SOCS1 as a Biomarker Candidate for HPV Infection and Prognosis of Head and Neck Squamous Cell Carcinomas

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

cimb-2411519

Title: “SOCS1 as a biomarker candidate for HPV infection and prognosis of head
and neck squamous cell carcinomas"

Authors: Guo et al.

Here, the authors presented a study addressing the molecular differences of HPV negative versus positive head and neck tumors (HNSCC). Therefore they analyzed the HNSCC dataset of The Cancer Genome Atlas (TCGA) to identify differentially expressed genes. In addition to the bioinformatics, they performed immunohistochemical staining of one potential marker protein in HNSCC tissue samples, as well as analysis of clinical data (survival). This workflow resulted in a potential biomarker candidate, namely SOCS1. SOCS1 downregulation was correlated with poor survival in the examined HNSCC cohort.

 

In general, objective of the study are relevant to the field aiming at elucidation of molecular mechanisms resulting in differing pathogenesis of HPV positive HNSCC. However, I feel that the results are quite sparse and also poorly presented providing not convincing evidence. Main criticism also concerns that authors overall argumentation line is not reflected by reality in the clinics where HPV-positive HNSCC patients exhibit better survival and prognosis compared to HPV negative (see below). In summary, I am unfortunately unable not recommend acceptance of the report for publication in CIMB in its current form.

 

All parts of the manuscript need extensive revision:

1.     Introduction: very short, needs more information about SOCS1, differences in prognosis of HPV-related HNSCC, and so on

2.     Materials and Methods also lack details, eg for IHC staining procedure, and statistics

3.     Results: presentation and description of the results is really poor! Many formal defects: Figure legends below, not above Figure, resolution is poor, hardly readable (eg survival curves in Fig 6), complete results of DEGs should be provided as Suppl. Information, Fig.5A what is shown? No valuable information content!, and so on…

4.      Discussion: the line of argumentation is NOT transparent! Authors argue with invasive character, high recurrence rates, and low survival rates of HNSCC, but this is mainly NOT due to HPV infection! HPV positive patients exhibit significant better prognosis. Authors have to clarify that!

 

 

 

Comments on the Quality of English Language

English and writing in general should be improved.

Author Response

Manuscript Code: cimb-2411519

Manuscript Title: SOCS1 as a biomarker candidate for HPV infection and prognosis of head and neck squamous cell carcinomas

 

Authors: Manli Guo, Lijie Zhang, Huihui Wang, Qiaozhen Zhou, Xinrang Zhu, Xinyu Fu, Jinlong Yang, Shanhe Liu, Dingcheng Guo, Baoping Zhang.

 

We would like to thank the reviewers for their insightful comments. Those comments are all valuable and useful for revising and improving our paper. We have studied comments carefully and have made corrections accordingly. Modified portions are highlighted in the revised paper. Below is our point-to-point response to each specific comment. Please let us know if anything remains unclear.

 

A point-by-point response to the reviewers’ comments

Reviewer 1

Comment 1: Introduction: very short, needs more information about SOCS1, differences in prognosis of HPV-related HNSCC, and so on.

Response: Thank you for your kind suggestion. We have re-written the introduction section, please check it work or not.

We state “Head and neck cancers (HNC) are a group of malignant tumors originating from the oral cavity, oropharynx, hypopharynx and larynx (1). According to Global Cancer Statistics 2020, approximately 745,000 people are diagnosed with HNC worldwide, making HNC the 8th most common malignancy (2). Head and neck squamous cell carcinoma (HNSCC) is the most prevalent pathological type of HNC, which is strongly associated with heavy use of tobacco and alcohol, as well as genetics, exposure to toxic substances, diet, and the environment (3-5). Human papillomavirus (HPV) infection, particularly with the HPV16 subtype, is also a key factor in the development of HNSCC (6). There has been a marked increase in the incidence of HPV-positive HNSCC which now represents the most common form of oropharyngeal cancer in many western populations (7, 8). HPV-positive HNSCC is associated with improved prognosis compared with HPV-negative HNSCC (9-11). However, the exact mechanism by which HPV infection is involved in the development of HNSCC remains unclear. Many patients with HNSCC present with locally advanced disease, frequently with prominent involvement of the lymph nodes, which is associated with a poor prognosis (12). Therefore, it is vital to identify biomarkers for early diagnosis of HNSCC to improve treatment and prognosis.

The suppressor of cytokine signaling (SOCS) family of proteins are negative-feedback inhibitors of signaling induced by cytokines that act via the JAK/STAT pathway (13). SOCS1, the most potent member, has been the most extensively discussed. SOCS1 has been found to be silenced in primary tumors in >50% of hepatocellular carcinoma (14), 44% of gastric carcinoma (15), 75% of melanoma(16), 40% of hepatoblastoma cases , 53–71% of pancreatic cancers and in solid tumors in 60% of acute myeloid lymphoma (17) and 62% of multiple myeloma (18). However, the correlation between SOCS1 and HNSCC is rarely reported.

”.

References

1. Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. Head and neck squamous cell carcinoma. Nat Rev Dis Primers. 2020;6(1):92.
2. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021.
3. Marquard FE, Jücker M. PI3K/AKT/mTOR signaling as a molecular target in head and neck cancer. Biochem Pharmacol. 2020;172:113729.
4. Shahoumi LA, Yeudall WA. Targeted therapies for non-HPV-related head and neck cancer: challenges and opportunities in the context of predictive, preventive, and personalized medicine. EPMA J. 2019;10(3):291-305.
5. Cohen N, Fedewa S, Chen AY. Epidemiology and Demographics of the Head and Neck Cancer Population. Oral Maxillofac Surg Clin North Am. 2018;30(4):381-95.
6. von Witzleben A, Wang C, Laban S, Savelyeva N, Ottensmeier CH. HNSCC: Tumour Antigens and Their Targeting by Immunotherapy. Cells. 2020;9(9).
7. Chaturvedi AK, D'Souza G, Gillison ML, Katki HA. Burden of HPV-positive oropharynx cancers among ever and never smokers in the U.S. population. Oral Oncol. 2016;60:61-7.
8. Chaturvedi AK, Anderson WF, Lortet-Tieulent J, Curado MP, Ferlay J, Franceschi S, et al. Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers. J Clin Oncol. 2013;31(36):4550-9.
9. Rischin D, Young RJ, Fisher R, Fox SB, Le Q-T, Peters LJ, et al. Prognostic significance of p16INK4A and human papillomavirus in patients with oropharyngeal cancer treated on TROG 02.02 phase III trial. J Clin Oncol. 2010;28(27):4142-8.
10. Fakhry C, Westra WH, Li S, Cmelak A, Ridge JA, Pinto H, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008;100(4):261-9.
11. Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tân PF, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med. 2010;363(1):24-35.
12. Solomon B, Young RJ, Rischin D. Head and neck squamous cell carcinoma: Genomics and emerging biomarkers for immunomodulatory cancer treatments. Semin Cancer Biol. 2018;52(Pt 2):228-40.

Comment 2: Materials and Methods also lack details, eg for IHC staining procedure, and statistics.

Response: Thank you for careful comment. We feel sorry for this omission and added the detail description of methodology, please see below for revision.

We state “Analysis of differentially expressed genes (DEGs)

The annotations that were obtained from TCGA gene expression matrix were converted into standard gene names with official gene symbols using the cluster Profiler of R software (v 3.18.1) and org.Hs.eg.db (v 3.12.0). Furthermore, the gene expression matrix was converted to a standardized gene expression matrix file to facilitate differential gene expression analysis.

The DEGs between the HPV fluorescence in situ hybridization (FISH)-positive sample group and HPV FISH-negative sample group were analyzed using the limma package of R software (v 3.46.0). The log2 fold change (log2 FC) and P value for each gene were also obtained. The screening criterion for DEGs was P < 0.05. In order to investigate the diagnostic value of these top ten genes, we evaluated its diagnostic value using the ROC curve.

 

KEGG pathway and GO function enrichment analysis

To predict the biological function of DEGs between the HPV positive and the HPV negative samples further, the KOBAS-i database (kobas.cbi.pku.edu.cn) (1) and Gene Ontology (GO; http://geneontology.org/) (2) was used to carry out Kyoto encyclopedia of genes and genomes (KEGG) pathway and gene ontology (GO) function enrichment analyses. The screening thresholds were set to count > 2 and P < 0.05. The ggplot2 in the R software (v 3.3.3) was employed to visualize all important KEGG signaling pathways and GO biological processes.

 

Protein-protein interaction analysis of DEGs and Hub genes screening

DEGs were imported into the STRING database for analysis of protein-protein interactions (PPI) (23). The lowest interaction score was set to the highest confidence level (0.400) to obtain data on the interactions between proteins encoded by DEGs. PPI were visualized using Cytoscape (v 3.8.2) to create a functional protein association network, meanwhile, based on the top 10 results of the dependable maximum net capability (DMNC) algorithm, hub genes with high connectivity in the associated network were identified using the CytoHubba plug-in.

 

Survival analysis of hub genes

Interactive gene expression profiling interactive analysis (GEPIA) was used to investigate the relationship between hub gene expression and HNSCC prognosis (24). The survival curves of the hub gene in patients with HNSCC were plotted by survival analysis of hub genes screened using GEPIA. Statistical significance was set at P < 0.05.

 

Single sample gene set enrichment analysis (ssGSEA)

A single sample's gene expression profile can be changed into a gene set enrichment profile using ssGSEA. The density of immune cells that infiltrate tumors is represented by the enrichment score of a gene set associated with immune cells (25).

The gene set specific to the immune cell population was obtained from a molecular feature database. The GSVA package of R software (v 1.38.2) was used to characterize and quantify the difference in infiltrating immune cells between the HPV-positive and HPV-negative sample groups from the gene expression data of patients with head and neck cancer, and to determine the difference in infiltration of different immune cell populations. Metabolic pathway gene sets were obtained from a molecular signature database. Using the GSVA package of R, the metabolic differences between the HPV-positive and HPV-negative sample groups were explored.

 

Tissue preparation

The study protocol was approved by the Ethics Committee of First Hospital of Lanzhou University (No. LDYYLL-2022-321). HNSCC tissue samples were collected from 36 patients at the Department of Oral and Maxillofacial Surgery, First Hospital of Lanzhou University, after obtaining written informed consent. All specimens obtained from a typical area of the lesion were analyzed by two qualified pathologists to rule out additional diseases, fixed in 10% neutral-buffered formalin, and embedded in paraffin.

 

Immunohistochemistry

The expression of SOCS1 in the hematoxylin and eosin-stained sections was detected using the SP method. The sections were cut into 4 μm pieces and fixed in 4% neutral paraformaldehyde in 0.1 M phosphate buffer. They were then paraffin‐embedded, dewaxed and dehydrated with gradient ethanol. This was followed by incubation with rabbit anti-human polyclonal SOCS1 antibody (ab137384 at 1/100 dilution, Abcam) at 4°C and then with a secondary antibody (HistostainTM-Plus Kits, SP-9000) at 37°C for 1 h. 3, 3′-diaminobenzidine (DAB, Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) was used during dehydration, tissue clearing, and neutral resin sealing. An Olympus BX53 microscope was used to visualize staining.

 

Statistical analysis

Statistical analysis was performed using SPSS 26.0 (IBM, America). The data were represented as mean with standard error and statistically analyzed using one-way ANOVA. The correlation between clinical features and HPV status was examined by Pearson's chi-square test based on the computed odds ratios and 95% confidence intervals. The significance level was chosen at P < 0.05”.

References

[1] Bu D, Luo H, Huo P, Wang Z, Zhang S, He Z, et al. KOBAS-i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis. Nucleic Acids Res. 2021;49(W1):W317-w25.

[2] Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25-9.

 

Comment 3: Results: presentation and description of the results is really poor! Many formal defects: Figure legends below, not above Figure, resolution is poor, hardly readable (eg survival curves in Fig 6), complete results of DEGs should be provided as Suppl. Information, Fig.5A what is shown? No valuable information content! and so on.

Response: Thank you for insightful comments. We have done an effort to improve our presentation in result section. We have deleted the figures in manuscript because they will inevitably be compressed in word, please see the files we uploaded, which meet the requirement of journal, in submission system for revision. As well, we have provided results of DEGs in supplementary table S1 and remade the figure 5A. Please check it.

Comment 4: Discussion: the line of argumentation is NOT transparent! Authors argue with invasive character, high recurrence rates, and low survival rates of HNSCC, but this is mainly NOT due to HPV infection! HPV positive patients exhibit significant better prognosis. Authors have to clarify that!

Response: Thank you for constructive feedback. We have added some content to support our conclusion. Please see below for revision.

We state “Although high-risk HPV is an important oncogenic factor in the development of HNSCC, our clinical research affirmed that survival months increases with SOCS1 expression in the same HPV status HNSCC, which matches the results we described above. However, patients with HPV-positive HNSCC have a higher three-year survival rate, which may be because HPV-positive HNSCC is more sensitive to radiotherapy and chemotherapy via complete p53-mediated apoptotic response (1) and more patients are willing to receive multidisciplinary treatment (2, 3). Besides, the extant literature indicated that the risk factors and demographic and tumor characteristics of HPV-positive patients differed from those of HPV-negative patients (4), HPV-positive tumors were more likely than HPV-negative tumors to arise from the oropharynx, to be poorly differentiated, and to have basaloid features (5). By the way, SOCS1 can exhibited tumor in certain classification such as HPV (+) and HPV (-) in our study. These investigation may imply that HPV-positive HNSCC is a unique clinical classification from HPV-negative HNSCC (3)”.

References

1. Sun Y, Wang Z, Qiu S, Wang R. Therapeutic strategies of different HPV status in Head and Neck Squamous Cell Carcinoma. Int J Biol Sci. 2021;17(4):1104-18.
2. Göttgens EL, Ostheimer C, Span PN, Bussink J, Hammond EM. HPV, hypoxia and radiation response in head and neck cancer. Br J Radiol. 2019;92(1093):20180047
3. Leemans CR, Snijders PJF, Brakenhoff RH. The molecular landscape of head and neck cancer. Nature Reviews Cancer. 2018;18(5):269-82
4. Gillison ML. Human papillomavirus-associated head and neck cancer is a distinct epidemiologic, clinical, and molecular entity. Semin Oncol. 2004;31(6):744-54.
5. Fakhry C, Westra WH, Li S, Cmelak A, Ridge JA, Pinto H, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008;100(4):261-9.

 

 

 

 

 

We appreciate for Editors/Reviewers’ warm work, and hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Sincerely,

Baoping Zhang

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript Guo et. al. analyzed differential gene expression between HPV-positive versus HPV-negative HNSSC for TCGA gene expression datasets. DEG between two groups were further analyzed for KEGG and GO pathway, and protein-protein interaction (PPI) analysis. Using CytoHubba plug-in and MCC algorithm 10 hub protein-protein interactions genes, including SOCS1, were described. An association of overall survival was found with SOCS1 among the hub genes.  Further on, no difference in immune cells signature was found between groups, however, certain changes in metabolic signatures were noted. Additionally, Significant decreased in SOCS1 protein expression was found in an independent cohort of HNSSC. Overall, several bioinformatic analysis and IHC analysis in independent cohort highlighted SOCS1 as a potential predictor of HNSSC outcome and HPV status. The findings are interesting and may have potential implication in further studies on HNSSC. However, it is not clear why TOP hits for each analysis were not tested for subsequent analysis. For example, top DEGs were not tested for the survival association analysis. Same is true for TOP pathway hits. Additionally, the presentation of the manuscripts needs significant improvement.

General comment on the manuscript presentation:

1.     For all the figures: Most of the texts in Figure are illegible, therefore inaccessible to make proper conclusion of the data.

2.     Each result section should briefly describe why a specific analysis was done and how it is done before presenting the result and conclusions.

Specific comments:

3.     Figure 2A: There are only few genes above the cutoff values (p> -log10p1.3, Log2FC>+-1). Does this plot reflect 300 upregulated and 481 downregulated DEGs (P < 0.05) describe in line 140? What cutoffs were used for the respective list/plot? Cutoffs should be consistent in both instances. Figure legend should define color codes. Highlight SOCS1 on this volcano plot. Where does it fall in respect to the cutoffs?

4.     Table 1: What are the predictive values of these top 10 DEGs mRNA expression with patients’ survival?

5.     Figure 2B: This panel is also insufficiently described in the figure legend. For instance, the group assignment in green and purple are not defined in the legend.

6.     Figure 3-4: Both the KEGG and GO analysis highlighted “olfactory transduction” and “detection of chemical stimulus involved in sensory perception”, respectively, as top pathway in high expressing DEG. What are the genes in the DEGs overlap with these two pathways? An association analysis of these genes’ expression with overall patient survival should be tested.

Author Response

Manuscript Code: cimb-2411519

Manuscript Title: SOCS1 as a biomarker candidate for HPV infection and prognosis of head and neck squamous cell carcinomas

 

Authors: Manli Guo, Lijie Zhang, Huihui Wang, Qiaozhen Zhou, Xinrang Zhu, Xinyu Fu, Jinlong Yang, Shanhe Liu, Dingcheng Guo, Baoping Zhang.

 

We would like to thank the reviewers for their insightful comments. Those comments are all valuable and useful for revising and improving our paper. We have studied comments carefully and have made corrections accordingly. Modified portions are highlighted in the revised paper. Below is our point-to-point response to each specific comment. Please let us know if anything remains unclear.

 

Comment 1: For all the figures: Most of the texts in Figure are illegible, therefore inaccessible to make proper conclusion of the data.

Response: Thank you for your kind suggestion. We have deleted the figures in manuscript because they will inevitably be compressed in word, which leading to illegibility of figures, please see the files we uploaded, they meet the requirement of journal, in submission system for revision.

Comment 2: Each result section should briefly describe why a specific analysis was done and how it is done before presenting the result and conclusions.

Response: Thank you for careful comment. We feel sorry for this omission and rewrote the result section, please see the new manuscript for revision.

Comment 3: Figure 2A: There are only few genes above the cutoff values (p> -log10p1.3, Log2FC>+-1). Does this plot reflect 300 upregulated and 481 downregulated DEGs (P < 0.05) describe in line 140? What cutoffs were used for the respective list/plot? Cutoffs should be consistent in both instances. Figure legend should define color codes. Highlight SOCS1 on this volcano plot. Where does it fall in respect to the cutoffs?

Response: Thank you for helpful comments. |log2 FC| ≥ 0.6, P < 0.05 has been set for cutoff values in this work, we have remade the figure 2A so upregulated and downregulated DEGs can be seen, and SOCS1 and its relative position of cutoff has been marked, please see our new figure 2A for revision.

Comment 4: Table 1: What are the predictive values of these top 10 DEGs mRNA expression with patients’ survival?

Response: Thank you for constructive feedback. We have added the survival of top 10 DEGs in result section, please see Supplementary Figure S1, S2 for revision.

Comment 5: Figure 2B: This panel is also insufficiently described in the figure legend. For instance, the group assignment in green and purple are not defined in the legend.

Response: Thank you for kind suggestion, we have improved the figure legends and defined the meaning of different colors, please see the new figure 2B for revision.

Comment 6: Figure 3-4: Both the KEGG and GO analysis highlighted “olfactory transduction” and “detection of chemical stimulus involved in sensory perception”, respectively, as top pathway in high expressing DEG. What are the genes in the DEGs overlap with these two pathways? An association analysis of these genes’ expression with overall patient survival should be tested.

Response: Thank you for careful feedback. We have analyzed the survival of overlap DEGs in top pathways, please see “KEGG and GO analysis of DEGs” in result section and Supplementary Figure S3 for revision.

 

We appreciate for Editors/Reviewers’ warm work, and hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Sincerely,

Baoping Zhang

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors provided an improved manuscipt, and also addressed the concerns. However, there are still some minor concerns, especially concerning formalities like figure presentation, introduction lack something like a short decription of the aim of this study, and so on. After second extensive revision of these formalities, I would recommend to accept it for publication.

Comments on the Quality of English Language

Writting and English should be revised (e.g. punctuation errors)

Author Response

Manuscript Code: cimb-2411519

Manuscript Title: SOCS1 as a biomarker candidate for HPV infection and prognosis of head and neck squamous cell carcinomas

 

Authors: Manli Guo, Lijie Zhang, Huihui Wang, Qiaozhen Zhou, Xinrang Zhu, Xinyu Fu, Jinlong Yang, Shanhe Liu, Dingcheng Guo, Baoping Zhang.

 

We would like to thank the reviewers for their insightful comments. Those comments are all valuable and useful for revising and improving our paper. We have studied comments carefully and have made corrections accordingly. Modified portions are highlighted in the revised paper. Below is our point-to-point response to each specific comment. Please let us know if anything remains unclear.

 

A point-by-point response to the reviewers’ comments

Reviewer 1

Comment 1: The authors provided an improved manuscript, and also addressed the concerns. However, there are still some minor concerns, especially concerning formalities like figure presentation, introduction lack something like a short decription of the aim of this study, and so on. After second extensive revision of these formalities, I would recommend to accept it for publication.

Response: Thank you for your kind suggestion. We have added the content you mentioned in introduction section, please check it work or not.

We state “Through bioinformatic analysis of the HNSCC sample base, we aim to investigate the molecular regulatory mechanisms related to the occurrence of HPV and HNSCC, and develop a new direction in bioinformatics analysis that may be useful in the prevention, diagnosis, and treatment of HPV-related head and neck tumors.”

In addition, we have further improved the quality of figures and put them in appropriate place according to author instruction in submission system, please see our new manuscript for review.

Comment 2: Writing and English should be revised (e.g. punctuation errors)

Response: Thank you for careful comment. We have improved the English language through the help of editage.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Revised manuscript addressed all my comments and improved significantly. 

Author Response

Thank you a lot for your warm work and careful feedback in our manuscript.