Background We examined PD-L1 expression on tumor cells (TCs) and immune cells (ICs) and density of CD3(+)and CD8(+)tumor-infiltrating lymphocytes (TILs) in patients with oropharyngeal squamous cell carcinoma (OPSCC) and investigated their significance on clinicopathological characteristics and clinical outcomes. Methods In a cohort of 65 patients treated by definitive intensity-modulated radiotherapy (IMRT) with curative intent, immunohistochemical analysis of PD-L1 expression on TCs and ICs, and TIL subtyping was performed on primary biopsy tumor tissues, followed by prognostic evaluation of these immune response-related parameters including classification into four tumor immune microenvironment (TIM) types. To evaluate HPV status, p16 immunohistochemistry was performed. Results Densities of CD3(+)and CD8(+)TILs and PD-L1 expressions on TCs and ICs were significantly higher in p16+/HPV-mediated OPSCC. Patients with high densities of stromal CD8(+)TILs displayed significantly better overall survival (OS) and progression-free survival (PFS). PD-L1 expression neither on tumor cells nor on immune cells affected survival outcomes. Distribution of TIM types based on the combination of PD-L1 expression on TCs and densities of CD8(+)TILs is significantly different in p16+ compared with p16- OPSCC. In type III TIM (TC-PD-L1+/low CD8(+)TIL density), significantly better OS was shown in p16+ group compared with p16- OPSCC. Conclusion The prognostic and predictive role of tumor immune microenvironment was confirmed for patients with OPSCC. Combining HPV status with the evaluation of densities of CD8(+)TILs and PD-L1 expression including TIM classification might be of high clinical interest and warrants further prospective evaluation.
The tumor immune microenvironment and its implications for clinical outcome in patients with oropharyngeal squamous cell carcinoma
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2020/05/2727-May-2020
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Author Response
2020/05/24Dear reviewers,
Thank you very much for your time and effort necessary to read our manuscript. Your insightful comments and suggestions have been immensely helpful. We have addressed each comment individually as outlined below. The revision has been approved by all authors. All changes in the manuscript are highlighted using a "tracking changes" function.
Reviewers' Comments to the Author:
Reviewer: 1Comments to the Author:
I read the manuscript, which was recently submitted to J of oral pathology and medicine, entitled as "The Tumor Immune Microenvironment and Its Implications for Clinical Outcome in Patients with Oropharyngeal Squamous Cell Carcinoma" with great interest. The authors conducted immunohistochemical stains of PD-L1 and CD markers using 65 patients treated by IMRT. They concluded that Combining HPV status with evaluation of densities of CD8+ TILs and PD-L1 expression including TIM classification might be of high clinical interest. Study design of this manuscript was relatively well and the results showing here seem to be plausible. However, the following could be reconsidered.- As I mentioned, the results the authors showed here are plausible. Most of data is comparable with previous reports. However, the novel findings are quite limited in pathological aspect. The data authors showed here have already published in other reports.
Answer: Thank you very much for your review. Presented study emphasizes overall importance and relevance of reproducible research. Using similar data analyses, we aimed to validate the conclusions of previous authors and possibly reach new observations that could further undermine complexity of used data analyses. Our data were obtained from real world evidence, outside of clinical trials.
- Al patients of your cohort underwent IMRT based treatment. The authors did not mention the association between treatment types and several parameters in detail. This might give useful information to readers. Please consider the association especially of patient's prognosis.
Answer: Further analysis of each treatment type has not been performed due to a high risk of bias. Our main inclusion/exclusion criterium was definitive non-surgical treatment based on definitive IMRT with curative intent. The concomitant systemic treatment was under the discretion of applying physician mostly depending on performance status and no medical contraindications of systemic therapy. Some of the patients received definitive IMRT, some of them definitive IMRT with concomitant chemotherapy and the third group were those with definitive IMRT with concomitant cetuximab (especially in the presence of cisplatinum contraindications) as specified in the article. So we could emphasise that the main and curative treatment modality was the IMRT. Of course we must agree, that the treatment type - especially the concomitant platinum based chemotherapy has an impact on the treatment results (e.g overall at least 8% due to the widely accepted MACH –NC analysis), but in our case the further fragmentation of the group to smaller cohorts according to the type of concomitant systemic therapy unfortunately ends up with the small patient groups insufficient for reliable statistical analyses.
- In figure 4 D to F, patients with p16 negative in TIM III significantly showed worse prognosis in OS, but did not in PFS and LR. These results seem to be somewhat strange. I think that IMRT could contribute to LR. Compared with Kaplan-Meier curves of OS and PFS, the curve of OS is steeper than that of PFS. What is the main cause of death of the patients? Are all of death associated with cancer?
Answer: We highly appreciate this comprehensive insight on how to improve our work. We double checked all performed analyses and found incorrectness in the description of methodology – patients' death should be for any reason. Definition of overall survival was corrected in the manuscript. We also acknowledge general limitations in all retrospective studies, especially in the evaluation of progression-free survival. Considering no regular – per clinical trial protocol – schedule for follow up, there is an inherent bias in the diagnosis of progression. This correlates with real world data evidence of patients treated outside of clinical trials.
- p10 line 44, CD3+→CD3+
Answer: Corrected in the manuscript.
- Table3 is not arranged well. Blue area showed inappropriately.
Answer: Corrected in the manuscript.
Reviewer: 2
Comments to the Author:
The article is relevant and presents a good organization, with all the textual components required by the Journal of Oral Pathology and Medicine. The authors demonstrated the tumor immune microenvironment (TIM) implications for clinical outcome in patients with oropharyngeal squamous cell carcinoma (OPSCC). The findings are not new or exclusive (Sato et al., 2019), however, the article complies with one of the noblest precepts of the scientific method: reproducibility.
Thus, since the results may be valuable to the scientific and clinical community, I add some suggestions for a minor review.Regarding the Methods, I would like to understand the inclusion or exclusion criteria of patients and read in the main text the specific demographics and survival data collected. Regarding the immunohistochemical (IHC) analysis, should be informative include the hematoxylin type and the nature of positive and negative controls (What type of tissue? How was the procedure? Have the author used a previous scientific article as a methodological reference?). Also, inform the levels of kappa or the percent agreement between the pathologist during the ICH evaluation would increase the scientific rigor of the results (Flack et al., 1988).
Answer: Thank you very much for your valuable review and recommendations on how to improve our work. We appreciate all your suggestions and within our possibilities addressed them in the revised manuscript.
- The selection criteria have been addressed in the Methods.
- Hematoxylin type has been added to the manuscript.
- Regarding nature of positive and negative controls, fixed tissue samples from tonsils have been used. In the case of negative controls, buffer was applied on tonsils instead of antibody (https://www.abcam.com/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730.html). Regarding working protocol, we only got inspired by https://www.abcam.com/ps/products/205/ab205921/documents/ab205921%20IHC,%20WB%20&%20FC%20Protocol%20Booklet%20(clone%2028-8)v3%20(website).pdf. NordicQC up to this date does not state any recommended methods for our antibody. Due to that, the nature of positive and negative controls has not been fully addressed in the manuscript.
- Based on your suggestion, concordance between pathologists has been added to the manuscript.Minor observations for correction:
Results page 11, line 29, add a space: "p < 0.0001and"
Answer: Corrected in the manuscript.
Page 15, line 49 the authors can just use "LR" because the term was presented early in the methods.
Answer: Corrected in the manuscript.
Data analysis seems very adequate to the methods. Strictly applying similar methodologies from studies on the same topic may result in reproducibility. And to build high-impact knowledge, we need studies that bring together the most suitable methods and findings, as in this article.
The discussion is greatly structured, articulating the function of molecular components with the histological and clinical findings. Throughout every scientific argument, the authors guide the reader into the understanding of TIM classification as a possible valuable prognostic and predictive tool for patients with OPSCC. Thus, it would be relevant for the study to cite the limitation and benefits of a biopsy analysis similar to what is discussed in the study of Rasmussen et al., (2019).
Answer: Thank your very much for your highly valuable suggestion which has been accordingly addressed in the discussion.
We hope the revised manuscript will better suit the Journal of Oral Pathology & Medicine, and we thank you for your continued interest in our research.
Kindest regards,
Dominik Gurín
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- pre-publication peer review (ROUND 1)
Decision Letter
2020/04/2222-Apr-2020
Dear Dr Dominik Gurin,
We recognize that the impact of the COVID-19 pandemic may affect your ability to return your revised manuscript to us within the requested timeframe. If this is the case, please let us know.
Manuscript ID JOPM-03-20-OA-6009 entitled "The Tumor Immune Microenvironment and Its Implications for Clinical Outcome in Patients with Oropharyngeal Squamous Cell Carcinoma." which you submitted to the Journal of Oral Pathology and Medicine, has been reviewed. The comments of the reviewer(s) are included at the bottom of this letter or attached to this email.
Your paper does not appear to be acceptable for publication in the present form. However, this work seems of interest and if you can adequately meet the comments and suggestions made by the reviewer(s), we would be happy to re-review it for publication in Journal of Oral Pathology and Medicine. Therefore, I invite you to respond to the reviewer(s)' comments and revise your manuscript.
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Once again, thank you for submitting your manuscript to the Journal of Oral Pathology and Medicine and I look forward to receiving your revision.
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Professor Peter Brennan
Editor-in-Chief, Journal of Oral Pathology and MedicineReviewer(s)' Comments to Author:
Reviewer: 1Comments to the Author
I read the manuscript, which was recently submitted to J of oral pathology and medicine, entitled as “The Tumor Immune Microenvironment and Its Implications for Clinical Outcome in Patients with Oropharyngeal Squamous Cell Carcinoma” with great interest. The authors conducted immunohistochemical stains of PD-L1 and CD markers using 65 patients treated by IMRT. They concluded that Combining HPV status with evaluation of densities of CD8+ TILs and PD-L1 expression including TIM classification might be of high clinical interest. Study design of this manuscript was relatively well and the results showing here seem to be plausible. However, the following could be reconsidered.- As I mentioned, the results the authors showed here are plausible. Most of data is comparable with previous reports. However, the novel findings are quite limited in pathological aspect. The data authors showed here have already published in other reports.
- Al patients of your cohort underwent IMRT based treatment. The authors did not mention the association between treatment types and several parameters in detail. This might give useful information to readers. Please consider the association especially of patient’s prognosis.
- In figure 4 D to F, patients with p16 negative in TIM III significantly showed worse prognosis in OS, but did not in PFS and LR. These results seem to be somewhat strange. I think that IMRT could contribute to LR. Compared with Kaplan-Meier curves of OS and PFS, the curve of OS is steeper than that of PFS. What is the main cause of death of the patients? Are all of death associated with cancer?
- p10 line 44, CD3+→CD3+
- Table3 is not arranged well. Blue area showed inappropriately.
Reviewer: 2
Comments to the Author
The article is relevant and presents a good organization, with all the textual components required by the Journal of Oral Pathology and Medicine. The authors demonstrated the tumor immune microenvironment (TIM) implications for clinical outcome in patients with oropharyngeal squamous cell carcinoma (OPSCC). The findings are not new or exclusive (Sato et al., 2019), however, the article complies with one of the noblest precepts of the scientific method: reproducibility. Thus, since the results may be valuable to the scientific and clinical community, I add some suggestions for a minor review.Regarding the Methods, I would like to understand the inclusion or exclusion criteria of patients and read in the main text the specific demographics and survival data collected. Regarding the immunohistochemical (IHC) analysis, should be informative include the hematoxylin type and the nature of positive and negative controls (What type of tissue? How was the procedure? Have the author used a previous scientific article as a methodological reference?). Also, inform the levels of kappa or the percent agreement between the pathologist during the ICH evaluation would increase the scientific rigor of the results (Flack et al., 1988).
Minor observations for correction:
Results page 11, line 29, add a space: "p < 0.0001and"
Page 15, line 49 the authors can just use "LR" because the term was presented early in the methods.Data analysis seems very adequate to the methods. Strictly applying similar methodologies from studies on the same topic may result in reproducibility. And to build high-impact knowledge, we need studies that bring together the most suitable methods and findings, as in this article.
The discussion is greatly structured, articulating the function of molecular components with the histological and clinical findings. Throughout every scientific argument, the authors guide the reader into the understanding of TIM classification as a possible valuable prognostic and predictive tool for patients with OPSCC. Thus, it would be relevant for the study to cite the limitation and benefits of a biopsy analysis similar to what is discussed in the study of Rasmussen et al., (2019).
Finally, I congratulate the authors for the work done, and I appreciate the opportunity to review this paper.
References:
- Flack, V.F., Afifi, A.A., Lachenbruch, P.A. et al. Sample size determinations for the two rater kappa statistic. Psychometrika 53, 321–325 (1988). https://doi.org/10.1007/BF02294215
- Rasmussen, J.H., Lelkaitis, G., Håkansson, K. et al. Intratumor heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma. British Journal of Cancer 2019; 120, 1003–1006. https://doi.org/10.1038/s41416-019-0449-y.
- Sato F, Ono T, Kawahara A, et al. Prognostic impact of p16 and PD-L1 expression in patients with oropharyngeal squamous cell carcinoma receiving a definitive treatment. Journal of Clinical Pathology 2019;72(8):542-549.Decision letter by
Cite this decision letter
Reviewer report
2020/04/21The article is relevant and presents a good organization, with all the textual components required by the Journal of Oral Pathology and Medicine. The authors demonstrated the tumor immune microenvironment (TIM) implications for clinical outcome in patients with oropharyngeal squamous cell carcinoma (OPSCC). The findings are not new or exclusive (Sato et al., 2019), however, the article complies with one of the noblest precepts of the scientific method: reproducibility. Thus, since the results may be valuable to the scientific and clinical community, I add some suggestions for a minor review.
Regarding the Methods, I would like to understand the inclusion or exclusion criteria of patients and read in the main text the specific demographics and survival data collected. Regarding the immunohistochemical (IHC) analysis, should be informative include the hematoxylin type and the nature of positive and negative controls (What type of tissue? How was the procedure? Have the author used a previous scientific article as a methodological reference?). Also, inform the levels of kappa or the percent agreement between the pathologist during the ICH evaluation would increase the scientific rigor of the results (Flack et al., 1988).
Minor observations for correction:
Results page 11, line 29, add a space: "p < 0.0001and"
Page 15, line 49 the authors can just use "LR" because the term was presented early in the methods.Data analysis seems very adequate to the methods. Strictly applying similar methodologies from studies on the same topic may result in reproducibility. And to build high-impact knowledge, we need studies that bring together the most suitable methods and findings, as in this article.
The discussion is greatly structured, articulating the function of molecular components with the histological and clinical findings. Throughout every scientific argument, the authors guide the reader into the understanding of TIM classification as a possible valuable prognostic and predictive tool for patients with OPSCC. Thus, it would be relevant for the study to cite the limitation and benefits of a biopsy analysis similar to what is discussed in the study of Rasmussen et al., (2019).
Finally, I congratulate the authors for the work done, and I appreciate the opportunity to review this paper.
References:
- Flack, V.F., Afifi, A.A., Lachenbruch, P.A. et al. Sample size determinations for the two rater kappa statistic. Psychometrika 53, 321–325 (1988). https://doi.org/10.1007/BF02294215
- Rasmussen, J.H., Lelkaitis, G., Håkansson, K. et al. Intratumor heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma. British Journal of Cancer 2019; 120, 1003–1006. https://doi.org/10.1038/s41416-019-0449-y.
- Sato F, Ono T, Kawahara A, et al. Prognostic impact of p16 and PD-L1 expression in patients with oropharyngeal squamous cell carcinoma receiving a definitive treatment. Journal of Clinical Pathology 2019;72(8):542-549.Reviewed by
Cite this review
Reviewer report
2020/04/18I read the manuscript, which was recently submitted to J of oral pathology and medicine, entitled as “The Tumor Immune Microenvironment and Its Implications for Clinical Outcome in Patients with Oropharyngeal Squamous Cell Carcinoma” with great interest. The authors conducted immunohistochemical stains of PD-L1 and CD markers using 65 patients treated by IMRT. They concluded that Combining HPV status with evaluation of densities of CD8+ TILs and PD-L1 expression including TIM classification might be of high clinical interest. Study design of this manuscript was relatively well and the results showing here seem to be plausible. However, the following could be reconsidered.
- As I mentioned, the results the authors showed here are plausible. Most of data is comparable with previous reports. However, the novel findings are quite limited in pathological aspect. The data authors showed here have already published in other reports.
- Al patients of your cohort underwent IMRT based treatment. The authors did not mention the association between treatment types and several parameters in detail. This might give useful information to readers. Please consider the association especially of patient’s prognosis.
- In figure 4 D to F, patients with p16 negative in TIM III significantly showed worse prognosis in OS, but did not in PFS and LR. These results seem to be somewhat strange. I think that IMRT could contribute to LR. Compared with Kaplan-Meier curves of OS and PFS, the curve of OS is steeper than that of PFS. What is the main cause of death of the patients? Are all of death associated with cancer?
- p10 line 44, CD3+→CD3+
- Table3 is not arranged well. Blue area showed inappropriately.
Reviewed by
Cite this review
