Prediction of the Aggressive Clinical Course of Papillary Thyroid Carcinoma Based on Fine Needle Aspiration Biopsy Molecular Testing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

I found this study interesting. The authors address a clinically relevant question that will add to the increasing information available to clinicians like me that deal with thyroid cancer.

The scientific language is good, the manuscript is well written and easy to understand.

The title is a very general title and does not pique the interest of the reader. I recommend they review the title and use one that would interest a clinician who is looking for up to date information regarding molecular testing of thyroid cancer.

The abstract gives a good summary of the findings.  Consider changing lines 33 and 37, so the abbreviations (FN1 and CKDN2A) can be used after the full words rather than before the full words. Unless limited by word count, would the authors consider adding a statement summarizing the significance of these 4 molecular markers in the abstract as discussed in the paragraph starting from line 205?

The authors have presented the results and discussion before presenting the study methods. I found this format confusing. It was difficult for me to understand the results before knowing the study population, methods and statistical methods. Is there some special reason the authors chose this format? Would they consider presenting the material/methods/analysis process earlier in the manuscript?

The study is only focused on PTC. The authors should consider adding a statement, possibly in section 4.1 (clinical material) that patients with follicular cancers were excluded so as to avoid any confusion that this study deals with DTC (differentiated thyroid cancer which would include follicular and Hurthle cell as well as PTC) or PTC only (Papillary thyroid cancer only)

The study was conducted on FNAB specimens preoperatively. It seems that all the patients underwent surgery. It would be helpful to know extent of surgery in the patients given that tumor size ranged from 0.6 cm to 8 cm.

Table 1- line 100- Did the authors assess for variants of PTC? If they did, it would be interesting to see the information related to the molecular markers? If no variants were identified, it should be reported that all PTC were assessed to be classical variants.

Based on my clinical experience, and perusal of scientific literature, it appears to me that there are too few low risk, and too many high risk patients in this group. Can the authors explain this? The study population is in Russia. Does this stratification reflect the prevalence of thyroid cancer in that population? 

It may be helpful to add information about how the patients presented initially (incidental finding, thyroid nodule, neck mass/ palpable lymphadenopathy) Information regarding radiation exposure may be helpful also given the high proportion of multifocal cancers. 

Line 105- please change minimal to minimum. 

Table 2 and line 119- Please review regarding BRAF information, the p value is placed in the wrong column for low risk and intermediate risk. 

Line 167- Do the authors have information regarding PTC/FTC/oncocytic, and variants of PTC, like tall cell variant regarding this finding? 

Line 372- The conclusion includes a statement by the authors regarding active surveillance compared to surgery. This is a good assumption but not an objective one based on this study where all patients underwent surgery.  I agree with the other conclusions made by the authors.

Author Response

The title is a very general title and does not pique the interest of the reader. I recommend they review the title and use one that would interest a clinician who is looking for up to date information regarding molecular testing of thyroid cancer.

Below, we propose some titles that might be better:

“Prediction of the aggressive clinical course of papillary thyroid carcinoma based on FNAB molecular testing”

Or

“Promising molecular markers for predicting the risk of papillary thyroid carcinoma recurrence: the miRNA-221, FN1 and CDKN2A genes”

Or

“FN1, CDKN2A and miRNA-221 expression reflects the risk of papillary thyroid carcinoma recurrence”

The abstract gives a good summary of the findings.  Consider changing lines 33 and 37, so the abbreviations (FN1 and CKDN2A) can be used after the full words rather than before the full words. Unless limited by word count, would the authors consider adding a statement summarizing the significance of these 4 molecular markers in the abstract as discussed in the paragraph starting from line 205?

We have made respective changes for the first sentence in Abstract.

Unfortunately, the word limit for Abstract is 200 words, and the word count is already 197. Therefore, we find it difficult to add even a single sentence to the Abstract.

The authors have presented the results and discussion before presenting the study methods. I found this format confusing. It was difficult for me to understand the results before knowing the study population, methods and statistical methods. Is there some special reason the authors chose this format? Would they consider presenting the material/methods/analysis process earlier in the manuscript?

Indeed, this manuscript structure is rather unusual (for us as well), but it is the format used in this journal. The journal provides a template that we fill out. In this template, the Materials and Methods section is found in the end of the manuscript and followed only by Conclusions and References.

The study is only focused on PTC. The authors should consider adding a statement, possibly in section 4.1 (clinical material) that patients with follicular cancers were excluded so as to avoid any confusion that this study deals with DTC (differentiated thyroid cancer which would include follicular and Hurthle cell as well as PTC) or PTC only (Papillary thyroid cancer only)

We have added the following statement to section 4.1:

Therefore, all the findings obtained in this study refer to PTC only rather than to the entire group of differentiated thyroid cancers.

The study was conducted on FNAB specimens preoperatively. It seems that all the patients underwent surgery. It would be helpful to know extent of surgery in the patients given that tumor size ranged from 0.6 cm to 8 cm.

A paragraph has been added to the description of patients:

The study included patients who had undergone total thyroidectomy. Lymph node dissection was performed for 53 of them. Central neck compartment lymph node dissection (level VI-VII) was performed for 36 of them, and central and lateral neck compartment lymph node dissection (level II-VII), for 17 patients.

Table 1- line 100- Did the authors assess for variants of PTC? If they did, it would be interesting to see the information related to the molecular markers? If no variants were identified, it should be reported that all PTC were assessed to be classical variants.

The information on subtypes of PTC is available and has been added to Table 1.

As for the association between PTC variants and molecular markers, we have not studied this association, since it actually is unrelated to the central idea of this study.

Based on my clinical experience, and perusal of scientific literature, it appears to me that there are too few low risk, and too many high risk patients in this group. Can the authors explain this? The study population is in Russia. Does this stratification reflect the prevalence of thyroid cancer in that population?

The ratio between patients with high/low risk of PTC used in our study is artificial: we were deliberately searching for patients with high risk of PTC; therefore, their relative number in our sample is so large.

It may be helpful to add information about how the patients presented initially (incidental finding, thyroid nodule, neck mass/ palpable lymphadenopathy) Information regarding radiation exposure may be helpful also given the high proportion of multifocal cancers.

This information about patients is unavailable (we have not collected it), since it is unclear how these data can be linked to the main idea of this study (searching for molecular markers of the recurrence risk of PTC).

Line 105- please change minimal to minimum.

We have changed this word, thank you for bringing it to our attention.

Table 2 and line 119- Please review regarding BRAF information, the p value is placed in the wrong column for low risk and intermediate risk.

The data in Table 2 are correct. The uncertainty can possibly be related to the fact that we have three groups (Low, Intermediate, and High) that we compare in a pairwise manner. Therefore, the p value needs to be correlated to the “Odds ratio” column, where these pairs are specified, rather than with the “Parameter” column.

Line 167- Do the authors have information regarding PTC/FTC/oncocytic, and variants of PTC, like tall cell variant regarding this finding?

We have not considered other DTCs or focused on different variants of PTC individually, since it lay beyond the concept of the study.

Generally speaking, we studied all these genes in different variants of DTCs, but almost all the associations were revealed for PTC. We present the approximate results below:

	ROC AUC
	PTC	DTC
TSP4	0.893	0.764
ECM1	0.796	0.748
HMGA	0.918	0.897
FN1	0.890	0.749
SRPN1	0.853	0.634
TIMP1	0.893	0.750
GMNN	0.681	0.599
CDKN2A	0.697	0.630
CITED1	0.929	0.765
NIS	0.601	0.733
AFAP1L2	0.570	0.642
TPO		0.883
TSHR		0.541
SLC26		0.804
CPQ	0.536	0.511
TFF3	0.789
RXRG	0.927	0.881
DIO1	0.771	0.696
APOE	0.688	0.573
CLU	0.844	0.812

Line 372- The conclusion includes a statement by the authors regarding active surveillance compared to surgery. This is a good assumption but not an objective one based on this study where all patients underwent surgery. I agree with the other conclusions made by the authors.

Indeed, this conclusion is not correct, so we have removed it.

Reviewer 2 Report

Comments and Suggestions for Authors

Summary:

In this original article, Lukyanov et al., assess the applicability of molecular markers in preoperative diagnosis of aggressive variants of papillary thyroid cancer. With the set of results obtained, the authors conclude that the recurrence risk of papillary thyroid carcinoma can be preoperatively predicted using miRNA-221, the fibronectin and cyclin-dependent kinase inhibitor 2A genes.

Comments:

The introduction section is very poor in content. Authors should use more recent references in the literature that address the topic discussed in the paper.

Regarding materials and methods, I suggest the authors describe in more detail the methodology carried out for each experiment. In addition, if the methods used have been previously published by the same group or by other research groups, please, at the end of each methodology, add the reference of the original paper.

Regarding the results, the authors should rewrite the figure captions with greater detail. The figures provide a lot of information, but the captions are poor in content. 

In table 1, please explain in more detail what "Value, n (%)" means.

Please explain better the results described in table 2. The way the table is presented, and the results are described, it is difficult to follow.

 

 

 

Comments on the Quality of English Language

I suggest authors reevaluate the text, with the help of a Native English Speaker

Author Response

The introduction section is very poor in content. Authors should use more recent references in the literature that address the topic discussed in the paper.

We have expanded the Introduction section and added some references.

Regarding materials and methods, I suggest the authors describe in more detail the methodology carried out for each experiment. In addition, if the methods used have been previously published by the same group or by other research groups, please, at the end of each methodology, add the reference of the original paper.

We have expanded the description of methods and cited earlier publications, since we actually used the same methods.

Regarding the results, the authors should rewrite the figure captions with greater detail. The figures provide a lot of information, but the captions are poor in content.

The new caption for Figure 1 is given below:

Box-whisker plots for the relative expression level of selected miRNAs (miR-146b, miR-221) and genes (FN1 and CDKN2A). Inner line, the median value; cross, the mean value; box, upper and lower quartiles; whisker, non-outlier range; circles, outliers. The data are presented for samples belonging to the low/intermediate/high ATA recurrence risk groups or to the groups with/without lymph node metastases.

The caption of Figure 2 has also been changed:

ROC curves for the three best markers that allow identifying samples with high ATA recurrence risk: miRNA-221, FN1 and CDKN2A genes. The curves are shown for comparison of either low versus high, or intermediate versus high ATA recurrence risk groups.

In table 1, please explain in more detail what "Value, n (%)" means.

Indeed, this denotation is far from optimal. We have changed it to “N (%)”, i.e., the number of specimens per sample with the respective characteristics and the percentage of the total sample.

Please explain better the results described in table 2. The way the table is presented, and the results are described, it is difficult to follow.

We find it difficult to answer this comment because it is unclear what was problematic: the way information is presented in the table or in the description of the table.

We have added the following sentence to the text of the manuscript:

“Thus, for BRAF mutation, no statistically significant association was found for sex, multifocality, extrathyroidal extension, metastasis or vascular invasion.”

This sentence is followed by a fragment that has already been there in the manuscript:

“The BRAF mutation was statistically significantly more frequent in the groups of patients with intermediate (80%, p = 0.03) and high (84%, р = 0.04) ATA recurrence risk compared to the low-risk group (56.5%).”

We hope that it will facilitate understanding.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for improving the quality of the manuscript. My intention was to help. I wish success to the entire team involved in this work.