ORIGINAL ARTICLE

A Randomized, Multicenter Study ComparingSteroid-Free Immunosuppression and StandardImmunosuppression for Liver TransplantRecipients With Chronic Hepatitis CGoran B. Klintmalm,1 Gary L. Davis,1 Lewis Teperman,2 George J. Netto,3 Kenneth Washburn,4

Stephen M. Rudich,5 Elizabeth A. Pomfret,6 Hugo E. Vargas,7 Robert Brown,8 Devin Eckhoff,9

Timothy L. Pruett,10 John Roberts,11 David C. Mulligan,7 Michael R. Charlton,12 Thomas G. Heffron,13

John M. Ham,14 David D. Douglas,7 Linda Sher,15 Prabhakar K. Baliga,16 Milan Kinkhabwala,8

Baburao Koneru,17 Michael Abecassis,18 Michael Millis,19 Linda W. Jennings,1 and Carlos G. Fasola13

1Baylor University Medical Center at Dallas, Dallas, TX; 2New York University Medical Center, New York, NY;3Johns Hopkins Medical Institutions, Baltimore, MD; 4University of Texas Health Science Center at San Antonio,San Antonio, TX; 5University of Cincinnati, Cincinnati, OH; 6Lahey Clinic, Burlington, MA; 7Mayo Clinic,Scottsdale, AZ; 8New York Presbyterian Hospital, New York, NY; 9University of Alabama at Birmingham,Birmingham, AL; 10University of Virginia, Charlottesville, VA; 11University of California San Francisco, SanFrancisco, CA; 12Mayo Clinic, Rochester, MN; 13Emory University School of Medicine, Atlanta, GA;14Oregon Health Sciences University, Portland, OR; 15University of Southern California, Los Angeles, CA;16Medical College of South Carolina, Charleston, SC; 17University of Medicine and Dentistry of New Jersey,Newark, NJ; 18Northwestern Memorial Hospital, Chicago, IL; and 19University of Chicago, Chicago, IL

This randomized, prospective, multicenter trial compared the safety and efficacy of steroid-free immunosuppression (IS) to thesafety and efficacy of 2 standard IS regimens in patients undergoing transplantation for hepatitis C virus (HCV) infection. The out-come measures were acute cellular rejection (ACR), severe HCV recurrence, and survival. The patients were randomized (1:1:2) totacrolimus (TAC) and corticosteroids (arm 1; n ¼ 77), mycophenolate mofetil (MMF), TAC, and corticosteroids (arm 2; n ¼ 72), orMMF, TAC, and daclizumab induction with no corticosteroids (arm 3; n¼ 146). In all, 295 HCV RNA–positive subjects were enrolled.At 2 years, there were no differences in ACR, HCV recurrence (biochemical evidence), patient survival, or graft survival rates. Theside effects of IS did not differ, although there was a trend toward less diabetes in the steroid-free group. Liver biopsy samplesrevealed no significant differences in the proportions of patients in arms 1, 2, and 3 with advanced HCV recurrence (ie, an inflamma-tion grade � 3 and/or a fibrosis stage � 2) in years 1 (48.2%, 50.4%, and 43.0%, respectively) and 2 (69.5%, 75.9%, and 68.1%,respectively). Although we have found that steroid-free IS is safe and effective for liver transplant recipients with chronic HCV, ste-roid sparing has no clear advantage in comparison with traditional IS. Liver Transpl 17:1394-1403, 2011. VC 2011 AASLD.

Received April 19, 2011; accepted August 5, 2011.

It has been estimated that 3 to 4 million people in theUnited States are chronically infected with hepatitis Cvirus (HCV). Complications of this infection, including

decompensated cirrhosis and hepatocellular carci-noma, are the main indications for liver transplanta-tion in this country and account for approximately

Abbreviations: ACR, acute cellular rejection; CI, confidence interval; CMV, cytomegalovirus; DAC, daclizumab; HCV, hepatitis Cvirus; IS, immunosuppression; MMF, mycophenolate mofetil; NS, not significant; OR, odds ratio; PRED, prednisone; RAI, rejectionactivity index; TAC, tacrolimus.

This study was supported by a grant from Roche.

Carlos G. Fasola is currently affiliated with the Scott and White Clinic, Temple, TX.

Address reprint requests to Goran B. Klintmalm, M.D., Ph.D., Baylor-Simmons Transplant Institute, 3410 Worth Street, Suite 950, Dallas, TX75246. Telephone: 214-820-2050; FAX: 214-820-4527; E-mail: gorank@baylorhealth.edu

DOI 10.1002/lt.22417View this article online at wileyonlinelibrary.com.LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

LIVER TRANSPLANTATION 17:1394-1403, 2011

VC 2011 American Association for the Study of Liver Diseases.

40% of cases. In comparison with other patients,those undergoing transplantation with chronic HCVinfections have worse long-term survival.1,2 Althoughsuccessful treatments with antivirals (pegylated inter-ferons and ribavirin) prevent liver failure and reducethe risk of hepatocellular carcinoma,3 only half oftreated patients respond to therapy.4 Furthermore,even fewer patients respond if the treatment isdelayed until decompensation has ensued or trans-plantation has been performed.5 Thus, HCV recur-rence remains a significant risk for patients whoundergo liver transplantation, and the diseaseappears to progress more rapidly in this setting.6

It has been suggested that more intensive immuno-suppression (IS)—particularly high-dose corticoste-roids or lymphocyte-depleting therapy for the treat-ment of acute cellular rejection (ACR)—accelerates theprogression of recurrent HCV. It has not, however,been determined whether the use of daily mainte-nance corticosteroid therapy (primarily in the first fewmonths after transplantation) presents the same riskof aggressive recurrent HCV.7,8 Because considerablecontroversy remains about the risks and benefits ofmaintenance corticosteroids after liver transplantationfor HCV,9-11 we conducted a randomized, prospective,multicenter trial to compare the safety and efficacy of3 IS regimens in HCV-positive liver transplant recipi-ents and specifically to determine whether a steroid-free regimen is safe and effective.

PATIENTS AND METHODS

Study Design

This open-label, randomized, prospective, multicentertrial compared the safety and efficacy of 3 IS treat-ment regimens in HCV-positive liver transplant recipi-ents. Because the main objective was to examine thefeasibility of steroid avoidance, subjects were random-ized with a weighted 1:1:2 ratio to 1 of 3 arms: arm 1(n ¼ 77), which received tacrolimus (TAC; Prograf,Astellas Pharma US, Inc., Deerfield, IL) and cortico-steroids; arm 2 (n ¼ 72), which received TAC, cortico-steroids, and mycophenolate mofetil (MMF; CellCept,Hoffmann-La Roche, Inc., Nutley, NJ); or arm 3 (n ¼146), which received daclizumab (DAC) induction(Zenapax, Hoffmann-La Roche), TAC, and MMF (a ste-roid-free regimen). The treatment regimens are sum-marized in Fig. 1. Specific doses are detailed in thelegend for Fig. 1. Notably, subjects received DAC ondays 0 (2 mg/kg), 3 (2 mg/kg), and 8 (1 mg/kg); thisdiffered from the standard 2-dose regimen typicallyused at that time.12 The study endpoints includedpatient and graft survival, ACR, advanced HCV recur-rence, and adverse events related to IS treatment.13

The study group initially consisted of 312 adultpatients (18 years old or older) with end-stage liverdisease due to an HCV infection who were going toreceive their first liver transplant. However, 17patients who were positive for anti-HCV antibodies

were found to be HCV RNA–negative at the time oftransplantation and on subsequent retesting. Theywere excluded from further analysis; this left 295evaluable subjects. Organ sources included bothdeceased and living donors. The exclusion criteriaincluded the following: previous transplantation; hep-atitis B core antibodies, hepatitis B surface antigen,or HCV antibodies in the donor; an ABO-incompatibledonor; and anti–human immunodeficiency virus anti-bodies in the donor or the recipient. Patients who hadcontraindications for the use of calcineurin inhibitorsor who were hospitalized in the intensive care unit atthe time of transplantation also were not eligible. Theprotocol was approved by the institutional reviewboards of each of the 18 participating US centers. Allsubjects agreed to participate and signed the consentform.

Liver biopsy was performed 90 days, 1 year, and 2years after transplantation. HCV recurrence was eval-uated according to the Batts and Ludwig system forchronic hepatitis,14 and advanced recurrent HCV wasdefined for the purposes of this study as an inflamma-tion grade � 3 and/or a fibrosis stage � 2. ACR wasdefined by the 1997 Banff criteria.15 Significant rejec-tion was defined as a global Banff grade � 2 and arejection activity index (RAI) score � 4. By necessity,day-to-day clinical decisions were based on liver bi-opsy interpretations by each study center’s local pa-thologist. However, histological data presented in thisreport were derived from a review of biopsy slides by asingle central hepatopathologist (G.J.N.). As previ-ously reported,16 significant interobserver agreementwas demonstrated between the center pathologistsand the central pathologist (j ¼ 0.76 for recurrentHCV and j ¼ 0.62 for ACR, P < 0.001).

Figure 1. Three IS protocols for liver transplant recipients withchronic HCV. TAC was administered at 0.08 to 0.12 mg/kg/dayin 2 equal doses; the first dose was 20% of that amount within72 hours, and the target trough levels were 10 to 15 ng/mLthrough week 6 and 5 to 12 ng/mL thereafter. As forcorticosteroids, 500 to 1000 mg of methylprednisolone (or theequivalent of intravenous hydrocortisone or dexamethasone) wasadministered intraoperatively; the dosage was orally tapered to10 mg/day by day 30 and to 5 mg/day by day 90. MMF wasadministered at 2 to 3 g/day in 2 divided doses, with the firstdose given within 12 hours; the dosage was adjusted for adverseevents. The dosage of DAC was 2 mg/kg on days 0 (within 12hours) and 3 and 1 mg/kg on day 8.

LIVER TRANSPLANTATION, Vol. 17, No. 12, 2011 KLINTMALM ET AL. 1395

Treatment of ACR

Liver biopsy for cause was performed when consecu-tive test results demonstrated a 1.5-fold elevation inthe serum aspartate aminotransferase or alanine ami-notransferase level above the upper limit of the nor-mal range or a serum bilirubin increase > 0.3 mg/dL.Because clear pathological differentiation betweenmild ACR and early recurrent HCV was difficult toachieve and because it was possible that the rejectiontreatment could trigger the activation of HCV, mildACR (Banff grade 1 or an RAI score of 1-3) was treatedwith an increase in the TAC level to 12 to 15 ng/mLwithout a corticosteroid bolus and recycle. Moderateto severe ACR (a Banff grade � 2 and an RAI score �4) was treated with a 1.0-g bolus of methylpredniso-lone, which was followed by a 6-day steroid taper ofintravenous methylprednisolone (200, 160, 120, 80,40, and 20 mg and then none) or oral prednisone.Although the 6-day steroid taper duration was sug-gested by the protocol, this was left to the investiga-tors’ discretion with the goal of returning patients totheir original IS regimen as soon as possible. Cortico-steroid-resistant ACR, which was defined as the histo-logical persistence of ACR (unchanged or worseaccording to repeat liver biopsy) after steroid therapy,could be treated with anti-lymphocyte antibody ther-apy at the discretion of the study site.

Assessment and Treatment of HCV Recurrence

Protocol liver biopsy was recommended on days 0(transplant date), 90, 365, and 730. A window of 630days was allowed for the 90-day biopsy, a window of660 days was allowed for the 1-year biopsy, and awindow of 690 days was allowed for the 2-year bi-opsy. Protocol biopsy samples were obtained for83.4%, 75.4%, and 62.0% of the living subjects withinthese 3 windows. However, later protocol liver biopsysamples remained relevant only if the subjects hadnot already reached the study’s histological endpointof advanced hepatitis. Year 2 protocol biopsy sampleswere available for 82% of these patients. Biopsy com-pliance did not differ between the groups. Event-driven biopsy was performed as clinically indicated,and the results of these additional biopsy procedureswere included in the analysis of the HCV recurrencerate. Advanced HCV recurrence was defined as thepresence of histological evidence of grade 3 (moderate)or higher necrosis/inflammatory activity and/or thepresence of stage 2 (periportal/septal) or higher fibro-sis. Advanced HCV recurrence was required for theconsideration of antiviral therapy, which was left tothe discretion of the investigators at each study site.Viral replication, which was reported as the log HCVRNA level, was measured with a branched DNA assay(Chiron Corp., Emeryville, CA) because this assayoffered the largest dynamic range for viral quantifica-tion that was available at the time of the study’s ini-tiation.17 HCV RNA was measured at the baseline(before transplantation) and 90, 365, and 730 days

after transplantation. HCV RNA levels did not affectpatient management decisions.

Safety Outcomes

New-onset diabetes mellitus was defined as the use ofinsulin for 30 or more days within the first posttrans-plant year among patients who did not have active di-abetes before transplantation. New-onset hyperten-sion was defined as the presentation of the conditionat the 1-year follow-up visit in patients who did nothave the condition at the baseline; new-onset hyper-lipidemia was defined in the same way. The rates ofinfections and malignancies were also reported.

Infection Prophylaxis

Intravenous ganciclovir or oral valganciclovir wasadministered for a minimum of 7 days after trans-plantation as prophylaxis against cytomegalovirus(CMV) infections. After 7 days, all patients were man-aged according to the institutional protocol. No CMVprophylaxis was given to CMV-positive patients whoreceived an organ from a seronegative donor. Prophy-laxis for Pneumocystis carinii, fungal, and other bacte-rial infections was administered to patients accordingto the institutional protocol.

Statistical Methods

A sample size of 312 subjects was deemed sufficientto discover a difference in the primary endpointbetween the groups (arms 1 and 2 versus arm 3) after1 year. Assumptions were based on a 2-sided v2 testwith continuity correction, a type I error rate of 0.05,a type II error rate of 0.20 (power ¼ 80%), a signifi-cance level of 0.013 (based on the Dunn-Sidak adjust-ment for multiple comparisons), a 10% dropout rate,and efficacies of 25%, 35%, and 50% in arms 1, 2,and 3, respectively.

With an intent-to-treat approach, all data were ana-lyzed with SAS software (version 9.1.3, SAS Institute,Inc., Cary, NC) with a 5% significance level. Follow-uppairwise comparisons between groups were performedwith the Bonferroni multiple-comparison adjustment.Continuous data were compared with the Kruskal-Wallis test and the Wilcoxon 2-sample test. Categori-cal data were compared with Fisher’s exact test (2-sided) for 2 � 2 tables and with the likelihood ratio v2

test for larger tables. A per-protocol analysis was alsoperformed but is not included here because the num-ber of assessable subjects at 2 years was reduced by39%, and the outcomes were not different from thoseof the intent-to-treat analysis.

Patient and graft survival, the time to the first epi-sode of ACR, clinically significant HCV recurrence,and other adverse events were estimated with theKaplan-Meier product-limit method18 and were com-pared with the log-rank statistic.19 Outcomes werecensored at the last biopsy for recurrence and at thelast follow-up or death for other events. Death and

1396 KLINTMALM ET AL. LIVER TRANSPLANTATION, December 2011

retransplantation were events for graft survival. If thelast biopsy occurred within a protocol biopsy visitwindow, the patient was censored on the last day ofthat window.

A Cox regression analysis was performed foradvanced HCV recurrence. We looked at both the studyendpoint (at least grade 3 or at least stage 2) andadvanced fibrosis alone (Metavir stage 3 or 4). Becauseof the significant potential for confounding, we includedall variables in the univariate analysis in the Cox analy-sis. This analysis looked at all subjects, regardless ofthe IS regimen, and at the treatment arms.

RESULTS

Study Groups

Two hundred ninety-five subjects at 18 liver trans-plant centers in the United States were randomized.Seventy-seven were randomized to arm 1 (TAC andprednisone), 72 were randomized to arm 2 (TAC, pred-nisone, and MMF), and 146 were randomized to arm3 (TAC, DAC, and MMF). The recipient and donorcharacteristics for the 3 study groups are summarizedin Table 1. Deceased donors accounted for most do-nor organs (88.1%). In comparison with recipients inarms 1 and 2, recipients in arm 3 received fewerorgans from male donors (P < 0.05). The groups werecomparable in all other categories.

The mean follow-up was 20.9 6 7.2 months (range¼ 0-32 months). Reasons that patients were with-

drawn over the 2-year study period (other than deathor graft loss) included withdrawal for safety and/orthe need to change to another IS regimen (cyclospo-rine or rapamycin; 15 in arm 1, 10 in arm 2, and27 in arm 3) and noncompliance or loss to follow-up(2 in arm 1, 4 in arm 2, and 9 in arm 3).

IS

The IS doses are summarized in Table 2. Although thedose of TAC was lower during the first 90 days in arm3 (P < 0.05), the mean trough levels were equivalentin the 3 arms. MMF was added as rescue therapy af-ter ACR at some point for 12 subjects in arm 1 (seeTable 2 for the time windows), mostly during the firstposttransplant year. Similarly, 18 subjects in arm 3received steroids after an episode of ACR. No differen-ces were observed in the use of concurrent medica-tions for antibacterial, antifungal, antiviral, lipid-low-ering, and antihypertensive treatments (data notshown). There was a tendency for subjects in arms 1and 2 to require insulin more often within the firstpostoperative month than subjects in arm 3 (16% and14% versus 6% with 1 month and 23% and 20% ver-sus 10% within 3 months), but this difference did notreach statistical significance (P ¼ 0.09).

ACR

Forty-four episodes of moderate to severe ACRoccurred during the study period. Most episodes

TABLE 1. Patient and Transplant Characteristics

Category

Arm 1: TAC/PRED

(n ¼ 77)

Arm 2: TAC/PRED/MMF

(n ¼ 72)

Arm 3: TAC/DAC/MMF

(n ¼ 143)

RecipientsSex, male [n (%)] 55 (71.4) 54 (75.0) 105 (71.9)Age (years)* 51.3 6 6.7 51.6 6 7.8 51.3 6 7.4Age > 55 years [n (%)] 18 (23.4) 16 (22.2) 35 (24.0)Race [n (%)]Caucasian 56 (72.7) 54 (75.0) 101 (69.2)African American 4 (5.2) 2 (2.8) 16 (11.0)Other 17 (22.1) 16 (22.2) 29 (19.9)

Weight (kg)* 82.3 6 16.5 86.8 6 17.2 86.0 6 18.5HCV genotype 1 [n (%)] 53 (68.8) 59 (81.9) 105 (71.9)Model for End-Stage Liver Disease score* 20.5 6 6.6 21.6 6 6.3 21.1 6 7.4

DonorsSource [n (%)]Deceased donor 70 (90.9) 62 (86.1) 128 (87.7)Living donor 7 (9.1) 10 (13.9) 18 (12.3)

Sex, male [n (%)] 52 (67.5) 50 (69.4) 78 (53.4)†

Sex mismatch, yes [n (%)] 29 (37.7) 32 (44.4) 57 (39.0)Cold ischemia time (hours)‡

Deceased donor 7.2 (0.5-12.6) 7.3 (0.9-18.8) 7.1 (0.5-14.4)Living donor 2.4 (1.5-3.5) 2.3 (0.7-5.2) 2.3 (0.5-4.5)

Age (years)* 39.8 6 19.0 38.2 6 15.3 41.5 6 16.2Age > 55 years [n (%)] 18 (23.4) 9 (12.5) 26 (18.2)

*The data are presented as means and standard deviations.†P < 0.05 for arm 3 versus arms 1 and 2.‡The data are presented as medians and ranges.

LIVER TRANSPLANTATION, Vol. 17, No. 12, 2011 KLINTMALM ET AL. 1397

(68.2%) occurred in the first 90 days after transplan-tation, and all but 1 episode (97.7%) occurred withinthe first postoperative year. Clinically significant ACRoccurred during the first 90 days in 14.3%, 6.9%, and9.6% of the subjects in arms 1, 2, and 3, respectively[not significant (NS)]. These episodes of ACR weremoderate (Banff grade 2) in 61.5%, 80.0%, and 75.0%of the subjects in arms 1, 2, and 3, respectively andwere severe (Banff grade 3) in the rest. By day 730,clinically significant ACR had occurred in 14.3%,12.5%, and 13.7% of the patients in the 3 arms. Noneof these differences between the groups were signifi-cant. Ductopenic rejection occurred in 4 subjects (2,1, and 1 in arms 1, 2, and 3, respectively).

HCV Recurrence

HCV RNA was detectable at the time of study entry inall subjects (17 anti-HCV–positive cases in whom thevirus was undetectable were excluded). HCV RNAspontaneously cleared in a single patient (0.3%); forthis subject, HCV RNA remained undetectable withouttreatment 90, 365, and 730 days after transplanta-tion. The mean HCV RNA levels were similar in the 3IS arms on days 0, 90, 365, and 730. However, mostviral levels exceeded the upper limitation of quantita-tion for the assay, so it is possible that differencesmight not have been detected. Biochemical hepatitiswas defined as the elevation of at least 1 of the 3 fol-lowing tests: alanine aminotransferase (>100 IU/mL),aspartate aminotransferase (>100 IU/mL), and totalbilirubin (>1.5 mg/dL). It was observed by day 90 in

43.8%, 39.1%, and 27.7% of the subjects in arms 1,2, and 3, respectively (P < 0.05 for arm 3 versus arms1 and 2). Biochemical abnormalities were present in49.2%, 42.4%, and 38.0% of the subjects on day 365(NS) and in 33.9%, 34.0%, and 29.9% of the subjectson day 730 (NS).

Clinically significant HCV (defined by protocol as aninflammation grade � 3 and/or a fibrosis stage � 2)occurred in 48.2%, 50.4%, and 43.0% of the subjectsin arms 1, 2, and 3, respectively, within 1 year and in69.5%, 75.9%, and 68.1% within 2 years (Fig. 2A).Grade 3 or higher inflammation was present in thefirst-year liver biopsy samples of 18.8%, 16.6%, and23.2% of the patients in arms 1, 2, and 3, respec-tively. By year 2, 35.4%, 24.3%, and 33.9% of thepatients had a similar inflammation grade (Fig. 2B).Stage 2 or higher fibrosis was found in 46.8%, 50.4%,and 43.1% of the patients in arms 1, 2, and 3, respec-tively, by year 1 and in 66.3%, 75.9%, and 68.2% byyear 2 (Fig. 2C). None of these differences were statis-tically significant. Fibrosing cholestatic hepatitisoccurred in 7 subjects (2, 2, and 3 in arms 1, 2, and3, respectively).

Ninety-two patients in the 3 arms (31.2%) receivedantiviral therapy [11 received pegylated interferonalone, and 81 received pegylated interferon and riba-virin (51.1% in year 1 and 32.6% in year 2)]. Treatmentwas more common in patients with stage 3 or 4 fibro-sis versus patients with lower stages of fibrosis (P <0.001 at years 1 and 2). However, because the treat-ment was not part of this protocol, we do not havedetails for the treatment doses, duration, or outcomes.

TABLE 2. IS Agents

Days

Arm 1:

TAC/PRED

Arm 2:

TAC/PRED/MMF

Arm 3:

TAC/DAC/MMF P Value

Oral TAC dose (mg/day) 1-90 7.7 6 3.8 7.7 6 3.7 6.7 6 3.9 —*91-365 5.2 6 2.8 5.7 6 4.6 5.1 6 3.1 NS

366-730 4.0 6 2.4 4.2 6 3.2 4.2 6 2.7 NSTAC trough level (ng/mL) 8 10.0 6 3.7 11.1 6 6.6 10.8 6 5.0 NS

30 12.2 6 5.8 11.1 6 4.6 11.1 6 4.7 NS90 9.5 6 4.6 11.0 6 5.5 9.6 6 4.2 NS

365 8.4 6 4.6 8.9 6 3.2 9.0 6 4.1 NS730 7.3 6 3.7 6.9 6 3.2 7.5 6 3.1 NS

Oral MMF dose (mg/day)† 1-90 218 6 563 1920 6 346 1811 6 453 —‡,§

91-365 245 6 600 1603 6 497 1381 6 570 —‡,§

366-730 156 6 480 1368 6 571 1301 6 553 —‡

Oral steroid dose (mg/day)k 1-90 12.1 6 5.5 11.0 6 3.2 2.0 6 9.7 —¶

91-365 6.0 6 4.7 4.9 6 3.5 2.1 6 8.6 —¶

366-730 6.2 6 6.0 5.0 6 4.5 0.8 6 3.9 —¶

NOTE: The data are presented as means and standard deviations.*P < 0.05 for arms 1 and 2 versus arm 3.†MMF was used in 11, 12, and 8 subjects in arm 1 for 1 to 90, 91 to 365, and 366 to 730 days, respectively (see the text).‡P < .001 for arm 1 versus arm 2 and/or arm 3.§P < 0.05 for arm 2 versus arm 3.kPRED was used in 11, 18, and 10 subjects in arm 3 for 1 to 90, 91 to 365, and 366 to 730 days, respectively (see thetext).

¶P < .001 for arm 3 versus arm 1 and/or arm 2.

1398 KLINTMALM ET AL. LIVER TRANSPLANTATION, December 2011

Nonetheless, the use of the treatment was similar inthe 3 arms and did not affect patient or graft survivalduring the 2-year study period (data not shown).

Risk Factors for Advanced Recurrent HCV

Advanced hepatitis (at least grade 3 and/or stage 2) isan ominous prognostic indicator for patients withHCV recurrence after liver transplantation and is typi-

cally considered to be the indication for the considera-tion of antiviral therapy (as it was in this study). ACox regression analysis was used to assess factors in-dependently associated with the development ofadvanced hepatitis after transplantation. A univariateanalysis found only the cumulative steroid dose andearly evidence of histological injury to be associatedwith the eventual achievement of this endpoint. Onthe other hand, a stepwise multivariate Cox regres-sion showed that male donors, male recipients, livingdonors, cold ischemia times, ACR, and early histologi-cal recurrence (grade 2 inflammation on the 90-dayliver biopsy sample or stage 1 fibrosis on the 1-yearbiopsy) were associated with the development ofadvanced hepatitis (Table 3).

Advanced fibrosis (stage 3 or 4) is an importantposttransplant milestone because it typically indicatesa high risk of subsequent hepatic decompensation.Thus, we also looked at advanced fibrosis alone (with-out any consideration of the grade) as an endpoint(Table 4). A univariate analysis showed that the donorage and early histological recurrence were associatedwith the development of advanced fibrosis. Accordingto a multivariate Cox analysis, the donor age andgrade 2 inflammation on the day 90 or year 1 liver bi-opsy sample were associated with the subsequent de-velopment of bridging fibrosis or cirrhosis; the admin-istration of OKT3 or thymoglobulin approachedsignificance.

Safety Data

Table 5 shows the rates of infection, new-onset diabe-tes, new-onset hypertension, new-onset hyperlipid-emia, and de novo malignancies. Although infections,diabetes, hypertension, and hyperlipidemia were quitecommon, there were no statistically significant differ-ences between the study arms. There was, however, atrend toward less diabetes in the steroid-free IS group(P ¼ 0.09). De novo malignancies were uncommonand also did not differ between the groups. Most post-transplant malignancies during the 2-year follow-upperiod were related to the recurrence or metastasis ofhepatocellular carcinoma. CMV infections occurred in18 cases (4, 6, and 8 in arms 1, 2, and 3,respectively).

Survival

The 1- and 2-year patient and graft survival rateswere similar in the 3 arms. At the end of the secondyear, the patient survival rates in arms 1, 2, and 3were 83.8%, 81.0%, and 86.1%, respectively (Fig. 3A).The 2-year graft survival rates were 79.1%, 79.8%,and 85.1%, respectively (Fig. 3B). The main causes ofdeath included sepsis [n ¼ 14 (3 in arm 1, 3 in arm 2,and 8 in arm 3)], malignancies [n ¼ 8 (2, 5, and 1)],HCV recurrence [n ¼ 5 (4, 1, and 0)], multiorgan fail-ure without sepsis [n ¼ 4 (2, 2, and 0)], intracerebralhemorrhaging [n ¼ 2 (1, 1, and 0)], cardiopulmonaryevents [n ¼ 3 (0, 1, and 2)], and unknown or other

Figure 2. Histological progression of recurrent HCV accordingto the treatment arm: (A) the probability of histologicalprogression according to the study endpoint (grade � 3 and/orstage � 2; P ¼ 0.60), (B) the probability of grade 3 or higherinflammation alone (P ¼ 0.31), and (C) the probability of stage 2or higher fibrosis alone (P ¼ 0.43).

LIVER TRANSPLANTATION, Vol. 17, No. 12, 2011 KLINTMALM ET AL. 1399

TABLE 3. Factors Associated With the Histological Study Endpoint (Inflammation Grade � 3 and/or Fibrosis Stage � 2)

Observations

(n) Variable

Univariate Analysis Multivariate Analysis

P Value OR (95% CI) P Value OR (95% CI)

280 Donor, male 0.69 0.94 (0.71-1.26) 0.04 0.7 (0.49-0.98)285 Donor, black 0.47 0.85 (0.55-1.32)283 Donor age (years) 0.17 1.01 (1-1.02)285 Recipient, male 0.96 1.01 (0.73-1.39) 0.001 2 (1.32-3.01)285 Recipient, black 0.08 1.56 (0.96-2.54)285 Recipient age (years) 0.83 1 (0.98-1.02)285 Living donor 0.27 1.29 (0.82-2.04) 0.002 2.79 (1.48-5.25)285 CMV 0.17 1.45 (0.86-2.46)243 Cold ischemia time (hours) 0.17 1.04 (0.98-1.09) 0.03 1.07 (1.01-1.14)285 Arm 1 0.95 0.99 (0.71-1.38)285 Arm 2 0.33 1.17 (0.85-1.62)285 Arm 3 0.44 0.89 (0.67-1.19)285 CellCept average dose until recurrence 0.52 1 (1-1)285 Steroid average dose until recurrence 0.03 1.01 (1-1.02)285 Grade 1 by day 90 <.001 3.18 (2.29-4.42)285 Grade 2 by day 90 <.001 4.59 (3.16-6.68) <.001 3.33 (2.11-5.26)285 Grade 1 by day 365 <.001 3.6 (2.56-5.05)285 Grade 2 by day 365 <.001 4 (2.91-5.48)285 Stage 1 by day 90 <.001 3.6 (2.55-5.07)285 Stage 1 by day 365 <.001 4.46 (3.21-6.21) <.001 5.19 (3.37-8)285 ACR during first 2 years 0.42 1.18 (0.79-1.76)285 ACR before recurrence 0.16 1.35 (0.89-2.03) 0.04 1.67 (1.03-2.7)285 OKT3/thymoglobulin for

ACR before recurrence0.41 1.45 (0.59-3.53)

NOTE: The analysis was performed with Cox regression.10 patients are excluded because they had no biopsies. Donor age and sex were missing in 3 additional patients, anddonor sex was missing in two additional patients.

TABLE 4. Factors Associated With Advanced Fibrosis (Metavir Stage 3 or 4) by Year 2

Observations

(n) Variable

Univariate Analysis Multivariate Analysis

P Value OR (95% CI) P Value OR (95% CI)

280 Donor, male 0.22 1.33 (0.84-2.11)285 Donor, black 0.14 0.54 (0.23-1.23)283 Donor age (years) <.001 1.03 (1.02-1.04) <.001 1.03 (1.01-1.04)285 Recipient, male 0.39 1.25 (0.75-2.08)285 Recipient, black 0.28 1.46 (0.73-2.91)285 Recipient age (years) 0.25 0.98 (0.95-1.01)285 Living donor 0.21 0.59 (0.26-1.35)285 CMV 0.15 1.72 (0.83-3.56)243 Cold ischemia time (hours) 0.32 1.04 (0.96-1.12)285 Arm 1 0.70 1.1 (0.68-1.77)285 Arm 2 0.99 1 (0.61-1.63)285 Arm 3 0.75 0.93 (0.61-1.43)285 CellCept average dose until recurrence 0.91 1 (1-1)285 Steroid average dose until recurrence 0.39 1.01 (0.99-1.02)285 Grade 1 by day 90 <.001 2.62 (1.65-4.14)285 Grade 2 by day 90 <.001 3.82 (2.36-6.19) <.001 2.87 (1.63-5.07)285 Grade 1 by day 365 <.001 2.68 (1.57-4.56)285 Grade 2 by day 365 <.001 3.92 (2.3-6.67) <.001 3.6 (1.79-7.24)285 Stage 1 by day 90 <.001 2.86 (1.78-4.59)285 Stage 1 by day 365 <.001 3.45 (2.02-5.87)285 ACR during first 2 years 0.58 1.19 (0.64-2.19)285 ACR before recurrence 0.51 0.81 (0.44-1.5)285 OKT3/thymoglobulin for ACR

before recurrence0.87 1.12 (0.28-4.57) 0.08 3.72 (0.86-16.2)

NOTE: The analysis was performed with Cox regression.10 patients are excluded because they had no biopsies. Donor age and sex were missing in 3 additional patients, anddonor sex was missing in 2 additional patients.

1400 KLINTMALM ET AL. LIVER TRANSPLANTATION, December 2011

causes [n ¼ 8 (0, 0, and 8)]. No statistically significantdifferences were observed between the arms.

DISCUSSION

The outcomes of liver transplantation for HCV-infected recipients are inferior to the outcomes forpatients undergoing transplantation for other indica-tions.1 HCV recurrence that leads to graft failure is amajor cause of posttransplant graft loss and death inHCV patients. Because antiviral therapy is poorly tol-erated in the posttransplant setting and we have notyet reached the point at which it can predictably alterthe course of recurrence, attention has mostly beenfocused on identifying and avoiding donor and hostfactors that are associated with progression toadvanced-stage HCV. Older donor age,20-22 high HCVRNA levels,23,24 CMV infections,25 lymphocyte-deplet-ing IS agents, corticosteroids, and other factors remaintopics of controversy. We confirmed the results of otherstudies with respect to the detrimental effects of donorage20-22 and sex20 on HCV progression. We found thatan organ from a male donor was a risk factor foradvanced hepatitis, although it was not for fibrosisalone. This was consistent with our observation that fi-brosis progression was similar in the 3 treatmentarms, despite the presence of fewer male donors in thesteroid-free arm. Although it is unlikely, it is conceiva-ble that this may have masked an otherwise higherrate of progression in this group. Nevertheless, none ofthe other aforementioned factors can be controlledwithin the current organ allocation system. What stoodout in our analysis was that early histological recur-rence, even after just 3 months, was highly associatedwith subsequent progression. This needs to be consid-ered when antiviral treatment is being timed.

Perhaps the one factor that can be modified toreduce the speed and severity of recurrence is theform of IS therapy. It remains unclear whether thechoice of the calcineurin inhibitor influences the ra-pidity of HCV recurrence after transplantation.Although cyclosporine and other cyclophilin inhibitorsdecrease HCV replication in vitro,26-28 cyclosporinedoes not confer a benefit in vivo. Indeed, TAC hasbeen associated with better patient and graft sur-vival.27 All patients in this study received TAC. MMF

also does not appear to have a significant antiviraleffect despite early reports to the contrary.29,30 Wecould find no evidence that MMF influenced HCV pro-gression in this study. Finally, it could be argued thatit would have been more appropriate to use a lowerdose of TAC when it was combined with MMF in arms2 and 3. Although this has been debated for years,there is no consensus for lowering the TAC levelswhen 3 drugs are used. Thus, we made the targetdrug levels the same for all groups.

TABLE 5. Likelihood of Complications by Study Year 2

Complication

Arm 1:

TAC/PRED (%)

Arm 2:

TAC/PRED/MMF (%)

Arm 3:

TAC/DAC/MMF (%)

Infection requiring intravenousantibiotics/hospitalization

41.6 36.1 34.8

New-onset insulin-dependent diabetes 29.8 32.8 18.6New-onset hypertension 79.0 70.5 66.5New-onset hyperlipidemia 56.1 41.5 51.6De novo malignancy: not hepatocellular carcinoma 2.6 4.2 1.4De novo malignancy: hepatocellular carcinoma 6.5 6.9 2.0

NOTE: None of the differences between groups were statistically significant.

Figure 3. Kaplan-Meier graphs of (A) patient survival and (B)graft survival.

LIVER TRANSPLANTATION, Vol. 17, No. 12, 2011 KLINTMALM ET AL. 1401

The use of corticosteroids after liver transplantationhas been a topic of debate for many years. Severalstudies have suggested that steroid avoidance is asso-ciated with lower rates of bacterial, fungal, and CMVinfections, diabetes, hypertension, and hyperlipid-emia, but this is accomplished at the cost of increasedrejection.31 The risk of rejection is reduced when ste-roids are replaced by another IS agent, such as aninterleukin-2 receptor–inhibiting antibody.11 Wefound no advantage with steroid avoidance in ourstudy with respect to the posttransplant occurrence ofinfections, new-onset diabetes, hypertension, or hy-perlipidemia. Thus, the remaining unknown is theway in which steroid avoidance affects the course ofrecurrent HCV infections. Steroids might acceleratethe progression by driving viral replication and facili-tating CMV coinfections. Alternatively, they mightreduce the progression by diminishing the risk forACR. Although steroids do not increase HCV replica-tion in vitro,32 it has been suggested that steroidmaintenance and repeated steroid boluses for treatingrejection may be associated with more rapid progres-sion of HCV recurrence and fibrosis.33 Small studiescomparing steroid-free IS to standard therapy haveshown conflicting results.11,34-36 Our study, which isthe largest to look at the impact of steroid avoidanceon HCV recurrence, found no differences in the ratesof HCV progression with steroid avoidance and stand-ard IS regimens using steroids. However, our studyobserved subjects for only 2 years after transplanta-tion, and some patients refused liver biopsy in thesecond year. Thus, we cannot dismiss the possibilitythat differences might have been observed if all sub-jects had been biopsied or the follow-up had been lon-ger. We also cannot rule out the possibility that rejec-tion-driven changes in IS, such as steroid boluses orthe addition of MMF, might have masked differencesbetween the randomized treatment regimens. Becauseof the similarities in the recurrence severity andrejection rates in the 3 groups, however, we feel thatneither of these 2 possibilities is likely.

In summary, although we have shown no distinctadvantage of steroid avoidance, we have found thatsteroid-free IS is safe in HCV-positive liver transplantrecipients and does not increase the risk of rejectionor fibrosis progression. Thus, this might be the pre-ferred option in certain patients, such as those whoalready have obesity, diabetes, hypertension, or hy-perlipidemia. Obesity and diabetes are associatedwith a more rapid progression of liver injury fromHCV infection,37,38 so steroid avoidance might confera long-term benefit in this group that we were unableto uncover in this 2-year study.

Definitions of HCV recurrence in the posttransplantsetting have been inconsistent and confusing.Although nearly all HCV-infected patients experienceviral recurrence, not all experience a rapid progres-sion of liver disease. Therefore, it is more appropriateto focus our attention on biochemical and histologicalrecurrence, as we have in this study. We observed nodifferences between the 3 treatment arms with respect

to the posttransplant levels of viremia or biochemicalmarkers of hepatitis activity. It is important to note,however, that biopsy evidence of progressive HCV of-ten occurred in the absence of biochemical abnormal-ities. Indeed, although only 29.9% to 34.0% hadaspartate aminotransferase, alanine aminotransfer-ase, or bilirubin elevations at year 2, 68.1% to 75.9%met the histological criteria for advanced recurrenthepatitis. This emphasizes the importance of regularsurveillance liver biopsy in patients who undergo livertransplantation for HCV.

Until we have safer and more effective antiviraltherapy for recurrent HCV in liver transplant recipi-ents, the only viable options for reducing the rate of fi-brosis progression in HCV-positive recipients may be(1) avoiding the use of older donors and (2) controllingcomorbid host factors that may accelerate progression(eg, obesity, diabetes, and alcohol intake). Recentpublications reporting that interleukin-28B polymor-phisms might influence the rate of HCV progressionafter transplantation require confirmation.39,40 Wecertainly require a better understanding of whatmakes HCV progress more rapidly in the posttrans-plant setting, and we look forward to the availabilityof more effective antiviral agents, which now seemwithin reach.

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