perSpecTiveS

opinion

antifibrotic therapiesemerging biomarkers as treatment end points

Jayant A. Talwalkar
abstract | The emergence of novel pharmacologic therapies for hepatic fibrosis holds promise for the management of this condition. Furthermore, novel, biological indicesin addition to liver biopsymay improve our ability to assess the unique pharmacological effects generated by these agents. This article will attempt to highlight the potential for emerging biomarkers to serve as end points in clinical studies that target salient features of hepatic fibrogenesis.
Talkwalkar, J. A. Nat. Rev. Gastroenterol. Hepatol. 7, 5961 (2010); doi:10.1038/nrgastro.2009.197

(as some of the potential error can be over come by having more patients with obvious changes after therapy)which are difficult to conduct. 4 in this regard, the applica tion of novel approaches to existing histol ogy assessment with liver biopsy, such as computerized image morphometric analysis and immunochemical detection of activated smooth muscle cells, may be promising.5 a real problem for liver biopsy (or any pro posed technique to assess histology) is the ability to detect small changes in regression of fixed, fibrous lesions over the short time intervals associated with most controlled clinical trials.

Hepatic fibrosis

Hepatic fibrosis is characterized by the excessive deposition of extracellular matrix proteins resulting from chronic liver injury. Both necroinflammation and subsequent hypoxia are recognized as common media tors of hepatic stellate cell activation, which is the primary mediator of hepatic fibro genesis. 1 Hepatic stellate cell activation is also promoted by other factors, includ ing oxidative stress byproducts (such as reactive oxygen intermediates), apoptotic bodies, lipopolysaccharide exposure, and paracrine stimuli from neighboring constit uents, including Kupffer cells. subsequent accumulation of extracellular matrix results in progressively thickened fibrous scar tissue, which leads to vascular dysregula tion and regenerative nodule formation culminating in cirrhosis.2 regression of established fibrosis can be accomplished in selected individuals with chronic liver diseases that have effective therapies.3 However, a much larger group of individuals will have an incomplete response to conventional treatment and will remain at risk for progressive hepatic fibrosis and cirrhosis. additionally, there are as many patients whose underlying liver disease currently has no effective medical therapy. the single greatest limitation in bringing new drugs for hepatic fibrosis to the clinical
Competing interests The author declares associations with the following companies: Salix, Merck. See the article online for full details of the relationships.

setting is a lack of sufficient methodology for measuring clinical trial end points, rather than a limited number of promising agents. experimental animal and human tissue studies have identified several molecular processes that represent poten tial targets for therapeutic intervention. a range of treatments, including those devel oped for other indications as well as hepatic fibrosis, are currently in the drug develop ment process. most agents are focused on modulating specific features of hepatic injury and/or the functions of activated hepatic stellate cells. Different treatments may require the use of different techniques for assessing treatment efficacy. Biomarkers are emerging that could potentially act as end points in clinical trials of novel antifibrotic therapies.

Existing biomarkers

Liver biopsy

much has been written about the strengths and limitations of diagnostic liver biopsy in assessing stage of fibrosis. many of the issues relating to the accuracy of this technique can potentially complicate the conduct of clinical trials that use liver histology as a study end point. Quality of biopsy speci men, interpretation of histology, and the spatial heterogeneity of fibrosis deposition among individuals are all variables that can create sampling errors and lead to the inac curate assessment of fibrosis. Furthermore, the semiquantitative and categorical nature of fibrosis staging using accepted scoring systems results in the need to design studies with extremely large sample sizes

there has been great interest over the past decade in developing noninvasive methods that can accurately detect the stage of hepatic fibrosis defined by liver histology. serum fibrosis marker panels now exist that reflect (indirectly) or represent (directly) molecular features of hepatic fibrogenesis. However, a number of substantial limita tions with regard to the speci ficity and overall accuracy of these tests remain, despite having ideal characteristics for widespread use, such as convenience, low cost and availability.5 several studies have directly examined the clinical utility of serum marker panels in assessing treat ment response following interferonbased therapy for hepatitis C.6 Currently, no con sensus exists on what minimum change in serum marker level defines a clinically important change. the continued use of serum markers within proofofprinciple clinical trials is required to achieve a consensus decision. as with serum marker tests, there is growing evidence that novel imaging tech niques can also characterize the extent of hepatic fibrosis defined by liver biopsy. this work stems from the recognition that conventional imaging protocols remain insensitive to anatomic details of early tointermediate stages of hepatic fibrosis.7 work in the past 5 years has focused on refining ultrasound and mri techniques to improve the detection of hepatic fibrosis. For ultrasoundbased methods, a tech nique known as the hepatic vein transit time (Hvtt) has been proposed for detecting
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hepatic fibrosis in patients with chronic liver injury. Hvtt is measured by Doppler ultrasonography following bolus injection of microbubble contrast agent. in studies so far, a reduced Hvtt value implies the presence of vascular dysregulation (and especially intrahepatic shunting), which is normally associated with advanced hepatic fibrosis. Further studies to verify the accuracy of this method in detecting hepatic fibrosis are ongoing.8 a more developed approach is ultrasoundbased transient elastography, which measures the degree of liver tissue deformation in response to lowfrequency mechanical vibrations. this information is then used to calculate a quantitative measure of tissue elasticity or stiffness. 9 studies to date have demonstrated excel lent accuracy with transient elasto graphy for identifying severe hepatic fibrosis (that is, increased liver stiffness) among patients with chronic viral hepatitis.10 Further work is expected on defining the biologic and clinical implications of alterations in liver stiffness following therapy. among several mri techniques proposed for assessing hepatic fibrosis, magnetic resonance elastography (mre) has been used for preliminary research and clinical investigations. although the underlying principles of mre are similar to those of transient elastography, mre enables the examination of multiple crosssectional areas of hepatic parenchyma in order to determine the extent of hepatic fibrosis.11 in addition to excellent accuracy for detect ing advanced hepatic fibrosis, mre has the ability to discriminate between patients with moderatetosevere fibrosis and individuals with minimal or no fibrosis. the perform ance of mre does not seem to be signifi cantly influenced by an individuals habitus or weight.12 Data demonstrate that features of liver injury, such as inflammation and cholestasis,13 seem to influence liver stiff ness. the influence of these processes and conditions on measuring specific changes associated with the modulation of fibrosis with drug therapies will have to be defined. Diffusionweighted mri has also been proposed for categorizing stage of hepatic fibrosis, but further work is required before expanding its role to clinical studies.14 importantly, both serum and imaging markers of hepatic fibrosis have demon strated acceptable performance for the diagnosis of advanced fibrosis. whether these biomarkers will be accurate enough to evaluate the subtle changes expected from the pharmacologic treatment of
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fibrosis at earlier stages of fibrosis remains to be proven.

Potential biomarkers
Vascular endothelial injury Chronic injury to vascular endothelium is a common pathophysiologic process associated with many systemic disorders. although described nearly four decades ago, there is renewed interest in measuring circulating endothelial cells as markers of vascular dysfunction. studies have recog nized significantly higher serum circulat ing endothelial cell levels in patients with oncologic and cardiovascular diseases when compared to healthy populations.1517 notably, circulating endothelial cells exist in low quantities within the peripheral blood of healthy individuals, which highlights their potential use as disease biomarkers.15 Given that vascular endothelial injury is associated with portal hypertension, the use of circulating endothelial cells as a treatment end point would be of inter est in clinical trials for hepatic fibrosis. specifically, this biomarker could be used in compensated patients with advanced hepatic fibrosis and clinically significant portal hypertension to study the shortterm efficacy of therapies designed to halt fibrosis progression. Preliminary data suggest that circulating endothelial cells do seem to dis criminate between patients with cirrhosis and portal hypertension and healthy con trols (J. a. talwalkar, unpublished data); however, additional diagnostic accuracy studies are required before this biomarker can be incorporated into earlyphase clinical trials for validation. another potential biomarker that repre sents vascular endothelial injury is flow mediated dilation (FmD). FmD involves the use of highfrequency ultrasonographic imaging and transient occlusion of the bra chial artery with a sphygmomanometer. the reactive hyperemia caused by transient bra chial arterial occlusion induces the release of local nitric oxide, which results in vaso dilation that can be quantified as an index of vasomotor function. the percentage difference between brachial artery diameter measured after reactive hyperemia and the basal diameter is used to define FmD. For cardiovascular diseases, an increase in FmD following directed therapy seems to improve endothelial function and perhaps clinical outcome.18 FmD has already been used as a treatment end point to assess interven tions in trial settings. 19 in patients with congestive heart failure, the measurement

of FmD has the potential to provide complementary information on vascular function given its relationship with circu lating endothelial cell levels.20 However, this technique will also require further study to determine if it has any value or relevance in assessing treatment responses following antifibrotic therapy.

angiogenesis evidence continues to grow in support of pathological angiogenesis as a stimulatory and perpetuating mechanism of hepatic fibrogenesis. in turn, several experimental studies have demonstrated that blockade of angiogenic mechanisms with specific drug therapies (including sorafenib and sutent) is followed by improvements in histological fibrosis and portal venous pressure.21 Both serum and imaging biomarkers of angio genesis have been recognized for poten tial use as treatment end points in clinical trials of antiangiogenic agents. However, a variety of serum markers, including vas cular endothelial growth factor, do not have adequate sensitivity or specificity to be useful in clinical trials, although data are limited.22 in terms of imaging methods, mri based techniques currently have the great est advantage for use in humans because of their spatial resolution, lack of ionizing radiation, and widespread use in clinical practice. with dynamic contrastenhanced mri, the ability to measure quantitative features of angiogenesis using specialized pharmacokinetic models is possible. 23 the diagnostic value of these quantitative parameters, alone or together with more established measures, in the assessment of hepatic fibrogenesis remains unknown. Further research using mri and other crosssectional imaging techniques would be of great interestespecially multimodal imaging with mri and Pet when this becomes available, as these approaches integrate structure and function when assessing disease status. importantly, the pharmacodynamic assessment of novel anti fibrotic therapies could also be facilitated by imaging platforms such as mri. hypoxia Hypoxia within organ tissues results from acute and chronic injury. Hypoxiainducible factor 1 has been linked with inflammation, as well as the activation of angiogenic path ways through vascular endothelial growth factor, in the pathogenesis of hepatic fibro sis.21 while opticalbased techniques have
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been used in experimental studies, the application of similar techniques using imaging modalities available in clinical practice would also be of great interest. the application of bloodoxygenlevel dependent mri has shown promise in assessing tissue hypoxia among patients with chronic kidney disease.24 this technique remains an unexplored area of investigation with the potential for high applicability in assessing patients with chronic liver injury. important to characterize patients at a par ticular stage of fibrosis at baseline before study enrollment. liver biopsy could be complemented with noninvasive techniques (including serum fibrosis markers or elas ticity imaging) to verify fibrosis stage. serial assessments with additional novel bio markers during the study period could then be performed to provide information about reproducibility of results as well as potential diagnostic accuracy. importantly, the use of several biomarkers associated with par ticular molecular pathways would further strengthen the ability to recognize whether a novel agent is going to have potential effi cacy. these efforts should coincide with attempts to use biomarkers in earlyphase clinical trials to identify patients at high risk for severe liver disease in the future.
Center for Advanced Imaging Research, Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic, 200 First Street S. W., Rochester, MN 55905, USA. talwalkar.jayant@mayo.edu
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apoptosis apoptosis is a common and widespread process that occurs in the evolution of hepatobiliary diseases. advances in identi fying serum apoptosis markers, such as cytokeratin 18, may be helpful to iden tify patients with nonalcoholic fatty liver disease who have a potentially severe disease prognosis. 25 to date, the incorporation of serum apoptosis markers as treatment end points in early phase clinical studies remains limited. unique imaging strategies have also been proposed for detecting apoptosis in vivo. the molecular detection of radiolabeled annexinv, an endogenous intracellular human protein that binds to phospholipid phosphatidylserine expression on apop totic cells, has shown great promise for the assessment of oncologic and cardiovascular disease in animal and human studies. 26 refinements in technology, especially with the creation of delivery methods that target the liver, will be required if these types of approaches are to become valuable for assessing hepatic fibrosis in the future.

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Conclusions

there is an emerging consensus that the strict use of liver biopsy to assess degree of fibrosis will continue to hamper efforts toward developing novel antifibrotic drug therapies. Fortunately, there is an equally exciting and important field of development involving the recognition of biomarkers of hepatic fibrosis. How might novel and existing biomarkers be incorporated in the setting of earlyphase proofofprinciple clinical trials once they have achieved a sufficient level of diagnostic performance? it might be argued that histology remains

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