Pathology of Nonalcoholic Fatty Liver Disease

Transcription

1 Anatomic Pathology / NONALCOHOLIC FATTY LIVER DISEASE Pathology of Nonalcoholic Fatty Liver Disease Matthew M. Yeh, MD, PhD, 1 and Elizabeth M. Brunt, MD 2 Key Words: Nonalcoholic steatohepatitis; Nonalcoholic fatty liver disease; Liver disease Abstract Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are gaining increasing recognition as components of the emerging epidemic of obesity in North America and in other parts of the world. These entities are considered the hepatic manifestations of the insulin resistance syndrome and represent the spectra of fatty liver disease associated with it. All features of metabolic syndrome are associated with NAFLD/NASH, including obesity, type 2 diabetes, arterial hypertension, and hyperlipidemia in the form of elevated triglyceride levels. NAFLD/NASH can progress to liver cirrhosis and has been reported as a cause of hepatocellular carcinoma. In this review, the histopathologic features of NAFLD/NASH and differential diagnostic considerations are discussed. In addition, grading and staging schema proposed and currently in use are reviewed. Finally, other aspects for consideration by practicing pathologists, such as sampling issues, histopathologic findings after therapeutic interventions, and recurrence after liver transplantation, are addressed. Fatty liver disease that develops in the absence of alcohol abuse is gaining increasing recognition as a major health issue. It is estimated that 20% to 30% of adults in the United States and Western Europe have excessive fat in the liver, and up to 10% of these people (2% to 3% of adult population) may also meet current diagnostic criteria for the progressive lesion nonalcoholic steatohepatitis (NASH). Nonalcoholic fatty liver disease (NAFLD) may actually now be the most common form of chronic liver disease, 1 as this more embracing term spans the entire spectrum of metabolic fatty liver diseases. 1-4 It is now recognized NAFLD/NASH is a manifestation of the metabolic (insulin resistance) syndrome. 5 Central (truncal) obesity, hyperglycemia, type 2 diabetes, arterial hypertension, and hypertriglyceridemia, elements of the metabolic syndrome, are also the best known risk factors of NAFLD. 2,6,7 Notably, like insulin resistance, NASH is specifically associated with central (visceral)-type obesity, rather than general obesity 8,9 as central obesity is strongly correlated with insulin resistance. 10,11 Clinical predictors of more advanced histologic findings in the initial liver biopsy specimen, including age older than 40 to 50 years and the severity of obesity and diabetes, have been shown with compelling evidence in several studies. 2,6 Histopathologic Features of NASH Our understanding of diagnostic criteria for NASH continue to evolve but center on the steatosis, liver cell injury, and the unique pattern of fibrosis. 1 Although recognized by several pathologists as an entity resembling alcoholic hepatitis in a nonalcoholic setting, NASH was the term coined by Am J Clin Pathol 2007;128:

2 Yeh and Brunt / NONALCOHOLIC FATTY LIVER DISEASE Ludwig et al12 in 1980 for the peculiar clinicopathologic entity that was characterized by lesions commonly associated with alcohol abuse (ie, fatty change, lobular hepatitis, and pericellular fibrosis) but with clinical evidence of absence of excess alcohol use. Thus, the article that serves as the foundation of our current pathologic diagnoses used the clinicopathologic criteria that remain in use today. By using these criteria, steatohepatitis may be separated from steatosis Image 1A. It may be difficult to distinguish NASH from simple steatosis if using oversimplified inclusion criteria.13 Ballooning of hepatocytes is a form of liver cell injury that may result from intracellular fluid accumulation and other toxic cell injury and is characterized by swelling of hepatocytes with rarefied cytoplasm Image 1B and Image 1C.14 An early study in the evolution of our learning process found this to be a feature that distinguished progressive NAFLD from the less progressive forms.15 In their study, Matteoni et al15 found that patients with livers with fat accumulation, ballooning degeneration, and Mallory hyaline or fibrosis had a higher incidence of developing cirrhosis and liver-related death compared with patients with livers with fat accumulation alone or with fat accumulation and lobular inflammation only. Investigators from the same group evaluated interobserver and intraobserver concordance in the histologic assessment of A B C D Image 1 A, Nonspecific steatosis, predominantly macrovesicular, with occasional foci of inflammatory cells in the hepatic lobules and many hepatocytes with glycogenated nuclei (H&E, 200). B, Steatohepatitis with several hepatocytes showing ballooning degeneration intermixed with steatosis and foci of inflammatory cells in the hepatic lobules (H&E, 200). C, Mallory hyaline showing eosinophilic and ropy inclusions in the cytoplasm (H&E, 400). D, Perivenular/pericellular fibrosis in zone 3 (Masson trichrome, 200). 838 Am J Clin Pathol 2007;128: Downloaded 838 from

3 Anatomic Pathology / REVIEW ARTICLE NAFLD/NASH and found the features of interest in NAFLD/NASH (steatosis, fibrosis, ballooning degeneration, and the presence of vacuolated [glycogenated] nuclei) had significant agreement among experienced liver pathologists.13 The results suggested that these histologic features might be useful for the development of a standardized and reliable pathologic scoring scheme for NAFLD and its various clinical outcomes. It was almost at the same time that Brunt et al16 introduced a semiquantitative grading and staging scheme for NASH in which the grading system was based on incremental increase in a combination of lesions: steatosis, ballooning, and lobular and portal inflammation, and the stage was proposed to reflect the typical fibrosis pattern of NASH from the unique zone 3 perisinusoidal/pericellular fibrosis, through portal fibrosis, bridging fibrosis to cirrhosis. A subsequently small clinicopathologic correlative study based on blinded review of entry biopsy specimens for a treatment trial showed hepatocytic ballooning associated with higher serum cholesterol levels; there was also a trend toward the presence of ballooning in biopsy specimens from patients with abnormal glycemic control, greater insulin resistance, and increased serum markers of necroinflammation.17 Steatosis, ballooning, and lobular inflammation have been considered a common set of minimal criteria for the diagnosis of NASH.18 Multiple regression analysis has shown the diagnosis of NASH is not dependent on a single histologic feature, but rather involves assessment of multiple independent features.19 The recent results of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)sponsored NASH Clinical Research Network study using multivariant analysis have suggested that several features are A significantly associated with the diagnosis of NASH, including lobular inflammation, ballooning degeneration, fibrosis, and steatosis.20 Apparently the aforementioned histologic changes represent the most helpful features to diagnose NASH. Whether other existing features help in distinguishing NASH awaits further investigation. In some cases, ballooning degeneration of the hepatocytes has to be distinguished from large, swollen, glycogenrich hepatocytes that are characteristic of glycogen storage disease or glycogenic hepatopathy, a recently described entity that is commonly associated with type 1 diabetes and poorly controlled blood glucose levels.21 Biopsy specimens from patients with the latter usually lack significant fatty change, inflammation, lobular spotty necrosis, and fibrosis. Microvesicular fatty change of the hepatocytes may, at times, mimic ballooning degeneration in that the hepatocytes may also be enlarged. The nuclei remain centrally located and appear to be indented by the small fat droplets Image 2. In addition, it should be noted that in patients with Wilson disease, severe liver disease develops that is also characterized by steatosis and glycogenated nuclei, in addition to lipofuscin, acidophil bodies, bile, iron, anisonucleosis, portal chronic inflammation, Mallory hyaline, and swollen hepatocytes, some with megamitochondria. Megamitochondria, or giant mitochondria, may be seen in NASH.21 These are round or needle-shaped intracytoplasmic inclusions Image 2B. Ultrastructurally, they manifest as multilamellar membranes and paracrystalline inclusions and with loss of cristae. It has been suggested that megamitochondria reflect injury or adaptive change to the mitochondria.22,23 B Image 2 Patchy microvesicular fat is present in the hepatocytes. A, The hepatocytes appear enlarged (upper portion of the biopsy specimen; H&E, 100). B, The nuclei remain centrally located and are indented by the small fat droplets; megamitochondria are also present, showing the round intracytoplasmic inclusions (H&E, 400). Am J Clin Pathol 2007;128:

4 Yeh and Brunt / NONALCOHOLIC FATTY LIVER DISEASE Sampling Issues in the Assessment of NAFLD/NASH To date, no imaging modalities are capable of distinguishing NASH from simple steatosis or for assessing fibrosis and architectural alterations, and liver biopsy remains the gold standard to diagnose NASH and to evaluate the severity of hepatic fibrosis in a person with features of NAFLD. 2,16,20 Just as sampling in grading and staging for chronic viral hepatitis is critical, 24 it has been suggested that in NASH sampling is also critical: parenchymal injury and fibrosis may vary in different regions of the liver, 25 and the stage of fibrosis in patients with NASH might not be assessed accurately in biopsy specimens of suboptimal length or from suboptimal locations (ie, subcapsular). In fact, it has been shown that NASH biopsy specimens longer than 1.6 cm had a significantly lower heterogeneity of fibrosis than biopsy specimens 1.6 cm or shorter. 25 Although why such heterogeneity of fibrosis occurs in NASH is largely unknown, regional gradients of insulin delivery, intrahepatic free fatty acids, and/or antioxidant levels or oxygen saturation of sinusoidal blood are putative considerations. 25 Without an existing ideal animal model, these are merely postulations and difficult to prove. In a prospective study, paired percutaneous liver biopsies were performed with 2 samples collected from individual patients with NAFLD. The study showed significant sampling variability in different regions of the liver parenchyma that can lead to substantial misdiagnosis and inaccurate staging. 26 For example, the impact of sampling variability on the diagnosis of NASH is significant when the diagnosis of steatohepatitis relied on hepatocyte ballooning, which was present on one sample but not on the other. Along the same line, bridging fibrosis was present on one sample but not on the other in a substantial portion of cases, significantly altering the staging of fibrosis. Histopathologic Features of NASH Shared With Alcoholic Steatohepatitis Alcoholic steatohepatitis (ASH) and NASH share many histologic features, including steatosis, hepatocytic injury (ballooning degeneration, apoptosis, and lytic necrosis), lobular inflammation, Mallory hyaline, megamitochondria, and the pattern of fibrosis. 27 In terms of pathogenesis, the alcohol-inducible hepatic cytochrome P-450 2E1 (CYP2E1) can also be induced in one animal model of steatohepatitis (although not typical NASH), methionine choline deficient rats. 28 Similarly, hepatic CYP2E1 is increased in patients with NASH, suggesting the pathogenesis of NASH may be in part similar to ASH, and, like ASH, may involve induction of CYP2E1. 29 Although histologically it is almost impossible to distinguish ASH from NASH, features more common in ASH than NASH are canalicular cholestasis, marked ductular reaction, and acute inflammation in the portal regions. Histologic changes more commonly encountered in NASH than ASH are greater amounts of steatosis and nuclear vacuolization, 30 and, overall, the necroinflammatory activity tends to be milder in NASH than in ASH. 12,14,27,31,32 Moreover, Mallory hyaline and periportal fibrosis are more common in ASH. 27,33 In ASH, venous outflow occlusion, lymphocytic phlebitis, and sclerosing hyaline necrosis have been described involving the outflow veins; however, they are not reported findings in NASH. 27 Cholestasis is reported in many forms of alcoholic liver disease, but not in NAFLD, and alcoholic foamy degeneration does not have a known counterpart in NAFLD. Of note, steatosis is not always present in ASH, particularly in alcoholic hepatitis. On the contrary, steatosis is a necessary element to establish a diagnosis of NASH, particularly when the liver is not cirrhotic. The Usefulness of Immunohistochemical Analysis The presence of Mallory hyaline (Image 1C) in zone 3 in a biopsy specimen with steatosis may suggest a diagnosis of steatohepatitis, and abundant Mallory hyaline may even implicate excessive alcohol use. 34,35 Mallory hyaline is intracytoplasmic inclusions within hepatocytes that actually may be seen in several liver diseases. The major components of Mallory hyaline are abnormal cytokeratins, which consistently contain cytokeratin (CK) 8 and CK18 and frequently contain CK7, CK19, and CK Ubiquitin, an intracellular protein that binds to other proteins to target them for proteolysis, and p62, another component of the proteosomal degradative pathway, are also associated with Mallory hyaline. Immunostaining for ubiquitin and/or p62 may, thus, confirm Mallory hyaline in questionable cases, 37 but interpretation of any positive granule that is positive for p62 or ubiquitin must be done with care. The definitive diagnosis of NASH vs ASH relies on clinicopathologic correlation. In an attempt to determine whether liver biopsy specimens with steatohepatitis could distinguish ASH from NASH by immunohistochemical stains, Sanderson and Smyrk 38 demonstrated that liver biopsy specimens from patients with NASH showed increased expression of protein tyrosine phosphatase 1B (PTP1B) and decreased expression of insulin receptor (IR), whereas specimens from patients with ASH tended to have a lower level of expression of PTP1B and normal expression of IR. PTP1B is a regulatory protein that exerts its action on IR through a negative dephosphorylation effect. 39 Increased expression of PTP1B has been suggested to be associated with obesity and insulin resistance 40 ; although 840 Am J Clin Pathol 2007;128: Downloaded 840 from

5 Anatomic Pathology / REVIEW ARTICLE more studies are needed to further explore the usefulness of this immunohistochemical staining pattern in NASH, this study confirms the clinical studies that connect the pathogenesis of the metabolic syndrome to the morphologic link in diagnosis of steatohepatitis. The study also emphasizes the reliance on clinical input for diagnosis. Fibrosis in NASH The initial collagen deposition in NASH or NAFLD differs distinctively from viral hepatitis, chronic biliary disease, and autoimmune hepatitis, in that the fibrosis starts in the zone 3 region as perivenular and/or pericellular fibrosis, a pattern referred to by some as chicken-wire fibrosis Image 1D.41 Immunohistochemical study using α-smooth muscle actin, a marker for activated hepatic stellate cells (HSCs), has shown the activated HSCs were predominantly distributed in zone 3 and the degree of HSC activation correlated with the severity of fibrosis,42 confirming HSC activation in NASH in the zone 3 distribution. These findings mirror the findings in viral hepatitis, in which the preferential zone of HSC activation is zone 1.43 The uneven zonal distribution of activated HSCs may reflect a heterogeneous population of HSCs that are differentially activated in various chronic liver diseases or may reflect the heterogeneity of stimuli within the acini of the liver for fibrogenesis. One study of bariatric patients suggested that some morbidly obese people may have portal fibrosis only without the other typical histologic features of NASH; this findings was associated with hyperglycemia.44 Whether this A observation suggests a variant pattern of fibrosis in NAFLD, reflects selective regression of fibrosis in zone 3, or is a result of sampling peculiar to bariatric procedures needs further investigation. One has to be cautious not to overcall NASH owing to a pattern of fibrosis mimicking chicken wire. For example, large lipogranuloma, composed of single or multiple fat droplets surrounded by chronic inflammatory cells and Kupffer cells, when present in the lobules, may be entrapped by fibrous tissue and mistaken for centrizonal perisinusoidal fibrosis that leads to overdiagnosis of NASH and even overestimation of the severity of fibrosis.27 One of the challenging issues in diagnosing NASH is in cirrhosis. When this remodeling occurs, the typical histologic features such as steatosis, lobular inflammation, and ballooned hepatocytes may persist or may no longer exist. In addition, the characteristic zonal distribution of steatosis in NASH may be difficult to discern because of the architectural distortion due to cirrhosis Image 3. Therefore, cautious consideration should be practiced to identify other lesions that may help in establishing a diagnosis, eg, the presence of Mallory hyaline, glycogenated nuclei, and the characteristic chicken-wire fibrosis. However, none of these features are specific for NASH and may be no longer recognizable in the cirrhotic phase. Exactly how often this occurs in NASH is yet to be established, and the assumption of NASH as the cause of all cases of otherwise cryptogenic cirrhosis is to be discouraged because many other forms of liver disease may burn out. In particular, autoimmune hepatitis and alcoholic liver disease may result in cirrhosis without other discernible histologic features. B Image 3 A, This liver biopsy specimen shows active nonalcoholic steatohepatitis in the cirrhotic phase with moderate steatosis. The distribution of steatosis is accentuated around the fibrous septa, without the obvious zone 3 pattern, as the hepatic architecture is largely distorted owing to cirrhosis (H&E, 40). B, At higher magnification, foci of inflammatory cell aggregates in the lobules and ballooned hepatocytes containing Mallory hyaline are evident (H&E, 100). Am J Clin Pathol 2007;128:

6 Yeh and Brunt / NONALCOHOLIC FATTY LIVER DISEASE Differential Diagnosis With Other Liver Diseases A host of chronic liver diseases have histologic features that may mimic NAFLD/NASH, and it is necessary to distinguish them from fatty liver disease to arrive at a precise diagnosis for appropriate management. In particular, up to 70% of chronic hepatitis C shows at least a mild degree of macrovesicular fatty change. 45 This is not typically restricted to zone 3, however. Specifically, hepatitis C virus (HCV) genotype 3a is tightly associated with hepatic steatosis Investigators have found that hepatitis C may induce insulin resistance, and this effect seems to be specific to the genotype. 19 Researchers have indicated that steatosis in hepatocytes is a cytopathic effect of HCV genotype 3, 49 and HCV core protein induces hepatic steatosis in transgenic mice. 50 Of note, when successfully treated with antiviral therapy, the degree of fat in genotype 3 is significantly ameliorated. 51 In a recent study of a large and geographically different group of patients with chronic hepatitis C, steatosis was shown as significantly and independently associated with fibrosis in chronic hepatitis C. 52 Hepatitis C can be diagnosed by serology and HCV RNA, tends to have more inflammation in the portal tracts than in the lobules, generally does not have ballooned hepatocytes, and lacks the distinct pericellular/perivenular fibrosis. Elevated antinuclear antibody titers have been reported in up to 34% of patients with biopsy-proven NASH. 53 However, anti smooth muscle antibody titers were not particularly elevated in this cohort, and the liver biopsies did not show features of autoimmune hepatitis. It is, therefore, strongly recommended that prudent evaluation of the serologic results and liver biopsy specimen be thoroughly practiced before making a diagnosis of autoimmune hepatitis because the therapeutic modalities for autoimmune hepatitis and NASH differ significantly. Alternatively, autoimmune hepatitis and NASH may reasonably coexist, given the overlapping demographic features in these entities Besides chronic hepatitis C and autoimmune hepatitis, it has been documented that histologic features of steatohepatitis coexisted in 1% to 7% of liver biopsy specimens (nonallograft) from patients with other liver diseases, including primary biliary cirrhosis, α 1 -antitrypsin deficiency, hepatitis B infection, and others. The frequency of the concurrence may have been underestimated because more stringent criteria for steatohepatitis were applied in the study so as not to overestimate NASH. 56 Grading and Staging of NASH A very relevant issue of NAFLD to treating clinicians is whether the histologic grade and stage in fibrosis correlate with the natural history, clinical parameters, and outcome. As with the development of grading schemes for chronic viral and autoimmune hepatitis, drug-induced liver disease, and chronic biliary diseases (primary sclerosing cholangitis and primary biliary cirrhosis), which have been widely applied in daily pathology reports and in large clinical trials and research, once NAFLD/NASH was recognized and accepted as a bona fide form of liver disease, it was clear there was no systematic method for reporting necroinflammatory activity (grade) and architectural remodeling (stage). In 1999, Brunt et al 16 first proposed a semiquantitative grading and staging scheme for NASH to parallel the concepts and terminology used in chronic hepatitis for semiquantitative evaluation, 57 in which the grade was based on the constellation of lesions considered important in steatohepatitis: steatosis, ballooning, and lobular and portal inflammatory infiltrates, and the stage was determined on the progression of fibrosis that starts from the unique zone 3 perisinusoidal/pericellular fibrosis, through portal fibrosis and bridging fibrosis to cirrhosis. 16 Since its seminal publication, this scheme (often referred to as the Brunt system) has been gradually adopted and widely used, including in clinicopathologic studies and in the setting of clinical trials to assess histologic changes after treatment Of note, this system was designed for NASH and was not developed to encompass the full spectrum of NAFLD as defined by Matteoni et al. 15 Through a recent joint effort and consensus on histopathologic characteristics, the Pathology Committee of the NIDDKsponsored NASH Clinical Research Network proposed and validated a feature-based scoring system that comprised 14 histologic features. This system was based on and further refined the scoring scheme proposed by Brunt et al. 16 After blindly reviewing biopsy specimens and generating diagnoses, features that statistically correlated with diagnoses were used to derive a scoring system. The unweighted sum of scores for each lesion steatosis, lobular inflammation, and hepatocellular ballooning was used to generate an NAFLD activity score (NAS). The fibrosis score derives from the fibrosis score in the Brunt scheme but with subclassification of stage 1 to separate delicate or dense sinusoidal fibrosis and addition of stage 1C to include portal-only fibrosis (eg, pediatric NASH; see the next section). A NAS of more than 5 was almost universally associated with the diagnosis of NASH, and cases with a NAS of less than 3 were all diagnosed as definitely not NASH. It should be emphasized that as stated in the publication, the NAS was originally designed to assess overall histologic change before and after therapeutic intervention trials and was not intended as numeric values to replace a pathologist s diagnostic determination of steatohepatitis. 20 Pediatric NASH As the prevalence of overweight, obesity, and insulin resistance among children has increased during past decades, 62,63 it is conceivable NAFLD affects a substantial 842 Am J Clin Pathol 2007;128: Downloaded 842 from

7 Anatomic Pathology / REVIEW ARTICLE proportion of children and is likely to reach epidemic proportions in children worldwide in the next decade It has been documented that NAFLD is more prevalent in boys than in girls.63,67-69 A unique histologic pattern of NAFLD/NASH in the pediatric population has been observed in that the inflammation and fibrosis are accentuated in the portal regions Image 4, in contrast with the zone 3 injury pattern typically seen in adult NAFLD/NASH. This distinct pattern (type 2 pattern) had been seen in 51% of a series of 100 children or adolescents with NAFLD, whereas only 17% showed the typical histologic features of NASH seen in the adult population, ie, steatosis, ballooning degeneration, and perisinusoidal fibrosis.70 Of note, in this study, boys and children of Asian, Native American, and Hispanic ethnicity were significantly more likely than girls and children of white race, respectively, to have these portal-based changes. A more recent study from Italy showed that although the classic zone 3 accentuated NASH was only 2.4% in their pediatric population (mean ± SD age, 11.7 ± 3.3 years), the portal-based injury was also only seen in 28.6%, whereas overlap of both patterns constituted the majority of cases, 52.4%.66 There was a lesser degree of interobserver agreement in scoring the histologic features of pediatric NAFLD.20 It is likely that a spectrum or different patterns of disease exist in pediatric NAFLD, and further studies are needed to elucidate the pathologic features in the pediatric population. Recurrence of NAFLD After Liver Transplantation Recurrence or occurrence of NASH after orthotopic liver transplantation (OLT) has been well described It has been suggested that in a time-dependent manner, fatty liver occurs A universally after OLT in patients with cryptogenic cirrhosis in whom a clinical and histologic profile consistent with NAFLD existed before transplantation. It has also been shown that cumulative steroid exposure correlates with the development of fatty liver in the allograft.75 The pre-olt diagnosis in most of these patients was cryptogenic cirrhosis, and they were included in the studies based on clinical and histologic features suggestive of NASH. The observation that NAFLD/NASH recurs after OLT supports the notion that the disease is a hepatic manifestation of the metabolic syndrome.1,6 However, caution is suggested in attributing all causes of cryptogenic cirrhosis to NASH based on occurrence of the lesions post-olt because liver allograft recipients are at increased risk of obesity, diabetes, and hyperlipidemia in the post-olt period. Liver Histologic Findings After Therapeutic Intervention The liver injury in NASH may improve or resolve spontaneously over time without any specific intervention; this has been observed directly in a randomized controlled trial76 and indirectly in a review of series of biopsy specimens.6,60 This phenomenon may be attributed to the consequences of subconscious lifestyle changes by patients, realizing the significant medical conditions that they may have.77 The histologic changes after therapeutic interventions, such as clinical trials using insulin-sensitizing agents, include improved steatosis, inflammation, ballooning, and fibrosis, some with complete resolution of NASH.60,61,76,78 In a study of laparoscopic gastricbanding surgery, 83% of patients no longer had NASH, and B Image 4 Typical pediatric nonalcoholic steatohepatitis (type 2 pattern) with inflammation and fibrosis accentuated in zone 1 region (A, H&E, 100; B, Masson trichrome, 100). Am J Clin Pathol 2007;128:

8 Yeh and Brunt / NONALCOHOLIC FATTY LIVER DISEASE there was a significant reduction in fibrosis, besides steatosis and inflammation. 79 Some studies have shown that there was a shift in the proportion of portal to lobular inflammation in that lobular inflammation resolved, whereas portal inflammation remained (or increased) after rosiglitazone or pioglitazone (peroxisome proliferator-activated receptor γ ligand) treatment or gastric-banding surgery. 60,78,79 Therefore, when pathologists encounter disproportionately denser portal inflammation in patients with NAFLD, posttreatment effect needs to be considered. Another consideration, of course, is concurrent disease. Ductular Reaction in NASH and Its Differential Diagnosis A correlation of the number of hepatic progenitor cells (HPCs) with fibrosis has been suggested in NASH in that a periportal ductular reaction has been observed in relation with the expansion of HPCs. 80 Similarly, ductular reaction has also been noted to be associated with increased fibrosis in alcoholic liver disease. 80,81 In a study of a series of biopsy specimens from patients with chronic hepatitis C infection, increasing steatosis was associated with a greater ductular reaction and an increased number of HPCs. 82 Therefore, activation of HPCs in NASH and steatosis, manifested as a ductular reaction, suggests liver cell injury, and hepatic fibrosis in chronic liver disease due to metabolic syndrome may induce the expansion of the progenitor cell compartment. Because the ductular reaction reflects a response to injury and likely is a phenomenon of regeneration in the liver, 83 pathologists should be reminded that it can be seen in various conditions, including NASH, alcoholic liver disease, extrahepatic obstruction, and chronic cholestatic disease (primary sclerosing cholangitis and primary biliary cirrhosis). Iron Deposition in NASH Iron deposition in NASH is an interesting and incompletely understood topic. It has been observed that deficient insulin secretion and insulin resistance were significantly associated with the severity of hepatic iron deposition in patients with familial hemochromatosis in the precirrhotic phase. 84 Various studies have also shown that iron deposition in NASH ranges from 15% to 55%. 12,16,85,86 In addition, George et al 85 demonstrated that an increased hepatic iron concentration in NASH is associated with increased portal hepatic fibrosis. Because hepatic steatosis and iron overload can result in hepatic fibrosis, whether the association of hepatic iron accumulation with NASH is due to unique iron deposition in fatty liver per se, is linked to a genetic mutation, or is a result of increased fibrosis warrants further clarification. NASH and Hepatocellular Carcinoma The association of hepatocellular carcinoma (HCC) with NAFLD or NASH is a growing concern. The incidence of HCC ranged from 18% in cryptogenic cirrhosis 87 to 27% in cryptogenic cirrhosis in obese patients. 88 Since NAFLD was implicated as the cause of cryptogenic cirrhosis in these studies, based on well-characterized associated clinical features of metabolic syndrome, the potential role of NAFLD in hepatocarcinogenesis was suggested. However, in a prospective study of wellcharacterized cases of NASH-related cirrhosis, no cases of HCC were noted during a 5-year follow-up, in comparison with a 17% incidence in hepatitis C related cirrhosis. 89 In addition, in the study by Powell et al 90 of up to 20 years in 42 patients with histologically defined NASH, only 1 case of HCC developed. Whether NASH per se is a significant cause of HCC or it is just an epiphenomenon due to liver cirrhosis or cycles of hepatic inflammation is not conclusive, and more prospective, long-term studies are needed to answer this question. 2 Future Perspectives There are many arenas that await extensive study in the diagnosis and therapeutic intervention of NASH and its pathogenesis. Previous studies using gene microarray tools in cirrhotic liver samples from patients with NASH have revealed overexpression or underexpression of genes that lead to decreased insulin sensitivity and defective mitochondrial function. 91 Recent results also showed that several differentially expressed genes in patients with NASH were related to lipid metabolism and extracellular matrix remodeling. In addition, genes related to liver regeneration, apoptosis, and the detoxification process were differentially expressed. 92 Although more work is certainly needed, these findings may help clarify the molecular pathogenesis of NASH and identify potential targets for therapeutic intervention. From the perspective of pathologists, there is an emerging need for diagnostic distinction in predicting outcome in NASH and in distinguishing the etiology of various liver diseases that morphologically resemble or share features with NASH, such as alcohol and various drug injuries, particularly in the phase of cryptogenic cirrhosis. From the Departments of Pathology, 1 University of Washington School of Medicine, Seattle; and 2 Saint Louis University Health Sciences Center, St Louis, MO. Address reprint requests to Dr Yeh: Dept of Pathology, University of Washington School of Medicine, 1959 NE Pacific St, NE 140D, Box , Seattle, WA References 1. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology. 2003;37: Am J Clin Pathol 2007;128: Downloaded 844 from

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