Hepatitis in Long-Term Liver Graft Recipients

Unexplained chronic graft inflammation (idiopathic posttransplant hepatitis) is characterized by a predominantly portal infiltrate of mononuclear inflammatory cells. Inflammation of bile ducts and portal vessels is typically minimal or absent. Portal inflammation is regularly associated with mild to moderate degrees of interface hepatitis ( Fig. 44.3 ). Lobular inflammatory changes may also be present; they characteristically involve centrilobular regions and may be associated with central perivenulitis (CP).

Patient 1: Protocol biopsy 5 years after pediatric liver transplantation (hematoxylin and eosin [HE] staining, 100 × magnification) showing portal inflammation and periportal fibrosis (liver allograft fibrosis score, 3/9) with interface activity.

(Courtesy of Dr. Bosmüller/Prof. Fend; University of Tübingen.)

Scoring systems used to grade chronic hepatitis in the native liver (e.g., METAVIR, Ishak) are only suitable for grading portal and periportal inflammatory changes in the liver allograft biopsies. The severity of CP can be better scored according to the system proposed by the Banff Working Group. Otherwise, chronic graft hepatitis with dominant CP and only minor or even absent portal inflammation may be underestimated. Severe, late CP can be associated with adverse long-term outcomes, even if there is only mild portal inflammation.

Fibrosis in Long-Term Liver Graft Recipients

Three main patterns of liver graft fibrosis were recognized in posttransplant biopsies from pediatric liver allograft recipients: periportal ( Fig. 44.4 ) , perisinusoidal, and centrilobular fibrosis. Preliminary findings suggest different pathogenic mechanisms for these patterns of fibrosis. Periportal fibrosis can develop following initial portal inflammation and interface hepatitis that is present in children with chronic hepatitis ( Figs. 44.4 and 44.5 ). The typical picture of centrilobular fibrosis has been described as a complication of CP in children with chronic rejection (CR). Several studies found evidence to suggest that perisinusoidal fibrosis occurring in the centrilobular region was associated with chronic antibody-mediated rejection (AMR).

Patient 1: Protocol biopsy 5 years after pediatric liver transplantation (Masson trichrome staining, 100 × magnification) showing portal inflammation with interface activity and periportal fibrosis with porto-portal bridging (liver allograft fibrosis score, 3/9).

(Courtesy of Dr. Bosmüller/Prof. Fend, University of Tübingen.)

Patient 2: Protocol biopsy 6 years after pediatric liver transplantation (hematoxylin and eosin staining, 100 × magnification) showing advanced periportal fibrosis (liver allograft fibrosis score, 5/9) and mild to moderate portal inflammation.

(Courtesy of Dr. Bosmüller/Prof. Fend, University of Tübingen.)

Standard scoring systems used to stage fibrosis in the native liver fail in staging the distinctive patterns of fibrosis in pediatric allografts. A specific scoring system focusing on the three patterns of allograft fibrosis, the liver allograft fibrosis (LAF) score, was developed by Venturi and colleagues ( Fig. 44.2 ) . The severity of fibrosis is assessed semiquantitatively in separate histological compartments: around portal tracts, in sinusoids, and around centrilobular veins. These three compartments contribute to a total LAF score. This LAF score helps to describe and quantify stages of allograft fibrosis specifically and is additionally useful to describe dynamics of the fibrosis progression in serial biopsies of pediatric liver transplant recipients.

Liver allograft semiquantitative scoring system (Venturi score) for assessment of fibrosis in pediatric liver transplant biopsies: histologic features and staging definitions.

(From Venturi C, et al. Novel histologic scoring system for long-term allograft fibrosis after liver transplantation in children. Am J Transplant. 2012;12(11):2986–2996.)

Recurrent Liver Disease

Recurrent liver disease plays an important role in the management of adult liver transplant recipients. In children, however, recurrence of underlying liver disease is a less prominent complication in the long-term post-LT management. A minority of children and adolescents have received a liver transplant for diseases that may relapse, including immunological (e.g., primary sclerosing cholangitis, autoimmune hepatitis, autoimmune sclerosing cholangitis, and bile salt export pump deficiency) and oncological (e.g., hepatoblastoma and hepatocellular carcinoma) disease. In patients transplanted for these indications, the recurrent liver disease can affect graft function and long-term outcome, so that this potential risk should be considered. The diagnosis of recurrent autoimmune liver disease can be problematic because of the histological overlap with T-cell-mediated rejection and difficulties in interpreting serum autoantibody levels after transplantation. Diagnosis and treatment of recurrent liver disease are described elsewhere (see Chapter 27 ).

Hepatitis E Virus Infection

Hepatitis E virus (HEV) is a recently described cause of chronic graft hepatitis with progressive fibrosis in adults and children. Post-LT, most patients are infected by genotype 3. HEV infection is usually acquired enterally and has been more prevalent in some geographic regions, (e.g., India). More recently, HEV has been identified as an emerging pathogen in developed countries as well, particularly among immunosuppressed solid organ transplant recipients. In those, HEV infection is most likely caused by foodborne transmission. Blood transmission of HEV or even organ-related transmission of HEV has rarely been reported in LT recipients. In the case of transmission, approximately 50% to 80% of adult LT patients infected with HEV develop chronic infection, and 10% to 15% of cases progress to cirrhosis. HEV is also prevalent in pediatric LT recipients, and 8.3% of children are immunoglobulin (Ig) G positive. Cases of chronic HEV infection with progressive allograft hepatopathy have also been observed in pediatric LT recipients. In chronic liver graft dysfunction, HEV RNA test by reverse transcriptase-polymerase chain reaction in blood and/or stool is recommended. The treatment should include modification of immunosuppression and antivirals. Initially, the immunosuppression should be reduced, but if this is not possible, or if there is no HEV RNA clearance within 3 months, treatment with ribavirin (15 mg/kg/day) is recommended for 3 months. Close monitoring of anemia and renal function, as well as HEV load, is required. Ribavirin was shown to be successful in treating HEV infection and preventing graft failure because of acute HEV infection.

Chronic and Antibody-Mediated Rejection

As already mentioned, there is a particular clinical and histopathological overlap between insidious, chronic graft injury, and chronic rejection (CR), or chronic antibody-mediated rejection (AMR). However, many of these patients with apparent or inapparent chronic graft injury do not fulfill the diagnostic criteria for CR or chronic AMR.

CR may itself cause long-term graft dysfunction and fibrosis. Its clinical presentation usually is associated with jaundice, pruritus, elevation of bilirubin, aspartate aminotransferase (AST), alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase levels, which is usually in contrast to the patients with subclinical graft injury. The Banff group defined the histological criteria of CR, including biliary epithelial senescence changes affecting a majority of the bile ducts with or without bile duct loss, foam cell obliterative arteriopathy, and bile duct loss (at least two criteria have to be fulfilled). In their latest update, early and late CR were discriminated, and criteria were more specifically detailed. In a recent study, CR was observed in 37.5% (48 of 128) of 20-year survivors after pediatric LT. In a SPLIT registry study analyzing patients with late graft loss (35 of 872 patients), CR was the cause for 37% of the lost grafts. Some reports have suggested that tacrolimus may markedly reduce CR in comparison with the cyclosporine regimens. Treatment of CR can be challenging, and graft loss is frequent. Of note, rescue treatment with mammalian target of rapamycin (mTOR) inhibitor may be of benefit in some cases.

Chronic AMR of the liver allograft is an emerging entity. The histological picture has a substantial overlap with the silent graft injury caused by idiopathic graft hepatitis. Diagnosis of chronic AMR can be made according to Banff working group criteria as follows:

• 1.
  

  Histological pattern of injury consistent with chronic AMR, requiring both of the following:

  
  
  
  
  • a.
    

    Otherwise unexplained and at least mild mononuclear portal and/or perivenular inflammation with an interface and/or perivenular necroinflammatory activity.

    
    
  • b.
    

    At least moderate portal/periportal, sinusoidal, and/or perivenular fibrosis.

  
  
  
  
• 2.
  

  Detection of circulating HLA DSAs in serum samples of patients with subclinical graft injury.

  
  
• 3.
  

  At least focal C4d positivity (> 10% portal tracts, microvascular and endothelial). However, in cases of so-called “possible chronic active AMR” C4d staining can be minimal or absent.

  
  
• 4.
  

  Exclusion of other insults that might cause a similar pattern of graft injury (e.g., hepatitis E infection).

Silent chronic allograft injury lacks specific/typical clinical or biochemical features; many histopathological features observed in protocol biopsies overlap with diagnostic criteria of chronic AMR and/or T-cell-mediated rejection.

Protocol Liver Biopsy

Liver biopsies are the gold standard for the evaluation of liver graft integrity. As previous studies have documented, abnormal histopathological findings are common in clinically and biochemically unremarkable patients after pediatric LT. Therefore, the biopsies are important to document the condition of the graft and to identify graft hepatitis or fibrosis more than 1 year after pediatric LT. Furthermore, immunohistochemical biomarkers like the expression of cytoplasmic alpha-smooth muscle actin (ASMA) could be considered for early prediction of the development of graft fibrosis. Assessment of the graft during the process of presumed operational tolerance and weaning chronic immunosuppression are additional reasons to encourage protocol liver biopsies in clinical management.

Protocol liver biopsies may provide relevant information about inflammatory changes or fibrosis. These results, in turn, can lead to modification of immunosuppressive treatment. The benefits of the biopsies as a diagnostic screening tool must be weighed against potential complications. The disadvantages of routine biopsies, including bleeding complications and significant costs, are well known. In current studies after pediatric LT, the rate of major complications (hemorrhage requiring blood transfusions) was lower than 1% with no mortality. In a large retrospective study of 68,276 percutaneous liver biopsies in adults, the risk of mortality was also low (0.9/10,000 biopsies). It is essential to identify patients with an increased risk of complications. Currently, protocol biopsies are the only available tool to assess subclinical chronic graft injury. However, uncertainties related to the clinical significance and a lack of guidance for therapeutic interventions require individualized consideration for the protocol biopsy. Further evidence of the long-term outcome of chronic allograft injury and the effectiveness of consequent treatment strategies is required before definite general recommendations can be made.

In the context of minimization and withdrawal of immunosuppression, however, the ESPGHAN Hepatology Committee and the Banff Working Group recommend a liver biopsy before and after modifying immunosuppression. Liver histology is necessary to discriminate patients under increased risk for allograft rejection. Monitoring of blood tests, liver ultrasonography, or noninvasive fibrosis markers are not considered sufficiently sensitive in assessing chronic damage to the liver allograft.

Noninvasive Assessment of Liver Graft Integrity

In the light of the risk for post-biopsy complications, including hemorrhage and the requirements for sedation/anesthesia, several noninvasive tests for assessment of chronic graft injury have been proposed. Apart from HLA and non-HLA antibodies, serological markers of liver transplant fibrosis were suggested (see Chapter 30 ). Furthermore, imaging studies including different modes of ultrasound and magnetic resonance elastography have been explored for assessment of liver fibrosis in transplant and non-transplant patients.

Dynamic ultrasound elastography is based on the assessment of the propagation of shear waves within tissues to calculate the viscoelastic properties. Transient elastography (FibroScan) is a first-generation dynamic ultrasound elastography method and does not offer a conventional B-mode image. It relies on the application of a short external shear wave pulse that is tracked by using one-dimensional ultrasound imaging. The test can be applied rapidly and repeatedly, and children tolerate it well. However, obesity, ascites, or limited operator experience can impair results in up to 20% of cases. Its diagnostic accuracy is better for higher-degree fibrosis/cirrhosis than for mild or moderate fibrosis. The technique has been evaluated in adult LT recipients showing excellent results for the detection of advanced fibrosis/cirrhosis (area under the receiver operating characteristic curve [AUROC], 0.86–0.98), but transient elastography was less useful for mild fibrosis (AUROC, 0.81). In pediatric LT, small studies aiming at correlating transient elastography to post-LT fibrosis have confirmed the potential of the technique to detect advanced fibrosis. Assessment of split or reduced grafts can be difficult because of a lack of defined cutoff values.

Acoustic radiation force imaging (ARFI) belongs to the second generation of ultrasound elastography methods measuring shear wave speed while offering conventional ultrasound B-mode imaging. Thereby, stiffness can be measured in a selected region of interest, making the assessment feasible in almost all patient groups. Comparable to transient elastography, ARFI accurately detected advanced fibrosis (AUROC, 0.98) in children and adults with chronic liver disease, but ARFI was less compelling in detecting mild fibrosis (AUROC, 0.83). Small studies in pediatric liver transplant recipients showed a good correlation of liver stiffness measurements by ARFI imaging with liver graft fibrosis. ARFI had good accuracy for diagnosing significant portal or moderate perisinusoidal graft fibrosis after pediatric living donor LT.

Magnetic resonance elastography (MRE) is a highly accurate, noninvasive technique for diagnosis and staging of liver fibrosis in the LT recipients, independent of body mass index and degree of inflammation. MRE has already been validated for the detection of liver fibrosis, including mild to moderate fibrosis in adult LT recipients and chronic pediatric liver disease. MRE may also be promising in pediatric LT recipients, but requires a prospective evaluation. Disadvantages of MRE include costs and the need for anesthesia in small children.

AST-to-platelet ratio index (APRI) is a straightforward, noninvasive score to assess liver fibrosis, which has been well validated for hepatic fibrosis in hepatitis C. In pediatric LT recipients, a higher APRI was associated with significant fibrosis (Ishak score ≥ 3; AUROC, 0.74). However, other studies did not show a correlation between APRI and stage of fibrosis. Of note, most of the patients with chronic graft injury initially show normal biochemistry despite advanced graft fibrosis.

In conclusion, noninvasive investigations, including imaging studies and biomarkers, can help monitor progressive fibrosis over time. Nevertheless, these tests cannot substitute the protocol liver biopsies entirely at this stage because they cannot identify patients who exhibit early stages of graft fibrosis and inflammation, pointing to their increased risk for progressive graft injury and graft loss.

Donor-Specific HLA Antibodies

In the last decade, donor-specific HLA antibodies (DSAs) and chronic AMR have been identified as important risk factors for long-term allograft dysfunction and chronic allograft failure in pediatric LT (see Chapter 17 ). A meta-analysis showed that the prevalence of DSAs was 41% after pediatric LT. Risk factors for the emergence of de novo DSAs include low trough levels of calcineurin inhibitors, cyclosporine versus tacrolimus use, younger age, and previous transplants.

Although the pathogenic role of DSAs in LT is still debatable, evidence is accumulating that preformed DSAs with high mean fluorescence intensity (MFI) values and de novo DSAs developing post-transplant are associated with chronic graft injury. Higher DSAs levels with MFI values over 5000 increase the risk of associated graft injury. There is emerging data suggesting that subsets of DSAs—those of the IgG3 subclass and/or those that bind the first subcomponent of complement complex C1 (C1q)—are particularly damaging. A recent study demonstrated an association between HLA class II DSAs and portal inflammation, which, over time, drives progressive fibrosis in the portal areas. However, it remains unclear whether DSAs are directly responsible for the chronic graft inflammation or are instead just a surrogate marker of chronic alloimmune recognition and injury of the graft. Importantly, severe graft injury may also occur with negative DSAs.

Complement activation in the liver tissue plays a significant role in the pathogenesis of AMR. The deposition of C4d in the portal microvascular endothelium with extension into inlet venules or periportal sinusoids in severe cases is the histochemical correlate of humoral graft inflammation in the liver tissue. This suggestion of antibody-mediated subclinical inflammation is more conceivable, as the C4d deposition in the hepatic tissue has been shown to correlate with the antibody presence. Therefore, C4d staining is an additional essential marker of antibody mediation in chronic liver graft injury. C4d positivity could be associated with chronic graft damage with or without a clinical picture of AMR. However, nonspecific staining is possible, and negative staining does not exclude antibody-mediated injury.

Different methods of analysis have been used for testing HLA antibodies, making interpretation and their comparison difficult. An isolated MFI value can be misleading. Therefore, prospective longitudinal studies of DSAs following pediatric transplantation are needed to assess the emergence and dynamics of DSA positivity to correlate the DSAs presence with clinical conditions and to draw conclusions relevant to clinical management.

Non-HLA Autoantibodies

In 40% to 60% of the liver allograft recipients, liver autoantibodies (usually anti-nuclear and smooth muscle actin antibodies) were detected by 5 to 10 years after LT. In most studies, the autoantibody positivity was associated with chronic graft inflammation. Liver autoantibodies, however, appear not to be the cause of the graft injury, but rather a reflection of its damage and could potentially be a helpful marker for chronic allograft hepatitis. Liver autoantibodies, de novo DSAs, and antibodies to glutathione S-transferase T1 are associated with plasma cell–rich rejection, also known as de novo autoimmune hepatitis. This entity is an uncommon cause of late graft dysfunction. The histopathological picture is very typical, showing portal and perivenular plasma cell infiltrates with interface activity. In pediatric LT recipients, de novo autoimmune hepatitis or plasma cell-rich rejection was found in about 2% to 7% of post-LT patients. This type of graft injury needs modified immunosuppression and can be associated with progressive graft dysfunction and the risk of graft failure (see Chapter 27 ).

More recently, non-hepatocyte autoantibodies were suggested to play a role in chronic graft injury. Detection of anti-ATR1 and anti-endothelin-1 type A receptor (ETAR) autoantibodies was associated with an increased risk of rejection and progression of liver allograft fibrosis in adult patients. Similarly, in pediatric transplant recipients, anti-ATR1 autoantibodies were associated with advanced fibrosis. However, the role and pathogenic mechanism of anti-ATR1 and anti-ETAR autoantibodies in chronic graft injury need further evaluation.