BCL11A Expression in Breast Cancer

Round 1

Reviewer 1 Report

In this study, authors have examined the expression pattern of BCL11A in breast cancer. For that, they analysed a patient cohort with breast cancer and mastophaty samples as control. In addition, they tried to correlate BCL11A expression with clinicopathological information. Unfortunately, they were not able to confirm the results that were obtained from patients with those from cell lines. Despite there are previous studies in breast cancer with the same aim, I consider that the present work is interesting because the N population is large (200 breast cancer cases + 13 mastophaty samples).

This study fits the scopus of Current Issues in Molecular Biology Journal in part, but it is really translational in nature. In addition, several changes should be performed prior acceptance for publication:

- Line 11. Taking into account that no importance is given to the location of BCL11A, I suggest to modify “The aim of this study was to investigate the location and the level of the expression of BCL11A” to “The aim of this study was to investigate the expression pattern of BCL11A” in Abstract section.

- Abstract section does not include anything about experiments with cell lines.

- Introduction is quite extensive. It could be summarized because some information is repeated in Discussion section.

- Line 155. Please, you should better describe “Tissue microarrays were made from selected cases of BC fixed”.

- Line 182. Please, you should better describe “The assessment of the intensity of BCL11A expression was carried out by two independent researchers”. How many images per condition were considered? How many cells were considered?

- Line 207. Please, you should better describe “probes were used for BCL11A (Hs00256254_m1, Hs01093196_m1, Hs00250581_s1)”. Why are you using different probes? Where do these probes map? Are you considering the different transcriptional isoforms (BCL11A-XL, BCL11A-L and BCL11A-S)?

- Line 226. Please, you should better describe “Evaluation of BCL11A ex-226 pression in cell lines was performed using Confocal Laser Scanning Microscope Fluoview 227 FV3000”.

- The order of figures must be corrected because the current version of the manuscript is really difficult to understand. You should to reorganize figures: all figures and tables should appear in the order of first mention in the text. Please, use a logic pattern. For example, current figure 2A should be Figure 1E.

- Results from cell lines (current figures 1E-J) should be separated in a different figure, for example Figure 2.

- Line 268-274. Statistical analysis should be perfomed to validate your affirmations. In fact, I observe a higher expression of BCL11A in non-tumoral MCF-10A cells than in other tumoral ones. Therefore, data from cell lines are contradictory with data from the patient cohort. Please consider to omit these results or discuss such discrepancy.

- Line 281-285. Idem as line 268-274: Statistical analysis should be perfomed to validate your affirmations. In fact, I observe a higher expression of BCL11A in non-tumoral MCF-10A cells than in other tumoral ones. Therefore, data from cell lines are contradictory with data from the patient cohort. Please consider to omit these results or discuss such discrepancy.

- Line 286-289. Figures and tables should appear in the order of first mention in the text.

- Line 294-302. Figures and tables should appear in the order of first mention in the text.

- Line 302-304. BCL11A expression level between the cases with HER2+/HER2- is not shown.

- Line 304. Please, you should better describe or omit: “All the above analyses were related to the cytoplasmic 304 expression of BCL11A”.

- Line 321-325 and Figure 3. You should try to be less assertive or consider to omit these data because BCL11A expression is not significantly associated with survival.

- Please revise all figure legends. 

- Line 348-349. You should remove this sentence because differences are not significant: “BCL11A mRNA levels showed a 348 similar trend, but these differences were not statistically significant”.

- Line 352. Please correct “were confirmed by studies on cell lines. Our results are in line with…”, because your results are the opposite.

- In discussion section, you should discuss the possible hypothesis to explain the significant increase of BCL11A at protein level, but not at mRNA level. Post-transcriptional regulation?

- Line 408-409. You should remove this sentence because differences are not significant.

- Line 417. You should remove this sentence because differences are not significant.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The aim of the article is identification of the localization and level of BCL11A expression in breast cancer, compared with mastopathy samples. The main finding was that the expression of BCL11A was negatively correlated with the grade of the histological malignancy.

From my point of view the introduction is a little bit to long and some of the presented data could be moved in discussion part of the article.

Materials and methods section is well written, but it wasn't very clear for me why were chosen two groups, one for IHC and one for molecular biology, and why wasn't possible to take samples for both investigations. The number of patients for IHC (200) was considerable higher than in the case of the patients (22) selected for molecular biology.

In the case of cell cultures, please indicate the criteria or a reference for establishment of grades of malignacy.

In the results part, the images from Figure 1 from E to I aren't very clear and a little bit confusing regarding the "colours" (blue, I assume is DAPI, and green is fluorescence from Ab against BCL11A). Also, the scale should be more clearer, because now it is unreadable.

Is any explanation about the inverse correlation between expression levels and degree of histological malignacy?

It is possible to indicate/speculate some cut-off values for separation between normal and pathological?

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed all my comments. Apart from minor changes (please, panels of Figure 1 should appear in the order of first mention in the text), I consider that this manuscript has been improved and it could be accepted for publication at Current Issues in Molecular Biology Journal.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

After some minor text corrections (line 14 ...was), the article is ready for publication.

Author Response

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Author Response File: Author Response.pdf