Prognostic Impact of Low-Level p53 Expression on Brain Astrocytomas Immunopositive for Epidermal Growth Factor Receptor

Round 1

Reviewer 1 Report

Astrocytomas, which are one of the most common tumors of the brain. Although surgical resection of brain astrocytomas can provide timely relief of the mass effect, high-grade astrocytomas unavoidably recur even after concurrent chemoradiotherapy. In this study, the authors showed that the concomitant expressions of EGFR and p53 in astrocytomas patients is a biomarker to anticipate a preferable treatment response.

I have several comments for the authors below:

Major comments:

1. The authors should show higher magnitude images for Figure 1 and Figure 2 to show clearer the location of p53 in the nucleus and EGFR in the membrane. With current magnification, it is difficult to conclude that EGFR is located in the membrane.

2. The authors should mention in the text whether these patients (82 people) received any other treatments such as irradiation or chemotherapy within 60 months post-observation after the surgery. 

3. There is an error in the content and grammar in this sentence. Please correct it “There were no significant differences between the p53 protein expressions for GBM8401, GBM8901, U87MG, G5T, and DBTRG-05MG is not significantly different than that of SVGq12 (Figure 5).”

 Minor comments:

1. Line 62, remove one period at the end of that sentence.

2. Line 113, CO2 – replace to CO2

3. For the proliferation assay, can the author write down the Cat# of the kit or reagents that they used for the experiment?

4. Table 2, For the p value = “1” : what does it mean?

Author Response

Comments and Suggestions for Authors

Astrocytomas, which are one of the most common tumors of the brain. Although surgical resection of brain astrocytomas can provide timely relief of the mass effect, high-grade astrocytomas unavoidably recur even after concurrent chemoradiotherapy. In this study, the authors showed that the concomitant expressions of EGFR and p53 in astrocytomas patients is a biomarker to anticipate a preferable treatment response.

I have several comments for the authors below:

Major comments:

The authors should show higher magnitude images for Figure 1 and Figure 2 to show clearer the location of p53 in the nucleus and EGFR in the membrane. With current magnification, it is difficult to conclude that EGFR is located in the membrane.

Thanks for your reminder. We change new data.

The authors should mention in the text whether these patients (82 people) received any other treatments such as irradiation or chemotherapy within 60 months post-observation after the surgery.

Thanks for your reminder. We had added “Thirty-seven (45.1%) patients had radiotherapy and twenty-eight (34.1%) patients had chemotherapy, such as temozolomide or bevacizumab.” in 3.1.

There is an error in the content and grammar in this sentence. Please correct it “There were no significant differences between the p53 protein expressions for GBM8401, GBM8901, U87MG, G5T, and DBTRG-05MG is not significantly different than that of SVGq12 (Figure 5).”

Thanks for your reminder. We modified it with “No significant differences in EGFR protein expression were detected in GBM8401, GBM8901, U87MG, G5T, DBTRG-05MG, and SVGp12 cells (Figure 5). However, p53 protein levels were significantly lower in GBM8401, GBM8901, U87MG, G5T, and DBTRG-05MG cells compared with the expression in SVGp12 cells (Figure 5).”.

Minor comments:

Line 62, remove one period at the end of that sentence.

Thanks for your reminder. We had finished it.

Line 113, CO2 – replace to CO2

Thanks for your reminder. We modified it.

For the proliferation assay, can the author write down the Cat# of the kit or reagents that they used for the experiment?

Thanks for your reminder. We had added “M5655, Sigma–Aldrich, USA” in 2.4.

Table 2, For the p value = “1” : what does it mean?

Thanks for your reminder. We had deleted table 2 and showed the statistical results in Figure 5. Because the p53 expression of GBM8401 was similar with that of GBM8901, the p value was 1.

Reviewer 2 Report

 In this manuscript Tsai and colleagues have addressed the “Prognostic impact of low-level p53 expression on Brain Astrocytomas with Immunopositivity for epidermal growth factor (EGFR)” after follow-up of 82 enrolled patients. The manuscript is interesting and will forward the knowledge in the medical community, however some issues require addressing before acceptance for publication.

My specific comments are:

Major concerns: 

 1. There is some sort of technical issue with the WB analysis, because the bands corresponding to EGFR expression are actually not bands, please provide a better image or a less cropped version of it. The crop of the Western Blot images should be performed with at least 1 cm distance from the interest bands for all the analysed markers. If a nonspecific band is captured in the crop, please indicate this nonspecific band with an asterix, but perform a proper crop. 

2. Figures 1 and 2- the legend does not specify which samples were analysed and presented (which stage and imaging area-if applicable).

3. The authors should motivate how the expression of EGFR and p53 in different brain tumors cell lines is correlated with the analysis of patient samples, and why the authors have not used at least for the WB analysis tumor tissue to show comparative expression levels with the selected cell lines. 

4. It is unclear how the p values presented in the table 1 have been calculated and furthermore why they are presented since only one value is statistically significant.

5. The authors should revise the statement from row 193-195, pag 7: “There were no significant differences between the p53 protein expressions for GBM8401, GBM8901, U87MG, G5T, and DBTRG-05MG is not significantly different than that of SVGq12 (Figure 5)” since the Western blot showed clear visible differences between p53 expression level from GBM cell lines and control cell line- glial (SVGq12). My feeling is that the sentences refer to EGFR expression not to p53 that is addressed further in the sentences by the authors. So please perform the proper change in the sentence. 

6. Please specify the number of replicates performed for Western blot experiments in the figure legend. 

7. Figure 6-Please specify the number of replicates performed for the MTT experiment in the figure legend.

8. The introduction should be expanded a bit and place better in the context the presented study.

 Minor concerns:

Figure 1 and Figure 2:

The authors should specify in the figure legends that a representative image is showed. 

Overall readability and written English should be moderately revised to make the manuscript more fluent.

Author Response

Major concerns: 

1. There is some sort of technical issue with the WB analysis, because the bands corresponding to EGFR expression are actually not bands, please provide a better image or a less cropped version of it. The crop of the Western Blot images should be performed with at least 1 cm distance from the interest bands for all the analysed markers. If a nonspecific band is captured in the crop, please indicate this nonspecific band with an asterix, but perform a proper crop. 

Thanks for your reminder. We change a new data.

2. Figures 1 and 2- the legend does not specify which samples were analysed and presented (which stage and imaging area-if applicable).

Thanks for your reminder. We had added it in Figure legend.

3. The authors should motivate how the expression of EGFR and p53 in different brain tumors cell lines is correlated with the analysis of patient samples, and why the authors have not used at least for the WB analysis tumor tissue to show comparative expression levels with the selected cell lines. 

Thanks for your reminder. These samples were in paraffin, so the quality from these samples was poor for western blot.

4. It is unclear how the p values presented in the table 1 have been calculated and furthermore why they are presented since only one value is statistically significant.

Thanks for your reminder. These data were calculated using chi-square tests, and we presented these data following our previously study, such as Liu PC, Lieu AS, Lin CJ, Tsai HP, Chai CY, Kwan AL. High Expression of Sp1 is Associated with Recurrence of Meningioma. World Neurosurg. 2021 May;149:e1056-e1060. doi: 10.1016/j.wneu.2021.01.016. and Chen YT, Tsai HP, Wu CC, Chen CY, Chai CY, Kwan AL. High-level Sp1 is Associated with Proliferation, Invasion, and Poor Prognosis in Astrocytoma. Pathol Oncol Res. 2019 Jul;25(3):1003-1013. doi: 10.1007/s12253-018-0422-8.

5. The authors should revise the statement from row 193-195, pag 7: “There were no significant differences between the p53 protein expressions for GBM8401, GBM8901, U87MG, G5T, and DBTRG-05MG is not significantly different than that of SVGq12 (Figure 5)” since the Western blot showed clear visible differences between p53 expression level from GBM cell lines and control cell line- glial (SVGq12). My feeling is that the sentences refer to EGFR expression not to p53 that is addressed further in the sentences by the authors. So please perform the proper change in the sentence. 

Thanks for your reminder. We modified it with “No significant differences in EGFR protein expression were detected in GBM8401, GBM8901, U87MG, G5T, DBTRG-05MG, and SVGp12 cells (Figure 5). However, p53 protein levels were significantly lower in GBM8401, GBM8901, U87MG, G5T, and DBTRG-05MG cells compared with the expression in SVGp12 cells (Figure 5).”.

6. Please specify the number of replicates performed for Western blot experiments in the figure legend. 

Thanks for your reminder. We had added “n=6” in the figure legend.

7. Figure 6-Please specify the number of replicates performed for the MTT experiment in the figure legend.

Thanks for your reminder. We had added “n=4” in the figure legend.

8. The introduction should be expanded a bit and place better in the context the presented study.

 Thanks for your reminder. We had added “Gliomas are identified more and more by molecular pathology in The 2021 WHO Classification of Tumors of the Central Nervous System, including isocitrate dehydrogenase1/2 (IDH1/2), 1p/19q-codeleted, phosphatase and tensin homolog (PTEN) alterations, telomerase reverse transcriptase promoter gene mutations, EGFR amplification and mutation, and TP53 (which encodes the p53 tumor suppressor protein) mutation [3, 4]. EGFR amplification and TP53 mutation are the most common genetic alterations in glioblastomas (GBMs).” in the introduction.

Minor concerns:

Figure 1 and Figure 2:

The authors should specify in the figure legends that a representative image is showed. 

 Thanks for your reminder. We had added it in Figure legend.

Overall readability and written English should be moderately revised to make the manuscript more fluent.

 Thanks for your reminder. We had revised our written English through Enago https://www.enago.tw/?gclid=Cj0KCQjwjbyYBhCdARIsAArC6LJq61WqMPQ6gqsXJ5g3Oc_8FIN_heWJnTkmdTJ1hO1guE5LNjJ7usEaArA2EALw_wcB .

Reviewer 3 Report

Dear authors,

your research is very interesting in a field with limited evidence about the prognostic significance of any biomarkers. I think that your results could better address the need for diversifying the therapeutic options based on the findings you retrieved. In particular, your results could justify the lack of significant benefit from EGFR inhibitors in unselected glioma cohorts. The fact demonstrated by you that, among patients with low p53 expression, those with EGFR positivity present significantly worse survival than those with EGFR negativity while no impact of EGFR status was detected among patients with high expression could help in better-selecting patients who potentially benefit most from EGFR inhibitors use. for this reason, I believe that your work is valuable and deserves publication after minor revision.

In particular, I met some typos throughout the paper:

line 62 - double full stop.

line 74 - please, check the position of "has" in the sentence.

line 174 - add "is" after "there".

line 199 - add "have" between "to" and "the".

line 280 - the repetition "shown/show", so near each other, does not sound good.

Moreover, I think you should enrich the references with further 3 glioma papers, which you can cite in the introduction or discussion, as you want:

1 - Palmisciano P, Ferini G, Watanabe G, Ogasawara C, Lesha E, Bin-Alamer O, Umana GE, Yu K, Cohen-Gadol AA, El Ahmadieh TY, Haider AS. Gliomas Infiltrating the Corpus Callosum: A Systematic Review of the Literature. Cancers (Basel). 2022 May 19;14(10):2507. doi: 10.3390/cancers14102507. PMID: 35626112; PMCID: PMC9139932.

2 - Bonosi L, Ferini G, Giammalva GR, Benigno UE, Porzio M, Giovannini EA, Musso S, Gerardi RM, Brunasso L, Costanzo R, Paolini F, Graziano F, Scalia G, Umana GE, Di Bonaventura R, Sturiale CL, Iacopino DG, Maugeri R. Liquid Biopsy in Diagnosis and Prognosis of High-Grade Gliomas; State-of-the-Art and Literature Review. Life (Basel). 2022 Mar 11;12(3):407. doi: 10.3390/life12030407. PMID: 35330158; PMCID: PMC8950809.

3 - Ahmed N, Ferini G, Barua KK, Halder R, Barua S, Priola S, Tomasi O, Umana GE, Shlobin NA, Scalia G, Garg K, Chaurasia B. Adult-Onset Pilocytic Astrocytoma Predilecting Temporal Lobe: A Brief Review. Life. 2022; 12(7):931. https://doi.org/10.3390/life12070931

Lastly, cite the following in the discussion section about lung cancer:

4 - Vadalà RE, Santacaterina A, Sindoni A, Platania A, Arcudi A, Ferini G, Mazzei MM, Marletta D, Rifatto C, Risoleti EV, Severo C, Pontoriero A, Iatì G, Pergolizzi S. Stereotactic body radiotherapy in non-operable lung cancer patients. Clin Transl Oncol. 2016 Nov;18(11):1158-1159. doi: 10.1007/s12094-016-1552-7. Epub 2016 Sep 29. PMID: 27686231.

5 - Puglisi C, Giuffrida R, Borzì G, Illari S, Caronia FP, Di Mattia P, Colarossi C, Ferini G, Martorana E, Sette G, Eramo A, Lorico A, Di Grazia A, Forte S. Ex Vivo Irradiation of Lung Cancer Stem Cells Identifies the Lowest Therapeutic Dose Needed for Tumor Growth Arrest and Mass Reduction In Vivo. Front Oncol. 2022 May 12;12:837400. doi: 10.3389/fonc.2022.837400. PMID: 35646627; PMCID: PMC9133629.

For example, citing the first (PMID: 27686231), you could write "The EGFR status might help to predict what lung cancers are radioresistant and therefore require a radiation boost to any residual at an early response assessment after a normofractionated radiotherapy course."

Please, cite all 5 suggested references.

Congratulations on your work!

Author Response

In particular, I met some typos throughout the paper:

line 62 - double full stop.

Thanks for your reminder. We had finished it.

line 74 - please, check the position of "has" in the sentence.

Thanks for your reminder. We had finished it.

line 174 - add "is" after "there".

Thanks for your reminder. We had finished it.

line 199 - add "have" between "to" and "the".

Thanks for your reminder. We had finished it.

line 280 - the repetition "shown/show", so near each other, does not sound good.

Thanks for your reminder. We had finished it.

Moreover, I think you should enrich the references with further 3 glioma papers, which you can cite in the introduction or discussion, as you want:

1 - Palmisciano P, Ferini G, Watanabe G, Ogasawara C, Lesha E, Bin-Alamer O, Umana GE, Yu K, Cohen-Gadol AA, El Ahmadieh TY, Haider AS. Gliomas Infiltrating the Corpus Callosum: A Systematic Review of the Literature. Cancers (Basel). 2022 May 19;14(10):2507. doi: 10.3390/cancers14102507. PMID: 35626112; PMCID: PMC9139932.

2 - Bonosi L, Ferini G, Giammalva GR, Benigno UE, Porzio M, Giovannini EA, Musso S, Gerardi RM, Brunasso L, Costanzo R, Paolini F, Graziano F, Scalia G, Umana GE, Di Bonaventura R, Sturiale CL, Iacopino DG, Maugeri R. Liquid Biopsy in Diagnosis and Prognosis of High-Grade Gliomas; State-of-the-Art and Literature Review. Life (Basel). 2022 Mar 11;12(3):407. doi: 10.3390/life12030407. PMID: 35330158; PMCID: PMC8950809.

3 - Ahmed N, Ferini G, Barua KK, Halder R, Barua S, Priola S, Tomasi O, Umana GE, Shlobin NA, Scalia G, Garg K, Chaurasia B. Adult-Onset Pilocytic Astrocytoma Predilecting Temporal Lobe: A Brief Review. Life. 2022; 12(7):931. https://doi.org/10.3390/life12070931

Lastly, cite the following in the discussion section about lung cancer:

4 - Vadalà RE, Santacaterina A, Sindoni A, Platania A, Arcudi A, Ferini G, Mazzei MM, Marletta D, Rifatto C, Risoleti EV, Severo C, Pontoriero A, Iatì G, Pergolizzi S. Stereotactic body radiotherapy in non-operable lung cancer patients. Clin Transl Oncol. 2016 Nov;18(11):1158-1159. doi: 10.1007/s12094-016-1552-7. Epub 2016 Sep 29. PMID: 27686231.

5 - Puglisi C, Giuffrida R, Borzì G, Illari S, Caronia FP, Di Mattia P, Colarossi C, Ferini G, Martorana E, Sette G, Eramo A, Lorico A, Di Grazia A, Forte S. Ex Vivo Irradiation of Lung Cancer Stem Cells Identifies the Lowest Therapeutic Dose Needed for Tumor Growth Arrest and Mass Reduction In Vivo. Front Oncol. 2022 May 12;12:837400. doi: 10.3389/fonc.2022.837400. PMID: 35646627; PMCID: PMC9133629.

For example, citing the first (PMID: 27686231), you could write "The EGFR status might help to predict what lung cancers are radioresistant and therefore require a radiation boost to any residual at an early response assessment after a normofractionated radiotherapy course."

Please, cite all 5 suggested references.

Thanks for your reminder. We had cited the ref 1, 2, and 4 in manuscript. Ref 3 discussed the pilocytic astrocytoma. In this study, the number of pilocytic astrocytoma patients was 3 (data not show) and specially discussed it, so we did not cite it. Ref 5 showed the correlation between radiation and lung cancer stem cells. However, no report showed the relationship between EGFR and lung cancer stem cells, so we did not cite it.

Round 2

Reviewer 2 Report

Dear authors, 

 Congratulations for your improvements to the manuscript!