Are Microsatellite Patterns Specific for Tumor Types? A Pilot Investigation

Round 1

Reviewer 1 Report

The authors compare the results of a previously published MSI cohort, an externally conducted MSI testing of selected cases and a commercially available MSI testing system.

Case numbers are low for the stated strong hypothesis.

MSI testings should be completed for all cases with all methods. Quality parameters like tumor cell content should be provided for all subcohorts.

The numbers of non-colorectal cases should be equally high as the number of colorectal cases and non-colorectal MSS cases should be included as well.

In addition, it remains unclear which method is the gold standard - Promega or QiaXcel? How was the correlation with IHC staining results? These could be easily obtained, also for the round robin cases.

The authors investigated 8 cases of a German round robin trial which they also have published in their study in 2018. However, a statement whether there has been any kind of expicit approval by the QuiP to use these cases is missing.

Author Response

Many thanks for the in depth comments that helped to improve our study. We have done our best to address all issue, but, unfortunately, are not able to add further expimental data to to budgetary reasons. Below are the detailed replies:

 

Reviewer 1

The authors compare the results of a previously published MSI cohort, an externally conducted MSI testing of selected cases and a commercially available MSI testing system.

Case numbers are low for the stated strong hypothesis.

Our reply: We agree with this comment and have turned down our conclusion by using more subjunctives

MSI testings should be completed for all cases with all methods. Quality parameters like tumor cell content should be provided for all subcohorts.

Our reply:

We included a statement on the tumor cell contents in the Methods chapter. However, unfortunately, we do not have enough residual specimens left to perform additional experiments nor do we have any further funding available. Whilst this was the major reason to choose the manuscript category “Hypothesis” we acknowledge this reviewer’s comment and included an additional statement that further studies are required to systematically compare more samples and more tumor types with different panels for MSI testing.

The numbers of non-colorectal cases should be equally high as the number of colorectal cases and non-colorectal MSS cases should be included as well.

Our reply:

We in principle agree with this statement, but due to budget limitations do not have the chance to improve the study at this point.

In addition, it remains unclear which method is the gold standard - Promega or QiaXcel? How was the correlation with IHC staining results? These could be easily obtained, also for the round robin cases.

Our reply:

We apologize for not being more precise and have included the respective statement in the Methods chapter.

We also agree that a correlation between IHC and molecular results would be benefcial to compare, but unfortunately cannot include them. We have added the following sentence to the discussion:

However, for the samples used in this study no IHC testing was available as for the round robin samples solely molecular information was provided and for tonsil samples these testing were not performed as limited sample volume/slides were available for study purposes. Thus, future studies should also address the correlation if IHC and molecular markers more in depth.

 

The authors investigated 8 cases of a German round robin trial which they also have published in their study in 2018. However, a statement whether there has been any kind of expicit approval by the QuiP to use these cases is missing.

Our reply:

The corresponding author is in direct contact with the QUIP headquarter in Berlin and will send the informed consent as soon as possible to the publisher.

Reviewer 2 Report

JMP-889134: Are Microsatellite patterns specific for tumor types? A pilot investigation Considering that multiple studies on microsatellites have been published, recent observations suggest that the microsatellites that define instability differ between tumor entities. This is a pilot study that compared different MSI assays validated for colorectal cancer. Whereas all assays carry the same MSI/MSS status for colorectal cancers, they differ for tonsillar tumors, leading to the hypothesis that MSI patterns are tumor-type specific. Comments This is an interesting pilot study regarding MSI/MSS status comparing different assays for colorectal cancer and tonsillar tumors using PCR amplification and detection using QiaXcel and Idylla™ and Promega in FFPE specimens. The procedure can be very useful in routine practice since is in house method and not expensive, although requires long hands-on time. The results found that the commercially available methods for MSI testing, favor the Idylla-system because it not require complex pre-analytical steps. Although the authors tested these methods in colorectal cancer and tonsil tumors it would be important to include other tumors, such as lung cancer and malignant mesothelioma. In both tumors to detect MSI/MSS usually we need to perform TMB, a procedure not accessible to laboratories in general, in addition to being expensive and requiring standardization.

Author Response

Many thanks for the in depth comments that helped to improve our study. We have done our best to address all issue, but, unfortunately, are not able to add further expimental data to to budgetary reasons. Below are the detailed replies:

 

Reviewer 2

JMP-889134: Are Microsatellite patterns specific for tumor types? A pilot investigation Considering that multiple studies on microsatellites have been published, recent observations suggest that the microsatellites that define instability differ between tumor entities. This is a pilot study that compared different MSI assays validated for colorectal cancer. Whereas all assays carry the same MSI/MSS status for colorectal cancers, they differ for tonsillar tumors, leading to the hypothesis that MSI patterns are tumor-type specific.

 

Comments

This is an interesting pilot study regarding MSI/MSS status comparing different assays for colorectal cancer and tonsillar tumors using PCR amplification and detection using QiaXcel and Idylla™ and Promega in FFPE specimens. The procedure can be very useful in routine practice since is in house method and not expensive, although requires long hands-on time. The results found that the commercially available methods for MSI testing, favor the Idylla-system because it not require complex pre-analytical steps. Although the authors tested these methods in colorectal cancer and tonsil tumors it would be important to include other tumors, such as lung cancer and malignant mesothelioma.

 

Our reply:

We fully agree with the comments of the reviewer and acknowledge the positive feedback. Regarding the testing of other tumors we currently do not have the financial resources to perform a broader screening and therefore have named the work “pilot study”. In order to address this comment we have added a short paragraph to include this important issue.

The following sentence was included in the discussion:

Future studies should systematically test tumors from different origin, metastases of those tumors, and make use of broad, empiric approaches including a wide spectrum of microsatellites in order to elucidate the phenomenon observed in the present study.

 

In both tumors to detect MSI/MSS usually we need to perform TMB, a procedure not accessible to laboratories in general, in addition to being expensive and requiring standardization.

 

Our reply:

This was also added to the discussion as follows:

MSI/MSS testing may alternatively performed also by NGS protocols and may require additional analyses of the tumor mutational burden (TMB), however, these assays are not available in all laboratories, thus making the rapid detection methods such as the Idylla assay a rapid and broadly available tool for therapy relevant daily routine diagnostics.

 

Reviewer 3 Report

The presnted study has potential interest for readers and impact on clinical practice, however it should be partially redesigned and overally improved.

Third system for comparison covers too small portion of samples. There should be third or even fourth system to cover all samples.

Material and method part should be rewritten to contain more details on methodology as well as samples. Pathologist's assessment of tumor tissue presence in all samples is essential to avoid false negativity. Ethical committee statement is needed too. Any known MSI or MSS status by IHC? Follow up data available ?

Huge discrepancy in results should bediscussed with clinical output cosequences.

Author Response

Many thanks for the detailed and helpful comments that helped to improve our manuscript. Please find the detailed reply below. Please note that due to a lack of further grant budgets we were not able to perform additional experiments, but have replied as best as possible to the queries:

 

Reviewer 3

The presnted study has potential interest for readers and impact on clinical practice, however it should be partially redesigned and overally improved.

Third system for comparison covers too small portion of samples. There should be third or even fourth system to cover all samples.

Our reply:

We agree that such an extension is desirable and should be performed, but, however, we do not have the financial resources for this as mentioned above. This is also the reason why we entitled the manuscript with the phrase “A pilot study” and have chosen the manuscript category “Hypothesis” rather than “Short Communication”.

Material and method part should be rewritten to contain more details on methodology as well as samples. Pathologist's assessment of tumor tissue presence in all samples is essential to avoid false negativity. Ethical committee statement is needed too. Any known MSI or MSS status by IHC? Follow up data available ?

Our reply:

We fully agree with these comments, and - also in reply to reviewers 1 and 2 – have added relevant information to the Methods section.

Huge discrepancy in results should bediscussed with clinical output cosequences.

Our reply:

From this comment we learned that the intention of our conclusion did not explain what we believe to be the consequences for the clinical output. We have therefore added the final conclusions sentences (while using subjunctives in order not to interfere with reviewer 1 who recommended to turn down the statements):

A putative consequence for the current clinical and pathological practice could be that MSI testing algorithms established for distinct entities such as colorectal cancer may lead to false positive or, more likely, false negative results in another tumor entity unless specific patterns have been established.

 

Round 2

Reviewer 1 Report

The authors improved the presentation of data. In addition, the sharpened the conclusions that can be drawn from the data. The revised manuscript will be better understandable now and is of interest for molecular pathologists.

 

Just one thing:

Please check the introduction once more:

"Patients with MSI tumors usually have a lower overall survival and progression free survival. "

-> prognosis is in fact better

Author Response

Many thanks to the reviewer for this comment, the sentence was deleted and we fully agree.

Reviewer 3 Report

The authors replied to all my questions reasonably and followed the suggestions to improve the text. I appreciate their sincere reply with financial issue.

Author Response

We thank the reviewer for this reply and are grateful for the understanding