The Fagerström and AUDIT Tests as Probable Screening Tools in Oral Cancer and Their Correlation with CYP1A1, GSTM1, GSTP1, and GSTT1 Gene Expression

Round 1

Reviewer 1 Report

1. A table with the results of the Mann-Whitney test should be added that will facilitate the text reading.
2. In addition, adding charts and diagrams will improve the final shape of the manuscript.

Author Response

Comments from Reviewer 1

• English language and style are fine/minor spell check required
  • Response: We agree, and in addition to the above recommendation the entire manuscript was checked concerning the language, style, and spelling.

Reviewer 2 Report

The authors presented an interesting and clinically relevant work on risk assessment of oral cancer based on questionnaires and gene expression tests. 

The novelty of the study is a combined investigation of behavioral habits and molecular factors to estimate a risk level of oral cancer.

The paper is well and clear written and is easy to read. All conclusions are consistent with presented data. 

 

Major revisions: no specific recommendations.

 

Minor revisions:

I would recommend to add a short explanation why exactly the genes CYP1A1, GSTM1, GSTP1 and GSTT1 were selected for the investigation.

As a suggestion, a number of cases might be mentioned as another limitation of the study. 

Author Response

Comments from Reviewer 2 

• English language and style are fine/minor spell check required
  • Response: We agree, and in addition to the above recommendation the entire manuscript was checked concerning the language, style, and spelling.

• Are the results clearly presented? – Can be improved
  • Response: This is correct. A new table has been added to complete the results

 

Table 2. Description of FTCD and AUDIT test scores results.

FTCD Score of dependence	Cancer Group n (%)	Control Group n (%)
0 – 2 = very low dependence	3 (8.33)	2 (5.56)
3 – 4 = low dependence	6 (16.66)	1 (2.78)
5 = moderate dependence	4 (11.11)	3 (8.33)
6 – 7 = high dependence	11 (30.555)	2 (5.56)
8 – 10 = very high dependence	3 (8,33)	1 (2.78)
Total	27	9
AUDIT Score
0 – 7 points = low risk use	10 (26.32)	4 (10.53)
8 – 15 = risk use	14 (3.84)	2 (5.26)
16 – 19 points = harmful use	5 (13.16)	1 (2.63)
≥ 20 points = probable dependence	2 (5.26)	-
Total	31*	7

• * 2 subjects with cancer declared not to use tobacco and alcohol.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript made by Bandeira CM et al is interesting since they are evaluating two important tests related to habits of tobacco and alcohol consumption, associating those tests with important genes related to development of malignant neoplasms, in which they focused on oral cancer. Despite the introduction is interesting, I have two questions that the authors need to resolve. 

1. Would it be possible to make a short introduction of each gene studied, and relate them with risk of oral cancer?  
   
   
2. Page 2; Lines 66 to 68... "These enzymes...." In these lines highly polymorphism of p450 and GSTs enzymes that are related with CYP1A1, GSTM1, GSTP1 and GSTT1 were described. Would it be possible to explain the relationship between all these? 
   
   
3. Page 2; Lines 66 to 68... "These enzymes...."  What are the low polymorphisms related to Brazilian population and how can these affect oral cancer development related to smoking and alcoholism?
   
   
4. Material and Methods
   
   

The materials and methods are well done and explained. However, I have some questions in which the authors need to explain in a better manner the following:

1. Page 2; Line 93 to 100; "As the control..." 
   
   
2. a) From what type of benign lesions were the healthy borders taken? 
   
   
3. b) How many centimeters were taken from the benign lesion to the healthy borders?
   
   
4. c) Why did the authors decide to exclude lip cancer?
   
   

Results

As well as in the Material and Methods section, the results are well explained, however, I have some suggestions to the authors. 

The Audit and FTCD score are interesting to evaluate alcoholism and smoking, they have interesting questions related to habits of consume. Due to this, would it be possible to make a table to explain the percentage of each question of Audit and FTCD test?

 

Discussion

Page 6; Lines 221 to 223: "Multicenter studies....." In these lines the authors include some multicenter studies " Gencapo, Arcage, Inhance, InterChange and HeadSpace, could it be possible to provide the website URL or any references to consult these studies?

Page 6; Line 224: "Although..." Would it be possible to explain the limitations about those Multicenter studies?

Page 6; Lines 228 to 230: "The Audit test..." I consider this paragraph is like to introduction. Please review and redact it again. 

Page 7; Lines 249 to 254: "We did not...." This paragraph is interesting but it generates several doubts.

1. Could it be possible to establish some hypotheses about the no differences between genes studied in the cancer group?
   
   
   
2. What was the influence of CYP1A1 gene on their results?
   
   
   
3. Would it be possible to establish some hypotheses about the interaction of genes CYP1A1 GSTM1 and GSTP1 and oral cancer?
   
   
4. What types of polymorphisms are present in the Brazilian population related to genes?
   
   

Page 7; Lines 255 to 260: The "CYP1A1 gene..." Would it be possible to deepen more into CYP1A1, oral cancer, AUDIT score and Brazilian population? Since this paragraph does not provide anything new about oral cancer and smoking.

Overall, the discussion is interesting, but it seems to be descriptive, and it is not completely related with the interesting, obtained results. Thus, I recommend to deepen more into the genes studied and discuss in more detail the interaction between AUDIT, Fagerström, genes, alcoholism, and smoking. Moreover, would it be possible to compare with other studies related to cancer and genes?

Overall comments 

The manuscript is interesting and well done in the methodology and results, however the discussion it seems weak and descriptive in which authors does not discussing properly their results. I recommend to authors respond each of my suggestion and deepen into discussion and to relate it more properly with their results.  

Author Response

Comments from Reviewer 3

• English language and style are fine/minor spell check required
  • Response: We agree, and in addition to the above recommendation the entire manuscript was checked concerning the language, style, and spelling.

Introduction:

• Does the introduction provide sufficient background and include all relevant references? – Can be improved.
  • Response: We agree with this and have incorporated new information into this topic which is highlighted in the manuscript and described below.

 

• Are the methods adequately described? – Can be improved.
  • Response: Thank you for your suggestion. We´ve added new data about this comment (shown below).

 

• Are the results clearly presented? – Can be improved.
  • Response: This is correct. A new table has been added to complete the results (shown below).

 

• Are the conclusions supported by the results? – Must be improved.
  • Response: Thank you for your suggestion. We have revised and changed the paragraph.
    • Motivating the health professionals, especially oncologists, to evaluate smokers’ nicotine dependence and encouraging them to participate in cessation programs are important educational measures and can undoubtedly impact the outcomes not only for diseases prevention, whether malignant or not, related to tobacco and alcohol dependence, but also to the treatment response. The FTCD and AUDIT questionnaires designed to evaluate and estimate the degree of exposure and dependence to tobacco and alcohol combined with simple gene expression tests can be useful tools to assess the risks of developing oral cancer.

 

• Would it be possible to make a short introduction of each gene studied, and relate them with risk of oral cancer?
  • Response: We agreed with this observation and included a new paragraph (page 2, lines 58 – 63) and references (16- 19).
    • CYP1A1 is one of the most important genes responsible for the bioactivation of tobacco carcinogenic substances including polycyclic aromatic hydrocarbons (PAH) and nitrosamines and benzopyrenes as well as alcohol metabolism (16). Phase II of Glutathione S-transferases (GSTs) enzymes has an important role in the detoxification of carcinogens specially the polycyclic aromatic hydrocarbons (PAH) found in tobacco smoke (17-19)

 

16 - Rendic SP, Guengerich FP. Human Family 1–4 cytochrome P450 enzymes involved in the metabolic activation of xenobiotic and physiological chemicals: an update. Archives of Toxicology 2021 95:2 [Internet]. 2021 Jan 18 [cited 2022 Feb 19];95(2):395–472. Available from: https://link.springer.com/article/10.1007/s00204-020-02971-4

17 - Varela-Lema L, Taioli E, Ruano-Ravina A, Barros-Dios JM, Anantharaman D, Benhamou S, et al. Meta-analysis and pooled analysis of GSTM1 and CYP1A1 polymorphisms and oral and pharyngeal cancers: a HuGE-GSEC review. Genet Med [Internet]. 2008/05/23. 2008;10(6):369–84. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18496222

18 - Singh RR, Reindl KM. Glutathione S-Transferases in Cancer. Antioxidants (Basel, Switzerland) [Internet]. 2021 [cited 2022 Feb 19];10(5). Available from: https://pubmed.ncbi.nlm.nih.gov/33946704/

19 - Bongers V, Snow GB, Braakhuis BJM. The role of glutathione S-transferases in head and neck squamous cell carcinogenesis. European journal of cancer Part B, Oral oncology [Internet]. 1995 [cited 2022 Feb 19];31B(6):349–54. Available from: https://pubmed.ncbi.nlm.nih.gov/8746263/

 

• Page 2; Lines 66 to 68... "These enzymes...." In these lines highly polymorphism of p450 and GSTs enzymes that are related with CYP1A1, GSTM1, GSTT1 and GSTT1 were described. Would it be possible to explain the relationship between all these?
  • Response: Thank you for your observation and due this relevance we included this aspect in the discussion with some new references.
    • This correlation between CYP1A1, GSTM1, GSTT1 and GSTT1 seems to be important, once this balance between activation and detoxification of carcinogens can influence the development, progression, and prevention of cancer. This relationship was found with the polymorphic forms and has been associated with other types of malignancies like breast, colon, and lung. Besides that, the research of polymorphic genes in head and neck cancer is more prevalent in specific countries like India, China, Japan, Spain, and Germany. Unfortunately, Brazil did not have scientific data regarding the prevalence of these polymorphisms and due to our population characteristics, we decide not to include polymorphic forms of those genes in our study. (48-52)

 

• Page 2; Lines 66 to 68... "These enzymes...." What are the low polymorphisms related to Brazilian population and how can these affect oral cancer development related to smoking and alcoholism?
  • Response: This is an interesting comment, but due to the fact, there are few national papers evaluating the same genes as we studied and most of them are with other phase I polymorphisms or other diseases, it was not possible to include this specific information about the Brazilian population.

 

Material and Methods

 

• Page 2; Line 93 to 100; "As the control..." From what type of benign lesions were the healthy borders taken? How many centimetres were taken from the benign lesion to the healthy borders?
  • Response: Thank you for this observation. It was included a piece of complementary sentence information regarding this aspect in the first paragraph of Materials and Methods.
    • As the control group, tissue fragments were collected from the healthy border at least 1 cm away from a benign lesion such as mucocele and fibroma...

 

• Why did the authors decide to exclude lip cancer?
  • Response: We appreciate your comment, but the authors decide not to include lip cancer samples as it is related to sun exposure and not tobacco and alcohol factors. We added this information on Pag. 3 – line 115.

 

Results

 

• The Audit and FTCD score are interesting to evaluate alcoholism and smoking, they have interesting questions related to habits of consume. Due to this, would it be possible to make a table to explain the percentage of each question of Audit and FTCD test?
  • Response: Thank you for your recommendation. We incorporate a new table describing the scores results and percentages of the Audit and FTCD tests

 

Table 2. Description of FTCD and AUDIT test scores results.

FTCD Score of dependence	Cancer Group n (%)	Control Group n (%)
0 – 2 = very low dependence	3 (8.33)	2 (5.56)
3 – 4 = low dependence	6 (16.66)	1 (2.78)
5 = moderate dependence	4 (11.11)	3 (8.33)
6 – 7 = high dependence	11 (30.555)	2 (5.56)
8 – 10 = very high dependence	3 (8,33)	1 (2.78)
Total	27	9
AUDIT Score
0 – 7 points = low risk use	10 (26.32)	4 (10.53)
8 – 15 = risk use	14 (3.84)	2 (5.26)
16 – 19 points = harmful use	5 (13.16)	1 (2.63)
≥ 20 points = probable dependence	2 (5.26)	-
Total	31*	7

* 2 subjects with cancer declared not to use tobacco and alcohol.

 

Discussion

 

• Page 6; Lines 221 to 223: "Multicenter studies....." In these lines the authors include some multicenter studies " Gencapo, Arcage, Inhance, InterChange and, could it be possible to provide the website URL or any references to consult these studies?
  • Response: Thank you for this observation. All the websites and paper references have been included and listed below (references 34-38).

34 - GENCAPO [Internet]. [cited 2022 Feb 19]. Available from: http://www.gencapo.famerp.br/lmmb/

35- Canova C, Hashibe M, Simonato L, Nelis M, Metspalu A, Lagiou P, et al. Genetic associations of 115 polymorphisms with cancers of the upper aerodigestive tract across 10 European countries: the ARCAGE project. Cancer research [Internet]. 2009 Apr 1 [cited 2022 Feb 19];69(7):2956–65. Available from: https://pubmed.ncbi.nlm.nih.gov/19339270/

36 - International Head and Neck Cancer Epidemiology Consortium - | University of Utah [Internet]. [cited 2022 Feb 19]. Available from: https://medicine.utah.edu/inhance/

37 - IARCRP: Home [Internet]. [cited 2022 Feb 19]. Available from: https://interchange.iarc.fr/

38 - HEADSpace: Home [Internet]. [cited 2022 Feb 19]. Available from: https://headspace.iarc.fr/

 

• Page 6; Line 224: "Although..." Would it be possible to explain the limitations about those Multicenter studies?
  • Response: Thank you! A new sentence has been included.
    • Although biomolecular science has got incredible advances to cancer study, unfortunately, its usefulness in daily medical care is available for a few countries or institutions, becoming more used in research centers.

 

• Page 6; Lines 228 to 230: "The Audit test..." I consider this paragraph is like to introduction. Please review and redact it again.
  • Response: Thank you for your advice. We revised the sentence for context in the discussion paragraph.
  • Before:
    • Introduction: The Alcohol Use Disorder Identification Test (AUDIT) was developed by the WHO as a simple and validated tool for alcohol use screening (23).
    • Discussion: We chose to use internationally validated and accessible questionnaires to assess the degree of tobacco and alcohol dependence and to compare it with the expression of genes involved in the metabolism of carcinogens. The AUDIT test, used to assess risky drinking behaviour, and the FTCD are important instruments to quantify the consumption and the degree of exposure to these products in patients with oral cancer
  • After:
    • We chose to use internationally validated and accessible questionnaires to assess the degree of tobacco and alcohol dependence and compare it with the expression of genes involved in carcinogens metabolism. (39) In this way, those questionnaires could be a useful tool to estimate indirectly the degree of exposure to tobacco and alcohol harmful products as well as those gene expression in patients with oral cancer. (40)

 

• Page 7; Lines 249 to 254: "We did not...." This paragraph is interesting, but it generates several doubts.
  • Response: We agree. The entire paragraph is confusing, so we revised and changed it.
    • We did not observe differences in GSTM1, GSTP1 or GSTT1 gene expression in the cancer group, but as Phase II enzymes are responsible for detoxification of carcinogens, once cancer is present, it possibly means that the role of these enzymes is not proceeded to neutralize the toxic substances. The greater correlation found between CYP1A1 expression and tumor size, and regional metastases reinforce the hypothesis that the more bioactivation and presence of carcinogens, the poorest outcome can be found (48).

 

• Could it be possible to establish some hypotheses about the no differences between genes studied in the cancer group?
  • Response: Thank you for this question. We have inserted a paragraph on this point.
    • Our hypothesis was that Phase I genes that metabolize and activate tobacco and alcohol carcinogens should be more expressed in cancer and, on the other hand, Phase II genes that are responsible for the detoxification process would be more present in the control group. The CYP1A1 and GSTP1 results found in our study may indicate that this hypothesis may be true.

 

• What was the influence of CYP1A1 gene on their results?
  • Response: We thank the reviewer for pointing out this issue. This point of view was included in the discussion. For the authors, it seems that, once cancer is present, the bioactivation of tobacco substances into carcinogens by Phase I enzymes were effective and Phase II detoxification enzymes were unable to metabolize and eliminate these products. This is a possible justificative for our finds regards CYP1A1expression in the cancer group.

 

• Would it be possible to establish some hypotheses about the interaction of genes CYP1A1 GSTM1 and GSTT1 and oral cancer?
  • Response: This is an interesting comment. We included this observation in the discussion.
    • Since Phase I and II enzymes show an opposite reaction regarding activation and detoxification of the carcinogens it was expected that the CYP1A1 expression was higher in cancer samples. On the other hand, GSTP1 was predominant among benign lesion and significantly correlated smoking parameters like number of cigarettes smoked per day, duration of tobacco consumed, and nicotine dependence evaluated by TFCD, giving a probable protective action to smokers. The GSTP1 results found in our study could indicate that this hypothesis may be true.

 

• What types of polymorphisms are present in the Brazilian population related to genes?
  • Response: Important aspect highlighted. Although there are few studies of CYP1A1, GSTM1, GSTP1 and GSTT1 polymorphisms in Brazil, they were all published almost two decades ago, used different methods and the results were controversial or without statistical significance.
  • References:
    • Leichsenring A, Losi-Guembarovski R, Maciel ME, Losi-Guembarovski A, Oliveira BW, Ramos G, et al. CYP1A1 and GSTT1 polymorphisms in an oral cancer case-control study. Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biologicas [Internet]. 2006 Dec [cited 2022 Feb 21];39(12):1569–74. Available from: https://pubmed.ncbi.nlm.nih.gov/17160265/
    • Marques CFS, Koifman S, Koifman RJ, Boffetta P, Brennan P, Hatagima A. Influence of CYP1A1, CYP2E1, GSTM3 and NAT2 genetic polymorphisms in oral cancer susceptibility: results from a case-control study in Rio de Janeiro. Oral oncology [Internet]. 2006 Jul [cited 2022 Feb 21];42(6):632–7. Available from: https://pubmed.ncbi.nlm.nih.gov/16488179/
    • Figaro Gattá GJ, de Carvalho MB, Siraque MS, Curioni OA, Kohler P, Eluf-Neto J, et al. Genetic polymorphisms of CYP1A1, CYP2E1, GSTM1, and GSTT1 associated with head and neck cancer. Head & neck [Internet]. 2006 Sep [cited 2022 Feb 21];28(9):819–26. Available from: https://pubmed.ncbi.nlm.nih.gov/16721740/
    • Duarte ECB, Ribeiro DC, Gomez MV, Ramos-Jorge ML, Gomez RS. Genetic polymorphisms of carcinogen metabolizing enzymes are associated with oral leukoplakia development and p53 overexpression. Anticancer research [Internet]. 2008 Mar [cited 2022 Feb 21];28(2A):1101–6. Available from: https://pubmed.ncbi.nlm.nih.gov/18507060/
    • Olivieri EHR, da Silva SD, Mendonça FF, Urata YN, Vidal DO, Faria M de AM, et al. CYP1A2*1C, CYP2E1*5B, and GSTM1 polymorphisms are predictors of risk and poor outcome in head and neck squamous cell carcinoma patients. Oral oncology [Internet]. 2009 Sep [cited 2022 Feb 21];45(9). Available from: https://pubmed.ncbi.nlm.nih.gov/19442564/
    • Losi-Guembarovski R, Cólus IMDS, de Menezes RP, Poliseli F, Chaves VN, Kuasne H, et al. Lack of association among polymorphic xenobiotic-metabolizing enzyme genotypes and the occurrence and progression of oral carcinoma in a Brazilian population. Anticancer research [Internet]. 2008 Mar [cited 2022 Feb 21];28(2A):1023–8. Available from: https://pubmed.ncbi.nlm.nih.gov/18507050/

 

• Page 7; Lines 255 to 260: The "CYP1A1 gene..." Would it be possible to deepen more into CYP1A1, oral cancer, AUDIT score and Brazilian population? Since this paragraph does not provide anything new about oral cancer and smoking.
  • Response: This observation is important. To the best of our knowledge, there is no Brazilian study that includes these variables. This result motivated us to investigate these aspects.
    • The CYP1A1 gene was significantly correlated with the instruments chosen to estimate the parameters of tobacco and alcohol consumption (TFCD and AUDIT scores), confirming the already known statement that the dual use of these substances substantially increases the risk of developing cancer. To the best of our knowledge, this correlation has not been evaluated before in Brazil. Our results partially disagree with the findings of Massod et al. (53) who observed reduced expression of CYP1A1 in some patients with oral cancer, but higher expression in cases in advanced stages. In our series, the number of cigarettes smoked per day and the exposure time were determinant for tumor stage, represented by the variable’s tumor size and regional metastasis.

 

• Overall, the discussion is interesting, but it seems to be descriptive, and it is not completely related with the interesting, obtained results. Thus, I recommend to deepen more into the genes studied and discuss in more detail the interaction between AUDIT, Fagerström, genes, alcoholism, and smoking. Moreover, would it be possible to compare with other studies related to cancer and genes?
  • Response: Thank you for your assessment. We are very pleased to receive these encouragements. As we did not find many papers with the same methodology or variables, it was not possible to make a comparative analysis for all the results obtained in our study.

 

• The manuscript is interesting and well done in the methodology and results, however the discussion it seems weak and descriptive in which authors does not discussing properly their results. I recommend to authors respond each of my suggestion and deepen into discussion and to relate it more properly with their results.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The manuscript has improved considerably in comparison to the prior version. Now is more understandable and better explained.