THE EPIDEMIOLOGY, PATHOGENESIS AND HISTOPATHOLOGY OF FATTY

LIVER DISEASE

Adam P Levene and Robert D GoldinDepartment of Histopathology,

Imperial College Faculty of Medicine at St Mary’s Hospital,London.UK(Histopathology 2012, 61, 141-152)

INTRODUCTION

• Fatty liver disease consists of alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), with a subset of these groups developing alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatits (NASH), respectively.

• The increasing prevalence of obesity and diabetes, and the associated metabolic syndrome is leading to an increased incidence of NAFLD.

• The increasing prevalence of NAFLD is particular worrying because patients appear to have a higher mortality from non-liver related and recent data have also highlighted an increased risk for cardiovascular disease.

• Underlying pathogenesis of NAFLD is still incompletely understood.

• Majority of patients with NAFLD have a stable disease, with isolated steatosis and an indolent course with no progression to advanced liver disease or clinical sequelae.

• However, a subset develop NASH and are at risk progressive fibrosis, cirrhosis and liver failure, or hepatocellular carcinoma (HCC).

• The distinction between isolated steatosis and NASH is important, as their prognoses and management are different

• The gold standard for identification of patients with NASH is liver biopsy.

EPIDEMIOLOGY AND DISEASE NATURAL HISTORY

NAFLD:

• Estimates of NAFLD prevalence based on ‘cryptogenic’ abnormal liver function test results, autopsy samples, ultrasound and magnetic resonance spectroscopy range between 3% and 37%, with the usual figure quoted being around 30%.

• Studies suggest that ethnic background plays a role in the incidence of NAFLD were provided by a study that revealed a significantly higher prevalence of NAFLD in Hispanics than in non-Hispanic whites, even after controlling for obesity and body fat distribution.

• Studies show that NAFLD is not always associated with raised alanine transaminase (ALT) or gamma-glutamyl transpeptide positivity.

• NAFLD was more likely in the presence of obesity, hyperglycemia,hyperinsulinaemia, hypertriglyceridaemia, and systolic hypertension, all features of the metabolic syndrome.

• Although total fat consumption is not significantly associated with the risk of cirrhosis or liver cancer, cholesterol consumption is.

• Recent clinical and experimental studies have shown that coffee protects the liver against the development of fat-induced liver damage.

• The prevalence of NASH is difficult to determine, as large population-based studies are not possible, because a liver biopsy is still, currently, required for diagnosis.

• An autopsy study found NASH in 18.5% of markedly obese patients and 2.7% of lean patients.

• This highlights the possible effect that weight has on the incidence of NASH.

Normal liver

Fatty liver

NASH

Progressive fibrosis

cirrhosis

Liver failure Liver cell cancer

Stable disease

30%

30%

20%

0-10%30-40%

• A well recognized complication of cirrhosis resulting from NAFLD is HCC.

• HCC has also been noted as a rare complication of NAFLD prior to cirrhosis.

• This may be explained by the association of diabetes and obesity with the development of HCC, as well as the carcinogenic factors associated with cirrhosis in general.

ALD:

• Studies in unselected heavy drinkers of alcohol suggest that 80% develop steatosis, which is the earliest and most common histopathological manifestation of ALD.

• Steatosis occurs in most people consuming alcohol in excess of 80 g/day, and can resolve within 2-4 weeks of abstinence.

Normal liver

Fatty liver

ASH

fibrosis

cirrhosis

Alcohol intake Alcohol abstinence?Abstinence effect

80%

20-40%

40%

• Severe ASH has a very poor prognosis, whereas patients with mild and moderate ASH can improve with abstinence.

• It is extremely difficult to predict histological stage clinically in ALD patients before the development of decompensated cirrhosis.

• A small subset of ALD patients presenting with decompensated chronic liver disease has severe ASH without cirrhosis on biopsy.

• Conversely, some ALD patients who are clinically well but have abnormal liver function test results and undergo liver biopsy are found to have advanced ALD with severe ASH.

• Staging Of ALD is most accurately performed histologically by performing liver biopsy, which may need to be via the transjugular route in patients with impaired clotting.

PATHOGENESIS

NAFLD:

• NAFLD is closely linked to obesity, insulin resistance, and the metabolic syndrome.

• The initial theory for the pathogenesis of NAFLD was the ‘two-hit’ hypothesis.

• The first hit, steatosis, sensitives the liver to the induction of inflammation by a second insult that promotes oxidative stress and steatohepatitis.

• This model has subsequently been revised, in recognition that a combination of ‘second hits’ (both environmental and genetic) may lead to the development of steatohepatitis.

• Currently, it is not fully understood why some patients develop isolated steatosis and others develop steatohepatitis.

• However, it appears that insulin resistance and increased levels of free fatty acids in the liver are strongly associated with NASH.

• When insulin resistance develops, free fatty acids are inappropriately moved to non-adipose tissues such as the liver by decreased inhibition of lipolysis and increased de novo lipogenesis.

• There is currently increasing work on the interaction between cytokines and adipokines (cytokines secreted by adipose tissue) in an attempt to understand the mechanisms involved in NAFLD development.

• Insulin resistance is thought to be regulated by proinflammatory cytokines, such as tumour necrosis factor-a (TNF-a), and adipokines such as adiponectin and leptin.

• Oxidative stress and apoptosis also appear to contribute to the development and progression of NASH.

ALD:

• Pathogenesis is still incompletely understood.

• Recent discoveries - Many pathways leading to oxidative stress, and the new mechanism of endoplasmic reticulum stress.

• There are many similarities with NAFLD, such as the proinflammatory cytokine TNF-a and the adipokines adiponectin and leptin.

• Obesity being implicated as a risk factor for the development of ASH and cirrhosis in people with heavy alcohol consumption.

HISTOPATHOLOGY

NAFLD

• Histological analysis of a liver biopsy remains the gold standard for and only accurate way of assessing the degree of steatosis, necroinflammatory changes and fibrosis of NASH, and therefore distinguishing NASH from isolated steatosis.

• The pathology committee of the NASH CRN group developed and histological scoring system for use in NAFLD.

• For each case, an NAFLD activity score (NAS) and a separate fibrosis stage was given.

• The NAS comprised 14 histological features, nine of which were recorded as present or absent (such as Mallory-Denk bodies), and three of which were semiquantitatively [steatosis (0-3), lobular inflammation (0-3), and hepatocellular ballooning (0-2)], to give a score of between 0 and 8.

Steatosis grade (0-3) Lobular inflammation (0-3) Hepatocyte ballooning (0-2)

0: <5% 0: None 0: None

1: 5-33% 1: <2 foci/*20 field 1: Mild, few

2: 34-66% 2: 2-4 foci/*20 field 2: Moderate-many

3: >66% 3: >4 foci/*20 field

NAFLD activity score (NAS): 0-8 0-2 not NASH

3-4 uncertain for NASH

5-8 NASH

Non-alcoholic steatohepatitis (NASH) Clinical Research Network Fibrosis Staging System

Fibrosis: based on the use of Masson’s trichrome stain

0: None

1a: Mild zone 3 perisinusoidal fibrosis Requires trichrome stain to identify

1b: Moderate zone 3 perisinusoidal fibrosis May be appreciated on haemotoxylin and eosin

1c: Portal fibrosis only

2: Zone 3 perisinusoidal fibrosis andd periportal fibrosis

3: Bridging fibrosis

4: Cirrhosis

Non-alcoholic steatohepatitis (NASH) Clinical Research Network Fibrosis Staging System

• The lobular inflammatory infiltrate is usually composed predominantly of neutrophils, but lymphocytes and macrophages are commonly seen.

• In practice, the presence of ballooned hepatocytes is a sine qua non for the diagnosis of NASH rather than steatosis.

• In a meticuluos histological study, serial staining consistently demonstrated that hepatocellular ballooning was associated with fat droplets, as shown by oil red O positivity and CK-18-positive Mallory-Denk bodies.

• Histological features recorded but not scored in NAS include Mallory-Denk bodies, megamitochondria, and nuclear vacuolation.

• In NAFLD, Mallory-Denk bodies are often small and poorly formed, and may be difficult to detect in routinely stained sections.

• Immunohistochemistry for ubiquitin, p62, CK-8 and CK-18 can be used to demonstrate antigens associated with Mallory-Denk bodies and ballooned hepatocytes.

• Summary, Histological spectrum of NAFLD is characterized by steatosis, lobular inflammation, ballooning of hepatocytes, fibrosis, and other features that may or may not be present, such as Mallory-Denk bodies and portal inflammation.

• The NASH CRN grading and staging system of NASH is based on the use of haematoxylin and eosin and Masson’s trichrome stain, so it can be used routinely by histopathologies.

Nonspecific steatosis, predominantly macrovesicular, with occasional foci of inflammatory cells in the hepatic lobules and many hepatocytes with glycogenated nuclei (H&E, ×200).

Steatohepatitis with several hepatocytes showing ballooning degeneration intermixed with steatosis and foci of inflammatory cells in the hepatic lobules (H&E, ×200).

Mallory hyaline showing eosinophilic and ropy inclusions in the cytoplasm (H&E, ×400).

Perivenular/pericellular fibrosis in zone 3 (Masson trichrome, ×200).

ALD:

• The diagnosis of ALD is usually easy to make with a clear history of excessive alcohol consumption and negative markers for other chronic liver diseases.

• However, one needs to remember that the liver disease seen in patients who drink excessively is not always caused by alcohol

• The role of Liver biopsy in ALD is controversial but it is still the gold standard investigation and used to

• clarify cases with an unusual clinical course,

• to better define the contribution of alcohol in patients with possible non-alcohol-related comorbidity (e.g. in hepatitis C or use of lipid-lowering medications),

• and in some patients, to determine the severity of liver disease.

• It can also be used to specifically assess the amount neutrophils within the liver parenchyma to act as a guide to whether the patient would benefit from steroid treatment.

• Liver biopsy is not used in advanced ALD where there is evidence of decompensated cirrhosis, as the risk outweigh any potential benefits.

• The histopathology of ALD is similar to that of NAFLD, and occurs mainly in the liver parenchyma in the perivenular areas.

• Characteristic early lesions in ALD include perivenular and pericellular fibrosis.

• The development of ASH is dominated by a neutrophilic infiltrate, ballooning degeneration, Mallory-Denk bodies, and hepatocyte necrosis.

• In most cases, a distinction between ALD and NAFLD cannot be made on morphological criteria and the diagnosis has to rely on clinicopathological correlation.

• Overall features in favour of ALD include canalicular cholestasis, cholangiolitis, florid zone 3 changes such as dense infiltration by neutrophils, prominent Mallory-Denk bodies, and extensive zone 3 fibrosis associated with sinusoidal obliteration and hepatic veno-occlusive lesions.

• NAFLD tends to have more marked steatosis and less severe steatohepatitis changes

• One of the most helpful changes, and one that strongly favours NAFLD, is nuclear vacuolation, which is seen in 70-80% of NAFLD cases and in only 5-10% of ALD cases.

• The glycogenated nuclei can be seen in NAFLD-related cirrhosis even when other changes of NASH have disaappeared.

• Immunohistochemistry for protein tyrosine phosphate 1B (PTP1B) and insulin receptor on hepatocytes has been suggested to be useful in differentiating NAFLD from ALD.

• PTP1B was shown to be upregulated in the cytoplasm of hepatocytes in NASH biopsies as compared with ASH biopsies.

• Insulin receptor showed loss of membranous staining in hepatocytes from NASH biopsies, but was still present on ASH biopsies.

CONCLUSION• NAFLD and ALD are increasingly common and

recognized diseases.

• The risk factors and possible courses of the two diseases are well known, with many areas of ovelap, although it is still unclear why some people progress to NASH/ ASH and cirrhosis and others do not.

• There is an increasing knowledge of the pathogenesis of both diseases.

• In NAFLD especially, and in ALD to a lesser degree, liver biopsy and histopathological assessment are key for determining whether a patient has steatosis or steatohepatitis, and also for assessing the degree of liver damage and fibrosis.

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