Often regarded as the gold standard for fibrosis assessment, liver biopsy does carry associated risks given its invasive nature. Moreover, liver biopsy is not a true gold standard owing to interobserver and intra-observer variability and the small amount of tissue that is typically obtained with this procedure. Advances in the development of serologic tests and conventional imaging techniques have been shown to reduce the need for liver biopsy for diagnosing hepatic fibrosis. More commonly, it is a tool that is now reserved for evaluating indeterminate noninvasive tests or excluding features of particular diseases (eg, autoimmune hepatitis, steatohepatitis).
The noninvasive assessment of hepatic fibrosis has been a popular topic of discussion over the past decade. The ideal properties of a noninvasive test include widespread availability, ease of use, cost-efficiency, reproducibility, and the ability to detect changes in fibrosis over time. Furthermore, the ability of noninvasive testing to identify intermediate to advanced histologic stages of fibrosis (stage F2 or higher) without liver biopsy is important as this is the usual threshold to start treatment in eligible subjects with chronic hepatitis C (HCV), for example. In addition, the identification of early stage cirrhosis by noninvasive testing allows for the timely implementation of disease management strategies (hepatocellular carcinoma and variceal screening) to reduce the likelihood of complications.
This article reviews the salient aspects of hepatic fibrogenesis as well as the diagnostic performance of serum markers and imaging techniques that are currently available for detecting hepatic fibrosis. Finally, will provide suggestions as to how these noninvasive methods can be incorporated into routine clinical practice.
Pathogenesis of Hepatic Fibrosis
The hepatic stellate cell is thought to play an integral role in hepatic fibrosis. When in a quiescent state, stellate cells serve as storage reservoirs for retinol (a precursor of vitamin A) and other lipid soluble substances. Moreover, they control extracellular matrix (ECM) turnover and regulate sinusoidal blood flow. Additional fibrogenic cells involved with hepatic fibrogenesis are derived from portal fibroblasts, circulating fibrocytes, bone marrow, and epithelial–mesenchymal cell transition. The proportion of fibrogenic cells from these various sources likely depends on the etiology of liver disease. For example, stellate cells are mainly involved when the damage is centered within the hepatic lobule. On the other hand, portal fibroblasts are observed to contribute more in cholestatic liver disease and ischemia.
A variety of mediators have been shown to promote ongoing stellate cell activation and fibrogenesis, with platelet-derived growth factor and transforming growth factor-β described as 2 of the major cytokines involved with this process. Stellate cell activation occurs through several additional pathways as well. Oxidative stress in the form of reactive oxygen species can activate stellate cells. This pathway may be of particular relevance in alcoholic liver injury, nonalcoholic fatty liver disease, and iron overload syndromes. Parenchymal cell apoptotic bodies can induce an inflammatory response that activates stellate cells as well. Bacterial lipopolysaccharide can elicit a fibrogenic response by binding to stellate cells via Toll-like receptor 4. Lastly, paracrine stimuli from adjacent cell types (macrophages, sinusoidal endothelium, and hepatocytes) can also aid in the transformation of stellate cells from a quiescent to an activated state.
Once activated, stellate cells and fibroblasts transform into myofibroblasts, which are cells that contain contractile filaments. Myofibroblasts have the capacity to alter the composition of the ECM. Progressive changes in the ECM include a change from type IV collagen, heparan sulfate proteoglycan, and laminin to types I and III collagen. Subsequently, ECM accumulates owing to its increased synthesis and decreased degradation. Fibrogenesis is further propagated by positive feedback mechanisms that arise from changes in ECM composition. Changes in membrane receptors (eg, integrins), activation of cellular matrix metalloproteases and increasing matrix stiffness serve as stimuli to perpetuate stellate cell activation. It is this dynamic production and turnover of matrix, as well as increases in matrix stiffness, that form the basis for clinical techniques to noninvasively assess the extent of hepatic fibrosis.
Clinical Implications of Hepatic Fibrosis
The accurate detection of hepatic fibrosis stage has important clinical decision-making implications for the management of chronic liver disease. For example, the presence of clinically significant hepatic fibrosis (defined as histologic stage ≥2) influences the timing for antiviral treatment for patients with chronic hepatitis B and C. In patients with nonalcoholic fatty liver disease (NAFLD), the presence of fibrosis overall is suggestive of nonalcoholic steatohepatitis (NASH) and thereby identifies a subset of patients who require closer monitoring and follow-up. Perhaps most important, the presence of early stage or compensated cirrhosis necessitates interval screening for disease-related complications, including esophageal varices and hepatocellular carcinoma.
Serial measurements to evaluate for progression or regression of hepatic fibrosis also have clinical utility. The successful medical treatment of various chronic liver diseases, including viral hepatitis, autoimmune hepatitis, and primary biliary cirrhosis, is often associated with histologic regression of fibrosis in addition to clinical and biochemical improvement. Conversely, monitoring for disease progression allows clinicians to implement treatment in eligible individuals as soon as possible to maximize the probability for a complete response.
Clinical Implications of Hepatic Fibrosis
The accurate detection of hepatic fibrosis stage has important clinical decision-making implications for the management of chronic liver disease. For example, the presence of clinically significant hepatic fibrosis (defined as histologic stage ≥2) influences the timing for antiviral treatment for patients with chronic hepatitis B and C. In patients with nonalcoholic fatty liver disease (NAFLD), the presence of fibrosis overall is suggestive of nonalcoholic steatohepatitis (NASH) and thereby identifies a subset of patients who require closer monitoring and follow-up. Perhaps most important, the presence of early stage or compensated cirrhosis necessitates interval screening for disease-related complications, including esophageal varices and hepatocellular carcinoma.
Serial measurements to evaluate for progression or regression of hepatic fibrosis also have clinical utility. The successful medical treatment of various chronic liver diseases, including viral hepatitis, autoimmune hepatitis, and primary biliary cirrhosis, is often associated with histologic regression of fibrosis in addition to clinical and biochemical improvement. Conversely, monitoring for disease progression allows clinicians to implement treatment in eligible individuals as soon as possible to maximize the probability for a complete response.
Liver Biopsy
The introduction of a liver biopsy technique proposed by Menghini changed the field of hepatology in the 1960s. Subsequently, liver biopsy has been considered the gold standard for detecting hepatic fibrosis. In recent times, the indications for liver biopsy have shifted from diagnosis of chronic liver disease to staging and monitoring for disease progression owing to improvements in serum laboratory testing.
Despite its widespread availability and use, there has been an increasing focus on the disadvantages of liver biopsy, which call its value into question. As an invasive procedure, procedure-related complications such as pain are reported in an estimated 20% of patients, and major complications including bleeding, hospitalization, and death are higher than 0.5%. The risk for complications increases further among patients with advanced liver disease affected by thrombocytopenia, coagulopathy, or ascites. For these reasons, patients are becoming more reluctant to undergo repeat liver biopsies to assess disease progression as noninvasive test modalities become available.
Sampling error remains a major limitation for liver biopsy, and is difficult to overcome; only 1/50,000 of the liver is analyzed. Even when the specimen size is adequate (25 mm long), the probability for underestimating fibrosis stage remains as high as 25%. It is important to be aware of this concept when determining the accuracy of liver biopsy. Many studies that evaluate the diagnostic accuracy of noninvasive measurements of fibrosis utilize the area under the receiver operating characteristic curve (AUROC) to gauge performance. Unfortunately, even in the best case scenario, an AUROC or more than 0.90 cannot be achieved for the perfect marker because liver biopsy cannot achieve 100% accuracy based on inherent features of the technique.
A variety of histologic scoring systems have been used to stage hepatic fibrosis. Some systems have 5 stages (0–4) whereas others (eg, Ishak fibrosis score) have 7 stages (0–6). Although systems that include additional stages convey more information, the likelihood of having excellent reproducibility declines. The fibrosis stages identified by classification systems are determined by both the quantity and location of the fibrosis. In other words, the numbered stages do not reflect equal units of severity, but represent categories describing the histologic changes. This semiquantitative nature of histologic scoring systems is also an important consideration when comparing liver biopsy with noninvasive measures, which can provide a continuous quantitative assessment of liver fibrosis.
Serum Fibrosis Markers
Indirect serum fibrosis markers
Significant attention has been paid to the development of serum fibrosis markers over the past 2 decades ( Table 1 ). Routine serum biochemical tests to assess hepatic fibrosis were first examined given their wide availability. One simple model proposed that an aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio of greater than 1 is indicative of cirrhosis. However, the sensitivity (53%) and negative predictive value (NPV; 81%) for detecting cirrhosis is not robust enough for use in clinical practice. The most commonly studied indirect serum marker test using widely available variables is the AST to platelet count ratio index (APRI). The APRI is calculated as AST (U/L)/upper limit of normal × 100/platelet count (10 9 /L). The performance characteristics of the APRI depend on the cutoff value used (<0.5 to >2.0). Many studies have been conducted to externally validate initial results, but they have been conflicting, in part, because of differences in study methodology.
| Panel | Liver Disease | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) |
|---|---|---|---|---|---|
| AST/ALT ratio | AST/ALT | 50 | 100 | 100 | 81 |
| Forns test | Platelets, GGT, cholesterol | 94 | 54 | 40 | 96 |
| APRI | AST, platelets | 41 | 95 | 88 | 64 |
| Fibrotest | GGT, haptoglobin, bilirubin, APO-A, α2-macroglobulin | 87 | 59 | 63 | 85 |
| Fibrospect | Hyaluronic acid, TIMP-1, α2-macroglobulin | 83 | 66 | 72 | 78 |
| ELF | Numerous ECM protein and proteinases | 90 | 41 | 35 | 92 |
From a systematic review of 22 studies that examined the APRI in patients with chronic HCV, the average prevalence of clinically significant fibrosis (stages F2–F4) was 47%. With an APRI cutoff set at 0.5 for detecting clinically significant hepatic fibrosis (stages 2–4), the summary sensitivity and specificity values were 81% and 50%, respectively. The positive predictive value (PPV) and NPV of the 0.5 cutoff were 59% and 75%, respectively. At a higher cutoff of 1.5, the sensitivity and specificity were 35% and 91%, respectively. The 1.5 threshold led to a tradeoff between PPV (77%) and NPV (61%). With regard to cirrhosis, 12 studies were examined using thresholds of 1.0 and 2.0. The NPVs were excellent at 91% and 94%, respectively, for each threshold value. However, the PPVs for these cutoffs were not high enough to allow one to “rule in” cirrhosis with high accuracy. Thus, the use of APRI in different populations seems to have variable performance in detecting clinically significant hepatic fibrosis in patients with chronic HCV. Fewer studies have been performed using APRI in other chronic liver diseases, but are expected in the future.
FibroTest (Biopredictive, Paris, France) is a proprietary panel developed in 2001 that combines several indirect serum fibrosis markers including α2-macroglobulin, haptoglobin, γ-glutamyl transferase (GGT), apolipoprotein A 1 , and total bilirubin. Values of FibroTest range from 0 to 1; higher values indicate a greater probability of significant fibrosis. Serum α2-macroglobulin is an acute phase protein present at sites of inflammation. Haptoglobin is negatively associated with fibrosis because its synthesis is decreased by hepatocyte growth factor. GGT production could be caused by early cholestasis or an increase in epidermal growth factor. The initial work by Imbert-Bismut and colleagues identified these 5 variables as the most informative markers for staging fibrosis in patients with chronic HCV. Subsequently, a meta-analysis of 16 publications demonstrated that a Fibrotest diagnostic cutoff value of 0.31 was associated with a NPV of 91% for excluding significant fibrosis. In the United States, a version of the Fibrotest assay called FibroSURE (Laboratory Corporation of America, Raritan, NJ) is available for use. FibroSURE contains the same markers as Fibrotest in addition to ALT, patient age, and gender. The FibroSURE assay is mainly used for patients with chronic HCV.
Although FibroTest was initially validated in patients with chronic HCV, further studies showed that it is an effective alternative to liver biopsy in populations with chronic hepatitis B, alcoholic liver disease, and NAFLD. With regard to chronic hepatitis B, Sebastiani and colleagues examined 110 consecutive patients who underwent assessment using multiple noninvasive methods including FibroTest and APRI. With a prevalence of 68% for significant fibrosis, FibroTest had a sensitivity of 81% and a specificity of 90%. However, the NPV was only 64%, which severely limits its utility in clinical practice for these patients. The authors concluded that noninvasive serum markers alone may not be good enough to obviate the need for liver biopsy in staging chronic hepatitis B.
Given the epidemic of obesity and the number of patients at risk for NAFLD in developed countries, 1 study evaluated FibroTest in 170 patients from a secondary care center and 97 patients from multiple centers who were at risk for NAFLD. The authors found that FibroTest reliably detected advanced fibrosis (F2–F4) in patients with NAFLD; the AUROC ranged from 0.75 to 0.86 in the 2 study groups.
False-positive results using FibroTest can occur when elevations in total bilirubin owing to hemolysis, Gilbert syndrome, or other causes of cholestasis are present. Furthermore, test results can be impacted by acute hepatic and/or systemic inflammation, which leads to spurious increases in serum α2-macroglobulin and haptoglobin levels. These factors need to be accounted for when deciding to utilize this noninvasive test modality.
Other indirect serum fibrosis tests which have been studied include the Forns index, which incorporates age, GGT, cholesterol, and platelets. This model has mainly been utilized in patients with chronic HCV. Similarly, the Hepascore combines laboratory (bilirubin, GGT, hyaluronic acid, α-macroglobulin) and demographic data (age, gender) to predict fibrosis in chronic HCV. The FIB-4 index, which uses age, ALT, AST, and platelets, has also been evaluated in HIV/HCV-coinfected individuals and found to be accurate in detecting hepatic fibrosis. These scores are likely to be used less often than FibroTest in clinical practice mainly because of comparatively fewer supporting studies.
Direct Serum Fibrosis Markers
In contrast with indirect markers, the combined use of variables representing unique molecular aspects of hepatic fibrogenesis have been termed “direct” serum fibrosis marker panels. Two commonly available methods are the FIBROSpect II and European Liver Fibrosis (ELF) panels. The FIBROSpect II panel (Prometheus Laboratories Inc., San Diego, CA) is a proprietary technique composed of hyaluronic acid, tissue inhibitor of metalloproteinase 1, and α2- macroglobulin. Patel and colleagues evaluated the diagnostic accuracy of FIBROSpect II in 294 patients with chronic HCV and validated the results in an external cohort of 402 patients. The sensitivity, specificity, and AUC in the external cohort was 77%, 73%, and 0.82, respectively. Their results showed relatively good diagnostic accuracy for the detection of moderate-to-severe fibrosis in patients with chronic HCV. A subsequent investigation among 108 patients validated these initial findings with sensitivity, specificity, and AUROC values of 72%, 74%, and 0.826, respectively.
The ELF panel group consists of age, hyaluronic acid, amino-terminal propeptide of type III collagen, and tissue inhibitor of metalloproteinase-1 for the detection of hepatic fibrosis. In a large cohort study examining patients with various chronic liver disease etiologies, the ELF panel also demonstrated good accuracy in detecting clinically significant hepatic fibrosis (stages F2–F4) in patients with alcoholic and NAFLD as well as chronic HCV. By adopting different test thresholds, sensitivities and specificities of over 90% could be obtained allowing the panel to reliably exclude or detect significant fibrosis.
The use of serum fibrosis markers to assess hepatic effects following antiviral treatment response has been a recent area of interest. Within the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) trial, a prospective study of maintenance pegylated interferon in chronic HCV patients with advanced fibrosis who failed prior treatment, a number of serum fibrosis markers were serially measured over the duration of the study. More specifically, serum samples of YKL-40, tissue inhibitor of metalloproteinase-1, amino-terminal peptide of type III procollagen, and hyaluronic acid were assessed at weeks 0, 24, 48, and 72. Among the markers examined, it was shown that a reduced YKL-40 level at baseline was an independent predictor of a virologic response at week 20. Although levels of some markers increased on antiviral treatment, there were significant reductions in all 4 markers noted among patients achieving a sustained virologic response (SVR) at week 72. Patel and colleagues also studied changes in noninvasive serum markers (FibroSURE and FIBROSpect II) in treatment-naïve patients with chronic HCV given albinterferon alfa-2b and ribavirin. Similar to the HALT-C study, patients achieving SVR had lower baseline scores than nonresponders. Moreover, there were significant declines in HCV FibroSURE and FIBROSpect II scores in those patients who achieved a SVR. One drawback to these studies is that a second comparative liver biopsy was not performed after treatment to document fibrosis regression. Future studies are expected to define the minimum change in serum marker panel score that is clinically significant.
Additional studies have evaluated the prognostic value of noninvasive serum biomarkers. A prospective cohort study compared the 5-year prognostic value of FibroTest with liver biopsy for predicting cirrhosis decompensation and survival among 537 HCV-infected patients. FibroTest values were used to classify those with minimal fibrosis (FibroTest < 0.32), moderate fibrosis (FibroTest 0.32–0.58), and severe fibrosis (FibroTest > 0.58). The authors found FibroTest to be a better predictor of both HCV-related complications (AUROC 0.96 vs 0.91) and HCV-related deaths (AUROC 0.96 vs 0.87) compared with liver biopsy. A similar study evaluated the ability of ELF to predict clinical outcomes (liver-related morbidity and mortality) in 457 patients with chronic liver disease of various etiologies. A unit change in ELF score was associated with a doubling of risk of liver-related outcome and ELF predicted outcome at least as well as liver biopsy. Again, future studies are expected to confirm these initial findings and to see if treatment response influencing prognosis can also be measured by serum markers.
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Noninvasive Tools to Assess Hepatic Fibrosis: Ready for Prime Time?
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