Steatosis

Both micro- and macrovesicular steatosis are common findings in donor liver biopsies; analysis of the combined U.S. and European databases report a prevalence of 20% for microvesicular steatosis and 23% for macrovesicular steatosis, and others find both even more frequently. They often coexist. Macrovesicular steatosis is defined as one or more cytoplasmic fat vacuoles that are larger than the hepatocyte nucleus and that typically displace it peripherally. The vacuoles in microvesicular steatosis are smaller than the nucleus, and the nucleus remains central. Steatosis is graded as mild (5–30%), moderate (30–60%) or severe (>60%), according to the estimated percentage of liver parenchyma affected. It should be noted that the small fat droplets, which are referred to as ‘microvesicular’ in the context of assessing donor liver biopsies, are probably best regarded as a small-droplet variant of macrovesicular steatosis ; the term ‘mediovesicular steatosis’ has been proposed to describe such cases. ‘True’ microvesicular steatosis, as seen in conditions associated with defects in heptocellular β-mitochondrial oxidation, rarely occurs in this setting. Macrovesicular steatosis is the more important finding in pretransplant biopsies; implicated donor factors include age, increased BMI and alcohol consumption, although in other cases, no definite cause can be found.

Because macroscopic appearances may not be reliable in assessing the severity of steatosis, a frozen section of the donor liver is often obtained in cases where fatty change is suspected clinically. Fatty change is usually distributed fairly uniformly within the liver, and one or two needle biopsies are sufficient to grade the severity of steatosis, two being more useful if there is clinical suspicion. Although frozen-section assessments are useful and reproducible in providing an estimate of overall amount of fat, they lack sensitivity in the ‘boundary zones’ between the different grades of steatosis severity. The use of specific fat stains such as Oil Red O generally increases the amount of fat visualized, but these methods are prone to considerable technical variation. There is often a discrepancy between the severity of steatosis determined on frozen section and that seen in subsequent paraffin histology, but this discrepancy appears to have little clinical impact. Because of problems with the subjective assessment of steatosis, the use of image analysis has been suggested as providing more objective and reproducible findings, but the utility of this alternative approach in clinical practice requires further study.

Donor grafts with up to 30% macrovesicular steatosis can be safely used and represent 97% of all steatotic grafts transplanted in the United States. Moderate steatosis is often associated with graft dysfunction in the early post-transplant period, usually manifest as elevation of serum transaminases, sometimes also with prolongation of prothrombin times, and may result in reduced graft survival; however, these grafts can be used in appropriate clinical circumstances. The majority of fat is cleared by macrophages over 1–3 weeks following transplantation.

Clinically, most transplant surgeons will not use donor livers with severe fatty change. This is not universal practice, however, and some report similar long-term outcomes for severely steatotic grafts if carefully managed. These grafts have an increased risk of graft nonfunction in the immediate post-transplant period (‘primary nonfunction’), resulting in death or retransplantation within the first week. The pathogenesis is multifactorial. Hepatocytes distended with large fat droplets can obstruct the sinusoidal microvasculature, resulting in problems with perfusion of the donor liver at retrieval and subsequent reperfusion in the recipient. Steatotic hepatocytes are significantly more sensitive to oxidative injury and death, causing the release of free lipid from hepatocytes, further compromising sinusoidal blood flow and enhancing lipid peroxidation in sinusoidal endothelial cells (SECs). Other consequences of fatty change on graft function include an increased susceptibility of SECs to cold and warm ischaemic damage, depletion in glycogen content and mitochondrial abnormalities in hepatocytes, a shift to necrosis as the main mechanism for cell death after reperfusion (compared with apoptosis as the predominant pathway in nonfatty livers) and an impaired regenerative capacity after cell loss caused by preservation/reperfusion injury. Complement also plays a role in the development of ischaemia/reperfusion injury in steatotic livers. Histological studies of failed liver allografts with severe fatty change have shown large extracellular fatty aggregates associated with areas of hepatocyte necrosis, haemorrhage and disruption of the sinusoidal framework. The release of fat droplets from necrotic hepatocytes may be associated with the formation of cystic lesions resembling peliosis hepatis (‘lipopeliosis’) ( Fig. 14.2 ). However, electron microscopic analysis has shown that the free lipid is extrasinusoidal, with only more severe cases resulting in rupture into the sinusoidal space.

Lipopeliosis. Centrilobular fat microcysts are surrounded by macrophages in this biopsy specimen taken 10 days following insertion of a steatotic donor liver. Subsequent biopsy specimens have shown resorption of the fat with only minimal residual perisinusoidal fibrosis. A, H&E stain; B, immunostaining for CD68.

Microvesicular (small-droplet) steatosis is also seen frequently in donor liver biopsies. As noted earlier, ‘true’ microvesicular steatosis with innumerable tiny steatotic vacuoles filling most or all of the hepatocytes is rare and was seen in only 1% of donors in a recent series. Small fat droplets can be difficult to detect in conventional haematoxylin and eosin (H&E)-stained sections, and the incidence and severity of microvesicular steatosis increase if special stains for fat (e.g. Sudan) are used on frozen sections, but in practice this is not generally done. Although microvesicular steatosis is well recognized as a rare cause of liver failure in the nontransplanted liver (e.g. Reye syndrome), its presence in donor livers, even to a marked degree, is not generally associated with poor graft function. A recent study suggested that small-droplet steatosis does not exacerbate the effects of macrovesicular steatosis.

Donor biopsy in living-related liver transplantation

Because of cadaveric donor shortages, particularly for paediatric livers and in some regions where deceased organ donation is negligible, a number of liver transplant units are utilizing living-related liver allografts for paediatric and adult transplantation. Donor safety is of paramount importance, and careful selection of donors is thus required to minimize the risks involved with liver resection. Although donors are generally in good health, the diagnostic pretransplant workup aims to detect occult hepatic or extrahepatic disease. Most centres do not routinely biopsy all potential donors, but guidelines on when to biopsy are lacking. In studies where biopsies have been obtained from potential living donors (some routinely, others after progressing beyond initial screening process), <50% are normal. The most common abnormality is macrovesicular steatosis, generally a manifestation of NAFLD and usually associated with an increased BMI. Steatosis is present in 20–60% of patients and is typically mild in severity. About 15–30% have either moderate or severe steatosis, and 1–15% have steatohepatitis. As with cadaveric liver grafts, mild degrees of steatosis are tolerated, but donors are excluded if macrovesicular steatosis is >25% or 33%, because of the risk to both donor and recipient of compromised regeneration. Steatohepatitis is also regarded as a contraindication to donation. A number of other histological findings have been found in a smaller proportion of donor biopsies. Portal/periportal and lobular inflammatory changes, in some cases producing a chronic hepatitis-like appearance, have been observed in up to 25% of donors but are usually less common. These are typically mild in severity and have no obvious cause. Unexplained portal fibrosis of variable degree may also be seen. Other rare lesions described include occult primary liver disease (PBC, PSC, AAT deficiency), nodular regenerative hyperplasia, sinusoidal dilation, portal eosinophilia, granulomas of uncertain cause, schistosomiasis and siderosis.

Other donor factors

Older donors are increasingly being considered for use. Particularly over 60 years of age, these have an increased risk of graft loss that is most evident after transplantation for hepatitis C, but there is also an increased risk of primary nonfunction, hepatic artery thrombosis, preservation/reperfusion injury and biliary complications. Avoidance of long cold ischaemic time (<7 hr) and performance of protocol liver biopsy have been shown to improve outcomes. Conventional microscopy shows no difference between young and older liver, but there is reduced hepatocyte volume, thickening and defenestration of the SECs and an increased susceptibility to preservation/reperfusion injury.

A number of other factors may cause damage to the donor liver before its removal for transplantation, without necessarily resulting in abnormalities that are recognizable morphologically. These include episodes of hypotension (leading to ischaemic graft injury), poor nutritional state (resulting in depletion of hepatic glycogen stores) and endotoxaemia, possibly related to mucosal injury of the gut. Moderate or severe hepatocellular siderosis, presumably related to genetic haemochromatosis, has been reported as an incidental finding in a small number of donor liver biopsies. Donor HFE mutations have been demonstrated in a few cases. In most cases, siderosis diminishes after LT, but in some patients, iron stores are slow to mobilize, and occasionally, severe siderosis has persisted for years after LT, suggesting that the liver itself may be abnormal. AAT deficiency can be transmitted with a liver graft, and this becomes problematic if there is concurrent hepatitis, which accelerates globule deposition even in the MZ phenotype.

Definition and related terms

Primary AMR is caused by preformed antidonor antibodies, with the greatest and most predictable risk being with ABO-incompatible grafts. Severe cases present with very early graft failure within days ( hyperacute rejection). The liver appears to have a relative resistance to antibody-mediated injury, and often, less severe changes occur in the setting of preformed blood group or anti-HLA antibodies, presenting with early graft dysfunction in the first month ( acute AMR). DSAs directed against HLA class II antigens appear to be more strongly associated with the development of acute AMR, particularly if they have a mean fluorescence intensity (MFI) >10,000. However, the sensitivity for detecting DSAs varies from one laboratory to another, and further standardization is required. DSAs can also develop de novo after LT and have been implicated in the pathogenesis of acute and chronic rejection. De novo DSAs directed at HLA class II antigens, especially HLA-DQ, appear to be particularly important in the pathogenesis of chronic AMR.

Incidence

Hyperacute rejection with rapid haemorrhagic necrosis of the allograft is distinctly rare in the liver. Further, in contemporary series it is no longer seen even in ABO-incompatible grafts with appropriate management, including rituximab administration and plasma exchange. A recent study reporting outcomes in 235 adults receiving ABO-incompatible grafts showed 3-year graft and patient survival rates of about 90%, comparable to that seen in recipients of ABO-compatible grafts. Acute AMR was the more common expression of ABO incompatibility in the 1980s, before the advent of effective immunosuppressive regimens, and caused graft failure within 30 days in about half the recipients. Currently, acute AMR is rare, probably occurring in about 5% of the 10–20% of allograft recipients with high titres of DSA and in approximately 7% of ABO-incompatible grafts, which amounts to ≤1% of liver transplants in total. However, this incidence increases to ≥10% when considering idiopathic early allograft failure before 3 months.

Chronic AMR is still an evolving concept in liver allograft pathology, so its significance remains uncertain. However, several studies in children, for whom recurrent liver disease is not a confounder, have shown the presence of late DSAs in about half of recipients and a significant association with increased graft inflammation and fibrosis in many of these. A lower frequency was found in a recent study of adult patients (20% had DSAs, with mean fluorescence intensity >5000), and this was also associated with increased graft fibrosis and graft failure after controlling for hepatitis C.

Clinical features and diagnostic criteria

AMR is of variable severity. Hyperacute AMR, now exceptionally rare due to the use of pretreatment for ABO-incompatible grafts, presents with severe graft dysfunction within the first week of transplantation. In contrast to renal allografts, where signs of hyperacute rejection are visible within a few minutes of reperfusion, changes in liver allografts may take several hours or even days to become manifest. An initial period of stable graft function is followed by a rapid rise in serum transaminases, coagulopathy and signs of acute liver failure. Decreased platelet count and total serum complement activity also occur from consumption and are indirect signs of humoral-mediated injury.

Acute AMR is more common and is characterized by unexplained graft dysfunction in the first few weeks, accompanied by falling platelet count and complement activity. Hyperbilirubinaemia is typically present, and transaminase levels may also be disproportionately high compared with the anticipated severity of PRI in the early post-transplant period. Acute AMR may manifest as severe steroid-resistant rejection, and correlation with DSA results is useful in these patients. Bortezomib, a proteasome inhibitor effective in depleting plasma cells, has been used in a small number of cases. With the increased use of effective anti-B lymphocyte pretreatment, AMR is more often milder and delayed and may be associated with late complications, including biliary strictures. The following four recommended diagnostic criteria for acute AMR include a combination of clinical, histological and laboratory findings :

• 1.
  

  Histopathological pattern of injury consistent with acute AMR

  
  
• 2.
  

  Positive serum DSAs

  
  
• 3.
  

  Presence of diffuse microvascular C4d deposition (C4d score = 3; see later)

  
  
• 4.
  

  Exclusion of other insults that might cause a similar pattern of graft injury

The clinical and biochemical features of chronic AMR are not well characterized. Many of the histological changes occurring in late biopsies that are potentially related to chronic AMR (e.g. unexplained graft inflammation and/or fibrosis) have been observed in protocol biopsies obtained from patients who appear to be clinically well and have normal liver biochemistry. The Banff Working Group recently proposed the following four features as suggestive of chronic AMR :

• 1.
  

  Histopathological pattern of injury consistent with chronic AMR (both a and b required)

  
  
  
  
  • a.
    

    At least mild portal inflammation with interface hepatitis and/or perivenular inflammation with necroinflammatory activity

    
    
  • b.
    

    At least moderate fibrosis (periportal/sinusoidal/perivenular)

  
  
  
  
• 2.
  

  Recent circulating DSAs

  
  
• 3.
  

  At least focal microvascular C4d deposition (C4d score ≥2)

  
  
• 4.
  

  Exclusion of other insults that might cause a similar pattern of graft injury

Histological features

Hyperacute rejection

Hyperacute AMR is largely confined to historical studies when ABO-incompatible grafts were transplanted without pretreatment. Histopathological findings vary according to the severity of the humoral reaction and the time when tissue samples are obtained. In clinical LT, severe coagulopathy precluded liver biopsy in most patients. In patients for whom liver biopsy was feasible, a typical sequence of events has been observed. The earliest changes in biopsies obtained 2–6 h after implantation include the deposition of fibrin, platelets, neutrophils and red blood cells in small vessels and hepatic sinusoids. As a consequence of endothelial injury, there is widespread neutrophilic exudation, congestion and coagulative hepatocyte necrosis, which can be seen in biopsies obtained 1–2 days after implantation. In severe cases this results in a characteristic picture of massive haemorrhagic necrosis throughout the liver ( Fig. 14.4 ). Lack of lymphocyte infiltration, or other typical features of cellular rejection, is another characteristic feature.

Hyperacute (humoral) rejection of liver allograft. A, Hepatectomy specimen obtained at retransplantation showing massive haemorrhagic necrosis. B, Histology shows panacinar haemorrhage and hepatocyte necrosis without an accompanying inflammatory reaction. (H&E stain.)

Immunohistological studies have shown deposition of immunoglobulins (IgG and IgM), complement (C1q, C3, C4) and fibrinogen in vascular and sinusoidal endothelium. These deposits tend to be most marked during the earlier stages of hyperacute AMR, between 2 h and 2 days after graft insertion, and rapidly diminish thereafter. Examination of failed allografts reveals more focal deposits of IgM and C1q, mostly confined to arteries. One case of possible hyperacute rejection was associated with strong C4d staining of the endothelium of portal vessels, particularly hepatic arteries, with focal staining of central venules and minimal staining of sinusoids.

Acute antibody-mediated rejection

The presence of preformed antidonor antibodies has been associated with a higher incidence and greater severity of acute cellular rejection. Importantly, there may also be atypical histological features that indicate the presence of AMR, such as portal/periportal oedema, portal haemorrhage, neutrophil-rich inflammatory infiltration in portal tracts and prominent ductular reaction, producing changes resembling those seen in biliary obstruction ( Fig. 14.5 A ). Careful inspection also shows more diagnostic changes in dilated portal microvessels, including capillaries of the peribiliary plexus and the portal venules that extend from the portal vein for a short distance into the lobule (inlet venules). Endothelial cells in these vessels show hypertrophy and cytoplasmic eosinophilia indicating activation, which is accompanied by microvasculitis. This differs from the endothelial injury seen in acute cellular rejection, because the inflammatory cells are within the lumen rather than beneath the endothelium, and the infiltrating cells are macrophages/monocytes, neutrophils and eosinophils rather than activated lymphocytes. Injury to tiny vessels forming the vasa vasorum of the biliary tract most likely underlies the later development of biliary strictures. There are usually changes of acute cellular rejection coexisting, sometimes progressing to chronic (ductopenic) rejection if not controlled. Changes described in the liver parenchyma include centrilobular hepatocellular swelling and canalicular cholestasis and sinusoidal neutrophil infiltrates.

Antibody-mediated rejection. A, Liver biopsy 13 days after transplant from patient with lymphocytotoxic-positive cross-match. Portal tract is oedematous with a mixed infiltrate of inflammatory cells. B, Marginal ductular reaction is also present. (H&E stain.) Immunostaining reveals C4d in small portal vessels.

(Courtesy of Dr. C. Bellamy, University of Edinburgh.)

Immunostaining for the complement component C4d, which is deposited at sites of classical complement pathway activation, can be done on routinely processed tissues and is used as a marker of AMR in the renal allograft and more recently in liver. However, interpretation of C4d staining in liver is more difficult than in other organs because of nonspecific reactivity and differing staining protocols. Deposition of C4d in liver biopsies from both ABO-incompatible and ABO-compatible grafts with features suggestive of AMR has been described ( Fig. 14.5 B ). The C4d deposits persist for at least several days but become more patchy. Positive staining is typically present in a strong linear or finely granular pattern, which may be present on both the lumenal and the basal surfaces of endothelial cells. Several different patterns have been described, the significance of which is still incompletely understood. Reviewing these in the context of their own extensive experience, Demetris et al. have suggested that typically, deposition should be both strong and diffuse (present in >50% of tracts), most frequently staining the endothelium in the proximal microvasculature (i.e. portal veins, portal capillaries, hepatic arteries, periportal sinusoids). The Banff Working Group recently proposed a similar approach for scoring C4d staining on a scale of 0–3; a C4d score of 3, which is needed to make a definitive diagnosis of AMR in a recipient of an ABO-compatible graft, requires the presence of C4d staining in >50% of portal microvascular endothelia (portal veins, capillaries, often extending into portal inlet venules or periportal sinusoids) involving >50% of portal tracts. Care is needed interpreting arteries because of nonspecific staining of the elastic lamina. Portal stromal staining has also been described in ABO-incompatible grafts, possibly reflecting more severe microvascular injury and release of immune complexes from the vessels. Staining of central vein endothelium and sinusoids is less frequently seen. Sinusoidal C4d expression may be the most reliable marker of AMR when immunofluorescence staining is done on frozen sections, although this has not been confirmed in a more recent study. Diffuse cytoplasmic C4d staining is seen in hepatocytes undergoing necrosis, regardless of the underlying cause, and therefore is not useful in the diagnosis of AMR. As discussed earlier, C4d immunoreactivity cannot be interpreted in isolation, since it can be seen in native livers with a variety of disorders, as well as in allografts with a variety of insults, including PRI, otherwise typical acute cellular rejection, chronic rejection, vascular thrombosis, biliary obstruction, recurrent viral hepatitis (hepatitis B and C), recurrent autoimmune disease (autoimmune hepatitis [AIH] and PBC) and de novo AIH.

Differential diagnosis

In isolation, the histological features previously described are not specific for AMR, and the clinical, histological and laboratory findings must be considered together. Massive haemorrhagic graft necrosis now occurs infrequently, and a clinical diagnosis of hyperacute rejection requires exclusion of other causes of graft failure occurring in the early postoperative period, in particular those associated with the syndrome of primary nonfunction. In contrast to PNF, most cases of hyperacute rejection follow an initial period of graft function. Acute graft failure in the early post-transplant period associated with the histological picture of massive haemorrhagic necrosis (MHN) has also been described in the absence of any demonstrable humoral mechanism. Nonhumoral factors implicated in this setting include ischaemia related to hepatic arterial kinking, gram-negative sepsis, opportunistic viral infections (e.g. herpes simplex, herpes zoster, adenovirus, enterovirus), recurrent infection with togavirus-like particles and a single-organ Shwartzman reaction. Although idiopathic MHN is rarely seen now, occasional cases with a similar pattern of graft injury are still being reported.

Acute AMR can mimic changes seen in bile duct obstruction, with a ductular reaction, portal oedema and a neutrophil-rich inflammatory infiltrate. The presence of interstitial haemorrhage, portal oedema, microvasculitis and interstitial rather than periductal neutrophilia should prompt C4d staining and testing for DSA; in this context, portal endothelial or stromal C4d staining favours a diagnosis of AMR. There is an increasing recognition that acute AMR interacts with T cell-mediated acute rejection, and overlapping histological features may thus occur. Features which favour AMR as the main diagnosis include portal vein endothelial cell hypertrophy, portal eosinophilia and eosinophilic venulitis, whereas lymphocytic portal inflammation and lymphocytic venulitis are less prominent in AMR. Similarly, acute cellular rejection with atypical features (e.g. prominent macrophage, neutrophil and eosinophil infiltrates) or ACR unresponsive to steroids should suggest that AMR is contributing to the graft injury.

Pathogenesis

In comparison with other organs such as the kidney, the liver is unusually resistant to AMR, as evidenced by the ability to carry out transplantation successfully in the face of positive antidonor crossmatching, including ABO incompatibility. Postulated reasons for the reduced susceptibility of the liver to AMR include (1) the presence of a dual blood supply, which may protect the organ from ischaemic damage; (2) a large sinusoidal vascular bed, which has more limited endothelial HLA expression compared with other solid organs and can dilute antibody binding across a larger endothelial surface; (3) release of soluble class I major histocompatibility complex (MHC) antigens into the circulation, which can bind to preformed antidonor antibodies; (4) the capacity for Kupffer cells and SECs to scavenge immune complexes and platelets and (5) the high regenerative capacity of the liver. Further evidence for the liver having a privileged immunological status in the context of organ transplantation has come from studies showing that transplantation of liver allografts into sensitized recipients is able to protect kidneys transplanted into the same individuals from developing hyperacute rejection.

The antibodies mediating AMR are heterogeneous. Isoagglutinins directed against blood group antigens are potent at inducing injury. The early and severe vascular injury that occurred in some patients has been modified with the increased use of novel therapeutic strategies, including anti-B lymphocyte therapies such as rituximab and plasmapheresis with or without splenectomy, so that most patients affected by AMR present later and less dramatically. In ABO-compatible grafts, DSAs are generally directed against HLA, usually class II, with variability in titre, complement-binding ability and immunoglobulin subclass that impacts their pathogenicity. The DSAs are now detected by flow cytometry and solid-phase immunoassays with increased sensitivity. Preformed DSAs disappear from the circulation in the majority of patients within the first week but can persist, especially when present at high titre. De novo DSAs, most frequently anti-HLA class II antibodies, develop at high titre in just under 10% of patients later after transplantation, usually in the context of reduced immunosuppression. Acute injury occurs through complement activation, causing coagulation and recruitment of innate immune cells including macrophages, neutrophils and eosinophils. Simultaneous development of acute cellular rejection is common. In other organs, complement-independent direct antibody binding to endothelial cells may have a role in chronic allograft injury, and a similar mechanism, possibly including direct binding of DSAs to activate hepatic stellate cells, may occur in liver to stimulate gradual fibrosis in the liver allograft.

Definition and related terms

Acute cellular rejection (ACR) can be defined as T cell-mediated damage to the liver allograft characterized by cellular infiltrates, principally present in portal areas and associated with damage to bile ducts and vascular structures. Inflammatory changes are also usually seen in the liver parenchyma, mainly around terminal hepatic venules. Most cases occur in the early postoperative period and are responsive to immunosuppression. ACR has also been called ‘acute rejection’ or ‘cellular rejection’ in the past, but the term ACR allows unambiguous distinction from acute AMR. Other terms include nonductopenic rejection, rejection without duct loss, early rejection and reversible rejection. As mentioned earlier, ‘T cell-mediated rejection’ (TCMR) has recently been suggested as the most appropriate term to use.

Incidence and risk factors

ACR is the most common form of liver allograft rejection. The incidence varies according to whether ACR is defined on the basis of clinically significant rejection (i.e. rejection accompanied by graft dysfunction requiring additional immunosuppression) or whether it is defined simply on the basis of histological abnormalities. In early studies, histological features compatible with a diagnosis of ACR have been observed in up to 80% of protocol biopsies obtained about the end of the first week after LT and it was recognized even then that not all these patients required treatment. A similar frequency of histological rejection (77%) was observed in a more recent study where protocol biopsies were obtained at the end of the first week. The incidence of clinically significant rejection is lower and appears to be declining, probably related to improvements in immunosuppressive therapy. In 2002 a systematic review showed biopsy-proven ACR with graft dysfunction in 35% of patients, but more recently the incidence under current immunosuppressive regimens has been 11–25% within the first year. A higher incidence of ACR has been noted in patients undergoing LT for autoimmune liver diseases and in those transplanted for hepatitis C, although the latter may reflect different approaches to the use of immunosuppression and the assessment of post-transplant biopsies in HCV-positive patients. Conversely, a lower incidence of ACR has been documented in patients undergoing transplantation for alcoholic liver disease and chronic HBV infection and in recipients of HLA-zero-mismatched grafts. A putative lower risk after LRLT remains controversial. Late ACR, occurring >3–6 months after LT, also occurs in 7–19% of recipients.

Clinical features

The majority of ACR episodes occur within the first month of transplantation. Clinical manifestations of acute rejection include pyrexia, graft enlargement and tenderness and reduced bile flow. Biochemical abnormalities typically have a predominantly cholestatic pattern. A sudden rise in serum transaminases may be a manifestation of parenchymal-based rejection changes. Peripheral blood leukocytosis and eosinophilia are also usually present. Clinical and biochemical abnormalities are nonspecific, and the diagnosis therefore requires histological confirmation. Late ACR cases often have atypical features, as discussed next.

Histological features

Liver biopsy specimens show various combinations of a diagnostic portal-based triad, described by Snover et al. and subsequently confirmed in other studies of post-transplant liver biopsies (reviewed by the Banff Working Group). In recent years, there has been increased interest in a spectrum of changes involving terminal hepatic venules and the surrounding liver parenchyma.

Portal tract lesions in acute cellular rejection

The three components of the diagnostic triad are portal inflammation, bile duct damage and venular endothelial inflammation (also known as endothelitis, endotheliitis or endothelialitis). At least two of these three features are required for a diagnosis of acute rejection. Because the inflammatory lesions occurring in acute rejection can vary considerably in intensity in different parts of a single biopsy specimen, it is recommended that sections be obtained from a series of levels and that a minimum of five portal tracts be available for examination.

Portal inflammation begins as a lymphocytic infiltrate. By the time that rejection presents clinically, there is typically a mixed infiltrate of cells, including lymphocytes (mostly T cells), large activated ‘blast’ cells, macrophages, neutrophils and eosinophils ( Fig. 14.6 A ). All these cell types are also involved in mediating damage to bile ducts and endothelial cells. The presence of large numbers of eosinophils may be helpful in identifying a more severe form of rejection less likely to respond to additional immunosuppressive therapy. Plasma cells occur in some biopsies and increase with severity of rejection. The presence of prominent interface hepatitis with spillover of inflammatory cells into the lobule is also a feature of more severe cellular rejection. Mast cells may be present in varying numbers. Portal tract granulomas are rarely seen in acute rejection.

Portal tract lesions in acute liver allograft rejection. A, Portal tract contains a dense, mixed inflammatory infiltrate, including lymphocytes, blast cells, neutrophils and eosinophils. B, An interlobular bile duct shows prominent inflammatory infiltration, mainly with neutrophils. C, Portal venule shows subendothelial inflammatory infiltration associated with lifting and focal disruption of the endothelium. D, Inflammatory infiltration of a small arterial branch. These changes are rarely seen in needle biopsy specimens. (H&E stain.)

The initial damage to bile ducts is probably mediated by lymphocytes, but by the time rejection is clinically evident, there is usually a mixed infiltrate, in some cases including a prominent component of neutrophils ( Fig. 14.6 B ). Bile ducts are typically cuffed and focally infiltrated by inflammatory cells and may show degenerative changes in the form of cytoplasmic vacuolation, pyknosis and focal disruption of the basement membrane. In cases where portal inflammation is particularly intense, bile ducts can be effaced by inflammatory cells and are difficult to identify in routinely stained sections. Immunostaining for bile duct keratins is useful in demonstrating that bile ducts are still present in this situation. In some cases the presence of large numbers of neutrophils, including lumenal aggregates of pus cells, may mimic changes seen in ascending infective cholangitis. Large numbers of neutrophils have also been identified in samples of bile obtained from patients with ACR.

Venular inflammatory changes are seen in portal and hepatic vein branches. In early or mild cases there is focal lymphoid attachment to the lumenal surface of endothelial cells. In more advanced or severe cases there is subendothelial infiltration, associated with lifting and sometimes disruption of endothelial cells ( Fig. 14.6 C ). Cells associated with endothelial damage are mostly lymphocytes. However, a mixed infiltrate resembling that seen in bile ducts may also be present. In most cases, endothelial inflammation only affects a small segment of the vessel. Involvement of the entire circumference of the venule is generally confined to cases with severe rejection. Venular endothelial inflammation has generally been regarded as the most specific feature of liver allograft rejection, but it is not invariably present, particularly in cases occurring beyond the early post-transplant period. Furthermore, venular endothelial inflammation can be seen in many other conditions in which there is inflammatory infiltration of portal tracts or the liver parenchyma, including viral hepatitis, PBC, AIH and lymphoproliferative diseases.

Arterial lesions including endothelial inflammation and fibrinoid necrosis have been reported but are rarely seen in needle biopsy specimens ( Fig. 14.6 D ). When present, these have been regarded as a sign of severe damage, possibly reflecting concomitant AMR, with an increased likelihood of progression to chronic rejection. Angiographic studies demonstrating attenuation of large and medium-sized arteries suggest that these vessels may also be affected in acute rejection.

Bile ductular reaction is often seen in biopsies showing features of ACR and may in part be a response to other portal tract changes, especially bile duct damage. Other possible causes of bile ductular reaction in the early post-transplant period include delayed effect of PRI, acute AMR and SFSS.

Parenchymal changes in acute cellular rejection including central perivenulitis

Lobular inflammatory lesions comprise a spectrum of changes, principally involving hepatic venules and the surrounding liver parenchyma for which the term ‘central perivenulitis’ (CP) is now most widely used. In some cases there may be a more diffuse lobular hepatitis or a predominantly sinusoidal pattern of lymphocytic infiltration. Other terms used to describe these changes include central venulitis, centrilobular necrosis, centrilobular necroinflammation, centrilobular alterations, centrilobular changes and ‘hepatitic phase’ of rejection. Other parenchymal changes frequently seen in association with acute rejection include cholestasis, hepatocyte ballooning, fatty change and focal apoptotic (acidophil) body formation. These lesions tend to be most marked in perivenular regions and in some cases may be causally related to ACR. Particularly in the early post-transplant period, however, much of the parenchymal damage is more likely to be related to PRI than rejection.

The histological features and significance of CP depend on when it is seen (early or late) and whether portal changes of ACR are also present. At one end of the spectrum, usually seen in early post-transplant biopsies, central vein endotheliitis is a prominent feature ( Fig. 14.7 A ). Portal tract changes of ACR are typically also present, usually at least moderate in severity. The diagnosis and grading of rejection in these cases are relatively straightforward. In other cases, usually occurring significantly later after transplant, centrilobular necroinflammatory lesions are present with minimal or no central vein inflammation ( Fig. 14.7 B ) and sometimes also with minimal or mild portal inflammatory changes, also referred to as ‘isolated central perivenulitis’(ICP). A diagnosis of rejection in such cases is less easily established, and other causes of centrilobular damage also need to be considered ( Table 14.5 ) (see later discussion). In two studies, features of ICP were observed in 22% of children biopsied >3 months after LT and 28% of adults undergoing protocol biopsy >3 years after transplant. The inflammatory infiltrate is mainly mononuclear with lymphocytes predominating, sometimes with conspicuous plasma cells. Foci of mild congestion and pigmented macrophages may also be seen. Perivenular necroinflammatory lesions may also be associated with the gradual development of parenchymal fibrosis in zone 3, with linkage in more severe cases.

Centrilobular lesions in acute liver allograft rejection (central perivenulitis). A, Hepatic venule shows subendothelial inflammation. Inflammatory cells extend into the surrounding liver parenchyma, where there is a narrow zone of hepatocyte dropout. B, Perivenular necroinflammatory lesion with normal hepatic venule. (H&E stain.)

Table 14.5

Possible causes of centrilobular (acinar zone 3) necrosis in the liver allograft

Cause	Type/example
Ischaemia	Preservation/reperfusion injury
	Vascular occlusion (hepatic artery, portal vein, hepatic vein)
Rejection	Acute
	Chronic
Viral hepatitis (recurrent or acquired)	Hepatitis B
	Hepatitis C
Autoimmune hepatitis (recurrent or acquired)
Drugs	Azathioprine
Other	‘Idiopathic’ chronic hepatitis

 

The prognostic significance of centrilobular inflammation and dropout depends on the histological context. Evidence suggests that the presence of perivenular necroinflammatory lesions with portal features of ACR in the first few months after LT indicates a more severe form of acute rejection, which is less likely to respond to immunosuppression and is more likely to progress to chronic rejection. In many patients the centrilobular changes appear to be present at an early stage, before bile duct loss is evident; recognition of this process and initation of appropriate immunosuppressive therapy may prevent progression to irreversible changes of chronic rejection. On the other hand, when present as ICP, the prognosis is generally favourable. Most patients, if treated at all, respond to bolstered immunosuppression, but the lesion may persist, recur or fluctuate. If there is no response to increased baseline immunosuppression or a single steroid bolus, the inflammation may behave more like de novo AIH and may respond to the reintroduction of steroids or mycophenolate mofetil for a time rather than more aggressive depleting therapies such as OKT3. When untreated, there may be progression to centrilobular fibrosis. A grading scheme for the severity of CP proposed by the Banff Working Group has been shown to correlate with adverse outcomes in one study of ICP.

In some patients, inflammation of hepatic venular endothelium may be associated with the development of veno-occlusive lesions and more severe congestive changes resembling venous outflow obstruction ( Fig. 14.8 ). Many of these patients have ACR with particularly severe endothelial inflammation. However, the lesion can also develop more insidiously, without overt portal changes of ACR, although it still appears to be responsive to optimization of immunosuppression.

Rejection related veno-occlusive disease. A, Severe congestion and hepatocyte loss are present in the centrilobular region, mimicking the changes seen in venous outflow obstruction. (H&E stain.) B, Hepatic vein is occluded by a mixture of inflammatory cells and immature fibrous tissue. (Haematoxylin and van Gieson stain.)

Grading of acute cellular rejection

The Banff Schema devised by an international panel of liver transplant pathologists, physicians, surgeons and scientists has been widely used for grading the severity of ACR. A slightly modified version of the original grading scheme, which incorporates perivenular necroinflammatory changes as well as portal-based inflammatory lesions, appears in the latest consensus document produced by the Banff Working Group. The schema incorporates two components: a global assessment of the overall rejection grade ( Table 14.6 ) and scoring of the three main features of liver allograft rejection semiquantitatively on a scale of 0 (absent) to 3 (severe), to produce an overall rejection activity index (RAI) ( Table 14.7 ). Studies have shown that the Banff Schema is simple to use, reproducible and clinically useful in making decisions regarding therapy and may also be helpful prognostically. In one study of 575 ACR episodes, the presence of moderate or severe rejection correlated with higher transaminase levels, development of perivenular fibrosis and increased risk for developing chronic rejection. However, another study in a similar-sized cohort receiving tacrolimus immunosuppression did not find any significant association of RAI with either steroid responsiveness or progression to chronic rejection.

Problems with applying the Banff Schema may be encountered when biopsies are taken beyond the early post-transplant period, partly because the histological features at this stage can be different, but also because there are other potential causes for cellular infiltration in the liver allograft. This particularly applies to biopsies obtained from HCV-positive individuals (discussed later) and those with unexplained chronic hepatitis. Importantly, when any uncertainty exists regarding the overall diagnosis of rejection, grading should not be done in these patients.

Differential diagnosis

The diagnosis of ACR rarely poses problems during the first month after transplant, because other causes of graft inflammation are infrequently seen during this period. Greater problems exist beyond the early post-transplant period, when other causes of graft inflammation become more common. Particularly important in this respect is recurrent hepatitis C infection, discussed later. Other diseases associated with a predominantly portal-based inflammatory infiltrate include AIH (recurrent or de novo ) and Epstein–Barr virus-associated post-transplant lymphoproliferative disease. In addition to the combination of changes seen in the typical diagnostic triad, a useful feature pointing to a diagnosis of ACR is the presence of a mixed population of inflammatory cells, which is rarely seen to a marked degree in the other allograft conditions associated with portal inflammation.

Another problem with the assessment of late acute rejection is that the features may mimic chronic hepatitis. When centrilobular necroinflammatory changes predominate, usually beyond the early post-transplant period, the diagnosis of ACR is straightforward if the triad of diagnostic portal changes is also present. Many cases can still be attributed to rejection in the absence of typical portal tract changes, although other causes of centrilobular injury should first be excluded (see Table 14.5 ), as discussed later. The presence of persistent centrilobular necrosis should raise the suspicion of progression to chronic rejection and warrants careful examination of the bile ducts.

In cases where bile ductular reaction appears unduly prominent, the possibility of biliary tract pathology should be considered, particularly if there is also portal oedema and an infiltrate disproportionately rich in neutrophil polymorphs. Large bile duct obstruction should be relatively easy to exclude radiologically. However, in some cases, subtle biliary features may represent the early stages of ischaemic bile duct damage, which may escape radiological recognition. Biliary features may also be a manifestation of acute AMR. If suggestive portal microvasculitis with macrophages, eosinophils and neutrophils rather than lymphocytes is present, it should prompt C4d staining and assessment of DSAs.

Response to treatment

Mild (often subclinical) ACR often resolves spontaneously without the need for additional immunosuppression. The development of mild rejection in the early post-transplant period may have a beneficial effect in inducing long-term graft tolerance. A small number of patients with more severe forms of ACR, including clinical and biochemical signs of graft dysfunction, have resolved spontaneously without additional immunosuppression. The concept of ‘self-limiting rejection’ is well recognized in animal models of LT and may also be relevant to human LT.

In the majority of patients in whom histological features of ACR are accompanied by clinical/biochemical signs of graft dysfunction, the administration of additional immunosuppression results in resolution, with no adverse impact on long-term graft function. In early studies of follow-up biopsies obtained after treatment for acute rejection, these showed bile duct atypia and features resembling those seen in large bile duct obstruction. Repeat biopsies are no longer obtained if there is adequate biochemical response to additional immunosuppression. In cases where features of cellular rejection persist despite treatment with corticosteroids (steroid-resistant rejection), treatment with other drugs (e.g. OKT3 or mycophenolate mofetil) may result in resolution of rejection. A small number of patients are unresponsive to all forms of immunosuppression (‘intractable rejection’), and many of these either have or will develop features of chronic rejection. A role for acute AMR in some cases of severe rejection unresponsive to conventional immunosuppression is now recognized.

Definition and related terms

Chronic rejection (CR) can be defined as immune-mediated damage to the liver allograft that is associated with potentially irreversible injury to the bile ducts, arteries and veins. It is characterized histologically by two main features: dystrophic epithelial changes and the loss of small bile ducts and an obliterative vasculopathy affecting large and medium-sized arteries. CR occurs later than acute rejection; many cases evolve from ACR incompletely or unresponsive to immunosuppression. Because bile duct loss is generally considered to be the most important diagnostic feature in needle biopsy specimens, the term ‘ductopenic rejection’ has been most widely used as an alternative to chronic rejection. Other terms that have been used but fail to recognize the full scope of the changes seen include late rejection, irreversible rejection, vanishing bile duct syndrome, rejection with bile duct loss and vascular rejection.

Incidence and risk factors

CR is considerably less common than acute rejection. The incidence in series reporting patients transplanted before 1991 ranged from 2% to 20%, a wide variation that may partly reflect different criteria used to define CR. The incidence of CR is declining, presumably because of more effective immunosuppression, and now results in graft failure in <2% of cases.

Risk factors identified for CR can be divided into two main categories, as follows:

• 1.
  

  Donor/recipient factors include transplantation for autoimmune liver disease, male-to-female sex mismatching of donor to recipient, non-European recipient ethnic origin, young recipient age, old donor age and presence of high-titre DSAs of IgG3 subclass. A lower rate of CR has been observed in recipients of living-related grafts than in those with cadaveric donor organs.

  
  
• 2.
  

  Post-transplant factors include the severity and number of episodes of acute rejection, late presentation of acute rejection (>1 month post-transplant), cyclosporine use (versus tacrolimus), cytomegalovirus infection, HBV and HCV infection and interferon therapy for viral hepatitis. Those undergoing retransplantation for CR have an increased risk of developing CR in subsequent grafts.

Clinical features

CR often occurs as a consequence of repeated episodes of ACR that are unresponsive to immunosuppression. Many cases occurred in the first year with a peak incidence at 2–6 months after LT in studies from the 1980s and 1990s. In some cases there was a more acute presentation, with rapid progression to graft failure within a few weeks of transplantation (‘acute vanishing bile duct syndrome’). Classic cases of CR presenting with graft failure during the first year after transplant are now less common, reflecting improvements in immunosuppression. Instead, more cases are diagnosed later, often in the context of poor compliance or reduced immunosuppression for infection. These patients may have different clinical features, including a more insidious presentation and more gradual graft damage. Histological features may also be different and, in some cases, are further modified by interaction with other graft complications, such as recurrent HCV infection, making liver biopsy assessment difficult.

Clinically, CR is characterized by progressive jaundice accompanied by cholestatic liver biochemistry. The transition from acute to chronic rejection may be associated with an elevation in aspartate transaminase (AST) levels, most likely related to the presence of central perivenulitis. In common with acute cellular rejection, the clinical and biochemical manifestations of CR are nonspecific, and the diagnosis therefore requires histological confirmation.

Histological features

Portal tract changes

Two main diagnostic features have been described for CR: (1) dystrophic or ‘dysplastic-like’ biliary epithelial changes or loss of bile ducts from >50% of portal tracts and (2) foam cell arteriop­athy. Characteristic but less specific changes are also present in the liver parenchyma. Some cases of late CR may have different histological features, including chronic hepatitis-like changes.

During the early stages of CR, bile ducts show inflammatory infiltration, indistinguishable from that seen during potentially reversible acute cellular rejection. When there has been an incomplete biochemical response to additional immunosuppression, the overall degree of inflammation in portal tracts may be reduced. However, bile ducts show continued lymphocytic infiltration associated with nuclear pleomorphism, disordered polarity and focal attenuation and/or disruption of biliary epithelium. The cytoplasm is often eosinophilic, and with the irregular nuclei, this produces a ‘dysplastic-like’ or atrophic appearance ( Fig. 14.9 A ). These changes are associated with features of ‘replicative senescence’ (e.g. nuclear p21 expression) and are widely regarded as an early sign of impending bile duct loss. When these appearances are seen without loss of the majority of bile ducts, there is a greater likelihood of reversal. Interestingly, a recent study suggested that the expression on biliary epithelial cells of the senescence-associated factor p21 and a marker of mesenchymal differentiation (S100A4) may be occurring as a relatively early stress-related response in liver allograft rejection, which is potentially reversible if the hostile environment is modulated.

Bile duct lesions in chronic rejection. A, Early chronic rejection. Liver biopsy obtained 5 months after transplant following unsuccessful treatment of an acute rejection episode. An interlobular bile duct shows nuclear pleomorphism and disordered polarity, producing a ‘dysplastic’ appearance. (H&E stain.) B, Late-stage chronic rejection. Portal tract has a characteristic ‘burnt-out’ appearance with no recognizable bile duct branch. Only mild inflammatory changes are present. There is no obvious ductular reaction. (H&E stain.) C, Large bile duct lesion in end-stage chronic rejection. In this hepatectomy specimen obtained at retransplantation, the lumen of a large bile duct is occluded by immature fibrous tissue. No residual biliary epithelium is identified. (Haematoxylin and van Gieson stain.) D, Keratin 7 immunostaining confirms bile duct loss and the absence of a ductular reaction. Numerous K7-positive cells with an intermediate hepatobiliary phenotype are present in the periportal region. P, Portal tract.

As the disease progresses to a later stage, there is loss of bile ducts, typically associated with a diminishing cellular infiltrate that eventually produces a characteristic ‘burnt-out’ appearance in end-stage livers ( Fig. 14.9 B ). Bile duct loss principally affects the small interlobular bile ducts and is thus readily diagnosed in needle biopsy specimens. In normal liver allografts, at least 70–80% of portal tracts should contain a bile duct of equivalent diameter to the hepatic artery branch. Bile duct loss should be seen in >50% of portal tracts to make a firm diagnosis of late CR. However, there are problems in counting bile ducts accurately, particularly in small needle biopsy specimens. Loss of both the artery and the bile duct from a portal tract may also impede counting. An adequate sample, usually 16G passes with at least 11 portal tracts and 20–30 mm combined length and ideally containing 20 or more portal tracts, and/or the demonstration of ductopenia in several biopsies may be required before a confident diagnosis of bile duct loss can be made. In hepatectomy specimens obtained at retransplantation, there may be loss of epithelium from medium-sized (septal) and large (hilar) ducts. The latter sometimes show a distinctive pattern of lumenal obliteration by fibrous tissue and inflammatory cells ( Fig. 14.9 C ).

A notable feature is the absence of bile ductular reaction or periportal fibrous expansion in the majority of cases, the main exception being an associated distal biliary stricture. This is in contrast to other diseases associated with loss of bile ducts, in which these secondary changes are almost always present. Studies have attributed the lack of ductular reaction in CR to an increase in apoptosis or a reduction in proliferative activity within the ductular compartment. A close relationship has been observed between ductular reaction and periportal neovessel formation in a number of liver diseases, and a lack of these two reparative responses has been implicated in the pathogenesis of irreversible bile duct loss in CR. Staining for biliary keratins such as K7 helps to confirm the absence of bile ducts and lack of a ductular reaction in CR and often also shows prominent positive staining of periportal cells with an intermediate hepatobiliary phenotype ( Fig. 14.9 D ).

The characteristic vascular lesions of CR are seen in large and medium-sized arteries and are typically manifest as intimal aggregates of lipid-laden foamy macrophages ( Fig. 14.10 A ), although other layers of the arterial wall can also be affected. These occlusive arterial foam cell lesions may produce abnormalities that can be detected angiographically. In some cases with a more acute presentation, there is a prominent infiltrate of inflammatory cells ( Fig. 14.10 B ), mainly T lymphocytes, suggesting an overlap with ACR. Conversely, in cases with a more prolonged course, there are increasing numbers of myofibroblasts associated with varying degrees of intimal fibrosis as well as fragmentation of the internal elastic lamina. However, advanced fibromuscular intimal thickening of the type classically seen in end-stage CR affecting renal or cardiac allografts is only rarely found in liver allograft rejection ( Fig. 14.10 C ). The macrophages and mesenchymal cells in arterial lesions are of recipient origin. Because these arterial lesions rarely affect small vessels of the size sampled in needle biopsy specimens, the definitive diagnosis of chronic vascular rejection is usually only made when the whole liver is available for examination ( Fig. 14.10 D ). However, smaller portal tracts may show a reduced number of small arterial branches and other microvascular channels. These changes can occur during the early stages of CR, before bile duct loss is present.

Arterial lesions in chronic rejection. A, Medium-sized muscular artery contains an intimal foam cell lesion resulting in lumenal occlusion. B, Medium-sized muscular artery shows prominent inflammatory infiltration involving all layers of the vessel wall. The majority of infiltrating cells are T lymphocytes. C, Fibromuscular intimal thickening in chronic liver allograft rejection. These lesions are seen less often than intimal foam cell lesions. When present, they probably reflect longstanding damage. D, Bisected liver allograft removed 4 months after transplantation. Occluded arterial branches stand up as yellow cords or nodules within large, perihilar portal tracts. One well-opened portal vein branch to the left shows yellow thickening of its wall. ( A and B, H&E stain; C, Elastic haematoxylin and van Gieson stain.)

Inflammatory and/or foam cell lesions are also seen in portal and hepatic venules in some cases of CR, particularly those associated with a more acute presentation, again suggesting areas of overlap with ACR. Inflammatory lesions in hepatic venules may also result in fibrous lumenal obliteration, producing changes similar to those seen in hepatic veno-occlusive disease. Similar changes can also occur in small portal vein branches ( Fig. 14.11 ). A combination of fibroinflammatory occlusive lesions involving portal and hepatic veins appears to be important in the pathogenesis of the parenchymal fibrosis in CR, in some cases resulting in a cirrhosis-like appearance, with a venocentric pattern. A similar mechanism has been postulated for the development of fibrosis and cirrhosis in nontransplanted liver.

Portal veno-occlusive disease in chronic rejection. A portal vein branch shows occlusion by fibrous tissue. The presence of this lesion in combination with hepatic veno-occlusive lesions may be important in the pathogenesis of parenchymal fibrosis in chronic rejection. (Elastic haematoxylin and van Gieson stain.)

In the majority of CR cases, bile duct loss and occlusive arteriopathy are both present. Morphometric studies have demonstrated a parallelism between the severity of these two components of CR and have suggested that ischaemia may be a factor contributing to bile duct loss in the liver allograft. However, there are well-documented cases with a purely ductopenic or a purely vascular form of CR. In a combined series of 72 cases from three centres, 51 (71%) had both lesions, 10 (14%) had ductopenia alone and 11 (15%) had a purely vascular form of CR.

Parenchymal changes

Perivenular bilirubinostasis is a prominent finding in CR and in most cases is presumably related to bile duct loss. Cholestasis can also be seen with the purely vascular form of CR, suggesting that ischaemia may also be a factor in some cases. Sinusoidal foam cells are frequently seen and are probably also a response to cholestasis.

Perivenular necrosis is also a common finding in CR ( Fig. 14.12 A ) and typically occurs as a sequela to the necroinflammatory lesions, which are seen during the preceding phase of acute cellular rejection. Humoral mechanisms may also be involved; the deposition of C4d has been described in portal and central venules as well as perivenular sinusoids. In cases with incomplete biochemical response to additional immunosuppression, the cellular infiltration in perivenular regions often subsides, but hepatocellular dropout persists. Necrosis typically has a lytic pattern and is accompanied by reticulin collapse and immature collagen fibre deposition. In some cases there may be more extensive necrosis with bridging and nodule formation ( Fig. 14.12 B ). Even in cases where zonal necrosis has been detected in serial biopsies over several months, the lesions frequently retain an appearance suggesting acute damage, without the formation of mature collagen or elastic fibres. This suggests a dynamic equilibrium between hepatocyte loss and regeneration in perivenular regions. However, in some cases there is development of more mature fibrous lesions, which may ultimately progress to cirrhosis-like changes. A range of rejection-related ischaemic mechanisms has also been implicated in the pathogenesis of parenchymal necrosis and fibrosis; these include occlusive lesions in large and medium-sized arteries, loss of small arterial branches and occlusive lesions involving portal and hepatic veins. Although centrilobular necrosis has been regarded as a ‘surrogate marker’ of rejection-related arteriopathy, a definite association between these two processes has not been convincingly demonstrated.

A, Parenchymal damage in chronic rejection. There is an area of lytic necrosis involving acinar zone 3. Mild congestion is also present in this area, along with severe cholestasis. B, More extensive parenchymal damage with areas of bridging necrosis accompanied by a moderately dense infiltrate of inflammatory cells. (H&E stain.)

Staging of chronic rejection

The Banff Schema was updated by the Banff Working Group in 2000 to incorporate features related to the staging of CR. A slightly modified version of the 2000 staging scheme, which recognizes different patterns of fibrosis that may be related to CR, appears in the latest consensus document produced by the group ( Table 14.8 ). In staging changes related to CR, a distinction is made between changes occurring during the early phase, when the disease is potentially still reversible, from those indicating advanced or irreversible disease, for which retransplantation is generally the only therapeutic option. In the liver biopsy diagnosis of early CR, particular emphasis is placed on the recognition of biliary epithelial atypia in the majority of bile ducts, which can be seen before bile duct loss has occurred, and zone 3 necroinflammatory lesions, which also occur at an early stage in the disease process. Application of Banff staging schema has been shown to improve greatly the sensitivity of needle biopsies for diagnosing CR at an early stage. More importantly, the use of potent immunosuppressive agents at an early stage in the disease process may prevent the development of irreversible graft damage. Late CR is recognized if at least two of the advanced features listed in Table 14.8 are present. Although bile duct loss has been the most widely used feature indicating progression to late (irreversible) CR, there are problems in counting bile ducts accurately in needle biopsy specimens, particularly concerning sampling variation. Examination of hepatectomy specimens with end-stage disease sometimes reveals marked variation in the degree of bile duct loss from one part of the liver to another. Perivenular fibrosis with bridging may also be indicative of irreversible graft damage, although this is not always a reliable criterion. As discussed earlier, areas of overlap exist between acute and chronic rejection, and an individual biopsy may show features of ongoing inflammatory activity, which can be graded, as well as signs of progressive liver injury (e.g. bile duct loss), which can be staged.

Some of the other features listed in Table 14.8 (e.g. lesions affecting large arteries and bile ducts) can only be diagnosed reliably in hepatectomy specimens. For these lesions, the distinction between ‘early’ and ‘late’ CR is based on theoretical concepts, which cannot be applied to needle biopsy assessment. Other features (e.g. cholestasis, sinusoidal foam cells) are nonspecific findings and can be seen in a variety of other conditions.

Differential diagnosis

A number of diseases in the liver allograft may be associated with portal inflammatory infiltration, bile duct damage and in some cases bile duct loss. These include recurrent viral hepatitis (in particular hepatitis C), recurrent biliary disease (PBC, PSC) and ischaemic cholangiopathy which, as discussed later, has histological features closely resembling those seen in PSC.

The relationship between recurrent HCV infection and liver allograft rejection is considered in more detail later. Cases with combined features of hepatitis C and CR pose particular problems for diagnosis and management. Although CR is similar to PBC and PSC (and ischaemic cholangiopathy) in that all three are vanishing bile duct diseases, there are important histological differences. The granulomatous bile duct lesions in PBC and the sclerosing duct lesions in PSC are different from the inflammatory and ‘dysplastic-like’ bile duct lesions seen during the early stages of CR. Perhaps the most useful feature distinguishing typical cases of CR from other diseases with bile duct loss is the absence of secondary biliary features such as ductular reaction, periportal fibrosis and deposition of copper-associated protein. However, as discussed earlier, these secondary biliary features may be seen in rare cases of late CR developing several years after LT. Bilirubinostasis, as seen in CR, is rarely prominent in other biliary tract diseases unless there is advanced fibrosis/cirrhosis, severe ductopenia or, in the case of PSC, a major bile duct stricture associated with features of large duct obstruction. The finding of severe unexplained cholestasis occurring beyond the early post-transplant period unaccompanied by portal tract changes suggesting biliary obstruction should therefore prompt a careful search for subtle bile duct lesions that might suggest a diagnosis of early CR.

The differential diagnosis of the centrilobular lesions which occur in chronic (and acute) rejection is more difficult. Although many cases are rejection related, a number of other factors, including vascular problems, viral hepatitis, AIH (recurrent or acquired) and drug toxicity may also be implicated (see Table 14.5 ).

Among the vascular causes of centrilobular necrosis (CLN), preservation/reperfusion injury is unlikely to result in lesions persisting beyond the early postoperative period. Even in biopsy specimens obtained the first week after transplant, CLN is most likely to be rejection related, particularly if accompanied by a lymphocytic inflammatory infiltrate. Rejection-associated changes resulting in hepatic veno-occlusive lesions and surrounding congestion and necrosis may be indistinguishable from other causes of venous outflow obstruction. In many cases the presence of rejection-related inflammatory changes, particularly with marked endothelial inflammation, either with or preceding histological features suggesting venous outflow obstruction, is helpful in showing rejection as the likely cause.

Centrilobular inflammation and CLN may be seen in association with recurrent or, more rarely, acquired viral or autoimmune hepatitis. Knowledge of the original indication for LT and post-transplant viral and immunology studies are helpful in the diagnosis of recurrent viral or autoimmune hepatitis. Features favouring a diagnosis of rejection include prominent hepatic venular endothelial inflammation together with bilirubinostasis and/or ballooning in surrounding viable hepatocytes. The presence of conspicuous bile duct injury and bile duct loss and portal venular endothelial inflammation are also features that favour a diagnosis of rejection. Conversely, the presence of a plasma cell-rich infiltrate, portal interface hepatitis and serological detection of autoantibodies indicates a diagnosis of AIH.

CLN and hepatic veno-occlusive lesions can also be seen as a toxic injury related to use of the immunosuppressive drug azathioprine and as a manifestation of ‘idiopathic’ post-transplant chronic hepatitis.

Response to treatment and outcome

The treatment of CR depends on the stage at which the disease is diagnosed. Patients with early CR, in whom bile duct atypia is present with minimal or no duct loss, may respond to a range of immunosuppressive agents, including tacrolimus, OKT3, mycophenolate mofetil and rapamycin (sirolimus). In contrast, patients with advanced CR, typically associated with severe biochemical cholestasis and advanced ductopenia, are generally unresponsive to immunosuppression and often require retransplantation. However, the exact point of irreversibility cannot be defined histologically. A small number of patients with apparently advanced CR (including bile duct loss in >50% of portal tracts) have recovered spontaneously or with the use of additional immunosuppression. Interestingly, some of these patients have had follow-up liver biopsies showing a persistent paucity of bile ducts without other histological features of CR. Ductopenia has also been noted as an incidental finding in protocol biopsies taken at annual review from patients who are clinically well with no previous biopsies suggesting CR. Other cases may have gradual bile duct loss over 5–10 years without progressing to graft failure. These observations suggest that some patients may have permanent duct loss as a result of rejection, but that sufficient ducts remain to allow the graft to function well. Because histological features in needle biopsy specimens are not reliable in identifying irreversible graft damage caused by CR, clinical features and biochemical markers are also used to determine the point at which retransplantation is required.

Targets for immune damage in liver allograft

Allograft rejection is a vigorous immunological reaction to foreign antigens of another member of the same species and is a complex interplay between the innate and adaptive arms of the immune system. This reaction is primarily to foreign, polymorphic major histocompatibility complex (MHC) antigens that are present on all nucleated cells. In uncontrolled ACR, mediated mainly by T cells, the vigour of the response results from the high frequency of antigraft lymphocytes, which are 100–1000 times more numerous than in usual immune responses. All the nucleated cells of the liver are potential targets for immune-mediated damage in acute and chronic rejection. Lesions involving bile ducts, vascular endothelial cells and hepatocytes (mainly in centrilobular zones) are well recognized, and Kupffer cells and other sinusoidal cells may also be destroyed in liver allograft rejection, to be replaced by cells of recipient phenotype.

The MHC complexes have a central peptide groove that holds endogenous or exogenous peptide fragments for presentation to the T-cell receptor (TCR). In the alloresponse the polymorphic MHC proteins are recognized as foreign. Additionally, because of varying shape and charge of the peptide groove and side pockets, the polymorphic MHC molecules bind different peptide fragments, even from the same protein, and these can also be recognized as ‘foreign’. Thus, alloreactive T cells may recognize the allogeneic MHC, the allogeneic peptide or in most cases, both. The diversity of MHC molecules and the large array of different peptides that they can bind in their grooves likely explain the vigour of the alloreaction. The alloreactive T cells in acute rejection are a combination of T cells specifically reactive against the donor MHC, as well as significant numbers of pathogen-specific T cells that cross-react with the foreign MHC.

Stages in allograft rejection

The stages in allograft rejection include allorecognition, T-cell activation and the alloresponse. Initially, allorecognition, or the afferent arm, involves the presentation of graft alloantigens and the recognition of these antigens by recipient T lymphocytes. Following T-cell activation and cytokine release, the alloresponse, or efferent arm, involves recruitment and activation of effector cells that mediate damage to target structures within the allograft.

Afferent arm of immune response: allorecognition

The initial phase of the allograft response involves the presentation of graft alloantigens and the recognition of these antigens by recipient T cells. Central to this process is the antigen-specific interaction between the MHC molecules expressed on the surface of antigen-presenting cells (APCs) and the TCR expressed on the surface of recipient T lymphocytes ( Fig. 14.13 ). Interactions involving CD4+ T cells and MHC class II-expressing ‘professional’ APCs are primarily involved in the initial stages of graft recognition. The principle APCs in normal liver are dendritic cells (DCs) located in portal areas and, to a lesser extent, in centrilobular regions of the liver parenchyma.

Interaction between T cells and graft cells. Three main components are illustrated. The first involves the interaction between T-cell receptor (TCR) and major histocompatibility complex (MHC) molecules on donor liver cells. Additional factors are adhesion molecules and co-stimulatory molecules.

Three main pathways of antigen presentation are described: direct, indirect and semidirect. In the direct pathway, the main pathway involved in the early post-transplant period, donor DCs transferred as passenger leukocytes in the liver at transplantation migrate to regional lymph nodes and spleen, maturing during transit, where they present donor MHC antigens and activate alloreactive T lymphocytes. The DC mobilization occurs as a nonspecific reaction to surgical and ischaemic injury, stimulated by the release of cytokines such as interleukin-1 (IL-1), IL-6 and tumour necrosis factor α (TNFα).

Later, the donor APC population is depleted and replaced by host APCs. These process and present other donor alloantigens shed from the graft, but this time in the context of host MHC molecules. This indirect pathway is slower and less vigorous than the direct pathway, but still appears to be important in sustaining an ongoing persistent immune response, particularly a humoral response. The semidirect pathway has been characterized more recently, following the recognition that host APCs could acquire whole allogeneic MHC-peptide complexes through exosomes released by graft cells and could express them on their surface. The semidirect pathway can stimulate T-cell responses and potentially links the direct and indirect pathways.

The interaction between T cells and graft cells involves other important signalling pathways, including adhesion molecules, which enable T cells to bind to graft cells, and co-stimulatory molecules, which are required for naive T-cell activation ( Fig. 14.13 ). These other factors are not antigen specific. Two important co-stimulatory molecule-ligand pairs are CD28/B7 and CD40/CD154. In the absence of appropriate co-stimulatory signals, T cells do not become fully activated and either become unresponsive to further antigenic stimuli (anergic) or undergo apoptosis. Co-inhibitory pathways involving interactions between programmed death 1 (PD-1) receptor, which is expressed on graft-infiltrating T cells in acute rejection, and its ligand PD-L1, expressed on hepatocytes, cholangiocytes and sinusoidal cells, may also be important in regulating immune responses in the liver allograft.

Cells in the normal liver have a limited expression of MHC antigens, adhesion molecules and other molecules involved in the alloimmune response. After LT, there is cytokine-mediated upregulation of many of these molecules on bile ducts, hepatocytes and endothelial cells. The functional significance of these changes is uncertain, but they may be involved in both enhancing antigen presentation and promoting T-cell activation and effector mechanisms.

Diagnosis and monitoring of graft tolerance

It has long been recognized that a small number of liver allograft recipients are able to cease immunosuppression after transplantation. As theoretical studies of tolerance move to clinical practice, pathology is likely to have an expanded role in monitoring patients before, during and after weaning from immunosuppression. However, although many patients worldwide are currently free of immunosuppression, the long-term benefits of immunosuppression withdrawal are as yet unproved, and this remains an experimental procedure. Since much is still unknown in the practical application of operational tolerance, it has been recommended that protocol biopsy should be performed at baseline and at defined time points after weaning, even in apparently stable patients, although this is not done in all centres. Weaning has generally been carried out over months, beginning more than 2–3 years after LT, in highly select patients with stable graft function and no evidence of significant pathology apart from recurrent hepatitis C.

With regard to factors associated with successful immunosuppression weaning, time since transplantation appears to be particularly important. A recent European study of operational tolerance in liver recipients found that weaning was possible in 12.5% of patients transplanted for <5.7 years, but this increased to 79.2% in patients transplanted for ≥10.6 years. Younger children and older adults appear to be more favourably disposed to immunosuppression withdrawal, although probably for differing reasons. Other factors reported to be associated with a more favourable outcome include the absence of early rejection and the presence of low levels of baseline immunosuppression before weaning. Factors associated with a reduced likelihood of successful weaning include initial transplantation for autoimmune disease, presence of C4d staining in baseline biopsies and presence of DSAs. However, a recent study of children with follow-up 5 years after withdrawal of immunosuppression suggested that DSAs (persistent or de novo ) are frequently present in operationally tolerant individuals without impacting graft histology.

Biopsies taken before weaning allow a baseline assessment of inflammation (portal/interface and centrilobular), fibrosis or other features that might indicate subclinical rejection. The presence of more than a mild degree of any of these changes may preclude proceeding with immunosuppression withdrawal. Conversely, features observed in preweaning biopsies found to be predictive of tolerance include absence of portal inflammation, absence of lobular CD3+ and CD8+ T cells, higher stage of fibrosis in HCV-positive patients and presence of portal FoxP3+ T cells. Studies on peripheral blood and liver samples have identified a range of putative biomarkers associated with a ‘tolerogenic profile’ which are under continued evaluation with prospective analyses and validation cohorts.

Postweaning biopsies have been obtained to investigate episodes of graft dysfunction and on a protocol basis. Rejection is the most common cause of graft dysfunction after immunosuppression withdrawal and is the most common reason for failure to achieve graft tolerance. Most cases present during the period of immunosuppression weaning or within the first few months thereafter. Liver biochemistry is not reliable in distinguishing rejection from other possible causes of graft dysfunction, and this remains an important indication for liver biopsy. Most cases have features resembling typical acute cellular rejection (ACR), are relatively mild in severity and respond well to reinitiation of baseline immunosuppression. A smaller number of cases with more severe ACR or features suggestive of early chronic rejection (CR) may require more aggressive immunosuppressive therapy. Rare cases have progressed to CR leading to graft failure. Some cases of rejection occurring after immunosuppression withdrawal lack the typical histological features seen in ACR and instead have features resembling the hepatitic picture more typically seen in late acute rejection. Comparison with baseline preweaning biopsies may be particularly helpful in this setting. Later protocol biopsies in an LRLT cohort have shown a significant increase of portal fibrosis in some operationally tolerant patients compared with those on immunosuppression. It was initially postulated that the higher numbers of Tregs seen in these patients, thought to be involved in mediating tolerance, may also be associated with profibrogenic cytokine profile. However, the observation that portal fibrosis reversed with the reintroduction of immunosuppression suggests that some of these apparently tolerant patients may have had a mild, subclinical form of rejection.

A more recent study showed that increased numbers of portal T lymphocytes, with enrichment of Tregs, were present in biopsies obtained 1–3 years after weaning from operationally tolerant patients with no signs of rejection and no evidence of fibrosis. Minor degrees of bile duct atypia or ductopenia have also been observed in postweaning biopsies, suggesting the possibility of low-grade CR. Histological features thought to be of concern in postweaning biopsies have been identified by the Banff Working Group and include the following :

• •
  

  Increased portal or lobular inflammation compared with baseline preweaning biopsy, especially if accompanied by lymphocytic bile duct inflammation or interface or venous endothelitis

  
  
• •
  

  New-onset central perivenulitis that is increased compared with baseline

  
  
• •
  

  Bile duct changes, including dystrophic/senescent changes or ductopenia, if other causes such as bile duct obstruction or stenosis are reasonably excluded

  
  
• •
  

  Fibrosis that is increased over baseline and is not explained by another cause

  
  
• •
  

  Arterial changes, such as foam cell or obliterative arteriopathy

Further investigation in this area is required. As experience grows, pathologists will have an important role in clinical decision making through biopsy diagnosis, documenting new patterns of injury and providing insights into the microanatomical processes occurring during operational tolerance.

Cytomegalovirus

Cytomegalovirus (CMV) was a common cause of graft infection in the 1980s and early 1990s, when it occurred in 30–50% of patients, was symptomatic in up to 25% of cases and was associated with significant morbidity and mortality. The clinical frequency and mortality have considerably declined due to the use of prophylactic and pre-emptive antiviral therapy, and symptomatic disease now occurs in only 2–10% of patients. Although the peak time of occurrence in untreated patients is between 4 and 12 weeks after transplantation, the use of antiviral prophylaxis with ganciclovir or valganciclovir for the first 3 months after transplantation has seen an increasing prevalence of delayed-onset infection, which may present up to 1 year or more after discontinuation of prophylaxis. Although most of these late infections are associated with mild disease, occasional studies still implicate CMV infection as an important cause of death at 1 year.

The most important risk factor for the development of CMV disease is the combination of a seropositive donor and a CMV-seronegative recipient (D ⁺ R ⁻ ). Late disease still occurs in this group, even with antiviral prophylaxis, with a prevalence of 19–27% at 1 year. Other risk factors include transplantation for acute fulminant hepatitis, amount and type of immunosuppressive therapy (particularly antilymphocyte antibodies, mycophenolate or high-dose steroids), previous episodes of acute rejection, hepatitis C infection, infection with human herpesviruses 6 and 7, female gender and polymorphisms associated with innate immunity.

CMV infection is common and is distinguished from CMV disease. Infection indicates the presence of virus in blood, fluids or tissues regardless of symptoms, but the presence of clinical symptoms or signs is termed ‘CMV disease’ and is generally associated with higher levels of virus. CMV disease manifests as CMV syndrome (fever and marrow suppression) in up to two-thirds of affected patients, with the remainder having tissue-invasive CMV. In solid-organ transplantation the allograft is particularly prone to CMV infection, so the liver has been the most common site of organ involvement in liver allograft recipients. Other manifestations of CMV disease include gastrointestinal (GI) disease, pneumonia, neurological complications and chorioretinitis. As delayed-onset infection becomes more common, the incidence of infection outside the liver has increased, particularly in the GI tract.

The characteristic and most common histological finding in CMV hepatitis is the presence of scattered neutrophil polymorph aggregates (microabscesses) within the liver parenchyma ( Fig. 14.15 A ). Parenchymal microgranulomas can also be seen. There is occasionally a picture of spotty lobular inflammation and lobular disarray, which is indistinguishable from other forms of viral hepatitis. Other reported features include mild portal inflammation—sometimes associated with mild inflammation of bile duct and portal venules, suggesting mild cellular rejection—Kupffer cell enlargement and hepatocellular changes, including anisocytosis, anisokaryosis, nuclear hyperchromatism and increased numbers of mitoses. Viral inclusions are most often seen within the infected hepatocytes, usually in the vicinity of inflammatory lesions, and less often in biliary epithelial cells and endothelial cells of sinusoids and vessels. Examination of serial sections and deeper levels is useful because inclusions can be rare. In one study of serial post-transplant liver biopsies, CMV infection was first detected within sinusoidal cells, several days before typical histological features of CMV hepatitis became apparent. Increasingly, hepatitis with suggestive inflammatory lesions but no obvious viral inclusions is seen. Immunohistochemical (IHC) staining for CMV antigens is useful in confirming the presence of the virus ( Fig. 14.15 B ). In situ hybridization (ISH) techniques have also been used but are not widely available and do not appear to be more sensitive than IHC staining.

Cytomegalovirus (CMV) hepatitis in the liver allograft. A, Dense aggregate of neutrophils (microabscess) surrounds a cell containing a characteristic eosinophilic inclusion. B, Immunostaining with antibodies reacting with early CMV antigens detects positive nuclei in absence of obvious inclusions. (H&E stain.)

Although parenchymal neutrophil microabscesses are highly suggestive of CMV hepatitis, these lesions may also occur in cases when no CMV infection can be detected. Suggested associations include other infections (bacterial, viral or fungal), graft ischaemia and biliary obstruction/cholangitis. In some cases, no other obvious disease can be found. One group found that neutrophil aggregates in CMV-negative cases were smaller and more numerous than those occurring in association with CMV infection, prompting the term ‘mini microabscesses’. Conversely, a subsequent study found that greater numbers of microabscesses (>9) indicated CMV infection.

CMV infection has been implicated as an indirect factor contributing to an increased risk of other allograft complications, such as acute and chronic rejection, hepatic artery thrombosis and biliary complications. The relationship between CMV infection and rejection is complex and remains poorly understood. Whereas the use of high-dose immunosuppression to treat episodes of rejection is a risk factor for the emergence of opportunistic infection, CMV can infect bile ducts and vascular endothelial cells (VECs), which are targets for immune-mediated damage in allograft rejection. Infection also stimulates immune responses such as inflammatory cell recruitment and the release of cytokines, which may augment rejection. However, CMV-infected sinusoidal endothelial cells (SECs) can also recruit Tregs and may also affect T-cell differentiation, since recent evidence suggests that primary CMV infection in seronegative recipients (D ⁺ R ⁻ ) leads to the emergence of hyporesponsive T cells and reduced late acute rejection. Some of the other complications may also relate to the capacity of the virus to infect VECs and biliary epithelium. For example, CMV infection of VECs is associated with a rapid procoagulant response, which may predispose to thrombosis. whereas CMV infection of biliary epithelium has been implicated in causing cholangitis, which may lead to the development of nonanastomotic biliary strictures.

Epstein–Barr virus and post-transplant lymphoproliferative disorder

Epstein–Barr virus (EBV) is an important pathogen and is associated with a broad spectrum disorders, from asymptomatic viraemia to a range of lymphoproliferative diseases, usually of B-cell origin, in immunocompromised individuals. Most adults (up to 95%) have been exposed to EBV, with latent viral infection usually controlled by NK and T cells. Impaired immune responses to primary and reactivation EBV infection with immunosuppression can result in post-transplant lymphoproliferative disorder (PTLD). At greatest risk are EBV-naive allograft recipients, which includes 50% of children, who may be unable to control the primary infection adequately and may develop subsequent B-lymphocyte lymphoproliferation. Monitoring of patients at high risk of primary infection by longitudinal EBV viral load measurement using quantitative polymerase chain reaction (PCR) shows seroconversion of 60% by 3 months. Detection of increased viral load does not necessarily indicate clinically significant disease, but a sustained viral detection for ≥6 months identifies a group at increased risk of PTLD. Other risk factors for PTLD include high-dose immunosuppression. Patients receiving grafts after 2000 fare better because of improved EBV monitoring. Other suggested risk factors, such as older age, transplantation for cholestatic liver disease, hepatitis C and CMV infection, are more controversial.

PTLD develops in 2–5% of liver transplant recipients overall, with a higher incidence of 5–10% in children without antiviral prophylaxis. PTLD usually develops within the first year in children, but later in adults. Up to one-third of patients present with allograft dysfunction and infiltration of the graft, usually in early-presenting cases; in others, lymphadenopathy, tonsillar enlargement or infiltration of other organs is seen. Most PTLD cases occurring in liver allograft recipients are derived from recipient lymphoid cells, although some cases of donor or mixed donor and recipient origin have been described. The last group was EBV negative and showed multiple clonal paraproteins. It is increasingly recognized that EBV may not be causative in up to a half or more of contemporary cases. A recent study using comparative genomic hybridization analysis showed that post-transplant cases of EBV-negative diffuse large B-cell lymphoma (DLBCL) had a genomic profile that was different to that seen in EBV-positive DLBCL and more closely resembled the profile seen in DLBCL occurring in immunocompetent individuals, suggesting that different molecular pathways are involved in the pathogenesis of EBV-positive and EBV-negative PTLD.

Clinically, typical signs of EBV infection, in particular fever, jaundice and lymphadenopathy, are only variably seen at presentation. There is a wide spectrum of disease, from mild hepatitis to polyclonal proliferations and monomorphic PTLD, as well as EBV-associated smooth muscle tumours. This spectrum is continuous, and clinicians should remember that there may be evolution in a single patient from benign to aggressive disease, as well as heterogeneity between multiple lesions.

Histologically, EBV hepatitis is characterized by portal mononuclear cell infiltrates with lobular disarray, swollen and apoptotic hepatocytes and accumulation of lymphoid cells in the sinusoids. Lymphoid cells can also be seen beneath the endothelium of portal and central vein branches. In some cases, sinusoidal mononuclear cells predominate with a mononucleosis-like pattern. The lymphoid cells in EBV infection are predominantly B cells, with plasma cells and immunoblasts present in the infiltrate. B cells can be highlighted by CD20 or CD79a immunostaining. Eosinophils and neutrophils are rarely seen. These features help to distinguish EBV hepatitis from the major differential diagnosis of acute rejection, which usually has a T-cell predominant infiltrate, few plasma cells or immunoblasts and conspicuous eosinophils. Additionally, although subtle duct injury can be seen in EBV hepatitis, it is only minor compared with the degree of inflammation, whereas in rejection, duct injury increases in proportion with the inflammatory infiltrate. Patients with mixed features of EBV hepatitis and rejection pose problems for diagnosis and management.

PTLDs are most often B-cell proliferations, with <15% showing T/NK cell differentiation, the latter less often EBV associated. The World Health Organization (WHO) classification of neoplastic lymphoid diseases recognizes four main categories of PTLD :

• 1.
  

  Early lesions: plasmacytic hyperplasia, infectious mononucleosis-like PTLD

  
  
• 2.
  

  Polymorphic PTLD: polyclonal (rare) or monoclonal

  
  
• 3.
  

  Monomorphic PTLD: B-cell and T/NK-cell types

  
  
• 4.
  

  Classic Hodgkin lymphoma-type PTLD

Early lesions are more typically seen in lymph node and tonsillar biopsies. In most cases, hepatic infiltration is characterized by a diffuse and heavy, predominantly portal infiltrate of lymphoid and plasma cells. The polymorphic PTLD is considered to be distinctive and only seen in immunocompromised individuals. There is a heavy portal infiltrate of mixed cells showing the full range of B-lymphocyte maturation; this effaces the portal architecture. Necrosis and conspicuous mitotic activity may be present, and some more monomorphous areas can occur. The pattern has a lymphomatous appearance, and the cells may be monoclonal and light chain-restricted despite the mixed pattern of the infiltrate. Histological workup should include routine histology, immunophenotyping, including assessment of light-chain restriction, and EBV-encoded nuclear RNAs (EBER) ISH. If only paraffin-embedded tissue is available, IHC assessment of light-chain restriction will generally fail unless plasmacytic differentiation is present. Genetic studies to determine clonality are a useful adjunct.

Monomorphic PTLD has essentially the same features as lymphoma occurring in immunocompetent individuals, with most of the infiltrating cells having an atypical appearance, prominent nucleoli, a high mitotic rate and areas of necrosis. Most are of B-cell type with morphology of DLBCL, Burkitt lymphoma or rarely, plasma cell myeloma. A small number of cases of EBV-associated T-cell lymphoma have also been documented in liver allograft recipients, and these may appear as peripheral T-cell lymphoma–not otherwise specified (NOS) or hepatosplenic T-cell lymphoma. In some patients, particularly those in whom PTLD is donor derived, the infiltrate is confined to the liver itself. These patients may show formation of localized masses, particularly in the porta hepatis, which can present with features of biliary obstruction.

Distinction from other causes of portal inflammation, particularly rejection, may be difficult, and in some cases, EBV-related PTLD may coexist with rejection or precipitate it when immunosuppression is reduced. As discussed, features favouring an EBV-related lymphoid infiltration are the presence of an infiltrate with many plasmacytoid cells, immunoblasts and frequent mitoses and sparse eosinophils ( Fig. 14.16 A ). IHC demonstration of B-lymphocyte predominance with CD20 or CD79a is also helpful. Immunohistochemistry or ISH can be used to demonstrate light- or heavy-chain restriction if plasmacytic differentiation is present, but immunoglobulin gene rearrangement analysis is more sensitive for demonstrating clonal proliferation of B cells in PTLD. IHC staining for virus-associated proteins (e.g. latent membrane protein 1) or EBER-ISH can also be used to detect the virus in tissue sections ( Fig. 14.16 B ); ISH is the more sensitive. A small number of EBV-positive cells may be found among portal inflammatory cells in other conditions, including acute and chronic rejection, and in inflammatory diseases occurring in a nontransplanted liver. Lymphoid infiltration should therefore only be ascribed to EBV infection if a substantial proportion of the cells can be shown to contain the virus.

Epstein–Barr virus (EBV) associated post-transplant lymphoproliferative disease. A, Portal tract contains a dense infiltrate of lymphoid cells. These include numerous blast cells, a feature which is helpful in distinguishing lymphoproliferative disease from other causes of portal infiltration, including rejection. (H&E stain.) B, In situ hybridization for EBV-encoded nuclear RNAs (EBERs) confirms that a large proportion of infiltrating lymphoid cells are EBV positive.

Guidelines for management of PTLD have been published. During the early stages of polyclonal B-cell proliferation, the disease is frequently reversible with a reduction in immunosuppression. Treatment with antiviral agents may also be of benefit at this stage, although most of the EBV in transformed B cells is latent and not accessible to antivirals. Progression to a monoclonal and monomorphic B-cell proliferation indicates more aggressive disease, which is less likely to be responsive to changes in immunosuppression and requires other therapeutic approaches, including combination chemotherapy, anti-B-cell monoclonal antibodies and, in some specialized centres, adoptive immunotherapy with in vitro expanded virus-specific T cells. Recent data showing a 90% complete or partial response rate to sequential anti-B-cell antibodies followed by chemotherapy suggest improving outcomes.

Other rare opportunistic viruses

Adenovirus is a common pathogen that can cause significant hepatitis in immunocompromised patients. A recent review found that 48% of reported cases were associated with LT, usually in the first 6 months after grafting. It is more often seen in paediatric liver transplant recipients, but cases have also been described in adults. In the early stages there may be parenchymal inflammatory lesions resembling those seen in CMV hepatitis, with more severe cases showing small or larger areas of confluent parenchymal necrosis that may have a prominent haemorrhagic picture. The characteristic nuclear inclusions have an irregular contour, producing a ‘smudge cell’ effect ( Fig. 14.17 A ). Immunohistochemistry using an anti-adenovirus group antibody is useful to confirm the diagnosis ( Fig. 14.17 B ). Adenovirus infection can also involve the biliary tree with necrotizing cholangitis and loss of interlobular bile ducts.

Adenovirus hepatitis. A, Several hepatocytes have characteristic basophilic nuclear inclusions (‘smudge cells’). Adjacent to these, there is an irregular area of haemorrhage and hepatocyte necrosis. (H&E stain.) B, Immunostaining with an anti-adenovirus antibody confirms the presence of numerous nuclear inclusions.

Herpes simplex virus (HSV) infection is a rare cause of allograft hepatitis. It is characterized by foci of hepatocyte necrosis containing neutrophils and macrophages ( Fig. 14.18 A ). These may develop into more extensive areas of confluent necrosis associated with high mortality. Similar changes have also been observed with varicella-zoster virus (VZV) infection. Dense nuclear inclusions are seen in viable cells at the edge of necrotic areas ( Fig. 14.18 B ), only rarely detected in liver biopsy specimens. IHC staining for HSV antigens is helpful to confirm the diagnosis ( Fig. 14.18 C ).

Herpes simplex virus (HSV) hepatitis. A, Two sharply circumscribed areas of coagulative necrosis are surrounded by a zone of haemorrhage. B, Many surviving hepatocytes surrounding an area of necrosis contain characteristic eosinophilic nuclear inclusions. (H&E stain.) C, Immunohistochemical staining for HSV antigens demonstrates nuclear and cytoplasmic inclusions.

Human herpesviruses 6 and 7 (HHV6 and HHV7) are relatively recently recognized members of the Betaherpesvirinae subfamily and are closely related to CMV. Infection with these viruses is common after organ transplantation, but clinical disease develops in only 1% of patients. HHV6 infection, present as a latent infection of lymphocytes and monocytes in 95% of humans, occurs in up to 80% of liver allograft recipients, usually between 2 and 8 weeks after transplant. Most cases represent reactivation and result in subclinical disease or a mild febrile illness. Hepatic involvement may be associated with a mild degree of graft dysfunction, a mild/moderate portal lymphocytic inflammation (without damage to bile ducts or portal vessels) and foci of parenchymal inflammation, including neutrophil aggregates similar to those seen in CMV hepatitis. The HHV6 antigens can also be demonstrated on IHC staining in portal lymphocytes. Rarely reported features include syncytial giant cell hepatitis, giant cell transformation of biliary epithelium (after cardiac transplantation) and periportal confluent necrosis/apoptosis causing a targetoid appearance around portal tracts. Although direct effects of HHV6 infection on the liver allograft appear fairly mild, the virus is an immunomodulator and has been implicated as potentiating other graft complications, including CMV disease, invasive fungal infection, possibly more aggressive recurrent HCV infection, rejection and an increased risk for graft loss in patients with unexplained graft hepatitis. HHV6-induced upregulation of adhesion molecules and lymphocyte activation may have a role in the pathogenesis of liver allograft rejection.

HHV7 infection has been implicated in the pathogenesis of CMV infection but does not appear to have any direct effects on the liver allograft itself.

HHV8 varies in frequency geographically and can cause viraemia in a minority of patients. Rare cases of Kaposi sarcoma associated with HHV8 infection have been reported in liver graft recipients. One case of donor-transmitted dengue viral infection developed 6 days after living-donor transplantation. The liver graft biopsy showed cholestasis.

Hepatitis B

Typical features of recurrent HBV infection

Three main phases of reinfection have been identified. In the initial incubation phase (first 3 months post-transplant), changes directly attributable to HBV are rarely present, although a slight disarray of hepatocytes is sometimes seen. Positive IHC staining for HBV-associated antigens is usually the first sign of recurrent infection, typically in the form of focal cytoplasmic or nuclear staining for core antigen ( Fig. 14.21 ). During the second phase of early reinfection (usually 1–6 months post-transplant), biopsies typically show features of lobular hepatitis, which is usually mild. Varying degrees of portal inflammation may also be present. Portal and periportal inflammatory changes generally become more prominent as the disease progresses to the third stage of established ( chronic ) infection (>6 months post-transplant) and resemble those seen in chronic hepatitis B involving the nontransplanted liver. In early studies the severity and speed of progression were considerably greater in the liver allograft than in the native liver. In a combined series of 42 patients reinfected with HBV prior to 1991, 41 developed acute hepatitis, 20 progressed to chronic hepatitis and eight had become cirrhotic within a period ranging from 7 to 70 months.

Recurrent hepatitis B infection in the liver allograft. A, Early infection. Focal immunoreactivity for HBcAg (nuclear and cytoplasmic) is present in a liver biopsy that otherwise appears normal. B, Diffuse hepatocyte ballooning and mild fatty change are present, without an accompanying inflammatory reaction. (H&E stain.) C, Diffuse immunoreactivity for HBcAg (nuclear and cytoplasmic) is present (same case as B ). These changes suggest that there is massive viral replication resulting in direct cytopathic damage to hepatocytes. D, Fibrosing cholestatic hepatitis B infection (H&E stain). Ballooned or ‘ground-glass’ hepatocytes are shown intermingled with small ductular structures. E, The latter are highlighted by immunostaining for biliary keratin (K7). F, Lower magnification shows widespread perisinusoidal fibrosis (Gordon & Sweet reticulin stain).

Hepatitis C

Typical features of recurrent HCV infection

Three main phases can be identified. During the initial stage of graft reinfection (0–2 months post-transplant), when viral RNA levels are already high, the assessment of HCV-related changes is difficult due to the frequent presence of other causes of graft damage, such as preservation/reperfusion injury and acute rejection. HCV-related inflammation is rarely seen at this stage. Instead, there is typically a picture of lobular disarray associated with hepatocyte ballooning, acidophil bodies and sometimes increased mitotic activity. Fatty change may also be seen at this stage. Severe steatosis may be the initial histological manifestation of recurrent hepatitis in cases of HCV genotype 3. The second stage of established graft infection (2–4 months post-transplant) is characterized by more typical features of acute hepatitis. Inflammation is generally mild, predominantly lobular in location and is associated with varying degrees of lobular disarray, hepatocellular ballooning, acidophil bodies and Kupffer cell enlargement ( Fig. 14.22 A ). There may also be evidence of increased hepatocyte turnover, with variation in hepatocyte size, scattered mitotic figures and an increased Ki-67 labelling index ( Fig. 14.22 B ). Sinusoidal lymphocytosis is sometimes a feature. Varying degrees of portal inflammation may also be seen at this stage. HCV hepatitis occasionally occurs within the first 2 months, with cases documenting inflammatory changes attributed to recurrent HCV infection as early as 9 days after transplant. The third stage of progressive liver damage (>6 months post-transplant) is characterized by histological features of chronic hepatitis resembling those seen in chronic HCV infection of the nontransplanted liver ( Fig. 14.22 C ). There is a predominantly mononuclear portal inflammatory infiltrate, typically associated with lymphoid aggregates and focal lymphocytic infiltration of bile ducts. Variable interface hepatitis is seen. There is also usually spotty lobular inflammation, fatty change (usually macrovesicular) and focal acidophil body formation. Approximately 50% of patients have histological evidence of chronic hepatitis at 1 year after transplant, this figure increasing with longer follow-up.

Recurrent hepatitis C virus (HCV) infection. A and B, Early recurrent HCV in a liver biopsy obtained 3 months after transplantation. A, There is spotty lobular inflammation with lobular disarray, scattered acidophil bodies and variation in hepatocyte size (H&E stain). B, Immunohistochemical staining for Ki-67 confirms the presence of increased hepatocyte proliferation. C, Chronic HCV infection in the liver allograft. This biopsy obtained 6 months after transplant shows typical histological features of chronic hepatitis C, including portal inflammation with lymphoid aggregates, mild interface hepatitis, spotty parenchymal inflammation and mild fatty change. (H&E stain.) D, Recurrent HCV infection with centrilobular necroinflammation (central perivenulitis) in a biopsy obtained 21 months after transplant from a patient in whom antiviral therapy had recently been discontinued. This was associated with a rise in serum transaminases (AST 15× normal) and HCV-RNA levels. Portal tracts (not shown) had no signs of rejection. (H&E stain.) E and F, Recurrent HCV infection with cholestatic features. E, Portal tract contains a moderately dense inflammatory infiltrate, which is associated with prominent marginal ductular reaction, a feature not typically seen in chronic HCV infection involving the nontransplanted liver. There is also ballooning of periportal hepatocytes. (H&E stain.) F, Immunoperoxidase staining for K7 confirms the presence of extensive ductular reaction. No K7-positive ‘intermediate’ hepatobiliary cells are present in the liver parenchyma. (H&E stain.) G, Early fibrosis in cholestatic HCV infection. In this biopsy obtained 3 months after transplant, there is early periportal fibrosis and a characteristic pattern of perisinusoidal fibrosis, which is present in both periportal and centrilobular regions. (Haematoxylin and van Gieson stain.) H, Rapidly progressive recurrent hepatitis C. Marked granular staining of hepatocyte cytoplasm treated with an anti HCV core antibody. Double immunostaining for HCV core antigen (grey-brown) and CD8 antigen (blue) .

There is increasing evidence that HCV infection behaves more aggressively in the liver allograft than in the nontransplanted liver. Histologically, more severe degrees of necroinflammatory activity have been noted, including areas of confluent and bridging necrosis, which are very rarely, if at all, seen in nonimmunocompromised individuals. In such cases, the possibility of an additional cause for centrilobular necroinflammation (‘central perivenulitis’) should be considered, the main ones being acute rejection and de novo AIH. However, in some cases these changes can be attributed to HCV infection alone ( Fig. 14.22 D ). When the diagnosis is uncertain histologically, information relating to recent changes in immunosuppression and/or antiviral therapy and HCV-RNA levels usually help in deciding whether changes seen are related to viral infection or an allo/autoimmune response. The severity of inflammation correlates with fibrosis stage at biopsy and may be important in the pathogenesis of the more rapid progression of fibrosis seen with HCV infection in the liver allograft compared with the native liver. Fibrosis is frequently present in biopsies obtained 1 year after LT, and a few patients are already cirrhotic by this time. Approximately 20–40% are cirrhotic 5 years after transplant, and 50% by 7–10 years. Connective tissue stains are required for accurate assessment of fibrosis ; morphometry may also be helpful in assessing the severity of fibrosis. There have been occasional cases of de novo HCC occurring as a complication of recurrent HCV infection with cirrhosis. Most occurred within 7 years of transplantation, again supporting an accelerated disease progression in the liver allograft, although one has been described >10 years after interferon treatment and viral clearance in a noncirrhotic graft. Recurrent HCV infection is now recognized as a major cause of late graft failure and in some centres has become the leading indication for retransplantation in patients surviving >12 months post-transplant.

Hepatitis C and rejection

There is a complex relationship between HCV and rejection. On the one hand, the immunosuppressive therapy used to treat episodes of ACR predisposes to more severe HCV infection. On the other hand, a higher incidence of acute and chronic rejection has been identified in HCV-positive patients compared with those transplanted for other diseases. This association may reflect different approaches to immunosuppression in HCV-positive individuals, shared pathways of inflammatory damage or the effects of antiviral therapy used to treat HCV infection. After IFN treatment, up to 25% of patients have episodes of acute rejection and up to 17% develop features of chronic rejection, in a small proportion of cases leading to graft failure. IFN therapy is thought to predispose to rejection by a number of mechanisms, including nonspecific stimulation of the recipient’s immune system, release of both viral and alloantigens from lysed cells and accelerated hepatocellular metabolism of immunosuppressive drugs occurring as a result of viral clearance from hepatocytes. However, it has been suggested that the higher reported frequency of rejection in HCV-positive patients may partly reflect confounding factors such as a tendency to biopsy these patients more frequently and difficulties in distinguishing recurrent HCV from acute rejection. Although patients treated with direct-acting antiviral agents (DAAs) might theoretically be at risk of developing rejection, for similar reasons to those postulated for IFN-induced rejection, episodes of rejection are rarely seen in patients treated with nucleotide analogues; the overall frequency of rejection was only 1% (3/262) in two recent studies.

The distinction between recurrent HCV infection and ACR continues to be a common problem in the assessment of post-transplant biopsies, because the two conditions have overlapping histological features, including predominantly portal-based inflammation, bile duct inflammation, portal venous endothelial inflammation and portal tract eosinophils. Table 14.13 summarizes features that are helpful in distinguishing recurrent HCV hepatitis from ACR. Although no absolutely specific features exist for either condition, in most cases a careful assessment of the pattern of inflammation and extent of damage allows identification of the main cause of graft damage. Changes such as bile duct and endothelial inflammation or reactive changes in the biliary epithelium should be present in the majority (>50%) of structures to diagnose rejection. The timing of events is helpful; most ACR episodes occur in the first month after transplant, and HCV-related inflammatory changes, particularly in portal tracts, are unlikely to be present at this time. A recent study showed that the combination of venous endothelial inflammation and time <6 weeks post-transplant excluded HCV as a cause of graft dysfunction. Conversely, ACR is rare >12 months post-transplant, when portal inflammatory changes related to recurrent HCV infection are likely to be present. Serum HCV-RNA levels tend to be higher in HCV patients with recurrent disease than in those with pure rejection, but there is considerable overlap between the two groups. In a small proportion of biopsies, distinction between hepatitis C and ACR may be difficult or impossible. It is likely that the changes present in many such cases reflect a combination of HCV- and rejection-related graft damage. In the majority of cases where a dual pathology is suspected, rejection-related changes are at worst ‘mild’ in severity; additional immunosuppression is not indicated in these patients, and recurrent HCV is best regarded as the primary diagnosis. Increased immunosuppression should only be considered as a treatment option if features of ACR are at least ‘moderate’ in severity, or if there are features suggestive of progression to chronic rejection.

Table 14.13

Comparison of histological changes occurring in hepatitis C infection and acute cellular rejection of the liver allograft

	Hepatitis C infection	Acute cellular rejection
Portal and periportal changes
Portal inflammation	Mononuclear cells (lymphoid aggregates)	Mixed infiltrate (lymphocytes, macrophages, blast cells, neutrophils, eosinophils)
Interface hepatitis	Variable	Mild
Bile duct inflammation	Mild (lymphocytes)	Prominent (mixed infiltrate)
Bile duct loss	None	Variable (in cases progressing to chronic rejection)
Venous endothelial inflammation	None/mild	Yes
Fibrosis	Yes	No
Parenchymal changes
Parenchymal inflammation	Generally mild	Variable
Pattern	Spotty	Confluent
Distribution	Random	Perivenular
Associated features	Lobular disarray	Hepatic vein endothelial inflammation
Cholestasis	Rare (except FCH-like cases)	Common
Fatty change	Yes (macrovesicular)	No
Acidophil bodies	Common	Less numerous

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