HLA-G 14 bp Ins/Del (rs66554220) Variant Is Not Associated with Breast Cancer in Women from Western Mexico

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

This is a resubmission of the manuscript were authors tried to answer all reviewer queries. The queries were answered and inserted in the text. So the manuscript now is acceptable for publication.

Author Response

RESPONSE 1: Thank you very much for your comments and acceptance of the revised version of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

In this study, the authors analyzed the association of 14-bp Ins/Del (rs66554220), a HLA-G variant, with clinical outcomes of breast cancer in western Mexico population. The cohort comprises the disease group of 182 female breast cancer (BC) patients and the control group of 221 sex-age matched disease-free females. The authors found that the 14-bp Ins/Del  variant of HLA-G was not associated with BC in the Mexican population, however, the Ins allele was a possible risk factor for developing BC in clinical stage IV.

For the study groups, there are some concerns in the study design. It is not clear on the control group's clinical parameters, e.g. the menopause status, and the body mass index, where the author provided detailed statistics for the patient group. The authors are advised to show the details of the control group to ensure the robustness of the comparison between groups. The stage IV BC group is limited to 11 patients, which is considerably small. The authors need to justify the statistics for making the conclusion that Ins variant is a risk factor for BC stage IV, and provided more details on the Ins/Del variant statistics in all stages.

The author described how they performed genotyping to detect Ins/Del variants in methodologies. Representative genotyping results should be shown in the results.

Comments on the Quality of English Language

The authors are advised to thoroughly check grammar and typos throughout the manuscript. For instance, the sentence in the abstract ‘In this report was analyzed the association of 14-bp Ins/Del…’ needs to be rewritten.

Author Response

FOR THE ATTENTION OF REVIEWER 2

 POINT 1: In this study, the authors analyzed the association of 14-bp Ins/Del (rs66554220), a HLA-G variant, with clinical outcomes of breast cancer in western Mexico population. The cohort comprises the disease group of 182 female breast cancer (BC) patients and the control group of 221 sex-age matched disease-free females. The authors found that the 14-bp Ins/Del variant of HLA-G was not associated with BC in the Mexican population, however, the Ins allele was a possible risk factor for developing BC in clinical stage IV.

For the study groups, there are some concerns in the study design. It is not clear on the control group's clinical parameters, e.g., the menopause status, and the body mass index, where the author provided detailed statistics for the patient group.

RESPONSE 1: Thank you very much for your valuable contribution, in the case of the control group, only age was captured as a clinical variable; other data such as body mass index and menopause status were not available.

POINT 2: The authors are advised to show the details of the control group to ensure the robustness of the comparison between groups.

RESPONSE 2: Thank you very much for the comment. In the Materials and Methods section, details of the control group are shown (lines 101-106), additionally more information was added in the same paragraph (lines 102-103).

POINT 3: The stage IV BC group is limited to 11 patients, which is considerably small. The authors need to justify the statistics for making the conclusion that Ins variant is a risk factor for BC stage IV, and provided more details on the Ins/Del variant statistics in all stages.

RESPONSE 3: Thank you very much for the comment, despite the statistical significance and given the small sample size, the statistical power was calculated and cited at foot of the table 2, resulting in a half of expecting, for that, this is mentioned as a weakness in the discussion section (lines 245-248). Also, to emphasize this first approach, needed to be corroborated in a bigger size sample in future studies, the following modifications were made in the manuscript:

• Abstract: “However we suggest the Ins allele as a possible risk factor to develop BC in clinical stage IV (OR = 3.05, CI 95% = 1.16 – 7.96, p = 0.01), nevertheless, given the small stratified sample size (n=11, statistical power = 41%) is inconclusive”. (lines 41-43).
• Results: “…however, the statistical power is insufficient (41%) to be a conclusive association.” (lines 158-159).
• Discussion: “…we suggest that the Ins allele could granted up…” (line 208); “suggest of association between Ins allele” (line 222).
• Conclusion: “…but might be related to advanced breast tumors; further studies are required. We suggest the Ins allele as a possible risk factor to develop BC in clinical stage IV, nevertheless, a bigger stratified sample size might be verify this. We propose the integration of clinical…” (lines 261-264).

POINT 4: The author described how they performed genotyping to detect Ins/Del variants in methodologies. Representative genotyping results should be shown in the results.

RESPONSE 4: Thank you very much for the comment. In the Results section an image of a polyacrylamide gel electrophoresis (Figure 1) and its description (lines 153-154 and 168-174). were added, where it is visualized the three genotypes corresponding to three different samples.

 

 

POINT 5: The authors are advised to thoroughly check grammar and typos throughout the manuscript. For instance, the sentence in the abstract ‘In this report was analyzed the association of 14-bp Ins/Del…’ needs to be rewritten.

RESPONSE 5: Thank you very much for your valuable contribution, the sentence that you mention in the abstract was rewritten and is shown below: “Herein, the relationship of 14-bp Ins/Del variant (rs66554220) with breast cancer (BC) and its clinical characteristics were analyzed in 182 women with non-familial BC and 221 disease-free women as a reference group. Both groups from western Mexico and sex-age matched (sm-RG”. (lines 34-36).

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. English writing should be improved.
2. Control (Reference) group should have some proper recruitments.
For instance, the age of reference group (RG, mean age 38.2 y/o) or sex-matched reference group (sm-RG, mean age 39.6 y/o) were too much younger compared to the case group (mean age 54.5 y/o). Additionally, most of breast cancer patients are female, the ratio of sex in the RG did not reflect the real world prevalence in this study.  

Author Response

For the attention of reviewer 1

Comments and responses:

1. English writing should be improved.

Response: At Universidad de Guadalajara it has trained personnel who supported us with the revision of the English language in the manuscripts, hence english proofreading in all documents was made (lines 75-77, 79-80, 330, 345 and table 1).

 

1. Control (Reference) group should have some proper recruitments. For instance, the age of reference group (RG, mean age 38.2 y/o) or sex-matched reference group (sm-RG, mean age 39.6 y/o) were too much younger compared to the case group (mean age 54.5 y/o). Additionally, most of breast cancer patients are female, the ratio of sex in the RG did not reflect the real-world prevalence in this study.

Response: Referent to the groups’ ages, we changed the mean ± SD for median (interquartile range (IQR) 25 - 75) which is more representative, showing as follows: BC = 54 (46 – 62.50) (line 139-140), sm-RG = 42 (22 - 51) (line 104), RG = 39 (22 - 50) (line 103). In this sense, the age distribution is more representative despite continuing to be a limitation in the recruitment criteria, however, based on the reported by Rodríguez-Cuevas et al., (2001) median age of Mexican women with breast cancer is 51 years, nevertheless, the most frequently affected age group is 40 - 49 in a 29.5% and groups from 30 – 39 and from 60 – 69 years of age in a 14%, contrasting with women from the United States and Europe (Rodríguez-Cuevas et al., 2000).

On the other hand, the objective of including an RG with both sexes in them was to show and corroborate the allelic and genotypic frequencies in our population already reported by other authors mentioned in the manuscript (references 23,28). Also, according to the paragraph included in the discussion (lines 226 - 231) “Nevertheless, we included men and women (RG) due to that the genetic structure population is mixed, and this is how genes are distributed. In this context, the sex di-morphism, mutation, and selection have effects on the phenotypic expression of different clinical traits [39]. However, according to Greif et al., the clinical characteristics between men and women with BC are more similar than different [40].

References

Agrawal AF. Evolution of sex: why do organisms shuffle their genotypes? Curr Biol. 2006 Sep 5;16(17):R696-704.

Rodríguez-Cuevas S, Macías CG, Franceschi D, Labastida S. Breast carcinoma presents a decade earlier in Mexican women than in women in the United States or European countries. Cancer. 2001;91(4):863-868.

Rodríguez-Cuevas S, Macías Martínez CG, Labastida Almendaro S. Cáncer de mama en México. Enfermedad de mujeres jóvenes? [Breast cancer in Mexico. Is it a young women disease?]. Ginecol Obstet Mex. 2000;68:185-190.

Sincerely,

PhD. Antonio Quintero Ramos,

Corresponding author.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

 

Thank you for the ideea of your research.Our knowledge about Genetics alteration and theirs clinical significance have to be improved in the future and this will be possible just by future reserch.

I have some coments about your article:

- You have to explain why did you compare the two  different groups of populaționale with high numeric difference: the control group had almost double number of persons than the BC group.

- you have to give explanation about statistical method for comparation the two groups with different numbers and gender. 
- on the conclusion secțion, you shoud comment how your studie’s founding could be usefull and could be applied in the future in the clinical practice setting.

Author Response

Comments and responses to reviewer 2:

Dear authors,

Thank you for the idea of your research. Our knowledge about Genetics alteration and theirs clinical significance have to be improved in the future and this will be possible just by future research.

I have some comments about your article:

1. You have to explain why did you compare the two different groups of populations with high numeric difference: the control group had almost double number of persons than the BC group.

Response: Ideally, in genetic association studies, 2:1 comparison (controls vs cases) is suggested. In this proposal, an attempt was made to approach this stipulation, despite the age ranges being limiting (Tenny et al., 2022).

Added: “On the other hand, and due to the fact that in genetic association studies, 2:1 comparison (controls vs cases) is suggested, we included 357 individuals, named as…” (lines 94-95).

2. you have to give explanation about statistical method for comparation the two groups with different numbers and gender.

Response: The statistical analysis in the case of BC vs RG, and BC vs sm-RG was made globally, using 2x2 comparisons tables as is mentioned “χ2 and logistic regression were performed in the online DeFinetti software (https://ihg.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl)” (lines 131-133).

3. On the conclusion section, you should comment how your studies’ founding could be useful and could be applied in the future in the clinical practice setting.

Response: Added: “This could be useful in the future clinical practice setting or in generating new strategies of diagnosis-prognosis of cancer” (lines 247-249).

References

Tenny S, Kerndt CC, Hoffman MR. Case Control Studies. [Updated 2022 Mar 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK448143/

 

Sincerely,

PhD. Antonio Quintero Ramos,

Corresponding author.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Becerra-Loaiza et al presents the analysis of the presence of a 14bp insertion or deletion on HLA-G in women with non-familial Breast cancer compared with healthy women from Western Mexico. This insertion is related to the level of expression of the gene and has already been described as a risk factor for BC in some populations. 

Authors used SSP-PCR and fragment analysis in gel to determine the insertion or deletion genotype.

This is an important issue to be discussed. However, some points need to be clarified.

1-In Material and Methods section, item Subjects, authors describe the mean age of RG and sm-RG as 38.2+/-16.6 and 39.6 +/-16.9 respectively and in Results they describe the BC patients mean age as 54.5 +/- 12.53 years. This means that the two groups are submitted to different risk factors. Authors should discuss this principally when they discuss the possibility of a risk factor for stage IV.

2- Pathological stage IV is an evolution of earlier stages. How to explain a genotype change when you are looking germline genotype? Could it be a different allele and not only an insertion or deletion? More reflexition and future investigations with new methodologies will be needed. Include this in the discussion section.

 

Author Response

Comments and responses to reviewer 3:

The manuscript by Becerra-Loaiza et al presents the analysis of the presence of a 14bp insertion or deletion on HLA-G in women with non-familial Breast cancer compared with healthy women from Western Mexico. This insertion is related to the level of expression of the gene and has already been described as a risk factor for BC in some populations.

Authors used SSP-PCR and fragment analysis in gel to determine the insertion or deletion genotype.

This is an important issue to be discussed. However, some points need to be clarified.

1. In Material and Methods section, item Subjects, authors describe the mean age of RG and sm-RG as 38.2+/-16.6 and 39.6 +/-16.9 respectively and in Results they describe the BC patients mean age as 54.5 +/- 12.53 years. This means that the two groups are submitted to different risk factors. Authors should discuss this principally when they discuss the possibility of a risk factor for stage IV.

Response: The average age ± SD was changed to median age (percentile 25-75), showing as follows: BC = 54 (46 – 62.50) (line 139-140), sm-RG = 42 (22 - 51) (line 104), RG = 39 (22 - 50) (line 103).

This is the most appropriate way to show the age ranges in the study groups, which despite being a limitation, proves to be within the age ranges reported associated with risk for the presentation of breast cancer in the Mexican population, which, is 10 years younger compared to the European population and the United States, that is, in Mexicans it occurs in people under 50 years of age (Salinas-Martínez et al., 2014).

According to Rodríguez-Cuevas et al., 2000 breast cancer diagnosis in Mexico is made in advanced stages (III, IV, N.C) in two out of three of the patients and a great proportion of them are younger than 50 years old.

The following was added to discussion: “taking in consideration that, in Mexican population, the diagnostic rate of BC is above <50 years compared with the United States and Europe, and it is also made mostly in advances stages (III, IV, N.C) in two out of three patients [41,42]” (lines 236 - 239).

1.  Pathological stage IV is an evolution of earlier stages. How to explain a genotype change when you are looking germline genotype? Could it be a different allele and not only an insertion or deletion? More reflexition and future investigations with new methodologies will be needed. Include this in the discussion section.

Response: The genetic variant evaluated, being from the germ line, does not change in individuals, which is why it is being evaluated as a risk factor for progression, in this sense, the genotypes do not vary over time.

The sentence: “Also, future investigations with new methodologies will be needed”, was added in lines 233-234 of discussion.

 

References

Rodríguez-Cuevas S, Macías Martínez CG, Labastida Almendaro S. Cáncer de mama en México. Enfermedad de mujeres jóvenes? [Breast cancer in Mexico. Is it a young women disease?]. Ginecol Obstet Mex. 2000;68:185-190.

Salinas-Martínez AM, Juárez-Ruiz A, Mathiew-Quirós Á, Guzmán-De la Garza FJ, Santos-Lartigue A, Escobar-Moreno C. [Breast cancer in México: a 10-year trend analysis on incidence and age at diagnosis]. Rev Invest Clin. 2014;66(3):210-217.

Sincerely,

PhD. Antonio Quintero Ramos,

Corresponding author.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Though the author used median and IQR instead of the mean to represent the age of each group, the difference of age in case groups were still older more than 10 y/o than reference groups. Total of 11 patients were stage 4 in the Table 3, author should check the statistic power. And the result of Table 3 did not do any adjustment for the age in the analysis especially the difference was so obvious.