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Study Identifies Protein Markers for Early Diagnosis of Biliary Atresia

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NEW YORK (360Dx) – A team led by researchers at Cincinnati Children's Hospital Medical Center has identified a protein marker for the early diagnosis of biliary atresia, a childhood disease of the liver.

Detailed in a study published this week in Science Translational Medicine, the marker could help speed detection of the condition, for which patient outcomes hinge on rapid intervention, said Jorge Bezerra, medical director of the pediatric liver care center at CCHMC and senior author on the paper.

Biliary atresia occurs when injury to extrahepatic bile ducts disrupts the flow of bile, leading to severe liver injury. The condition can be treated by surgery in which bile is rerouted to drain directly from the liver into the intestine, without which, Bezerra said, affected children are unlikely to survive beyond one year.

Even among children who undergo surgery, roughly half need a liver transplant by the time they are two years old. Outcomes are significantly improved by quick diagnosis and surgery. Early detection of biliary atresia has been difficult, however, due to the low incidence of the condition and the fact that symptoms like neonatal jaundice are common to a number of conditions.

"It's very common to have jaundice in newborn babies," Bezerra said. "But by two weeks of age, [it] should go away, and when it doesn't, that's when you begin to have an increased risk for these liver diseases.

Typically, in such cases, doctors measure patient bilirubin levels by looking at the direct bilirubin fraction, in particular, which Bezerra said rises in cases where bile is not being excreted properly.

But, he added, "even when the pediatrician sees the baby has an increase in the direct bilirubin level, the question is still, what is causing that?"

Often, he said, children undergo a time-consuming series of blood and urine tests along with ultrasound, X-rays, and liver biopsies before they are diagnosed with biliary atresia and moved on to surgery.  

"It takes a couple of weeks before someone is able to make the final diagnosis and indicate surgery," Bezerra said. "If we can simplify that, there's a major potential positive impact on the outcome of surgery. So the sooner we can do it the better."

He said that he and his colleagues have for years been looking for biomarkers that might enable earlier diagnosis of the condition, using hypothesis-guided approaches in both humans and animal models. They developed several small panels of cytokines and other markers through this approach, but none of them performed better than existing clinical measures when put through validation experiments.

Their failure to identify markers based on existing hypothesis around biliary atresia led them to move to an unbiased proteomic approach.

"We decided that we should be humble and realize that we just don't know what [all the mechanisms underlying the condition] are," Bezerra said.

In the STM study, he and his colleagues used Somalogic's SomaScan platform, which uses an array of the company's Somamer affinity reagents to measure up to 1,310 proteins per sample. The researchers analyzed 175 subjects with biliary atresia and 70 subjects with neonatal intrahepatic cholestasis splitting them into one discovery cohort and two validation cohorts.

In the initial discovery experiment they identified 76 proteins whose expression differed significantly between the biliary atresia and IHC groups. Using multivariate logistic regression, they found that the matrix metalloproteinase-7 (MMP-7) combined with ectonucleotide pyrophosphatase/phosphodiesterase 7 (ENPP7) distinguished between the two groups with an area under the curve (AUC) of .99

MMP-7 was particularly strong diagnostically, by itself distinguishing between the two groups with an AUC of .97. This observation led the researchers to combine that marker with the protein GCT, which previous studies had found is typically higher expressed in babies with biliary atresia.

The researchers then tested MMP-7, ENPP7, and GCT singly and in combinations in the two validation cohorts, determining that the combination of MMP-7 and GCT could detect biliary atresia with sensitivity of 97 percent, specificity of 94 percent, and positive and negative predictive values of 85 percent and 99 percent, respectively.

Those levels of performance suggest the test could be useful in "increasing the diagnostic tempo," a key goal for treating biliary atresia, Bezerra said.

"You don't have to spend a lot of time with very expensive testing to rule out other diseases," he said. "The way we envision a practitioner using it in the clinic is you get the liver function test and the MMP-7, and if they are high you can very quickly send the baby for a liver biopsy and then surgery."

The study also suggested that MMP-7 could be a therapeutic target in biliary atresia, involved in tissue repair pathways that could be linked to the inflammation and fibrosis that characterizes the condition. As proof of principle, the researchers used antibodies and the drug batimastat to inhibit the activity of MMP-7 in mouse models of biliary atresia, finding that this treatment prevented the extrahepatic bile duct obstruction that characterizes the condition in more than 80 percent of the mice they studied.

Bezerra cautioned that the STM findings need further validation and added that he and his colleagues are currently working to establish more quantitative cut-offs for MMP-7, measuring levels in 50 healthy babies to see if there is a natural post-natal curve of expression for the protein.

They are also planning a prospective validation study and looking to see if the marker might be useful in other biliary diseases, including several common adult conditions.

Bezerra said he and his colleagues are in contact with potential industry partners to explore developing kits for measuring MMP-7 that could be distributed to other hospitals and laboratories.