Nonalcoholic fatty liver disease (NAFLD), the most common chronic liver condition, is on the rise. Consequently, so is the number of people progressing to the next, more aggressive stage—nonalcoholic steatohepatitis (NASH). With NASH in particular a setup for cirrhosis and liver cancer, there’s a paramount need for methods that quickly identify individuals most at risk for these complications.

Worldwide, NAFLD affects approximately 25% of adults. But, according to the National Institute of Diabetes and Digestive and Kidney Diseases, the prevalence is more significant in the U.S. NAFLD affects between 30% and 40% of American adults and up to 10% of children. Approximately 12% of adults have developed NASH, characterized by scarring and fibrosis.

Currently, liver biopsy is the routine method for diagnosing NAFLD and NASH, but it’s suboptimal. Not only invasive with associated pain and bleeding, this approach also is expensive, carrying a price tag between $1,500 and $2,700. Additionally, biopsy is merely a pixel in a snapshot of the diseased organ, said Scott Friedman, MD, professor of medicine, liver diseases, and pharmacological sciences at Icahn School of Medicine at Mount Sinai in New York City.

“Liver biopsy, itself, isn’t a gold standard. It’s a tin standard,” explained Friedman, who also serves as Icahn’s dean for therapeutic discovery. “You’re only capturing a tiny piece of a very large organ, so you get significant sampling variability.”

Replacing liver biopsy hasn’t been easy, though. To address the need for less invasive, less costly, and more accurate diagnostics, several imaging and serum biomarker tests have come to market. Others are under development. However, there’s still no consensus on which method is most effective and efficient at identifying individuals with either NAFLD or NASH.

The Case for Better Diagnostics

NASH is an especially challenging condition, Friedman said, because the hierarchy of disease is unclear. Several factors, such as fat accumulation, inflammation, and changes to the gut microbiome caused by high sugar and antibiotic-laden diets, make diagnosing disease progression difficult. This lack of understanding is concerning, he said, because up to 50% of liver cancers appear before NASH patients even learn they have cirrhosis.

Unfortunately, existing noninvasive tests are more effective in pinpointing NAFLD patients than those with NASH. And, without a diagnostic that specifically targets NASH, millions of people will lose out on life-saving therapies in earlier disease stages, said Mary Rinella, MD, professor of medicine at Northwestern Feinberg School of Medicine in Chicago.

“These patients are the ones that are the low-hanging fruit that we really need to capture,” she said. “If we can identify them, hopefully, we can reverse their trajectory.”

Imaging: An Imperfect Modality

Despite offering noninvasive views of the liver and being good clinical options for diagnosing steatosis and liver stiffness, imaging modalities, such as ultrasound and magnetic resonance imaging (MRI), fall short in identifying NASH, advised Rohit Loomba, MD, MHSc, director for the NAFLD Research Center at the University of California at San Diego School of Medicine.

Diagnosis can be particularly difficult in obese patients, he added. “The problem with ultrasound comes when a patient is overweight or obese with a [body mass index] of 35 to 40,” Loomba explained. “These tests don’t perform as well because of the fat content in the liver, and the penetration of the ultrasonic waves into the liver is also affected by fibrosis.”

Alongside lower sensitivity, imaging modalities can cost more than blood tests, and some are difficult to access, such as the MRI-estimated proton density fat fraction (MRI-PDFF). While MRI-PDFF maps the entire liver in seconds, it’s time consuming and requires special instrument coils and staff training.

Existing Tests

Given imaging tests’ shortcomings, the search continues for an optimal noninvasive serum biomarker test to quickly and accurately identify patients with NASH, said Brent Neuschwander-Tetri, MD, director of the division of gastroenterology and hepatology at Saint Louis University School of Medicine in St. Louis.

“The ideal test must have a good negative predictive value of at least 90 percent,” Neuschwander-Tetri said. “We want a blood test that can tell us a patient doesn’t have the disease. That way, they don’t have to spend more money for unnecessary testing.”

To date, many blood biomarker and panel tests are moving toward greater sensitivity and specificity. Used to assess a variety of liver conditions, the aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio also has clinical utility in workups for NAFLD and NASH. Some of the more promising biomarkers already used to some extent for this purpose or on the horizon as diagnostic tools include Fibrosis-4 index, the BARD score, Enhanced Liver Fibrosis test, and cytokeratin-18 (CK-18) (See Box on the left). Of these, CK-18 is the most extensively evaluated analyte for NASH diagnosis, according to Lawrence de Koning, PhD, DABCC, FACB, FCACB, a clinical biochemist at Calgary Laboratory Services in Calgary, Alberta, Canada.

On the Horizon

Even with myriad test options, experts agree much more work remains to identify efficacious and practical NAFLD and NASH diagnostics. Consequently, the pipeline is stocked with burgeoning work in lipidomics, metabolomics, and genetics.

Recent research combined genetics, lipidomics, metabolomics, and clinical data to create the NASH ClinLipMet score, unaffected by statin medications or obesity, to identify with high accuracy patients with NASH (Clin Gastroenterol Hepatol 2016;14:1463-72).

Other investigations also used meta-genomic sequencing of stool microbiota from NAFLD patients to identify, with high accuracy, 37 species present in the gut microbiome associated with advanced fibrosis (Cell Metab 2017;25:1054-62). Recently, Loomba’s lab developed a test using gene sequencing to analyze gut bacteria from stool samples to identify individuals with some level of NAFLD (Nat Commun 2019; 10:1406).

These types of multiomic investigations hold the most promise, de Koning offered. “Metabolomics is where the real gains will be made,” he said. “It allows for research into multiple different markers of different pathways simultaneously. Researchers can measure biomarker levels and be agnostic about whether they’re predictors of NAFLD or NASH.”

Ultimately, he said, improving the specificity and sensitivity of existing biomarker tests and cultivating new diagnostic tools is critical to addressing the growing menace of NAFLD and NASH.

“Early detection means early treatment and better patient outcomes. It means people will be more responsive to treatment,” de Koning concluded. “Having biomarkers can tell us how well a patient is doing in terms of the treatment we’re giving them, possibly replacing the need for other diagnostics.”

Whitney J. Palmer is a freelance journalist in Holly Springs, North Carolina. +Email: whitneyljhowell@gmail.com

Author disclosures on separate page.

 


Candidate Biomarkers for Diagnosing and Monitoring NAFLD and NASH

With nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) conservatively affecting at least one-quarter of the adult population worldwide, a vibrant quest is underway to find sensitive and specific noninvasive tests to diagnose and monitor these diseases. Many candidate biomarkers have been proposed and continue to be investigated. Some of the more useful and promising include:

AST/ALT ratio: The aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio helps distinguish alcoholic hepatitis from NAFLD and NASH. An AST/ALT ratio greater than 2 indicates alcoholic hepatitis. However, ALT levels higher than AST levels—without the presence of cirrhosis—point to NASH. AST levels are higher in NASH with cirrhosis.

However, in some cases elevated ALT levels are reported erroneously as normal because laboratory reference populations don’t exclude obese patients, according to Brent Neuschwander-Tetri, MD, director of the division of gastroenterology and hepatology at Saint Louis University School of Medicine in St. Louis. Appropriate ALT levels for men and women are <30 IU/L and <9 IU/L respectively, he suggested.

Fibrosis-4 index: This scoring system combines age, AST, ALT, and platelet count. It has a negative predictive value of more than 90% for advanced liver fibrosis, according to experts. Results can be subject to rapid AST and ALT changes, though.

BARD score: Calculated from body mass index, the ALT/AST ratio, and the presence of diabetes, this score, reported on a 0-4 scale, is routinely used to predict liver fibrosis in NAFLD patients. Scores less than 2 have a strong negative predictive value for advanced liver fibrosis associated with NAFLD.

Enhanced Liver Fibrosis (ELF) test: Though not yet approved by the Food and Drug Administration and not sensitive to early-stage fibrosis, this panel is an algorithm comprised of three fibrosis markers—amino-terminal propeptide of type III procollagen, hyaluronic acid, and tissue inhibitor of metalloproteinase 1. It detects advanced fibrosis with high accuracy in both adult and pediatric patients. U.K. National Institute for Health and Care Excellence guidelines recommend retesting every 3 years for adults and every 2 years for children with ELF scores less than 10.51.

Cytokeratin-18: One of the most scrutinized analytes in assessing NAFLD and NASH, this protein accumulates in the bloodstream as liver cells die. High levels could indicate NASH, but elevated levels are possible without disease, making the test only moderately accurate, according to Lawrence de Koning, PhD, DABCC, FACB, FCACB, a clinical biochemist at Calgary Laboratory Services in Calgary, Alberta, Canada.