overexpression and downregulation of particular genes, analysed? That is,
how many showed 2, 3 or 4 genes simultaneously over expressed and/with down regulation of
CDKN2A and SMAD4 together or independently and in combination? This is to determine the
relative roles of those genes in GBC. May be presented in a tabular form. It is concerned more
with their expression pattern (might be synergistic or antagonistic or else?) in individual sample,
may be useful in personalized diagnosis/therapy, in future. Only percentage gives an arbitrary
picture. If any correlation appears between expression pattern and gender/sex or tumour stage?
5. Discussion: Some overlapping statements between introduction and discussion may be
omitted to reduce the text length. For example, the second para in discussion section.
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