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- Patients, Material and Methods
Hepatitis C is the most common indication for liver transplantation. Recurrence of HCV is universal leading to graft failure in up to 40% of all patients. The differentiation between acute rejection and recurrent hepatitis C is crucial as rejection treatments are likely to aggravate HCV recurrence. Histological examination of liver biopsy remains the gold standard for diagnosis of acute rejection but has failed in the past to distinguish between acute rejection and recurrent hepatitis C. We have recently reported that C4d as a marker of the activated complement cascade is detectable in hepatic specimen in acute rejection after liver transplantation. In this study, we investigate whether C4d may serve as a specific marker for differential diagnosis in hepatitis C reinfection cases. Immunohistochemical analysis of 97 patients was performed. A total of 67.7% of patients with acute cellular rejection displayed C4d-positive staining in liver biopsy whereas 11.8% of patients with hepatitis C reinfection tested positive for C4d. In the control group, 6.9% showed C4d positivity. For the first time we were able to clearly demonstrate that humoral components, represented by C4d deposition, play a role in acute cellular rejection after LTX. Consequently C4d may be helpful to distinguish between acute rejection and reinfection after LTX for HCV.
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- Patients, Material and Methods
Liver transplantation today is a well-established procedure for curative treatment of various liver diseases. In spite of continuously improving immunosuppressive protocols, acute rejection remains a rather frequent (25–40%) and potentially hazardous complication that is mostly treated by steroid-pulse therapy and/or increased doses of calcineurin inhibitors as well as the introduction of alternative immunosuppressive substances such as MMF or sirolimus (1–3).
Hepatitis C-induced liver cirrhosis is one of the most frequent pathologies requiring liver transplantation (4–6), associated with the high risk of post-transplantation HCV reinfection and consecutive development of liver fibrosis and cirrhosis.
Both acute rejection and hepatitis C reinfection often display the same clinical picture with rising serum transaminases, elevated bilirubin levels and deterioration of productive liver function in the absence of perfusion deficits.
Liver biopsy represents the gold standard for diagnosis of both acute rejection and HCV reinfection, nevertheless, discrimination can be highly difficult due to quite similar display of alterations in the liver specimen (7–9).
False treatment of suspected acute rejection with high-dose pulse steroid therapy in HCV-positive patients can have deleterious effects as hepatitis C virus activity may be severely increased by steroid treatment. Many authors advocate a very restricted use of steroid-pulse treatment even in cases of validated rejection in HCV-positive patients in order to avoid activation of virus replication (8,10–12).
Therefore a specific marker expressed only in rejection but not in HCV reinfection cases would be a great asset to differential diagnosis for HCV-positive patients with clinically suspicious symptoms in order to validate rejection diagnosis.
C4d is an end-product of the activated classical complement cascade, typically detectable in infectious situations and autoimmune disorders stimulating the complement system.
In recent years C4d has become a highly valued tool for diagnosis of acute rejection following kidney transplantation where humoral mechanisms play a by far greater role than after liver transplantation (13–16). Early humoral rejection with consecutive graft loss remains a rather exotic complication following liver transplantation where T-cell-mediated mechanisms are mainly held responsible for organ damage. Only incidental reports of acute humoral rejection, often associated with fatal outcome, are described (17).
In this study we investigated whether profound humoral mechanisms not leading to hyper-acute dramatic rejection do play a role in mainly T-cell-mediated rejection episodes after liver transplantation. Furthermore, we evaluated the potential role of C4d as a valid parameter in differential diagnosis between acute rejection and hepatitis C reinfection in HCV-positive patients (18).
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- Patients, Material and Methods
Twenty-three of 34 patients who by conventional histological examination had been diagnosed with acute cellular rejection displayed C4d-positive staining in their liver biopsy (67.7%) whereas 4 of 34 patients with hepatitis C reinfection were tested positive for C4d (11.8%). In the control group 2 of 29 specimens showed C4d positivity (6.9%).
C4d expression was significantly increased in acute rejection compared to hepatitis C recurrence (p < 0.001) and controls (p < 0.001).
In C4d, positive samples were found along endothelial cells of portal veins, portal arteries and portal capillaries. No distinct distribution pattern of C4d regarding portal vascular structures could be observed. Additionally, no C4d deposits were detected along hepatic veins or sinusoids. Furthermore no difference concerning the distribution of C4d was found between acute rejection and C4d-positive HCV recurrence cases.
Group 1: Nine patients in the rejection group had received LTX for HCV-induced cirrhosis; therefore C4d detection in these cases was especially interesting in order to discriminate between true rejection and possible HCV reinfection. At the time of biopsy, HCV reinfection was apparent in none of the 9 patients, C4d staining was positive in 6 of these 9 patients.
Two of the 6 HCV patients with positive retrospective C4d detection had biopsies 11 days after LTX, a high-risk period for acute rejection that could be confirmed by C4d positivity now. One other patient experienced rejection 3 months after transplantation, a time point when HCV reinfection also is likely to occur. C4d positivity was consistent with the clinical course after steroid-pulse treatment proving rejection. Hepatitis C reinfection in this case could be proved only as late as 27 months after LTX. The fourth patient experienced steroid-resistant rejection 2 months after LTX requiring OKT 3 therapy. Patient numbers 5 and 6 developed hepatitis C reinfection only years later. In summary, the further clinical course proved rejection diagnosis and associated C4d positivity to be correct in these six cases.
The remaining 3 of the 9 HCV patients in the rejection group also proved to have undergone acute rejection as response to anti-rejection therapy and evaluation of the further clinical course revealed. However, C4d staining was negative in these cases, well in line with the overall percentage of C4d positivity among all rejection cases (66.6% and 67.7%).
No correlation between presence and intensity of C4d and severity grade of rejection could be observed (18,19).
Group 2: In the HCV recurrence group 4 of 34 patients (11.8%) presented with C4d-positive liver biopsies. Of these four cases three had received interferon therapy prior to biopsy due to previously proven recurrence of hepatitis C.
Group 3: In the control group, 2 of 29 patients (6.9%) had C4d-positive biopsies, one of them having been transplanted for cryptogenic cirrhosis 2 years before biopsy and never having experienced rejection. However, 3 years later, portal fibrosis and chronic rejection were detected without exact classification of origin. The second patient had received LTX for polycystic liver disease and now displayed a C4d-positive result in the 1 year protocol biopsy showing a metabolic toxic lesion of the parenchyma yet had never experienced rejection episodes (Figure 1).
The significance for C4d expression as a positive marker of acute rejection displayed a specificity of 90.5% with a sensitivity of 67.7%. The positive predictive value of C4d accurately labeling acute rejection was 75.128% and the negative predictive value was 86.807%. These data were calculated with an area under the curve value of 0.791 (ROC curve, 95% confidence interval 0.696 to 0.867) and a standard deviation of 0.051.
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- Patients, Material and Methods
In this study we were able to show for the first time that reasonable amounts of C4d are present in liver tissue in cases of acute rejection following liver transplantation. Significant differences could be detected compared to C4d presence in control biopsies and hepatitis C reinfection patients. These data suggest the activation and involvement of B-cell-related mechanisms in acute rejection episodes after LTX to a far greater extent than previously expected. In previous studies, we were able to show increased presence of complement factors as well as macrophages and plasma cells in liver tissue undergoing acute rejection (20,21). However, no increase of other proliferating cells could be found suggesting migration of plasma cells and macrophages to the spot of immunological activation. Additionally, up-regulation of IgVH genes in liver allograft rejection specimen has demonstrated selected accumulation of B-cells and plasma cells (22).
C4d as the activated component of C4, a central factor of the classical complement pathway, has been established as an indispensable marker tool for acute as well as chronic rejection in kidney transplant patients (15,23,24).
Since its first description by Feucht et al. (25,26) C4d has proven to be a reliable indicator of rejection mechanisms present in the transplanted kidney tissue (16,27–30). After activation of the complement component C4, its degradation product C4d covalently binds to protein and carbon structures directly in the area where it is expressed enabling us to detect it in tissue biopsies. In NTX cases with de novo donor-specific antibodies C4d is expressed in 95% and humoral rejection accounts for as much as 25% of all rejection episodes (15,21).
Humoral mechanisms have been known to play a far greater role in acute rejection following kidney transplantation than in liver transplantation. Steroid-resistant rejection episodes are often of B-cell-related origin and susceptible to plasmapheresis, IVIG or anti-CD 20 antibody treatment (13).
The liver has widely been accepted as the immunologically more “tolerant” organ where rejection mechanisms are almost always based on T-cell-mediated immune response (13). Cases of pure humoral rejection are extremely rare, most often of fulminant nature and reports rather anecdotal (17,22). However, Sawada et al. demonstrated humoral immunity to be partially responsible for allograft damage in acute rejection after LTX (31).
It was our aim to evaluate whether C4d can be detected in liver biopsy specimen of patients with acute rejection (AR) episodes and may, therefore, serve as a specific marker supporting diagnosis of AR. Our special interest was dedicated to the differences in C4d expression compared to hepatitis C reinfection patients after LTX. Due to the immunologic processes within the transplanted organ following HCV recurrence differential diagnosis between acute rejection and HCV reinfection can be of great difficulty to the pathologist, especially in cases of mild rejection and de novo HCV reinfection (7,8). Mononuclear infiltrates of the portal tract are widely present in both cases challenging accurate differential diagnosis (32–34). In the past, this phenomenon has led to false histological diagnosis of acute rejection in HCV reinfection cases with consecutive steroid-pulse therapy and fatal effects on virus activation (4,10).
Many authors advocate steroid-free rejection therapy regimes for hepatitis C patients in order to minimize viral stimulation as steroid application for presumed rejection has been associated with significantly impaired outcome for HCV patients (8,35).
Several potential markers have been investigated to improve differential diagnostic criteria (36–39) yet no clinically applicable routine could be established. Ciccorossi et al. demonstrated anti-HCV IgM to be a highly specific marker of HCV recurrence when measured quantitatively. However, continuous HCV serological data need to be available in order to set quantitative levels into the right context. HCV-RNA was found to be expressed to a greater extent in patients transplanted for hepatitis C if HCV reinfection was present than in acute rejection yet no decisive quantitative value can be determined (39).
Srekunar et al. were able to detect a significantly greater expression of MHC class I and II associated genes in liver specimen in case of acute rejection than in HCV recurrence.
Unfortunately, genetic characterization is a complicated and time-consuming procedure that cannot serve for immediately required diagnosis in the clinical situation (36).
C4d staining is a fast and widely available procedure well established in the histological evaluation of kidney transplant rejection today. Hence liver biopsy specimen can be immediately stained for C4d either in cryopreserved or paraffinized tissue granting C4d-associated evaluation within hours (20).
We therefore looked at C4d as a potential marker for diagnostic discrimination between acute rejection and HCV reinfection in patients who had received LTX for HCV-induced cirrhosis.
Our results clearly show great differences in C4d expression between the respective groups. We were able to demonstrate a specificity of 90% for C4d positivity indicating acute rejection. However, sensitivity was only 67.7%, therefore we advocate the application of C4d as a supportive yet not secure marker of acute rejection after LTX. These numbers are well in line with expression rates after kidney transplantation where detection of C4d in rejection cases varies between 50% and 60% (12,26,27). As discussed above, retrospective scrutinization of individual cases has led us to suspect misled diagnosis due to insufficient conventional biopsy in specific cases.
Analysis of the 97 cases included in this study revealed false diagnosis by conventional histological staining for at least 6 patients; 4 individuals with hepatitis C reinfection who were misdiagnosed as undergoing acute rejection and 2 HCV patients who did not experience hepatitis C recurrence but acute rejection instead. Availability of C4D staining at the time of diagnosis might have spared these patients from false diagnosis followed by inadequate and potentially hazardous treatment.
Among the 4 C4d-positive HCV patients 3 had received interferon treatment prior to biopsy possibly inducing humoral mechanisms including complement activation in hepatitis C-positive patients. Baid et al. could show a remarkable increase in C4d-positive rejection episodes in kidney transplant patients suffering from chronic hepatitis C infection, which is possibly due to enhanced cell surface expression of HLA alloantigens (40). However, the question whether interferon treatment may induce susceptibility to acute or chronic rejection in solid organ transplantation is being discussed controversially. Cosimi et al. have shown interferon not to increase frequency of acute rejection after LTX (41) whereas many other authors advocate an increased risk for rejection episodes under interferon treatment (42,43).
Jain et al. performed a large retrospective study analyzing 105 LTX patients with hepatitis B and C who had received interferon-alpha treatment and could not detect a significant increase in rejection rates. However, patients in this study had received slightly higher steroid doses than control patients without interferon treatment (44).
In conclusion, we state that C4d as an established marker for acute rejection in kidney transplantation also plays a considerable role in acute rejection following liver transplantation suggesting the involvement of humoral mechanisms to a greater extent than currently accepted.
For the first time we were able to detect significantly stronger C4d deposits in liver biopsies of patients suffering from acute rejection than in rejection-free individuals.
Especially in differential diagnosis to HCV recurrence, which in conventional histological staining can be rather demanding, C4d may be able to contribute to more accuracy in specifying exact diagnosis in the future.