**2.2 Diagnostics**

206 Liver Transplantation – Basic Issues

Regarding the recurrence of pre-transplant diseases after LT, HCV-re-infection may represent one of the most important threats to graft and patient survival after primarily successful transplantation. Although remarkable differences in the course of HCV-infection exist between pre- and post-transplant settings, the uniform picture of liver or graft cirrhosis

HCV-recurrence after LT is one of the most important issues regarding the spectrum of current graft diseases. Despite comparable pathophysiological processes, the course of graft hepatitis is usually more progressive compared to the natural setting of HCV-infection [11-15].

Most of the patients either show biochemical or histological signs of inflammation, whereas 30% of all graft recipients develop graft cirrhosis within 5 years after LT, leading to an impaired patient survival and a dubious transplant success. Progressive loss of graft function may even require re-transplantation despite of consistent antiviral treatment [16]. Arbitrary in use and manifold in extent, the term "HCV-recurrence" implies the whole spectrum of graft disease such as asymptomatic infection, graft-hepatitis, fibrosis and eventually transplant cirrhosis. The uniform picture of end stage graft disease comprises scar formation and replacement of liver parenchyma by connective tissue as a result of accelerated fibrogenesis [1, 17, 18]. Clinical presentation of graft function loss is very similar to the natural setting though more rapid and progressive. Ascites, variceal bleeding,

Patient survival with HCV-recurrence is dramatically compromised compared to non-HCVrelated transplants [15]. Several studies pointed out significantly lower survival rates in HCV-positive recipients due to accelerated fibrosis development. Survival analysis performed in a cohort of 2294 patients (Charité, Berlin, Germany since 1988) demonstrated highly significant differences (unpublished data) comparing 455 HCV-positive to 1839 HCV-

encephalopathy and jaundice are common results of graft decompensation.

is similar and comparable to a certain extent.

**2.1 Epidemiological and etiological aspects** 

unrelated transplants (p<0.001; fig.1).

Fig. 1. Disease-related post-transplant survival

**2. HCV-recurrence after liver transplantation** 

HCV-recurrence, ranging from asymptomatic viremia to rapid fibrosis progression requires an exact description and diagnostic assessment of injury extent. Clinical presentation of HCV-recurrence is frequently unspectacular, basically corresponding to HCV-infection in the natural setting. Though highly variable and unreliable, general discomfort and fatigues may appear as first symptoms of HCV-recurrence. In contrast, advanced stages may clinically result in jaundice, hemorrhage, edema, ascites, encephalopathy, infection, secondary organ failure according to the degree of functional deterioration. Therefore, standardized quantification of graft damage and disease extent must be performed, in order to identify high-risk-patients, initiate antiviral treatment and monitor further development.

#### **2.2.1 Clinical and biochemical aspects**

After successful LT, graft function is usually followed-up according to local protocols. Clinical presentation after LT is usually unremarkable unless advanced graft disease has already developed and symptoms of liver insufficiency become apparent [21]. Elevated aminotransferases (2-4-fold) are frequently observed along with normal parameters of synthesis and excretion as a biochemical expression of inflammatory activity in parenchyma. Severe HCV-recurrence may lead to variably impaired detoxification and synthesis similar to the natural course of HCV-associated liver disease [16]. However, the differentiation of HCV-associated graft hepatitis from acute cellular rejection (ACR) is frequently impossible, based on laboratory data, only. Taken a sufficient level of immunosuppression and a detectable HCV-load, HCV-recurrence seems to be probable after the exclusion of immunological, metabolic, vascular and biliary causes for biochemical abnormalities. Therefore, graft biopsy must urgently be performed as diagnostic gold standard [21].

#### **2.2.2 Histology**

As a very reliable method, histological analysis of graft tissue usually helps to determine the etiology of graft malfunction especially in combination with supportive results of other paraclinical examinations such as laboratory analysis, cholangiography and Dopplersonography. The histological picture of HCV-re-infection usually implies a mild sinusoidal infiltration by lymphocytes and mononuclear cells resulting in a variable degree of portal inflammation. HCV-associated inflammation may trigger an excessive synthesis of ECMcomponents and result in the accumulation of collagens.

The imbalance between synthesis and degradation of connective tissue defines the progression of fibrosis [1]. Once the diagnosis of HCV-related graft hepatitis is made, the indication to antiviral treatment should be evaluated in order to prevent aggravation. The development of fibrosis is not linear [21, 23]. Since the accumulation of connective tissue and pathologic alteration of histological structure are definitive endpoints of HCV- recurrence, serial biopsies and long-term follow-up are highly indispensable for the assessment of HCVrelated damage (fig. 2) [21, 24, 25].

Fig. 2. Post-transplant dynamics of fibrosis

Along with several available and currently accepted scores, hepatic fibrosis is frequently characterized by a semiquantitative score, proposed by Scheuer & Desmet [26]. Using a scale (0-4), fibrosis is staged as ordinal data values (0: absent, 1: mild without septa, 2: moderate with few septa, 3: numerous septa without cirrhosis and 4: cirrhosis). Although no arithmetic procedures can be performed with ordinal data, Desmet and Scheuer-score appears to be superior in reproducibility over other semiquantitative systems in fibrosis assessment [27]. In a recent analysis according to Desmet and Scheuer, accepted time-related rates of graft cirrhosis development (30% after 5 and 50% after 10 years) could be confirmed in a representative cohort of more than 400 transplants with HCV-recurrence (Charité, Berlin, Germany). Advanced fibrosis stages (3-4) were observed in 39.2% after 5 and 47.5% after 10 post-transplant years, respectively, emphasizing the importance of a universal term definition (fig. 3).

Apart from fibrosis quantification, microscopic evaluation of inflammatory pattern helps to differentiate severe cellular rejection from HCV-infection in spite of significant biochemical similarities [28]. Low levels of immunosuppression may induce an ACR-event. Due to frequently simultaneous occurrence of acute cellular rejection and HCV-re-infection in the early post-operative period, these entities tend to be easily misdiagnosed. Classified according

inflammation. HCV-associated inflammation may trigger an excessive synthesis of ECM-

The imbalance between synthesis and degradation of connective tissue defines the progression of fibrosis [1]. Once the diagnosis of HCV-related graft hepatitis is made, the indication to antiviral treatment should be evaluated in order to prevent aggravation. The development of fibrosis is not linear [21, 23]. Since the accumulation of connective tissue and pathologic alteration of histological structure are definitive endpoints of HCV- recurrence, serial biopsies and long-term follow-up are highly indispensable for the assessment of HCV-

Along with several available and currently accepted scores, hepatic fibrosis is frequently characterized by a semiquantitative score, proposed by Scheuer & Desmet [26]. Using a scale (0-4), fibrosis is staged as ordinal data values (0: absent, 1: mild without septa, 2: moderate with few septa, 3: numerous septa without cirrhosis and 4: cirrhosis). Although no arithmetic procedures can be performed with ordinal data, Desmet and Scheuer-score appears to be superior in reproducibility over other semiquantitative systems in fibrosis assessment [27]. In a recent analysis according to Desmet and Scheuer, accepted time-related rates of graft cirrhosis development (30% after 5 and 50% after 10 years) could be confirmed in a representative cohort of more than 400 transplants with HCV-recurrence (Charité, Berlin, Germany). Advanced fibrosis stages (3-4) were observed in 39.2% after 5 and 47.5% after 10 post-transplant years, respectively, emphasizing the importance of a universal term

Apart from fibrosis quantification, microscopic evaluation of inflammatory pattern helps to differentiate severe cellular rejection from HCV-infection in spite of significant biochemical similarities [28]. Low levels of immunosuppression may induce an ACR-event. Due to frequently simultaneous occurrence of acute cellular rejection and HCV-re-infection in the early post-operative period, these entities tend to be easily misdiagnosed. Classified according

components and result in the accumulation of collagens.

related damage (fig. 2) [21, 24, 25].

Fig. 2. Post-transplant dynamics of fibrosis

definition (fig. 3).

to Banff-criteria, ACR may present microscopically as an accumulation of mononuclear cells (lymphocytes, eosinophil and neutrophil granulocytes) including endothelitis, portal, centrolubular inflammation and biliary alterations [28].

Fig. 3. Progression to advanced fibrosis stages

Currently ACR is treated by the application of intravenous steroid pulses for 3-5 days. In few cases, mono- or polyclonal antibodies are administered in case of steroid resistant rejection [21]. HCV-exacerbation is widely accepted as an inevitable side effect of ACRtreatment due to limited alternatives. Morphologically, mild forms of ACR hardly differ from HCV-re-infection due to principal differences in the pathogenesis. Regarding the danger of steroid-associated HCV-exacerbation, steroid-based treatment is recommended in moderate and severe degrees of ACR in HCV-positive transplants according to current standards. In contrast, mild ACR should be treated by the administration of higher calcineurin inhibitor doses (CNIs) and MMF-complementation as dual immunosuppressive medication. Therefore, the diagnosis must be based on the histological analysis of graft biopsy as most reliable method [21, 27].

#### **3. Risk factors**

The progression of HCV-associated graft disease is influenced by a whole range of virus, donor, recipient and environmental factors. The variety of relevant confounders exhibiting variable impact, their interaction in genetically unique living individuals resulting from a successful LT have been in the center of attention for many decades. Some risk factors for the development of graft fibrosis have been identified during the scientific attempt to unravel the mystery and to understand the substance of HCV-recurrence in detail [21, 29]. However, the majority of observations were based on low sample size. Nevertheless, the existence of virological, immunological, surgery-related and even historical confounders illustrates the complexity and variability of the issue (table 1).


Table 1. Variables related to the severity of HCV-recurrence

#### **3.1 Donor- and surgery-related factors**

Liver graft has been quickly suspected to affect HCV-recurrence as a dominant location of pathologic events. Several studies detected a negative effect on HCV-recurrence regarding donor age, organ quality, histocompatibility matching, steatosis and iron concentration [30, 31]. Furthermore, transplant-related factors such as duration of organ harvesting, warm and cold ischemia time (transport and implantation) have been shown to contribute to HCVrelated post-transplant events and processes [21]. Genetic variance of growth factors and cytokines in donor is currently suspected to impact on the progression and treatment of HCV-associated graft disease [32, 33].

#### **3.2 Host-related factors**

The development of fibrosis seems to be twice as fast in males compared to female recipients. In contrast to the controversially discussed role of recipient age, black race and male gender have been shown to negatively affect fibrosis progression [22]. Cytokines and growth factors are final effectors in the pathogenesis and may theoretically play a key role in fibrogenesis [1, 34]. Analogously to donor, genetic polymorphisms of recipient cytokines are being currently intensively investigated and seem so far to modulate the course of HCVrecurrence and antiviral treatment success. This issue will be presented below.
