AGED Diagnostics Clinical Validation Study

Study Overview:

This multicenter, prospective, observational study is designed to validate investigational blood-based diagnostic tests developed by AGED Diagnostics for the detection of metabolic dysfunction-associated steatohepatitis (MASH) and the staging of liver fibrosis.

MASH is a progressive form of metabolic dysfunction-associated steatotic liver disease (MASLD) characterized by fat accumulation, inflammation and injury to liver cells. Over time, MASH can lead to liver fibrosis, cirrhosis and serious liver-related complications. Early identification of MASH and accurate assessment of liver fibrosis are important for determining appropriate clinical management.

Current methods for definitively diagnosing MASH generally rely on liver biopsy, an invasive procedure that is not practical for widespread screening or routine disease monitoring. AGED Diagnostics is developing blood-based tests that analyze circulating cell-free DNA (cfDNA) DNA methylation patterns to identify biological signals associated with MASH and different stages of liver fibrosis.

Study Objectives:

The primary objective of this study is to independently validate candidate diagnostic prediction models developed during an earlier Verification Study. The study will assess the ability of these models to distinguish patients with simple steatosis from those with MASH and to classify liver fibrosis across stages F0 through F4, ranging from no fibrosis to cirrhosis.

A second objective is to identify the best-performing prediction model for each investigational blood test. The selected models will undergo further evaluation in a subsequent Clinical Utility Study to determine how the tests may support clinical decision-making and improve patient care.

The investigational tests are being developed with a target overall diagnostic accuracy of at least 90%. This is a development objective and does not represent established clinical performance.

Study Population and Design:

Approximately 400-500 participants with suspected MASLD will be enrolled across multiple clinical research sites. Eligible participants will have undergone or be scheduled to undergo a liver biopsy as part of their routine clinical evaluation, in accordance with the study's eligibility criteria.

All participants will be independent of those enrolled in AGED's previous studies.

Participants will be categorized according to their clinical and liver biopsy findings, including simple steatosis, MASH and relevant additional subgroups. Participants will also be classified according to their stage of liver fibrosis, from F0 to F4. A subset of participants may have metabolic dysfunction and alcohol-associated liver disease (MetALD).

The study is observational. Blood samples and relevant clinical information will be collected for research purposes. The investigational blood tests will not be used to direct participants' clinical care during this study.

Blood Sample Collection and Analysis:

Blood samples will be collected by trained healthcare professionals using standard venipuncture and study-provided collection kits. Samples will be transported to AGED Diagnostics' central laboratory for processing and analysis.

Researchers will isolate plasma and extract circulating cell-free DNA from the collected blood samples. The DNA will be analyzed using advanced sequencing technology capable of detecting DNA methylation patterns associated with liver disease.

DNA methylation is a naturally occurring chemical modification of DNA that can provide information about the tissues from which circulating DNA originates and their biological state. AGED's investigational tests use selected DNA methylation biomarkers and computational prediction models to identify patterns associated with MASH and different stages of liver fibrosis.

Laboratory processing and data analysis will follow standardized procedures and quality control requirements to support the reliability and consistency of the results.

Relevant demographic information, medical history, laboratory findings and liver biopsy results will be collected through secure electronic case report forms.

Study Procedures and Analysis:

The study will be conducted in two principal stages.

Stage 1: An interim analysis will be conducted after approximately half of the participants' blood samples have been processed and sequenced. Candidate prediction models will be evaluated for their ability to distinguish simple steatosis from MASH and classify liver fibrosis severity (F0-F4). Based on diagnostic performance, one or two models for each investigational test will be selected for further evaluation, with limited optimization if necessary.

Stage 2: The selected models will be evaluated using the remaining participants as an independent validation cohort, with predictions compared against liver biopsy findings. A subsequent analysis of the entire study population will assess overall diagnostic performance across relevant clinical subgroups. Laboratory personnel and the primary analysis team will remain blinded to clinical diagnoses and cohort assignments until the relevant analyses are completed.

Expected Outcomes and Future Research:

The study will assess the diagnostic performance of AGED's investigational blood tests for identifying MASH and determining the stage of liver fibrosis. The findings will support the selection of final prediction models for further clinical development.

The selected tests are intended to undergo subsequent clinical utility evaluation to determine whether blood-based diagnosis of MASH and liver fibrosis can improve clinical decision-making, facilitate earlier identification of patients with progressive liver disease and reduce reliance on invasive diagnostic procedures.

This study is intended to evaluate diagnostic performance and will not assess the effectiveness of a treatment or use investigational test results to guide clinical care.