Transplant Immunology 20 (2008) 43–47

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Transplant Immunology

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The Tor Vergata weaning off immunosuppression protocol in stable HCV livertransplant patients: The updated follow up at 78 months

Giuseppe Orlando b,c, Tommaso Manzia a, Leonardo Baiocchi d, Alberto Sanchez-Fueyo e,Mario Angelico d, Giuseppe Tisone a,⁎a Transplant and General Surgery, Tor Vergata University of Rome, S. Eugenio Hospital, Rome, Italyb Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USAc Department of Surgery, Nuffield University Hospital, University of Oxford, Oxford, UKd Chair of Gastroenterology and Hepatology, Tor Vergata University of Rome, Rome, Italye Liver Transplant Unit, Hospital Clinic Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain

Abbreviations: LT, liver transplantation; HCV, hepatitsion; LFT, liver function tests; AST, aspartate aminotratransferase; GGT, gamma glutamyl transferase; AP, alkal⁎ Corresponding author. Tor Vergata University of R

Surgery, Ospedale S. Eugenio Piazzale dellUmanesimo 1051002280; fax: +39 06 5922681.

E-mail address: tisone@med.uniroma2.it (G. Tisone)

0966-3274/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.trim.2008.08.007

a b s t r a c t

a r t i c l e i n f o

Article history:

Background: We report the Received 5 August 2008Accepted 7 August 2008

Keywords:Liver transplantWeaningImmunosuppressionToleranceHCV recurrenceQuality of life

update of the Tor Vergata immunosuppression (IS) weaning protocol in stablehepatitis C virus (HCV) liver transplant (LT) recipients.Methods: The weaning off IS was attempted in 34 patients who had received a LT 63.5±20.1months earlier,for HCV-related end stage liver disease. Patients were observed over a period of 6.5years. During this time,yearly protocol liver biopsies were performed. Primary endpoints were determined as the feasibility ofweaning off IS and its impact on the long term disease progression. Secondary endpoints were defined as theimpact on patient morbidity and quality of life.Results: Of the 8 originally tolerant patients, 7 remain alive and in good condition, while 1 died of severe HCVrecurrence 10years post-LT and 6years after complete removal of IS. Four out of 26 intolerant individuals diedof HCV recurrence (2×), lung carcinoma (1×) and acute myocardial infarction (1×), after a mean follow upperiod from LT of 115 (range 100–124). The 10-year survival from LT was comparable (89% vs. 87.5%). Livergraft pathology showed no significant differences between the two groups in terms of staging, fibrosisprogression rate, and grading. Quantitative HCV RNA assay showed a significant non-logarithmic differencebetween the two groups (p = 0.03). The two groups were comparable in terms of liver function tests and lipidprofile, whereas they differed with regards to glycaemia. While all tolerant individuals were euglicemic, 11intolerant individuals developed new onset diabetes that required specific treatment (p = 0.03). Finally,significantly more intolerant patients are suffering from either cardiovascular (14/22 vs. 0/7, p = 0.01) orinfectious diseases (13/22 vs. 0/7, p = 0.01).Conclusions: After a 6.5-year follow up, the complete withdrawal of IS in HCV LT recipient remains safe andbeneficial to patients, because it reduces the IS-related morbidity and increases the quality of life. The impacton HCV disease recurrence is less marked than after 3.5years.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

An earlier study published in 2006 by the current authorsdisclosed the results of a 4year prospective trial in which immuno-suppressive drugs werewithdrawn from stable hepatitis C virus (HCV)liver transplant (LT) patients [1]. It was shown that the progressionof HCV disease recurrence is slowed down in response to the

is C virus; IS, immunosuppres-nsferase; ALT, alanine amino-ine phosphatase.ome, General and Transplant00144 Rome, Italy. Tel.: +39 06

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l rights reserved.

reconstitution of the immune surveillance in the host. In fact, patientswho could permanently be weaned off immunosuppression (IS)showed a significant reduction in the fibrosis progression rate and amarked improvement of liver function tests (LFT). In addition, it wasdemonstrated that the occurrence of acute rejection was not harmfulto the patient, as it did not have any impact on patient outcome andLFT normalized after prompt resumption of IS.

The present study describes the results over the course of78months post IS withdrawal. Specifically, these findings demonstratethat the complete withdrawal of IS in HCV LT recipients remains safeand is also beneficial to patients because it reduces the IS-relatedmorbidity and increases the quality of life. However, the impact onHCV disease recurrence is less marked than after 4years. Importantly,our data provide further evidence that HCV-related end stage liver

Table 1Synoptic view of the main clinical findings at 6.5-year follow up

Tolerant Intolerant p value

Number of patients 7 2210-year survival rate 89% 87.5% n.s.Median follow up (months) 80±15.1 74.5±7.1 n.s.ALT (n.v. 5–31 IU/L) 74 40 (10–85) n.sAST (n.v. 5–31 IU/L) 43 35 (8–45) n.sAlkaline phosphatase (n.v. 40–120 IU/L) 113 (58–249) 154 (51–287) n.s.GGT (n.v. 5–36 IU/L) 86 (23–156) 57 (20–138) n.s.Total bilirubin (n.v. 0.2–1.1 mg/dl) 1.1 (1.0–2.0) 0.9 (0.6–2.0) n.s.Serum creatinine (n.v. 0.4–1.1 mg/dl) 1.3 (0.8–6) 1.3 (0.8–7.2) n.s.Cholesterol (n.v. 110–200 mg/dl) 127 (107–219) 170 (107–270) n.s.Triglicerydes (n.v. 40–160 mg/dl) 137(55–221) 130 (53–393) n.s.Glycaemia (n.v. 50–110 mg/dl) 112 (85–197) 129 (86–333) n.s.Recurrent infection disease 0 13 0.01Cardiovascular disease 0 14 0.01Diabetes 0 11 0.03Other drugs 1 16 0.03Staging at 6 years 1.5±0.9 2.8±1.5 n.s.Grading at 6 years 3.1±0.7 3.7±1.6 n.s.Final yearly fibrosis progression rate 0.23±0.16 0.48±0.26 n.s.

Legend: n.v. normal values.

44 G. Orlando et al. / Transplant Immunology 20 (2008) 43–47

disease does not represent a contraindication to the withdrawal of IS[2,3], but instead it should be considered as one of the most valuableindications.

2. Materials and methods

Test subjects consisted of 34 individuals whose baseline char-acteristics have been extensively described earlier [1]. Throughout the78months of the follow up evaluation, patients were assessed throughroutine blood tests including LFT, renal function tests, lipemic profile(cholesterol and triglycerides), glycaemia and immunosuppressantblood trough levels. Liver biopsies were taken annually to establishstaging, grading, and fibrosis progression rate (defined as thedifference between the staging score in the last and the baselineliver biopsy divided by the years of follow up [1]), and to excludefeatures of acute or chronic rejection. Additional biopsies were takenwhenever there was suspicion of acute rejection. Grading and stagingwere assessed according to Ishak [4]. Acute rejection was definedaccording to standard criteria [5]. Chronic rejection was assessedaccording to Banff classification [6]. We received written approvalfrom all patients involved for this study.

Patients who could be permanently weaned off IS are referred to astolerant, whereas those who developed acute rejection and thusrequired the resumption of IS are referred to as intolerant.

3. Endpoints

The primary endpoint was the long term feasibility and safety ofthe weaning protocol. The secondary endpoint was to assess whetherthe achievement of a sustained IS-free state have any impact on theprogression of HCV recurrence in the future. The tertiary endpoint wasto verify the impact of the IS-free state on patient quality of life, asexpressed by IS-related morbidity (namely, the onset of cardiovas-cular, tumoral, infectious and metabolic complications) and theprescribed number of medications per patient.

4. Statistical analysis

We used the survival analysis to assess mortality. Survival rateswere calculated using the Kaplan–Meier method. For normaldistribution continuous data analysis, we used the parametric test(Student t-test); categorical variables were evaluated according to thenon-parametric test (Fisher exact test). A p-value of ≤0.05 wasconsidered significant.

The program used for statistical analysis was SPSS® 13.0 (233South Wacker Drive, Chicago, USA).

5. Results

5.1. Survival

No patient was lost to follow up. After amean follow up period of 78 (range 57–109)months, the participation to the weaning protocol did not cause either patient or graftloss. Of the original 8 patients whom the IS could be successfully and steadily weanedoff (tolerant group), 1 died due to severe cholestatic HCV recurrence 10years aftertransplantation, 6years following the complete withdrawal of cyclosporine. Out of the26 individuals who did not tolerate the tapering of IS (intolerant group) (76.5%), 4 diedafter a mean of 115 (range 100–124) months following LT: 2 due to severe cholestaticHCV recurrence, 1 from lung carcinoma, and 1 due to acute myocardial infarction.Overall, the 10-year survival rate from the LT was 88%, with no significant differencebetween the tolerant and intolerant groups (Table 1).

5.2. Liver pathology

At least 7 consecutive yearly liver biopsies were available from each patient, inaddition to the one performed at the time of enrollment in the study.

Overall, the significant differences observed 3 years following withdraw from ISwere unable to be confirmed 3years later (i.e., 6years after the weaning). In fact, withinthe tolerant group, the comparison of 3-year versus 6-year biopsies showed an

improvement of grading in 5 patients (71%) and a stabilization in 3 patients. In addition,an improvement (namely, a reduction) of at least 1 unit of the staging score wasobserved in 4 patients (57%), while the score did not vary in the remaining 3 patients.Similar features were detected in the intolerant patient population, where the gradingworsened in 4 (18%) patients, improved in 9 (41%) and remained unchanged in 9 (41%);the staging deteriorated in 8 (30%) patients and remained unchanged in the remaining14.

In contrast to previous data recorded after 4-year follow up [1], the fibrosisprogression rates calculated for the tolerant and intolerant groups at 72months offollow up were comparable, despite tolerant patients showed a slight trend toward aslower progression rate (0.26±0.13 and 0.43±0.29 respectively, p = n.s.). However, focalductopeniawas occasionally observed in protocol liver biopsies, always being limited toless than 20% of portal spaces (usually less than 10%). Of the patients of the presentseries, there was no evidence of early or late chronic rejection, in consistence with ourdata at 4years [1].

5.3. HCV virology

At 6years from study entry, tolerant patients presented significantly lower non-logarithmic mean HCV RNA titers compared to intolerant individuals [205,800 (range0–993,000) IU/L vs. 3,129,588 (0–16,400,000) IU/L] (p = 0.03), in consistence with ourprevious report [1].

5.4. Blood biochemistry

Both tolerant and intolerant groups scored similarly in liver and renal functiontests, as well as their lipid profile (Table 1). Although glycaemia tests were alsocomparable between the two groups,11 of the 22 surviving intolerant patients (50%) arecurrently under hypoglicemizing drugs following the onset of de novo diabetesmellitus.

5.5. IS-related morbidity

Out of 22 intolerant patients who are still alive, 14 (63%) suffer from cardiovasculardisturbances and are currently taking at least one medication active on thecardiovascular system. On the contrary, none of the tolerant individuals are beingtreated with any similar medication, nor have they suffered from any arterialhypertension (p = 0.01).

In addition, none of the tolerant patients suffer from recurrent infections, whereas13 (59%) intolerant patients have developed urinary and/or pulmonary (bacterialand/or viral and/or fungal) infections requiring specific treatment during the studyperiod (p = 0.01).

Sixteen out of 22 (72%) intolerant patients are also receiving other medications,specifically allopurinol, levothyroxine, antipsychotics, or bisphosphonates, whereasonly 1 tolerant (14%) is taking allopurinol (p = 0.03).

6. Discussion

The study is peculiar for two reasons. First, we did not administerany drug, nor did we adopt any specific regimen, expected to betolerogenic, but – more simply and pragmatically – we based our

45G. Orlando et al. / Transplant Immunology 20 (2008) 43–47

working hypothesis on the well documented and still largelyunexplored immunomodulatory properties of the liver, as well as onthe most recent insights on the natural history of HCV diseaserecurrence after LT [7–10]. Second, the endpoint of the study was notthe achievement of the IS-free state itself, which had already beenlargely documented earlier [2,11–17]; instead, the endpoint of thestudy was assessed according to the improvement of patient clinicalcondition, with regard to the recurrence of HCV disease after thetransplant. Finally, it was hypothesized that there would also be asignificant amelioration of the patients' quality of life, as well as amarked reduction of IS-related long term morbidity in successfullyweaned off patients.

In our first report [1], it was demonstrated that maintenance IS canbe safely withdrawn in 1 out of 4 LT recipients with established HCVdisease recurrence, and that the reconstitution of the immunesurveillance is followed by a significantly reduced disease progressionas indicated by a combination of histological, biochemical andvirological findings [1]. In fact, weaned patients showed eitherstabilization or improvement of necro-inflammatory changes in thegraft, resulting in a marked reduction of fibrosis progression and theimprovement of liver function tests. In contrast, the majority ofpatients unable to achieve an IS-free state underwent deterioration ofhistological and biochemical parameters. Strikingly, in tolerantpatients, the yearly fibrosis progression rate indicated fibrosis regres-sion, which is rarely observed in immune-competent non-trans-planted patients with chronic hepatitis C. In contrast, the mean yearlyfibrosis progression rate for weaning intolerant patients was con-sistent with continued disease progression.

After a mean follow up of 6.5 years following the study entry, theobtained results from this study confirm the original workinghypothesis. The impact of the IS-free state on the progression ofHCV recurrence is not as remarkable as reported after 3.5 years, asdemonstrated by the similar histological and biochemical profilesdepicted in both groups. This notwithstanding, tolerant individualshave a better quality of life, deriving from higher individualsatisfaction, lower number of medications prescribed, and reducedIS-related morbidity [18]. It has been demonstrated that quality of lifeis regarded as an important measure of outcome after solid organtransplantation, and is used similarly to rejection rates to determineimmunosuppressive regimens and to guide other therapeutic choices[19]. In fact, it is regarded as an important measure of outcome aftersolid organ transplantation and is used similarly to rejection rates todetermine immunosuppressive regimens or to guide other therapeu-tic choices. This due to the fact that patients are now aware of theresults achievable with transplantation, thus they are not only expectto be healed, but also to be able to enjoy the same lifestyle of a non-transplant individual. Consequently, transplant physicians haveshifted their focus to achieve long term survival, free of morbid andmortal complications associated with an acceptable quality of life. It isof note that the cost of treatment and the frequency and dosage ofprescribed medications are among the factors that patients contributeas having the most impact on quality of life [18]. In fact, it has beendemonstrated that the higher number of medications and dosagerequired by the patient corresponds to a lower quality of life. Inaddition, patient non-compliance, which is one the most importantdeterminants of quality of life and one of the major causes of organfailure, is significantly influenced by the number of medications.Therefore, the optimal immunosuppressive regimen in terms ofpatient satisfaction should include the prevention of organ rejection,disease recurrence, andmaintenance of graft function, while using thefewest agents at the lowest effective dosage.

New-onset, post-LT diabetes mellitus occurs in up to 20% of LTrecipients, is a major risk factor for cardiovascular diseases andinfections, and negatively impacts on graft function [20]. Intolerantpatients in this study presented a significantly higher incidence ofdiabetes mellitus after the study entry. The finding that, in HCV LT

individuals under standard IS, the incidence of de novo diabetesmellitus is 42% compared to 19% observed in non-HCV patients,provides striking evidence of the intimate interplay between HCVinfection, IS and the development of diabetes mellitus. In our series,there were no observed cases of diabetes mellitus in tolerant patients,and such data should be interpreted as a proof-of-concept that the IS-free state protects LT recipients from the side effects usually ex-perienced by chronic administration of immunosuppressants.

The results from the present study suggest that the weaning off ofIS can be attempted safely and provide additional benefits to patients.All episodes of acute rejection were easily and conservativelymanaged, and none were severe nor required steroid pulses. Inaddition, intolerant patients did not develop a worsened diseaseprogression attributable to the weaning itself, while tolerant indivi-duals did not show any higher risk for ductopenia, generallyinterpreted that the IS-free state did not expose tolerant individualsto a higher risk for chronic rejection. The deaths recorded in bothgroups (tolerant and intolerant) were attributable to factors unrelatedto the withdrawal of IS and, in all cases, are consistent with the wellcharacterized natural history of LT, the original hepatic disease and thelong term drug-derived toxicity. According to the results mentionedabove, the concerns expressed by many authors with regard to thepossibility of attempted IS withdrawal in HCV patients, are notjustifiable; on the contrary, HCV represents a valuable indication forpossible weaning off of IS after LT.

Of interest, tolerant patients from the current study were enrolledin a European study designed by the University of Barcelona, in which17 tolerant LT patients from 4 different European centers wereinvestigated and compared with 63 controls, in order to identify animmune profile of operational tolerance and to design a clinicallyapplicable molecular test of tolerance [21,22]. The investigators havefound and validated several gene signatures capable of identifyingtolerant and intolerant LT recipients with high accuracy [23]. Using acombination of quantitative real-time PCR and flow cytometrytechniques, specific interest was focused on increases in the numbersof peripheral T cells using the GD antigen receptor, in particular thoseexpressing the d1 form of the receptor, and to a difference in theactivation state of circulating NK cells, as shown by a small set ofdifferentially expressed genes. It is noteworthy to point out that thepredictive biomarkers were derived using a training set of samplesand then validated in an independently gathered cohort of test-setpatients. These findings are consistent with previously publishedreports of increased da-gdTCR+ T cells in the blood of tolerant livertransplant patients [21,24,25], thereby giving further and robust creditto this measure as a valid biomarker of tolerance in this application.Given the relative ease with which these assays can be performed andtheir targeted list of differentially expressed genes (26 in total), theseassessments can be easily validated for use in the clinic.

Transplant tolerance is defined as the conditionwhere a functioninggraft lacks histological signs of rejection in the absence of anyimmunosuppressive drugs in an immunocompetent host capable ofaccepting a second graft of the same donor origin, while being able toreject a third-party graft [26]. The clinical era of transplantation beganon December 23rd 1953, when Dr. Joseph Murray performed the firstsuccessful renal transplant between the identical Herrick twins [27].Because of the patient and donor's identical genetic makeup of the twosiblings, the 23-year old patient did not receive any IS after theoperation, thus representing the first case of clinical operationaltolerance after solid organ transplantation. More than 50 years later,tolerance remains a very difficult goal to achieve, and most transplantphysicians still believe that it cannot be attempted as an intention-to-treat manner in any clinical transplant setting. The present experience,as well as that reported by many other centers worldwide (Table 2)[2,11–17,28–52], demonstrate that this attitude is no longer appropriateand perhaps outdated in the field of LT. Incontrovertible evidence hasbeen provided that –when driven by experienced hands – a permanent

Table 2Synoptic view of the available literature reporting on LT patients weaned off IS, up to July 2008

Center Study type No of pts Mean follow-up fromLT (months)

Accidentalfinding

Intention-to-treat Success rate Follow-up time fromwithdrawal (months)

Pittsburgh [11–13,29,36,42] Prospective, single arm 95 70 Yes Yes 28 (29%) 180London [2,38] Prospective, single arm 18 NA Yes No 2 (11%) 120Kyoto [16,17,24,25,34,39,43] Mixed, single arm 63 ≥24 No Yes 24 (38%) 24Pittsburgh [30] Prospective, single arm 82 Ab initio No Yes 0 (0%) 15Murcia [32] Prospective, single arm 9 ≥60 No Yes 3 (33%) 20New Orleans [37] Prospective, single arm 18 ≥6 No Yes 1 (6%) 12Miami [35] Prospective, controlled 104 ≥12 No Yes 20 (19%) 26Rome [1,21,22] Prospective, controlled 34 63.5 No Yes 8 (24%) 45Gent–Brussels [33,40,44] Prospective 3 Ab initio No Yes 3 (100%), but 2 patients

died after 561 and356 days for tumourrecurrence

543, 356, 498 daysrespectively

Safed–London (Ontario)–Winnipeg [45,46]

Prospective 26 56 months Yes No 2 (8%) 12

Zurich [31] Prospective 1 Ab initio No Yes – 12Columbus–Madison [14,15] Observational 1 36 Yes No – 36Innsbruck [28] Observational 1 71 No Yes – 30Kaohsiung [41] Observational 1 60 No Yes – 60Sidney [47] Observational 1 14 No Yes – 46Brussels–UCL [49] Prospective 1 2 No Yes – 55Inssbruck–Vienna [50] Observational 1 Ab initio No Yes – 4Gothenburgh [51] Observational 1 Ab initio No Yes – 60Chapel Hill [52] Observational 1 Ab initio No Yes – 24

46 G. Orlando et al. / Transplant Immunology 20 (2008) 43–47

and stable IS-free state can be achieved in individuals who had receiveda LT for treatment of non-immune mediated liver diseases. Conse-quently, thebelief that theweaningof IS after LT isharmful hasnoreasonto be anymore, shouldbe consideredas superstition, and reveals a lackofin-depth knowledge of LT immunology and IS management after LT.

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