MET Exon 14 Variants in Non-Small Cell Lung Carcinoma: Prevalence, Clinicopathologic and Molecular Features

Round 1

Reviewer 1 Report

In the manuscript entitled "MET exon 14 variants in non-small cell lung carcinoma: prevalence, clinicopathologic and molecular features" by Lisi Yuan et al., the authors aimed to further characterize the clinicopathologic features and mutational profile of 44 cases harboring MET gene alterations, including MET ex14 variants, in patients with non-small cell lung cancer (NSCLC) by using a DNA-only next-generation sequencing (NGS)-based assay. The authors also assessed the prevalence of such genomic alterations in the context of PD-L1 expression. The authors found that 1.9% of the 2296 NSCLCs tested between 2017-7/2019 harbored a MET gene alteration and that 72.7% of those variants corresponded to canonical MET exon 14 skipping mutations. A small subgroup of cases analyzed had histological types and growth pattern data available and a high percentage of tumors with MET ex14 mutations were positive for PD-L1.

The novelty of this study is somewhat weak, as a search in PubMed renders at least 18 publications from 2016 to 2022 covering this topic. In addition, it would be desirable that the authors address some revisions in order to consider publication:

Minor Revision

1-      Please correct the statement about the gold standard testing for PD-L1 in the “Discussion” section of the manuscript (page 7, line 228). The actual gold-standard test for quantifying PD-L1 expression is IHC, not FISH, as indicated by the reference No. 28.

Major Revision

1- As noted by the authors, several studies have demonstrated the superiority of RNA based NGS assays in detecting MET exon 14 skipping mutations compared to DNA based NGS assays. Moreover, amplicon-based RNA NGS panels can detect gene fusions in poor quality RNA samples, such as those obtained from FFPE tissue samples. Thus, using RNA based NGS assays would have allowed the authors to better classify the samples with non-canonical DNA mutations, including missense and VUS variants identified in their patient cohort, and that would be of interest for the readers of this journal.

Author Response

Thank you for the opportunity to respond to your comments regarding our manuscript entitled “MET exon 14 variants in non-small cell lung carcinoma: prevalence, clinicopathologic and molecular features”.  We appreciate your time and effort in making suggestions, and we have adjusted our manuscript accordingly.  Specific changes were made as follows:

Minor Revision

1-      Please correct the statement about the gold standard testing for PD-L1 in the “Discussion” section of the manuscript (page 7, line 228). The actual gold-standard test for quantifying PD-L1 expression is IHC, not FISH, as indicated by the reference No. 28.

Thank you for catching this error. It was corrected. 

Major Revision

1- As noted by the authors, several studies have demonstrated the superiority of RNA based NGS assays in detecting MET exon 14 skipping mutations compared to DNA based NGS assays. Moreover, amplicon-based RNA NGS panels can detect gene fusions in poor quality RNA samples, such as those obtained from FFPE tissue samples. Thus, using RNA based NGS assays would have allowed the authors to better classify the samples with non-canonical DNA mutations, including missense and VUS variants identified in their patient cohort, and that would be of interest for the readers of this journal.

We acknowledge that this is a major limitation of our study, which we will work on in our future projects. We have switched to combined DNA-RNA testing in 2022. We revised the paragraph (Page 6 line 147) discussing this issue accordingly:

Recent studies by Poirot et. al. and Davies et. al have demonstrated the superiority of RNA based assays in detecting MET exon 14 skipping mutations compared to DNA based assays [13,14]. In both these studies, DNA based assays were able to identify only approximately 60% of the mutations as any variant outside the amplified area or preventing binding due to mutation of the primer would not be detected. In contrast, in RNA based assays, any variant causing skipping of exon 14 in-vivo is identified as fusion on exon 13 to exon 15. In general, RNA-based targeted approach analyses and quantifies directly fusion transcripts and is more accurate than DNA panels on tumor tissue, but it can be limited by RNA quality and quantity. However, amplicon-based RNA NGS panels can detect gene fusions in poor quality RNA samples, such as those obtained from FFPE tissue samples (PMID: 32726941). This is a major limitation of our study, as using RNA based NGS assays or combined DNA-RNA testing would have allowed us to better classify the samples with non-canonical DNA mutations, including missense and VUS variants in this cohort. 

Reviewer 2 Report

Interesting study describing the frequency and characteristics of MetEx14 alterations in NSCLC tested by NGS. The findings are largely in line with other published studies.

Suggestions:

1-Do the authors have any clinical follow-up data for this cohort of MetEx14 patients, namely response to targeted therapies (or ICI therapy given they assessed PD-L1 status)? Any difference in response based on they type of MetEx14 alteration? Some clinical data would strengthen the impact of this cohort.

2-Table on Page 4. Would strongly suggest formatting this data into a stand-alone table and not a screenshot of an Excel spreadsheet as it currently looks.

3-More for the journal layout/editors: need to ensure the figure legends are placed next to the figure/table and not separated.

4-Table 1 (page 5) is difficult to read, as the authors have combined different variables/outputs into a single table. Would suggest re-formatting the data in this table.

5-Line 228. Do the authors mean immunohistochemistry (and not FISH) as the gold standard for quantifying PD-L1 in tumor samples?

6-Line 231. The authors could use the current NCCN guideline for NSCLC (ver 1.2023)

7-Figure 2 does not really add anything, as it just shows the different histologic patterns seen in NSCLC/adenocarcinoma. If the authors were to keep a histologic image, would suggest either a correlation/comparison with PD-L1 staining, or perhaps highlighting other histopathologic features that may be more/less commonly encountered in MetEx14 altered NSCLC tumors. Also, the red spell-check autocorrect needs to be removed from the labels for each panel (presumably figure created in PPT).

 

Author Response

Thank you for the opportunity to respond to your comments regarding our manuscript entitled “MET exon 14 variants in non-small cell lung carcinoma: prevalence, clinicopathologic and molecular features”.  We appreciate your time and effort in making suggestions, and we have adjusted our manuscript accordingly.  Specific changes were made as follows:

Suggestions:

1-Do the authors have any clinical follow-up data for this cohort of MetEx14 patients, namely response to targeted therapies (or ICI therapy given they assessed PD-L1 status)? Any difference in response based on they type of MetEx14 alteration? Some clinical data would strengthen the impact of this cohort.

Thank you for this suggestion. We agree that clinical follow-up data such as the response rate to ICI therapy or targeted therapies would be helpful and strengthen the impact of our cohort. However, as our molecular lab is also a reference lab for the regional hospitals and a nationwide reference lab, a significant number of patients were not treated at our hospital and were followed up elsewhere. In addition, only 22 cases were positive for PD-L1 and 11 cases were at the level of > 50% and likely treated, we fear the small number of patients in this cohort might cause significant bias and might not be able to contribute to the literature. We now switched to combined DNA and RNA testing, and we'd like to propose a larger study in future to better investigate and answer this clinically relevant question. Thanks again for this wonderful idea! 

2-Table on Page 4. Would strongly suggest formatting this data into a stand-alone table and not a screenshot of an Excel spreadsheet as it currently looks.

Thank you for the suggestion. We have formatted this data into a standalone table. We re-submitted this table to the editorial office email box (we can only attach one table in revision, we attached Table 1 for Q4)

3-More for the journal layout/editors: need to ensure the figure legends are placed next to the figure/table and not separated.

Thank you.

4-Table 1 (page 5) is difficult to read, as the authors have combined different variables/outputs into a single table. Would suggest re-formatting the data in this table.

The table's formatting was unfortunately lost during the process of uploading. We uploaded the table in word format here. 

5-Line 228. Do the authors mean immunohistochemistry (and not FISH) as the gold standard for quantifying PD-L1 in tumor samples?

Thank you for catching this error. It was corrected. Line 235.

6-Line 231. The authors could use the current NCCN guideline for NSCLC (ver 1.2023)

Thank you for pointing it out. The manuscript was updated accordingly. "Patients with METex14 skipping mutations have a modest response (16%, single-agent ICIs) to immunotherapy, even those with high PD-L1 levels." (NCCN NSCLC ver 1.2023, MS-21). Line 237. 

7-Figure 2 does not really add anything, as it just shows the different histologic patterns seen in NSCLC/adenocarcinoma. If the authors were to keep a histologic image, would suggest either a correlation/comparison with PD-L1 staining, or perhaps highlighting other histopathologic features that may be more/less commonly encountered in MetEx14 altered NSCLC tumors. Also, the red spell-check autocorrect needs to be removed from the labels for each panel (presumably figure created in PPT).

We have corrected the red spell-check autocorrect. 

We have added correlations with PD-L1 staining. In general, it seems like the poorly differentiated tumors had higher PD-L1 expression levels, while the lepidic and acinar predominant ones had lower PD-L1 expression levels, which correlates with the stages, although there are exceptions. Thank you for this suggestion. 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

This reviewer appreciates the effort done by the authors to include the paragraph about the advantages of using RNA-based NGS assays and acknowledging the limitations of their study in their revised Discussion. Since the authors have now switched to combined DNA-RNA testing (as they mention doing in 2022), I would strongly recommend to run at least the few samples with VUS mutations in their new assay to assess if those mutations lead to exon 14 skipping or not.

Author Response

Thank you for your suggestion. We have checked the five VUS cases that might lead to METex14 skipping, they are case #13 (FFPE), case #20 (FNA), case #21 (FFPE), case #23 (FNA), and case #25 (FFPE). We found out that we don't have enough material left for testing for case #20 and #23 (cell blocks were depleted). We could run RNA-based NGS on the rest of cases; however, our IRB was approved as retrospective chart review only and does not allow us to run additional tests which may lead to additional findings and even legal issues. In addition, we fear testing on small sample size may cause significant sampling bias. We checked current NCCN guidelines (Version 1.2023, page NSCL-H 5 OF 7): ◊ Testing Methodologies: NGS-based testing is the primary method for detection of METex14 skipping events; RNA-based NGS may have improved detection.  Although RNA-based NGS is not currently required and mandatory, we can design a new study to compare these two methods, especially in FNA samples; however, this is beyond the scope of this study. To clarify that we did not use RNA-based NGS, we updated page 2 line 87 under Results to "Of the 2296 cases of NSCLC analyzed by DNA-based NGS between 2017-7/2019, MET ex14 variants were present in 44 cases (1.9%)".

Reviewer 2 Report

The  authors have addressed the majority of the issues raised by the reviewers, and in doing so have improved the paper overall. 

Author Response

Thank you. We appreciate your time and effort in making suggestions and comments. 

Round 3

Reviewer 1 Report

No further comments