Nonalcoholic Steatohepatitis

Transcription

1 Nonalcoholic Steatohepatitis Elizabeth M. Brunt, M.D. 1 ABSTRACT Nonalcoholic fatty liver disease (NAFLD) is being increasingly recognized as a common liver disorder that represents the hepatic manifestation of the metabolic syndrome, a variably defined aggregate of disorders related to obesity, insulin resistance, type II diabetes, hypertension and hyperlipidemia. Nonalcoholic steatohepatitis (NASH) is the progressive form of liver injury that carries a risk for progressive fibrosis, cirrhosis, and end-stage liver disease. Hepatocellular carcinoma (HCC) is a documented complication in an as yet unknown percentage of cases of NASH cirrhosis. The diagnosis of nonalcoholic steatohepatitis requires histopathologic evaluation because the lesions of parenchymal injury and fibrosis cannot be detected by imaging studies or laboratory tests. This article will briefly discuss prevalence studies and the pathophysiology of NAFLD and focus on current discussions related to the specific lesions in the pathology of NASH, including the challenges of pediatric NASH and NASH-related cirrhosis. KEYWORDS: Nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, obesity, metabolic syndrome, insulin resistance Objectives: On completion of this article, the reader should be able to recognize (1) the microscopic lesions that characterize adult and pediatric NASH as well as (2) the areas of pathology that are under study. Accreditation: Tufts University School of Medicine (TUSM) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. TUSM takes responsibility for the content, quality, and scientific integrity of this CME activity. Credit: TUSM designates this educational activity for a maximum of 1 Category 1 credit toward the AMA Physicians Recognition Award. Each physician should claim only those credits that he/she actually spent in the educational activity. The central role of insulin resistance in NASH has been confirmed in clinical correlation studies and animal models and in recent therapeutic intervention trials. Multifaceted functions of visceral adipose tissue and the polygenic and interrelated pathways that link obesity, insulin resistance, hepatic steatosis, and the subsequent progression to parenchymal necroinflammation and fibrosis continue to be elucidated. With all the progress that has occurred, it is interesting that liver biopsy evaluation has retained an essential role in the clinical diagnosis of NASH and is recognized as a significant component for analysis in the growing number of therapeutic trials. This article will focus on pathology while providing an overview of current advances in the field. BACKGROUND The diagnosis of NAFLD and NASH remains one of clinicopathologic correlation. 1 3 In evaluation of subjects suspected to have NAFLD or NASH, standard laboratory tests may document liver test elevations and exclude Issues in Hepatic Pathology; Editor in Chief, Paul D. Berk, M.D.; Guest Editors, Maria Isabel Fiel, M.D., and Swan N. Thung, M.D. Seminars in Liver Disease, volume 24, number 1, Address for correspondence and reprint requests: Elizabeth M. Brunt, M.D., 4th Floor FDT, Saint Louis University Hospital, 3635 Vista Avenue at Grand Boulevard, St. Louis, MO [email protected]. 1 Associate Professor of Pathology, Saint Louis University Liver Center, Saint Louis University Health Sciences Center, Department of Pathology, St. Louis, Missouri. Copyright # 2004 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) ,p;2004, 24,01,003,020,ftx,en;sld00247x. 3

2 4 SEMINARS IN LIVER DISEASE/VOLUME 24, NUMBER a variety of potential underlying causes of liver dysfunction. Careful clinical evaluation may exclude alcohol as a significant underlying factor and radiological findings may confirm hepatic steatosis but may not be reliable with small amounts of steatosis. However, there are no tests that replace liver tissue analysis to confirm or exclude the diagnosis of steatohepatitis. Biopsy analysis is also necessary to determine the extent of active injury, the presence and character of fibrosis, and the degree of architectural remodeling. Biopsy evaluation is considered the gold standard. Finally, although the pathologist may document the lesions of steatohepatitis, histological evaluation often cannot reliably distinguish between alcoholic and nonalcoholic origin. Just as the clinician cannot diagnose steatohepatitis, the pathologist cannot always discern nonalcoholic origin. The terminology of NAFLD and NASH has been questioned by pathologists and clinicians alike. 2,4,5 In current hepatologic practice, no other liver disease is diagnosed as having a non association. Furthermore, rather than being a disease process that is characterized primarily by nonuse of alcohol, there now are recognized underlying disorders associated with NAFLD and NASH. Thus, the appeal for upgraded nomenclature such as metabolic syndrome steatohepatitis (MSSH 4 or MESH 2 ) has been made; current practice, however, retains the original nomenclature and thus is used in this article, with the anticipation that nomenclature will shift with our growth in scientific observations. Hepatopathologists are aware that prior to Ludwig s seminal manuscript 6 in which the rubric nonalcoholic steatohepatitis (NASH) for a hitherto unnamed disease was formally introduced, numerous histological studies of the liver in obese patients, with and without diabetes, had documented the lesions that we now associate with NASH. These studies had also noted that the microscopic findings were similar to many of those of alcoholic liver disease However, the 1980 study of Ludwig et al 6 is credited with having solidified the concept that subjects who did not consume alcohol could have liver disease with lesions similar to those seen with alcoholic liver disease. The study was based on 20 cases selected by biopsy findings and documented clinical correlations that are now familiar in NASH: most of the subjects were obese (90%) and were women (65%), and hyperlipidemia or diabetes, or both, were noted in 25% of the patients. Over the ensuing two decades, the associations of NAFLD and NASH with obesity, insulin resistance, and dyslipidemia have been strengthened, and the insightful nature of the initial clinicopathologic study by Ludwig et al 6 was confirmed. Studies of natural history of NAFLD are inherently challenging because of the reliance on histology and the inability to document the beginning of the process. To date, a spectrum of clinical outcomes that parallels that of histological features ranges from the benign, nonprogressive nature of simple steatosis 11 to advanced fibrosis and cirrhosis in 15 to 19% of patients, presumably because of steatohepatitis. 12 The retrospective study of 98 subjects with 10-year follow-up data by Matteoni et al 13 found that cirrhosis and liver-related mortality were related to histological categories in NAFL. Hepatic steatosis was defined as type 1 and was a common denominator in the remainder; inflammation (type 2), ballooning (type 3), and ballooning and either Mallory s hyaline (MH) or fibrosis (type 4) defined the groups. The study showed that prevalence of severe liver disease (cirrhosis) progressed from patients with types 1 (1%) and 2 (0%) to those with types 3 (21%) and 4 (28%) and to death in less than 2% with simple fatty liver compared with 11% in types 3 and 4. The term NAFL has gradually been modified as terminology to include D for disease in recognition of the fact that patients with NAFLD are indeed at risk for progressive liver disease NASH, with its potential longterm complications of cirrhosis; liver failure; and HCC. 14,15 PREVALENCE OF NAFLD AND NASH The true prevalence of both NAFLD and NASH also remains elusive, largely because of the lack of a definitive laboratory test and hence dependence on different definitions of these entities. Ascertainment of cases for investigation, inclusion criteria, and consequent results vary. However, the reality of the increasing long-term burden of NAFLD and NASH to society and medical practice remains the driving force behind studies to determine prevalence and pathogenesis. Published series of biopsy-confirmed NAFLD and NASH are reporting selected cases that have presented to medical attention, but it is acknowledged that NAFLD and NASH may be clinically silent and indolent in progression, and therefore the number of affected patients is likely underrecognized. Without serological assays to confirm the existence of NAFLD and NASH, the most commonly cited prevalence studies have used a variety of databases: screening ultrasound in general populations, clinical databases from population screening, and histological studies. Fundamental to all studies is the concept of nonalcoholic, yet, interestingly, in the clinical literature, the exact definition of insignificant alcohol use is not applied uniformly. 12 Values in published studies range from < 20 g/week to < 140 g/week. One recent study in morbidly obese patients showed a decreased incidence of histological NASH in subjects who used up to 210 g/week compared with the 0 to 200 g/week users, likely because of the effects on hyperinsulinemia. 16 There have also been suggestions that gut-derived endogenously produced alcohol may participate in cellular events that result in hepatic manifestations of NASH. 17,18

3 NONALCOHOLIC STEATOHEPATITIS/BRUNT 5 Lonardo et al 19 reported prevalence of bright liver (hepatic steatosis detected by ultrasound) in 19.8% of 363 unselected Italian patients who had had diagnostic abdominal ultrasound. The Dionysos Study 20,21 focused on the prevalence of hepatic steatosis as detected by ultrasound in a well-defined study group of 257 adults without evidence of liver disease, diabetes, hypertriglyceridemia and known medications in northern Italy; the study was not restricted to teetotalers. Hepatic steatosis was associated with obesity and with heavy alcohol consumption, from 16% of healthy nonobese, nondrinkers and 46% of heavy drinkers to 76% of obese subjects and 95% obese heavy drinkers. The strongest association for steatosis was with obesity. Interestingly, elevated liver tests were found in 22% of otherwise normal, healthy controls. The value and the shortcomings of current imaging studies for screening and diagnosis in fatty liver disease has been reviewed 14 ; Saadeh et al 22 reported that state of the art imaging procedures do not reliably detect steatosis below 33% and confirmed lack of detection of features of necroinflammation (ballooning, MH) and fibrosis. Recently, two groups used clinical data and serological results from more than 15,000 subjects for the third U.S. National Health and Nutrition Examination Survey (1988 to 1994) (NHANES III); elevated liver chemistry tests were considered surrogate markers of NAFLD in subjects without other forms of clinically documentable liver disease or significant alcohol use (> 2 drinks/day). Ruhl and Everhart 23 found that 2.8% of nondiabetic adults in the United States have elevated alanine aminotransferase (ALT) (fasting level > 43U/L); 65% of elevated ALT (and possibly NAFLD) was attributed to overweight (body mass index [BMI] 25 kg/m 2 ) and obesity (BMI 30 kg/m 2 ). This association was more pronounced in the higher BMI ranges. The investigators noted that if newer, lower values for normal ALT had been applied (19 U/L for women, 30 U/L for men), 24 the total values for elevated ALT would have risen to 12.4% for men and 13.9% for women. Other associations with nondiabetic elevated ALT were younger age (39 years versus 45 years), male gender (4.3% versus 1.6% for women), and Mexican-American ethnicity (8.4% versus 2.6% non-hispanic whites and 1.9% non-hispanic blacks). In addition, impaired fasting glucose, higher BMI and waist to hip ratio (WHR) (used as a measure of visceral adiposity), increased fasting serum leptin, triglycerides, and insulin levels, as well as recently defined criteria of metabolic syndrome were univariate factors associate with elevated ALT. The five components that compose the metabolic syndrome are central (truncal) obesity, hyperglycemia, low levels of high-density lipoprotein cholesterol, hypertriglyceridemia, and hypertension. Subjects with specified values for at least three of these components are considered to have metabolic syndrome. 25 The difference between the results for men and women could be explained by the differences in WHR. Fasting serum insulin levels and other markers of insulin resistance were associated with elevated ALT independent of BMI and WHR. Of subjects with elevated ALT, > 40% fit criteria for metabolic syndrome. The authors further reported that subjects with metabolic syndrome were five times as likely to have elevated ALT compared with the normal population. Clark et al 26 used the same database but applied different criteria for analysis; diabetics were not excluded from their analysis, both aspartate aminotransferase (AST) and ALT were included, and ALT levels considered elevated were lower than the 43 U/L of the study by Ruhl and Everhart. 23 The values used were AST > 37 IU/L, ALT > 40 IU/L for men, and AST or ALT > 31 IU/L for women. Exclusion criteria were markers for viral hepatitis, transferrin saturation > 50%, alcohol use (> 1 drink/day for women, > 2 drinks/day for men). Of the 7.9% of the total study population with elevated aminotransferases, 69% were considered unexplained and therefore considered as NAFLD. This results in a total of 5.5% adults in the NHANES population. If the newly recommended values for upper limits of ALT (> 19 IU/L for women, > 30 IU/L for men) had been used, 24 the prevalence of elevated transaminases would have been 26%, and those that were unexplained 81.7%; this results in a total of 21.2% of the NHANES study population with unexplained liver test elevations. When the data were analyzed to include AST, ALT, and gamma glutamyltransferase, this group found 27% of adults had elevated liver tests, 79% of which were unexplained. 14 Features associated with elevated ALT similar to that of Ruhl and Everhart 23 were younger age, male gender, Mexican-American ethnicity, increased BMI, fasting triglycerides, insulin levels, type 2 diabetes, and hypertension. One notable difference was the increased prevalence of elevated ALT among non-hispanic blacks (8.1%) compared with 1.9% in the study by Ruhl and Everhart. 23 Elevated aminotransferases were analyzed among various subgroups: among absolute nondrinkers, liver test elevation was 6.4% of men, 7.7% of women, 7.4% overall. Ruhl and Everhart showed no significant difference in results comparing nondrinkers (< 1 drink/ day) (2.7%) to the total group (2.9%). 23 Acknowledged difficulties with the studies based on NHANES data 14,23,26 include the fact that presumed NAFLD cases were not histologically proved to be NAFLD or NASH. In addition, there remain uncertainties as to aminotransferase values considered to be normal in the increasingly obese population of the United States. 24 Finally, the data for these analyses were derived from previously frozen sera, in which a proportion of aminotransferase activity may have been lost. 15 Even considering the difficulties in establishing prevalence, NAFLD may be the most common cause of asymptomatic

4 6 SEMINARS IN LIVER DISEASE/VOLUME 24, NUMBER chronic liver test elevation. 14,15,23,27 NAFLD is likely more common in the U.S. population than are hepatitis C(1.3to2.0%),alcoholicliverdisease(1%),hepatitisB (0.3 to 0.4%), hereditary hemochromatosis (1:200 to 1:400 of northern European descent), and the chronic liver diseases with low prevalence: autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), alpha 1 -antitrypsin (A 1 AT) deficiency, and Wilson disease. 28 Serological liver tests are not necessarily adequate screening tests because they may be normal in patients with chronic liver disease(s), including hepatitis C. 29 A recent study based on biopsy findings documented lesions of NAFLD and NASH in subjects evaluated for unexplained hepatomegaly or for living donor transplantation but not elevated liver tests. 30 Of note in this study, however, liver aminotransferases considered to be within normal range for the reference laboratory were considerably higher than they were for the two NHANES studies or current recommendations, < 52 IU/ L for women, <75 IU/L for men. Histologically based studies have shown the prevalence of nonalcoholic fatty liver, defined as the presence of hepatic steatosis, ranging from 15 to 39%. 12,31 Hilden et al 32 found steatosis in 24% of 503 livers from car accident victims at postmortem examination; only 6 of the 120 cases had features of alcoholic hepatitis. Wanless et al 33 are credited with documenting the association of histological steatosis and steatohepatitis with obesity and diabetes. In an unselected autopsy series of 351 nonalcoholic patients, steatosis was noted in 70% of obese and 35% of lean patients; steatohepatitis was found in 2.7% of lean patients and 18.5% of markedly obese patients. Advanced fibrosis was greater in obese (13.8%) than in lean (6.6%) patients, and the difference was associated with the concomitant increased prevalence of diabetes. In a highly selected patient population, Charlton et al 34 documented histological NASH in 2.6% of 1207 native hepatectomies of patients with end-stage liver disease undergoing liver transplant. The increasing incidence of obesity, currently calculated to approach 40% of the U.S. population by the year 2025, and the attendant rise in insulin resistance and diabetes in up to 10% of obese subjects 35,36 raise serious concerns for occurrence and progression of liver disease due to NAFLD and NASH. Disconcertingly, prevalence rates for pediatric obesity and type 2 diabetes are also briskly increasing and show an imbalance favoring ethnic minorities in the United States. The full impact of liver disease in these children as they develop cirrhosis and complications of end-stage liver disease, including HCC, is predicted to be significant even if relatively unknown. 37 Other problems in assessing true prevalence of this marker-negative disease include the unusual patient settings. The first is cryptogenic cirrhosis. The recognition that NAFLD may be a source of a large number of cases of otherwise cryptogenic cirrhosis was introduced by Caldwell et al. 38 The true prevalence of this association is unknown, but the difficulties inherent in documenting both cryptogenic cirrhosis and NAFLD raise a further concern of underestimation of the prevalence. The second, broadly, is several unique clinical settings: nonobese, lean patients 39 ; subjects with normal ALT values, 30 varying forms of lipodystrophy, 2 and insulin resistance syndromes such as polycystic ovary syndrome and Bardet-Biedl and Alström syndromes in children. 37 Finally, various drugs and occupational toxins have been implicated in NASH. 40,41 Farrell s thorough article 41 details known and possible mechanisms related to drugs reported with phospholipidosis and NASH: mitochondrial injury and direct hepatotoxicity (coralgil, amiodarone, and possibly tamoxifen); aggravating underlying conditions of central obesity, hyperinsulinemia, or hyperlipidemia (tamoxifen, estrogens, corticosteroids); or exacerbation of fibrosis (methotrexate). Highly active antiretroviral therapy (HAART) for human immunodeficiency virus (HIV) has been associated with lipodystrophy and insulin resistance, but histological lesions, steatosis, cholestasis, and hepatocellular injury are not characteristic of NASH. 41 Furthermore, some reported cases may be spurious associations in which alcohol has not been adequately excluded or circumstantial in patients with other conditions of metabolic syndrome (calcium channel blockers). Farrell stresses that definitive histological studies are sparse. In his opinion, < 2% of NASH is drug induced. Estimates of NASH in NAFLD subjects in published series range from 2 to 5% 3,12,35 to 20 to 30%. 36 As discussed by some in the field, differing criteria for diagnosis of NASH are recognized but perhaps underappreciated 12 ; this has been implicated as a potential source of variances in study results. 4,5,42 As an example, one study found histologically confirmed NASH in 25% of 105 morbidly obese patients, 16 whereas another study documented NASH in 69.5% of 46 obese patients. 43 The value of liver biopsy evaluation for confirmation of the diagnosis of steatohepatitis is unquestioned. 44 Of equal importance, but less recognized, is the value of biopsy for exclusion of other causes of liver disease in the clinical setting of marker-negative enzyme elevations. 45 The study by Skelly et al 46 of 354 biopsies for unexplained liver test elevation showed that more than one third of biopsies had clinically meaningful yet previously undiagnosed features other than steatosis (seen in 34%) or steatohepatitis (seen in 32%). The former included subsequently confirmed autoimmune hepatitis, hereditary hemochromatosis, PBC, PSC, or sarcoid (which collectively accounted for 5.1%); 7.6% were drug-related injury; normal histology was seen in 5.9%; and 9% remained unexplained.

5 NONALCOHOLIC STEATOHEPATITIS/BRUNT 7 OVERVIEW OF PATHOGENESIS: CLINICAL The strong associations of NAFLD and NASH with obesity, various disorders that include insulin resistance, and the metabolic syndrome are documented in a growing body of literature. 36,37,44,47 53 Investigators have suggested that NAFLD and NASH are hepatic manifestations of the metabolic syndrome. 23,50 Clinicopathologic studies in patients with NASH, 51,52 as well as recent treatment trials with insulin-sensitizing agents, have confirmed that insulin resistance is a cause, not a result, of the liver disease. Biopsy studies have correlated clinical features of metabolic syndrome in patients with NAFLD or biopsy-proven NASH. In a study of 551 biopsies from morbidly obese patients, Marceau et al 47 noted an increased risk of steatosis with accrual of each of the clinical features of metabolic syndrome. Fibrosis (zone 3 perisinusoidal or portal based) correlated with steatosis, BMI, and WHR and showed a sevenfold increase with diabetes or impaired glucose tolerance. Cirrhosis correlated with steatosis, diabetes, and age. In a recent study of 304 nondiabetic patients with clinical NAFLD by Marchesini et al, 36 metabolic syndrome was present in 36% overall, with increasing prevalences found in overweight (29%) and obese patients (67%) compared with those of normal weight (18%); other features that were strongly associated were female sex and age. After adjustment for the latter two factors, fasting glucose > 110 mg/dl was the most predictive feature, and insulin resistance was also strongly associated with metabolic syndrome in these patients with NAFLD. Of 163 patients who also had liver biopsy, 51 had criteria of metabolic syndrome. NASH (defined as steatosis with the presence of fibrosis or hepatocyte ballooning) was diagnosed in 120 biopsies (73.6%) overall; the remainder had fatty liver. Although not associated with gender, age, or BMI, 38% of biopsyproven NASH subjects had criteria of metabolic syndrome compared with only 14% of those with fatty liver only. When comparing the 51 patients with metabolic syndrome to the 112 without, the former had more severe fibrosis. Significant fibrosis (stages 3 and 4) was noted in 33% of cases with metabolic syndrome, compared with only 15% of cases without criteria of metabolic syndrome; 41% of the latter had no fibrosis whereas only 12% of cases with metabolic syndrome lacked fibrosis. Steatosis and necroinflammatory grades did not correlate with classification of subjects into metabolic syndrome. PATHOGENESIS: LABORATORY RESEARCH It is clear that not all obese, hyperinsulinemic patients have NAFLD or NASH. The multifactorial and interrelated metabolic mechanisms and inflammatory mediators that link obesity, insulin resistance, and, in selected subjects, the end result of hepatic steatohepatitis with fibrosis are topics of intense investigation using a variety of animal models and sophisticated human metabolic studies. Recent concepts of the cellular, subcellular, and molecular pathways known or proposed to promote and perpetuate the cycle(s) of hepatocellular injury and fibrogenesis in NASH have been reviewed. 40,58 60 Broadly, hepatic steatosis is derived from a combination of factors: aberrations in insulin-related postprandial lipolysis with increased free fatty acid delivery to the liver, excess dietary carbohydrate resulting in de novo fatty acid synthesis in the liver, and impairment of mitochondrial b oxidation and the complex mechanisms of triglyceride assembly and export. Steatosis is both cause and result of pathways related to generation of reactive oxygen species (ROS), lipid peroxidation and oxidative stress, impaired mitochondrial respiratory chain function and adenosine triphosphate depletion, and production of the proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-a) A self-perpetuating cycle of insulin resistance and inflammation due to chronic activation of inhibitor kinase kappa beta (IKKb) and interactions with nuclear transcription factor NF k b has been inferred from animal models. 14,61 Studies have also shown the profibrogenic nature of hyperinsulinemia and hyperglycemia 62 and leptin. 63 Scientific observations serve as the basis for development of therapeutic agents in treatment trials such as insulin-sensitizing agents, lipid-lowering drugs, antioxidants, and obesity-related manipulations or lifestyle modifications that promote regulated weight loss. To date, treatment interventions have met with varied success. 2,44,64,65 Investigations into mechanisms of obesity have paralleled the recognition of its significant health impact. 66 To date, gut peptides known to have roles in the regulation of hunger and satiety by the central nervous system include cholecystokinin, gherlin, and PYY-36; redundancy in their actions appears to favor energy intake and accumulation for survivial. 67 Laboratory studies have shown that central, visceral adipose tissue is more significant than is peripheral fat in the risks of cardiovascular disease related to metabolic syndrome 61 ; the same is suggested in NAFLD. 14,68 Recent articles highlight current concepts from rodent and human studies for the known and putative links for visceral adipose tissue in deleterious manifestations, lipotoxicity, of metabolic syndrome. 61,69 71 Obesity 72 and metabolic syndrome 73 may be inflammatory conditions akin to atherosclerosis. 72 No longer considered inert tissue for storage of energy or excess free fatty acids, visceral adipose tissue is highly active as an endocrine organ and the source of numerous biologically active secretory products, collectively referred to as adipokines or adipocytokines. These products variably act in endocrine, autocrine, and paracrine fashions and include inflammatory cytokines and immune system molecules (TNF-a, interleukin-6, transforming

6 8 SEMINARS IN LIVER DISEASE/VOLUME 24, NUMBER growth factor-beta, alternate complement proteins); proteins that affect vascular tone, fibrinolysis, and vascular hemostasis (angiotensinogen, other proteins of the reninangiotensin system, and plasminogen-activator inhibitor); hormones that affect glucose homeostasis and energy regulation (adiponectin, resistin, and leptin); lipid and lipoprotein metabolism (free fatty acids, lipoprotein lipase, fatty acid transport protein, cholesteryl ester transfer protein, apolipoprotein E, retinal-binding protein); and endocrine and reproductive factors (corticosteroids and sex steroids). 69,70 The thiazolidinediones (TZDs) are a newer class of drugs effective in treatment of type II diabetes. Rosiglitazone and pioglitazone also differ structurally from troglitazone and have not been associated with similar severe liver damage. The mechanisms of these agents have been reviewed. 69,70,72 TZDs result in reduction of insulin resistance, increased glucose uptake in skeletal muscle and adipose tissue, and reduced hepatic glucose output. TZDs act through agonist binding to the nuclear factor peroxisome proliferator activated receptor g (PPARg). PPARg regulates adipose tissue differentiation (and endocrine function), affects lipid metabolism (free fatty acid disposition and the effects on insulin signaling), insulin sensitivity (and the effects of TNF-a), and inflammatory events through activation of regulatory genes in a variety of tissues including fat cells, endothelial cells, mononuclear cells, and vascular smooth muscle cells. PPARg are present but less abundant in liver, pancreas, and skeletal muscle, as well as in colon, breast, and prostate. Human and animal studies of TZDs have shown not only increased insulin sensitivity in muscle and liver but also redistribution of fat from metabolically active visceral adipose tissue, skeletal muscle, and pancreatic b cells to inert subcutaneous fat stores. Anti-inflammatory properties involved in atherogenesis are also shown for TZDs. TZDs have shown promise in human pilot trials in NASH. 54,55,57 Adipokines resistin, leptin, and adiponectin are under intense laboratory investigation in obesity and metabolic syndrome. 69 Adiponectin is currently seen as a central mediator of pathogenic processes in metabolic syndrome Adiponectin plasma concentrations are inversely related to obesity and positively correlate with increased insulin sensitivity, suppression of hepatic glucose production, and lessening of hyperlipidemia. Adiponectin, structurally similar to TNF-a, has opposite biological effects, specifically abrogation of anti-insulin and proinflammatory effects of TNF-a. 74 Recently, the role of adiponectin in a rodent model of steatohepatitis was reported. Xu et al 74 showed improvement of hepatic steatosis; a variety of biochemical markers; and insulin, glucose, and lipid metabolic parameters in both nonalcoholic steatohepatitis in an obese mouse model and in alcoholic hepatitis in ethanol-fed mice by adiponectin infusion. A recent publication of microarray analysis in liver biopsies from patients with NASH-related cirrhosis compared with other causes of cirrhosis and controls has provided evidence of altered expression in several genes related to insulin and mitochondrial function in patients with NASH 75 ; this is a new area of investigation that will likely expand in the future. Finally, just as not all individuals who consume large quantities of alcohol develop alcoholic liver disease, multiple host genetic factors and genetic polymorphisms are recognized as significant modifying factors relating to individual risk in the metabolic and inflammatory processes of NASH. 59,60 Potential candidate genes proposed include those associated with patterns of obesity; those that affect insulin sensitivity, hepatic lipid storage, and export; and those involved in fatty acid oxidation, oxidative stress, antioxidants, and cytokines. 59 HISTOPATHOLOGIC FINDINGS IN NAFLD AND NASH Clinical studies have shown correlations of clinical features of metabolic syndrome with histological features of NAFLD and NASH. 30,36,47 Laboratory research has identified or proposed underlying metabolic and inflammatory mechanisms that correlate with several of the significant lesions of NASH, including steatosis, lobular inflammation that includes polymorphonuclear (PMN) leukocytes, and fibrosis. 58,59 As treatment trials for NASH develop, detailed histopathologic analysis after treatment has become an accepted, if not expected, assessment in determining efficacy and potential toxicity and will likely provide additional insights. Alcoholic and Nonalcoholic Steatohepatitis Although the lesions of nonalcoholic steatosis, steatohepatitis, and end-stage cirrhosis share many lesions of alcoholic liver disease, contrary to the commonly stated theme in the literature, hepatopathologists recognize that NAFLD and NASH are not necessarily histologically identical to alcoholic liver disease. The initial descriptions of nonalcoholic steatohepatitis 6,33,76 79 noted the features that are similar to many of those seen in alcoholic steatohepatitis, including macrovesicular steatosis, hepatocellular ballooning, lobular inflammation that included a component of PMN leukocytes, zone 3 perisinusoidal fibrosis, and MH. There are lesions of alcoholic hepatitis not reported to date in nonalcoholic fatty liver diseases, however, 80,81 and they include sclerosing hyaline necrosis, the veno-occlusive lesion of alcoholic liver disease, ductular proliferation (ductular reaction), cholangiolitis, and acute cholestasis. Furthermore, Ludwig et al 76 and others 82 have commented that steatohepatitis with numerous MHs is more likely of alcoholic than of nonalcoholic origin.

7 NONALCOHOLIC STEATOHEPATITIS/BRUNT 9 The recognition that some cases may be difficult to distinguish from alcoholic steatohepatitis on the one hand and that steatohepatitis may be present as a result of metabolic disorder(s) of fatty acid metabolism and insulin sensitivity on the other provides a compelling impetus for revision of the pathologists nomenclature in diagnoses. A recommendation to discontinue the use of non association with alcohol and to conform to current practice in other forms of chronic hepatitis has been suggested. 2,4,5 In this manner, pathologists diagnoses reflect the presence of microscopic lesions and pattern of injury (steatohepatitis) and can state a clinical association if provided (i.e., obesity, diabetes, as examples). Indications of assessment of necroinflammatory activity and fibrosis may be additional components. Table 1 categorizes histological lesions of NASH and lesions that raise concerns of a separate liver disease. A review of the literature detailed the frequency of specific features noted by the initial studies in adults and in children. 5 As noted earlier, in the markedly increased numbers of clinicopathologic studies of NAFLD and NASH, the histopathologic lesions delineated for study and requirements for inclusion have shown significant variability in all histological aspects of NASH, including presence and amounts of individual features and requirements for inclusion of specific lesions (steatosis, ballooning, lobular inflammation, MH, perisinusoidal fibrosis) and presence of portal-based fibrosis in the absence of perisinuoidal fibrosis. To assess variability of lesions used for evaluating adult NASH, a survey of 10 international hepatopathologists who have published articles on the subject of NAFLD and NASH was electronically circulated. The survey included 23 histopathologic lesions and options for separating the lesions into categories of necessary, common but not sufficient or necessary, helpful but not sufficient or necessary, not common but may occur, and unusual. It was recognized from the outset that, as with any histopathologic analysis, the exercise of deriving a microscopic diagnosis comprises more than simple addition of features and that an overall pattern of injury is also important. The respondents also supplied comments as to personal requirements for a diagnosis of NASH, and a unifying theme was that NASH is a combination of lesions. Steatosis was noted as either a required feature or one of a choice of required features, along with ballooning. Table 2 summarizes the diagnostic requirements, and specific results are further discussed later. Noncirrhotic Steatohepatitis STEATOSIS In early studies of NASH, steatosis was reported in 100% of cases in published studies. 5 Two survey pathologists stated that steatosis was not a required feature and emphasized the importance of cell injury in the form of ballooning or MH. The type of steatosis that predominates in adults and in children is macrovesicular, with a single droplet that results in nuclear eccentricity because it occupies the entire cell cytoplasm or with several smaller but well-defined droplets within the cytoplasm. Special stains for fat are not required for microscopic detection of macrovesicular steatosis; studies that use oil red O for steatosis estimates often report much greater amounts than those found with standard hematoxylin-eosin (H & E) staining. On the other hand, it is not uncommon for there to be a mixture of hepatocytes with combinations of large single droplets and multiple small droplets. Clusters of hepatocytes with intracytoplasmic delicate septations due to microvesicular steatosis may also be present. Criteria for assessing steatosis, thirds, quartiles, or written descriptors, are not uniform in the literature. Further amounts of steatosis to be considered pathological are not known and yet would seemingly be a critical determination. An early autopsy study suggested that steatosis in small amounts may be present in otherwise normal, healthy hepatic parenchyma, and the finding increased with age. 32 The commonly accepted normal value for liver steatosis of 5% is based on lipid content measurements. 82 An opposing view is that even this small amount is questioned as normal based on a study with current imaging techniques that detected no steatosis in normal subjects. 83 Of the survey pathologists who noted a requirement for steatosis, the value considered necessary ranged from any amount to 15 to 30% minimum. Distribution of acinar lesions of steatohepatitis is frequently noted, as in alcoholic steatohepatitis, as zone 3 predominant, rather than nonzonal. 5,80 Of the survey respondents, 80% found zone 3 predominance to be necessary or common; 20% noted this as helpful. BALLOONING Hepatocyte ballooning, a feature denoting cellular injury, is considered a required feature of NASH in adults by 60% of surveyed pathologists. As with steatosis, the pathologists who do not require ballooning note that ballooning is one of three features to be considered in diagnostic criteria. Ballooning is a feature noted by Matteoni et al 13 that denotes progressive NAFL (types 3 and 4). The term applies to enlarged, swollen-appearing hepatocytes; the affected cells are often intermixed in areas of steatosis and, in classic steatohepatitis, in the perivenular regions. Hepatocellular ballooning is most commonly noted in regions of H & E detectable perisinusoidal fibrosis. Ballooned hepatocytes are most easily noted when they contain MH (either typical or poorly formed). Hepatocyte ballooning is a structural manifestation of microtubular disruption and severe cell injury 82 and is not unique to alcoholic or nonalcoholic steatohepatitis but is likely a representation of cells undergoing lytic necrosis. 84

8 10 SEMINARS IN LIVER DISEASE/VOLUME 24, NUMBER Table 1 Lesions of Steatohepatitis Necessarily Present Usually Present, but Not Necessary for Diagnosis May be Present but Not Necessary for Diagnosis Unusual for NASH; Consider Other Causes of Liver Disease Steatosis, macrovesicular > microvesicular; most commonly concentrated in acinar zone 3 Minimum quantity not known but at least > 5 10% Mixed, mild, lobular inflammation; scattered PMN leukocytes as well as mononuclear cells Hepatocellular ballooning; most apparent near steatotic liver cells, typically in zone 3 Zone 3 perisinusoidal fibrosis; eventually is component of central-portal bridging fibrosis Zone 1 hepatocellular glycogenated nuclei 1 þ granular periportal (zone 1) hepatocellular iron or mild panacinar sinusoidal lining cell iron by Prussian Blue Lipogranulomas in the lobules; of varying, but usually small, size Occasional acidophil bodies; occasional PAS-d Kupffer cells MH in Zone 3 hepatocytes; typically Purely or predominantly microvesicular steatosis poorly formed; may require antibodies to ubiquitin, p62, or CK 8 and18 to detect Very little macrosteatosis in a nonzonal distribution or zone 1 predominant Portal inflammation greater than lobular inflammation; lymphoid aggregates; numerous plasma cells Megamitochondria in hepatocytes Sclerosing hyaline necrosis; veno-occlusive lesions; perivenular fibrosis, phlebosclerosis Portal/periportal fibrosis in absence of, or markedly greater than, zone 3 perisinusoidal fibrosis; portal-based bridging fibrosis Fat cysts Lobular disarray and marked inflammation; endophlebitis; confluent or bridging necrosis Acute or chronic cholestasis; bile duct lesions, bile duct loss, or ductular proliferation (reaction) PAS-d globules in hepatocytes Epithelioid granulomas (Modified from Brunt 5 )

9 NONALCOHOLIC STEATOHEPATITIS/BRUNT 11 Table 2 Results of Survey of 10 Hepatopathologists* Lesions Steatosis Ballooning Lobular Inflammation MH Perisinusoidal Sinusoidal Fibrosis Zone 3 Accentuation All three þ þ þ Common Any two of three þ þ þ All three þ þ þ (CI or PAS-d KCs) Required Required þ or þ Common Required Required þ or þ Steatosis plus two of three Required þ (with or without MH) þ þ Required Required Required Steatosis plus two of three Required Required þ Common þ Required Common *The author wishes to thank the following pathologists, listed in alphabetic order, for their participation in the survey and their willingness to be included in this article (the table does not correspond with the order of names): Prithi Bhatal, M.D.; Alastair Burt, M.D.; Zachary Goodman, M.D.; Terry Gramlich, M.D.; Joel Greenson, M.D.; Julia Iezzoni, M.D.; Jay Lefkowitch, M.D.; Dina Tiniakos, M.D.; Kay Washington, M.D.; Ian Wanless, M.D. CI, chronic inflamation; PAS-d, periodic acid stain after diastase; KC, Kuppfer cell. Apoptotic cell death results in formation of acidophil bodies and is a recognized process in many forms of chronic progressive liver disease, including alcoholic liver disease. 85,86 Fibrogenesis has recently been shown to be stimulated by cultured hepatic stellate cells (HSCs) following engulfment of apoptotic hepatocytes. 87 In a recent study of human biopsies, Feldstein et al 88 used Tdt-mediated d UTP-x nick end labeling (TUNEL) assay and immunohistochemical markers for caspases 3 and 7 as markers of apoptosis and demonstrated that apoptotic hepatocytes not only are detectable in nonalcoholic steatohepatitis, but also outnumbered those in cases of alcoholic hepatitis or simple steatosis. Apoptosis also corresponded with histological and biochemical determinations of disease severity. Upregulated expression of Fas (death-receptor) ligand was shown in NASH cases, and the authors discussed putative mechanisms of the proapoptotic pathogenesis of NASH. This is an under-explored lesion in NASH that may receive further attention. Rarely, subacute necrosis and liver failure have been reported in histologically proven NASH 89,90 or previously unrecognized NASH cirrhosis 91 ; all seven reported cases were women. Two cases, reported as letters, were women without prior liver disease who developed liver failure following prednisolone therapy for systemic lupus erythematosis. 89,90 The Caldwell series 91 documented NASH-related cirrhosis in liver explants or autopsies of five obese women with a clinical course of subacute liver failure for whom no other etiology or prior diagnosis of liver disease had been made. INFLAMMATION Lobular inflammation is usually mild but is integral to the diagnosis of steatohepatitis; 50% of survey respondents noted this as a requirement, and 50% noted it as common. Studies of pathogenesis have delineated or proposed numerous proinflammatory cytokines involved in the progressive lipotoxicity of NAFLD and NASH. 40 A characteristic form of lobular inflammation in which PMN leukocytes are a component is mixed. In the early descriptions of NASH, PMN leukocytes were noted in 56 to 100% of cases in which this feature was specifically examined. 5 The survey results highlight an area of divergence among pathologists: 20% noted PMNs as a requirement, whereas the remainder were divided between common (40%) or helpful (30%); one respondent noted PMNs were uncommon. Results relating to chronic inflammation also varied from necessary (10%) to common (60%) to helpful (10%). One respondent noted this as uncommon. Other forms of lobular inflammation in steatohepatitis include small lipogranulomas, some with an associated eosinophil, microgranulomas, and single pigmented Kupffer cells. Portal inflammation either was not specifically addressed in several of the early studies of NASH or was noted as present in small amounts. 5 The survey results confirm that 80% of respondents consider the latter, but 20% noted portal inflammation as uncommon. Further, portal inflammation that is greater compared with lobular inflammation is considered not common (40%) or unusual (60%) and may represent concurrent inflammation of another origin, such as chronic hepatitis. The presence of steatohepatitis and other forms of liver disease, most specifically hepatitis C, is an area of active discussion not only for pathologists, but also for hepatology research involving pathogenesis of hepatitis C, treatment, and outcomes of affected subjects FIBROSIS Activated HSCs are a recognized source of fibrosis in the liver; activation of HSCs is characterized by expression of a-smooth muscle actin. Two immunohistochemical studies have demonstrated activated HSC in human

10 12 SEMINARS IN LIVER DISEASE/VOLUME 24, NUMBER tissue with NASH with differing results. 96,97 Washington et al 96 found positive HSC in zone 3 in 74 of 76 cases of steatosis or NASH, compared with none in controls; Cortez-Pinto et al 97 found HSC reactivity in zone 3 in all liver biopsies examined, including normal controls, with greater numbers of positive cells in the 15 NASH biopsies. Greater numbers of activated stellate cells were associated higher fibrosis scores in one study 96 but not in the other. 97 One study showed a trend toward increased numbers of HSC with increased steatosis, but no correlation with inflammatory grade, 96 whereas the other noted a significant correlation with lobular and portal inflammation, but no association with degree of steatosis. 97 The authors conclusions varied along with their results. One group concluded that HSC activation in NASH is correlated with steatosis and is significant in fibrosis. 96 The other group concluded that inflammation likely drives the fibrogenesis of NASH, but the immunohistochemical detection of zone 3 HSC is of questionable specificity for NASH because it has been documented in normal livers and in other forms of chronic liver disease. 97 Larger studies of this nature may further clarify these considerations. The necessity of fibrosis for a diagnosis in NASH is an area of discussion. Although it is considered common or helpful, 80% of respondents to the survey do not require fibrosis to be present for the diagnosis. The characteristic pattern of fibrosis that distinguishes noncirrhotic steatohepatitis (alcoholic and nonalcoholic) from other forms of chronic liver disease is the initial deposition of collagen in perisinusoidal spaces in acinar zone 3; 20% of respondents require perisinusoidal fibrosis, whereas 80% find it common. When dense, the fibrosis may be observed by H & E, but in all cases it is readily detected with specific histochemical stains for collagen, such as Masson trichrome. A source of inconsistency in observations of fibrosis in published studies of a variety of liver diseases is derived from the different uses of histochemical stains to detect collagen; Sweet reticulin and picrosirius red stains highlight more types of collagen that are present in the sinusoids of the liver than the Masson stain, and use of these stains may result in observation of greater involvement than detected by Masson. With progression, periportal fibrous spurs may be noted; bridging fibrosis and cirrhosis may eventually develop. With extensive bridging fibrosis, cirrhosis, and architectural remodeling of the parenchyma, perisinusoidal fibrosis may no longer be present. Portal-based fibrosis in the absence of zone 3 perisinusoidal fibrosis is considered not common (20% of respondents) or unusual (80% of respondents) in adult NASH. The use of portal-based scoring systems may account for differences in clinicopathologic studies; a modification of METAVIR portal-based fibrosis score to account for zone 3 perisinusoidal fibrosis was used in a recent study. 62 Finally, although perisinusoidal fibrosis is often associated with active lesions of steatohepatitis as described earlier, zone 3 perisinusoidal fibrosis in the absence of active lesions may be indicative of prior episodes of steatohepatitis. MALLORY S HYALINE The intracellular ropy inclusions known as MH, a recognized histological hallmark of alcoholic hepatitis, are also noted in zone 1 hepatocytes in chronic cholestatic liver diseases, copper toxicity, and certain drugs (griseofulvin in mice and phospholipidosis-associated amiodarone toxicity) and in hepatocellular proliferative lesions, focal nodular hyperplasia, and HCC. 82,98 In alcoholic hepatitis, MH is most commonly found in zone 3 in ballooned or apoptotic hepatocytes; in nonalcoholic steatohepatitis, MH may be poorly formed cytoplasmic aggregates noted in zone 3 ballooned hepatocytes. 6,99 MH is chemotactic, and thus affected hepatocytes may be rimmed by neutrophils (satellitosis). In the study by Matteoni et al, 13 MH was a lesion that delineated types 3 and 4 from types 1 and 2; other published studies of NASH have shown that MH is more commonly noted in higher grades of steatohepatitis 100 and is often present in foci of hepatocytes involved by perisinusoidal fibrosis. Abundant MH is strongly suggestive of alcoholic hepatitis. 82,99 Early studies of NASH reported MH from zero (in pediatric cases) to 90% frequency in one series; most series reported MH in 10 to 70% of cases. 5 MH is unquestionably useful for a diagnosis of steatohepatitis when present. Survey pathologists and published series agree, however, that the lesions of MH are not required, and, by definition, absence of MH does not mitigate against the diagnosis. As noted in Table 3, MH has been identified variably as one of two required lesions (along with either steatosis or perisinusoidal fibrosis) or, in combination with steatosis, as one of three lesions required (zone 3 mixed inflammation, ballooning, pericellular fibrosis). Extensive literature exists regarding formation and significance of MH. 98,101 Rather than representing passive collapse of cytoplasmic structures, concerted active metabolic pathways are required for formation of the hyperphosphorylated, ubiquitinated, abnormally folded intermediate filaments cytokeratin (CK) 8 and 18 that are associated with heat shock proteins. 98 p62 is a protein product of an early response gene that associates with MH; this association is significant in the hepatoprotective nature of sequestration of the abnormal proteins into biologically inert inclusions. 101 One study has shown elevated serum levels of ubiquitin in NASH patients compared with controls, and this marker was proposed as an indication of cytoprotection against oxidative stress rather than a marker of inflammation in NASH. 102 Antibodies to ubiquitin, CK 8 or 18, and

11 NONALCOHOLIC STEATOHEPATITIS/BRUNT 13 Table 3 Pediatric Pathology: Review of the Literature Reference n Male:Female Ballooning/MH Inflammation Fibrosis Schwimmer et al. Hepatology 2002;36:412A Molleston et al. Am J Gastroenterol 2002;97;2460 Rashid et al. J Pediatr Gastroenterol Nutr 2000;30:48 Manton et al. Med J Austral 2000; 173:476 Baldrige et al. J Pediatr 1995;127:700 Moran et al. Am J Gastroenterol 1983;6: :13 5%/2% Portal>>Lobular Portal only: 70% Psf: 19% 2 (3 bxs) 2:0 / Portal ¼ lobular in Mild, NOS (1) noncirrhotic Cirrhosis (2) 36 (bx in 24) 21:15 /0 Not specified (21/24) None: 25% Portal, mild: 42% Portal, moderate severe: 30% Cirrhosis: 4% (1 case) 17 11:6 /0 Present in 8; 7 were portal Present in 53% Portal only/bridging: 35% Psf: 18% 14 10:4 /1 Portal: all Lobular: Portal: 100% Psf: 21% mild in some 3 2:1 / Foci of acute: 1 Portal only: 66% Portal, chronic: 1 indicates this lesion was not mentioned psf, perisinusoidal fibrosis, bx, biopsy. p62 are useful for confirmation or identification of MH by immunohistochemistry. OTHER LESIONS OF NASH Megamitochondria may be observed in hepatocytes in NASH; the relative frequency of this finding has not been rigorously studied. Megamitochondria appear as rounded or needle-shaped intracytoplasmic inclusions. When rounded, they are approximately the size of a red blood cell. In one study of alcoholic hepatitis, megamitochondria were identified in combination with mixed steatosis as a marker of progression to fibrosis. 103 Ultrastructural studies of human liver in NAFLD and NASH have documented a variety of structural lesions, including paracrystalline inclusions, loss of cristae and multilamellar membranes. 104,105 As opposed to alcoholic liver disease, however, mitochondrial DNA deletion is uncommon in NASH. 104 Caldwell et al 104 speculate that megamitochondria are a manifestation of either cell injury or adaptation. Granular iron pigment detected in hepatocytes (typically graded as 1 þ to 2þ) sinusoidal lining cells with Perls histochemical stain are considered an unusual feature by survey pathologists; this may be a reflection of the practice of routine evaluation of liver biopsies with iron stain. Iron deposition in NASH has been reported in studies that evaluated results specifically for it: 15%, 6 18%, %, 106 and 55%. 39 One study showed positive correlation of increased iron detected by stains or biochemical determination with increased portal-based fibrosis. 106 The significance of iron deposition, aberrant iron genetics, and development of fibrosis are ongoing topics of debate Glycogenated nuclei are well-recognized in pediatric liver tissue, as well as Wilson disease, diabetes, and NASH. One study found 10 to 15% glycogenated nuclei in biopsies with alcoholic hepatitis compared with 75% in biopsies from NASH patients; there was an association with diabetes and obesity. 110 In summary, pathologists agree that adult NASH is characterized by a combination of lesions in which steatosis, ballooning, lobular inflammation, and perisinusoidal fibrosis are the primary components of interest. For some, a zone 3 predominance of the lesions is emphasized. Minimal criteria for diagnosis vary in terms of the specific combination of lesions (see Table 2). A consideration not often discussed is that variances in histological lesions may actually reflect variances in the individual patient s metabolic status at the time of the biopsy. This area may receive further consideration because current studies in NASH are including more detailed studies of insulin resistance related parameters. HISTOLOGICAL RESOLUTION Current standards for modern therapeutic trials in NASH include histopathologic evaluation for entry into the trial. Most trials also require histological comparisons of a posttreatment biopsy as one of the measures of efficacy. One benefit of this practice is characterization of histological features that may represent resolution of NASH. Neuschwander-Tetri et al 54 recently reported detailed clinicopathologic results of a 48-week treatment trial of rosiglitazone in 30 subjects with NASH. The biopsies were reviewed in a single group in a randomized, coded fashion for adequate blinding. In addition

12 14 SEMINARS IN LIVER DISEASE/VOLUME 24, NUMBER to biochemical improvement in the patients, histological features that showed improvement included decrease in global activity grade, either improvement or no change in the components of grade (steatosis, ballooning, lobular inflammation), loss of zone 3 accentuation, and a shift in proportion of lobular and portal inflammation toward a relatively greater portal component. Fibrosis scores did not change, but zone 3 perisinusoidal fibrosis changed from a qualitative assessment of dense to delicate in 33% of cases. Reported histological results of early treatment trials in NASH have varied, likely because of differences in patient selection, agents used, and treatment protocols. 2,44,64,65 Results range from improvement in steatosis, inflammation, and fibrosis with betaine 111 ; improvement in steatosis in some cases with ursodeoxycholic acid but no change in steatosis, inflammation, or fibrosis with ursodeoxycholic acid or clofibrate 112 improved but persistent steatohepatitis, with no change in fibrosis with troglitazone 57 ; and improvement in steatosis with increased portal inflammation and fibrosis in weight loss. 113 The growing numbers of treatment trials will likely increase protocol uniformity and specific histological features to be examined. HISTOLOGICAL FEATURES OF PROGRESSION Numerous clinicopathologic studies have examined markers for prognosis in patients with NASH. An early study identified age, obesity, diabetes and AST to ALT ratio greater than 1 as markers of advanced fibrosis. 114 Two recent studies derived scores from clinical and laboratory values to predict fibrosis, 115 the BAAT (BMI, ALT, Age, Triglycerides) score or the presence of NASH 116 and the HAIR (Hypertension, ALT, Insulin Resistance) score in obese subjects. Studies of prognostic lesions for advanced fibrosis in NASH have reported variable findings; this may be related to inclusion criteria and variances in methods of assessment. Findings that have been reported to statistically correspond with advanced fibrosis have included MH but not amounts of steatosis or lobular inflammation 114 ; lobular inflammation, steatosis grade, and stainable iron 106 ; steatosis amounts and MH, but not inflammation 117 ; steatosis amounts, MH, and lobular inflammation with PMN leukocytes 115 ; amount of steatosis and inflammation. 43 Interestingly, Evans et al 118 failed to detect any progression of fibrosis in repeat biopsies of seven patients with NASH after mean follow-up of 8.2 years. GRADING AND STAGING IN NASH Pathologists are currently familiar with the concepts of evaluating chronic viral hepatitis for necroinflammatory grade and for fibrosis stage. Methods currently in use for diagnostic practice and for clinical studies vary in some details but are based on assessments of portal inflammation and interface activity, lobular necroses, and portalbased fibrosis. 119 Matteoni et al 13 based their clinically relevant definitions for four categories of fatty liver disease on the presence of specific histological lesions, as noted earlier. The system does not, however, include assessments of severity or acinar localization of lesions. A proposed grading and staging system for NASH was developed to reflect the significant lesions, to provide a method of deriving a semiquantitative global activity grade from a combination of the lesions, and to recognize the pattern of fibrosis. 100 The system is summarized in Table 4. The Pathology Committee of the NASH Clinical Research Network will present a system for analysis of NASH biopsies prepared for treatment trials within the Clinical Research Network at the 2003 American Association for the Study of Liver Disease meeting. PEDIATRIC NAFLD AND NASH The rising incidence of NAFLD and NASH in children has paralleled the dramatic increase in obesity in this age group. Lifestyle changes for American children, including increased use of computers, sedentary forms of entertainment, and dietary exposure to high fats and refined sugars have all been implicated. 37 Recent studies have brought to light the magnitude of the problem as well as the potential for the future burden of liver disease in affected subjects. Roberts 37 has reviewed current clinical correlations and metabolic considerations in pediatric NASH. Distinctive findings are male predominance and strong association with Hispanic ethnicity and with insulin resistance. An insulin resistance associated cutaneous disorder, acanthosis nigricans, characterized by darkening in flexural areas, the posterior neck folds, axillae, and groins, is a clinical feature described in series of pediatric NAFLD. Table 4 summarizes the histological lesions reported in published biopsy series from North American and Australian pediatric centers and highlights variances with in adult subjects. These include more severe steatosis, little or no ballooning or MH, less lobular inflammation, little to no involvement with PMN leukocytes, and more portal-based chronic inflammation. Portal fibrosis without concomitant perisinusoidal fibrosis and the absence of perisinusoidal fibrosis are commonly reported. A subtler feature not often mentioned in pediatric series is the apparent lack of zone 3 predominance, which is where the combination of lesions of steatosis, ballooning, and MH are most commonly noted in adult biopsies. Anecdotal experience with pediatric NASH suggests that some cases may more closely resemble adult histology than in published articles.

13 NONALCOHOLIC STEATOHEPATITIS/BRUNT 15 Table 4 Grading and Staging the Histopathologic Lesions of NASH GRADE 1, MILD Steatosis: predominantly macrovesicular, involves < 33 up to 66% of the lobules Ballooning: occasionally observed; zone 3 hepatocytes Lobular inflammation: scattered and mild acute (polymorphs) inflammation and occasional chronic inflammation (mononuclear cells) Portal inflammation: none or mild GRADE 2, MODERATE Steatosis: any degree and usually mixed macrovesicular and microvesicular Ballooning: obvious and present in zone 3 Lobular inflammation: polymorphs may be noted associated with ballooned hepatocytes, pericellular fibrosis; mild chronic inflammation may be seen Portal inflammation: mild to moderate GRADE 3, SEVERE Steatosis: typically > 66% (panacinar); commonly mixed steatosis Ballooning: predominantly zone 3; marked Lobular inflammation: scattered acute and chronic inflammation; polymorphs may appear concentrated in zone 3 areas of ballooning and perisinusoidal fibrosis Portal inflammation: mild or moderate Steatosis: grade 1 ¼ 0 33%, 2 ¼ 33% 66%, 3 ¼ >66% Ballooning: zonal location noted and severity (mild or marked) recorded according to estimate of numbers of hepatocytes involved Lobular inflammation: 0 3 based on observations of foci per 20 field; 1 ¼ 1 2 foci, 2 ¼ up to 4 foci, 3 ¼ > 4 foci. In addition, cell types (acute or chronic) and location were noted Portal inflammation: 0 3, 1 ¼ mild, 2 ¼ moderate, 3 ¼ severe STAGING FIBROSIS IN NASH Stage 1: Zone 3 perivenular perisinusoidal/pericellular fibrosis, focal or extensive Stage 2: As above with focal or extensive periportal fibrosis Stage 3: Bridging fibrosis, focal or extensive Stage 4: Cirrhosis (Modified from Brunt 5 ) Compared with adult NASH, the experience in pediatric NASH is still relatively small; studies to date, however, indicate that application of histological criteria used for adults may not be appropriate. Further documentation of pediatric NASH histology, as well as possible explanations for the differences, will likely evolve from the increased research in this area. STEATOHEPATITIS-RELATED CIRRHOSIS The biopsy findings of cirrhotic NAFLD and NASH are another area of investigation in pathology. Abdelmalek et al 120 documented that biopsy-proven NASH that has progressed to cirrhosis may no longer retain diagnostic histopathologic features of steatohepatitis in follow-up biopsy, which provides support for the concept that NASH may result in burned-out cirrhosis for which no histological hallmarks remain. Caldwell et al 38 are credited with the initial study suggesting that NAFLD may account for a large percentage of otherwise cryptogenic cirrhosis based on clinical parameters related to NAFLD. Others have speculated that histological evidence of NAFLD and NASH in allograft livers of patients transplanted for cryptogenic cirrhosis likely represents disease recurrence. 121 It is increasingly common in the literature for cryptogenic cirrhosis, with or without biopsy, to be attributed to NAFLD or NASH. At this time, unfortunately, this is an area in which definitional variances play a remarkable role in study results. For example, if prior biopsy-proven NASH were known, by definition the subsequent cirrhosis would not be cryptogenic. On the other hand, there may yet be undiscovered or undetected viral infections, autoimmune or metabolic diseases that may result in cirrhosis but are currently grouped under the rubric of cryptogenic. Practicing pathologists are also aware that cases of NASH cirrhosis may retain some or all of the characteristic histological features. Shimada et al 122 documented that all of the lesions of active steatohepatitis were present in each of six cirrhotic livers that also harbored HCC. Hui et al 123 stressed the importance of histological criteria in categorizing cases of NASH cirrhosis. This group proposed criteria for pathologic interpretation of cirrhotic livers with NASH: the categories were definite (steatosis with mixed lobular inflammation), probable (steatosis with only chronic inflammation),

14 16 SEMINARS IN LIVER DISEASE/VOLUME 24, NUMBER possible (with either steatosis only or mixed inflammation only), and cryptogenic (no steatosis or inflammation). In their 23 cases of NASH cirrhosis, 20 were definite and 3 were probable; ballooning, not a required feature, was noted in 91% of cases and residual perisinusoidal fibrosis was found in 78%. The frequency with which loss of each of the active lesions occurs has not been characterized because of the lack of necessary follow-up histological material to do so. Mechanisms for loss of specific histological lesions with progression of fibrosis and architectural remodeling are also only speculative at the current time. As the experience with NAFLD and NASH continue, the histological lesions of cirrhotic end-stage disease will likely become more adequately analyzed. HEPATOCELLULAR CARCINOMOA IN NASH The increased mortality from malignancies in multiple organ systems because of concurrent obesity has recently been calculated to be 14% for men and 20% for women. 124 A review of the United Network for Organ Sharing (UNOS) Database of 19,271 explant hepatectomies found obesity as an independent factor for the prevalence of HCC in cirrhosis due to alcoholic or cryptogenic cirrhosis but not other known etiologies of chronic liver disease and cirrhosis. 125 The documentation of HCC in NAFLD and NASH has only recently been reported. Studies have documented a range of 18% 126 to 27% 127 incidence of HCC in cryptogenic cirrhosis and cryptogenic cirrhosis in obese patients (but not lean patients with cryptogenic cirrhosis), respectively. Both groups noted this is similar to the incidence of HCC in hepatitis C related cirrhosis. In both studies, NAFLD was the inferred underlying pathogenesis of the cryptogenic cirrhosis based on wellcharacterized associated clinical features of metabolic syndrome. Shimada et al 122 reported that from 82 cases of biopsy-proven NASH, 6 of 13 patients with NASHrelated cirrhosis had HCC (7.3% of total cohort; 47% of patients with cirrhosis). This is in contrast with the Hui et al 123 study of 23 patients with biopsy-proven NASH related cirrhosis in which no cases of HCC occurred during the 10-year observation period, compared with 17% incidence in hepatitis C related cirrhosis. Whether the necroinflammatory lesions and fibrosis that result in cirrhosis are primarily implicated in progression to malignancy or the underlying factors related to obesity or metabolic syndrome, or both, may account for neoplastic transformation are currently under investigation. Hyperinsulinemia was associated with increased mortality due liver carcinoma but inversely related to mortality from gastric, oral, and laryngeal cancers in a prospectively studied cohort of nondiabetic men. 128 In ob/ob mice, an obesity animal model with genetic leptin deficiency, obesity-related hepatic steatosis and insulin resistance, hepatocyte hyperplasia, and decreased apoptosis have been documented in enlarged fatty livers without fibrosis or cirrhosis. 129 The investigators speculated that these results support considerations that factors related to obesity and associated insulin resistance may promote hepatocyte proliferation even in the absence of cirrhosis and, further, that the changes may represent a premalignant condition. CONCLUSIONS Significant work detailing the complexity of pathophysiology of NAFLD and NASH has been published in the past 10 years. Insulin resistance is currently recognized as a significant underlying mechanism that promotes and is promoted by the cascade of inflammatory events involved in hepatic steatosis and subsequent steatohepatitis. The role of central adiposity, products of adipose tissue, and metabolic aberrations of fatty acids are being elucidated. In spite of these scientific advances, clinical determination of steatohepatitis continues to rely on liver histology. Biopsy evaluation continues to play a central role in clinical trials as well. Although most of the histopathologic lesions of steatohepatitis have been agreed on, the qualitative amounts and acinar localization remain foci of interest in pathology in adults, in pediatric cases, and in cirrhosis. Because the clinical interest in NAFLD and NASH will only continue to grow, pathologists can anticipate abundant material to study. 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