A Case Series of Metastatic Malignant Gastrointestinal Neuroectodermal Tumors and Comprehensive Genomic Profiling Analysis of 20 Cases

Round 1

Reviewer 1 Report

Very interesting article. Please address the following comments:

• Sentence "Fluorescence in situ hybridization (FISH) was negative for EWSR1-ATF1 fusion which was further confirmed by NanoString and NGS" (lines 83-84) is ambiguous. Please clarify whether EWSR1-ATF1 fusion was not found with FISH but identified by NanoString and NGS.
• The part of the manuscript dealing with 20 samples from the reference lab should be clearly identified with title "Clinicopathological characterization and comprehensive genomic profiling (CGP) of 20 additional GNET cases" and should consist of merger of sections 3 and 4, while keeping the same subdivisions as in the original text.
• How do you know that the 2 cases of EWSR1-FLI1 fusions were not Ewing sarcomas?
• Were the 3 cases which were described in detail reviewed by an expert pathologist?

Author Response

Dear editors/reviewers

We greatly appreciate the editors and reviewers’ time and efforts to provide valuable suggestions and comments on our manuscript. We have taken these suggestions/comments seriously and modified our manuscript accordingly. Please see attached revised version of manuscript as well as below specific modifications to address these comments on a point-by-point basis.

Reviewer 1 :

• Sentence "Fluorescence in situ hybridization (FISH) was negative for EWSR1-ATF1 fusion which was further confirmed by NanoString and NGS" (lines 83-84) is ambiguous. Please clarify whether EWSR1-ATF1 fusion was not found with FISH but identified by NanoString and NGS.

Response: We agree completely that this sentence is ambiguous. We have modified this sentence to improve clarity to the readers “Interestingly, EWSR1 fusion was absent in this patient’s tumor confirmed by 3 methods including Fluorescence In Situ Hybridization (FISH), NanoString and NGS.” (Lines 93-95). Although EWSR1 fusion is a pathognomic feature for GNET, about 7-13% patients, depending on the case series, do not have this fusion therefore it is not an absolute diagnostic criterion (please see discussion section line 348-362).

• The part of the manuscript dealing with 20 samples from the reference lab should be clearly identified with title "Clinicopathological characterization and comprehensive genomic profiling (CGP) of 20 additional GNET cases" and should consist of merger of sections 3 and 4, while keeping the same subdivisions as in the original text.

Response: Thanks for the reviewer’s great suggestion. We have modified the manuscript as suggested (Page 3-14).

• How do you know that the 2 cases of EWSR1-FLI1 fusions were not Ewing sarcomas?

Response: Thanks for the reviewer for pointing out this important finding. We have discussed these two cases extensively among our two expert pathologists from Foundation Medicine. We are uncertain if these 2 cases of EWSR1-FLI1 fusions represent GNET or Ewing Sarcomas given all the information provided from outside pathologists from the originating labs. We have also highlighted the two cases in the oncoprint (figure 2) with and asterisk and added a note in the legend that cases are discussed in further detail in section 4.7. Please see lines 490-511 for additional discussion.

• Were the 3 cases which were described in detail reviewed by an expert pathologist?

Response: Again, thanks for the reviewer’s great question. Yes, all 3 clinical cases presented in this manuscript were reviewed by local expert sarcoma pathologists especially the first case which does not have EWSR1-ATF1 fusion. All 3 cases were discussed in local provincial sarcoma tumor board to confirm the diagnosis and treatment plan.

Reviewer 2 Report

Review (Remark to authors)  

    The authors describe in this manuscrpit a therapeutic future of high clinical and social interest, referring to a complex type of cancer but of high significance in today's society.

   In 2020, 111 cases of GNET were diagnosed. The authors demonstrate high capacity for work and collaboration by processing large number of samples, data analysis and innovation in omics sciences (NGS).

          Minor Considerations

1. The title is complex, difficult to understand and excessively long. I recommend your writing to attract the most future readers
2. Abstract: I recommend rewriting the abstract, emphasizing more on the conclusions. It is too general. In addition, there are phrases that require greater scientific or clinical emphasis, for example Lines 19-20 "due to the rarity of this disease" or line 21 "we believe" or lines 29-30, which is very general and does not provide any conclusion.
3. The authors should further develop line 136-137. Explain in a short paragraph if the comparison of the 20 patients includes the 3 clinical cases analyzed, if they are independent or if they will be compared with each other.
4. Table 2: is it posible to re-make this table in other format? Not neccesarially reducing data but showing in other format
5. Section 5.6. Title should be modify

Major Considerations

1. Figure 1: The authors must specify the staining shown in figure 1 in each case, could they elaborate a small material and methods, included in the figure legend?
2. The IHC assay needs a small section on material and methods. Specifying how the fixation, sample preparation and staining have been developed. If the author’s and/or editors considers this information excessive for the final format of the manuscript, the reviewer must receive this information personally.
3. Regarding the analysis of table 1, can the authors show any IHC that is representative and complementary to the figure?
4. In figure 2, the authors only show mutations landscape. Data obtained from NGS analysis. However the information that allows to analyze the NGS technique. The authors have considered showing genomic alterations (base substitutions, insertional/deletion, copy Number alterations and gene rearrangment) and genomic signatures (tumor mutational burden, microsatelite instability)
5. Have the authors performed RNA seq assays of tissues? The message of the manuscript would grow exponentially and would provide a key information of expression and possible therapeutic pathways.
6. The authors should perform protein expression assays (wbs) related to MAPk adaptation pathways, AMPK/mTORC1-like energy pathways, and DNA damage repair marker assays. Data from at least 2-3 patients should be shown to emphasize the final conclusion and increase the power of attraction towards future readers.

Author Response

Dear editors/reviewers

We greatly appreciate the editors and reviewers’ time and efforts to provide valuable suggestions and comments on our manuscript. We have taken these suggestions/comments seriously and modified our manuscript accordingly. Please see attached revised version of manuscript as well as below specific modifications to address these comments on a point-by-point basis.

Reviewer 2:

The authors describe in this manuscript a therapeutic future of high clinical and social interest, referring to a complex type of cancer but of high significance in today's society.

   In 2020, 111 cases of GNET were diagnosed. The authors demonstrate high capacity for work and collaboration by processing large number of samples, data analysis and innovation in omics sciences (NGS).

Response: Thanks for the comment and support.

          Minor Considerations

1. The title is complex, difficult to understand and excessively long. I recommend your writing to attract the most future readers

Response: We agree that this title is long as we would like it to reflect two main sections of this manuscript including case series of 3 GNET as well as pathological characterization and NGS of 20 additional cases. Based on journal requirement, the title of the manuscript needs to reflect the main content of the manuscript.  However we would like to kindly ask reviewer/editor’s suggestion to modify our title in a more succinct manner. We would be very happy to modify it to attract readers.

1. Abstract: I recommend rewriting the abstract, emphasizing more on the conclusions. It is too general. In addition, there are phrases that require greater scientific or clinical emphasis, for example Lines 19-20 "due to the rarity of this disease" or line 21 "we believe" or lines 29-30, which is very general and does not provide any conclusion.

Response: Thanks for the reviewer’s suggestion. We have modified phrases on lines 20-21 to reflect contribution/conclusion from our case series as well as literature review which we have humbly and modestly present as our unique opinion. We would be happy to modify further upon additional suggestions. We hope the abstract conveys our main messages that GNET are a spectrum of diseases and that EWRS1 fusions appear to be the main driver genomic event.

1. The authors should further develop line 136-137. Explain in a short paragraph if the comparison of the 20 patients includes the 3 clinical cases analyzed, if they are independent or if they will be compared with each other.

Response: Thanks for the reviewer’s suggestion. We have now added a separate section 3. “Clinicopathological characterization and comprehensive genomic profiling (CGP) of 20 additional GNET cases from Foundation Medicine.” to make it clearer to the readers that these 20 cases are different from 3 clinical cases presented in section 2.

1. Table 2: is it possible to re-make this table in other format? Not necessarily reducing data but showing in other format

Response: The current format of table 2 is commonly used in published systemic review. However we would be happy to modify it if the reviewer could kindly provide detailed suggestion to improve the clarity of table 2.

Section 5.6. Title should be modify

Response: Thank you for the suggestion. We have modified the title to Radiotherapy Potentially Beneficial in the Metastatic Setting (now in section 4.6) hoping that this reflects the current limited clinical data on the use of radiotherapy in GNET but the need to further evaluate its utility in this setting.

Major Considerations

1. Figure 1: The authors must specify the staining shown in figure 1 in each case, could they elaborate a small material and methods, included in the figure legend?

Response: Thanks for the suggestion. Please note that all 3 samples included in Figure 1 were prepared externally to the reference lab into FFPE blocks. Therefore, we do not know the exact fixation/processing methods utilized at the originating lab. The pathologists at the reference lab review H&E slides to determine how to process the specimen for NGS testing. Some of the H&E slides are provided by the originating lab. Others were cut in house and underwent the H&E standard staining procedure used in the reference lab. This information has now been added to the methods section (line 162-176). Additionally, we have edited the legend on figure 1 to highlight that all 3 panels show H&E stainings. We hope this will address the reviewer’s concern.

1. The IHC assay needs a small section on material and methods. Specifying how the fixation, sample preparation and staining have been developed. If the author’s and/or editors considers this information excessive for the final format of the manuscript, the reviewer must receive this information personally.

Response: Please see comment above which is relevant to this question as well. Please note that all IHC and FISH assays were performed and interpreted externally to the reference lab, and we do not know the exact fixation/staining/methods that the original lab utilized. All the information/results on these tests mentioned in the paper are based on pathology reports provided by the originating labs. We emphasized this in the methods section on lines 160-169 and lines 162-176 and again in the IHC/FISH section on lines 226-227. We hope this will address the reviewer’s concern.

1. Regarding the analysis of table 1, can the authors show any IHC that is representative and complementary to the figure?

Response: Table 1 summarizes all the previous GNET cases available in the literature. Figure 1 represents 3 examples (H&E) from the 20 cases submitted to Foundation Medicine. We do not have access to any of the IHC slides performed on these cases.  We only have what was written in the reports provided by the originating labs.

1. In figure 2, the authors only show mutations landscape. Data obtained from NGS analysis. However the information that allows to analyze the NGS technique. The authors have considered showing genomic alterations (base substitutions, insertional/deletion, copy Number alterations and gene rearrangment) and genomic signatures (tumor mutational burden, microsatelite instability)?

Response: Thank you for the suggestion. The oncoprint in figure 2 was generated in cBioPortal using the standard OncoPrinter function: step 1) input of genomic alteration data which considers only genes and respective class of genomic alteration. While TMB and Microsatellite information could potentially be embedded inan oncoprint plot to give a more comprehensive review of biomarker and mutational landscape, we would have to use different tools. Additionally, because all cases harbored a low level of TMB (<10 mutations/megabase) and were microsatellite stable we chose to include that information in more detail for each case in table S1.

1. Have the authors performed RNA seq assays of tissues? The message of the manuscript would grow exponentially and would provide a key information of expression and possible therapeutic pathways.

Response: Thank you for your question, this is an important point that we further clarified in the manuscript. The 20 samples submitted to Foundation Medicine where sequenced using either FoundationOne or FoundationOne CDx (DNA sequencing only, 8 cases) to detect all classes of alterations substitutions, indels, rearrangements and copy number alterations, or FoundationOne Heme (DNA and RNA sequencing, 12 cases). In addition to the DNAseq, the RNAseq component of FoundationOne Heme only allows detection of fusion transcripts but is not used for analysis of RNA expression. This is also the reason why we cannot perform pathway analysis. EWSR1 gene rearrangements were detected at the DNA and/or RNA levels. This information has been added to the manuscript lines 267-274.

1. The authors should perform protein expression assays (wbs) related to MAPk adaptation pathways, AMPK/mTORC1-like energy pathways, and DNA damage repair marker assays. Data from at least 2-3 patients should be shown to emphasize the final conclusion and increase the power of attraction towards future readers.

Response: Thanks for the reviewer’s great suggestion. We completely agree that future research should focus on elaborating molecular pathways to elucidate underlying mechanisms of development and progression of GNET and response to treatment. Unfortunately, it is not logistically possible to perform protein expression assay on the 3 clinical samples as well as 20 additional samples provided from originating labs. We have added this on our discussion (line 527-530).

Round 2

Reviewer 2 Report

I consider all my suggestions have been done. I accept in the present format

   Best Regards

   José Manuel Rodríguez