A Prospective Validation Study of Lung Cancer Gene Panel Testing Using Cytological Specimens

Round 1

Reviewer 1 Report

Comment to the author

Morikawa et al prospectively evaluated the feasibility of a lung cancer gene panel test in cytological specimens. In clinical practice, there are some cases where the tissue sample is insufficient for the driver mutation testing by the multiplex method. Therefore, this result, which showed high success of NGS in cytopathological specimens, is important in future clinical practice. However, this paper has some points to improve. 

Major comments

1.     The authors compare the results of the CDx and LCCP Panel tests, but it is not clear what CDx is used for. Therefore, author needs to clarify what CDx was used for each driver mutation in Method.

2.     The amount of nucleic acid from FFPE is affected by the amount of submitted sample. Therefore, Author needs to clarify the slice thickness and number of FFPE samples to be submitted for LCCP.

3.     Some cases of pleural effusion have been submitted for examination by FFPE. How is FFPE in the pleural effusion example processed? Is it a cell block? Please describe in the method.

Minor comments

1.　 Throughout the paper, genes should be italics. (e.g. EGFR, et al)

Author Response

Reviewer 1

Comments and Suggestions for Authors

Comment to the author

Morikawa et al prospectively evaluated the feasibility of a lung cancer gene panel test in cytological specimens. In clinical practice, there are some cases where the tissue sample is insufficient for the driver mutation testing by the multiplex method. Therefore, this result, which showed high success of NGS in cytopathological specimens, is important in future clinical practice. However, this paper has some points to improve.

The Authors would like to thank the Reviewer for raising the following comments. We believe these comments strengthen our manuscript and have answered each question below.

Answers

Major comments

1. The authors compare the results of the CDx and LCCP Panel tests, but it is not clear what CDx is used for. Therefore, author needs to clarify what CDx was used for each driver mutation in Method.

1) We have added the following section to Methods section.

Pathological diagnosis and companion diagnostic test (CDx)

A genetic test which was approved by medical insurance, was performed as a companion diagnostic. For cases in which sufficient sample amounts could be collected [13], samples were preferentially submitted for the Oncomine Dx Target Test Multi-CDx system, which is a gene panel test. As a single gene search, Cobas® EGFR mutation test was used to detect EGFR mutations, immunohistochemistry (IHC), Ventana OptiView ALK (D5F3) (Roche Molecular Systems, Pleasanton, CA, USA) [14] and FISH, Vysis® ALK Break Apart FISH probe kit (Abbott Japan LLC, Tokyo, Japan) for the ALK mutation, and Archer® MET (Invitae Corp., San Francisco, CA, USA) for the MET exon 14 skipping mutation. Other rare gene mutations were confirmed by Oncomine Dx Target Test Multi-CDx system.

2. The amount of nucleic acid from FFPE is affected by the amount of submitted sample. Therefore, Author needs to clarify the slice thickness and number of FFPE samples to be submitted for LCCP.

2) We added the following sentence to the Methods section.

Four FFPE slides were prepared per case with a 10 μm thickness, two slides for DNA extraction and two slides for RNA extraction.

3. Some cases of pleural effusion have been submitted for examination by FFPE. How is FFPE in the pleural effusion example processed? Is it a cell block? Please describe in the method.

3) We added the following sentence to the Methods section.

Those with sufficient pleural effusion were used as cell blocks as a substitute for tissue.

Minor comments

1.　 Throughout the paper, genes should be italics. (e.g. EGFR, et al)

1) As you have pointed out, we have revised all genes to italics.

Author Response File: Author Response.docx

Reviewer 2 Report

In the manuscript "A Prospective Validation Study of Lung Cancer Gene Panel Testing Using Cytological Specimens" by Kei Morikawa et al. the authors use a lung cancer gene panel and develop methods aimed at validating its usage to test cytological specimens. This work builds up on previous work performed in the field, including previous work by the same group. The approach is not innovative and the techniques are already being used on the daily basis with successful results. Yet, it is always relevant to have additional strategies reported across different parts of the world. The paper is well-organized and well-written. In addition, the research is conducted in a careful and well-thought manner. Even though the paper is of good quality, there are some aspects that merit the attention of the authors:

1. The information on the morphological diagnosis performed for each case is not present in the paper. That is fundamental to value the findings reported. Are all the cases studied lung adenocarcinomas?

2. The authors should give more details on the methodology used, including test sensitivity.

3. Table 1 needs to be re-designed and improved.

4. There is no detailed data on the testing performed. The paper would benefit from a comprehensive table containing the following information: diagnosis for each case, percentage of tumoral cells present in FFPE sample and cytology sample, global number of reads for each case (FFPE sample and cytology sample), allelic fraction for the mutations detected both for FFPE and cytology, among other aspects.

Therefore, it will be mandatory to perform additional changes in the present manuscript if it is considered relevant for publication in Cancers.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript entitled "A Prospective Validation Study of Lung Cancer Gene Panel Testing Using Cytological Specimens" highlighted that the success of gene analysis using cytological specimens was high, and the yield and quality of the extracted nucleic acid were sufficient for panel analysis.

- The Authors should provide the expand forms for all acronyms, including gene acronyms, through the text when they first appear.

- Gene acronyms should be written in italics.

- The Authors should better discuss the role of cytological samples and molecular testing by adding further literature experiences.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

In the revised version of the manuscript "A Prospective Validation Study of Lung Cancer Gene Panel Testing Using Cytological Specimens" by Kei Morikawa et al. the authors use a lung cancer gene panel and develop methods aimed at validating its usage to test cytological specimens. This work builds up on previous work performed in the field, including previous work by the same group. The approach is not innovative and the techniques are already being used on the daily basis with successful results. Yet, it is always relevant to have additional strategies reported across different parts of the world. The paper is well-organized and well-written. In addition, the research is conducted in a careful and well-thought manner. The authors addressed in a satisfactory way the points raised previously and, thus, I have no further objections towards the publication of the manuscript.