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Oral Cavity Beta-Defensin Levels Are Regulated Differently during Radiotherapy in Head and Neck Cancer Patients

Appl. Sci. 2023, 13(4), 2056; https://doi.org/10.3390/app13042056

by Mutlu Keskin¹

, Emma Suomi², İlknur Harmankaya³, Didem Karaçetin³, Timo Sorsa^4,5 and Ulvi Kahraman Gürsoy^2,*

Reviewer 1:

Sezin Yuce Sari

Reviewer 2: Anonymous

Appl. Sci. 2023, 13(4), 2056; https://doi.org/10.3390/app13042056

Submission received: 27 December 2022 / Revised: 27 January 2023 / Accepted: 3 February 2023 / Published: 5 February 2023

(This article belongs to the Special Issue Oral Microbial Communities and Oral Health: Volume II)

Round 1

Reviewer 1 Report

Abstract:

Line 22: ‘Oral rinses of patients collected before radiotherapy,1st ,3rd ,6th weeks of radiotherapy treatment and 1st month following the end of radiotherapy’. Please correct this sentence as: ‘Oral rinses of patients were collected before radiotherapy, on the 1st, 3rd, and 6th weeks of radiotherapy and the 1st month following the end of radiotherapy.’

Line 24: ‘Oral hBD-1 levels elevated during radiotherapy at week 6 (p=0.019).’ Please revise as: ‘Oral hBD-1 levels were elevated … OR ‘Oral hBD-1 levels increased during radiotherapy…’

Line 25: ‘hBD-1 levels return back to pretreatment levels after the finalization of radiotherapy.’ Please revise this sentence as: ‘hBD-1 levels returned to pretreatment levels after the end of radiotherapy.’

Introduction:

Line 47: ‘While hBD-2 is only found in this tissue, suggesting that its presence in the gingival epithelium may be triggered by interactions with the microflora living there’. When the sentence is started with ‘while’, one expects it would continue with an opposite fact. Therefore, the word ‘while’ is not adequate here so please omit it.

Materials and Methods:

Lines 116-117: Please add the information when these patients applied to the clinic.

Line 119: ‘This protocols of this study…’ Please revise as ‘The protocol of this study…’

Line 125: Please revise ‘NSSN’ as ‘NCCN’

Line 158: Please revise ‘which based’ as ‘which is based’

Lines 160-161: The open form of CAL is redundant here, please erase it

Lines 185-186: Please use the open form of CT in the first sentence and omit it in the second.

Line 190: Write the open form before the abbreviation please. Planning target volume (PTV)

Lines 192-199: The whole paragraph is written in bad English. Please use ‘the’ and quotation marks where needed. Some sentences are really difficult to understand. Also, what are the doses to the PTV1 and PTV2 in operated and non-operated patients? I can only see a 66 Gy but the dose should be different in the primary tumor and neck levels depending on the surgical margin, involved neck node levels and dissected node levels. Please, rewrite this paragraph.

Lines 217-218: ‘According to the Friedman test, only hBD-1 levels differed between the groups (p=0.034)’ This is a finding and should not be in the M&M section. Please omit it.

Results:

Line 223: We do not use ‘average’ for statistical results. Please use ‘mean’. Also add ‘years’ after the age numbers.

Lines 229-231: ‘The medication used by the patients is as follows: Four of the patients use Metformin hydrochloride, three use ipratropium bromide, three use acetylsalicylic acid, two use levothyroxine sodium, one uses atorvastatin, and one uses metoprolol.’ All this sentence should be in the past tense. Please correct.

Lines 232-234: ‘The patients have been exposed to an average dose of 6481.75 (+/-567.21) cGy of radiation therapy. The patients have received an average of 31.12 (+/- 3.44) days of radiation therapy.’ This sentence should be in the past tense also, not in present perfect. Please correct. Also, not ‘average’ again, please revise to ‘mean’.

Table 1:

The percentage sign (%) should come after the numbers, not before. Please correct.

Not ‘females’ and ‘males’. Please revise as ‘female’ and ‘male’

‘Systemical status’ is not correct. Please use ‘systemic condition’ instead.

Use only ‘ASA’ in the table and write the long form in the abbreviations section under the table. OR do not use the short form at all.

Add an ‘abbreviations’ section under the Table and add the long form of SD (and ASA if you will use it).

Table 2:

Please use a full stop between decimals, not a comma.

Write the stage and grade of periodontitis either in the text or in the table. Writing in both is redundant. Please erase in one.

Lines 243-244: How many samples were collected in total? Please add.

Lines 244-246: What do you mean by substitution? Please clearly define it in the M&M section.

Line 249: Please correct Figure 1; this is Figure 2.

Figure 2:

As seen in a, I would also like to see the separate p values for b and c between each variable. Please add on the figure.

Discussion:

Line 262: ‘may be suppressed’ Please revise as ‘can be suppressed’

So many repeats here. Same things were also stated in the ‘Introduction’. Please shorten the ‘Introduction’.

The ‘Discussion’ should actually discuss the effect of radiotherapy in hBD levels but I could not see any related sentences. For example, please read ‘Radiotherapy for oral cancer decreases the cutaneous expression of host defence peptides’ by S. Koerdt et al. published Journal of Cranio-Maxillo-Facial Surgery in 2016.

To speak in general, the number of patients is too low and there is no follow-up period. In the end, I do not think it adds anything new into the literature. Also, the result is written but what is means is unclear. There is no statistical analysis for whether other factors of the patients, tumors and treatment would affect the results. Therefore, we cannot say the result is related to radiotherapy per se. Besides, the English is not good and should be revised by a native speaker. I also could not understand the role of two Finnish scientists in this study.

Author Response

Dear reviewer, thank you very much for your valuable comments. Your suggestions are highly important. Our responses to your suggestions are as follows:

Abstract:

Answer: The section you mentioned has been corrected in the article. (Line 22)

Line 24: ‘Oral hBD-1 levels elevated during radiotherapy at week 6 (p=0.019).’ Please revise as: ‘Oral hBD-1 levels were elevated … OR ‘Oral hBD-1 levels increased during radiotherapy…’

Answer: The section you mentioned has been corrected in the article. (Line 24)

Answer: The section you mentioned has been corrected in the article. (Line 25)

Introduction:

Answer: The section you mentioned has been revised in the article. (Line 50)

Materials and Methods:

Lines 116-117: Please add the information when these patients applied to the clinic.

Answer: The section you mentioned has been revised in the article. (Line 113)

Line 119: ‘This protocols of this study…’ Please revise as ‘The protocol of this study…’

Answer: The section you mentioned has been corrected in the article. (Line 117)

Line 125: Please revise ‘NSSN’ as ‘NCCN’

Answer: The section you mentioned has been corrected in the article. (Line 123)

Line 158: Please revise ‘which based’ as ‘which is based’

Answer: The section you mentioned has been corrected in the article. (Line 147)

Lines 160-161: The open form of CAL is redundant here, please erase it

Answer: The section you mentioned has been corrected in the article. (Line 149)

Lines 185-186: Please use the open form of CT in the first sentence and omit it in the second.

Answer: All section regarding radiotherapy procedure has been revised. (Line 175-199)

Line 190: Write the open form before the abbreviation please. Planning target volume (PTV)

Answer: All section regarding radiotherapy procedure has been revised. (Line 175-199)

Lines 217-218: ‘According to the Friedman test, only hBD-1 levels differed between the groups (p=0.034)’ This is a finding and should not be in the M&M section. Please omit it.

Answer: The section you mentioned has been revised in the article. Information regarding hBD-1 levels was implemented into the Results section. (Line 252)

Results:

Line 223: We do not use ‘average’ for statistical results. Please use ‘mean’. Also add ‘years’ after the age numbers.

Answer: The section you mentioned has been corrected in the article.

Answer: The section you mentioned has been corrected in the article. (Line 230-233)

Answer: The section you mentioned has been corrected in the article. (Line 234-239)

Table 1:

The percentage sign (%) should come after the numbers, not before. Please correct.

Not ‘females’ and ‘males’. Please revise as ‘female’ and ‘male’

‘Systemical status’ is not correct. Please use ‘systemic condition’ instead.

Use only ‘ASA’ in the table and write the long form in the abbreviations section under the table. OR do not use the short form at all.

Add an ‘abbreviations’ section under the Table and add the long form of SD (and ASA if you will use it). Answer: The section you mentioned has been revised in the article. (Table 1 and Line 242)

Table 2:

Please use a full stop between decimals, not a comma.

Answer: The section you mentioned has been corrected in the article. (Table 2)

Write the stage and grade of periodontitis either in the text or in the table. Writing in both is redundant. Please erase in one.

Answer: The section you mentioned has been revised in the article. Information regarding staging and grading of periodontitis have been have been erased from main text.

Lines 243-244: How many samples were collected in total? Please add.

Answer: The section you mentioned has been revised in the article. Total number of samples have been mentioned. (Line 247)

Lines 244-246: What do you mean by substitution? Please clearly define it in the M&M section.

Answer: Substitution (or replacement the value with the half of the LOD value) was used when the level of a certain analyte (in this case, hBD-1, hBD-2, and hBD-3) is below the limit of detection (LOD) of the assay. In this particular case, the levels of hBD-1 that were below the LOD were replaced with 2 pg/ml, levels of hBD-2 below the LOD were replaced with 8 pg/ml, and levels of hBD-3 below the LOD were replaced with 31 pg/ml. This replacement value is half of the LOD for each analyte. This approach is used to avoid biasing the results by assuming that a value that is not detected is zero, and this is a common practice in the scientific field. It was defined in Materials & Methods Section. (Line 211-215)

Line 249: Please correct Figure 1; this is Figure 2.

Answer: The section you mentioned has been corrected in the article. (Line 250)

Figure 2:

As seen in a, I would also like to see the separate p values for b and c between each variable. Please add on the figure.

Answer: The section you mentioned has been revised in the figure legend. As friedman test did not indicate any statistical difference for hBD2 and hBD3, pairwise analysis was not performed. (Figure 2)

Discussion:

Line 262: ‘may be suppressed’ Please revise as ‘can be suppressed’

Answer: The section you mentioned has been corrected in the article. (Line 269)

So many repeats here. Same things were also stated in the ‘Introduction’. Please shorten the ‘Introduction’.

Answer: The passages (in introduction and discussion sections) has been revised in the article.

Answer:

Dear Reviewer,

Thank you for your valuable comments. The prevalence of head and neck cancers is quite low compared to general cancer cases. We are aware of the fact that our patient number is low and we mentioned this limitation in the second paragraph of the discussion section. However, the strongest aspect of this study is the long-term follow-ups that include the first month after the completion of radiotherapy. The patients were exposed to similar radiation doses and received the treatment for similar periods. Although it has been shown in many studies that radiotherapy applied to the head and neck region can induce oral inflammatory processes, oral inflammatory markers tend to return to pre-radiotherapy levels one month after the completion of radiotherapy (Sroussi et al., 2017, Keskin M. et al., 2020). We also added a one-month post-radiotherapy follow-up to our study protocol in order to directly monitor the effect of radiotherapy by minimizing the effects of other factors.

This study is a continuation of our previous study (Keskin et al., 2020) and our Finnish collegues played a role in the planning of the study, the analysis of the samples, and the finalization of the article.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments to the Author

The aim of this study was to investigate oral hBD-1, -2, and -3 levels in individuals with head and neck cancer and the changes in their levels in relation to radiotherapy. Sample comprised 16 individuals. Clinical periodontal parameters were recorded to define periodontal status of all patients in detail. Oral rinse samples (instead of saliva or gingival crevicular fluid) were collected before radiotherapy, at the 1st week, 3rd week, 6th week of radiotherapy, and at the 4th week following the completion of radiotherapy and analyzed through ELISA (hBDs 1-3) method. Findings demonstrated increased oral rinse levels of hBD-1 with the initiation of the radiotherapy and no difference levels of hBD-2 and hBD-3 during or after the radiotherapy.

The introduction is well designed, it presents a good introduction to the topic and the literature is relevant. The research is methodologically correctly appointed, and the presentation of the results (tables, figures) corresponds to the objectives of the research and the set hypothesis. The discussion part debates the results of the study, however, the possible effects of the drugs used and the periodontal status of the included patients on the study results should be discussed.

Comment 1:

Introduction, page 1 line 33-35 = The references for the sentences below should be added.

“Human beta defensins (hBDs) are a family of small antimicrobial peptides that are produced by cells in a variety of tissues, including skin, intestine, lung, and oral cavity. hBDs are the first line of defense against infections and are important for protecting the body against various pathogens. ‘’

Comment 2:

Introduction, page 2 line 49 = Delete the ref highlighted below. It should be the number.

‘’While hBD-2 is only found in this tissue, suggesting that its presence in the gingival epithelium may be triggered by interactions with the microflora living there (Schibli et al., 2002). ‘’

Comment 3:

There should be a simple definition of periodontitis in the introduction part.

Comment 4:

What have previous studies concluded about the interaction between periodontal status and head and neck cancers? Just one or two sentences could be added to introduction part.

Comment 5:

Material and Methods, page 3 line 123= The initials of expert oncologist should be written.

Comment 6:

Material and Methods, page 3 line 125= Give the reference for National Comprehensive Cancer Network® (NSSN®) guideline.

Comment 7:

Material and Methods, page 4 line 136= For the Michigan probe, please add footnote or information into parenthesis to identify manufacturers of equipment.

Comment 8:

Periodontal examination procedure, page 4: Is not it too much to give all details about periodontal indices such as ‘’ The CEJ is determined by reference to the exit profile of enamel over …………………………………………………. Bleeding of probing (as per- 147 centage) and plaque index (PI) were measured from the four surfaces (distal, buccal/labial, 148 mesial, lingual/palatinal) of each tooth. The probe is inserted into the periodontal pocket …………………………………………………………………….total number of sites examined and multiplying by? Please revise.

Comment 9:

Plaque index is determined by calculating the percentage, why not common used PI according to Loe and Sillness? That is today’s standard.

Comment 10:

Material and Methods, please indicate a reference for ‘Oral Rinse Collection Procedure.’

Comment 11:

Material and Methods, please indicate a reference for ‘The Radiotherapy Treatment Procedure. ‘

Comment 12:

What was the scientific assumption for the power calculation? Which study served as reference?

Comment 13:

Results, page 6 line 223: Correct sentence as ‘’This study included 16 head and neck cancer patients with an average age of 51.8 ±14.03 years ( …….. females and …. Males). ‘

Comment 14:

Results, page 6, Table 1 = The smoking n number was represented as 20 in table 1. Normally the study population is 16. Please check the study population numbers in table 1 and adjust.

Comment 15:

Results, Table 1= There is an information about chemotherapy of patients in table 1 but there is nothing about it at the method part. It will be better to revise the information about chemotherapy at both method and results part.

Comment 16:

Results part, page 7 line 237: Please revise the sentence as ‘’ The initial clinical periodontal parameters of the patients are specified in the Table 2. ‘’

Comment 17:

The data for CAL and PD should be represented as mean ± STD. Please revise table 1 or add this info into manuscript’s’ results part.

Comment 18:

Figure legends, Figure 2: The statistical methods for this figure and the significant p value should be given.

Comment 19:

According to your results, is there a correlation between beta defensins and tumor types, patients’ medical and periodontal status, or medication? If you think that adding this data has positive aspects, it can be added. This is just a recommendation, not an expectation.

Comment 20:

Discussion: For the limitations of the study, it can be stated that the salivary flow rate was not measured for this study group and the periodontally healthy group was not included.

Comment 21:

Discussion: Authors should interpret and discuss advantages and disadvantages of oral rinse evaluation over saliva (unstimulated or stimulated) or other oral liquids such as gingival crevicular fluid in the study.

Discussion Authors should discuss possible effects of periodontal disease status on the study results in terms of periodontitis inflammation magnitude on human beta defensins.

Discussion: Authors should discuss the effect of patients’ medication (especially metformin) on the study results. Please add some explanation also.

Comments for author File: Comments.pdf

Author Response

Dear reviewer, thank you very much for your valuable comments. Your suggestions are highly important. Our responses to your suggestions are as follows:

Comment 1:

Introduction, page 1 line 33-35 = The references for the sentences below should be added.

Answer to Comment 1: References have been added. (Line 34)

Comment 2:

Introduction, page 2 line 49 = Delete the ref highlighted below. It should be the number.

Answer to Comment 2: Related sentence has been revised. (Line 50-52)

Comment 3:

There should be a simple definition of periodontitis in the introduction part.

Answer to Comment 3: Periodontitis definition has been implemented into the article. (Line 45)

Comment 4:

What have previous studies concluded about the interaction between periodontal status and head and neck cancers? Just one or two sentences could be added to introduction part.

Answer to Comment 4: Possible periodontitis & Head and neck cancer link has been implemented into the introduction. (Line 87-90)

Comment 5:

Material and Methods, page 3 line 123= The initials of expert oncologist should be written.

Answer to Comment 5: The initials of expert oncologist has been written. (Line 121)

Comment 6:

Material and Methods, page 3 line 125= Give the reference for National Comprehensive Cancer Network® (NSSN®) guideline.

Answer to Comment 6: Reference has been given. (Line 125)

Comment 7:

Material and Methods, page 4 line 136= For the Michigan probe, please add footnote or information into parenthesis to identify manufacturers of equipment.

Answer to Comment 7: Manufacturer information has been provided. (Line 134)

Comment 8:

Answer to Comment 8: Periodontal examination procedure has been revised. (Line 130-145)

Comment 9:

Plaque index is determined by calculating the percentage, why not common used PI according to Loe and Sillness? That is today’s standard.

Answer to Comment 9: Patients with head and neck cancer are a group of patients for whom oral procedures are more difficult to perform due to their underlying disease. We chose this method because its scientific validity has been established and also because of the characteristics of our study group. The Visible Plaque Index (VPI) developed by Ainamo and Bay is an assessment tool used to evaluate the presence and amount of plaque on the teeth. It has several advantages over other similar assessment tools, such as the Silness and Löe Plaque Index (PI).

One of the advantages of VPI over PI is that the VPI is a simpler and faster method to use

as the VPI is based on the visual examination of the teeth, while the PI is based on the tactile examination, which can be affected by factors such as the skill and experience of the examiner.

In addition, the VPI allows for more accurate inter- and intra-examiner reproducibility, as it is a more objective method of assessment than the PI, which relies more on the subjective judgment of the examiner.

Comment 10:

Material and Methods, please indicate a reference for ‘Oral Rinse Collection Procedure.’

Answer to Comment 10: Reference has been indicated. (Line 168)

Comment 11:

Material and Methods, please indicate a reference for ‘The Radiotherapy Treatment Procedure. ‘

Answer to Comment 11: Reference has been indicated. (Line 175)

Comment 12:

What was the scientific assumption for the power calculation? Which study served as reference?

Answer to Comment 12: To the best of our knowledge, our study is the first to investigate changes in oral beta-defensin levels in relation to radiation therapy. A similar study protocol by Keskin et al. 2020, which investigated oral inflammatory changes, was taken into account as a reference in the power analysis. Keskin M, Lähteenmäki H, Rathnayake N, Räisänen IT, Tervahartiala T, Pärnänen P, Şenışık AM, Karaçetin D, Yentek Balkanay A, Heikkilä P, Hagström J, Rautava J, Haglund C, Gursoy UK, Silbereisen A, Bostanci N, Sorsa T. Active matrix metalloproteinase-8 and interleukin-6 detect periodontal degeneration caused by radiotherapy of head and neck cancer: a pilot study. Expert Rev Proteomics. 2020 Oct;17(10):777-784. doi: 10.1080/14789450.2020.1858056. Epub 2021 Jan 7. PMID: 33406924.

Comment 13:

Results, page 6 line 223: Correct sentence as ‘’This study included 16 head and neck cancer patients with an average age of 51.8 ±14.03 years ( …….. females and …. Males). ‘

Answer to Comment 13: The sentence has been revised. (Line 224-225)

Comment 14:

Results, page 6, Table 1 = The smoking n number was represented as 20 in table 1. Normally the study population is 16. Please check the study population numbers in table 1 and adjust.

Answer to Comment 14: It was mistakenly written. It has been corrected to 16. (Table 1)

Comment 15:

Answer to Comment 15: Information regarding chemotherapy has been implemented into the article. (Line 124-125, 235-239)

Comment 16:

Results part, page 7 line 237: Please revise the sentence as ‘’ The initial clinical periodontal parameters of the patients are specified in the Table 2. ‘’

Answer to Comment 16: The sentence has been revised. (Line 243)

Comment 17:

The data for CAL and PD should be represented as mean ± STD. Please revise table 1 or add this info into manuscript’s’ results part.

Answer to Comment 17: CAL and PD information has been implemented into the table 2.

Comment 18:

Figure legends, Figure 2: The statistical methods for this figure and the significant p value should be given.

Answer to Comment 17: Figure 2 legend has been revised.

Comment 19:

Answer to Comment 19: Thank you for your valuable suggestion. However, head and neck cancers can include cancers in different localizations and different types. Since we think that more cohort studies are needed to evaluate the correlations you mentioned, compared to our study group, we find it more appropriate not to include this information.

Comment 20:

Discussion: For the limitations of the study, it can be stated that the salivary flow rate was not measured for this study group and the periodontally healthy group was not included.

Answer to Comment 20: A part of discussion regarding salivary flow rate has been implemented. (Line 286)

Comment 21:

A part of discussion regarding advantages and disadvantages of oral rinse evaluation over saliva (unstimulated or stimulated) or other oral liquids has been implemented. (Line 285-290)

Discussion Authors should discuss possible effects of periodontal disease status on the study results in terms of periodontitis inflammation magnitude on human beta defensins.

A part of discussion regarding possible effects of periodontal disease status on the study results has been implemented. (Line 318-326)

Discussion: Authors should discuss the effect of patients’ medication (especially metformin) on the study results. Please add some explanation also.

A part of discussion regarding the effect of patients’ medication (especially metformin) on the study results has been implemented. (Line 291-297)

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for the edits.

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Oral Cavity Beta-Defensin Levels Are Regulated Differently during Radiotherapy in Head and Neck Cancer Patients

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