We found that most livers of infants with biliary atresia display some elements of inflammation and fibrosis at diagnosis, with a subset (30% of the biopsies) containing more predominant histological features of either inflammation or fibrosis based on a greater differential score for the phenotypes. Using a gene expression signature highly specific for this subset of livers, we were able to group 91% of the biopsies into molecular inflammation or fibrosis and found significant association with age at portoenterostomy and transplant-free survival. These findings suggest that molecular profiling at diagnosis may stage the liver disease by the identification of biological pathways that may not be easily distinguishable by standard histological approaches to quantify inflammation or fibrosis. This may be due to intrinsic limitations of morphological methods (that is, hematoxylin/eosin or trichrome staining) or to a sampling artifact caused by a non-uniform tissue injury that varies between anatomical lobes and, perhaps more importantly, among neighboring lobules and portal tracts. Both obstacles may be overcome by the molecular profiling described herein. First, it uses RNA isolated from a fragment of tissue that, although small, contains a much larger representation of lobules/portal tracts than individual histological sections. Second, it is based on a molecular signature that contains the collective expression behavior of gene groups, without a priori bias related to their biological affiliations.
In experiments to validate the grouping of liver biopsies based on molecular signatures, we found that some gene groups are functionally related to the population of portal tracts by inflammatory cells and to molecular circuits previously implicated in pathogenesis of disease. For example, livers with a molecular signature of inflammation had an increase in the number of T and NK lymphocytes, overexpressed genes related to the immune system, and contained a cluster of genes with NFκB transcription sites. The activation of NFκB was also reported in this mouse model [29, 30], but the enrichment of bindings sites for NFAT and other transcription factors in the list of genes that are differentially expressed suggests that molecular networks regulated by these factors may be important for the pathogenesis of disease. Despite the activation of these molecular pathways within the inflammation signature, we recognize that there might be distinctions between wedge and core liver biopsies. We were unable to make a direct comparison between these two types of biopsies due to the unavailability of tissues. Further, the isolation of RNA from a liver biopsy fragment may limit the potential implication of the findings with regards to disease pathogenesis because the biopsy includes several cell types and different regions of the liver lobule. This type of study will benefit from the use of laser-capture microdissection, which enables the analysis of specific cell types or anatomical regions (that is, portal tract versus lobule).
Gene expression profiling increases the number of available methods to quantify prominent biological processes in biliary atresia. A previous study used histological staining methods and reported that a high degree of syncytial giant cells, focal and bridging necrosis, and inflammation were associated with poor clinical outcome [18, 19]. These findings differ from the improved outcome of our subjects assigned to the inflammation group, but we recognize that our findings will require validation in a larger population. Other studies have investigated the association of hepatic fibrosis and clinical course after portoenterostomy, with poor outcome reported for children with advanced fibrosis, either quantified by standard methods or aided by computerized morphometry [18–21]. This association was reproduced in our study in the children assigned to the group of molecular fibrosis.
The temporal differences in age at diagnosis for the molecular groups raise the possibility that the gene expression signatures reflect two distinct but inter-related stages of disease. The first stage, represented by younger patients with an inflammatory signature (most often but not exclusively at younger age), is placed biologically earlier in pathogenesis of disease, while the other patients may have transitioned to a more advanced stage of fibrosis. Such a continuum in the pathogenesis of disease has been demonstrated in the rotavirus-induced mouse model of biliary atresia [7, 8, 34], which begins with prominent inflammation of the liver and extrahepatic bile ducts and progresses to less inflammation and persistent duct obstruction; however, in humans, the stages appear not to obey a strict temporal organization. The presence of fibrosis in a subset of younger infants suggests that age alone cannot stage the disease. In these patients, the liver injury may have started at an earlier age, or it may have undergone rapid progression to fibrosis. The possibility of a rapid progression to fibrosis is supported by a previous report showing greater fibrogenesis in the livers of neonatal rats when compared to adults in a model of cholestasis induced by bile duct ligation .
Molecular profiling of liver biopsies at the time of diagnosis has been shown to differentiate the embryonic and perinatal forms of biliary atresia and identified genes with potential roles in pathogenesis of the embryonic form of disease . For example, among the genes with unique expression patterns were five imprinted genes (Igf2, Peg3, Peg10, Meg3, and IPW) in infants with the embryonic form, suggesting that a failure to down-regulate embryonic gene programs may be involved in the non-hepatic malformations that are typical of this group of patients . In a separate study, molecular profiling also revealed the activation of an interferon-gamma-rich proinflammatory circuit . The biological relationship between this circuit and biliary injury was demonstrated in mechanistic studies showing that the in vivo depletion of interferon-gamma in mice prevented the obstruction of extrahepatic bile ducts . Despite the informative data produced by these two studies, we recognize that the approach described here to stage the disease using molecular profiles needs future studies to evaluate its relevance in clinical practice and in potential therapies. This can be pursued by prospective validation in a new group of patients adequately powered for statistical analysis to look at clinical correlates and at responses to clinical intervention. For example, will the 150-probe set be reproduced if the same statistical method is applied to new livers with histological scores ≥2 for inflammation or fibrosis? Will infants with an inflammation signature do better if treated with anti-inflammatory drugs (for example, corticosteroids)? Formal answers to these questions will ultimately reveal the clinical impact of staging of liver disease and open opportunities for new trials that take into account the patient's biological makeup.