Identification of novel biomarkers to distinguish clear cell and non-clear cell renal cell carcinoma using bioinformatics and machine learning

Chanita Panwoon; Wunchana Seubwai; Malinee Thanee; Sakkarn Sangkhamanon

doi:10.1371/journal.pone.0305252

Peer Review History

Original SubmissionApril 1, 2024
15 Apr 2024 Decision Letter - Giuseppe Lucarelli, Editor PONE-D-24-11162Identification of novel biomarkers to distinguish clear cell and non-clear cell renal cell carcinoma using bioinformatics and machine learningPLOS ONE Dear Dr. Sangkhamanon, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 30 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this study the authors identified NDUFA4L2 and DAT as novel biomarkers to differential diagnosis ccRCC from non-ccRCC usign a bioinformatic approach. I have some comments: -Renal cell carcinoma (RCC) is essentially a metabolic disease characterized by a reprogramming of energetic metabolism (PMID: 36960789; PMID: 30983433, PMID: 36430837,PMID: 36310399). In particular the metabolic flux through glycolysis is partitioned (PMID: 29371925, PMID: 28933387, PMID: 25945836), and mitochondrial bioenergetics and OxPhox are impaired , as well as lipid metabolism (PMID: 30538212; PMID: 32861643, PMID: 29371925, PMID: 36430448, PMID: 38540735). In this scenario it has been shown by Lucarelli et al. that NDUFA4L2 is an important regulator of cell metabolism in ccRCC and regulates many biological characteristics of renal cancer stem cells (PMID: 37685983). These findings should be referenced and discussed. -In addition, renal cell carcinoma is one of the most immune-infiltrated tumors (PMID: 31527133, PMID: 30738745; PMID: 27063186). Emerging evidence suggests that the activation of specific metabolic pathway have a role in regulating angiogenesis and inflammatory signatures (PMID: 32345771, PMID: 28359744). Features of the tumor microenvironment heavily affect disease biology and may affect responses to systemic therapy (PMID: 38003705; PMID: 37189689; PMID: 33265926; PMID: 36902242; PMID: 37373581). NDUFA4L2-induced metabolic alterations can modulate immune cell infiltration and regulate immunoflogosis. These processes should be explored and discussed. - The entire study was based on data mining, the lack of independent validation with wet lab experiments using cell lines or clinical specimens weakens this study. The author should specify this limitation in the discussion Reviewer #2: Renal cell carcinoma (RCC) is the most common type of urogenital cancer. . It comprises a diverse group of malignancies arising from the nephron. The World Health Organization (WHO) classifies RCC into different subtypes based on its morphologic, molecular, and genetic features. It’s important for the purpose of management and prognosis to differentiate clear cell RCC from nccRCC. This article provides a comprehensive investigation into find biomarkers that can differentiate between clear cell RCC and non-clear cell RCC using advanced computational techniques. The study utilizes gene expression data from The Cancer Genome Atlas (TCGA) to identify differential expression genes (DEGs) specific to each RCC subtype, including papillary RCC (pRCC) and chromophobe RCC (chRCC). The author identifies two new genes (NDUFA4L2 and DAT) as possible biomarkers in the differentiation of clear cell RCC and non-clear cell RCC. There are some limitations. Firstly, the sample size is inadequate to generalize the results. Also, more detailed discussions on the biological functions and clinical implications of the identified biomarkers is needed. I suggest adding the following scientific article links to the bibliography section for a more accurate representation of the references and the general topic of this study: - 10.3390/ijms24054615 , an interesting review on new prospective in the management of renal cancer. - 10.1177/17562872231164803, a literature review on the newest tools in kidney lesions evaluation - 10.3390/diagnostics13132308, enhancing the use of AI in the urological field. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. 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Revision 1
20 May 2024 Author Response Editor comments 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Answers: Thank you. We have adjusted the manuscript, including the file naming, according to the style requirements guideline. 2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. Answers: Thank you. We have uploaded code to GitHub with the following link below: https://github.com/CHaPAN6551/machine-learning-classification-algorithm 3. Please state what role the funders took in the study. Answers: Thank you. We have added “The funders had no role in study design, data collection, and analysis, manuscript preparation, or publication decisions” as a yellow highlight on Page no. 29, lines 439-440. 4. Third-party data Answers: Thank you. We have added the information to the supporting information section. ---------- Review Comments to the Author Reviewer #1: In this study the authors identified NDUFA4L2 and DAT as novel biomarkers to differential diagnosis ccRCC from non-ccRCC using a bioinformatic approach. I have some comments: 1) Renal cell carcinoma (RCC) is essentially a metabolic disease characterized by a reprogramming of energetic metabolism (PMID: 30983433, PMID: 36430837,PMID: 36310399). In particular the metabolic flux through glycolysis is partitioned (PMID: 29371925, PMID: 28933387, PMID: 25945836), and mitochondrial bioenergetics and OxPhox are impaired, as well as lipid metabolism (PMID: 30538212; PMID: 32861643, PMID: 29371925, PMID: 36430448, PMID: 38540735). In this scenario it has been shown by Lucarelli et al. that NDUFA4L2 is an important regulator of cell metabolism in ccRCC and regulates many biological characteristics of renal cancer stem cells (PMID: 37685983). These findings should be referenced and discussed. Response to Reviewer: Thank you for your great suggestion. According to the reviewer's comment, We added some information in the discussion section (references no. 17-19) with the following sentences “RCC is suggested as a metabolic disease, with alterations in various pathways like energy metabolism, fatty acid metabolism, and amino acid processing (Schaeffeler et al., 2019; Wettersten, Aboud, Lara, & Weiss, 2017). Wettersten et al. suggest a reprogramming of sugar and fat usage, along with changes in the tricarboxylic acid cycle, often linked to mutations in the VHL gene (Wettersten et al., 2017). Lucarelli et al. reported that a high level of glucose-6-phosphate dehydrogenase (G6PD) because of increasing pentose phosphate pathway (PPP)-derived metabolites led to enhanced activity in both glycolysis and the PPP in ccRCC (Lucarelli et al., 2015). These findings suggest energy metabolic and glycolysis reprogramming is key in promoting RCC. Additionally, Schaeffeler and co-workers explored that RCC subtypes had different biological and metabolic profiles based on the correlation of the original region (Schaeffeler et al., 2019).”as yellow highlighted on Page no. 24, lines 319-327. In addition, we added some discussion (reference no. 28) as the following sentences - “The high expression of NDUFA4L2 regulates glucose and energy metabolism alteration in cancer cells, promoting increased glucose uptake, a shift towards glycolysis, and altered mitochondrial function (Lucarelli et al., 2018).” as yellow highlighted on Page no. 26, line no. 374-376 - “Conversely, NDUFA4L2 silencing also disrupts cellular processes, including inhibition of the autophagic machine, increased mitochondrial mass, and induced overproduction of ROS, especially in hypoxic conditions (Lucarelli et al., 2018).” as yellow highlighted on Page no. 26, line no. 381-383 2) In addition, renal cell carcinoma is one of the most immune-infiltrated tumors (PMID: 31527133, PMID: 30738745; PMID: 27063186). Emerging evidence suggests that the activation of specific metabolic pathways has a role in regulating angiogenesis and inflammatory signatures (PMID: 32345771, PMID: 28359744). Features of the tumor microenvironment heavily affect disease biology and may affect responses to systemic therapy (PMID: 38003705; PMID: 37189689; PMID: 33265926; PMID: 36902242; PMID: 37373581). NDUFA4L2-induced metabolic alterations can modulate immune cell infiltration and regulate immunoflogosis. These processes should be explored and discussed. Response to Reviewer: Thank you for your kind suggestion. Regarding your advice, we added the text from references (references no. 32 and 33) in the discussion section with the following sentences: “ In particular, ccRCC stands out as a tumor with significant immune infiltration. This infiltration in tumors has been linked to the VHL tumor suppressor gene, triggering HIF-driven cellular responses, angiogenesis, and metabolic alterations (Vuong, Kotecha, Voss, & Hakimi, 2019). Furthermore, previous study has revealed that metabolic-associated genes like MUC1 can influence immunoflogosis within the ccRCC microenvironment by activating the classical pathway of the complement system and modulating immune infiltrates, thereby fostering an immune-silent microenvironment (Lucarelli et al., 2023). These findings displayed the hidden significance of NDUFA4L2 in potentially inducing modulation of immune cell infiltration and regulating immunoflogosis by controlling metabolic alterations in ccRCC. In summary, the evidence suggests that targeting NDUFA4L2 could be a potential immunotherapeutic target of ccRCC.” as yellow highlighted on Page no. 27, line no. 386-395 3) The entire study was based on data mining, the lack of independent validation with wet lab experiments using cell lines or clinical specimens weakens this study. The author should specify this limitation in the discussion. Response to Reviewer: Thank you for your kind suggestion. As the reviewer mentioned, “Limitation of this study”. We added the limitation of this study in the discussion section as follows; “The larger sample size should further validate the expression of these genes. The investigation of functional study on important genes in in vitro model might be used to be a potential target for RCC treatment in the future.” as yellow highlighted on Page no. 28, line no. 421-423 ---------- Reviewer #2: Renal cell carcinoma (RCC) is the most common type of urogenital cancer. It comprises a diverse group of malignancies arising from the nephron. The World Health Organization (WHO) classifies RCC into different subtypes based on its morphologic, molecular, and genetic features. It’s important for the purpose of management and prognosis to differentiate clear cell RCC from nccRCC. This article provides a comprehensive investigation into find biomarkers that can differentiate between clear cell RCC and non-clear cell RCC using advanced computational techniques. The study utilizes gene expression data from The Cancer Genome Atlas (TCGA) to identify differential expression genes (DEGs) specific to each RCC subtype, including papillary RCC (pRCC) and chromophobe RCC (chRCC). The author identifies two new genes (NDUFA4L2 and DAT) as possible biomarkers in the differentiation of clear cell RCC and non-clear cell RCC. There are some limitations. Firstly, the sample size is inadequate to generalize the results. Also, more detailed discussions on the biological functions and clinical implications of the identified biomarkers are needed. 1) Clinical implications of the identified biomarkers are needed. Response to Reviewer: Thank you for your kind suggestion. According to the reviewer's comment, we added some information in the discussion section on these two new genes (NDUFA4L2 and DAT) with the following sentences: 1.1) NDUFA4L2 - “This suggested NDUFA4L2 plays a role in resistant of chemotherapy of RCC.” as yellow highlighted on Page no. 27, line no. 385-386 - “Possibly, NDUFA4L2 might be a potential immunotherapeutic target in many cancers.” as yellow highlighted on Page no. 27, line no. 398-399 1.2) DAT - “Previous studies suggest that DAT was expected to be a specific biomarker of prognosis or targeted therapy for ccRCC.” As a yellow highlighted on Page no. 27, line no. 405-406 - “DAT might be a serological diagnostic marker of gastric cancer.” As a yellow highlighted on Page no. 28, line no. 415-416 2) I suggest adding the following scientific article links to the bibliography section for a more accurate representation of the references and the general topic of this study: 2.1) 10.3390/ijms24054615, an interesting review on new prospective in the management of renal cancer. Response to Reviewer: Thank you for your kind suggestion. According to the reviewer's comment, We added some information in the introduction section (reference no. 5) with the following sentences “Despite the promising approach of novel RCC management through radiogenomics, which offers the capability to characterize potentially malignant lesions in their genetic, epigenetic, and pathological heterogeneity via noninvasive methods such as advanced imaging study (Ferro, Musi, et al., 2023). The comprehensive understanding of the differences in transcriptomic and proteomic profiles between ccRCC and non-ccRCC remains unclear.” as yellow highlighted on Page no. 3, line no. 63-68 2.2) 10.1177/17562872231164803, a literature review on the newest tools in kidney lesions evaluation 2.3) 10.3390/diagnostics13132308, enhancing the use of AI in the urological field. Response to Reviewer: Thank you for your thoughtful recommendation. Following your guidance, we have incorporated the content from references 20 and 21 into the discussion section. We considered merging these two points (2.2 and 2.3) with the following sentences: “In the era of precision medicine, artificial intelligence (AI) and machine learning (ML) have been applied using a multi-omics approach to enhance performance in detecting and differentiating urological tumors. Particularly successful were radiography studies for kidney and bladder lesions (Ferro, Crocetto, et al., 2023; Ferro, Falagario, et al., 2023). Integrating radiologic diagnosis with AI and ML is currently undergoing widespread development. However, there are limited studies in the pathology field. Thus, there is an opportunity to integrate transcriptomic analysis and ML that can be applied in pathology practice.” as yellow highlighted on Page no. 24, line no. 328-334 https://doi.org/10.1371/journal.pone.0305252.r002
28 May 2024 Decision Letter - Giuseppe Lucarelli, Editor Identification of novel biomarkers to distinguish clear cell and non-clear cell renal cell carcinoma using bioinformatics and machine learning PONE-D-24-11162R1 Dear Dr. Sangkhamanon, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Giuseppe Lucarelli, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0305252.r003
Formally Accepted
31 May 2024 Acceptance Letter - Giuseppe Lucarelli, Editor PONE-D-24-11162R1 PLOS ONE Dear Dr. Sangkhamanon, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Giuseppe Lucarelli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0305252.r004

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