Prospective Surveillance for Very Early Hepatocellular Carcinoma (PRECAR): an Observational Cohort Trial
Conditions: Carcinoma, Hepatocellular
Sponsor: Eastern Hepatobiliary Surgery Hospital

Prospective surveillance for very early hepatocellular carcinoma 
(PRECAR): a study protocol for an observational multicenter cohort 
trial 
 
NCT number: 03588442 
 
Date:2020.4.30 
 
Version: v1.0 
 
 
 
 
 
 
 
 
 
 
 
 
 

Abstract 
Background: Approximately 20% patients are diagnosed to hepatocellular carcinoma at early 
stage, when effective treatments are feasible, and benefit from curative therapeutic approaches, 
such as hepatic resection and liver transplantation. Biannual ultrasound is recommended for 
patients with cirrhosis for early diagnosis of hepatocellular carcinoma. However, limited 
sensitivity and false-positivity call for development of a new surveillance tool with prior 
accuracy. This study aims to build a biosignature to stratify high risk population for developing 
hepatocellular carcinoma and to diagnosis hepatocellular carcinomas at very early stage using 
biological signature. 
Methods: This investigator-initiated, multicenter, observational, cohort trial will enroll 5000 
patients with liver cirrhosis and 5000 patients with hepatitis B virus infection. Exclusion criteria 
include human immunodeficiency virus infection, previous diagnosis of active pulmonary 
tuberculosis, and pregnancy. Patients will be recruited for 1 year and follow-up investigation 
will be performed biannually for 3 years to collect demographic and clinical characteristics, 
blood samples, disease status, and outcome data. Development of hepatocellular carcinoma and 
overall survival are the primary endpoints, which will be contributed to the development of the 
biosignature for risk stratification for incidence, diagnosis at very early stage, and survival 
prediction. The secondary endpoints include development of non-hepatocellular carcinoma 
hepatic malignancy, liver resection rate, postresection survival, and relapse-free survival. 
Discussion: The biosignature will be useful in clinical practice by stratifying patients with 
cirrhosis or hepatitis B virus infection at high risk for development of hepatocellular carcinoma 
and diagnosing at very early stage. 

Trial registration: ClinicalTrials.gov identifier, NCTxx, first received xx, xx 2018. 
Key words: liver cancer, diagnosis, early stage, observational cohort trial, biosignature, 
prediction model 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Introduction 
China has among the highest incidence and mortality of hepatocellular carcinoma (HCC) in the 
world with increasing trend.1,2 Prognosis of HCC is variable according to different stages due 
to feasibility of therapeutic options.3 Radical resection of tumor is recommended for patients 
with a solitary tumor and preserved liver function without extrahepatic metastasis, whereas 
liver transplantation provides survival benefits for unresectable cases within Milan criteria 
(solitary tumor ≤ 5 cm or up to three nodules ≤ 3 cm).4,5 In addition, image-guided ablation is 
the most frequently applied strategy with limitations of size and localization of the tumor.6 For 
multifocal disease without vascular invasion and extrahepatic metastasis, chemoembolization 
has been suggested to be promising therapy for improvement of prognosis.7 Despite candidate 
selection criteria according to the disease status, recent advances revealed that diagnosis at an 
early stage is the optimum approach for to achieve favorable prognosis by offering curative 
treatment options.8 
To date, approximately 20% of patients with HCC are diagnosed at early stage and benefit 
from potentially curative ablative treatments.9 According to a study involving 5 Dutch centers, 
surveillance (≥ 2 screening tests during 3 preceding years and at least once of imaging test 
within 18 months before development of HCC) was found to be an independent prognostic 
factor that predicts favorable outcomes.10 A randomized controlled trial including 18,816 
patients with hepatitis B virus (HBV) infection demonstrated that surveillance by biannual α-
fetoprotein test and ultrasonography examination ameliorated HCC-related mortality from 
83.2/100,000 (screening group) to 131.5/100,000 (control group) with a hazard ratio (HR) of 
0.63 (95% CI, 0.41 to 0.98), which indicates 37% of decrease in HCC-related mortality.11 

Although a validation trial is yet to be carried out, since the only opportunity to provide 
effective treatments with long-term survival is the detection of the tumor at an early stage, 
surveillance is crucial predictor for favorable prognosis. 
Justification of inclusion criteria for a surveillance program is determined by the risk of HCC 
life expectancy, and cost-effectiveness.12 Regarding absence of experimental data, the decision 
for patient inclusion is based on its efficacy evaluated by heterogeneous designs, indicating that 
the efficacy of a surveillance program is dependent to incidence of HCC. Accordingly, 
populations with liver cirrhosis and HBV infection are currently recommended for 
surveillance.13 
Ultrasonography and serum tumor markers are the preferred tests for surveillance of HCC 
based on toleration, availability, non-invasiveness, and sensitivity. Ultrasonography has a 
reported sensitivity of 60 to 80%, whereas α-fetoprotein was approximately 60% with the most 
efficient cut-off (10 to 20 ng/mL).14,15 Combination of ultrasonography and α-fetoprotein may 
increase accuracy, but also increases false-positivity and cost. Regarding importance of 
diagnosis of HCC at early stage, there is an unmet need for a well-made surveillance model to 
detect early HCC with priority in accuracy and sensitivity; identification of populations at high 
risk before development of HCC may be ideal if high sensitivity is achieved. In addition, it may 
prevent up to 6 months of disease progression and improve diagnostic sensitivity, ultimately 
improving prognosis. 
The Prospective Surveillance for Very Early Hepatocellular Carcinoma (PRECAR) trial is 
designed as an observational multicenter cohort trial to conduct a signature to detect and define 
very early HCC and individuals at high risk of HCC by analyzing genome sequencing, plasma-

free DNA sequencing, and high-throughput protein expression screening. 
 
Methods and Design 
Ethics 
The PRECAR trial will be conducted in accordance with the Declaration of Helsinki and current 
Good Clinical Practice guidelines. The relevant ethics committee approval will be obtained 
before enrolment of patients. Informed consents will be obtained from all participants before 
enrollment. 
 
Eligible population 
The inclusion and exclusion criteria for the PRECAR trial are described in Table 1. Briefly, two 
independent cohorts (cirrhosis cohort and HBV infection cohort) will be recruited for 
surveillance of HCC. The cirrhosis cohort will be consisted of patients with biopsy diagnosis 
or distinctive clinical characteristics of cirrhotic liver regardless etiology of cirrhosis. For HBV 
infection cohort, seropositivity of hepatitis B surface antigen is the basic condition for inclusion. 
 
Overview of trial design 
PRECAR is a prospective, observational, multicenter trial surveilling development of HCC in 
patients with cirrhosis or HBV infection. In PRECAR, the aim will be to recruit a total of 10,000 
patients (5000 each for the two cohorts) from a minimum of 10 sites. Follow-up will be carried 
out for eligible patients for collection of demographic, clinical, and serological characteristics 
and samples for analyses. For those who developed HCC, hepatic resection will be the standard 

of care unless unresectable. Data of interest and schedule for the PRECAR trial are summarized 
in Table 2.  
 
Outcome measures 
The primary endpoints of the PRECAR trial include development of HCC, overall survival 
(OS), and liver-related disease progression (LDP). Secondary endpoints are development of 
non-HCC malignant neoplasm, such as intrahepatic cholangiocarcinoma, liver resection rate 
(LRR), and postoperative overall (POS) and relapse-free survival (PRFS) after development of 
liver malignant neoplasm. 
 
Outcome definitions 
HCC – the time from the date of inclusion to development of HCC. 
OS – the time from the date of inclusion to death from any cause. 
LDP – the time from the date of inclusion to non-malignancy-associated liver-related disease 
progression or death, whichever is earlier. 
Non-HCC – the time from the date of inclusion to development of non-HCC malignant 
neoplasm. 
LRR – the number of malignancy-developed patients whose tumor is resectable without  
vascular invasion and extrahepatic metastasis. 
POS – the time from the date of liver resection to death from any cause. 
PRFS – the time from the date of liver resection to tumor progression in the liver or death, 
whichever is earlier. 

 
Blood collection for biosignature analysis 
Peripheral blood samples (25 ml) will be collected from all consenting patients at first 3 follow-
up (0, 6th, and 12th month), which will be used to develop a biosignature to stratify patients at 
high risk for development of HCC. After development of a biosignature, 20 ml of peripheral 
blood samples will be collected from patients at high risk for HCC at 4th to 7th follow-up for 
modification and validation of the biosignature. 
 
Statistical analysis 
All statistical analyses will be performed using R (http://www.r-project.org/) or SPSS (Chicago, 
IL). Cox hazards proportional model and logistic regression will be applied to screen factors 
for the inclusion in a biosignature. Correlation test will be performed between results of 
multivariate Cox and logistic regressions, and false-positive factors will be deleted. After 
screening covariates, receiver operating characteristic (ROC) curves will be constructed to 
calculate optimum threshold for setting of cut-off value to define high risk for HCC. 
Independent demographic and clinical prognostic factors may be combined with the 
biosignature to improve performance sensitivity and specificity. Predictive signatures for 
cirrhosis cohort and HBV infection cohort will be developed independently. A correction for 
multiple testing may be applied. 
 
Discussion 
The PRECAR trial will identify predictors for development of HCC and diagnosis of very early 

HCC by comparing HCC cases arising from cirrhosis or HBV infection with non-HCC 
developing cases. To the best of our knowledge, no prospective observational cohort trial has 
been published with intent to develop a biosignature to stratify patients at high risk for HCC. 
However, a case control study with similar design (miRNA as a diagnostic tool for HCC) is 
ongoing in South Africa, where HIV is hyper endemic, thus the etiology of HCC is different 
from the PRECAR.16 In addition, ideal aim of the PRECAR trial is to successfully stratify 
patients who have high potential to develop HCC and carry out intensive follow-up to prevent 
up to 6 months of disease progression, as well as to offer curative therapeutic options at very 
early stage to improve prognosis. 
Surveillance for HCC has been introduced as an effective strategy to improve prognosis of 
patients with HCC.17 However, this field remains to be considered immature due to absence of 
validation of previous studies and limited accuracy that reduced cost-effectiveness. In recent 
years, the development of powerful tools, such as genome sequencing, plasma-free DNA 
sequencing, and high-throughput protein expression screening, suggests that the establishment 
of a biosignature could improve sensitivity and specificity of current surveillance method. The 
current surveillance, which is consisted of biannual ultrasonography and α-fetoprotein, reveals 
limited accuracy, false-positivity, and not efficient in a timely manner. Especially, these are 
confined to detect already-developed HCC, indicating that if HCC occurred a month after 
routine surveillance, the tumor will progress for 5 followed months before the next surveillance. 
Regarding aggressive characteristics and rapid progression, advancement of the tumor is likely 
to derive patient a worse candidate for curative treatments and deteriorates survival outcomes. 
The biosignature developed from PRECAR trial will improve predictive and diagnostic 

accuracy for HCC in settings with cirrhosis and HBV infection. During the establishment of 
the PRECAR biosignature, developmental process of HCC will be further elucidated and 
translated to improve predictive and diagnostic accuracy. We believe that the PRECAR 
biosignature will be useful in stratifying patients with cirrhosis or HBV infection at high risk 
for HCC, as well as in diagnosing HCCs at very early stage. 
 
Trial status 
The PRECAR trial is currently ongoing. Patient recruitment will be closed on 15 July 2019 
with estimated participant number of 10,000 (5000 for cirrhosis cohort and 5000 for HBV 
infection cohort). The final analysis will be triggered after 3 years of additional follow-up for 
enrolled patients for validation of the PRECAR biosignature. 
 
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4. Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small 
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symptomatic treatment in patients with unresectable hepatocellular carcinoma: a 
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10. van Meer S, de Man RA, Coenraad MJ, et al. Surveillance for hepatocellular carcinoma is 
associated with increased survival: Results from a large cohort in the Netherlands. J Hepatol 
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stage hepatocellular carcinoma in patients with cirrhosis. Aliment Pharmacol Ther 2009;30: 
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and lectin-bound alpha-fetoprotein in early hepatocellular carcinoma. Gastroenterology 
2009;137: 110-18. 
16. Sartorius K, Sartorius B, Kramvis A, et al. Circulating microRNA’s as a diagnostic tool for 
hepatocellular carcinoma in a hyper endemic HIV setting, KwaZulu-Natal, South Africa: a 
case control study protocol focusing on viral etiology. BMC Cancer 2017;17(1): 894. 
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screening of patients with cirrhosis for hepatocellular carcinoma. Gastroenterology 
2017;152: 608-15.e4. 
 
 
 
 
 
 
 
 
 
 
 

Table 1. Patient eligibility criteria for PRECAR trial 
 
Cirrhosis cohort 
HBV infection cohort 
Inclusion 
Aged between 35 and 70 years 
Aged between 40 to 70 years 
 
Diagnosis of liver cirrhosis within recent 6 months 
Chronic HBV infection (≥6 months) 
 
Satisfying one of below conditions: 
ECOG performance status 0 to 1 
 
1. Metavir score of 4 
2. Ishank score of 5 to 6 
3. Ascites, hepatic encephalopathy, or variceal hemorrhage 
4. Satisfying ≥ 2 of below conditions 
⚫ 
Imaging study indicating 
⚫ 
characteristics of liver cirrhosis 
⚫ 
Platelet count < 200 × 109/L 
⚫ 
ALT < 5 folds and liver hardness > 12 kPa 
⚫ 
Gastoesophageal varices from endoscopy 
Written informed consent provided 
 
Child-Pugh score A to B 
 
 
ECOG performance status 0 to 1 
 
 
Written informed consent provided 
 
Exclusion 
Hereditary metabolic liver diseases 
Hereditary metabolic and autoimmune liver diseases 
 
HIV infection 
HCV, HDV, HEV, or HIV infection 
 
Previous diagnosis of active pulmonary tuberculosis 
Previous diagnosis of active pulmonary tuberculosis 
 
Diagnosis of malignant tumor before or during hospitalization 
Diagnosis of malignant tumor before or during hospitalization 
 
Allogenic blood transfusion or cell therapy within recent 1 year 
Allogenic blood transfusion or cell therapy within recent 1 year 
 
Pregnancy 
Pregnancy 
 
 
Liver cirrhosis 
ALT, alanine aminotransferase; ECOG, Eastern Cooperative Oncology Group; HIV, human 
immunodeficiency virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HEV, 
hepatitis E virus. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Table 2. PRECAR trial assessment schedule 
Schedule 
Follow-up 
 
1st 
2nd 
3rd 
4th 
5th 
6th 
7th 
 
(0 m) 
(6th m) 
(12th m) 
(18th m) 
(24th m) 
(30th m) 
(36th m) 
Informed consent 
x 
 
 
 
 
 
 
Demographics 
x 
 
 
 
 
 
 
General habits 
x 
 
 
 
 
 
 
Medical and surgical history 
x 
 
 
 
 
 
 
Concurrent diseases 
x 
 
 
 
 
 
 
Concomitant medications 
x 
 
 
 
 
 
 
Physical and clinical assessment 
Height 
Weight 
Blood pressure 
Body temperature 
Routine blood test 
Liver function test 
Renal function test 
Electrolyte test 
Blood glucose 
Glycosylated hemoglobin 
Blood lipid 
Hepatitis virus 
HBV DNAa 
HIV antibody 
Serum tumor markers 
Ascitesb 
INR 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
op 
x 
 
 
x 
x 
x 
x 
x 
op 
op 
 
 
 
 
x 
 
x 
op 
x 
 
 
x 
x 
x 
x 
x 
op 
op 
 
 
 
 
x 
 
x 
op 
x 
 
 
x 
x 
x 
x 
x 
op 
op 
 
 
 
 
x 
 
x 
op 
x 
 
 
x 
x 
x 
x 
x 
op 
op 
 
 
 
 
x 
 
x 
op 
x 
 
 
x 
x 
x 
x 
x 
op 
op 
 
 
 
 
x 
 
x 
op 
x 
 
 
x 
x 
x 
x 
x 
op 
op 
 
 
 
 
x 
 
x 
op 
x 
Ultrasonography 
x 
x 
x 
x 
x 
x 
x 
Contrast enhanced CT or MRIc 
x 
op 
op 
op 
op 
op 
op 
Liver hardness 
x 
x 
x 
x 
x 
x 
x 
Child-Pugh score 
x 
x 
x 
x 
x 
x 
x 
Endpoints evaluation 
 
x 
x 
x 
x 
x 
x 
Sample collection 
x 
x 
x 
x 
x 
x 
x 
HBV, hepatitis B virus; HIV, human immunodeficiency virus; INR, international normalized ratio; CT, 
computed tomography; MRI, magnetic resonance imaging.  
arequired for patients with HBV infection.  
brequired for patients with ultrasonography results indicating ascites.  
crequired for patients with ultrasonography results indicating hepatic nodules.