Research Article: Assay validation and clinical performance of chronic inflammatory and chemokine biomarkers of NASH fibrosis

Date Published: July 10, 2019

Publisher: Public Library of Science

Author(s): Sumit Kar, Sabina Paglialunga, Sharon H. Jaycox, Rafiqul Islam, Angelo H. Paredes, Rohit Kohli.


Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that can lead to cirrhosis, liver transplant, and even hepatocellular carcinoma. While liver biopsy remains the reference standard for disease diagnosis, analytical and clinical development of non-invasive soluble biomarkers of NASH are of great importance to advance the field. To this end, we performed analytical and clinical validation on a series of pro-inflammatory cytokines and chemokines implicated hepatic inflammation; IL-6, CRP, TNFα, MCP-1, MIP-1β, eotaxin, VCAM-1. Biomarker assays were validated for accuracy and precision. Clinical performance was evaluated in a random sample of 52 patients with biopsy-proven NAFLD/NASH. Patients were categorized into three groups according to their fibrosis stage; advanced (F3-F4), mild (F1-2) and no (F0) fibrosis. Serum IL-6 was increased in patients with advanced fibrosis (2.71 pg/mL; 1.26 pg/mL; 1.39 pg/mL p<0.01) compared to patients with mild or no fibrosis respectively. While, there was no significant difference noted in CRP, TNFα, MCP-1, MIP-1β, eotaxin among the three groups, VCAM-1 levels were increased by 55% (p<0.01) and 40% (p<0.05) in the advanced cohort compared to the mild and no fibrosis groups respectively. VCAM-1 also displayed good clinical performance as a biomarker of advanced fibrosis with an area under the receiver operating curve of 0.87. The VCAM-1 assay demonstrated robust accuracy and precision, and VCAM-1 outperformed IL-6, CRP, TNFα, and the chemokines MCP-1, MIP-1β, and eotaxin as a biomarker of advanced fibrosis in NASH. Addition of biomarkers such as IL-6 and VCAM-1 to panels may yield increased sensitivity and specificity for staging of NASH.

Partial Text

Nonalcoholic steatohepatitis (NASH) is a chronic metabolic liver disorder which can progress to hepatic fibrosis, cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Recent estimates indicate that the prevalence of NASH in the general adult population is 3–5% [1], making NASH the second leading etiology for a liver transplant [2]. Moreover, increased prevalence of underlying risk factors such as obesity and type 2 diabetes, along with greater recognition of the disease and diagnosis by primary care physicians [3], are anticipated to greatly impact the NASH diagnosis rates over the next several years. In this regard, by 2030 the number of NASH cases with advanced fibrosis is anticipated to be nearly 8 million in the US, an increase of over 160% from recent estimates [4].

The test population were subjects enrolled in an on-going tissue and serum repository at Brooke Army Medical Center, TX. The study protocol was approved by the Brooke Army Medical Center ethics review board and conducted according to the principles of the Declaration of Helsinki. All subjects gave their written informed consent to participate in the study. All subjects underwent a liver biopsy due to the clinical suspicion of NASH. NAFLD was defined by consumption of less than 20 and 30 grams of alcohol every day for women and men, respectively and a liver biopsy showing fatty liver disease [20]. Histology was assessed by a single hepatobiliary pathologist using the NASH-Clinical Research Network criteria [7]. The presence of steatohepatitis was defined by steatosis, inflammation and cytologic ballooning. NASH severity was categorized as liver grade ranging between 0 and 2. Participants were categorized based on hepatic fibrosis stage; no fibrosis (F0), mild fibrosis (F1-F2) and advanced fibrosis (F3-F4). Twelve-hour fasting serum was obtained from all subjects the day of their liver biopsy and processed for clinical laboratory evaluations including liver function tests, glucose, insulin, and lipids. An aliquot of serum was stored at -80°C for biomarker analysis. FIB-4 was calculated as previously reported [6].

In the present study, a suite of pro-inflammatory cytokine and chemokine assays were analytically validated as well as evaluated in a clinical setting to demonstrate use as a biomarker for fibrosis severity in NASH patients. Analytical validation examines the performance of a test or assay. The level of validation depends on the assay COU and risk associated with the biomarker. All soluble biomarker assays in the present study were developed according to FDA guidelines [16] to be used as an exploratory endpoint for regulatory submission of a clinical trial, though additional tests can be performed for other COUs such as a secondary endpoint. Clinical performance is typically measured against a reference standard. For many NASH biomarkers, this analysis is against histological biopsied results. Here we demonstrated that IL-6 and VCAM-1 are increased in NASH patients with advanced fibrosis stages, and displayed good performance in distinguishing advanced fibrosis from milder stages.

In a single study comparison, the role of several cytokines and chemokines as biomarkers for NASH fibrosis were examined. IL-6 and VCAM-1 are a potential soluble biomarker for NASH risk stratification, able to discriminate between mild and severe stages of fibrosis. Moreover, VCAM-1 outperforms the pro-inflammatory cytokines and chemokines examined in the present study. Analytical validation is also required to satisfy regulatory guidance for biomarker use in a clinical trial. To this end, we demonstrated good accuracy and assay robustness for VCAM-1 as well as clinical value.