Molecular Comparison of Imatinib-Naïve and Resistant Gastrointestinal Stromal Tumors: Differentially Expressed microRNAs and mRNAs
Round 1
Reviewer 1 Report
The paper presents an analysis of miRNA and mRNA differential expression in gastrointestinal stroll tumors that at imatinib-native and imatinib-resistant patients.
there are several points that I recommend be considered before publication that deal with the patient/sample selection and expression data analysis and interpretation relative to imatinib
there is limited data concerning the selection of patients and GIST diagnosis although there are different criteria/guidelines in the field. A comparison to the specific criteria used in this study and reference as to whether a centralized or center-specific diagnosis process should be included
as with many studies that use expression analysis, there is a focus on "fold-change"...but it is also known that the magnitude of the fold-change may not necessarily correlate with biological significance, i.e. extremely small changes in very tightly controlled genes may be more functionally significant than large changes in less strictly controlled genes
given the use of imatinib in other cancers, does any data exist regarding development of resistance, and if so, how do these studies compare with this study
Author Response
Point-to-point response to the reviewer’s comments
Reviewer 1
The paper presents an analysis of miRNA and mRNA differential expression in gastrointestinal stroll tumors that at imatinib-native and imatinib-resistant patients.
there are several points that I recommend be considered before publication that deal with the patient/sample selection and expression data analysis and interpretation relative to imatinib
there is limited data concerning the selection of patients and GIST diagnosis although there are different criteria/guidelines in the field. A comparison to the specific criteria used in this study and reference as to whether a centralized or center-specific diagnosis process should be included
as with many studies that use expression analysis, there is a focus on "fold-change"...but it is also known that the magnitude of the fold-change may not necessarily correlate with biological significance, i.e. extremely small changes in very tightly controlled genes may be more functionally significant than large changes in less strictly controlled genes
given the use of imatinib in other cancers, does any data exist regarding development of resistance, and if so, how do these studies compare with this study
For our research we have made use of a unique, fresh-frozen GIST sample set consisting of both primary GIST samples as well as samples from GISTs that progress on imatinib. We would like to stress that particularly, resection material of imatinib-resistant samples is hard to come by. All GIST samples come from patients treated in two well-known and highly experienced soft-tissue sarcoma centers. Before inclusion in our study all GIST samples were assessed by an expert pathologist experienced with GIST and further histopathologically examined for CD117/KIT and DOG1 expression. Furthermore, the diagnosis GIST was confirmed by the presence of specific KIT mutations. The pathological and initial diagnostic molecular evaluation of the GIST samples were all performed at KU Leuven. It must be noted that two of the participating institutions, KU Leuven as well as the Erasmus MC have been instrumental and front-runners in the development of imatinib treatment for GIST patients. The majority of the patients from whom the GIST samples are derived were diagnosed and treated from 2000 – 2011 according to the applicable guidelines in that time period. We have adapted the text in the Materials and Methods section to better reflect some of these issues.
We fully agree with the reviewer that small fold-changes in transcript levels may still have considerable biological significance. This is particularly true for microRNAs, a point we specifically address in the discussion section where we state “Although the fold changes observed were relatively small, they can still have a significant impact on protein levels because the regulation of multiple targets within the same pathway can amplify their biological effect [21] and different miRNAs may cooperate and have synergistic effects [22].”
The reviewer has a point when asking how our data compare to findings in cancers, other than GIST, that are being treated with imatinib. In the discussion section of the manuscript we do compare our miRNA findings to those in chronic myeloid leukemia (CML) another cancer that is being treated with imatinib. However, one has to bear in mind that CML is an entirely different cancer with a different biology and clinical behaviour. In addition, the target of imatinib in the CML setting is BCR-ABL an oncogenic fusion protein and not c-KIT as in GIST. This may limit the relevance of a GIST – CML comparison.
Reviewer 2 Report
The manuscript by Amirnasr et al. investigated 53 GIST cases to identify differentially expressed genes and miRNAs between IM-n and IM-r cases by using a combination of DNA microarray and sophisticated bioinformatics. The authors identified some clusters of mRNAs and miRNAs that characterized important signatures specific to the IM-r cases. Given the clinical significance of developing new strategies for the treatment of GIST cases that are resistant to imatinib, I believe that the present study provided valuable information to overcome the clinical issue, and should be considered for publication in the journal. However, as the authors have been aware of, the present study did not extend our understanding and knowledge on the molecular mechanisms or etiology of GIST progression. Therefore, the relevance of the findings revealed by the present study needs to be further investigated by experiments using cultured cancer cells and animal models in the future studies. I found the paper has been well written so that non-English speaking researchers could understand it. I invite the authors to address the following comments before publication.
1) The authors identified many genes and miRNAs that are specifically upregulated or downregulated in IM-r cases. Given that GIST tumor cells are derived from interstitial cells of Cajal (ICC) or PDGFRa-expressing fibroblastic cells, are there any genes or miRNAs that are specifically expressed in the normal ICC and PDGFRa-positive cells in the gene/miRNA list identified by the present study? I believe the authors, if possible, need to compare the gene list found by the authors in the present study with the genes that are specifically expressed in ICC and PDGFRa-positive cells in a physiological context, given the authors’ future perspective to find the best target(s) to treat the patients that became resistant to imatinib.
2) In 4.2 and 4.3 of the Materials and Methods, the number of GSE accession numbers have not been provided. Clarification of these numbers will be necessary to help readers confirm and follow up the findings revealed by the authors.
Author Response
Point-to-point response to the reviewer’s comments
Reviewer 2
The manuscript by Amirnasr et al. investigated 53 GIST cases to identify differentially expressed genes and miRNAs between IM-n and IM-r cases by using a combination of DNA microarray and sophisticated bioinformatics. The authors identified some clusters of mRNAs and miRNAs that characterized important signatures specific to the IM-r cases. Given the clinical significance of developing new strategies for the treatment of GIST cases that are resistant to imatinib, I believe that the present study provided valuable information to overcome the clinical issue, and should be considered for publication in the journal. However, as the authors have been aware of, the present study did not extend our understanding and knowledge on the molecular mechanisms or etiology of GIST progression. Therefore, the relevance of the findings revealed by the present study needs to be further investigated by experiments using cultured cancer cells and animal models in the future studies. I found the paper has been well written so that non-English speaking researchers could understand it. I invite the authors to address the following comments before publication.
1) The authors identified many genes and miRNAs that are specifically upregulated or downregulated in IM-r cases. Given that GIST tumor cells are derived from interstitial cells of Cajal (ICC) or PDGFRa-expressing fibroblastic cells, are there any genes or miRNAs that are specifically expressed in the normal ICC and PDGFRa-positive cells in the gene/miRNA list identified by the present study? I believe the authors, if possible, need to compare the gene list found by the authors in the present study with the genes that are specifically expressed in ICC and PDGFRa-positive cells in a physiological context, given the authors’ future perspective to find the best target(s) to treat the patients that became resistant to imatinib.
Reply:
The point made by the reviewer is well-taken. It may indeed be interesting to see if the genes found up- or downregulated in imatinib-resistant GISTs are expressed in the interstitial cells of Cajal or associated PDGFRA-expressing fibroblastic cells. If this is the case these genes may constitute a target for therapy in imatinib-resistant GISTs. The transcriptome of these cell types, the interstitial cells of Cajal and PDGFRA-expressing fibroblastic cells, has recently been well-characterized in mice (Lee, M.Y. et al. 2017 PLOS ONE 12:e0176031; Ha, S.E. et al. 2017 PLOS ONE 12:e182265). However, we do feel that an in silico comparison of our gene list with the murine transcriptomic data - which will undoubtedly need validation experiments in the human setting - is outside the scope of the current manuscript and needs to be addressed in a separate follow-up study.
2) In 4.2 and 4.3 of the Materials and Methods, the number of GSE accession numbers have not been provided. Clarification of these numbers will be necessary to help readers confirm and follow up the findings revealed by the authors.
Reply:
As suggested by the reviewer we included the Gene Expression Omnibus accession number (GSE132542) that lists our mRNA expression data obtained with the Affymetrix platform. Furthermore, we now present our miRNA expression data as supplementary information in Table S7.
