ATOM INDONESIA
Author's Responses
Article : #378
Name of All Authors : IIN KURNIA, ZUBAIDAH ALATAS, BUDININGSIH SIREGAR, TJACHYA KURJANA, ANDRI ANDRIJONO, MARINGAN DIAPARI LUMBAN TOBING, BETHY SURYAWATHI HERNOWO, TEJA KISNANTO, DEVITA TETRIANA, SETIAWAN SOETOPO Article Title : The Relationship between Expression of p70s6k with Radiotherapy Response
in Cervical Cancer
E-mail : [email protected]
Line # Referee’s Comments Author's Responses
14
we determined the association between the expressions of …in cervical cancer was determined.
MIB-1 and p53…
showed that p70s6k…
Ok, I have changed in abstract
14-15
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.
Ok, I have changed in abstract
23 This treatment, and sometimes, could be
combined… Ok, I have changed in line 23
19-25
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
Ok I have changed in line 26 - 44
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
order”especially The 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].
28 …with chemoradiotherapy are such as clinical… Ok. I have changed, in line 28
40-74
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 that carry carrying 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].
Ok I have changed, in line 45 -79
44-45 The activation target of p70s6k and its
downstream target are ribosomal… Ok I have changed in line 51 -52 50 …of apoptosis as one kind of death in cancer
cell… Ok I have changed in line 57
63 …synthesis (S), and gap2 (G2) and mitosis (M)… Ok I have changed in line 68-69
75-79
If it doesn’t change the actual meaning, the sentence should be revised into: “In our previous publication there were negative correlation between apoptosis cascape-3 with radiotherapy response, that is high level of Act expression related with poor or bad radiotherapy response, and there were…”
Ok, I have changed in line 80 – 83
75-84
In Our previous publication reported there were the correlation between apoptosis caspase3 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.
Ok, I have changed in line 80 – 89
82 The aim of the study is to investigate the
association… Ok, I have changed in line 86-87
75-84
In Our previous publication reported there were the correlation between apoptosis caspase3 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.
Ok, I have changed in line 80 -89
90-117
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
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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.
98-99 …external radiation and internal radiation. Ok, I have changed in line 105-106 100 …with C-60 of total doses… Ok, I have changed in line 107 117 Do you mean ‘written informed concern’? Yes, I rewrite in line 124-125
119- 130
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).
Ok, I have changed in line 127-137
131 AgNOR Staining and Immunohistochemical
Immunohistochemistry Ok, I have change in line 138
132- 148
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 was
conducted 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
We do not take the sample after radiotherapy.
Ok I agree with you, I have change in line 139 – 201
positive (++), and strongly strong positive (+++) [22]. The score from negative to slightly 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?)
146 …as low expression while moderate… Ok, I have change in line 200-201 152 …used to analyze the comparison… Ok, I have changed in line 203-204
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.
I used the ANOVA tes was used t to asses mean of AgNOR, MIB-1 index as numeric data with category data of expression of p70s6k. F-Test as categorical data test was used to asses categorical data expression of p70s6k and response of radiotherapy. We do not use the bias or uncertainty of our
measurement.
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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.
I have describe in figure legend.
Yes in all patient sample tissue.
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166-
167 …high expression (61.9%... and low expression.. Ok, I have changed in line 220
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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
We use the ANOVA because of normality data of of AgNOR mean, and MIB-1 and p53 indexes by Kolmogorov Smirnov Test.
F Test is used proportional test in small data (number patient 21, less than 30). If large sample/data can use Chi Square Test.
P value between Table 1 and Figure 2a,b,c is not different.
Ok, I have changed in line 222-233
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]..
187-
188 9 Gy irradiation Ok, I have changed in line 242
190 Where..where Ok, I have changed in line 245
204 …in mutant than in wild type… Ok, I have changed in line 261 205 …for this current data that explaining higher… Ok, I have changed in line 269
210 - 231
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
Ok, I have changed in line 268-290.
Using of Cisplatin is not separated but in concurrent chemoradiotherapy.
Ok. I have deleted the statement.
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.
212-
213 …as one of the way of cancer cell death… Ok, I have changed in line 271 230 ….process of death cell and should Ok, I have changed in line 289
232- 239
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.
We think about the proportion phase of radiosensitive cell in cell cycle
237- 238
…radiotherapy response was early response, we
think that major of cancer cell death was … Ok, I have changed in line 297
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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.
We think, response to chemoradiotherapy treatment related to p70s6k also influenced by the type of cancer cell.
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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
Ok, I have deleted this paragraph
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.)
258 Expressions of p70s6k have important role… Ok, I have changed in line 311 258-
263
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.
Ok, I have revise the title.
260 radioresistance Ok, I have changed in line 313
362- 363
Please revise Table 1. Give space between data and n.s. Please define n you used when you show standard deviation data
Ok, I have revised Table 1 373-
377
Figure need to be revised, particularly labels for x and y axes.Figure need to be revised, particularly labels for x and y axes.
Ok. I have revised the Figure 2a,b,c,d
Fig 2.
Please state clearly which expressed of high and low expressions of p70s6k in x-axis (in the figure just written as 1 and 2)?
Ok, I have modified the Figure 2
Fig 3.
Same with figure 2, which expressed of high or low expressions of p70s6k? And the picture is not clear enough. Please provide with another picture that more clearly.
Ok, I have write in line 414, Figure Legend 3
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This original sheet should be returned to:
Administrator Atom Indonesia, PPIKSN-BATAN, Gedung 71, Lantai 1, Kawasan Puspiptek Serpong, Tangerang, Indonesia 15310
Author Signature(s) Date
July, 2nd , 2015
IIN KURNIA
