Analysis of Genes Related to Invadopodia Formation and CTTN in Oral Squamous Cell Carcinoma—A Systematic Gene Expression Analysis
, Susanne Jung 1, Nikolai Purcz 2, Yahya Açil 2, Christoph Sproll 3, Johannes Kleinheinz 1 and Sonja Sielker 1,*Round 1
Reviewer 1 Report
Dear Authors,
It is a very interesting work, about possibilities of SCC spread.
There are only few conserns:
1. Please include previous treatment of patients in table 1, how many of them were recurrent?
2. Please write the exact version of UICC you are using in this work.
3. Please add exact locations of all tumors.
4. Please include HPV status of patients and check if it has an effect on gene expression.
Best regards
Author Response
We thank the reviewer for their helpful comments and will respond to each comment directly.
1.-3. Please include previous treatment of patients in table 1, how many of them were recurrent? Please write the exact version of UICC you are using in this work. Please add exact locations of all tumors.
In the 83 analysed tumour samples, one tumour was recurrent. We noted it in table 1. Further, patients had not received any adjuvant radiation or chemotherapy, as we wrote in 2.1 “Patient data”. The UICC classification we used was based on the 7th TNM edition from 2009. We added the information in table 1. In addition, the location of all tumours was added in table 1 also.
Table 1. Overview over observed OSCC parameters.
|
Group |
|
number |
(%) |
|
T-status |
T1*+T2 |
56 |
(67) |
|
|
T3+T4 |
27 |
(33) |
|
G-status |
G1 |
2 |
(2.4) |
|
|
G2 |
67 |
(80.7) |
|
|
G3 |
14 |
(16.9) |
|
N-status |
N- |
53 |
(64) |
|
|
N+ |
30 |
(36) |
|
UICC classification (TNM 7th edition; 2009)
|
UICC1 |
15 |
(18.1) |
|
UICC2 |
23 |
(27.7) |
|
|
UICC3 |
8 |
(9.6) |
|
|
UICC4 |
37 |
(44.6) |
|
|
Type of OSCC |
keratinized |
67 |
(81) |
|
|
not-keratinized |
9 |
(11) |
|
|
n/a |
7 |
(8) |
|
Smoker |
Yes |
49 |
(59) |
|
|
No |
31 |
(37) |
|
|
n/a |
3 |
(4) |
|
Alcohol abusus |
Yes |
48 |
(58) |
|
|
No |
32 |
(39) |
|
|
n/a |
3 |
(4) |
|
Smoker & alcohol |
|
40 |
(48) |
|
Localisation |
mouth floor |
23 |
(27.7) |
|
|
alveolar ridge |
22 |
(25.4) |
|
|
tongue |
20 |
(24) |
|
|
buccal plain |
8 |
(9.6) |
|
|
lip |
5 |
(6) |
|
|
palate |
3 |
(3.6) |
|
* one recurrent tumour in T1 group |
|||
- 4. Please include HPV status of patients and check if it has an effect on gene expression.
We do not have the information about the HPV status of the patients, unfortunately. During sample collection, the status of HPV was not as relevant as today. Therefore, this is a bias of our study.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper focus the attention on a very interesting topic, as , invadopodia mechanisms as well as CTTN genes are not widely studied in head and neck region.
It is a original production, which halps giving new data snd result to the previous literature on this topic.
It is a clear paper, with structured and solid data analysis
The results are reproducible, and the conclusions are consistent with the thesis and argument presented.
The conclusions are interesting and add advances in the current scientific knowledge.
No ethical problems are found in this study.
I would like to make some minor suggestions.
Specific comments:
· Line 32: I should delete “the” before oral squamous cell carcinoma.
· Line 74 and next “Included were patients with a histological diagnosed squamous cell carcinoma of the oral cavity”. The sentence is quite confusing. I suggest changing into “Inclusion criteria were ah histological diagnosis of oral cavity squamous cell carcinoma, age over than 18 year and no adjuvant radiotherapy or chemotherapy. Moreover, patients with recurrent disease were also included”.
Comments for author File: Comments.pdf
only minor english revision
Author Response
The paper focus the attention on a very interesting topic, as, invadopodia mechanisms as well as CTTN genes are not widely studied in head and neck region.
It is an original production, which helps giving new data and result to the previous literature on this topic.
It is a clear paper, with structured and solid data analysis
The results are reproducible, and the conclusions are consistent with the thesis and argument presented.
The conclusions are interesting and add advances in the current scientific knowledge.
No ethical problems are found in this study.
I would like to make some minor suggestions.
Specific comments:
- Line 32: I should delete “the” before oral squamous cell carcinoma.
- Line 74 and next “Included were patients with a histological diagnosed squamous cell carcinoma of the oral cavity”. The sentence is quite confusing. I suggest changing into “Inclusion criteria were ah histological diagnosis of oral cavity squamous cell carcinoma, age over than 18 year and no adjuvant radiotherapy or chemotherapy. Moreover, patients with recurrent disease were also included”.
We thank the reviewer for their positive feedback and commendation, as well as pointing out possible points of confusion for an uninitiated reader.
We changed the lines accordingly:
Line 32 deleted “the” before oral squamous cell carcinoma
Line 74 rewrote the section as follows:
Inclusion criteria were patients over the age of 18, with a histologically diagnosed oral squamous cell carcinoma, and no adjuvant radiation or chemotherapy. Included were patients with a histological diagnosed squamous cell carcinoma of the oral cavity. These patients were over 18 years old and had not received any adjuvant radiation or chemotherapy. Patients with recurrent disease were included.
Author Response File: Author Response.pdf
Reviewer 3 Report
In this study, the authors analyzed the gene expression patterns of CTTN and various other genes (n=36) associated with invadopodia formation, aiming to uncover relevant expression patterns and provide a comprehensive overview of their role in the context of OSCC. The analysis was performed on a whole genome data set comprising 83 OSCC samples, with a focus on tumor size, grading, lymph node status, and UICC classification. Their findings revealed significant overexpression of 18 genes, most notably CTTN, SRC, EGFR, SYK, WASL, and ARPC2. These genes exhibited a strong correlation with various tumor parameters, indicating their potential significance in OSCC progression.Nevertheless, given the methods employed to find the invadosome genes, the seeking is extremely biased. The value of human samples implies that analysis should be done along all genes, finding differentially expressed genes and not only those that fit in one functional annotation. Authors need to add information regarding other findings in their data.
Expression patterns could be not valid since the authors did not validate expression information of genes by qPCR. They have to validate at least 3 or 4 key genes.
English needs to be improved and paragraphs must be restructured since the authors used isolated sentences instead of solid clear idea in the results and disscusion sections.
English needs to be improved and paragraphs must be restructured since the authors used isolated sentences instead of solid clear idea in the results and disscusion sections.
Author Response
Comments reviewer 3
In this study, the authors analyzed the gene expression patterns of CTTN and various other genes (n=36) associated with invadopodia formation, aiming to uncover relevant expression patterns and provide a comprehensive overview of their role in the context of OSCC. The analysis was performed on a whole genome data set comprising 83 OSCC samples, with a focus on tumor size, grading, lymph node status, and UICC classification. Their findings revealed significant overexpression of 18 genes, most notably CTTN, SRC, EGFR, SYK, WASL, and ARPC2. These genes exhibited a strong correlation with various tumor parameters, indicating their potential significance in OSCC progression.Nevertheless, given the methods employed to find the invadosome genes, the seeking is extremely biased. The value of human samples implies that analysis should be done along all genes, finding differentially expressed genes and not only those that fit in one functional annotation. Authors need to add information regarding other findings in their data.
Expression patterns could be not valid since the authors did not validate expression information of genes by qPCR. They have to validate at least 3 or 4 key genes.
English needs to be improved and paragraphs must be restructured since the authors used isolated sentences instead of solid clear idea in the results and disscusion sections.
We thank the reviewer for the valuable comments. The reviewer unerringly pointed out the bias of our study. The principal proposal of our study was to present the gene expression patterns of cortactin and genes related directly to cortactin or the formation of invadosomes from our pool of OSCC samples. Furthermore, this paper serves as an entryway for a deeper expansion into this subject by our department for oro-maxillo-facial surgery. It should also help other researchers to compare their results with our data, as well as spark discussion. We agree with the reviewer, that an analysis of all genes is interesting and should be continuously pursued in the future by other or ourselves. With the presented data here, we can only hope that it will also lead to more focus on this topic and not only follow-ups by ourselves, but also the scientific community.
Regarding the validity of microarray data, the presented data pool is part of a former study. To avoid auto plagiarism, we summarized study design and data generation briefly. For the whole genome gene expression analysis, we isolated the total RNA from tissue samples and checked the quality with the Bioanalyzer. For sample preparation and microarray hybridization, we used the manufacturer’s standard protocols, chemistry, and platform. After scanning, the data was extracted with the manufacturer's software and uploaded to GeneSpring GX7.3.1 for further analysis. Here, normalisation of the data occurred. In a first step, the average values of the replicate spots were due to background and internal controls subtracted and normalized. With this step, a comparison of all 83 samples and controls was guaranteed. In a second step, we normalized all tumour samples against the data from the healthy pool. The gene expression level in the control group was set at 1. In tumour samples, gene expression fold lower than control was signed lower than 1 and with a higher gene expression fold higher than 1.
With this procedure, the microarray data are validated. Formation of invadopodia and epithelial-to-mesenchymal transition are an important factor in migration and the metastatic abilities of tumours. Especially for OSCC, since many patients die due to metastases. Cortactin is one of the key players, but it is not a loner. There are more genes and pathways involved. With the whole genome analysis, we have the possibilities to show the gene expression of interesting and related genes entirely. With RT-qPCR analysis, we focused just on single genes by ignoring their genomic interaction to give an overview.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
corrections have been made
corrections have been made
