—  SHORT COURSE   —

Disorders of the Intra- and Extrahepatic Bile Ducts

Case 5 - Acute Graft-versus-host Disease

Kay Washington, M.D., Ph.D.
Pathology, C-3321 MCN
Vanderbilt University Medical Center
Nashville, TN 37232-2561


Clinical History
This 16 month old male child with severe combined immunodeficiency received a haploidentical bone marrow transplant. Skin rash and diarrhea developed at day 10 post transplant. Marked abdominal distension developed on day 16. Chest x-ray showed worsening bilateral interstitial infiltrates. The patient died on day 21 post transplant.

Morphologic Findings
The overall hepatic architecture is normal. The portal tracts contain a relatively sparse inflammatory infiltrate that does not expand or fill portal areas. The infiltrate is composed of large and small lymphocytes, with very few eosinophils and neutrophils, and is centered around bile ducts. Mononuclear cells infiltrate the biliary epithelium. In some ducts, the epithelial cells are sloughed; others show degenerative changes such as nuclear pyknosis, cytoplasmic vacuolization, and irregular spacing of nuclei. The extent of bile duct injury is greater than one might expect from the relatively sparse inflammatory infiltrate. No endothelialitis is seen. The hepatic parenchyma is largely spared by the inflammatory process.

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Slide 13
Acute Graft-versus-host Disease
Interlobular bile ducts are the primary target in the liver in acute graft-versus-host disease. A mild lymphocytic infiltrate is often seen around the affected bile ducts, but inflammation is often minimal in relationship to the degree of bile duct injury. The bile duct epithelial cells in this example are sloughed and show degenerative changes. Lymphocytes may also infiltrate biliary epithelium.


Diagnosis: Acute Graft-versus-host Disease
Acute rejection of the hepatic allograft and acute graft-versus-host disease after bone marrow transplantation share morphologic features, as might be predicted, as both are the product of interaction between an immune system and a liver that differ at the major histocompatibility complex. In a sense, both are iatrogenic cholangiopathies. The bile ducts are the major target of injury in both processes, and small bile ducts are more severely affected than larger ducts. The pathogenesis of bile duct injury is not completely understood, but direct immunologic injury from invading lymphocytes and indirect damage due to cytokine release are plausible mechanisms. In acute rejection, damage to the peribiliary vascular plexus may result in ischemic injury to bile ducts, although this is less likely in GVHD. The presence of immunologically active molecules on the cell surface of biliary epithelium is also a factor in these post-transplant cholangiopathies. For instance, the major blood group antigens and Class I MHC antigens are normally expressed on biliary epithelial cells. Class II MHC antigens, ICAM-1, CD51, and LFA-3 are upregulated and expressed in the setting of inflammation and contribute to the immune-mediated injury .1 

Clinical Features
Acute rejection in the liver is more common in younger patients and patients mismatched at the HLA-DR locus. Patients who undergo liver transplantation because of alcoholic liver disease may have a lower incidence of acute rejection .2  Acute rejection generally develops 5 to 21 days after transplantation and occurs in ~50% of patients. Very late presentations are often due to inadequate immunosuppression, either from an attempt to decrease immunosuppression or due to poor patient compliance. Clinical presentation is highly varied, ranging from no symptoms and normal liver tests in patients undergoing protocol biopsies, to malaise and fever with elevated bilirubin, aminotransferases, and alkaline phosphatase .3 

Acute graft-versus-host disease in bone marrow transplantation is more likely to occur in patients receiving HLA-mismatched grafts. Histologic abnormalities of small bile ducts resembling acute hepatic GVHD have also been described in recipients of autologous hematopoietic cell transplants; the pathogenesis is unclear but may be related to failure to reconstitute and maintain tolerance for self antigens after transplantation .4  Acute GVHD usually occurs 3 to 6 weeks after transplantation and often presents first with skin involvement, followed by gastrointestinal manifestations. Liver involvement is manifested by increased serum alkaline phosphatase, hyperbilirubinemia with jaundice, and mild hepatomegaly. Approximately one-half of the 80% of bone marrow transplantation patients with abnormal liver function tests will have acute hepatic GVHD. Because most patients with hepatic involvement will also have skin and GI GVHD, liver biopsy is often not obtained for diagnosis but to rule out other causes of hepatic dysfunction .5 

Histopathologic Features
The most helpful diagnostic features in acute rejection are the presence of a mixed portal inflammatory infiltrate with eosinophils, largely sparing the hepatic parenchyma; infiltration and injury of bile ducts by lymphocytes; and endothelialitis. The inflammatory infiltrate expands the portal areas and may focally spillover into the adjacent parenchyma. Marked involvement of the hepatic parenchyma is not generally a feature of acute rejection, although this may be seen in very severe cases. The infiltrate is composed of small lymphocytes, large activated lymphocytes, macrophages, eosinophils, and varying numbers of neutrophils. Plasma cells are rare, and immunophenotyping shows that most of the portal lymphocytes are T cells, with both CD4+ and CD8+ cells represented. CD4+ cells mediate graft injury by releasing cytokines which activate other effector cells, while CD8+ cells probably cause injury by direct cytopathic attack on graft cells .3 

Bile duct injury may be focal in mild acute rejection, or bile ducts may be obscured by the inflammatory infiltrate and difficult to identify. The bile duct epithelium is infiltrated by lymphocytes, and the biliary epithelial cells show cell swelling, cytoplasmic vacuolization, nuclear crowding and reactive change such as prominent nucleoli and slight increase in the nuclear/cytoplasmic ratio, and irregular spacing of nuclei. In very mild cases, only cuffing of bile ducts by inflammatory cells, and slight reactive changes may be seen. In rejection treated with corticosteroids prior to biopsy, infiltration of bile ducts by neutrophils may be prominent and may mimic biliary obstruction.

Endothelialitis, infiltration of venular endothelium by mononuclear inflammatory cells, is probably overdiagnosed. In addition to the presence of lymphocytes in close proximity to vascular endothelium, there should be evidence of endothelial cell injury such as endothelial cell enlargement and detachment. Both portal veins and central veins may be affected. Occasionally central vein involvement is particularly striking and may be associated with centrilobular hepatocyte necrosis and perivenular hemorrhage. Portal changes typical of acute rejection are usually present in such cases. In pediatric liver transplant recipients, isolated central venulitis occurs in up to 16%, generally appearing later than portal rejection alone .6  Central venulitis may be seen in both acute and chronic rejection .7  Severe histologic endothelialitis may be associated with a poor response to antirejection therapy .8 

Graft dysfunction associated with autoimmune features has been described in both children and adults and may represent an unusual morphologic variant of acute rejection Features include development of ANA and atypical anti-liver-kidney-microsomal antibodies, high aminotransferase levels indicative of hepatocyte damage, and liver biopsy showing markedly active hepatitis with numerous plasma cells. Subjects in one study showed higher prevalence of HLA-DR3 than other liver transplant recipients. 10 

Portal inflammation is also a hallmark of acute graft-versus-host disease, although it is less intense than in acute rejection. As in rejection, the inflammatory infiltrate is predominantly mononuclear, and eosinophils and neutrophils are rare. The inflammatory infiltrate centers about bile ducts, which show the diagnostic alterations in hepatic GVHD. Interlobular bile ducts are distorted and angular, focally infiltrated by lymphocytes, and the epithelial cell nuclei are irregularly spaced and pleomorphic. As in acute rejection, the lymphocytes infiltrating the bile ducts in acute GVHD are T cells. The bile duct lumen may contain necrotic debris or sloughed epithelial cells. Biopsies taken early in the course of hepatic GVHD (before Day 35 post-transplant) may not show characteristic bile duct abnormalities, and may show only non-specific lobular changes such as spotty hepatocyte necrosis. In my experience, endothelialitis is rarely if ever seen in acute hepatic GVHD, although a rate of 40% has been reported. Cholestasis is a common finding in acute GVHD. Parenchymal necrosis may be seen, but is not specific.

Chronic Allograft Rejection
Chronic rejection in the hepatic allograft generally develops after multiple episodes of acute rejection, or evolves from an episode of unresolved acute rejection. It may rarely occur de novo in the patient who has never had clinical acute rejection. It is usually diagnosed months to years following transplantation. Two major sites of attack are recognized in the liver: interlobular bile ducts and hepatic arteries .3  The bile duct damage generally takes the form of "vanishing bile duct syndrome", in which the bile duct loss is accompanied by only a mild lymphoplasmacytic inflammatory infiltrate. Bile ductular proliferation is not seen, and the portal inflammatory infiltrate subsides with loss of bile ducts, leaving empty-appearing portal tracts. Loss of small hepatic artery branches occurs early, followed by bile duct loss .13  The foam cell arteriopathy of chronic rejection is rarely seen on liver biopsy, as it preferentially involves the large arteries near the hepatic hilum. This obliterative arteriopathy is characterized by the accumulation of foamy histiocytes in the thickened intimal layer. The resulting ischemia may contribute to bile duct loss. Centrilobular areas in both forms of chronic rejection show spotty hepatocyte necrosis, cholestasis, and perivenular inflammation or fibrosis. Transient lobular hepatitis may also be a manifestation of chronic rejection ,14  and the shift from a portal based inflammatory process towards lobular necroinflammatory activity may represent the transition from acute to chronic rejection 15 .

Minimal diagnostic criteria are the presence of bile duct atrophy/pyknosis, affecting a majority of the interlobular bile ducts; foam cell obliterative arteriopathy; or bile duct loss in over 50% of portal tracts .16  Chronic rejection has been divided into early and late phases; such information from the liver biopsy is used to guide patient management, as allografts with lesions of late chronic rejection without inflammatory activity are unlikely to respond to additional immunosuppressive therapy. In early chronic rejection, degenerative duct changes predominate and bile duct loss is seen in less than 50% of portal tracts. Intimal or lumenal inflammation may be seen in central veins, and mild perivenular fibrosis is seen in zone 3. Loss of portal tract arterioles is seen in less than 25% of portal tracts .16  In late chronic rejection, bile ducts are lost in over 50% of portal tracts. Inflammatory changes are variable. Centrilobular fibrosis is more prominent than in early chronic rejection, and hepatic arterioles are lost in over 25% of portal tracts .16  However, no histologic feature reliably predicts response to immunosuppression or outcome .14  Differential diagnosis of chronic ductopenic rejection includes bile duct stricture, particularly when the obstruction is partial; drug reaction; and cytomegalovirus infection.

Chronic Graft-versus-host Disease
Chronic GVHD usually occurs after bouts of acute GVHD but may be seen de novo in a minority of patients .17  The histopathology of chronic GVHD is not well delineated. Like chronic rejection, it is characterized by bile duct distortion and loss and cholestasis. Hepatic involvement occurs in about 90% of patients and the skin and oral mucosa are usually involved as well, resulting in scleroderma-like changes and Sjogren's syndrome. Liver biopsy shows a sparse portal mononuclear inflammatory infiltrate with severely distorted interlobular bile ducts. Canalicular cholestasis is seen in the lobule. With loss of bile ducts and continued disease progression, changes of chronic cholestasis may be seen and portal fibrosis followed by cirrhosis may develop. Unlike in chronic rejection, arterial changes are not seen. An unusual pattern of chronic hepatic GVHD in which the characteristic bile duct injury is combined with marked lobular hepatitis has recently been described .18  This pattern of injury was seen in patients receiving little or no immunosuppression after allogeneic transplantation. Treatment with cyclosporine and prednisone resulted in progressive improvement; those patients who were not treated with immunosuppressive therapy developed ductopenia and progressive cholestasis .18  Since the differential diagnosis for chronic GVHD includes chronic viral hepatitis, such cases may prove to be diagnostic dilemmas. The degree of portal inflammation, piecemeal necrosis, and bile ductular proliferation are generally greater in viral hepatitis, whereas bile duct injury and loss predominate in typical chronic GVHD.



Differential Diagnosis
Distinction between recurrent disease and acute rejection is often challenging. Histologic recurrence of primary biliary cirrhosis is probably quite common, although clinically significant disease is rare. A more pressing problem is the distinction of recurrent hepatitis C from rejection. Recurrence of hepatitis C after transplantation is unavoidable, although not all patients develop clinically significant hepatitis. Histologically evident recurrence begins with spotty hepatocyte necrosis and lobular mononuclear infiltrates .19  A mononuclear inflammatory infiltrate, generally mild, is present in the portal areas, and focal infiltration of bile ducts by lymphocytes may be seen. As the disease progresses, the typical dense nodular aggregates of small lymphocytes typical of hepatitis C infection may be seen in portal areas. Diagnostic problems occur because of temporal and histologic overlap with acute rejection. One approach to this dilemma is to compare portal and lobular changes. If lobular changes such as hepatocyte necrosis and lobular hepatitis are more prominent than portal changes, then hepatitis C is favored. Acidophilic bodies may be a more reliable indicator of recurrence than lobular inflammation .20  If the portal inflammatory infiltrate includes activated lymphocytes and prominent eosinophils and there is little lobular activity, acute rejection is favored.

Grading
Several grading schemes for acute rejection have been developed. Most rely on the density of the portal inflammatory infiltrate, the number of portal tracts involved, the extent of bile duct injury and the extent of endothelialitis. One of the more recent schemes is the Banff Consensus Schema, which relies upon a global assessment and generation of a rejection activity index by grading individual features .21  At least two of the following three features are required for a histologic diagnosis of acute rejection: mixed portal inflammatory infiltrate, predominantly mononuclear but also containing neutrophils and eosinophils; bile duct damage or inflammation; and endothelialitis involving portal vein branches or terminal hepatic venules. Once the diagnosis of acute rejection has been established in the Banff schema, the grade is assigned based on the global assessment, which is largely based on the portal inflammatory infiltrate. In addition, a rejection activity index may be generated and a total numerical score based on the sum of the individual component scores assigned. In comparison to other grading schemes, the Banff schema often results in up-grading of rejection .22 

Banff Schema: Grading of Acute Hepatic Allograft Rejection 21 
Global Assessment Criteria
Indeterminate Portal inflammatory infiltrate that fails to meet criteria for diagnosis of acute rejection
Mild Rejection infiltrate in a minority of triads that is generally mild and confined to the portal area
Moderate Rejection infiltrate that expands most or all of the triads
Severe As above for moderate, with spillover into periportal areas and moderate to severe perivenular inflammation that extends into the hepatic parenchyma and is associated with perivenular hepatocyte necrosis


Rejection Activity Index: Banff Schema 21 
Category Criteria Score
Portal Inflammation Mostly lymphocytic infiltrate involving but not expanding a minority of portal tracts 1
Expansion of most or all of triads by mixed inflammatory infiltrate containing activated lymphocytes, neutrophils, and eosinophils 2
Marked expansion of most or all triads by mixed infiltrate with numerous lymphoblasts and eosinophils with spillover into periportal parenchyma 3
Bile Duct Inflammation/Damage A minority of bile ducts are cuffed and infiltrated by inflammatory cells; mild reactive changes such as increased N:C ratio 1
Most or all duct infiltrated by inflammatory cells. More than an occasional duct shows degenerative changes such as nuclear pleomorphism, altered polarity, and cytoplasmic vacuolization 2
As above for 2, with most or all of the ducts showing degenerative changes or focal lumenal disruption 3
Venous Endothelial Inflammation Subendothelial lymphocytic infiltration involving some but not a majority of portal and/or hepatic venules 1
Subendothelial infiltration involving most or all or portal and/or hepatic venules 2
As above for 2, with moderate or severe perivenular inflammation that extends into the perivenular parenchyma and is associated with perivenular hepatocyte necrosis 3

Acute GVHD is graded clinically by assessing skin, liver, gastrointestinal involvement, and clinical performance status .5  Histopathologic grading of acute hepatic GVHD provides little prognostic information and is often not done, although various schemes have been proposed. Chronic GVHD is graded as limited or extensive based on organ involvement or the presence of severe liver disease. The hepatic involvement is regarded as limited if there is duct injury without piecemeal necrosis, fibrosis, or loss of bile ducts. The designation of extensive chronic hepatic GVHD is reserved for liver biopsies with these features .23 

References

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