Wet biomarkers commonly used in clinical trials
Mark W. Tengowski, DVM, MS, PhD – Medical Affairs Director, Musculoskeletal at Clario Medical Imaging
In a prior blog post, I provided some examples of tests that could be used in Nonalcoholic Steatohepatitis (NASH) trials. In this post, I will go into greater detail on just a few of those tests which are reported in recent clinical trials. These are wet biomarkers or combinations of tests that can be used with or without the conjunction of imaging across the entire NASH spectrum.
Enhanced Liver Fibrosis (ELF) Score
This is an extracellular matrix composite marker for fibrosis, comprised of the following test inputs:
- Hyaluronic Acid (HA)
- Procollagen III Amino-terminal Peptide (PIIINP)
- Tissue Inhibitor of Matrix Metalloproteinase 1 (TIMP-1)
The ELF scores in the 8.0-10.5 range indicate advanced liver disease, which can be from NASH or hepatitis. This test is available through Siemens.
This is a new biomarker which combines a blood test (FibroMeter, a collection of 5 markers: platelet count, AST, ALT, ferritin, and glucose) and the VCTE component for stiffness found on the Echosens FibroScan® system. This is a compelling combination biomarker, useful in advanced fibrosis where VCTE is superior to blood-based test, of which the FibroMeter appears to be the best of the blood tests. FM-VCTE is a possible diagnostic and/or clinical trial screening tool and an alternative to biopsy.
Another blood imaging combo test algorithm, this biomarker combines CAP and VCTE from FibroScan systems with the common blood tests, aspartate aminotransferase (AST), alanine aminotransferase (ALT), or AST:ALT ratio. FAST may be a biomarker that can non-invasively identify subjects at risk of progressive NASH in clinical trials, reducing the need for liver biopsies.
When you combine MRI-PDFF, MRE, and the serum test for aspartate aminotransferase, you get MAST. Algorithmically derived, MAST may have better NASH classification discrimination compared to FAST, FIB-4, or the NAFLD fibrosis score (NFS).
This is another algorithmic non-invasive blood-based diagnostic test requiring the NASH-associated biomarkers: miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated hemoglobin. NIS4 can be used as a screening tool in trials to rule in/out at-risk NASH subjects in subjects with metabolic risk factors and suspected disease or used to reduce unneeded biopsies in subjects with a low risk of disease progression. NIS4 can be easily combined with MRI-PDFF.
This test is effective in subjects with advanced fibrosis. It uses the chemistry markers, AST and ALT, the platelet count, and the subjects age in its algorithm. Originally developed for liver fibrosis in hepatitis C virus infections, it has found a place in NASH clinical trials. FIB-4 can be combined with a liver stiffness parameter such as MRE (sometimes reported as MEFIB).
For those interested in NASH histology, Fatty Liver Inhibition of Progression (FLIP) algorithm and the Steatosis, Activity and Fibrosis (SAF) scoring systems are new methods of morphological profiles of disease severity and activity.
Often used as a simple test to determine if a patient is on the NAFLD/NASH spectrum – patients usually have a mildly elevated AST and/or ALT, often with an AST:ALT ratio <1. In later stages of NASH, this ratio can flip so that AST:ALT >1. AST:ALT ratio can also be used to distinguish NASH from alcoholic liver disease and as an alternative to liver biopsy early in NASH.
In keeping with the idea that AST is an important liver enzyme, the APRI or AST-to-platelet ratio index can be used as an estimate of (advanced or severe) liver fibrosis using a simple formula. Since this could be useful is evaluating advanced fibrosis, changes in these biomarkers could reduce (or eliminate) the amount of biopsies needed to assess treatment.
OWLiver Care and OWLiver tests
Incorporating BMI with the metabolomic profile of 25 different fatty acids and triglycerides markers, these algorithms can be useful in stratifying NAFLD and NASH from those candidates/patients with no NAFLD.
The exact details of this test do not appear to be disclosed, but the company claims that SomaSignal tests measure specific proteins in a small blood sample and then use computer algorithms to provide health and wellness information from the ~95 differentially expressed proteins found in NASH subject, refining this to smaller, more specific protein classifiers.
As a complement to blood tests, genetic markers for use in the screening process or as druggable targets are on the rise in trials. A few of the markers of interest useful in stratifying or identifying subjects include:
- rs6834314, rs72613567, and rs62305723 in the gene, HSD17B13 (17-beta hydroxysteroid dehydrogenase 13)
- rs738409 in the gene, PNPLA3 (Patatin-like phospholipase domain protein 3)
- rs58542926 in the gene, TM6SF2 (transmembrane 6 superfamily member 2)
Since Type lll collagen is one of the most abundant proteins in fibrotic tissue, it can serve as a biomarker for liver fibrosis. PRO-C3, the pro-peptide of type lll collagen, may be a useful serological biomarker for the formation of fibrotic liver tissue. Since PRO-C3 levels are not affected by age, sex, BMI or ethnicity and increase as a function of liver fibrotic state, monitoring a subject with advanced fibrosis via changes in this biomarker of treatment take effect.
Encoded by the thrombospondin 2 (THBS2) gene, the matricellular glycoprotein thrombospondin-2 (TSP-2) has been identified through transcriptomic analysis as a protein up-regulated in NASH and/or advanced fibrosis (stage F3-F4). In NAFLD/NASH subjects, TSP-2 expression was found to be significantly higher in NASH than in NAFL and increased parallel to fibrosis stage yet independent of platelet count.
Just when you thought the amount of imaging biomarkers were set in NASH, ARFI may be useful as a way to stage fibrosis. ARFI is Acoustic Radiation Force Impulse Ultrasonography, and it uses conventional B-mode Ultrasonography to generate an ultrasonic pulse and measure the response of the liver tissue as shear wave velocity. Similar to VCTE and MRE, the median velocity of sound measured by ARFI increases with the degree of fibrosis.
Patient Reported Outcomes
Sometimes we focus too much on the physician’s assessments. Regulatory agencies recognize the need and value of collecting patient reported outcomes. NASH-CHECK aims to collect answers to different symptom questions in the domains such as pain, fatigue, itch, sleep and cognition. A second battery of questions addressed the quality-of-life domains such as activity limitations, social/psychological/relationship or economic impact, healthy eating, emotional impact and work.
There are a variety of biomarkers to identify patients at every stage of disease progression in NASH trials. By having single or combination tests to tailor to your study needs, this empowers sites and sponsors to choose the correct aggregate of biomarkers for the utmost accuracy in their clinical trials; therefore increasing the trustworthiness of trial data, which in turn leads to decreased trial duration and cost, ultimately expediting drug development with an auditable trail of endpoints.
Mark Tengowski, DVM, MS, PhD
Medical Affairs Director, Musculoskeletal at Clario Medical Imaging