Cholestatic Hepatitis C Following Liver Transplantation: An Outcome-Based Histological Definition, Clinical Predictors, and Prognosis

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1 LIVER TRANSPLANTATION 19:78 88, 2013 ORIGINAL ARTICLE Cholestatic Hepatitis C Following Liver Transplantation: An Outcome-Based Histological Definition, Clinical Predictors, and Prognosis Elizabeth C. Verna, 1 Rita Abdelmessih, 1 Marcela A. Salomao, 2 Jay Lefkowitch, 2 Roger K. Moreira, 2 and Robert S. Brown Jr 1 1 Center for Liver Disease and Transplantation, Division of Digestive and Liver Diseases, and 2 Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY Cholestatic hepatitis C virus (HCV) is a rare form of recurrent HCV following liver transplantation (LT) without specific diagnostic criteria. An outcome-based method to improve its diagnosis and a description of its prognosis are needed. All 1-year post-lt protocol liver biopsy samples and biopsy samples initially reported to show cholestatic HCV from patients transplanted with HCV between February 2002 and December 2009 were reviewed for the inflammation grade, the fibrosis stage, and 4 cholestatic HCV features: ductular proliferation, canalicular cholestasis with or without intracellular cholestasis, hepatocyte swelling with or without lobular disarray, and sinusoidal/pericellular fibrosis. We used patient and graft survival to define histological criteria for cholestatic HCV, and compared the clinical features of these patients to those of patients with minimal or significant post- LT fibrosis. One hundred seventy-nine patients were analyzed, the median age was 56 years, and 73% were male. Patients with 3 or more of the 4 cholestatic HCV criteria had significantly worse survival (log-rank P < 0.001) regardless of the fibrosis stage, and this was used as our novel definition of cholestatic HCV. Using this definition, we found that 27 patients (15%) had cholestatic HCV, 53 (30%) had significant fibrosis (stage 2/4), and 99 (55%) had minimal fibrosis (stage < 2/4). The final model for clinical predictors of cholestatic HCV included donor age [odds ratio (OR) per decade, P ] and previous rejection (Banff grade 5; OR , P ). Total bilirubin was the strongest laboratory predictor of cholestatic HCV (area under the curve ), whereas the HCV viral load was not a significant predictor. The final model of post-lt survival included the pathology group {cholestatic HCV [hazard ratio (HR) , P < 0.001] and significant fibrosis (HR , P )}, donor age (HR per decade, P < 0.001), and cold ischemia time (HR per hour, P ). In conclusion, we propose diagnostic criteria for cholestatic HCV that include specific criteria (the presence of at least 3 of the 4 histopathological features on biopsy) and other supportive and exclusionary criteria. Older donor age and rejection increase the risk of cholestatic HCV, and an elevation in the total bilirubin level may help to identify these patients. These criteria must be validated prospectively. Liver Transpl 19:78-88, VC 2012 AASLD. Received March 24, 2012; accepted September 12, Cholestatic hepatitis C virus (HCV) is a rare and severe form of viral hepatitis that affects patients with recurrent HCV after liver transplantation (LT). Although cholestatic hepatitis was first described in patients with hepatitis B virus, 1,2 cholestatic hepatitis due to HCV is now widely reported in the literature Abbreviations: CI, confidence interval; CIT, cold ischemia time; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HR, hazard ratio; IQR, interquartile range; LT, liver transplantation; MELD, Model for End-Stage Liver Disease; OR, odds ratio; PT, portal tract; ROC, receiver operating curve; SVR, sustained virological response; WIT, warm ischemia time. Address reprint requests to Roger K. Moreira, M.D., Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC 238B, New York, NY Telephone: ; FAX: ; rkm2124@ columbia.edu; or Robert S. Brown Jr, M.D., M.P.H., Center for Liver Disease and Transplantation, Division of Digestive and Liver Diseases, Columbia University College of Physicians and Surgeons, 622 West 168th Street, PH 14, New York, NY Telephone: ; FAX: ; rb464@columbia.edu DOI /lt View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI /lt. Published on behalf of the American Association for the Study of Liver Diseases VC 2012 American Association for the Study of Liver Diseases.

2 LIVER TRANSPLANTATION, Vol. 19, No. 1, 2013 VERNA ET AL. 79 and primarily affects patients with HCV who are immunosuppressed due to cytotoxic therapy, 3,4 human immunodeficiency virus infection, 5,6 or solid organ transplantation of the kidney, 7-12 heart, 13,14 or liver. 6,15-32 Early recognition is crucial to the successful treatment of cholestatic HCV, a disease that is characterized by a high risk of liver failure and mortality independent of cirrhosis or portal bridging fibrosis. Yet very little is known about the pathophysiology of cholestatic HCV and the ways in which it differs from other more conventional forms of aggressive HCV. Immunosuppression-deranged antiviral immune responses and high levels of HCV viral replication have been implicated. 33,34 The lack of a standardized definition has significantly limited investigations into the clinical features and prognosis of cholestatic HCV. In 2003, a consensus conference on post-lt HCV first attempted to define the disease as occurring greater than 1 month after transplantation with a serum bilirubin level > 6 mg/dl, serum alkaline phosphatase and gamma-glutamyl transpeptidase levels greater than 5 times the upper limits of normal, a characteristic histological state (including ballooning of hepatocytes predominantly in the perivenular zone, paucity of inflammation, and variable degrees of cholangiolar proliferation without bile duct loss), very high HCV RNA levels, and the absence of biliary complications or hepatic artery thrombosis. 35 However, little data on liver test or viral load cutoffs are available to justify this definition, which is sufficiently vague that it is difficult to apply clinically and is rarely used even in the literature. 33,36 One review that summarizes the varied definitions reported for published cases found that cholestasis was the most common biopsy finding, followed by ballooning degeneration, fibrosis, and ductal/cholangiolar proliferation, but the pathological criteria were not described in some cases. 36 In addition, viral loads and patient outcomes were not reported in all cases, and only 25% of the reviewed studies that were published after the consensus definition used its criteria. Other authors have attempted to characterize early cholestatic HCV biopsy findings with novel scoring systems for cholestasis and sinusoidal fibrosis with a small series of cases, 17 but this approach requires the application of additional complex scores and has not been widely adopted. The largest and most recent series of cholestatic HCV patients to be published 15 used alternate liver biopsy findings to define the syndrome (cholestasis, periportal fibrous septa, ductular reaction, and mild inflammation), but importantly, this study lacked the control groups with advanced portal-based fibrosis and minimal HCV recurrence required to draw conclusions about their clinical presentation and outcomes. We, therefore, aimed to identify outcome-based clinical and histopathological criteria for cholestatic HCV through a review of liver biopsy samples, clinical information, and detailed graft and patient outcome data for a large cohort composed of all patients with HCV who underwent transplantation at our center. In addition, we compared the prognosis of patients meeting the cholestatic HCV criteria to the prognosis of patients who had either minimal fibrosis or advanced fibrosis but did not meet the cholestatic HCV criteria. PATIENTS AND METHODS Patients All patients who underwent LT for HCV-related endstage liver disease at New York Presbyterian Hospital Columbia University between February 2002 and December 2009 were retrospectively assessed. Patients who had a human immunodeficiency virus or hepatitis B virus coinfection, patients who had no detectable HCV RNA at LT (because of successful treatment or spontaneous clearance) and were, therefore, not at risk for recurrent disease, and patients who died or underwent retransplantation within 30 days of LT were excluded. Patients with hepatic artery thrombosis at any time or significant biliary complications (defined as abnormal biliary imaging requiring endoscopic, percutaneous, or surgical intervention within 6 months of the study biopsy) were excluded. In addition, patients with no 1-year post-lt liver biopsy performed or available for review (with a window of 8-16 months after LT) were excluded. Data were collected from the United Network for Organ Sharing database and through chart and electronic medical record reviews. The standard immunosuppression regimen for LT recipients with HCV at our center includes a steroid bolus and taper, mycophenolate mofetil, and a calcineurin inhibitor (either cyclosporine or tacrolimus). This protocol was approved by the institutional review board of Columbia University. Development of Histological Criteria for Cholestatic HCV Our center performs protocol liver biopsy after LT for HCV patients at 3 months, at 1 year, and then annually, as well as to assess elevations in liver tests. The study liver biopsy samples included all 1-year post-lt liver biopsy samples (with a window of 8-16 months after LT) as well as 19 additional biopsy samples that were not within this window but were initially read as having features of cholestatic HCV. These were included to ensure that a significant number of possibly real cholestatic HCV cases were not excluded from the analysis, but they were reclassified for the purposes of this study according to their cholestatic HCV scores and fibrosis stages just as all other study biopsy samples were. Specifically for the purposes of this study, all study liver biopsy samples were rereviewed by 1 attending pathologist (R.K.M.) who was blinded to the clinical conditions and patient outcomes. Because these biopsy samples may have been taken under different clinical circumstances, a sensitivity analysis excluding the 19 patients with study biopsy samples outside the 1-year time point was performed to ensure that differences in mortality and

3 80 VERNA ET AL. LIVER TRANSPLANTATION, January 2013 Figure 1. Pathological findings for cholestatic HCV. (A) Portal tracts (PT) are markedly expanded by proliferating ductules and inflammatory cells (ductular reaction; hematoxylin and eosin stain, original magnification 3100). (B) The prominent ductular reaction seen in this case is highlighted by cytokeratin 7 immunohistochemistry (original magnification 3100). (C) Pericellular/ sinusoidal fibrosis is often seen in periportal regions (Masson s trichrome stain, original magnification 3400). (D) Hepatic lobules characteristically show significant hepatocyte swelling, lobular disarray, and marked cholestasis (circled areas; hematoxylin and eosin stain, original magnification 3400). graft loss were not solely due to the inclusion of these patients. Archival, formalin-fixed, paraffin-embedded, 3-lm tissue section slides stained with hematoxylin and eosin and Masson trichrome stains were retrieved from pathology files. Grading and staging were scored according to the Batts-Ludwig system.37 In addition, each case was evaluated for the following histopathological parameters proposed to be characteristic of cholestatic HCV (Fig. 1): (1) a prominent ductular reaction resembling a biliary obstruction in the majority of portal tracts (PTs), (2) cholestasis (defined as canalicular bile plugs and/or intracellular bile pigment), (3) prominent hepatocyte ballooning with lobular disarray, and (4) any degree of sinusoidal/ pericellular fibrosis. Each of these findings was assigned 1 point, and all study biopsy samples were assigned a score of 0 to 4. Each score was then assessed for its relationship to posttransplant time-dependent patient survival with Kaplan-Meier modeling in order to define cholestatic HCV cases on the basis of inferior recipient and graft outcomes, independent of the Batts-Ludwig fibrosis stage. Patients were then divided into 3 categories for analysis: (1) patients with cholestatic HCV (based on the criteria developed and defined later), (2) patients with significant fibrosis (defined as at least stage 2 of 4 according to the Batts-Ludwig staging system) on 1year post-lt liver biopsy samples who did not meet the criteria for cholestatic HCV, and (3) patients with minimal fibrosis (defined as stage 1 or lower) on 1-year post-lt liver biopsy samples who did not meet the final criteria for the diagnosis of cholestatic HCV. Statistical Analysis Between groups, continuous variables were compared with t test and an analysis of variance, and categorical variables were compared with chi-square and Kruskal-Wallis tests. Determination of cholestatic HCV criteria were used in the Kaplan-Meier survival analysis and log-rank testing. To identify clinical predictors of cholestatic HCV, the histological group was evaluated as a primary outcome in logistic regression and receiver operating curve (ROC) analysis. Predictors tested in univariate and multivariate models included the following: recipient age, sex, and calculated Model for End-Stage Liver Disease (MELD) score at LT; presence of hepatocellular carcinoma (HCC) on explant; donor age and HCV status; extended donor criteria; donation after cardiac death; living donor organ; cold ischemia time (CIT); warm ischemia time (WIT); initial calcineurin

4 LIVER TRANSPLANTATION, Vol. 19, No. 1, 2013 VERNA ET AL. 81 TABLE 1. Number of Histological Features Final Group Minimal fibrosis (n) Significant fibrosis (n) Cholestatic HCV (n) Figure 2. Cumulative survival by the number of histological criteria present. inhibitor; rejection (Banff grade 5) before the study biopsy; rejection requiring high-dose therapy (an intravenous steroid bolus or biological agent) before the study biopsy; and HCV viral load at the time of biopsy. The HCV viral load was defined as the closest value within 3 months of the biopsy date and was treated as a dichotomous variable (ie, or <600,000, 800,000, or 1,000,000 IU/mL). An ROC analysis was used to evaluate the ability of serum biomarkers of liver function (eg, bilirubin and aminotransferases) to differentiate cholestatic HCV patients from the other groups. Biomarker cutoffs were determined from the ROC analysis to maximize the sensitivity of the test. Kaplan-Meier survival curves were compared with a log-rank test to evaluate overall time-dependent patient and graft survival in each pathological group. Cox proportional hazards models were used to test multivariate models. The tested predictors included all those listed previously for modeling predictors of cholestatic HCV as well as the histological category according to the study biopsy. All multivariate models were evaluated in a stepwise fashion. All variables with P 0.25 in the univariate analysis were included in model building. Variables that were no longer significant at P < 0.05 were then sequentially eliminated. All analyses were performed with Stata 10.0 (StataCorp, College Station, TX). HCV Treatment HCV treatment was defined as a patient receiving any interferon or ribavirin in the posttransplant period. The criteria for HCV treatment at our center include 1 or more of the following in the absence of a contraindication to treatment: necroinflammation grade > 2/ 4, fibrosis stage 2/4 according to the Batts-Ludwig system, or the presence of features consistent with cholestatic HCV. Sustained virological response (SVR) was defined as a persistently undetectable viral load at least 6 months after the discontinuation of HCV antiviral therapy. For a proportion of the patients who received antiviral treatment in the post-lt period, treatment was initiated before the study biopsy date. In order to ensure that treatment before this biopsy did not significantly affect our survival model, a second sensitivity analysis excluding these patients was performed. RESULTS Three hundred seventy-nine patients underwent transplantation with HCV in the study period, and 179 patients met the study criteria and were included in the analysis. Exclusionary criteria included less than 1 month of follow-up (n 5 15), biliary complications within 6 months of the study biopsy (n 5 57), and no 1-year post-lt liver biopsy being performed (n 5 145) or available for re-review (n 5 41). There were no significant differences in the age at transplantation, sex, MELD score, HCC status, donor age, use of extended criteria donor grafts, CIT, WIT, initial calcineurin inhibitor, induction immunosuppression, or need for retransplantation between study patients and those patients excluded for the aforementioned reasons. Patients not included in the study were at a higher risk of death according to a Kaplan-Meier analysis (log-rank P ) because of deaths in the first year precluding an assessment of 1-year protocol biopsy samples. Definition of Cholestatic HCV All study biopsy samples were reviewed for the aforementioned 4 findings and were given a score of 0 to 4. Each score was then assessed for its relationship to time-dependent patient survival after transplantation (Fig. 2) in order to identify those patients with a poor prognosis regardless of bridging fibrosis or cirrhosis. The largest decrease in patient survival was seen between patients with at least 3 of these criteria and patients with 2 or fewer (log-rank P < 0.001), whereas the outcomes were statistically similar for patients with 3 or 4 of the criteria (log-rank P ) and for patients with 1 or 2 of the criteria (log-rank P ). Similarly, the greatest difference in graft survival was seen between patients with at least 3 of these criteria and patients with 2 or fewer (log-rank P < 0.001), although the outcomes were also statistically different for patients with 1 or 2 criteria (log-rank P ). Therefore, patients who had at least 3 of the 4

5 82 VERNA ET AL. LIVER TRANSPLANTATION, January 2013 TABLE 2. Patient Characteristics Cholestatic HCV Significant Fibrosis Minimal Fibrosis Variable (n 5 27) (n 5 53) (n 5 99) P Value Male [n (%)] 22 (81) 35 (66) 74 (75) 0.29 Age at LT (years)* 55.6 (9) 55.2 (11) 56.4 (8) 0.53 Race/ethnicity [n (%)] Non-Hispanic white 15 (56) 29 (55) 58 (59) 0.51 Hispanic white 8 (30) 14 (26) 28 (28) Black 3 (11) 5 (9) 6 (6) Other 1 (4) 5 (9) 7 (7) Genotype 1 HCV [n/n (%)] 16/19 (84) 26/34 (76) 47/61 (77) 0.78 HCC [n (%)] 10 (37) 15 (28) 56 (57) MELD score at LT* 21 (12) 22 (12) 16 (11) Living donor [n (%)] 0 (0) 6 (11) 9 (9) 0.21 Extended criteria donor [n (%)] 17/23 (74) 29/51 (57) 48/94 (51) 0.14 Donor after cardiac death [n (%)] 0 (0) 2 (4) 2 (2) 0.55 CIT (hours)* 8 (3.3) 7.1 (5.8) 8 (4.2) 0.37 WIT (minutes)* 45 (4) 42 (9) 42 (10) 0.32 Donor age (years)* 56.5 (24) 46 (16.8) 43 (21) 0.03 HCV-seropositive donor [n (%)] 6/26 (23) 15/49 (31) 19/95 (20) 0.36 Cyclosporine as initial calcineurin inhibitor [n (%)] 22 (81) 40 (75) 73 (74) 0.71 Any induction [n (%)] 6 (22) 13 (25) 16 (16) 0.54 Simulect 6 (22) 13 (25) 14 (14) Thymoglobulin 0 (0) 0 (0) 1 (1) Daclizumab 0 (0) 0 (0) 1 (1) Treated acute cellular rejection [n (%)] 4 (15) 7 (13) 12 (12) 0.93 Acute rejection: Banff grade 5 [n (%)] 13 (48) 10 (19) 16 (16) Laboratory values at study biopsy Total bilirubin (mg/dl)* 12.8 (16.6) 1.2 (2.4) 1 (0.9) <0.001 Direct bilirubin (mg/dl)* 6.9 (10.2) 0.4 (1.4) 0.3 (0.3) <0.001 Aspartate aminotransferase (U/L)* 199 (118) 112 (175) 48 (73) <0.001 Alanine aminotransferase (U/L)* 102 (94) 74 (161) 52.5 (92) Alkaline phosphatase (U/L)* 195 (180) 173 (129) 155 (173) 0.07 Gamma-glutamyl transpeptidase (U/L)* (506) 249 (498) 130 (266) 0.02 HCV viral load at biopsy [n/n (%)] >800,000 IU/mL 18/23 (78) 28/38 (74) 43/67 (64) 0.36 >1,000,000 IU/mL 17/23 (74) 27/38 (71) 40/67 (60) 0.33 Time to first biopsy (days)* 33 (81) 54 (94) 114 (123) <0.001 Total number of biopsies* 5 (3) 4 (3) 4 (3) 0.1 Time to study biopsy (days)* 281 (322) 356 (108) 381 (70) Follow-up after LT (years)* 2.1 (4.5) 4.0 (3.1) 3.4 (2.9) *The data are presented as medians and IQRs. The genotype and the HCV viral load at biopsy were not available for all patients. Acute rejection included only rejection episodes before the study biopsy. The HCV viral load upper limit of detection was 6.89 log IU/mL. histopathological criteria were defined as having cholestatic HCV for the purposes of this study. On the basis of the aforementioned criteria, 27 patients (15% of the study cohort) were classified as having cholestatic HCV, whereas 53 (30%) had significant fibrosis (stage 2/4) and 99 (55%) had minimal fibrosis (stage < 2/4) but did not meet our cholestatic HCV criteria. Cholestatic HCV by this definition was, therefore, diagnosed in 7.1% of the entire population of LT patients with HCV at our center in the study period. The breakdown of our novel histological scoring system for those patients in the minimal and significant fibrosis groups is displayed in Table 1. Sensitivity analyses were performed that excluded the 19 patients with study biopsy samples taken outside the defined 1-year posttransplant window. These patients included 14 patients in the cholestatic HCV group and 5 patients in the significant fibrosis group. When these patients were excluded, the largest decrease in patient survival was still seen between patients with at least 3 of the criteria and patients with 2 or fewer (log-rank P < 0.001), whereas outcomes were statistically similar for patients with 3 or 4 of the criteria (log-rank P ) and for patients with 1 or 2 of the criteria (log-rank P ). Similarly, the greatest difference in graft survival was seen

6 LIVER TRANSPLANTATION, Vol. 19, No. 1, 2013 VERNA ET AL. 83 TABLE 3. Multiple Logistic Regression Model for the Prediction of Cholestatic HCV Univariate Analysis Multivariate Analysis Predictor* OR 95% CI P Value OR 95% CI P Value Donor age (per decade) Banff grade 5 rejection *Tested predictors included the following: recipient age, sex, MELD score, and HCC status; donor age and HCV status; extended criteria organ; donation after cardiac death; living donor organ; CIT; WIT; initial calcineurin inhibitor; rejection (Banff grade 5); and treated rejection. between patients with at least 3 of these criteria and patients with 2 or fewer (log-rank P < 0.001). The exclusion of these patients, therefore, did not change our final model. Patient Characteristics The overall median age was 56 years [interquartile range (IQR) years], and 73% were male (Table 2). There were no statistically significant differences between the groups with respect to age, sex, race/ ethnicity, HCV genotype 1, graft type (living, extended criteria, or donation after cardiac death donors), CIT, WIT, or HCV-seropositive grafts. Overall, 83 (46%) of these patients underwent transplantation in the early part of our study period ( ), and they included 13 (48%) of the cholestatic HCV patients. The median MELD score at LT was significantly higher for patients with cholestatic HCV [21 (IQR 5 11), P ] and patients with significant fibrosis [22 (IQR 5 12), P ] in comparison with patients with minimal fibrosis [16 (IQR 5 11)]. The median donor age was significantly higher for patients with cholestatic HCV [56.5 years (IQR 5 24 years)] versus patients with minimal fibrosis [43 years (IQR 5 21 years), P ], and it tended to be higher for the cholestatic HCV patients versus patients with significant fibrosis, but this did not reach statistical significance [46 years (IQR years), P ]. Patients with cholestatic HCV were more likely to have experienced biopsy-proven rejection (Banff grade 5) in the period preceding the study biopsy (48%) than patients with minimal fibrosis (16%, P ) or significant fibrosis (19%, P ), but the percentages for rejection treated with high-dose therapy (including an intravenous steroid bolus or biological agent) were similar for the groups. The median time to a cholestatic HCV diagnosis was 281 days (IQR days). All groups had similar median numbers of total post-lt biopsies (Table 2). Clinical Predictors of Cholestatic HCV Clinical predictors of the development of cholestatic HCV were evaluated with univariate and multivariate logistic regressions (Table 3). The only significant predictors in the unadjusted analysis were donor age [odds ratio (OR) per decade, 95% confidence interval (CI) , P ] and biopsy-proven acute rejection (Banff grade 5) preceding the study biopsy (OR , 95% CI , P ). In the final adjusted model, donor age (OR per decade, 95% CI , P ) and Banff 5 rejection (OR , 95% CI , P ) remained the only significant predictors. Laboratory parameters at the time of study biopsy also differed between the groups (Table 2). The median total bilirubin level was higher in the cholestatic HCV group [12.8 mg/dl (IQR mg/dl)] versus the minimal fibrosis group [1.0 mg/dl (IQR mg/dl), P < 0.001] and the significant fibrosis group [1.2 mg/dl (IQR mg/dl), P < 0.001], as were the direct bilirubin, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transpeptidase levels. HCV viral loads within 3 months of the study biopsy were available for 128 patients (72%), and they did not differ significantly between the groups when they were classified as or <600,000, 800,000, or 1,000,000 IU/mL. In the ROC analysis, the total and direct bilirubin levels at the time of study biopsy were the strongest predictors of cholestatic HCV: each had an area under the curve of 0.93 (95% CI ). In comparison with total bilirubin, aspartate aminotransferase (0.78, 95% CI , P < 0.001), alanine aminotransferase (0.62, 95% CI , P < 0.001), alkaline phosphatase (0.64, 95% CI , P < 0.001), and gamma-glutamyl transpeptidase (0.70, 95% CI , P ) were significantly less predictive. At a cutoff of 2.0 mg/dl, total bilirubin was 100% sensitive and 60% specific for a diagnosis of cholestatic HCV. In addition, at a cutoff of 150 U/L, gamma-glutamyl transpeptidase was 96% sensitive and 27% specific for cholestatic HCV, whereas aspartate aminotransferase at a cutoff of 70 U/L was 96% sensitive and 42% specific. Patient Outcomes The 1-year, 3-year, and overall patient survival rates were significantly different between the groups (Table 4). According to a Kaplan-Meier analysis, cholestatic HCV patients experienced significantly worse timedependent survival than patients with significant (P < 0.001) or minimal fibrosis (P < 0.001; Table 4 and

7 84 VERNA ET AL. LIVER TRANSPLANTATION, January 2013 TABLE 4. Overall Patient Outcomes Cholestatic Significant Minimal Outcome HCV (n 5 27) Fibrosis (n 5 53) Fibrosis (n 5 99) P Value Patient survival [n (%)] 1 year 21 (78) 48 (91) 97 (98) < years* 10 (42) 31 (72) 53 (88) <0.001 Overall 9 (33) 35 (66) 84 (85) <0.001 Overall graft survival [n (%)] 5 (19) 33 (62) 82 (83) <0.001 HCV antiviral therapy [n (%)] 21 (78) 27 (51) 25 (25) <0.001 SVR [n (%)] 2 (7) 5 (9) 6 (6) Cause of death [n (%)] Recurrent HCV 8 (30) 8 (15) 4 (4) Sepsis 7 (26) 4 (8) 2 (2) HCC 1 (4) 0 (0) 2 (2) Other/unknown 2 (7) 6 (11) 7 (7) *Three-year survival is presented for patients with at least 3 years of possible follow-up (24, 43, and 60 patients in the cholestatic HCV, significant fibrosis, and minimal fibrosis groups, respectively). Figure 3. Cumulative survival of the final histological groups. (1.11 per hour, 95% CI , P ) independently predicted mortality (Table 5). Predictors of graft loss were also modeled. In the unadjusted analysis, the fibrosis group [cholestatic HCV (HR , 95% CI , P < 0.001) and significant fibrosis (HR , 95% CI , P ) versus minimal fibrosis], donor age (HR per decade, 95% CI , P < 0.001), and CIT (HR , 95% CI , P ) were the only significant predictors of graft loss. In the final adjusted model, the fibrosis group [cholestatic HCV (HR , 95% CI , P < 0.001) and significant fibrosis (HR , 95% CI , P )], donor age (HR per decade, 95% CI , P < 0.001), and CIT (HR per hour, 95% CI , P ) as well as female sex (HR 5 2.0, 95% CI , P ) and donation after cardiac death (HR , 95% CI , P ) were predictive of graft loss. Fig. 3). At a median follow-up of 3.3 years (IQR years), the mortality rate was higher for patients with cholestatic HCV (67%) versus patients with significant (34%, P ) or minimal fibrosis (15%, P < 0.001). The causes of death included most prominently liver failure due to recurrent HCV, sepsis, and recurrent HCC (Table 4). In the unadjusted analysis of mortality, the fibrosis group {cholestatic HCV [hazard ratio (HR) , 95% CI , P < 0.001] and significant fibrosis (HR , 95% CI , P ) versus minimal fibrosis (referent group)} and donor age (HR per decade, 95% CI , P < 0.001) were the only significant predictors. In the final adjusted model, the fibrosis group [cholestatic HCV (HR , 95% CI , P < 0.001) and significant fibrosis (HR , 95% CI , P ) versus minimal fibrosis], donor age (1.49 per decade, 95% CI , P < 0.001), and CIT HCV Treatment Seventy-three patients received HCV antiviral therapy. A significantly higher proportion of patients with cholestatic HCV [21 (78%)] received antiviral therapy in comparison with patients with significant [27 (51%), P ] or minimal fibrosis [25 (25%), P < 0.001]. SVR was achieved by 2, 5, and 6 patients in the cholestatic HCV, significant fibrosis, and minimal fibrosis groups, respectively. Those patients who achieved SVR experienced a significant survival advantage in comparison with those who were treated but did not achieve SVR (log-rank P ). Thirty-seven of the 73 treated patients (51%) underwent transplantation in the early half of the cohort ( ), whereas 36 (49%) underwent transplantation in the more recent era ( ). Twenty-four of these 73 patients received HCV treatment with a date of initiation before the date of the study biopsy (11 patients with cholestatic HCV, 7

8 LIVER TRANSPLANTATION, Vol. 19, No. 1, 2013 VERNA ET AL. 85 TABLE 5. Final Multivariate Cox Proportional Hazards Model for Predicting Mortality and Graft Loss Mortality Univariate Analysis Multivariate Analysis Predictor* HR 95% CI P Value HR 95% CI P Value Histological group Minimal fibrosis (reference) Significant fibrosis Cholestatic HCV < <0.001 Donor age (per decade) < <0.001 CIT (per hour) Graft Loss Univariate Analysis Multivariate Analysis Predictor* HR 95% CI P Value HR 95% CI P Value Histological group Minimal fibrosis (reference) Significant fibrosis Cholestatic HCV < <0.001 Female sex Donor age (per decade) < <0.001 Donation after cardiac death CIT (per hour) *Tested predictors included the following: histological group; recipient age, sex, MELD score, and HCC status; donor age and HCV status; extended criteria organ; donation after cardiac death; living donor organ; CIT; WIT; initial calcineurin inhibitor; rejection (Banff grade 5); and treated rejection. patients with significant fibrosis, and 6 patients with minimal fibrosis). In order to ensure that treatment before the study biopsy did not significantly affect our models, sensitivity analyses excluding these 24 patients were performed. As in our complete cohort, the largest decrease in patient survival was seen between patients with at least 3 of these criteria and patients with 2 or fewer (log-rank P < 0.001), whereas differences were less significant for patients with 1 or 2 criteria (log-rank P ). Similarly, the greatest difference in graft survival was seen between patients with at least 3 of these criteria and patients with 2 or fewer (log-rank P < 0.001); therefore, our overall model was not significantly altered. DISCUSSION This is the first study to our knowledge to attempt to objectively define clinical and histopathological criteria for the diagnosis of cholestatic HCV on the basis of poor patient outcomes independent of bridging fibrosis and cirrhosis. These criteria were chosen from the available literature and were tested in combination for the prediction of post-lt mortality. When they were applied to all patients who underwent transplantation with HCV at our center and had biopsy samples available for review, cholestatic HCV was found to have occurred in up to 15% of these patients (7.1% of the total transplant cohort), whereas standard significant fibrosis (at least stage 2) occurred in another 30%. The rate of cholestatic HCV in the study cohort was higher than the rates reported for other centers, but the actual rate of cholestatic HCV in our group was likely lower because the patients who were excluded for lack of post-lt biopsy samples may have been less likely to have significant HCV recurrence, and we also excluded patients with any signs of biliary obstruction. In addition, because we reviewed biopsy samples for all patients and not just those initially thought to have cholestatic HCV, we may have captured additional cases through this more sensitive approach. Several clinical parameters were significantly more abnormal for the patients who developed cholestatic HCV; these included higher MELD scores at LT, older donor age, and a higher incidence of biopsy-proven acute cellular rejection (Banff grade 5) before the study biopsy. Several laboratory parameters, including most significantly total and direct bilirubin, were also associated with cholestatic HCV. In the adjusted analysis, donor age and Banff grade 5 rejection remained the most significant clinical predictors. In contrast to what previous reports and guidelines have suggested, 15,18,25,30,35,36,38,39 the HCV viral load at traditional cutoffs was not predictive of cholestatic HCV, although the viral loads were high in all groups.

9 86 VERNA ET AL. LIVER TRANSPLANTATION, January 2013 TABLE 6. Proposed Diagnostic Criteria for Cholestatic HCV I. Specific criteria Histopathology: at least 3 of the following 4 lesions on biopsy: 1. Prominent ductular reaction resembling a biliary obstruction in the majority of portal tracts 2. Cholestasis (defined as canalicular bile plugs and/or intracellular bile pigment) 3. Prominent hepatocyte ballooning with lobular disarray 4. Any degree of periportal sinusoidal/pericellular fibrosis Timeframe: >1 month after transplantation II. Supportive criteria Total bilirubin level 2.0 mg/dl Gamma-glutamyl transpeptidase level 150 U/L Aspartate aminotransferase level 70 U/L Older donor age Recent biopsy-proven acute rejection (Banff rejection activity index 5) III. Exclusionary criteria Absence of biliary obstructions Absence of hepatic artery thrombosis However, this analysis of viral loads was limited by the lack of serial measurements over time, and these data were available for only 72% of the patients. In addition, several measurements were above the upper limit of quantification, and this limited our ability to analyze the viral load as a continuous variable. The impact of the viral load on cholestatic HCV and recurrent HCV warrants additional study. Cholestatic HCV was associated with a strong negative impact on patient and graft survival. Because we based our cholestatic HCV criteria on patient mortality, this is a predictable finding. In multivariate models, significant fibrosis as well as donor age and CIT also predicted mortality, and this is consistent with previous findings In the adjusted model for graft survival, in addition to these factors, female sex and donation after cardiac death significantly predicted poor outcomes. Female sex has recently been described as a risk factor for graft loss related to HCV in a multicenter, retrospective cohort study, 46 and this finding should be further confirmed prospectively. The strengths of this article include the very important evaluation of control groups in the description of the cholestatic HCV patients and the specific review of biopsy samples by 1 pathologist for the purposes of this investigation. This has allowed us to test combinations of cholestatic HCV criteria in a wide range of patients and to objectively evaluate the utility of serological testing in the setting of suspected cholestatic HCV. We have been able to demonstrate a bilirubin cutoff that may be clinically useful and raise questions regarding the diagnostic utility of the HCV viral load. Weaknesses of this study include the small sample size for cholestatic HCV patients (although it is among the larger series published to date) and the inability to reliably classify all liver biopsies into routine protocol biopsies and for-cause biopsies performed in the setting of a clinical event. In order to ensure that the differences seen in patient and graft outcomes were not merely the result of this limitation, sensitivity analyses were performed that excluded the 19 biopsy samples taken outside the 1-year posttransplant period, and they revealed an almost identical impact of cholestatic HCV histological characteristics on patient and graft survival. Furthermore, this is a retrospective study, so some clinical information such as the HCV genotype was inconsistently reported, and additional potential predictors such as the donor and recipient interleukin-28b status were not available for analysis. Finally, several patients did not have post-lt liver biopsy samples or slides available for review, and this possibly affected the results of our analysis, although the baseline demographics of these patients did not differ from those of the study group. Most patients with cholestatic HCV are likely to undergo clinically indicated biopsy because of abnormal liver tests. We, therefore, have proposed a novel set of diagnostic criteria for cholestatic HCV based on specific criteria (the presence of at least 3 of 4 histopathological lesions on biopsy and a timeframe of > 1 month after transplantation) in combination with supportive criteria (total serum bilirubin, gamma-glutamyl transpeptidase, and aspartate aminotransferase levels) and exclusionary criteria (an absence of biliary obstructions and hepatic artery thrombosis; Table 6). 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