Integrative Analysis of the Role of TP53 in Human Pan-Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript focuses on a review meta analysis about p53 expression in tumor patients is a timely relevant manuscript where moderate integrations should be approached to imrpove the readibility of the manuscript.

 

- In the introductio nsection, please, could the authors investigate the role of p53 in the tumorigenesis?

 

- In the methodological section, please, could the authors focus on the clinical meaning of p53 expression in different tumor types? In partcular, could the authors overview prognostic, diagnostic role in each scenario?

- In the text, could the authors add a brief analysis of the methodolgical approaches available to evaluate p53 expression?

- In the manuscript an experimental section may be considered the main limitation. Could the authros discuss this point?

Comments on the Quality of English Language

Major english editing is require

Author Response

The manuscript focuses on a review meta analysis about p53 expression in tumor patients is a timely relevant manuscript where moderate integrations should be approached to imrpove the readibility of the manuscript.

 

 

 

- In the introduction section, please, could the authors investigate the role of p53 in the tumorigenesis?

Thanks for the questions raised by experts, regarding the role of P53 in tumors, the following explanations are made: The high expression of TP53 in tumors is due to the fact that Hsp90 and Hsp70 maintain the stability of mutp53 in cancer by interacting with the DNA binding domain of mutp53. The functions of TP53 in tumors include genetic instability (promotes amplification and chromosomal instability), regulation of ferroptosis (which has been shown in most studies to promote the occurrence of iron death) and tumor  microenvironment,  acquisition of cancer stem cells (CSCs) phenotype. The hallmark feature of CSCs is their ability to produce  heterogeneous tumor cells, which are critical in the initiation and progression of cancer.

- In the methodological section, please, could the authors focus on the clinical meaning of p53 expression in different tumor types? In partcular, could the authors overview prognostic, diagnostic role in each scenario?

Thank you for pointing out the issue. According to the modification suggestions, the clinical significance, prognosis, and diagnostic analysis of TP53 in pan cancer have been added to the methodology. The analysis results are as follows: TP53 is highly expressed in most tumors. We analyzed the clinical significance of TP53 in tumors by clinicopathological T-, N-, and M-stages and found that there were differences in N-stage in COAD and KIRP, and differences in T- and N-stages in KIRC (Table 1, Figure 3A). Next, we analyzed the prognosis and diagnosis of TP53 in pancarcinoma, and the results showed that Finally observed in 5 types of tumors (TCGA - GBMLGG (N = 619, p = 4.2 e-9, HR = 1.65 (1.40, 1.94)), TCGA LGG (N = 474, p = 8.9 e-4, HR = 1.50 (1.18, 1.90)), TCGA KIPAN (N = 855 At HR = 1.19, p = 0.05 (1.00, 1.41)), TCGA - THCA (N = 501, p = 0.04, HR = 3.67 (1.10, 12.20)), the TCGA - ACC (N = 77, p = 0.02, HR = 1.81 (1.09, 3.01))) increased with poor prognosis (Figure 3B). Diagnostic analysis includes ROC curve and logistics analysis. T-, N-, M- clinicopathological staging and logistics model of TP53 are shown in Table 2. The value of the TP53 gene in diagnosis of pan-cancer was evaluated by receiver operating characteristic curves. Figure 4 show the diagnostic values of the TP53 gene in pan-cancer. The ROC analysis showed that the respective area under the curves (AUCs) of BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LIHC, LUAD, LUSC, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, THCA, THYM, and UCEC were 0.625, 0.537, 0.791, 0.987, 0.789, 0.759, 0.993, 0.550, 0.910, 0.767, 0.821, 0.723, 0.676, 0.641, 0.771, 0.507, 0.600, 0.747, 0.684, 0.638, 0.819, 0.693, 0.471, and 0.705. The results showed that AUC > 0.5 (except THYM), indicating that TP53 gene has good diagnostic value in the course of pan-cancer.

- In the text, could the authors add a brief analysis of the methodolgical approaches available to evaluate p53 expression?

Thanks for the questions raised by the experts, we conducted the following analysis to evaluate the expression of TP53 in pancarcinoma. First, by analyzing the data in the TCGA dataset, we analyzed the expression of TP53 in each tumor compared with the healthy control group. Meanwhile, we also analyzed the significance of TP53 in different clinicopathological stages in pancarcinoma and the diagnostic ROC. We analyzed the methylation, gene mutation, mRNA expression, and biological function of TP53 in pancarcinoma, and we also predicted the transcription factors that interact with TP53 to better evaluate various tumors. Finally, some potential therapeutic drugs that may target TP53 for cancer treatment are predicted, in order to lay a foundation for subsequent cancer research.

- In the manuscript an experimental section may be considered the main limitation. Could the authros discuss this point?

Thanks to the advice of the experts, the limitations have been described in the article, described below: And there are some limitations in this study. Bioinformatic analysis is an effective way to infer the role of a gene in a tumor, but it is not sufficient to fully confirm its role. Experimental verification can provide more reliable and concrete evidence to support the conclusions of bioinformatics analysis. For example, the expression of TP53 in tumor cells can be disrupted or enhanced by CRISPR/Cas9 gene editing technology, and then the characteristics of cell proliferation, apoptosis, and invasion can be observed to further confirm the role of TP53 in tumors.

 

Reviewer 2 Report

Comments and Suggestions for Authors

 

The work is potentially interesting but the manuscript is not well written, it is very difficult to read, and contains many inaccuracies.

 

“Highlight” or what should be the Introduction should “introduce” the reader to the topic. I would suggest to the authors to first provide a general description of TP53 under normal/physiological conditions and after describe the pathological conditions. Figures are generally barely readable, as for example Figure 1, Figure 4, Figure 5, Figure 6, etc. Relative Legends should provide the reader with a clear description of each panel. Acronyms/abbreviations should be opened when used for the first time and explained throughout the main text. “Material and Methods” should not include “Results”. Results are presented in a confused manner. Discussion instead to “discuss” the results/observations considered other aspects of TP53 that often are not in line with the aim of the paper. References are lacking.

 

Minor comments:

- Pag. 1, line 23: How many TP-53 genes? This sentence should read as TP-53 gene.

- I would suggest to the authors to rename the section “Method” with “Material and Methods”.

- Results should not be included in the Material and Methods section, as for example, pag. 2 line 72 and line 82.

- Pag. 4, lines 143-144: Please specify the tissue type.

- Pag. 5, lines 184-187: I would suggest to remove this paragraph since contains inaccuracies.

- Pag. 8, lines 259-262: I would suggest to remove this paragraph because it is not clear.

- Pag. 9, lines 283-285: Very confusing.

- Pag. 11, line 350: I would suggest to remove this paragraph since contains inaccuracies.

- Please provide reference for the panel B in Figure 9.

- Pag. 13, lines 376-377: This sentence should be accompanied by relative reference/s.

- Pag. 13, line 377: How did the authors make the prediction?

- Pag. 14, lines 401-407: This paragraph comes from the blue. The authors are focusing on TP53.

- Pag. 15, line 447: This sentence is incorrect. Please refer to PMC9856662.

 

 

 

Comments on the Quality of English Language

English editing is required.

Author Response

The work is potentially interesting but the manuscript is not well written, it is very difficult to read, and contains many inaccuracies.

 

 

 

“Highlight” or what should be the Introduction should “introduce” the reader to the topic. I would suggest to the authors to first provide a general description of TP53 under normal/physiological conditions and after describe the pathological conditions.

Thanks for the questions raised by the experts, the introduction part is reorganized according to the opinions of the experts, which is summarized as follows: . Under normal/physiological conditions, TP53 (also known as p53) is a tumor suppressor gene that plays a crucial role in maintaining the integrity of the genome.  It functions as a transcription factor that regulates the expression of numerous genes involved in cell cycle arrest, DNA repair, apoptosis, and senescence.  TP53 acts as a guardian of the genome by detecting DNA damage and initiating the appropriate response to prevent the propagation of damaged cells. In addition to mutations, TP53 activity can be modulated through other mechanisms. Some viruses can inactivate TP53, allowing infected cells to evade immune surveillance and promote viral replication. Additionally, various cellular stressors such as hypoxia, radiation, and chemotherapy can stabilize TP53, leading to the activation of its downstream target genes and inducing cell cycle arrest or apoptosis. Overall, TP53 plays a critical role in maintaining genomic stability and suppressing tumor formation under normal conditions. Its dysregulation through mutations or other mechanisms contributes to the development and progression of various pathological conditions, particularly cancer.

Figures are generally barely readable, as for example Figure 1, Figure 4, Figure 5, Figure 6, etc. Relative Legends should provide the reader with a clear description of each panel. Acronyms/abbreviations should be opened when used for the first time and explained throughout the main text.

Thanks for the problems pointed out by the experts. According to the opinions of the experts, the figure notes have been changed and explained in the paper, and the abbreviations have been re-marked. The full names of various cancers are shown in Supplementary Table 1, which is not added in the paper. 

Figure 1 Aberrant expression of TP53 in pan-cancer. (A) mRNA level of TP53 performed by the TIMER2 database, the blue represents the healthy control group, and the red represents the tumor patients. (B) Box plot of TP53 mRNA level in CHOL, COAD, DLBC, GBM, LAML, LCC, LUSC, OV, PAAD, READ, STAD, TGCT, THYM, and UCEC performed by the GEPIA2 database, the gray represents the healthy control group, and the red represents the tumor patients. (C) The expression of TP53 in normal tissue and OV, COAD, KIRC, LUAD, LUSC, HNSC, PAAD, and LIHC performed by CPTAC, the blue represents the healthy control group, and the red represents the tumor patients. (D) The expression of TP53 in each tumor pathological stage. Relationship between TP53 expression and tumor pathological stage performed in GEPIA2. Compared with the healthy control group, *P< 0.05; **P< 0.01; ***P < 0.001.

Figure 6 mRNA expression of TP53 in pan-cancer. (A) Mutation of TP53 mRNA expression in pan cancer, including Splice (Driver), Truncating (Driver), Inframe (Driver), Inframe (VUS), Missense (Driver), Missense (VUS), Not mutated, Not profiled for mutations, Amplification, Gain, Diploid, Shallow Deletion, Deep Deletion, Structural Variant, Not profiled for CNA and Structural Variants. (B) mRNA expression under different mutations of TP53, the horizontal coordinate represents TP53 mutation types, including Missense, Inframe, Truncating, Splice, Multiple, No mutation, and Not profiled, and the vertical coordinate represents mRNA expression. (C) Altered and mutations in TP53 fragments in pan cancer, the horizontal coordinate represents the various tumors, and the vertical coordinate represents the TP53 alters and the types of alters. (D) TP53 methylation affects mRNA expression, the horizontal coordinate represents TP53 methylation [WRAP53 (cg06587969): methylation (HM27 and HM450 merge)] and the vertical coordinate represents mRNA expression. (E) Relationship between mRNA and putative copy number of TP53, the horizontal coordinate represents the TP53 copy number, including Deep Deletion, Shallow Deletion, Diploid, Gain, and Amplification, and the vertical coordinate represents mRNA expression. (F) Relationship between TP53 mRNA expression and protein expression, the horizontal coordinate represents TP53 mRNA expression, and the vertical coordinate represents protein expression.. (G) TP53 mutations count in pan-cancer, the horizontal coordinate represents the various tumors, and the vertical coordinate represents the count of TP53 mutations.

Figure 7 Protein phosphorylation and DNA methylation of TP53 in pan-cancer. (A) CPTAC indicated the phosphorylation levels of PDHA1 at S315. (B) DNA methylation of TP53 between normal and primary tumor tissues was performed by the UALCAN database. The blue represents the healthy control group, and the red represents the tumor patients.

Figure 8 Roles of TP53 in the immune infifiltration and its score in all TCGA tumor types. (A-D) Correlation heatmap between TP53 expression and tumor infifiltrating immune cells across 33 cancer types was displayed, including T cell CD8+ (A), T cell CD4+ (B), B cell (C), and myeloid dendritic cells (D). A positive correlation was marked as red color, while a negative correlation was marked as blue color. Non significant correlations values were marked with a cross. (E-J) The correlation between TP53 expression and immune infiltration in HNSC (R=0.24) (E), LIHC (R=0.26) (F), THYM (R=0.17) (G), STAD (R=0.16) (H), LGG (R=0.01) (I), and KIRC (R=0.16) (J), the horizontal coordinate represents TP53 expression, and the vertical coordinate represents immune score.

“Material and Methods” should not include “Results”. Results are presented in a confused manner. Discussion instead to “discuss” the results/observations considered other aspects of TP53 that often are not in line with the aim of the paper. References are lacking.

Thank the experts for pointing this out. The supplementary result in the method has been removed and added to the result. The discussion has been reorganized. And add references. The changes are as follows: The high expression of TP53 in tumors is an important indicator. The significance of TNM staging for tumor is to determine the severity of the disease and predict the survival rate of patients. High expression of TP53 is associated with poor prognosis and tumor diagnosis, suggesting important information about patients' disease progression and treatment response. In addition, mutations and methylation of TP53 play an important role in the occurrence and development of tumors. The interaction and regulation between TP53 and immune infiltration and immune checkpoint further indicate the potential role of TP53 in tumor immunotherapy. TP53 may play a crucial role in the biological processes of cancer progression, including a positive correlation with differentiation, metastasis, inflammation, proliferation, and quiescence, and a negative correlation with DNA repair, DNA damage, cell cycle, and apoptosis. Many other transcription factors associated with TP53 include AURKA, BARD1, CDK2, CREBBP, DDX5, EP300, GSK3B, KAT5, MDM2, and RPA1. These transcription factors, together with TP53, are involved in many biological processes such as cell cycle regulation, DNA repair, apoptosis, cell migration and metastasis, and play an important regulatory role in the occurrence and development of tumors. For example, AURKA inhibited DNA damage response by suppressing the expression of various DNA damage repair genes in a TP53-dependent manner. MDM2 is a well-known factor that interacts with TP53 and can regulate cell life cycle and apoptosis by binding to TP53 and inhibiting its function. In conclusion, there is a complex interaction between TP53 and these transcription factors, which plays an important regulatory role in tumorigenesis. Studying the interaction mechanism between these transcription factors and TP53 is of great significance for understanding the tumorigenesis mechanism and providing targets and strategies for tumor therapy. In-depth research and comprehensive analysis of multiple aspects related to TP53 may help to further reveal the mechanism of tumor occurrence and development, and provide an important basis for precision treatment.

 

Minor comments:

 

- Pag. 1, line 23: How many TP-53 genes? This sentence should read as TP-53 gene.

Thanks for the problems pointed out by the experts, which have been corrected in the article.

- I would suggest to the authors to rename the section “Method” with “Material and Methods”.

Thanks to the expert's advice, the "Method" has been changed to "Material and Methods".

- Results should not be included in the Material and Methods section, as for example, pag. 2 line 72 and line 82.

Thanks for the problems pointed out by the experts. According to the suggestions of the experts, the supplementary information in the method part of the paper has been transferred to the result part. Thank you for the suggestions of the experts.

- Pag. 4, lines 143-144: Please specify the tissue type.

Thanks for the opinions of experts, according to the suggestions of experts, the organization type has been added to the article: BLCA (tissue type: bladder urothelial), CESC (cervix), CHOL (bile duct), COAD (colon), ESCA (esophageal), GBM (brain), KIRC (kidney), KIRP (kidney), LIHC (liver), LUAD (lung), LUSC (lung), PRAD (prostate), READ (rectum), STAD (stomach), THCA (thyroid), and UCEC (endometrial).

- Pag. 5, lines 184-187: I would suggest to remove this paragraph since contains inaccuracies.

Thanks to expert advice, the error statement has been removed.

- Pag. 8, lines 259-262: I would suggest to remove this paragraph because it is not clear.

Thanks to expert advice, the error statement has been removed.

- Pag. 9, lines 283-285: Very confusing.

Thanks to the problems pointed out by the experts, the original text has been changed as follows: The tumor suppressor p53 maintains an equilibrium between self-renewal and differentiation to sustain a limited repertoire of stem cells for proper development and maintenance of tissue homeostasis. Inactivation of p53 disrupts this balance and promotes pluripotency and somatic cell reprogramming. A few reports in recent years have indicated that prevalent TP53 oncogenic gain-of-function (GOF) mutations further boosts the stemness properties of cancer cells. Stem cells are a rare population of cells that can perpetuate themselves through self-renewal and can give rise to mature cells of a tissue by differentiation.

- Pag. 11, line 350: I would suggest to remove this paragraph since contains inaccuracies.

Thanks for the problems pointed out by the experts, which have been modified in the article as follows: The TP53 tumor suppressor protein is a major barrier to preventing cancer from occurring and developing. In biochemistry, TP53 functions primarily as a sequence-specific transcription factor capable of binding to DNA sequences identified within the genome (called TP53 response elements or TP53 binding sites) and activating transcription of adjacent genes, as well as transcription of more distant genes regulated by enhancers with TP53 binding sites. In addition, TP53 can inhibit the transcription of a large number of genes, often through indirect mechanisms. In normal, unstressed cells, TP53 protein levels are kept low by conformational proteomic degradation, which is indicated by the E3 ubiquitin ligase MDM2, a major inhibitor of TP53. In addition, the biochemical activity of TP53 as a transcription factor is also limited by the MDM4 protein (also known as MDMX), so it is an additional physiological inhibitor of TP53.

- Please provide reference for the panel B in Figure 9.

Thanks to the questions raised by the experts, the scale has been added to the diagram.

- Pag. 13, lines 376-377: This sentence should be accompanied by relative reference/s.

Thanks to experts for their comments, references have been added.

- Pag. 13, line 377: How did the authors make the prediction?

Thanks to the opinions of experts, proteins plus database (https://proteins.plus/) was used for molecular docking, and corresponding binding pockets were obtained by calculating DoGSite scores. The optimal binding position of drugs and TP53 was obtained by docking with different binding pockets.

 

 

- Pag. 14, lines 401-407: This paragraph comes from the blue. The authors are focusing on TP53.

Thanks for the problems pointed out by the experts, changes have been made in the article as follows: In many cancer processes, transcription factors can be mutated or dysregulated through various mechanisms of action, including chromosomal translocation, gene amplification or deletion, point mutations, and expression changes. The most prominent feature of TP53 is that it is a transcription factor, and many of these target genes are related to apoptosis or cell cycle regulation, such as TP21 encoding cyclin-dependent protein kinase inhibitor and BAX encoding apoptosis precursor protein. Transcription factors play important biological roles in diseases such as cancer, autoimmune diseases, diabetes, and cardiovascular diseases. However, transcription factors have traditionally been considered "untreatable" targets because of their severe structural disorganization and lack of well-defined small-molecule binding cavities.

- Pag. 15, line 447: This sentence is incorrect. Please refer to PMC9856662.

Thank you very much for the error pointed out by the experts, according to the reference PMC9856662, it has been modified and, citing the reference, changed as follows: At present, the US FDA has approved targeted drugs against TP53. Gene therapy, targeted tumor vaccines, and anti-cancer drugs targeting TP53 mutations are in early clinical trials, including APR-246 (eprenetapopt, PRIMA-1MET), PEITC (phenethyl isothiocyanate), ATO (arsenic trioxide/Trisenox), HSP90 inhibitor (ganetespib/STA-9090), Atorvastatin, ATO/Trisenox, Vorinostat/Zolinza/SAHA, Wee1 inhibitor (adavosertib/AZD1775/MK-1775), Lamivudine (3TC/Epivir/Zeffix/DELSTRIGO) , Zoledronic acid (ZA/Reclast/Zometa) and atorvastatin.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In their original article, Tingting Liu and colleagues present a comprehensive overview of the diverse roles played by the major tumor suppressor gene product, p53. The authors delve into the potential impact of mutations in p53 on cancer development and explore therapeutic opportunities arising from these mutations. Utilizing public databases, the authors conducted an extensive investigation into these mutations across various cancer types. Their findings consistently revealed correlations between p53 mutations at both genetic and protein levels in most tumors, with some cases indicating a worse prognosis, although not universally applicable.

While the reviewer did not conduct an exhaustive examination of the supplementary tables, the employed approach by the authors appears to be valid. The manuscript addresses various aspects of p53, cancer, and ongoing early clinical studies on innovative treatment approaches. However, the authors missed the opportunity to highlight the clinical significance of p53 mutational status in certain tumor types. Notably, the World Health Organization (WHO) incorporated the distinction between p53 mutation and wildtype in specific brain tumors, such as medulloblastoma, SHH-group, in 2016 (further refined in 2021). This inclusion signifies a crucial step in clinical diagnosis, with mutated p53 associated with a poorer outcome in this context, as elucidated in a relevant review (https://pubmed.ncbi.nlm.nih.gov/36829544/).

To enhance the manuscript, the following minor issues could be addressed:

• Line 23: Suggest using the singular form: TP53 gene.

• Line 35: Ensure clarity regarding the reference of "its."

• Line 51: Please review for grammar.

• Lines 52/53: Include a reference and consider adding a brief sentence on how the Hepatitis B virus may lead to p53 mutations in liver cancer.

• Line 55: Suggest incorporating the following points:

  • Highlight that p53 mutations are not always a late event in tumor development/progression, citing an example such as low-grade astrocytomas/gliomas/brain tumors (von Deimling, A. et al. 1992. https://pubmed.ncbi.nlm.nih.gov/1349850/).
  • Emphasize the use of p53 mutations in differentiating similar tumors in the new WHO classification of tumors of the nervous system (2016 and 2021), with specific reference to medulloblastoma (Ohgaki, H. et al. 1991. https://pubmed.ncbi.nlm.nih.gov/1933879/).
  • Highlight that p53 mutational status can have varying implications for different medulloblastoma groups, as discussed in the provided reference (https://pubmed.ncbi.nlm.nih.gov/36829544/).

• Supplementary Table 2: Verify the correctness of the data, specifically the repetition of the number 207 in brain tumors, GBM and low-grade glioma.

• Figure S5: Address the discrepancy in the correlation between GBMLGG and GBM, discussing how the studies differ.

• Line 410: Clarify and correct for accuracy: "There mutations are high probability of TP53 mutations in tumors."

 

Author Response

In their original article, Tingting Liu and colleagues present a comprehensive overview of the diverse roles played by the major tumor suppressor gene product, p53. The authors delve into the potential impact of mutations in p53 on cancer development and explore therapeutic opportunities arising from these mutations. Utilizing public databases, the authors conducted an extensive investigation into these mutations across various cancer types. Their findings consistently revealed correlations between p53 mutations at both genetic and protein levels in most tumors, with some cases indicating a worse prognosis, although not universally applicable.

 

While the reviewer did not conduct an exhaustive examination of the supplementary tables, the employed approach by the authors appears to be valid. The manuscript addresses various aspects of p53, cancer, and ongoing early clinical studies on innovative treatment approaches. However, the authors missed the opportunity to highlight the clinical significance of p53 mutational status in certain tumor types. Notably, the World Health Organization (WHO) incorporated the distinction between p53 mutation and wildtype in specific brain tumors, such as medulloblastoma, SHH-group, in 2016 (further refined in 2021). This inclusion signifies a crucial step in clinical diagnosis, with mutated p53 associated with a poorer outcome in this context, as elucidated in a relevant review (https://pubmed.ncbi.nlm.nih.gov/36829544/).

Thanks to the experts for their valuable opinions, relevant descriptions have been added to the article, which are described as follows: Notably, the World Health Organization (WHO) incorporated the distinction between TP53 mutation and wildtype in specific brain tumors, and TP53 mutational status can have varying implications for different medulloblastoma groups, for example, divided into sonic hedgehog (SHH)-activated and TP53-wildtype, SHH-activated and TP53-mutant.

To enhance the manuscript, the following minor issues could be addressed:

Line 23: Suggest using the singular form: TP53 gene.

Thanks for the problems pointed out by the experts, which have been corrected in the article.

Line 35: Ensure clarity regarding the reference of "its."

Thanks to the errors pointed out by experts, the first “its” refers to TP53 gene and the second “its” refers to tumor cells, which have been corrected in the paper.

Line 51: Please review for grammar.

Thanks to the grammar error pointed out by the experts, it has been changed as follows: Studies have shown that some environments and diets can directly cause mutations in the TP53 gene and aflatoxin B1 in foods, so it is necessary to avoid eating moldy foods, which may contain aflatoxin.

Lines 52/53: Include a reference and consider adding a brief sentence on how the Hepatitis B virus may lead to p53 mutations in liver cancer.

Thanks to the suggestions of experts, relevant descriptions have been added to the article, which are described as follows: The hepatitis B (HBV) and hepatitis C viruses (HCV)-related carcinogenesis initiates in the context of chronic hepatitis, and progresses to HCC in a multistep process lasting for as long as 30 years. During hepatocellular carcinoma (HCC) progression, several environmental factors  as well as host co-factors (elevated serum androgen levels, genetic polymorphisms, DNA repair enzymes) may synergize and lead to progressive accumulation of multiple genomic changes in the hepatocytes. Among these, non-synonymous mutations in TP53  gene are well known cancer drivers for HCC development with variable frequencies depending on the underlying etiology.

Line 55: Suggest incorporating the following points:

Highlight that p53 mutations are not always a late event in tumor development/progression, citing an example such as low-grade astrocytomas/gliomas/brain tumors (von Deimling, A. et al. 1992. https://pubmed.ncbi.nlm.nih.gov/1349850/).

Thanks for the problems pointed out by the experts, and thanks for the references provided by the experts, the following modifications have been made in the article: The studies show that p53 mutations are not restricted to glioblastoma multiforme and may be important in the tumorigenesis of lower-grade astrocytomas and that p53 mutations in lower-grade astrocytomas are associated with loss of chromosome 17p.

Emphasize the use of p53 mutations in differentiating similar tumors in the new WHO classification of tumors of the nervous system (2016 and 2021), with specific reference to medulloblastoma (Ohgaki, H. et al. 1991. https://pubmed.ncbi.nlm.nih.gov/1933879/).

Thanks for the problems pointed out by the experts, and thanks for the references provided by the experts, the following modifications have been made in the article: The use of p53 mutations in differentiating similar tumors in the new WHO classification of tumors of the nervous system. Moreover, previous studies have shown that the tumor suppressor gene TP53 mutates in primary human brain tumors.

Highlight that p53 mutational status can have varying implications for different medulloblastoma groups, as discussed in the provided reference (https://pubmed.ncbi.nlm.nih.gov/36829544/).

Thanks for the problems pointed out by the experts, and thanks for the references provided by the experts, the following modifications have been made in the article: TP53 mutational status can have varying implications for different medulloblastoma groups. For example, divided into SHH-activated and TP53-wildtype, SHH-activated and TP53-mutant.

Supplementary Table 2: Verify the correctness of the data, specifically the repetition of the number 207 in brain tumors, GBM and low-grade glioma.

Thanks to the expert who pointed out the problem, has been confirmed, correct.

Figure S5: Address the discrepancy in the correlation between GBMLGG and GBM, discussing how the studies differ.

Thank you to the experts for pointing out the issue. We have reanalyzed the correlation between TP53 and tumor stemness. The issue that needs to be pointed out is that previously included in the analysis were DNAss: DNA methylation based (Stem cell signature probes (219 probes), which combined the 3 signatures listed below. However, the reanalysis used RNA expression based (All set of available genes), which resulted in a wider gene set and more accurate results. After reanalysis, both GBMLGG and GBM showed a positive correlation.

Line 410: Clarify and correct for accuracy: "There mutations are high probability of TP53 mutations in tumors."

Thanks to the problems pointed out by the experts, the inaccurate description in the article has been deleted.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors greatly improved the manuscript.