Section 2 -

Moderate Steatosis and Steatohepatitis Neil Theise, M.D. Romil Saxena, M.D.

Case 2A 33 year old woman with morbid obesity. Liver biopsy during bariatric surgery. Diagnosis: Moderate steatosis and steatohepatitis, compatible with obesity-associated fatty liver disease. Case 2B 22 year old woman with morbid obesity. Liver biopsy during bariatric surgery. Diagnosis: Moderate steatosis with steatohepatitis and extensive steatofibrosis consistent with obesity-associated fatty liver disease. Case 2C 28 year old man with morbid obesity. Liver biopsy during bariatric surgery Diagnosis: No pathologic change Comments: Terminology The term NASH (non-alcoholic steatohepatitis) was coined in 1980 by Ludwig to describe biopsy findings resembling those seen in chronic alcohol-related liver disease in patients who did not drink. While the hepatic lesions associated with obesity and diabetes, and their similarity to alcoholic liver disease had been recognized for a long time, this landmark publication highlighted these findings and named a "hitherto unnamed disease". Recent improvement in our understanding of the pathophysiology of non-alcoholic fatty liver disease (NAFLD) has brought into question the appropriateness of the term NASH, as it defines a disease by what it is not. On the other hand, it has become increasingly clear that in most individuals, NAFLD is a metabolic syndrome in which obesity, diabetes, hyperinsulinemia and insulin resistance (metabolic syndrome or syndrome X) play an important role. The wide range of histological findings of non-alcoholic fatty liver disease ranging from steatosis to steatosis with inflammation and fibrosis, with variable progression from one stage to another, have further complicated the use of uniform terminology. Currently, two terms dominate medical literature, NAFLD (non-alcoholic fatty liver disease) and NASH. The former is an all-inclusive term encompassing the entire range of histological findings, while NASH is restricted to those cases that show inflammation and fibrosis. The surgical pathologist faces a third scenario of steatosis with fibrosis and no inflammation; "steatofibrosis" is often used for this situation. The advantage conferred on the surgical pathologist to visualize the changes of NAFLD, allows better terminology in the diagnostic report. We therefore use the following terms in our pathology reports: Steatosis - with an assessement of the extent of steatosis Steatohepatitis - reflects additional changes of cellular injury namely, Mallory hyaline, inflammatory infiltrate Steatofibrosis - reflects the distinctive pericellular fibrosis seen in NAFLD Prevalence: With an increasing trend towards obesity in both children and adults, and a projected incidence of 40% in adults by 2025, 10% of whom will have insulin resistance and diabetes, the focus on NASH is increasing. The difficulty in estimating the true incidence or prevalence of NAFLD arises from the following facts: 1) The disease is silent in the majority of patients and may only be discovered during routine medical examination (slightly elevated ALT) or investigation for other medical conditions (abnormal liver on imaging). 2) There are no diagnostic laboratory tests (no gold standard). LFTs are neither sensitive nor specific for NAFLD 3) Imaging studies similarly lack sensitivity and specificity for diagnosis of fatty liver disease. 4) There is uncertainty about the minimal histological criteria necessary for diagnosis of NAFLD. This reflects uncertainty regarding the natural history and progression of findings on the lower end of the spectrum, like minimal steatosis. 5) The diagnosis requires the exclusion of alcohol as an etiologic agent, and there is disagreement on what constitutes medically acceptable alcohol use. Furthermore, there is wide variability in individual metabolism of alcohol. 6) Studies on prevalence and incidence use variable criteria for diagnosis of NAFLD, as well as variable levels of alcohol use. With these limitations in mind, prevalence of NAFLD is either based on imaging studies, biochemical studies or autopsy studies on selected or unselected populations; the former have focused on patients most at risk, e.g. patients with diabetes or obesity. Two large population-based studies used data from 15,000 subjects in the third US National Health and Nutrition Examination Survey (NHANES III, 1988-1994). One group (Ruhl and Everhart) looked at non-diabetic patients with ALT >43 U/L, no clinically documented liver disease and alcohol consumption of not more than 2 drinks/ day. ALT was raised in 2.8% of adult non-diabetic subjects; of these 65% were overweight (BMI > 25 kg/m2) or obese (BMI >30kg/ m2). Other features associated with increased ALT in this analysis included younger age (39 yrs vs 45 yrs), male gender, Mexican American ethnicity, impaired fasting glucose, increased waist to hip ratio, increased fasting serum leptin, triglycerides and insulin levels. Forty subjects with increased ALT fit criteria for the metabolic syndrome which is defined by truncal obesity, hyperglycemia, low levels of HDL, hypertriglyceridemia and hypertension. A second study (Clark et al) used the same database but applied different criteria to identify patients with NAFLD: AST > 37 IU/L and ALT >40U/L for men, AST and ALT > 31 IU/L for women. They excluded patients with transferrin saturation > 50%, positive viral markers and alcohol consumption of > 1 drink per day for women and 2 drinks per day for men. The study included diabetic patients. NAFLD, defined by unexplained elevation in transaminases, was present in 5.5% of the population. The prevalence of NAFLD in studies based on histological examination varies, partly because of the varied populations studied and the varying criteria used for diagnosis. Hilde found steatosis in 24% of 503 livers from car accident victims at postmortem, of these 6 of 120 cases had features of alcoholic hepatitis. Wanless, in an autopsy series of 351 non-alcoholic patients, found steatosis in 70% obese and 35% lean patients; steatohepatitis in 18.5% obese and 2.7% lean patients, and fibrosis in 13.8% vs 6.6% obese and lean patients respectively. Charlton found NASH in 2.6% of 1207 hepatectomies with end-stage liver disease at transplantation. In a biopsy study for unexplained enzyme elevations, steatosis was found in 34% and steatohepatitis in 32% of 354 biopsy specimens. Steatosis was found in 14.5% of healthy donors for live related liver transplantation without enzyme elevations (Marcos, 2000). Conditions Associated with NAFLD (a non-comprehensive list of the commoner associations) Obesity Diabetes mellitus Hyperlipidemia Metabolic syndrome X Rapid weight loss (jejuno-ileal, gastric bypass) Total parenteral nutrition Drugs (Amiodarone) Etiopathogenesis Insulin resistance and hyperinsulinemia are central to the etiopathogenesis of NAFLD. In the liver, insulin promotes glycogen synthesis, and inhibits gluconeogenesis as well as mitochondrial β-oxidation of fatty acids. In peripheral tissues, insulin promotes lipogenesis and inhibits lipolysis. Insulin resistance is associated with a failure to suppress lipolysis, thus increasing the delivery and uptake of fatty acids in the liver. The net effect is to increase metabolic turnover of fatty acids either by mitochondrial β-oxidation, packaging and secretion into VLDL or storage in the liver as triglycerides. Free fatty acids in blood further worsen insulin resistance. Both free fatty acids and TNF-α are increased in obesity and both down regulate IRS-1 (insulin receptor substrate-1). IRS-1, which is activated by tyrosine phosphorylation, promotes translocation of the glucose transporter protein GLUT4 to the cell membrane where it facilitates uptake of glucose from blood. Both free fatty acids and TNF-a hinder phosphorylation, and therefore the activity of IRS-1. TNF-alpha is released from adipose tissue in the absence of inflammation or infection. The development of steatohepatitis requires factors additional to those that cause fatty liver - a "second hit". This second hit is mediated by oxidant stress and production of free radicals that cause lipid peroxidation within hepatocytes. The P450 cytochrome system, TNF, hepatic iron stores, and lipoxygenase, are all potential sources of free radicals. Fatty acids themselves induce the Cyp2E1 enzyme. In addition, the increased mitochondrial β-oxidation of fatty acids is a source of free radical injury. Fatty acids also exert a direct deleterious effect on hepatocytes, which explains the existence of myriad mechanisms to guard against excess accumulation of free fatty acids by binding, transforming, catabolizing and exporting free fatty acids. These mechanisms include FFA binding proteins, triglyceride synthesis, secretion as VLDL, mitochondrial β-oxidation and enzymatic removal of lipid peroxidation products. The Role of the Liver Biopsy in NAFLD In the absence of non-invasive markers with acceptable predictive and discriminative value for the diagnosis of NAFLD, liver biopsy offers the only means of making a definitive diagnosis. The clinical diagnosis of NAFLD is suspected in individuals with asymptomatic elevations of liver enzymes, increased echogenicity on ultrasonography, unexplained hepatomegaly, or the presence of diabetes, obesity or dyslipidemia. Exclusion of other causes of chronic liver disease, like viral, autoimmune, metabolic and genetic, is a necessary step in the clinical diagnosis of NAFLD. Elevations of liver enzymes are minimal and non-specific, and may be entirely absent in some cases. Imaging studies are similarly non-discriminatory, and cannot accurately detect hepatitis and fibrosis. Fibrosis, including septal fibrosis and cirrhosis, is present in a significant number of NAFLD patients in the initial biopsy. Therefore, besides being a diagnostic tool, the liver biopsy provides valuable prognostic information as well. Pathology Although the finding of steatosis was initially considered to be important for diagnosis of NASH, the greater awareness of this condition and the finding of NASH as a cause of end-stage cryptogenic cirrhosis, ahs led to a re-evaluation of the necessity of requiring steatosis for a diagnosis of NASH. It is now considered that steatosis is not an absolute necessity as long as there are other features of cellular injury like ballooning injury or MH. Steatosis when present is macrovesicular or large-droplet fat; the presence of exclusively or predominantly microvesicular fat should prompt exclusion of other pathologic processes. At least 5-10% of fat with a predominant zone 3 distribution is indicative of NAFLD. Ballooning degeneration is a useful, and in the opinion of most pathologists, a necessary feature for diagnosis of NASH. Ballooning change consists of enlarged hepatocytes that are usually intermixed with areas of steatosis and perisinusoidal fibrosis. They are a manifestation of cellular injury and microtubular disruption. Lobular inflammation is an integral part of the pathology of NASH. Since the term arose by extrapolation from alcohol-related liver disease, it was necessarily assumed that neutrophils should form an integral part of NASH. However, with increasing experience, it has become evident that neutrophils are not the only inflammatory component present in patients with NASH; the inflammatory component very often consists of lymphocytes, with or without neutrophils. In fact, portal inflammation with lymphocytes is the predominant component of NASH in children. Experts in the field are not unanimous about the requirement or the frequency of PMNs or lymphocytes in NASH. If portal inflammation is in excess of lobular inflammation, it is imperative to rule out concurrent liver disease, eg hepatitis C. The presence of perisinusoidal and perivenular fibrosis is an important diagnostic feature of NAFLD. This type of fibrosis is present predominantly in zone 3. However, it has been increasingly observed that NAFLD is also associated with portal fibrosis which may show thin incomplete radiating septa. Portal fibrosis predominates over perisinusoidal fibrosis in pediatric NAFLD. NAFLD in Children The incidence of NASH in children is increasing in concert with a sedentary lifestyle and exposure to diets rich in fats and refined sugars. As in adults, pediatric NAFLD is associated with obesity, hyperinsulinemia, insulin resistance and the metabolic syndrome. The exclusion of alcohol as an etiologic factor is a lesser problem in this group of patients, especially if adolescents are not included. Pediatric NAFLD shows male preponderance and strong association with Hispanic ethnicity. Histological differences with adults include more severe steatosis; little or no ballooning, Mallory hyaline, or neutrophils. In addition, pediatric NAFLD shows chronic portal inflammation in preference to lobular inflammation. Fibrosis occurs predominantly in a portal and periportal distribution, and central perisinusoidal distribution is seen less often than in adults. There is also a lesser incidence of centrilobular distribution of NAFLD changes like steatosis, ballooning and Mallory hyaline. Grading and Staging the Histopathologic Lesions of Nonalcoholic Steatohepatitis Brunt EM Seminars in Liver Disease 2001; 21: 3-16 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 to moderate 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 Suggested Reading: Adams LA, Sanderson S, Lindor KD et al. 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