ATOM INDONESIA JOURNAL
Referee’s Report
Article No. : #378
Title of Paper : Association between Expressions of p70s6k with Radiotherapy Response in Cervical Cancer
Referee Name :Abdul Mutalib
Comment on Descriptions 1. Title
[ ] Appropriate [√] Should be changed
2. Abstract
Yes[√] No[ ] Is the length reasonable?
Yes[√ ] No[] Is it an appropriate summary of the content?
3. Main Text
Yes[ ]No[√] Is there anything new in this work?
Yes[ ] No[√] Is the relation to previous studies adequately stated?
Yes[√] No[ ] Are the assumption(s) and/or method(s) described comprehensively?
Yes[ ] No[√] Are the new results adequately emphasized?
Line # Referee’s Comments
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There are many important prognostic factors in advanced stage cervical cancer primary primarily treated with radiotherapy. Besides clinical factor, many biomarkers have been studied in relation with to radiotherapy response. P70s6k is one of biomarker plays an important a significant role in cell proliferation. Increased levels of p70s6k also have associated with drug resistance of in cancer.
“
In the present study,
we determined the association between the expressions of p70s6k before treatment using chemo-radiotherapy with radiotherapy response in cervical cancer”.
…… After complete the treatment had completed, early radiotherapy response was observed by pelvic control method. The results show that p70s6k is highly expressed (61.9%, 13/21) and low expressed (38.1%, 8/21) in the cancer cells. They also show that there is and no significant difference was found on AgNOR mean, and MIB-1and p53 index indexes in the different degrees of p70s6k expression (p≥0.05). High expression of p70s6k had an association with good radiotherapy response compared to lower one
(p=0.05). In conclusion, degree of p70s6k expression before treatment has association with radiotherapy response.
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There are many important prognostic factors,one of which is clinical pathologic factor including stage and tumor histology,in advanced stage cervical cancer primarily treated with chemoradiotherapy are clinical pathologic factors, including stage and tumor histology [3]. Besides the clinical pathologic factors, many biomarkers have been studied in relation to survival and/or response to chemoradiotherapy. “Please define at here what is a
biomarker? Please explain also in one alinea the relationship between prognosis factors, pathologic factors and biomarkers in order your paragraph has a logical oder”especiallyThe markers, which mainly involve involved in tumorigenesis and tumor progression, such as genes associated with apoptosis, angiogenesis, and cell growth, that have been investigated extensively. At present the moment, the focus of improving survival rates is mainly on targeted therapies in combination with standard chemo-radiotherapy [4].
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P70s6k, AgNOR, MIB-1, and p53are biomarkers that are related to cell proliferation.
P70s6k, is a serine/threonine protein kinase responsible for ……….. In some in vitro studies, it was found that activity of p70s6k related to with a prevention of apoptosis as one of death processes kind in of cancer cell treatment with chemotherapy and in radiotherapy [8-11].AgNOR, MIB-1, and p53 are biomarkers that related to cell proliferation.AgNORs are Nucleolar organizer regions (NORs),which are chromosomal segments
thatcarrycarrying ribosomal genes, stained with silver stained NORs are called AgNORs and when examined using light microscopy they appear as well-defined black dots exclusively located within the nucleoli [12]. MIB-1 is a monoclonal antibody to detect a Ki-67 antigen. This antigen in the nucleus of cells can be detected during the gap1 (G1), synthesis (S), and gap2 (G2) and mitosis (M) phases of the cell cycle [13]. Thus, it can be only detected in the nucleus of proliferating cells in all active stages of the cell-division cycle but is absent in non-proliferating cells [14]. The p53 gene, located on chromosome 17, encodes a 53-kDa protein. Wild-type p53 protein is a transcription factor that, when activated, acts to arrest cells temporarily in the G1/G2 phase of the cell cycle allowing time for the repair of the damaged DNA, or to cause cells to proceed to apoptosis if the DNA damage is irreparable irrepairable [15,16].
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In Our previous publication reported there were the correlation between apoptosis caspase- 3 with better radiotherapy response. However, and contrary with the high level of Akt expression indicated that related with poor or bad radiotherapy response, but there were was no association between some proliferation markers like AgNOR, MIB-1, and p53, with radiotherapy response [17,18]. The aim of this the present study is to investigate association the relationship between p70s6k as a biomarker with radiotherapy response in cervical cancer.
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Twenty-one consecutive patients who have underwent achieved a complete treatment from a whole series 91 of 60 cases were subjected to this experiment , which were suffering from nonmetastatic localized cervical carcinoma in local advance stage (IIB-IIIB), were
prospectively conducted from July 2010 to March 2011. (“It is better you describe here what do you mean ‘series 91 of 60 cases’? What the reason did you choose this
criterion? ”). The patients were suffering from non-metastatic localized cervical carcinoma in local advance stage (IIB-IIIB). The radiation therapy treatment conducted was consists of external radiation and internal radiation. External Beam Radiotherapy (EBRT) with C-60 with total doses 50 Gy in 25 fractions and Internal radiation in with 192Ir High Dose-Rate Intracavitary Brachytherapy (HDR-ICBT) using a Microselectron (Nucletron International, Amsterdam, Netherlands) followed by EBRT in two fractions (850 cGy/fraction). Cisplatin as a chemotherapeutic agent was administered at dose of 40 mg/m2 on days 1, 8, 15, 22, and 29 that given concurrently with the EBRT in 2 hours or less before the treatment for the corresponding day. The process of diagnosis and treatment followed the Standard
Operational Operation Procedure (SOP) as shown in our previous publication [17].
Before the studies were carried out, the research proposal was approved by the Research and Ethical Committee from , Faculty of Medicine, the University of Indonesia and the patients received written informed consent.
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Tumor response to radiation therapy Radiation response was evaluated by radiotherapist and as grouped based on by Hong Criteria as follows [19]:
1. NRT response (no gross residual tumor) response as a good response is a complete or nearly complete regression of pelvic tumor, non specific fibrosis, or granulation over the cervix (called as good response).
2. GT response (gross residual tumor) response as a bad response is gross tumor or palpable nodularity on cervix, and/or palpable in duration on the parametrium (called as bad
response).
131 AgNOR Staining and Immunohistochemical Immunohistochemistry
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AgNOR staining and Immunohistochemical immunochemistry of MIB-1, p53, and p70s6k were performed on microscopic slide obtained from biopsy cancer tissue sample samples before treatment with radiotherapy. (Did you performed with the same method for biopsy cancer tissue samples after treatment with radiation therapy? If you did please notice at here). The detail detailed analysis method of MIB-1 and p53 wasconducted with followed procedures as described in our previous publication [20,21] (You can claim this statement like this, if the procedures is really your idea and invention. However, if the procedures come from literatures of others researchers or you have modified those procedures, you have to cite the original literatures not your publications. It looks the literature of 20 you refer is not your publication. Not all readers understand the procedure, so it is better you describe it briefly at here) . P70s6k expression is observed in the nucleus and cell
cytoplasm. The degree of its P70s6k expression in the nucleus and cell cytoplasm was estimated blindly of to the clinical patient characteristics and outcome, with using microscope magnification (10x40) semi-quantitatively. The scored score was ranged from negative (−) to slightly slight positive (+), moderately moderate positive (++), and strongly strong positive (+++) [22]. The score from negative toslightly slight positive is was
grouped classified as low expression, while and
the high expression one is indicated with
moderate to strong positive was grouped as high expression. (Please explain what do you meant with “estimate blindly”? How did you make score decisions as negative, slight positive, moderate positive, and strong positive?)151- 157
This sub-section needs to be revised. It is better in this sub-section you describe the purpose you perform statistical analysis and the reason you choose ANOVA and F-test. Please describe what kind of data you have and clarify your data before treatment and after treatment (? ). Please define the uncertainties of your measurement or experiment based on n (21) samples/patients.
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Expression of p70s6k was detectable identified as brown color spots in the nucleus and cytoplasm of cancer cell as shown in Figures 1a and b. (Please describe the Figure 1a and b and how you can make a judgment that the arrows in both Figures express p70s6k). Its expression can be was observed in all of 21 microscopic slides from cancer tissue slide before radiotherapy. (Does this mean all of 21 cervix cancer patients?) Then, they It can be classified grouped into high expressed (61.9%, 13/21) and low expressed (38.1%, 8/21). High expression of p70s6k is related with to cell proliferation. As a mitogen-activated Ser/Thr protein kinase, p70s6k is required for G1 cell cycle progression and cell proliferation [8]. In The active proliferation cell will show the amount of cell in mitotic (cycling cell) is higher than that of G0 phase.
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In Table 1, there was no statistical different (based on ANOVA or F-test? How many n (number of patients or experiment?). Please define it) of AgNOR mean, MIB-1 and p53 indexes in high and low expressions of p70s6k before treatment.
Different with In contrast to AgNOR mean that tends lower in high expression of p70s6k, either both of MIB-1 and or p53 index is seem appears higher in high expression of p70s6k than the lower one (Fig.2 a,b,c). (Please clarify data in the Table 1 and Figures 2a, b, and c. If they are the same data, what the reason the p values are different between Table and Figure and also in the Figures 2a, b, and c.
Please notify whether notation 1 and 2 on the x axis in the Figure 2 is low
expression and high expression of p70s6k). Low AgNOR mean in high expression of p70s6k is related with to the different activity site of p70s6k and the presence of AgNOR in part of a cell cycle. AgNOR can be detected identified in G1, S and G2 in the cell cycle, whereas and the presence of p70s6k expression is focused in observed in G1only. The number of AgNOR will increase from G1 and
accumulated in S, then decrease in G2 [23]. High MIB-1 indexes that appear in high expression of p70s6k (Table 1) are also relevant with Gabele, et.al. [24] and Fikret, et.al. [25]. High MIB-1 index indicates also mean that higher of the growing fraction of cancer cell is high[21]. The high p53 index in high expression of p70s6k in Table 1 was due to the presence of most expression of p53 in cervical cancer in mutant rather than in wild type, even though DNA sequencing analysis was not performed. In vitro investigation reported that wild-type p53 can decrease of p70s6k expression by through mTOR pathway [26,27,28].
After treatment with radiotherapy there was statistical different in proportion of
complete and partial radiotherapy response in high and low expression of p70s6k
(p=0.05) (Figure 3). (It is very difficult to evaluate this Figure 3 due to unreadable
labels. Please revise the Figure 3).). This was also depicted with the AgNOR mean
of 9 Gy irradiated cancer tissue that was lower than that of before irradiation, if G2
phase and S phase are considered [21]. A similar case was also indicated when
AgNOR is observed in stimulated mitotic of hepatocyte cell [20].
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This paragraph is moved to the above paragraph.
Tendency of start to be detected in G1, increased and accumulated in S and decreased again in G2 [23]. Another report also found that when compared in cancer tissue before treatment and after 9 Gy irradiated AgNOR mean is lower in after 9 Gy irradiated as considered in G2 phase and S phase before irradiation [21].
The similar data was also found whenre AgNOR observed in stimulated mitotic of hepatocyte cell [20]. As a mitogen-activated Ser/Thr protein kinase, p70s6k is required for G1 cell cycle progression and cell proliferation [8].
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This paragraph is moved to the above paragraph
High expression of p70s6k related with cell proliferation. Its clearly that higher expression of p70s6k also show higher of MIB-1 index [24,25]. Higher MIB-1 also mean that higher of growing fraction of cancer cell [21].
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This paragraph is moved to the above paragraph
Even though without DNA sequencing analysis, almost expression of p53 in cervical cancer was in mutant than wild type. It was the main reason for this current data explaining higher p53 index in high expression of p70s6k than in lower one. In vitro investigation reported that wild-type p53 can decrease of p70s6k expression by mTOR pathway [26,27,28].
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In this current result, The high expression of p70s6k shown in this experimental result made indicates the presence of the high proliferated cells that and also
prevent the process of apoptosis as one of a way the death ways of cancer cell after receiving exposure to ionizing radiation in radiotherapy. The high portion of a proliferating cells also made indicate the cells would be more radiosensitive rather than radioresistance. A number of studies have focused on the involvement of p70S6k in influencing cellular responses to apoptotic stimuli [please put here the references you referred]. Animal experiments using Treatment of Swiss 3T3 and RAT-1 cells treated with etoposide and staurosporine resulted in dephosphorylation that and decreases the activity of p70S6k in preventing (??? Please check again, not preventing but supporting) apoptosis preventing [8,9]. It has also been shown that cisplatin inhibited inhibits the phosphorylation of p70s6k in mouse myoblasts [10].
(Please explore the experimental results you performed associated with cisplatin you used in the Experimental Method section with this statement. You did not report the experimental results of cisplatin you used, even though you mention the use of cisplatin in the Experimental Method section.) (Please consider to delete these statements: “Rapamycin, an immune-suppressant blocked the activation of mTOR/p70s6k, thus compromising the cell’s ability to progress through the G1 phase of the cell cycle [10,11]. On other in vitro study, using breast cancer cell, inactivated of p70s6k also let the process of cell death and should sensitize cancer cell to chemotherapeutic agents [29]”, if there is close relationship with your experiment)
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It is difficult you use these argument “Cell in high proliferation were highly radiosensitive and gave good radiotherapy response, in otherwise lower
proliferation proportion will give the bad radiotherapy response. Because in this result the observation of radiotherapy response as the early response, we think that major of the death of cancer cell was almost caused by irradiation than cisplatin in concurrent therapy”, if you did not represent your experimental results associated with cisplatin.
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Please consider this paragraph (This Our present experimental results seem seems
have different with some reports explaining the relation between the expression of
p70s6k with the resistance of some cancer cells after treatment. They report that
high expression of p70s6k before treatment has relationship related with resistance or poor response of cancer, especially in cancer treated with chemotherapy [30- 32].) If you have experimental results of chemotherapy, it is Ok. You can compare it. Otherwise, it is better you delete that paragraph.
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This paragraph does not have relationship with experimental results and discussion, so it is better you move it to the conclusion section as suggestion (In the future, if this result will be able to be confirmed in a bigger sample, we suggested the higher expressed p70s6k patients as one of consideration parameter receiving earlier treatment priority than the lower one. It also has potential using anti p70s6k as target therapy in the form of adjuvant therapy after the patients completed the treatment of radiotherapy in cervical cancer.)
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It looks the conclusion does not have a close relationship with the title of this paper.
Please consider to revise either the title or the conclusion.
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Please revise Table 1. Give space between data and n.s. Please define n you used when you show standard deviation data
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Figure need to be revised, particularly labels for x and y axes.
Final comments and recommendations:
Logical order of each paragraph, particularly in the experimental results and discussion is not clear. Some paragraphs that do not support each other need to be deleted.
It looks there is no good relationship between title, the aim of the experiment, experimental methods, experimental results, and conclusion. Please revise it
Experimental results of cisplatin is not mentioned. Please show and discuss it
This paper is recommended to be [ ] Accepted without further revision [ ] Accepted with minor revision [√] Major Revision is required [ ] Rejected
