Protease-based liquid biopsy a promising approach for diagnosing fatty liver disease

A liquid biopsy platform that utilizes protease biosensors can detect nonalcoholic steatohepatitis (NASH) with pinpoint accuracy in both mice and humans.

In mouse model experiments, the test was able to distinguish NASH-induced from healthy mice with perfect accuracy. In humans, the test correctly differentiated between NASH patients and healthy individuals 97 percent of the time.

The data demonstrate that the novel liquid biopsy platform has highly reproducible results, according to Dr Arun Sanyal, of Virginia Commonwealth University, who presented their results at the American Association for the Study of Liver Diseases (AASLD) virtual annual meeting. 

Called LBx-NASH, the platform directly measures the activity between NASH-specific proteases and fluorogenic biosensors that contain peptide molecules. When the biosensors are applied to the blood sample, the fatty liver proteases will cleave or break down some of the peptides. Monitoring this cleavage process qualifies which proteases are active and ascertains the disease status.

Sanyal pointed out that such an approach can be a boon to patients, in that it improves accessibility and convenience in terms of diagnosing the disease.

“This diagnostic approach could potentially diagnose patients with NASH who require further clinical management and reduce unnecessary testing and invasive liver biopsies,” he said, adding that the next step in the research is to conduct prospective studies.

Animal, human studies

Sanyal and colleagues initially tested the platform in 10 C57BL/6 NASH mice fed a choline-deficient high-fat diet (CD-HFD) and 28 mice fed a healthy diet. The most significantly cleaved biosensor in NASH mice was N11, a peptide sensing cathepsin L (CTSL) activity.

Measuring the CTSL activity with the liquid biopsy proved far better than quantifying the CTSL abundance in plasma using a commercial ELISA kit for detecting NASH (area under the curve [AUC], 1.00 vs 0.52). [AASLD 2021, abstract L03]

The preclinical results were replicated in three independent human NASH pilot cohorts (n=35) and healthy controls (n=24), with N11 showing a significant discriminatory power (p=0.003).

Next, the team developed LBx-NASH by screening >600 peptide substrates. They identified a panel of 20 biosensors to query disease biology in NASH and tested them using plasma samples from 76 NASH patients (60.5 percent female, 77.3 percent had body mass index ≥30kg/m², 50.7 percent were diabetic) and 12 healthy lean or obese individuals.

The biosensor assay achieved high discriminatory accuracy (AUC, 0.97, 95 percent confidence interval, 0.93–1.00) and was able to distinguish NASH from healthy samples independently of gender, obesity status, and type 2 diabetes.

“[The present] data demonstrate the diagnostic potential of protease activity to accurately predict NASH disease state,” said Sanyal.

He believed that the liquid biopsy platform could greatly improve the quality of care for NASH patients.