Abstract

Objective Metabolic fatty liver disease drives chronic liver injury leading to fibrosis. This study aimed to establish a model utilising serum circulating fibroblast activation protein (cFAP) to diagnose advanced fibrosis in patients with fatty liver disease.

Design Two retrospective cohorts recruited from tertiary hepatology clinics were studied as training (n=160) and external validation cohorts (n=342), with prevalence of histologic advanced fibrosis (F3-F4) of 20% and 11%, respectively. A marker of activated mesenchymal fibrogenic cells, cFAP, was measured using our single-step enzyme assay. A predictive model, FAP Index, containing age, type 2 diabetes, alanine transaminase and ordinal cFAP was developed using logistic regression. Diagnostic accuracy of FAP Index was assessed on a single and then sequential basis.

Results FAP Index AUROC was 0.875 (95% CI 0.813-0.938) in the training cohort and 0.841 (95% CI 0.776-0.906) in the validation cohort. Low cut-off −1.68 (Sensitivity 80.0%, negative predictive value 95.5%) and high cut-off +0.953 values (Specificity 97.7%, positive predictive value 88.9%) excluded and diagnosed advanced fibrosis, respectively. In the validation cohort, FAP Index then FIB-4 reduced indeterminate results by one-third compared to FIB-4 alone. Whereas FAP Index followed by NFS (NAFLD Fibrosis Score) resulted in a reduction of indeterminate results by 70% compared to NFS alone.

Conclusion FAP Index is a novel, rapid, robust, inexpensive diagnostic tool for advanced fibrosis in metabolic fatty liver disease. Applying FAP Index followed by FIB-4 or NFS facilitates accurate risk-stratification of patients by greatly reducing the frequency of indeterminate results compared to FIB-4 or NFS alone, without compromising negative predictive value.

What is already known on this topic Fatty liver disease affects one quarter of the global population. Current screening algorithms to triage those at high risk of advanced fibrosis use a dual cut-off approach that results in a proportion of patients that cannot be classified (indeterminate result) and hence need further and more costly testing.

What this study adds We have developed the FAP Index, which is a model using a simple circulating fibroblast activation protein enzyme assay and routinely available clinical variables. Using FAP Index as a first-line test followed by the current recommended screening tests (FIB-4 and NFS [NAFLD Fibrosis Score]) can reduce indeterminate results by up to 70% compared to the current first-line standard of care tests alone, without compromising diagnostic accuracy.

How this study might affect research, practice or policy With recently approved pharmacotherapy for fatty liver disease, improved tools for triaging people with metabolic fatty liver disease has increasing urgency. Use of FAP Index could have a dramatic effect on screening for advanced fibrosis by reducing fruitless referrals to tertiary care and/or further testing. Furthermore, our single-step enzymatic cFAP assay can be adapted to point of care or reflex testing settings, allowing for low-cost and high throughput FAP Index screening.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

This project was funded by National Health and Medical Research Council (NHMRC) of Australia, grants APP1053206, APP1105238, APP1107178, APP1108422, APP1196492, APP2001692, Rebecca L Cooper Medical Research Foundation, Centenary Institute Foundation, a Gilead Fellowship, the Robert W. Storr Bequest to the Sydney Medical Foundation of The University of Sydney, Cancer Institute NSW grant 2021/ATRG2028, the Hollywood Private Hospital Research Foundation and the Sir Charles Gairdner Osborne Parke Health Care Group.

Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Human Ethics Committee of Sydney Local Health District gave ethical approval for this work. Approval HREC_X18-0241. Human Ethics Committee of Western Sydney Local Health District gave ethical approval for this work. Approval 2019/ETH02319. Human Ethics Committee of Sir Charles Gairdner Hospital, Perth, gave ethical approval for this work. Approval RGS 01287. Human Ethics Committee of The Alfred Hospital, Melbourne, gave ethical approval for this work. Approval 195/15.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Data Availability

All data produced in the present study are available upon reasonable request to the authors

ABBREVIATIONSALPalkaline phosphataseALTAlanine transaminaseAPRIAST to Platelet Ratio Index; AST to Platelet Ratio IndexASTAspartate transaminaseAUROCarea under the receiver operating characteristics curveBMIbody mass indexcFAPcirculating fibroblast activation proteinECMextracellular matrixESALequivalent single axle loadFAPfibroblast activation proteinFGF-21fibroblast growth factor 21GGTGamma-glutamyl transferaseHSChepatic stellate cellIQRinter-quartile rangeMAFLDmetabolic associated fatty liver diseaseMREmagnetic resonance elastographyNFSNAFLD Fibrosis ScoreNITnon-invasive testNPVnegative predictive valuePLTplateletPPVpositive predictive valueROCreceiver operating characteristics curveT2DMtype 2 diabetes mellitus.