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[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
The manuscript contains a limited number of tumour samples (both discovery and validation samples). The in silico validation carried out on TCGA data did not confirm their findings. Thus, to provide transparent and comparable data to the scientific literature, the inclusion of control/normal lung samples is critical.
In addition, the following points should be addressed:
1. Fig 3 B. Sample dendrogram should be included.
2. QRT-PCR is performed in two-step thermal cycling conditions. Is this the fact, or is there any missing information there?
3. The authors should take out the DUOX1 parameter from the nomogram and see if the nomogram still predicts survival probability to show the impact of DUOX1 expression on prognosis.
**PeerJ Staff Note**: Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
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The article by Zhang et al performed a transcriptomic profiling to identify the molecular differences in lung cancer samples (synchronous multiple primary lung adenocarcinoma and single primary lung adenocarcinoma). The present study have performed molecular profiling has identified a couple of genes that can determine the pathological implication of MPLC.
Minor comments-
1. Data availability should be mentioned.
2. Typos throughout the manuscript.
3. Out of 22 upregulated genes, why were the five genes (DUOX1, CACNA2D2, GPX8, COL1A2, and COL1A1) selected for further analysis.
4. As mentioned by the authors, DUOX1 is silenced in lung cancer (line#280); therefore it is unclear how DUOX1 might be involved in differentiating the sMP-LUAD and SP-LUAD phenotype. Also highlighted by authors (ref#31) that DUOX1 silencing results in oncogenic behavior which is contrary to their findings- DUOX1 is upregulated in sMP-LUAD.
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This manuscript investigates the molecular genetic differences between synchronous multiple primary lung adenocarcinoma (sMP-LUAD) and single primary lung adenocarcinoma (SP-LUAD) using transcriptome sequencing technology. The study employed RNA-seq technology to analyze 16 tissue samples from 8 sMP-LUAD patients and 8 tissue samples from 8 SP-LUAD patients, identifying 194 differentially expressed genes and validating the expression levels of 5 candidate genes through qRT-PCR. The results showed that DUOX1 expression was significantly elevated in the sMP-LUAD group, and GO and KEGG analyses suggested it may participate in tumor development through oxidative stress and epithelial-mesenchymal transition pathways. While this study provides new insights into the molecular mechanisms of MPLC, the manuscript still has the following areas that need improvement.
Major Comments
1. The authors should include more control groups in the study design, particularly benign lung nodule tissues as negative controls, to better distinguish malignant tumor-specific molecular changes. Additionally, the authors could consider including intrapulmonary metastasis (IPM) cases as control groups, which would more directly validate the diagnostic value of transcriptomic differences in differentiating sMP-LUAD from IPM.
2. Although the authors predicted the biological functions of differentially expressed genes through GO and KEGG analyses, experimental validation is lacking. The authors should design functional experiments to verify the specific mechanisms of DUOX1 in lung cancer cells, including effects on cellular phenotypes such as proliferation, apoptosis, migration, and invasion. Recent studies have shown that oxidative stress-related genes play important roles in lung cancer progression, and DUOX1, as a dual oxidase, may affect the tumor microenvironment and epithelial-mesenchymal transition processes through reactive oxygen species (ROS) production (PMID: 38389415; 37489792).
3. The authors could use the GseaVis package and IOBR package for subsequent immune mechanism-related analyses (DOI: 10.1002/mdr2.70001; 10.1002/mdr2.70000). Furthermore, the authors should analyze the correlation between differential gene expression and patients' clinicopathological characteristics, including tumor stage, histological grade, lymph node metastasis status, etc. The authors could also evaluate the clinical significance of these molecular markers in predicting patient prognosis through survival analysis to validate their potential as prognostic biomarkers.
4. The authors should validate their research findings using public databases (such as TCGA, GEO, etc.), particularly the expression patterns and clinical significance of DUOX1 in lung adenocarcinoma. Additionally, the authors could seek independent external cohorts to validate their findings, improving the reproducibility and clinical translation potential of the results.
5. Although the authors mentioned study limitations in the discussion, the analysis was not sufficiently thorough. The authors should provide detailed discussion of the potential impacts of sample heterogeneity, technical bias, selection bias, etc., on the results and propose corresponding solutions. The authors could also discuss the application prospects of transcriptomic analysis in precision medicine in conjunction with the latest multi-omics technology development trends.
Minor Comments
1. The authors should carefully check the full names before abbreviations throughout the manuscript to ensure all abbreviations are spelled out upon first appearance.
2. The article needs professional native English editing to reduce grammatical errors and improve idiomatic expressions.
3. The abstract section needs to be rewritten to better summarize the main content and clinical significance of the article.
4. The figure legends need to be written in more detail to allow readers to understand them more clearly.
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