**1. Introduction**

#### **1.1 Hepatitis C**

224 Liver Transplantation – Basic Issues

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transplant with silibinin in nonresponders to pegylated interferon-based therapy.

retransplantation for allograft failure caused by recurrent hepatitis C infection.

Sequels of chronic HCV infection such as end-stage liver cirrhosis and hepatocellular carcinoma (HCC) are the leading indications for liver transplantation (LT) in Europe and in the United States. According to the United Network for Organ Sharing (UNOS) database the proportion of transplants performed due to the decompensated cirrhosis secondary to hepatitis C infection slightly declined in the last few years from 34% in 2002 to 29% in 2007, but at the same time the increased number of candidates for LT with HCC was noted [*www.unos.org*]. This trend will be observed until year 2030. Generally, one third of LTs worldwide is performed in HCV-positive patients. Given that recurrence of HCV infection is almost universal and the natural history of HCV hepatitis in allograft is more rapid than in the immunocompetent patient, liver failure secondary to recurrent HCV infection has a significant impact on post-transplant survival and will soon become the most common cause of liver retransplantation. Organ shortage and increasing evidence of poorer outcome in retransplanted patients makes this procedure a controversial issue, not accepted in many centers. Therefore efforts of transplant physicians to manage recurrent HCV infection in order to optimize outcomes and to slow down the progression of HCV-related liver disease are the greatest challenge. Most widely explored areas of interest include timing and schedule of antiviral treatment, immunosuppression regimens and matching in donor and recipient-related factors influencing outcomes.

#### **1.2 Natural history of HCV recurrence**

Natural history of HCV infection in the immunocompetent host since the acute HCV infection until the end-stage liver cirrhosis and eventually hepatocellular carcinoma covers approximately 30 years of progressive fibrosis developing in liver parenchyma (Hu & Tong, 1999). After liver transplantation the chronic HCV disease, albeit not fully understood and highly variable in different recipients, seems to be far more aggressive and significantly impacts the overall poorer post-LT survival in comparison with HCV-negative patients. Liver transplantation performed in viremic recipients results in rapid allograft reinfection in nearly 100% of cases. In the anhepatic phase HCV viremia dramatically declines to the very low or even undetectable levels, but within a few days after transplantation increases to the load 10 to 20-fold higher than pretransplant ones. That gives too narrow window of opportunity for any potential intervention such as passive immunization or preemptive antiviral treatment to make it effective. The higher the pretransplant viral load the bigger the chance for faster and more severe HCV reinfection. Immunosuppressive treatment is responsible for the weaker immune control of HCV infection and a distinct natural history in post-transplant setting. Typically viremia peaks 1–3 months after LT, clinically apparent acute hepatitis develops after a median time of 4 to 6 months in more than 60% of patients and almost 100% of recipients show histological evidence of chronic C hepatitis between 1 to 4 years after LT (Gane et al., 1996). Protocol liver biopsies performed every year after LT show progression of fibrosis in the reinfected liver rating from 0.3 to 0.6 points per year (Pelletier et al., 2000) whereas in the immunocompetent hosts it scores 0.1 to 0.2 points per year (Poynard et al., 1997). According to the accessible database the mean time between acute HCV reinfection and the decompensation of liver disease, presented as variceal bleeding, refractory ascites or encephalopathy, is approximately 9.5 years (Berenguer et al., 2000). Once decompensation of liver function is established, one year survival probability decreases dramatically below 50% and 3-year survival does not exceed 10% of recipients (Berenguer et al, 2000; Roche & Samuel, 2007). This is significantly inferior than survival in the immunocompetent patients. It is estimated that 20 to 30% of HCV-positive recipients will progress to the liver cirrhosis within five years after LT. A small proportion of patients (appr. 3%) will present with a particularly severe form of HCV recurrence known as fibrosing cholestatic hepatitis with a fatal outcome (or retransplantation) in the first year post-LT (Gane, 2008). According to the UNOS database 3-year survival after LT in HCVpositive recipients is inferior to the survival rate in the other etiologies (78% vs. 82%) and 5 year survival is also lower (56.7% vs. 65.6%, p<0.05) (Forman et al., 2002). All the above mentioned data explain why liver disease secondary to HCV infection is currently one of the worst and most challenging indications for LT.

#### **1.3 Factors influencing severity of recurrent HCV infection**

To date a number of factors has been described as having potential influence on the natural history of post-transplant recurrent C hepatitis. This debate has a practical aspect since a few of them are modifiable by the transplant team, for example optimization of immunosuppression therapy, anti-HCV therapy prior to LT, aggressive and efficient treatment of post-transplant diabetes or the appropriate donor selection. These factors are routinely divided into four groups: donor, recipient, virus and transplant procedure related (Table 1).

In the era of organ shortage careful donor-recipient matching is not always possible and poses some controversy, but there is a growing evidence that donor related variables such as younger age (< 40 years), male sex and lack or minimal steatosis in the liver (in < 30% of hepatocytes) are associated with a significantly better survival and slower progression of chronic C hepatitis (Aytaman et al., 2010). Factors related to the transplant procedure with a potential influence on hepatitis C progression include prolonged cold ischemia time, treatment of acute rejection episodes within the first months of LT, acute CMV infection post-LT, type of immunosuppressive regimen and type of liver transplantation (deceaseddonor vs. living-donor LT). Initially, there was some concern that HCV recurrence was more severe with live donor transplant, but a growing experience did not confirm these findings (Gallegos-Orozco et al., 2009).

load 10 to 20-fold higher than pretransplant ones. That gives too narrow window of opportunity for any potential intervention such as passive immunization or preemptive antiviral treatment to make it effective. The higher the pretransplant viral load the bigger the chance for faster and more severe HCV reinfection. Immunosuppressive treatment is responsible for the weaker immune control of HCV infection and a distinct natural history in post-transplant setting. Typically viremia peaks 1–3 months after LT, clinically apparent acute hepatitis develops after a median time of 4 to 6 months in more than 60% of patients and almost 100% of recipients show histological evidence of chronic C hepatitis between 1 to 4 years after LT (Gane et al., 1996). Protocol liver biopsies performed every year after LT show progression of fibrosis in the reinfected liver rating from 0.3 to 0.6 points per year (Pelletier et al., 2000) whereas in the immunocompetent hosts it scores 0.1 to 0.2 points per year (Poynard et al., 1997). According to the accessible database the mean time between acute HCV reinfection and the decompensation of liver disease, presented as variceal bleeding, refractory ascites or encephalopathy, is approximately 9.5 years (Berenguer et al., 2000). Once decompensation of liver function is established, one year survival probability decreases dramatically below 50% and 3-year survival does not exceed 10% of recipients (Berenguer et al, 2000; Roche & Samuel, 2007). This is significantly inferior than survival in the immunocompetent patients. It is estimated that 20 to 30% of HCV-positive recipients will progress to the liver cirrhosis within five years after LT. A small proportion of patients (appr. 3%) will present with a particularly severe form of HCV recurrence known as fibrosing cholestatic hepatitis with a fatal outcome (or retransplantation) in the first year post-LT (Gane, 2008). According to the UNOS database 3-year survival after LT in HCVpositive recipients is inferior to the survival rate in the other etiologies (78% vs. 82%) and 5 year survival is also lower (56.7% vs. 65.6%, p<0.05) (Forman et al., 2002). All the above mentioned data explain why liver disease secondary to HCV infection is currently one of the

worst and most challenging indications for LT.

(Table 1).

(Gallegos-Orozco et al., 2009).

**1.3 Factors influencing severity of recurrent HCV infection** 

To date a number of factors has been described as having potential influence on the natural history of post-transplant recurrent C hepatitis. This debate has a practical aspect since a few of them are modifiable by the transplant team, for example optimization of immunosuppression therapy, anti-HCV therapy prior to LT, aggressive and efficient treatment of post-transplant diabetes or the appropriate donor selection. These factors are routinely divided into four groups: donor, recipient, virus and transplant procedure related

In the era of organ shortage careful donor-recipient matching is not always possible and poses some controversy, but there is a growing evidence that donor related variables such as younger age (< 40 years), male sex and lack or minimal steatosis in the liver (in < 30% of hepatocytes) are associated with a significantly better survival and slower progression of chronic C hepatitis (Aytaman et al., 2010). Factors related to the transplant procedure with a potential influence on hepatitis C progression include prolonged cold ischemia time, treatment of acute rejection episodes within the first months of LT, acute CMV infection post-LT, type of immunosuppressive regimen and type of liver transplantation (deceaseddonor vs. living-donor LT). Initially, there was some concern that HCV recurrence was more severe with live donor transplant, but a growing experience did not confirm these findings


Table 1. Predictors of severe HCV recurrence. BMI- body mass index, CNI – calcineurin inhibitor, ACR – acute cellular rejection, LT – liver transplantation, AZA – azathioprine, CMV - cytomegalovirus

Among the baseline viral characteristics some relationship was noted between genotype 1 of the virus and the patient and graft survival, but the predominance of genotype 1 among transplanted patients can be explained by the worse antiviral treatment results before and after transplantation in comparison with other genotypes, therefore negative impact of genotype 1 on the severity of HCV recurrence was not clearly determined. It was already mentioned that high pretransplant viremia, but also persistence of the same HCV variants are responsible for more severe picture of acute HCV reinfection and more progressive course of chronic HCV disease (Berenguer, 2003).

The hottest debate concerns influence of immunosuppressive drugs on the evolution of HCV infection, two issues being discussed the most: how to optimally handle corticosteroids and which calcineurin inhibitor has an advantage in HCV-positive recipients. Some transplant centers favor corticosteroid-sparing regimens whereas in the others lowdose steroid maintenance is preferred over steroids avoidance or early withdrawal. The clear benefit of one of these approaches was not concluded. The most notable influence of corticosteroids on HCV infection is seen in the case of acute cellular rejection (ACR) treatment. It was proved that pulses of methylprednisolone commonly used for such treatment are associated with a transient increase in HCV-RNA concentration of 4- to 100 fold (Gane et al., 1996). While episodes of ACR treated with steroid pulses are associated with decreased mortality in HCV-negative patients, in the group of HCV infected recipients the relative risk of mortality or graft loss increases almost three times (p=0.04) and is even higher when ACR is steroid refractory. Therefore, in HCV-positive recipients empiric treatment of presumed rejection episodes without histological confirmation should be avoided to avert unnecessary exposure to corticosteroids. In contrast with convincing association of steroid pulses with increased severity of HCV recurrence, detrimental effect of low-dose steroids versus no steroids is less obvious and requires further observations. According to the study of Manousou P. et al., lack of steroids in the immunosuppressive protocol was an independent factor affecting fibrosis (Manousou et al., 2009). Some authors suggest that a low maintenance dose of prednisone (usually 5mg daily) has no deleterious effect on HCV infection, but instead, is associated with better LT outcome, while early withdrawal of steroids (3 months beyond LT) can be detrimental and should be avoided. Some other researchers favor complete avoidance of steroids in HCV infected recipients undergoing LT. Maintenance of a low-dose and no steroids options are currently best supported.

Another hot topic is a choice of calcineurin inhibitor (CNI) in HCV-positive recipients. This discussion was initiated by the clinical observation that results of LT for cirrhosis type C in the last two decades of twentieth century did not differ from results of LTs performed for other etiologies, whereas nowadays they are clearly inferior in patients with HCV infection (Forman et al., 2002; Berenguer, 2005). One of the possible explanations was introduction of more potent immunosuppressive drugs such as tacrolimus or mycophenolate mofetil which practically replaced cyclosporine and azathioprine in the immunosuppression protocols used for liver transplant patients. The role of cyclosporine in the post-transplant recurrent C hepatitis is a subject of controversy. This drug is known as having an inhibiting effect on HCV replication *in vitro* (Watashi et al., 2003). It was also used in combination with interferon alpha-2b to treat chronic HCV infection and proved to be significantly more effective than interferon monotherapy (Inoue et al., 2003). For anti-HCV effect some authors combined cyclosporine A with interferon to treat established HCV-related graft disease (Inoue & Yoshiba, 2005). Conflicting results regarding effects of CNI on HCV recurrence may be explained by the previous study design (mostly retrospective), small groups of patients, the lack of histological examinations, variety of confounding factors and multitude of immunosuppressive protocols. However, in the prospective randomized trial and in metaanalysis that were aimed to compare influence of tacrolimus and cyclosporine on HCV recurrence, no difference in the outcome was demonstrated (Berenguer et al., 2006a; Berenguer, 2007). It was concluded that the course of recurrent HCV hepatitis is not related to the CNI used after LT. Similar observation was made in HCV-positive patients with the end-stage renal disease subjected for kidney transplantation (Kahraman et al., 2011). The only difference noted by Berenguer et al. was the shorter time between LT and recurrent acute C hepatitis in tacrolimus treated patients (59 days vs. 92 days, p=0.02). On the other hand, introduction of tacrolimus improved results of LT (Cholongitas et al., 2011). Used in HCV-negative liver recipients, TAC is associated with fewer rejection episodes and significantly better survival. It cannot be ruled out that this effect is counterbalanced by anti-HCV effect of cyclosporine in HCV-infected recipients, therefore none of CNIs is favored in this specific group of patients.

Another type of immunosuppressive drugs that exert a specific antiviral effect on *Flaviviridae* is a group of antimetabolites such as azathioprine (AZA) and mycophenolate mofetil (MMF). As immunosuppressants they arrest T-cell proliferation by inhibiting inosine monophosphate dehydrogenase and by the same mechanism exert an anti-HCV effect (like ribavirin). AZA is one of the oldest immunosuppressive drugs, commonly used in combination with cyclosporine and steroids, but it was substituted a decade ago by more

association of steroid pulses with increased severity of HCV recurrence, detrimental effect of low-dose steroids versus no steroids is less obvious and requires further observations. According to the study of Manousou P. et al., lack of steroids in the immunosuppressive protocol was an independent factor affecting fibrosis (Manousou et al., 2009). Some authors suggest that a low maintenance dose of prednisone (usually 5mg daily) has no deleterious effect on HCV infection, but instead, is associated with better LT outcome, while early withdrawal of steroids (3 months beyond LT) can be detrimental and should be avoided. Some other researchers favor complete avoidance of steroids in HCV infected recipients undergoing LT. Maintenance of a low-dose and no steroids options are currently best

Another hot topic is a choice of calcineurin inhibitor (CNI) in HCV-positive recipients. This discussion was initiated by the clinical observation that results of LT for cirrhosis type C in the last two decades of twentieth century did not differ from results of LTs performed for other etiologies, whereas nowadays they are clearly inferior in patients with HCV infection (Forman et al., 2002; Berenguer, 2005). One of the possible explanations was introduction of more potent immunosuppressive drugs such as tacrolimus or mycophenolate mofetil which practically replaced cyclosporine and azathioprine in the immunosuppression protocols used for liver transplant patients. The role of cyclosporine in the post-transplant recurrent C hepatitis is a subject of controversy. This drug is known as having an inhibiting effect on HCV replication *in vitro* (Watashi et al., 2003). It was also used in combination with interferon alpha-2b to treat chronic HCV infection and proved to be significantly more effective than interferon monotherapy (Inoue et al., 2003). For anti-HCV effect some authors combined cyclosporine A with interferon to treat established HCV-related graft disease (Inoue & Yoshiba, 2005). Conflicting results regarding effects of CNI on HCV recurrence may be explained by the previous study design (mostly retrospective), small groups of patients, the lack of histological examinations, variety of confounding factors and multitude of immunosuppressive protocols. However, in the prospective randomized trial and in metaanalysis that were aimed to compare influence of tacrolimus and cyclosporine on HCV recurrence, no difference in the outcome was demonstrated (Berenguer et al., 2006a; Berenguer, 2007). It was concluded that the course of recurrent HCV hepatitis is not related to the CNI used after LT. Similar observation was made in HCV-positive patients with the end-stage renal disease subjected for kidney transplantation (Kahraman et al., 2011). The only difference noted by Berenguer et al. was the shorter time between LT and recurrent acute C hepatitis in tacrolimus treated patients (59 days vs. 92 days, p=0.02). On the other hand, introduction of tacrolimus improved results of LT (Cholongitas et al., 2011). Used in HCV-negative liver recipients, TAC is associated with fewer rejection episodes and significantly better survival. It cannot be ruled out that this effect is counterbalanced by anti-HCV effect of cyclosporine in HCV-infected recipients, therefore none of CNIs is favored in

Another type of immunosuppressive drugs that exert a specific antiviral effect on *Flaviviridae* is a group of antimetabolites such as azathioprine (AZA) and mycophenolate mofetil (MMF). As immunosuppressants they arrest T-cell proliferation by inhibiting inosine monophosphate dehydrogenase and by the same mechanism exert an anti-HCV effect (like ribavirin). AZA is one of the oldest immunosuppressive drugs, commonly used in combination with cyclosporine and steroids, but it was substituted a decade ago by more

supported.

this specific group of patients.

potent MMF. However, the role of MMF in HCV-infected liver recipients is controversial. Addition of an effective immunosuppressive drug with antiviral properties to the immunosuppression protocol seemed very attractive. Some authors demonstrated that MMF not only improved allograft function but was also associated with reduction of HCV viral load and delayed histological recurrence. In a study of Fasola et al., it was shown that high doses of MMF efficiently reduced HCV-RNA concentration and liver fibrosis one year post-transplant, but after 2 years the extent of fibrosis did not differ along all studied groups (Fasola et al., 2002). Other groups demonstrated no superiority of MMF over AZA in decreasing HCV viremia, histological slow down and better survival. Moreover, it was reported that AZA decreased replication of flaviviruses 10 times more effectively than MMF and was as potent as ribavirin in HCV inhibition in a replicon model (Stangl et al., 2004). Lately, use of AZA in liver transplant recipient, chronically infected with HCV, was reevaluated. It was concluded that MMF shows little, if any, clinical benefit in LT versus AZA and that HCV recurrence was less severe in patients treated with AZA in contrast to MMF (Germani et al., 2009). Further randomized controlled trials are warranted to solve this issue.

mTOR inhibitors such as sirolimus and everolimus are not licensed in liver transplantation. Sirolimus, however, due to its antifibrotic, antiproliferative and renal sparing properties has been recently used by some transplant teams with encouraging results (Harper et al., 2011). Different mode of action by inhibiting proangiogenic factors (i.e. VEGF, vascular endothelial growth factor) is associated with a decreased risk of cancer recurrence or *de novo* development. The main indications for sirolimus in liver recipients are CNI nephrotoxicity, hepatocellular carcinoma (as an indication for LT, *de novo* or recurrent) and fibrosis related to chronic HCV infection. Some authors suggest conversion from CNI to sirolimus-based immune suppression in case of fibrosis progression > 2. Further trials are necessary to confirm the beneficial role of sirolimus in this indication.

In conclusion, the best immunosuppressive regimen for recurrent C hepatitis is not known. Despite some reports on beneficial effect of cyclosporine in this setting, the type of CNI does not seem to matter (Berenguer, 2011), and tacrolimus remains the major immunosuppressant in the protocol. Temporary conversion to CsA is suggested during antiviral treatment to combine cyclosporine with interferon and ribavirin. As soon as fibrosis progresses to the moderate–severe stages, switching to sirolimus-based suppression can be considered, but currently this is an off-label approach. A role of MMF in the HCV recurrence is unclear. As grafted liver usually does not require very potent immunosuppression, replacement of MMF by AZA is feasible and recommended. As far as steroids are considered, steroid-free protocols or low-dose maintenance steroids seem to be the best option.

#### **1.4 Treatment of recurrent HCV infection**

Because many of the above mentioned strategies aiming to slow down progression of posttransplant HCV disease fail, the best option is to introduce anti-HCV treatment in order to attempt eradication of the virus prior to LT or after surgery to prevent recurrence or liver damage. Three approaches are possible: pre-transplant anti-viral therapy, early post-LT treatment (preemptive or in the acute phase) and treatment of the established disease. Each strategy has its pros and cons (Table 2), but the overall outcomes are rather disappointing. There are several reasons for worse results in transplant patients in comparison with the non-transplant setting: history of unsuccessful antiviral treatment pre-LT, predominance of genotype 1 patients, significant increase in viral load following transplantation, concomitant immunosuppressive treatment, frequent dose reductions due to numerous side effects, mostly cytopenias and infections, frequent peritransplant renal impairment, limiting ribavirin dosing, and ocassionally poor general status of the patient.

Firm conclusions on the role of interferon and ribavirin in the transplant setting are hard to be driven from clinical studies due to many methodological limitations such as a small number of patients, mostly retrospective character of the trials, lack of randomization and control, differences in immunosuppressive protocols, variability in patient selection, different doses, schedules and types of anti-viral therapy, different study end-points and scarce number of control liver biopsies. Nevertheless, in a limited and carefully selected number of patients anti-viral treatment can be strongly considered and is currently recommended (Wiesner et al., 2003).


Table 2. Advantages and disadvantages of anti-HCV treatment in the transplant setting
