Bioinformatics-Based Characterization of ATP-Binding Cassette Subfamily B Member 1 (ABCB1) Gene Expression in Non-Small-Cell Lung Cancer (NSCLC)

Round 1

Reviewer 1 Report

I only have minor comments:

1. I would increase the font size for all figures.

2. Define TPM+1 in Fig1A; Use log or segmented y-axis in Fig. 1B. Figure 1 legend should specify the difference between 1A and 1B.

3. Specify the difference between transcript and gene expression in Fig. 2.

4. What is the smoking status in "Normal" in Figure 4? If possible, Normal vs cancer should be compared at the same smoking status.

5. Similarly, Normal vs cancer should be compaired at the same age, gender, and race (Figure 5).

6. discuss the potential reason why stage 4 has elevated ABCB1 expression.

7. Specify the difference between Figures 8B-D, and 8F. Specify the difference between panels with the same label. 

8. This study is about correlation analysis. I would not say "DNA methylation is the cause of ABCB1 expression" on page 9.

9.  On page 13, the author claimed that elevated ABCB1 in stage 4 could suggest MDR. Please specify the rationale or provide references.

 

NA

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript has been well presented. There is clarity in the methodology. However, the introduction could be streamlined further. The authors have made significant effort in interpretations of the data. However, we have few queries that has been mentioned below:

1.     The title requires rephrasing as in “bioinformatics-based characterization…”

2.     Introduction can be further streamlined by removing general cancer-related sentences, and adding lung cancer-related information as suggested in Query no. 6 below.

3.     In figure 2, we see that ABCB1 gene expression is downregulated in tumor but overexpressed in metastasis. What could be the probable reason/ interpretation, with regards to LUSC. If mRNA levels (figure 5) are decreasing then how come protein expression increasing in metastatic cells? This also contradicts with subheading “Physiologically low levels of ABCB1 expression in lung tissue are significantly downregulated in cancer cells”

4.     Citations is required for the sentences “among smokers there is a tendency to develop squamous histological type of this cancer. Nonsmokers are more likely to be diagnosed with the second type of NSCLC, adenocarcinoma”

5.     In figure 5, it appears that transcript per million is increases/ progressively restoring in cancer cells with age. This seems to be quite a contradiction and requires a scientific explanation or interpretation.

6.     A preliminary data of diagnosis and mortality of lung cancer based on age, ethnicity, and stage could be helpful from discussion perspective.

7.     Methylation patterns of p-gp seems to be reciprocal in between LUSC and LUAD in a number of categories. What could be the possible interpretation?

Author Response

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Reviewer 3 Report

The study of Jeleń et al. provided an investigation on the changes of ABCB1 gene in non-small cell lung cancers. The authors present the gene expression changes of ABCB1 in LUAD and LUSC cancers, and found that the expression levels are reduced in cancers. The changes are also related with multiple factors and associated with survival majorly in LUAD. The results above suggested that ABCB1 could be an important prognostic factor contributing to alteration of disease progression and survival of cancer patients.

 

There are a few issues the authors should address:

1. Since only 7 samples available, authors may plot the figure without metastatic tissue in TNMplot. The increase in metastatic tissue is not convincing.  

2. It would be much better that authors replotted the figures with different y-axis setting. In Figure 1B, it is hard to see the details in most of the tissues. For Figures 4, 5, 6, 9, and 10, if the normal samples are not described in the results, the boxes of normal could be removed and the figures could be re-scaled. 

3. The expression of ABCB1 is always at a lower level. Authors may comment on that.

4. The statistical tests based on the UALCAN are always performed by T-test on TPM. Authors can do gene expression analysis on the read count with more accurate model and considering the multiple factors in the model, such as age, smoking status, and gender. Current results can’t provide the information on the intersection between the factors and can’t exclude the possibilities like the Caucasian patients are older ones and male, which means the results could be biased by the cohort surveyed.

5. Are there any other database including recent datasets for evaluating the prognostic value in addition to PrognoScan? Because in PrognoScan the datasets are not updated for 12 years.

Author Response

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Round 2

Reviewer 2 Report

The authors have incorporated significant modifications to the manuscript. They have also answered all the queries satisfactorily.