Biliary hCGβ Is a Potential Novel Marker for Prediction of Biliary Neoplasia in Primary Sclerosing Cholangitis Patients

Round 1

Reviewer 1 Report

The authors present an original single center study with the aim of identifying novel potential markers for prediction of biliary neoplasia in PSC patients. Bile samples from  214 patients referred for ERCP were evaluated for sin(ogen)-2 and -3, SPINK1 and hCGβ. Based on their outcomes the authors conclude that monitoring of hCGβ may be justified in PSC patients.

This is a very interesting and well-written study. It is original, concerns a fairly good sample of patients and is of clinical value.

Some minor points that need to be addessed by the authors:

• Methods section: "The study included 214 patients, referred for endoscopic retrograde cholangiography 76 (ERC), from Helsinki University Hospital PSC registry. Patient demographics are shown 77 in Table 1....." all of this information constitute outcomes and should be moved towards the results section. Equally "During the follow-up of 9.1 yrs (median, IQR 0.9 yrs), five patients were diagnosed 98 for CCA (median 1.6 yrs, range 0.1-8.8 yrs, after the sample collection), five for hepatocel-99 lular carcinoma (5.8 yrs, 0.0-9.1 yrs), 22 (three of whom had a hepatocellular carcinoma 100 diagnosis) underwent liver transplantation..." this is a descriptive result which should be moved to the relevant section as well.
• IRB approval should be provided if needed for this study
• Please add a limitation section, this is crucial for the readership in order to interpret these outcomes

Author Response

We thank for constructive comments, which, we feel, have allowed us to significantly improve the manuscript. Please, find below our response (in italics and blue).

The authors present an original single center study with the aim of identifying novel potential markers for prediction of biliary neoplasia in PSC patients. Bile samples from 214 patients referred for ERCP were evaluated for trypsin(ogen)-2 and -3, SPINK1 and hCGβ. Based on their outcomes the authors conclude that monitoring of hCGβ may be justified in PSC patients.

This is a very interesting and well-written study. It is original, concerns a fairly good sample of patients and is of clinical value.

Some minor points that need to be addessed by the authors:

1. Methods section: "The study included 214 patients, referred for endoscopic retrograde cholangiography 76 (ERC), from Helsinki University Hospital PSC registry. Patient demographics are shown 77 in Table 1....." all of this information constitute outcomes and should be moved towards the results section. Equally "During the follow-up of 9.1 yrs (median, IQR 0.9 yrs), five patients were diagnosed 98 for CCA (median 1.6 yrs, range 0.1-8.8 yrs, after the sample collection), five for hepatocel-99 lular carcinoma (5.8 yrs, 0.0-9.1 yrs), 22 (three of whom had a hepatocellular carcinoma 100 diagnosis) underwent liver transplantation..." this is a descriptive result which should be moved to the relevant section as well.

As suggested, we have moved most of the patient details in the Results section in a new paragraph 3.1. Patients.

2. IRB approval should be provided if needed for this study.

This information can be found before References, but since this is important we have added the following also in the Methods section in the end of paragraph 2.1. Patients: “All the patients included in the PSC registry gave written informed consent. The study was performed following the principles of good clinical practice and in accordance to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008). The study protocol was approved by Helsinki University Hospital Ethical Committee IV, HUS/1566/2020.”

3. Please add a limitation section, this is crucial for the readership in order to interpret these outcomes.

We agree that this would be very helpful for the readers. While we already discussed about the limitations of the bile samples, we added in Discussion: “The study has also some other limitations, mostly due to small number of patients with biliary neoplasia, reflecting the disease prevalence. Thus, we were not able to fully evaluate the clinical performance of the biliary hCGβ for prediction or early detection of biliary neoplasia and, especially, CCA. Furthermore, the commonly used serum markers, CEA and CA19-9, were not measured from biliary samples and, at present, their performance in bile, compared to that of hCGβ, is unknown.” However, as already stated in the end of Discussion, monitoring of hCGβ may be justified in PSC patients and our results warrant larger clinical studies.

Reviewer 2 Report

The paper is interesting. The introduction chapter is well written and quite explicative of the study hypothesis

The disease is rare and probably justified The study period is not indicated in the “patients” subchapter of the chapter “Patients and Methods”. It is also not specified the method of 182 patients had diagnosis of sclerosing cholangitis done. Did all the patients have a histological diagnosis?

Can Authors comment on the presence of 5 patients with hepatocellular carcinoma included in this study? Why did these patients undergo ERC? In which of the two indications for ERC did they fell?

Twenty-two out of 214 included patients underwent liver transplantation. Which information could be retrieved from the obtained entire biliary duct in the view of the present study?

How these patients were selected for entering the study? Were all of them consecutive patients?

Did the Authors measure the level of hCGβ in the blood samples they collected at the time of ERC?

Did Authors find any correlations between hCGβ bile levels and blood laboratory tests? Accordingly to references #7 and #10 this should be possible. If this was not done, can Authors explain the reason?

Discussion is somehow poor.

Author Response

We thank for constructive comments, which, we feel, have allowed us to significantly improve the manuscript. Please, find below our response (in italics and blue).

The paper is interesting. The introduction chapter is well written and quite explicative of the study hypothesis

The disease is rare and probably justified The study period is not indicated in the “patients” subchapter of the chapter “Patients and Methods”.

Thank you for pointing this out. We have added in section 2.1 Patients that: “Study period was 2010-2012, with follow up to 2020 for hard end points such as CCA, liver transplantation or death.

It is also not specified the method of 182 patients had diagnosis of sclerosing cholangitis done. Did all the patients have a histological diagnosis?

We have added in section 2.1 Patients: “While the PSC diagnoses were mainly based on ERC findings, liver biopsy was performed in 82% (150) of PSC patients. According to present guidelines, liver biopsy is recommended only for the diagnosis of PSC with AIH–like features (PSC-AIH overlap syndrome) and in suspicion of so called 'small duct PSC'. The absence of PSC was confirmed histologically in 22 (66%) of control patients in addition to ERC.”

Can Authors comment on the presence of 5 patients with hepatocellular carcinoma included in this study? Why did these patients undergo ERC? In which of the two indications for ERC did they fell?

Patients included into the study participated in systematic biliary dysplasia surveillance program at Helsinki University Hospital. The indication for ERC was dysplasia surveillance and HCC were found at MRI.

Twenty-two out of 214 included patients underwent liver transplantation. Which information could be retrieved from the obtained entire biliary duct in the view of the present study?

We have added in Results in section 3.1. Patients: “Of the patients who underwent liver transplantation, five were transplanted for suspicion of malignancy. In explant, dysplasia was found in three of them. None of the patients transplanted for end stage liver disease (n=12) had dysplasia in the explant, but one had three small HCC-lesions, all less than 20 mm. In patients transplanted for symptoms (n=5) no dysplasia was found either in the explant, and 4 of them had cirrhosis.”

How these patients were selected for entering the study? Were all of them consecutive patients?

We have modified the description of patients to address this (in 2.1 Patients): “The study included 214 consecutive patients from Helsinki University Hospital PSC registry, referred for endoscopic retrograde cholangiography (ERC), and from whom bile samples were available.”

Did the Authors measure the level of hCGβ in the blood samples they collected at the time of ERC?

While this would have been good, we did not measure serum hCGβ as we assumed that marker levels in bile reflect the disease state and risk for CCA/neoplasia more accurately than those in serum. One of the purposes of the present study was to obtain data that would justify, in future, larger studies, also with serum samples.

Did Authors find any correlations between hCGβ bile levels and blood laboratory tests? Accordingly to references #7 and #10 this should be possible. If this was not done, can Authors explain the reason?

Indeed, biliary hCGβ weakly correlated with some of the laboratory blood tests. We added these results in the end of 3.2 Detection of markers in bile: “While no correlation between biliary hCGβ and serum CA19-9 was observed in PSC patients (ρ=0.10, p=0.22, n=161), biliary hCGβ correlated with plasma ALT and ALP, and serum bilirubin (for all ρ>0.16, p<0.05, n=177-179).”

Discussion is somehow poor.

Also based on the comment by Reviewers #1 and #3, we have added in Discussion a section about limitations of the study and discussion about peroral cholangioscopy (POCS) compared to biliary markers.

 

Reviewer 3 Report

This is an interesting article to detect biliary neoplasms in PSC cases by biomarker in bile juice.

 

Main

1. This study included various cases included various timing. Let us know the details of patient character by flow chart or table.

 

2. Detection of malignancy in PSC was still difficult and POCS was routinely performed in PSC
   Please discuss about POCS in diagnosing cholangiocarcinoma in PSC cases, and compare with bile juice biomarker.

・Arnelo U, et al. Prospective evaluation of the clinical utility of single-operator peroral cholangioscopy in patients with primary sclerosing cholangitis. Endoscopy.　2015;47:696–702.

・Fujisawa T, et al. Role of Peroral Cholangioscopy in the Diagnosis of Primary Sclerosing Cholangitis. Diagnostics (Basel). 2020 Apr 29;10(5):268.

 

3. The authors mention about the relation between hCGβand cholangiocarcinoma in previous literature.

 

4. The reviewer thinks that intrahepatic CCX which showing mass-forming and extrahepatic cholangiocarcinoma which existed in the bile duct were different. Please divide the biliary neoplasia into these 2 categories, and perform additional analysis.

Author Response

We thank for constructive comments, which, we feel, have allowed us to significantly improve the manuscript. Please, find below our response (in italics and blue).

This is an interesting article to detect biliary neoplasms in PSC cases by biomarker in bile juice.

Main

This study included various cases included various timing. Let us know the details of patient character by flow chart or table.

We have modified the description of patients to address the timing and selection: “The study included 214 consecutive patients from Helsinki University Hospital PSC registry, referred for endoscopic retrograde cholangiography (ERC), and from whom bile samples were available. Study period was 2010-2012, with follow up to 2020 for hard end points such as CCA, liver transplantation or death.” We hope that this togther with the main patient groups described in the Table 1, and details in the new section 3.1 Patients in Results give sufficient information.

Detection of malignancy in PSC was still difficult and POCS was routinely performed in PSC. Please discuss about POCS in diagnosing cholangiocarcinoma in PSC cases, and compare with bile juice biomarker. (Arnelo U, et al. Prospective evaluation of the clinical utility of single-operator peroral cholangioscopy in patients with primary sclerosing cholangitis. Endoscopy. 2015;47:696–702.; Fujisawa T, et al. Role of Peroral Cholangioscopy in the Diagnosis of Primary Sclerosing Cholangitis. Diagnostics (Basel). 2020 Apr 29;10(5):268.)

We added a short paragraph in Discussion and three references, including the ones suggested by the reviewer: “In addition to biomarkers and brush cytology, methods like peroral cholangioscopy (POCS) with targeted biopsies have been suggested for diagnosis of PSC and provide information about PSC-associated biliary neoplasia [20,21]. However, brush cytology with a possibility to reach bile ducts, including intrahepatic ones, more widely has been found to have better diagnostic accuracy than POCS for indeterminate biliary strictures [22]. Furthermore, biomarkers that are functionally involved or associated with the development of neoplasia may be able to de-tect or predict development of neoplasia at much earlier state than visualization-based approaches. However, all this warrants further studies.”

The authors mention about the relation between hCGβ and cholangiocarcinoma in previous literature.

This literature shows that hCGβ is a potential marker for detection of CCA, and is associated also with adverse prognosis in CCA patients. In these earlier studies the patients already had diagnosed CCA, while in the present study we showed that biliary hCGβ levels are elevated already before the detection of CCA by routine clinical methods.

The reviewer thinks that intrahepatic CCX which showing mass-forming and extrahepatic cholangiocarcinoma which existed in the bile duct were different. Please divide the biliary neoplasia into these 2 categories, and perform additional analysis.

In our whole PSC registry, only 16% of CCAs are intrahepatic, so, although the comment is highly relevant, it is unfortunately not possible to address given the small number of CCA patients in the study.

Round 2

Reviewer 3 Report

This article improved well after revise. Thanks for accepting our suggestions.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.