Discovery of Serotransferrin Glycoforms: Novel Markers for Diagnosis of Liver Periductal Fibrosis and Prediction of Cholangiocarcinoma

Round 1

Reviewer 1 Report

The Manuscript entitled "Discovery of Serotransferrin Glycoforms; Novel Markers for Diagnosis of Liver Periductal Fibrosis  and Prediction of Cholangiocarcinoma"  by Jamnongkan et al identify novel bio-markers of CCA which can be used to distinguish CCA from HCC. Manuscript is interesting and gives some important information that can aid in diagnose of CCA based on glycosylated form of serum Transferrin. manuscript is written well. There are few places needs editing with some minor errors.

My comments:

If authors can categorise and perform statistical analysis of their data based on age and gender could give some more information on the findings. If there is a figure number limit with journal, submit as supplemental figure. Also provide how many samples you have analysed for each figure.

Author Response

Review#1 comments

The Manuscript entitled "Discovery of Serotransferrin Glycoforms; Novel Markers for Diagnosis of Liver Periductal Fibrosis and Prediction of Cholangiocarcinoma"  by Jamnongkan et al identify novel bio-markers of CCA which can be used to distinguish CCA from HCC. Manuscript is interesting and gives some important information that can aid in diagnose of CCA based on glycosylated form of serum Transferrin. Manuscript is written well. There are few places needs editing with some minor errors.

Minnor comments:

If authors can categorise and perform statistical analysis of their data based on age and gender could give some more information on the findings. If there is a figure number limit with journal, submit as supplemental figure. Also provide how many samples you have analysed for each figure.

Authors reply to the comments:

Thank you very much for your supportive comments and suggestions.

We provided sample number (n) that analyzed for each figure.

We also performed statistical analysis of the 9 glycoforms and the clinical data including ages, genders, histological types and metastasis status. The degrees of 9 glycopeptides were no correlated with ages, genders, histological types and metastasis statuses as shown in Table S3 (Revised version). Also, the combinations of the 3 glycopeptides including 6503, 6502 and 6501 were also no significantly correlated with ages, genders, histological types and metastasis statuses as shown in Table S4 (Revised version). Moreover, the increasing of 6503, 6502 and 6501 glycopeptides were significantly correlated with poor prognosis as shown in Figure 3.

For the tumor staging, we need the data of T (the size of the origin tumor), M (nearby lymph node metastasis) and N (distant metastasis) for calculation of the staging. However, 15 of CCA tissues are biopsy samples. We don’t have the complete data set for clarifying the TMN staging of the 15 cases. Moreover, 85 tissues had complete TNM staging data.  The statistical analysis showed significantly correlation between the tumor stages and the 6501 glycoform levels as shown in Table S3 (Revised manuscript). Therefore, we included tumor stages of the 85 cases in the Table S3 and Table S4.

Taken all together, our results indicated that the increasing of the 9 glycoforms of serotransferrin can be used for CCA diagnosis and the increasing of the 6503, 6502 and 6501 glycoforms can be used as the prognosis marker of CCA patients. Moreover, the high 6501 glycoform levels can be used for CCA staging marker.

Reviewer 2 Report

There is almost no study related to Serotransferrin Glycoforms in CCA ,  so this study might give some help on CCA diagnosis.

Some sentences have grammar problems, so the author should better modify carefully on the writing.

Some abbreviation shouldn’t put full name again after the first time, such as  “ infinity ultrahigh-performance liquid chromatography (UPLC)” should better be put in the introduction part instead of in the method part.

About the serotransferrin glycoforms in CCA patients, does it have any relationship to other signal pathways involving in the liver flukes infection? And is there any correlation between different CCA disease stages with serotransferrin glycoforms?

Could the author add various serotransferrin glycoforms NMR pictures in the supplementary?

Author Response

Reviewer#2 Comments

1. 1. There is almost no study related to Serotransferrin Glycoforms in CCA ,  so this study might give some help on CCA diagnosis.

Authors reply to the comments: Thank you very much for your supportive comments.

2. 2. Some sentences have grammar problems, so the author should better modify carefully on the writing.

Authors reply to the comments: We intensively checked through the manuscript and edited the manuscript as shown by Track Change. We hope that this revised manuscript will be accepted for publication.

3. 3. Some abbreviation shouldn’t put full name again after the first time, such as  “ infinity ultrahigh-performance liquid chromatography (UPLC)” should better be put in the introduction part instead of in the method part.

Authors reply to the comments: Thank you very much for your suggestion. We added “the Agilent 1290 infinity ultrahigh-performance liquid chromatography (UPLC) system and electrospray ionization (ESI) coupled to an Agilent 6490 triple quadrupoles (QqQ) mass spectrometer (UPLC-ESI-QqQ) with MRM analysis” in the introduction part.

4. 4. About the serotransferrin glycoforms in CCA patients, does it have any relationship to other signal pathways involving in the liver flukes infection?

Authors reply to the comments: N-glycofroms of the Ov parasite were identified [1, 2]. The glycoforms that were found in the parasite are different from our results. Up to date, there are no evidences describing any relationship between the liver fluke (Ov) infection and signal pathways involving in glycosylation. According to our finding, sialylated glycoforms were highly detected in PDF and CCA subjects. Sialylated glycoforms are the products of sialyltransferases.  Sialytransferases can be up-regulated by inflammation [3]. Therefore, we hypothesized that inflammation induced by the parasite infection, PDF formation and cancer progression may up-regulate sialytransferases leading to increasing of sialylated glycoforms of serotransferrin and other glycoproteins in Ov-infected subjects as well as PDF and CCA subjects.

Reference

1. 1. Talabnin, K.; Aoki, K.; Saichua, P.; Wongkham, S.; Kaewkes, S.; Boons, G. J.; Sripa, B.; Tiemeyer, M., Stage-specific expression and antigenicity of glycoprotein glycans isolated from the human liver fluke, Opisthorchis viverrini. Int J Parasitol 2013, 43, (1), 37-50.
2. 2. Talabnin, K.; Yagi, H.; Takahashi, N.; Suzuki, T.; Kato, K.; Uemura, H.; Saichua, P.; Kaewkes, S.; Wongkham, S.; Suzuki, Y.; Sripa, B., Glycobiological study of adult Opisthorchis viverrini: characterization of N-linked oligosaccharides. Mol Biochem Parasitol 2006, 147, (2), 230-3.
3. 3. Rodrigues, E.; Macauley, M. S., Hypersialylation in Cancer: Modulation of Inflammation and Therapeutic Opportunities. Cancers (Basel) 2018, 10, (6).

5. 5. Is there any correlation between different CCA disease stages with serotransferrin glycoforms?

Authors reply to the comments:  

85 tissues had complete TNM staging data.  The statistical analysis showed significantly correlation between TMN staging of the tumors and the 6501 glycoform levels as shown in Table S3 (Revised manuscript). For the TMN staging, we need the data of T (the size of the origin tumor), M (nearby lymph node metastasis) and N (distant metastasis) for calculation of the staging. However, 15 of CCA tissues are biopsy samples. We don’t have the complete data for clarifying the TMN staging. Therefore, we included the tumor stages of the 85 cases in the Table S3 and Table S4.

We also performed statistical analysis of the 9 glycoforms and the clinical data including ages, genders, histological types and metastasis status. The degrees of 9 glycopeptides were no correlated with ages, genders, histological types and metastasis statuses as shown in Table S3 (Revised version). Also, the combinations of the 3 glycopeptides including 6503, 6502 and 6501 were also no significantly correlated with ages, genders, histological types, tumor stages and metastasis statuses as shown in Table S4 (Revised version). Moreover, the increasing of 6503, 6502 and 6501 glycopeptides were significantly correlated with poor prognosis as shown in Figure 3.

Taken all together, our results indicated that the increasing of the 9 glycoforms of serotransferrin can be used for CCA diagnosis and the increasing of the 6503, 6502 and 6501 glycoforms can be used as the prognosis marker of CCA patients. Moreover, the high 6501 glycoform levels can be used for CCA staging marker.

6. 6. Could the author add various serotransferrin glycoforms MRM pictures in the supplementary?

 Authors reply to the comments: We used standard serotransferrin protein for setting up the instruments. Therefore, we have only the MRM chromatogram of the standard protein as shown in Figure S1.