Reviewer’s report
Title: 
Tissue Microarrey: A potential cost-effective approach for mismatch
repair testing in colorectal cancer
Version: 
0 
Date: 
31 Mar 2022
Reviewer's report:
Thank you very much for this work. 
This is an interesting study and very timely
as the role of MMR IHC continues to expand, including reflexive staining for
colorectal and other tumours.
There are several issues with the study which would need to be addressed before
the work is suitable for publication.
The work starts with a bias in selection - as the TMA was guaranteed to have
reasonable IHC staining. 
Only cases with IHC staining were selected for the
TMA. 
This, in part, explains the higher sensitivity 
/ specificity of this work
compared to previous work. 
The need for larger and multiple cores is addressed in the discussion. 
The need
for normal tissue is questionable and should be addressed, including the impact
of not having the normal cores on the interpretation of the tumour MMR IHC
should be discussed.
The exclusion of PMS2 staining from over-fixation is questionable. 
In the study
337 cores were excluded because they showed lack of PMS2 nuclear staining.
I'm assuming that this lack of nuclear staining was in tumour and normal tissue.
There is variability in the staining quality in large tissue sections and this
exclusion speaks to the overall use of TMAs to assess MMR IHC in colorectal
cancer. 
More explanation around this exclusion and its impact is needed,
including some reference to the explanation that this is due to over fixation.
While the image analysis of the sections is interesting, the results are
disappointing with a lower sensitivity than hoped. 
Given the ability of deep
learning to identify dMMR / MSI tumours based on H&E, it would be hoped
that an algorithm could detect abnormal MMR-IHC with a higher degree of
accuracy.
The workflow is hard to visualize in practice. 
There is evidence to suggest that
MMR IHC should be done on biopsy samples. 
While the work flow may suit
identification of potential CRC associated with Lynch Syndrome, MMR status is
also used to determine treatment. 
The delays involved in the creation of a TMA,
accruing sufficient cases and then having the results reviewed (centrally),
reported and incorporated back with the original case may not occur in a timely
manner. 
Further logistic reviews and economic analysis would be required
before being able to consider the proposed workflow.
Minor Points -
In the materials and methods section, the reference to the research ethics could
be shortened. 
The line "Institutional ethical approval was granted for the
performance of this study" was repeated with different sites / IDs.
There are many spelling mistakes and typographic issues which need to be
addressed. 
The section - TMA accuracy measurements of IHC for MMR proteins
is missing a key word - "If at least of these proteins does not show nuclear
staining of tumor cells, the test is considered pathologic and the tumor potentially
dMMR." 
The spelling mistakes are especially evident in the discussion section.
In the 1st 2 paragraphs of page 13 alone there are at least 6 spelling mistakes
("evalute", "heterogenity", :decreaase", "suggeste", "comparisson", "fasion"). 
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons after the final decision on the manuscript has been made. If you have
already registered at Publons, they will notify you of the receipt of this review
and update your profile 
as per your settings and their policy. If you are not
registered with Publons, you will receive an email from them asking you to
register in order for them to be able to recognize your review on your new
profile page. Publons may use the Review Data to generate derivative metadata
for the benefit of Publons and you as a reviewer, carefully considering the
sensitivity of such information. For example, Publons may verify your record as
a reviewer by updating your profile published on its webservice if you have
registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
https://publons.com/about/terms
Yes
Declaration of competing interests
Please complete a declaration of competing
interests, considering the following questions:
1
. 
Have you in the past five years received reimbursements, fees, funding, or
salary from an organisation that may in any way gain or lose financially
from the publication of this manuscript, either now or in the future?
2
. 
Do you hold any stocks or shares in an organisation that may in any way
gain or lose financially from the publication of this manuscript, either now
or in the future?
3
. 
Do you hold or are you currently applying for any patents relating to the
content of the manuscript?
4
. 
Have you received reimbursements, fees, funding, or salary from an
organization that holds or has applied for patents relating to the content of
the manuscript?
5
. 
Do you have any other financial competing interests?
6
. 
Do you have any non-financial competing interests in relation to this
paper?
If you can answer no to all of the above, write 'I declare that I have no competing
interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests
Reviewer Publication Consent.
 I agree for my report to be made available
under an Open Access Creative Commons CC-BY License (
http://creativecommons.org/licenses/by/4.0
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the
editorial office before completing the review.
If you wish, you can include your
name in your published report. Please note you must decide whether to include
your name at the start of the process and confirm this decision whenever
prompted. If you change your decision later, we will not be able to publish your
name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
Yes
Are the methods sufficiently described to allow the study to be repeated?
Yes
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
Yes
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
Reviewer’s report
Title: 
Tissue Microarrey: A potential cost-effective approach for mismatch
repair testing in colorectal cancer
Version: 
0 
Date: 
31 Mar 2022
Reviewer's report:
Thank you very much for this work. 
This is an interesting study and very timely
as the role of MMR IHC continues to expand, including reflexive staining for
colorectal and other tumours.
There are several issues with the study which would need to be addressed before
the work is suitable for publication.
The work starts with a bias in selection - as the TMA was guaranteed to have
reasonable IHC staining. 
Only cases with IHC staining were selected for the
TMA. 
This, in part, explains the higher sensitivity 
/ specificity of this work
compared to previous work. 
The need for larger and multiple cores is addressed in the discussion. 
The need
for normal tissue is questionable and should be addressed, including the impact
of not having the normal cores on the interpretation of the tumour MMR IHC
should be discussed.
The exclusion of PMS2 staining from over-fixation is questionable. 
In the study
337 cores were excluded because they showed lack of PMS2 nuclear staining.
I'm assuming that this lack of nuclear staining was in tumour and normal tissue.
There is variability in the staining quality in large tissue sections and this
exclusion speaks to the overall use of TMAs to assess MMR IHC in colorectal
cancer. 
More explanation around this exclusion and its impact is needed,
including some reference to the explanation that this is due to over fixation.
While the image analysis of the sections is interesting, the results are
disappointing with a lower sensitivity than hoped. 
Given the ability of deep
learning to identify dMMR / MSI tumours based on H&E, it would be hoped
that an algorithm could detect abnormal MMR-IHC with a higher degree of
accuracy.
The workflow is hard to visualize in practice. 
There is evidence to suggest that
MMR IHC should be done on biopsy samples. 
While the work flow may suit
identification of potential CRC associated with Lynch Syndrome, MMR status is
also used to determine treatment. 
The delays involved in the creation of a TMA,
accruing sufficient cases and then having the results reviewed (centrally),
reported and incorporated back with the original case may not occur in a timely
manner. 
Further logistic reviews and economic analysis would be required
before being able to consider the proposed workflow.
Minor Points -
In the materials and methods section, the reference to the research ethics could
be shortened. 
The line "Institutional ethical approval was granted for the
performance of this study" was repeated with different sites / IDs.
There are many spelling mistakes and typographic issues which need to be
addressed. 
The section - TMA accuracy measurements of IHC for MMR proteins
is missing a key word - "If at least of these proteins does not show nuclear
staining of tumor cells, the test is considered pathologic and the tumor potentially
dMMR." 
The spelling mistakes are especially evident in the discussion section.
In the 1st 2 paragraphs of page 13 alone there are at least 6 spelling mistakes
("evalute", "heterogenity", :decreaase", "suggeste", "comparisson", "fasion"). 
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons after the final decision on the manuscript has been made. If you have
already registered at Publons, they will notify you of the receipt of this review
and update your profile 
as per your settings and their policy. If you are not
registered with Publons, you will receive an email from them asking you to
register in order for them to be able to recognize your review on your new
profile page. Publons may use the Review Data to generate derivative metadata
for the benefit of Publons and you as a reviewer, carefully considering the
sensitivity of such information. For example, Publons may verify your record as
a reviewer by updating your profile published on its webservice if you have
registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
https://publons.com/about/terms
Yes
Declaration of competing interests
Please complete a declaration of competing
interests, considering the following questions:
1
. 
Have you in the past five years received reimbursements, fees, funding, or
salary from an organisation that may in any way gain or lose financially
from the publication of this manuscript, either now or in the future?
2
. 
Do you hold any stocks or shares in an organisation that may in any way
gain or lose financially from the publication of this manuscript, either now
or in the future?
3
. 
Do you hold or are you currently applying for any patents relating to the
content of the manuscript?
4
. 
Have you received reimbursements, fees, funding, or salary from an
organization that holds or has applied for patents relating to the content of
the manuscript?
5
. 
Do you have any other financial competing interests?
6
. 
Do you have any non-financial competing interests in relation to this
paper?
If you can answer no to all of the above, write 'I declare that I have no competing
interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests
Reviewer Publication Consent.
 I agree for my report to be made available
under an Open Access Creative Commons CC-BY License (
http://creativecommons.org/licenses/by/4.0
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the
editorial office before completing the review.
If you wish, you can include your
name in your published report. Please note you must decide whether to include
your name at the start of the process and confirm this decision whenever
prompted. If you change your decision later, we will not be able to publish your
name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
Yes
Are the methods sufficiently described to allow the study to be repeated?
Yes
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
Yes
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
Please review this paper.
user
Reviewer’s report
Title: 
Tissue Microarrey: A potential cost-effective approach for mismatch
repair testing in colorectal cancer
Version: 
0 
Date: 
31 Mar 2022
Reviewer's report:
Thank you very much for this work. 
This is an interesting study and very timely
as the role of MMR IHC continues to expand, including reflexive staining for
colorectal and other tumours.
There are several issues with the study which would need to be addressed before
the work is suitable for publication.
The work starts with a bias in selection - as the TMA was guaranteed to have
reasonable IHC staining. 
Only cases with IHC staining were selected for the
TMA. 
This, in part, explains the higher sensitivity 
/ specificity of this work
compared to previous work. 
The need for larger and multiple cores is addressed in the discussion. 
The need
for normal tissue is questionable and should be addressed, including the impact
of not having the normal cores on the interpretation of the tumour MMR IHC
should be discussed.
The exclusion of PMS2 staining from over-fixation is questionable. 
In the study
337 cores were excluded because they showed lack of PMS2 nuclear staining.
I'm assuming that this lack of nuclear staining was in tumour and normal tissue.
There is variability in the staining quality in large tissue sections and this
exclusion speaks to the overall use of TMAs to assess MMR IHC in colorectal
cancer. 
More explanation around this exclusion and its impact is needed,
including some reference to the explanation that this is due to over fixation.
While the image analysis of the sections is interesting, the results are
disappointing with a lower sensitivity than hoped. 
Given the ability of deep
learning to identify dMMR / MSI tumours based on H&E, it would be hoped
that an algorithm could detect abnormal MMR-IHC with a higher degree of
accuracy.
The workflow is hard to visualize in practice. 
There is evidence to suggest that
MMR IHC should be done on biopsy samples. 
While the work flow may suit
identification of potential CRC associated with Lynch Syndrome, MMR status is
also used to determine treatment. 
The delays involved in the creation of a TMA,
accruing sufficient cases and then having the results reviewed (centrally),
reported and incorporated back with the original case may not occur in a timely
manner. 
Further logistic reviews and economic analysis would be required
before being able to consider the proposed workflow.
Minor Points -
In the materials and methods section, the reference to the research ethics could
be shortened. 
The line "Institutional ethical approval was granted for the
performance of this study" was repeated with different sites / IDs.
There are many spelling mistakes and typographic issues which need to be
addressed. 
The section - TMA accuracy measurements of IHC for MMR proteins
is missing a key word - "If at least of these proteins does not show nuclear
staining of tumor cells, the test is considered pathologic and the tumor potentially
dMMR." 
The spelling mistakes are especially evident in the discussion section.
In the 1st 2 paragraphs of page 13 alone there are at least 6 spelling mistakes
("evalute", "heterogenity", :decreaase", "suggeste", "comparisson", "fasion"). 
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons after the final decision on the manuscript has been made. If you have
already registered at Publons, they will notify you of the receipt of this review
and update your profile 
as per your settings and their policy. If you are not
registered with Publons, you will receive an email from them asking you to
register in order for them to be able to recognize your review on your new
profile page. Publons may use the Review Data to generate derivative metadata
for the benefit of Publons and you as a reviewer, carefully considering the
sensitivity of such information. For example, Publons may verify your record as
a reviewer by updating your profile published on its webservice if you have
registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
https://publons.com/about/terms
Yes
Declaration of competing interests
Please complete a declaration of competing
interests, considering the following questions:
1
. 
Have you in the past five years received reimbursements, fees, funding, or
salary from an organisation that may in any way gain or lose financially
from the publication of this manuscript, either now or in the future?
2
. 
Do you hold any stocks or shares in an organisation that may in any way
gain or lose financially from the publication of this manuscript, either now
or in the future?
3
. 
Do you hold or are you currently applying for any patents relating to the
content of the manuscript?
4
. 
Have you received reimbursements, fees, funding, or salary from an
organization that holds or has applied for patents relating to the content of
the manuscript?
5
. 
Do you have any other financial competing interests?
6
. 
Do you have any non-financial competing interests in relation to this
paper?
If you can answer no to all of the above, write 'I declare that I have no competing
interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests
Reviewer Publication Consent.
 I agree for my report to be made available
under an Open Access Creative Commons CC-BY License (
http://creativecommons.org/licenses/by/4.0
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the
editorial office before completing the review.
If you wish, you can include your
name in your published report. Please note you must decide whether to include
your name at the start of the process and confirm this decision whenever
prompted. If you change your decision later, we will not be able to publish your
name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
Yes
Are the methods sufficiently described to allow the study to be repeated?
Yes
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
Yes
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
assistant