Reviewer's report
Title:Heterogeneity of Amplification of HER2, EGFR, CCND1 and MYC in
Gastric Cancer
Version:2Date:5 November 2014
Reviewer: Luca Mastracci
Reviewer's report:
This manuscript focuses on tumor heterogeneity in FISH amplification of four
different genes, which play a role in establishing prognosis or predicting
response to treatment.
The manuscript is well written, easily understandable and with an important
message which has not yet been adequately studied or published.
For these reason I recommend publication with revisions.
Major Revisions:
1) Abstract section, first paragraph.
The following sentence: “intra-tumor heterogeneity is a potential cause of failure
of targeted therapy in gastric cancer, but the extent of heterogeneity…..had not
been systematically studied” . This sentence is not clear; the manuscript refers
mainly to heterogeneity in gene amplification studied by FISH; also the
immunohistochemical heterogeneity for HER2 in gastric cancer is considered as
part of the work. This aspect has been previously and extensively studied by
other authors and heterogeneity of immunohistochemical expression of HER2 in
GC is well known. This aspect renders the sentence unclear; it would be better to
specify in the text that the extent of gene amplification by FISH has not been
systematically studied. Moreover, the problem is not limited only to the potential
failure of targeted therapy but also may lead to the exclusion of patients who
could benefit from targeted therapy. This last point is as important as the first
argument and merits separate mention (both in the abstract if enough space is
available and, most of all, in the introduction section).
2) Abstract section, last paragraph.
Last sentence: “ …. And may potentially limit treatment decisions based on the
analysis of a single cancer biopsy”. The underlined part of this sentence has to
be modified for several reasons:
a) The term “biopsy” is not clear in this context: if it refers to tissue core used to
construct TMA, it is preferable to use the term “single tissue core” rather than the
term biopsy to avoid confusion with endoscopic /surgical biopsy that often, in
advanced GC, represents the only available material for FISH analysis.
b) if the term biopsy however applies to endoscopic or surgical biopsy, then it is
necessary to underline that results derived from TMAs do not necessarily apply
to biopsy scenarios. In this case, the authors should modify their conclusions.
3) Introduction section, last paragraph.
The last sentence: “The results of our study demonstrate a variable but generally
high degree of heterogeneity for all four analyzed genes” should be avoided. It is
a conclusion and there is no reason to anticipate it in the introduction.
4) Discussion section, end of the first paragraph.
The last sentence “this clearly limits the potential of treatment decisions based on
the amplification status determined on one single cancer biopsy”. See comments
reported previously (point 2).
5) Conclusions section.
Also in this conclusion the sentence concerning the single cancer biopsy should
be modified. See comments reported previously (point 2).
6) The message of the study is clear and of translational importance (mainly for
the product of those genes which are currently studied as target of tailored
therapies). I think that it is important that authors try to transfer the results they
have found on TMA in a useful and practical message for routine practice.
Statistical analysis (ie Poisson regression model to establish the minimum
number of tissue cores able to predict amplification status for different examined
genes) was not performed. Why? Which is the suggestion that authors can give
in order to solve the problem of cancer heterogeneity (in a practical way a
sentence more precise than “a single cancer biopsy is not enough”).
Excluding clinical trials in which tissue is centralized to one center for TMA
construction, in routine practice, TMAs are not a common facility used to study
prognostic/predictive parameters.
Furthermore guidelines suggest the use of multiple endoscopic GC biopsies
(usually from 5 to 10) for evaluation of predictive parameters such as HER2.
Cancer heterogeneity demonstrated by this study is for sure of great importance
in selecting possible candidates for tailored therapy. A comment on this aspect is
than required.
A sentence on possible limitations of this work is missing. In particular it seems
important that authors underline that TMA are not interchangeable with biopsies
(mainly endoscopic). In this study no mention is made on the exact position in
which the sampling from donor block was performed for TMA construction. I
guess that authors sampled randomly, selecting from both superficial and deep
areas of GC. In advanced GC only endoscopic biopsies are often available, but
these are representative only of the luminal part of the cancer.
Discretionary Revision
The tables (1-3-4-5) are easily readable and easily understandable. I presume
that authors gave a number to each case in an arbitrary way. In fact, only the first
50 cases (on 109 examined) have amplification of 1 or more genes. The only
difficulty in reading the tables is that cases are not consecutive. Colors
(independently of stratification of cases on the basis of amplification or not)
clearly identify homogeneous and heterogeneous cases, so probably the use of
consecutiveness in numeration should render cases described in the text more
easily detectable.
Minor essential revision:
Table 5: Tumor n° 2 is in “bold”.
Table 6: tumor n° 15 (first line) is unformatted respect to other numbers.
Table 6: Tumor n° 7 (first line, HER 2) is missing.
Table 5: tumor n° 42; 4/9 is in green (but it should be in orange since it’s a low
amplification following the definition in material and methods where high and low
amplification is based on more or less than 10 copy of genes)
Table 5: tumor n° 44; 3/8 is in green but it’s a low amplification (in case number
35 in fact is in orange colour).
Table 5: tumor n° 9; 3/7 is a low amplification (it’s in blue color and not in orange)
Supplementary table 2: correct “amplification” instead of “amplifcation”
Supplementary table 2: “*” means?? (not referred in the notes). Why it’s not
present also for EGFR?
Supplementary Table 1: “* amp. vs no amp.” in the footnote. Please specify
better without using abbreviation.
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests
Reviewer's report
Title:Heterogeneity of Amplification of HER2, EGFR, CCND1 and MYC in
Gastric Cancer
Version:2Date:5 November 2014
Reviewer: Luca Mastracci
Reviewer's report:
This manuscript focuses on tumor heterogeneity in FISH amplification of four
different genes, which play a role in establishing prognosis or predicting
response to treatment.
The manuscript is well written, easily understandable and with an important
message which has not yet been adequately studied or published.
For these reason I recommend publication with revisions.
Major Revisions:
1) Abstract section, first paragraph.
The following sentence: “intra-tumor heterogeneity is a potential cause of failure
of targeted therapy in gastric cancer, but the extent of heterogeneity…..had not
been systematically studied” . This sentence is not clear; the manuscript refers
mainly to heterogeneity in gene amplification studied by FISH; also the
immunohistochemical heterogeneity for HER2 in gastric cancer is considered as
part of the work. This aspect has been previously and extensively studied by
other authors and heterogeneity of immunohistochemical expression of HER2 in
GC is well known. This aspect renders the sentence unclear; it would be better to
specify in the text that the extent of gene amplification by FISH has not been
systematically studied. Moreover, the problem is not limited only to the potential
failure of targeted therapy but also may lead to the exclusion of patients who
could benefit from targeted therapy. This last point is as important as the first
argument and merits separate mention (both in the abstract if enough space is
available and, most of all, in the introduction section).
2) Abstract section, last paragraph.
Last sentence: “ …. And may potentially limit treatment decisions based on the
analysis of a single cancer biopsy”. The underlined part of this sentence has to
be modified for several reasons:
a) The term “biopsy” is not clear in this context: if it refers to tissue core used to
construct TMA, it is preferable to use the term “single tissue core” rather than the
term biopsy to avoid confusion with endoscopic /surgical biopsy that often, in
advanced GC, represents the only available material for FISH analysis.
b) if the term biopsy however applies to endoscopic or surgical biopsy, then it is
necessary to underline that results derived from TMAs do not necessarily apply
to biopsy scenarios. In this case, the authors should modify their conclusions.
3) Introduction section, last paragraph.
The last sentence: “The results of our study demonstrate a variable but generally
high degree of heterogeneity for all four analyzed genes” should be avoided. It is
a conclusion and there is no reason to anticipate it in the introduction.
4) Discussion section, end of the first paragraph.
The last sentence “this clearly limits the potential of treatment decisions based on
the amplification status determined on one single cancer biopsy”. See comments
reported previously (point 2).
5) Conclusions section.
Also in this conclusion the sentence concerning the single cancer biopsy should
be modified. See comments reported previously (point 2).
6) The message of the study is clear and of translational importance (mainly for
the product of those genes which are currently studied as target of tailored
therapies). I think that it is important that authors try to transfer the results they
have found on TMA in a useful and practical message for routine practice.
Statistical analysis (ie Poisson regression model to establish the minimum
number of tissue cores able to predict amplification status for different examined
genes) was not performed. Why? Which is the suggestion that authors can give
in order to solve the problem of cancer heterogeneity (in a practical way a
sentence more precise than “a single cancer biopsy is not enough”).
Excluding clinical trials in which tissue is centralized to one center for TMA
construction, in routine practice, TMAs are not a common facility used to study
prognostic/predictive parameters.
Furthermore guidelines suggest the use of multiple endoscopic GC biopsies
(usually from 5 to 10) for evaluation of predictive parameters such as HER2.
Cancer heterogeneity demonstrated by this study is for sure of great importance
in selecting possible candidates for tailored therapy. A comment on this aspect is
than required.
A sentence on possible limitations of this work is missing. In particular it seems
important that authors underline that TMA are not interchangeable with biopsies
(mainly endoscopic). In this study no mention is made on the exact position in
which the sampling from donor block was performed for TMA construction. I
guess that authors sampled randomly, selecting from both superficial and deep
areas of GC. In advanced GC only endoscopic biopsies are often available, but
these are representative only of the luminal part of the cancer.
Discretionary Revision
The tables (1-3-4-5) are easily readable and easily understandable. I presume
that authors gave a number to each case in an arbitrary way. In fact, only the first
50 cases (on 109 examined) have amplification of 1 or more genes. The only
difficulty in reading the tables is that cases are not consecutive. Colors
(independently of stratification of cases on the basis of amplification or not)
clearly identify homogeneous and heterogeneous cases, so probably the use of
consecutiveness in numeration should render cases described in the text more
easily detectable.
Minor essential revision:
Table 5: Tumor n° 2 is in “bold”.
Table 6: tumor n° 15 (first line) is unformatted respect to other numbers.
Table 6: Tumor n° 7 (first line, HER 2) is missing.
Table 5: tumor n° 42; 4/9 is in green (but it should be in orange since it’s a low
amplification following the definition in material and methods where high and low
amplification is based on more or less than 10 copy of genes)
Table 5: tumor n° 44; 3/8 is in green but it’s a low amplification (in case number
35 in fact is in orange colour).
Table 5: tumor n° 9; 3/7 is a low amplification (it’s in blue color and not in orange)
Supplementary table 2: correct “amplification” instead of “amplifcation”
Supplementary table 2: “*” means?? (not referred in the notes). Why it’s not
present also for EGFR?
Supplementary Table 1: “* amp. vs no amp.” in the footnote. Please specify
better without using abbreviation.
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests
Please review this paper.
user
Reviewer's report
Title:Heterogeneity of Amplification of HER2, EGFR, CCND1 and MYC in
Gastric Cancer
Version:2Date:5 November 2014
Reviewer: Luca Mastracci
Reviewer's report:
This manuscript focuses on tumor heterogeneity in FISH amplification of four
different genes, which play a role in establishing prognosis or predicting
response to treatment.
The manuscript is well written, easily understandable and with an important
message which has not yet been adequately studied or published.
For these reason I recommend publication with revisions.
Major Revisions:
1) Abstract section, first paragraph.
The following sentence: “intra-tumor heterogeneity is a potential cause of failure
of targeted therapy in gastric cancer, but the extent of heterogeneity…..had not
been systematically studied” . This sentence is not clear; the manuscript refers
mainly to heterogeneity in gene amplification studied by FISH; also the
immunohistochemical heterogeneity for HER2 in gastric cancer is considered as
part of the work. This aspect has been previously and extensively studied by
other authors and heterogeneity of immunohistochemical expression of HER2 in
GC is well known. This aspect renders the sentence unclear; it would be better to
specify in the text that the extent of gene amplification by FISH has not been
systematically studied. Moreover, the problem is not limited only to the potential
failure of targeted therapy but also may lead to the exclusion of patients who
could benefit from targeted therapy. This last point is as important as the first
argument and merits separate mention (both in the abstract if enough space is
available and, most of all, in the introduction section).
2) Abstract section, last paragraph.
Last sentence: “ …. And may potentially limit treatment decisions based on the
analysis of a single cancer biopsy”. The underlined part of this sentence has to
be modified for several reasons:
a) The term “biopsy” is not clear in this context: if it refers to tissue core used to
construct TMA, it is preferable to use the term “single tissue core” rather than the
term biopsy to avoid confusion with endoscopic /surgical biopsy that often, in
advanced GC, represents the only available material for FISH analysis.
b) if the term biopsy however applies to endoscopic or surgical biopsy, then it is
necessary to underline that results derived from TMAs do not necessarily apply
to biopsy scenarios. In this case, the authors should modify their conclusions.
3) Introduction section, last paragraph.
The last sentence: “The results of our study demonstrate a variable but generally
high degree of heterogeneity for all four analyzed genes” should be avoided. It is
a conclusion and there is no reason to anticipate it in the introduction.
4) Discussion section, end of the first paragraph.
The last sentence “this clearly limits the potential of treatment decisions based on
the amplification status determined on one single cancer biopsy”. See comments
reported previously (point 2).
5) Conclusions section.
Also in this conclusion the sentence concerning the single cancer biopsy should
be modified. See comments reported previously (point 2).
6) The message of the study is clear and of translational importance (mainly for
the product of those genes which are currently studied as target of tailored
therapies). I think that it is important that authors try to transfer the results they
have found on TMA in a useful and practical message for routine practice.
Statistical analysis (ie Poisson regression model to establish the minimum
number of tissue cores able to predict amplification status for different examined
genes) was not performed. Why? Which is the suggestion that authors can give
in order to solve the problem of cancer heterogeneity (in a practical way a
sentence more precise than “a single cancer biopsy is not enough”).
Excluding clinical trials in which tissue is centralized to one center for TMA
construction, in routine practice, TMAs are not a common facility used to study
prognostic/predictive parameters.
Furthermore guidelines suggest the use of multiple endoscopic GC biopsies
(usually from 5 to 10) for evaluation of predictive parameters such as HER2.
Cancer heterogeneity demonstrated by this study is for sure of great importance
in selecting possible candidates for tailored therapy. A comment on this aspect is
than required.
A sentence on possible limitations of this work is missing. In particular it seems
important that authors underline that TMA are not interchangeable with biopsies
(mainly endoscopic). In this study no mention is made on the exact position in
which the sampling from donor block was performed for TMA construction. I
guess that authors sampled randomly, selecting from both superficial and deep
areas of GC. In advanced GC only endoscopic biopsies are often available, but
these are representative only of the luminal part of the cancer.
Discretionary Revision
The tables (1-3-4-5) are easily readable and easily understandable. I presume
that authors gave a number to each case in an arbitrary way. In fact, only the first
50 cases (on 109 examined) have amplification of 1 or more genes. The only
difficulty in reading the tables is that cases are not consecutive. Colors
(independently of stratification of cases on the basis of amplification or not)
clearly identify homogeneous and heterogeneous cases, so probably the use of
consecutiveness in numeration should render cases described in the text more
easily detectable.
Minor essential revision:
Table 5: Tumor n° 2 is in “bold”.
Table 6: tumor n° 15 (first line) is unformatted respect to other numbers.
Table 6: Tumor n° 7 (first line, HER 2) is missing.
Table 5: tumor n° 42; 4/9 is in green (but it should be in orange since it’s a low
amplification following the definition in material and methods where high and low
amplification is based on more or less than 10 copy of genes)
Table 5: tumor n° 44; 3/8 is in green but it’s a low amplification (in case number
35 in fact is in orange colour).
Table 5: tumor n° 9; 3/7 is a low amplification (it’s in blue color and not in orange)
Supplementary table 2: correct “amplification” instead of “amplifcation”
Supplementary table 2: “*” means?? (not referred in the notes). Why it’s not
present also for EGFR?
Supplementary Table 1: “* amp. vs no amp.” in the footnote. Please specify
better without using abbreviation.
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests
assistant