Tacrolimus Trough Levels, Rejection and Renal Impairment in Liver Transplantation: A Systematic...

American Journal of TransplantationWiley Periodicals Inc.

C© Copyright 2012 The American Society of Transplantationand the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2012.04140.x

Tacrolimus Trough Levels, Rejection and RenalImpairment in Liver Transplantation: A SystematicReview and Meta-Analysis

M. Rodrıguez-Peralvareza, G. Germania,

T. Dariusb, J. Lerutb, E. Tsochatzisa

and A. K. Burroughsa,∗aThe Royal Free Sheila Sherlock Liver Centre andUniversity Department of Surgery. UCL, and Royal FreeHospital. Pond Street, London, UKbStarzl Unit of Abdominal Transplantation, CliniquesUniversitaires Saint Luc, Universite Catholique de Louvain(UCL), Brussels, Belgium*Corresponding author: Andrew K. Burroughs,[email protected]

We hypothesized that current trough concentrations oftacrolimus after liver transplantation are set too high,considering that clinical consequences of rejection arenot severe while side effects are increased. We system-atically reviewed 64 studies (32 randomized controlledtrials and 32 observational studies) to determine howlower tacrolimus trough concentrations than currentlyrecommended affect acute rejection rates and renalimpairment. Among randomized trials the mean oftacrolimus trough concentration during the first monthwas positively correlated with renal impairment within1 year (r = 0.73; p = 0.003), but not with acute rejection,either defined using protocol biopsies (r = −0.37; p =0.32) or not (r = 0.11; p = 0.49). A meta-analysis of ran-domized trials directly comparing tacrolimus troughconcentrations (five trials for acute rejection [n = 957]and two trials for renal impairment [n = 712]) showedthat “reduced tacrolimus” trough concentrations(<10 ng/mL) within the first month after liver trans-plantation were associated with less renal impairmentat 1 year (RR = 0.51 [0.38–0.69]), with no significantinfluence on acute rejection (RR = 0.92 [0.65–1.31])compared to “conventional tacrolimus” trough levels(>10 ng/mL). Lower trough concentrations oftacrolimus (6–10 ng/mL during the first month) wouldbe more appropriate after liver transplantation. Reg-ulatory authorities and the pharmaceutical industryshould allow changes of regulatory drug information.

Key words: Acute cellular rejection, liver transplan-tation, renal impairment, tacrolimus, toxicity, troughconcentration

Abbreviations: ACR, acute cellular rejection; LT, livertransplantation; TC, trough concentration; RCTs, ran-domized controlled trials; HCV, Hepatitis C virus; IQR,

interquartile range; ROC, receiver operating character-istic.

Received 17 February 2012, revised 25 April 2012 andaccepted for publication 27 April 2012

Introduction

The incidence of acute cellular rejection (ACR) after livertransplantation (LT) has decreased since tacrolimus wasintroduced (1,2). Tacrolimus is used in most transplantunits as it results in less ACR and better survival than cy-closporine (3). Significant ACR, which includes moderate–severe histological grades, is most frequent within 2 weeksafter LT and decreases progressively thereafter. In LT therisk of ACR is similar to other solid-organ transplants butit has less clinical relevance (4). Moreover the responseto specific therapy (i.e. corticosteroids boluses) is satisfac-tory in most cases (5), thus evolution to chronic rejectionand graft loss is an infrequent event (<5%) (6,7).

Despite this the conventional and regulatory targets fortacrolimus trough concentrations (TC) used in immunosup-pression trials in LT are high (i.e. 10–15 ng/mL during thefirst 4–6 weeks and 5–10 ng/mL thereafter), since theseare transposed from kidney transplantation (8). Adverse ef-fects of immunosuppression are also similar being early ex-posure to tacrolimus especially associated with long-termchronic renal dysfunction (9,10). The increasing need to useolder donors, donation after cardiac death and the morefrequent renal dysfunction in recipients, has increased re-nal impairment after LT (11). This emphasizes the need tohallmark the early transplant period, in which changes inimmunosuppression may lead to long-term gains.

Nevertheless calcineurin inhibitors are critical in pre-venting ACR and graft loss; clinical trials testing theircomplete avoidance were stopped prematurely due to in-creased ACR (12,13). The main focus in immunosuppres-sion still is optimizing tacrolimus dosing, especially withinthe first weeks after transplantation, to balance preventionof moderate–severe ACR versus adverse effects, particu-larly on the kidney. However although the relationship be-tween tacrolimus TC and toxicity is well established (9,14–16), the relationship between TC and ACR is controversial

Rodrıguez-Peralvarez et al.

Figure 1: Immunosuppression in liver transplantation. Cur-rently the consequences of rejection are much reduced whiletoxicity and complications derived from immunosuppression areincreased.

(14,15,17,18). The latter is contributed by the heterogene-ity in how ACR is diagnosed: most studies do not use pro-tocol biopsies, but use “clinical suspicion” based on liverfunction tests to perform a biopsy, misdiagnosing 32% ofpatients (7,19).

Indeed in a scenario characterized by less rejection andworse renal outcomes (9), LT recipients are overimmuno-suppressed (20) (Figure 1). Therefore, it should be possi-ble to decrease target TC of tacrolimus. In this review weevaluate the evidence related to tacrolimus TC, ACR andrenal dysfunction following adult LT, and draw conclusionson what TC should be, so as to influence current clinicalpractice and future trial design.

Materials and Methods

Literature search, study selection and data extraction

A search on MEDLINE, Cochrane Controlled Trial Register (CENTRAL), EM-BASE and Science Citation Index databases was done from January 2002 toJanuary 2012, to assess the more current usage of tacrolimus. We identifiedstudies using these key words: “liver transplantation”, “tacrolimus”, “troughconcentration”, “rejection” and “renal impairment”, also using equivalentfree-text terms, and no language restrictions. The search resulted in 204records which were categorized and screened independently by MRP andGG (differences resolved by AKB). Duplicate records, reviews and not re-lated articles were removed resulting in 78 eligible studies including 46 ran-domized controlled trials (RCTs). Another 14 RCT were excluded becauserandomization occurred late after LT, or concerned pediatric population, orwere preliminary reports not providing enough data about renal impairment,rejection or tacrolimus exposure. We finally selected 64 articles comprising32 RCT and 32 observational studies (Figure 2).

The following data were extracted from each study independently by MRPand ET using a predefined form: year of publication, number of patients,immunosuppression regimen, tacrolimus TC target, mean tacrolimus TCwithin the first month after LT, ACR rates, renal impairment rates andmethodological quality. Evaluated outcomes were biopsy-proven ACR afterclinical suspicion or by protocol biopsies, renal impairment at 3–6 monthsand renal impairment at 1 year after LT.

Methodological quality

Meta-analysis was appropriate for RCT in which tacrolimus with conven-tional and reduced TC (>10 ng/mL and <10 ng/mL, respectively) werecompared in opposite arms. The methodological quality was defined as thecontrol of bias in the treatment comparison. The randomization methodswere classified as the primary measure of bias control (21), being evaluatedby the allocation sequence generation (classified as adequate if based ona table of random numbers, computer-generated random numbers or sim-ilar) and allocation concealment (classified as adequate if based on central

Figure 2: Flow diagram illustrat-

ing the search strategy used

according to PRISMA (preferred

reporting items for systematic re-

views and meta-analyses) state-

ment (62).

American Journal of Transplantationdoi: 10.1111/j.1600-6143.2012.04140.x

Tacrolimus Levels after Liver Transplantation

randomization, identically appearing coded drug containers, serially num-bered opaque sealed envelopes or similar). We also extracted blinding,the risk of attrition bias and whether the primary outcome measure wasdefined and reported. MRP and ET independently evaluated the method-ological quality (any differences resolved by AKB).

Statistical analysis

The analyses were performed using RevMan version 5.1 (Nordic CochraneCentre, Copenhagen, Denmark). Meta-analyses were performed to allowintention-to-treat analyses, using random effects models due to expectedclinical heterogeneity. The number of events and number of patients ineach intervention arm were used to calculate relative risks (RR) and 95%confident intervals (CI). I2 values were indicators of the degree of intertrialheterogeneity. The possibility of publication bias was explored with funnelplots (supplementary Figure 1).

Results

Pharmacokinetics of tacrolimus in liver transplant

recipients

A small fraction of tacrolimus binds to leukocytes (0.61± 0.4%) which are the therapeutic target (22), leading toa narrow therapeutic index, coupled with a long-half life(43 h) and slow renal clearance. Thus monitoring blood TCis necessary to minimize overdosage. In LT the variabil-ity of tacrolimus dosage is increased due to changes inblood count, protein concentrations and albumin supple-mentation (23). Hepatitis C virus (HCV) decreases hepaticCYP3A4 activity, requiring lower doses of tacrolimus overtime (24).

Current recommendations

The currently recommended tacrolimus TC after LT comefrom RCTs (Table 1), in which the stipulated targets forTC were transposed from kidney transplantation (8), andreflect the registration details filed by manufacturers. Ac-cording to these, the conventional initial tacrolimus dosesare adjusted to maintain TC of 10–15 ng/mL during the first4–6 weeks with a progressive reduction thereafter, achiev-ing 5–10 ng/mL in the long term, with minimal variationsbetween studies (see Table 1). With this regimen adverseeffects are common, leading to dose reduction or stop-ping tacrolimus, which explains the discordance betweentacrolimus TC aimed for and actually achieved in somestudies (25–27). Indeed in a recent study (28) a target TC of>12 ng/mL was used within the “standard therapy” groupduring the first 6 weeks after LT: 45% (83/183) of patientsneeded to stop, mostly due to adverse effects. In contrasta clinical trial reported within the same period, aiming attacrolimus TC 6–8 ng/mL, only had one withdrawal (0.64%)due to toxicity (29).

In current clinical practice, most centers target lowertacrolimus TC, often combined with other agents. IndeedTC in RCT of “tacrolimus sparing” are usually lower, buthave added agents in the “mistaken” premise to compen-sate for tacrolimus reduction, thus favoring overimmuno-suppression, and which may not be necessary. In the liter-

ature a significant difference exists between TC targetedin RCTs (Table 1) during the first 4 weeks (>10–12 ng/mL)compared to those in observational studies (14,15,18,24)(7–10 ng/mL).

Rejection rates and tacrolimus TC

Using lower tacrolimus TC could result in more ACR, espe-cially early after transplantation, when there is increasedgraft immunogenicity. Nevertheless ACR after LT is oftenmild without significant detriment to graft and patient sur-vival (7,30). Furthermore lower tacrolimus concentrationscould translate into more immune activation, which is ben-eficial to induce tolerance in the long term (31). Only moder-ate/severe ACR is most likely to require specific treatmentto prevent chronic rejection and graft loss.

Data from interventional studies: overall analysis: Fig-ure 3 shows rates of ACR in RCTs, which vary depend-ing on how ACR is defined: most studies use histologicalconfirmation after clinical suspicion, with ACR 20%–30%,while if protocol biopsies were used this was 20–50%.Rejection rates are unchanged over the last decade. Therewere 20 RCTs comprising 39 comparison arms and 5000patients, defining ACR as histologically proven after clini-cal suspicion. In 21 arms the mean tacrolimus TC duringthe first month was 10–15 ng/mL, while 18 arms reported6–10 ng/mL. In 5 RCTs (9 comparison arms; n = 719)which used protocol biopsies, 7 arms had tacrolimus TC<10 ng/mL during the first month after LT, and 2 arms hadhigher levels. Figure 4 shows no correlation between themean TC and the occurrence of ACR either defined withprotocol biopsies (r = −0.37; p = 0.32) or not (r = 0.11;p = 0.49). As this could be due to using other immuno-suppressant drugs combined with tacrolimus, we furthersubdivided the analysis.

Interventional studies aiming at lower TC of tacrolimus

without differences in other immunosuppression:

“minimizing strategy”: Hitherto only one study hascompared identical immunosuppressive regimens usingdifferent tacrolimus TC (32). A standard regimen usingtacrolimus (TC 10–15 ng/mL), mycophenolate and taperingsteroids was compared with an identical regimen but tar-geting tacrolimus TC at 5–8 ng/mL. ACR rates were similar(17%) (biopsy proven after biochemical suspicion requiringtreatment) by 26 weeks after transplantation. As the pri-mary objective was to evaluate pharmacokinetics, only 30patients/arms were included, so a type 2 error is possible.However, results suggest that lower tacrolimus TC may beadequate to prevent ACR.

Other studies have also used lower tacrolimus TC with-out large differences in ACR. One (33) allowed a widerTC range (5–15 ng/mL) in both arms: ACR in patients re-ceiving only tacrolimus and steroids was 26.6%. Another(29) compared tacrolimus monotherapy with tacrolimusand steroids using a target of 6–8 ng/mL in both arms:



Figure 3: Incidence of acute cellular rejection in liver transplant recipients among randomized clinical trials between 2002 and

2012 comparing protocols with tacrolimus (with standard or reduced dosage and in association or not with other drugs).

Figure 4: Relationship between mean trough tacrolimus concentrations during the first month after liver transplantation and

the occurrence of acute cellular rejection (defined by using protocol biopsies or histologically confirmed after clinical suspicion).

Each data point represents one arm from the included trials (2002–2012).



moderate/severe ACR using protocol biopsies at day 7 was50% and 48.7%, respectively, rates within the range ofother studies using protocol biopsies. However approxi-mately half of patients had TC <6 ng/mL at the time ofbiopsy. Another study from the same group also using pro-tocol biopsies and aiming for identical tacrolimus TC hadACR rates between 0% and 50% (only 20%–25% of pa-tients had <6 ng/mL) (34). Importantly both studies (29,34)showed that the higher ACR rates did not affect patient orgraft survival, and lower rates of renal dysfunction (creati-nine ≥1.5 mg/dL) were seen (5% to 10%). Thus tacrolimusused in monotherapy or combined with steroids (withoutother interventional drugs) could be safe for long-term out-comes.

In HCV transplanted patients, tacrolimus monotherapy wascompared with tacrolimus, azathioprine and steroids aim-ing at lower tacrolimus TC in both arms (5–10 ng/mL),and ACR was diagnosed by protocol biopsies (35,36).There were higher rates of moderate–severe ACR withinthe tacrolimus-based triple therapy group (48% vs. 29%)(35,36). Again these rates are within the range seen in stud-ies using protocol biopsies, and the higher rate seen in thetriple therapy arm was likely to be due to lower tacrolimusTC. Graft and patient survival were similar (36).

Interventional studies aiming at lower tacrolimus TC

by adding another immunosuppressive drug: “renal

sparing strategy”: Five RCT (13,28,32,37,38) and onenonrandomized trial (39) compared protocols in which con-ventional and reduced TC of tacrolimus were in opposingarms (see Table 1), evaluating anti-IL2 or antithymocyteglobulin, or adding mycophenolate. ACR was defined solelyby histological confirmation after biochemical suspicion ofrejection, which is an important limitation as liver functiontests are not specific (40), and are only late markers (41) ofhistological ACR. Furthermore, the “clinical” definition ofsuspected rejection is not mentioned, or is different, be-tween studies. With these caveats the meta-analysis of thefive RCT including 917 patients (13,28,32,37,38) (quality as-sessment in Table 2) showed no significant differences forbiopsy-proven ACR (RR = 0.92 [95% CI = 0.65–1.31]), be-tween reduced (6–10 ng/mL) and conventional (>10 ng/mL)tacrolimus TC (Figure 5). The I2 test for heterogeneity was54% (p = 0.07). The funnel plot did not reveal publicationbias (Supporting Figure S1). Results did not substantiallychange when we repeated the analysis including only stud-ies with optimal methodological quality (28,37) (RR = 0.82[95% CI = 0.66–1.01]; I2 = 59%, p = 0.12).

Overall among the six trials (these five RCT and the not ran-domized study), four (37–39) showed no statistical differ-ences in ACR: 11%–23.2% in standard, and 14.9%–27%in reduced dose arms. In another RCT standard tacrolimus(TC 10–15 ng/mL during the first 3 months and 7–12 ng/mLthereafter), was compared to induction with antithymocyteglobulin and reduced tacrolimus (TC 5–12 ng/mL during thefirst 3 months and slow weaning thereafter to achieve very

low doses at 12 months) (13). As there was a significantlyhigher rejection rate in the antithymocyte induction group(66.7% vs. 31.2%; p = 0.03) the trial was prematurelystopped. Interestingly all patients developing rejection dur-ing weaning had TC <5 ng/mL suggesting that completeweaning is not possible, and TC under this threshold within12 months are not safe. In the sixth study, 100 patients with“full dose tacrolimus” (TC >12 ng/mL for the first 6 weeks)were compared with 95 patients given mycophenolate and“reduced dose tacrolimus” (TC <10 ng/mL for the first 6weeks). Both groups also received tapering steroids. ACRwas decreased with lower tacrolimus TC (30% vs. 46%;p = 0.024) (28).

Data from observational studies: The data here are het-erogeneous. In a retrospective study comprising 248 pa-tients who received a liver, kidney, lung or combined trans-plant the relationship between tacrolimus TC and ACR wassignificant but weak (14). In a prospective analysis of 111 LTpatients tacrolimus TC (over a 7-day period before diagno-sis of histological rejection) independently predicted ACR(RR = 0.86; p = 0.049), but as the AUROC was <0.5 nooptimal predictive threshold could be derived (15). In con-trast, a systematic review of three RCTs in LT (n = 721)(42–44) did not find any relationship between tacrolimusTC and ACR (17). Nevertheless, these results are barelyinterpretable because of different definitions of ACR be-tween studies and the use of plasma levels of tacrolimusinstead of whole-blood levels. Furthermore the first andonly study which evaluated protocol biopsies in 90 patients(18), documented moderate–severe ACR in 41.1% at day7, without correlation between tacrolimus TC and histolog-ical ACR (mean TC was 7.3 ± 4.4 with no-mild rejectionand 7.5 ± 3.7 with moderate–severe rejection).

There are several important limitations in interpreting theseresults. Firstly, both the populations and immunosuppres-sion protocols were heterogeneous; secondly most stud-ies did not use protocol biopsies; thirdly the threshold forliver function test abnormalities defining a biochemical sus-picion of ACR, varied between studies and even betweencenters within the same study (1). Lastly using a 7-dayperiod (15) between measuring TC and correlating withhistological confirmation of ACR, does not mirror clinicalpractice, and has little applicability.

Renal dysfunction related to tacrolimus after liver

transplantation

Chronic renal failure occurs in 18% of liver transplantpatients by 5 years: only intestinal transplantation isworse (9). In contrast to the weak relationship betweentacrolimus TC and ACR, the relationship with acute andchronic renal dysfunction is well established. Indeed earlyexposure to tacrolimus after LT is particularly deleterious,affecting long-term outcomes (9,10).

Data from interventional studies: overall analysis:

Among RCTs, renal dysfunction is documented in 20–50%



Figure 5: Forest plot diagram illustrating the risk of acute cellular rejection among five randomized controlled trials (n = 957)

using reduced (< 10 ng/mL) and conventional (>10 ng/mL) tacrolimus trough concentrations in opposite arms.

(Figure 6), although three studies reported over 60%(36,39,45), two of them (39,45) had very high exposureto tacrolimus (>12 ng/mL) during the first month after LT.As shown in Figure 6 there is an increasing trend of higherrates of renal dysfunction at 1 year after LT.

There were 16 RCTs including 31 comparison arms and4114 patients, documenting renal impairment between 3and 6 months (17 arms), and at 1 year (14 arms). In 18 com-parison arms the mean tacrolimus TC recorded (during thefirst month after LT) was within current recommended lev-els (10–15 ng/mL; mean 11.7 ± 1.1 ng/mL). In the other 13comparison arms tacrolimus TC were lower (6–9.9 ng/mL;mean 8.96 ± 0.9 ng/mL). Rates of renal impairment inRCTs positively correlated with mean tacrolimus TC dur-ing the first month after LT (r = 0.58; p = 0.001), especiallyfor studies reporting renal dysfunction at 1 year (r = 0.73;p = 0.003) (Figure 7).

Interventional studies aiming at lower TC of tacrolimus

without differences in other immunosuppression:

“minimizing strategy”: The single study using identi-cal protocols evaluating 60 patients either with standardtacrolimus TC (aiming 10–15 ng/mL), or reduced dose (TC5–8 ng/mL), found that creatinine clearance was higherwith reduced TC, but this difference was already at thebaseline so this result is uninterpretable (32). Interestinglytwo further RCTs from the same group (29,34) reported re-nal impairment (serum creatinine ≥1.5 ng/mL) under 11%when aiming for lower tacrolimus TC (6–8 ng/mL).

Interventional studies aiming at lower tacrolimus TC

by adding another immunosuppressive drug: “renal

sparing strategy”: Two RCT (28, 37) (n = 712) comparedreduced and conventional tacrolimus TC within the firstmonth after LT and reported rates of renal impairment,both at 1 year (quality assessment in Table 2). In the meta-analysis (Figure 8), lower tacrolimus TC (6–10 ng/mL) ledto less renal impairment (RR = 0.51 [95% CI = 0.38–0.69])

compared with conventional tacrolimus TC (>10 ng/mL). I2

was 0% (p = 0.44). The funnel plot did not reveal publica-tion bias (Supporting Figure S1).

In other RCT renal function was compared between stan-dard immunosuppression (n = 76) comprising tacrolimus(TC 10–15 ng/mL during the first month), mycophenolateand steroids, and an experimental arm (n = 72) receivingthe same protocol but adding daclizumab and delaying useof tacrolimus until days 4–6 after LT (TC 4–8 ng/mL) (38). Byday 3 after LT, the glomerular filtration rate had decreased25 mL/min from baseline in the standard arm, while the ex-perimental arm (who had not received tacrolimus as yet)had an improved glomerular filtration rate by 12 mL/min(p = 0.001), a difference that remained statistically signifi-cant at 1 month after LT.

With a similar design without mycophenolate, and sub-stituting daclizumab for basiliximab in 45 patients, a notrandomized trial showed renal impairment rates of 67% at3 months in the standard arm, and 26% in the delayedreduced arm (TC 5–10 ng/mL), (p < 0.001)(39). In 195patients aiming for tacrolimus TC <10 ng/mL early afterLT, a beneficial effect on renal function was still presentat 1 year after (42% to 24%; p = 0.009) (28) confirmingthe importance of the early period after LT with respectto renal toxicity. Figure 6 shows that another RCT whichused a reduced tacrolimus TC within the experimentalarms (comprising daclizumab, tacrolimus, mycophenolateand steroids in one, and tacrolimus, mycophenolate andsteroids in the other) (37), found a nonstatistical trend forless renal impairment in this group, a result likely due to asimilar tacrolimus TC in both arms (differences <1 ng/mL)(37). Thus there was no appropriate evaluation of lowertacrolimus TC, and the effect of the additional immunosup-pressive agent made the regimen more immunopotent andpotentially increased adverse effects. Similar results wereobtained in a RCT allowing reduced tacrolimus TC but onlyafter the first month after LT (33).



Figure 6: Incidence of renal

impairment in liver transplant

recipients among randomized

clinical trials between 2002 and

2012 comparing protocols with

tacrolimus (with standard or re-

duced dosage and in associa-

tion or not with other drugs).

Data from observational studies: In 111 patients with afollow-up of 12 weeks, 43.2% experienced adverse effectsdue to tacrolimus. Multivariately tacrolimus TC were inde-pendently related to nephrotoxicity (OR 1.27; p < 0.001),and when higher than 11.6 ng/mL (common in the earlypostransplant period using standard target TC) the posi-tive predictive value was 64% (15).

A retrospective study of 96 patients evaluated the rela-tionship between nonalcoholic steatohepatitis (NASH) asthe indication for LT and subsequent renal dysfunction(46). Acute kidney injury within the first month was higheramong NASH patients (50% vs. 29%), as was persistentkidney injury after 3 months (31.2% vs. 8.3%). Interest-ingly despite more use of mycophenolate among the NASH



Figure 7: Relationship between

mean trough tacrolimus concen-

trations during the first month af-

ter liver transplantation and renal

impairment in randomized con-

trolled trials (2002–2012). Eachdata point represents one study armfrom the included trials.

group (40% vs. 21%), tacrolimus TCs were similar be-tween the groups at 3 months (8.4 ng/mL vs. 7.7 ng/mL;p = 0.14) and 2 years (8.0 ng/mL vs. 8.1 ng/mL; p = 0.95)after LT. Thus, NASH patients may be particularly suscep-tible to renal dysfunction and should be considered fortacrolimus TC reduction. Again adding another drug (my-cophenolate) did not result in lower tacrolimus TC, so thata real “test” of renal sparing was not achieved. In addition,the early exposure to tacrolimus was not recorded and dif-ferences in this aspect were not evaluated regarding renaloutcomes.

Outcomes were reported in 124 patients receivingtacrolimus monotherapy (47) (30 from a RCT (48) and94 from a consecutive prospective cohort), with a me-dian follow-up of 8 years. Mean tacrolimus TC were be-tween 7 ng/mL and 10.3 ng/mL during the first 3 weeksbut 34% had peak tacrolimus TC >15 ng/mL, and 45%required dose reduction. Renal dysfunction (glomerular fil-tration rate <60 mL/min) occurred in 36% of patients. How-ever long-term renal support was only needed in one pa-tient (0.8%), a rate substantially lower than that reportedby Ojo et al. (9) (18.1%) for patients transplanted duringthe same period with similar follow-up.

Discussion

In LT there is a different clinical impact of rejection com-pared to other organs (4,5), but recommended tacrolimusTC remain identical to kidney transplantation, in which ACRoften carries higher risk of irreversible graft damage. Animmediate consequence is that tacrolimus-related compli-cations are frequent, which is particularly important giventhe correlation between early exposure to tacrolimus andrenal impairment (Figure 7). Our systematic review of RCTswith 1-year follow-up showed an even stronger correlation.This confirms a premise for this review that tacrolimusearly exposure is critical in determining long-term renaldysfunction. Indeed renal impairment occurs in up to 60%of patients (28,39,49,50) and is independently related tomortality (9). Preventing renal impairment is vital, as onceestablished, reducing tacrolimus dosage (even by 50%) orconversion to non nephrotoxic drugs such as sirolimus ormycophenolate, only stabilizes renal dysfunction but rarelyreverses it (51–55). The meta-analysis showed that lowertacrolimus TC after LT (6–10 ng/mL) led to a twofold reduc-tion in renal impairment. Thus lower tacrolimus TC couldcounteract the recent trend of increasing rates of renaldysfunction at 1 year after transplantation (Figure 5).

Figure 8: Forest plot diagram illustrating the risk of renal impairment at 1 year among two randomized controlled trials (n = 712)

using reduced (< 10 ng/mL) and conventional (> 10 ng/mL) tacrolimus trough concentrations in opposite arms.



Our review shows that lower tacrolimus TC do not ad-versely affect ACR. Several RCTs comparing standard andlower tacrolimus TC show no difference in terms of rejec-tion (32,37–39), or even an advantage for the lower TC arm(28). Importantly in the only study directly comparing twoidentical protocols but varying tacrolimus TC, there was nodifference in ACR (32). In our evaluation of RCTs there wasno significant correlation between the mean tacrolimusTC and ACR (Figure 4). The meta-analysis showed no dif-ferences in ACR when targeting tacrolimus TC <10 ng/mLor >10 ng/mL within the first month after LT. There wasa trend toward significant heterogeneity (I2 = 54%; p =0.07) which may be explained by the lack of consensusin the definition of clinical suspicion of rejection and alsobecause one study aimed to use very low tacrolimus TC(<6 ng/mL) in the “reduced dose” arm (13). The completeweaning of tacrolimus, or achieving very low concentra-tions during the first weeks after LT carries a higher risk ofrejection (12,13), which could be deleterious.

There are other potential benefits of using lower tacrolimusTC early after transplantation. Several animal models, andone prospective study in humans documented a positivecorrelation between tacrolimus exposure and hepatocellu-lar carcinoma recurrence (56–58). Secondly HCV patientsmay develop less fibrosis as shown in a RCT using lowertacrolimus TC (5–10 ng/mL) (36), compared to other stud-ies (59,60). There may be a benefit in not suppressing ACRcompletely in terms of graft tolerance, since rejection andtolerance are along a continuum (31,61).

We searched to find evidence for the optimal tacrolimusTC after LT. This proved to be difficult: most RCTs did notuse equipotent regimes, as they were designed to testnew agents, and not specifically lower doses of tacrolimus.Whether results would be similar without additional agentscannot be definitely ascertained, but it is likely from the ev-idence taken as a whole. However, in observational stud-ies, there was great variability in the relationship betweentacrolimus TC and the risk of ACR. This may be due tothe definition of ACR used, which was based on clinicalsuspicion in most studies. Protocol biopsies would pro-vide a more objective assessment but these are seldomperformed.

However this systematic review suggests that to re-duce renal impairment and not to increase moderate–severe ACR simultaneously, it would be preferable to tar-get tacrolimus TC between 6 and 10 ng/mL during thefirst 4–6 weeks after LT. Then a progressive reduction ofdosage should be accomplished to achieve TC between 4ng/mL and 8 ng/mL in the long term. Studies with largecohorts of liver transplant patients with protocol biopsiesare needed to determine the lowest threshold of TC possi-ble, which may vary for different etiologies (24,46) and im-munosuppression protocols. Future RCTs using tacrolimusshould target lower TC, which will require regulatorychanges.

Acknowledgments

M.R.P. is a recipient of a grant from the Institute Maimonides for Biomedicalresearch of Cordoba (IMIBIC).

Disclosure

The authors of this manuscript have no conflicts of inter-est to disclose as described by the American Journal ofTransplantation.

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Supporting Information

Additional Supporting Information may be found in the on-line version of this article.

Figure S1: Funnel plot representing randomized trials in-cluded in the meta-analysis to address the relationship be-tween tacrolimus trough concentrations (<10 ng/mL vs.>10 ng/mL) within the first month after liver transplanta-tion and acute cellular rejection (A) and renal impairment(B).

Please note: Wiley-Blackwell is not responsible for the con-tent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for thearticle.



Tab

le1:

Ran

dom

ized

cont

rolle

dtr

ials

com

parin

gdi

ffere

ntim

mun

osup

pres

sive

prot

ocol

sus

ing

tacr

olim

usin

liver

tran

spla

ntat

ion

betw

een

2002

and

2012

.Onl

yco

mpa

rison

arm

sus

ing

tacr

olim

usar

ede

scrib

ed

Con

tinue

d



Tab

le1:

Con

tinue

d

Con

tinue

d



Tab

le1:

Con

tinue

d

AC

R=

acut

ece

llula

rre

ject

ion;

ATG

=an

tithy

moc

yte

glob

ulin

;DA

C=

dacl

izum

ab;M

MF

=m

ycop

heno

late

;TA

C=

tacr

olim

us;S

TD=

ster

oids

;NS

=no

tsi

gnifi

cant

.∗ T

acro

limus

trou

ghco

ncen

trat

ions

wer

em

easu

red

inw

hole

-blo

odsa

mpl

esin

alls

tudi

es.T

hem

etho

dw

asta

ndem

mas

ssp

ectr

osco

pyin

Fisc

her(

63)a

ndN

asha

n(3

2).I

mm

unoa

ssay

was

used

byM

anou

sou

(36)

,Ler

ut(2

9)an

dS

heno

y(6

7).T

here

mai

ning

rand

omiz

edtr

ials

did

not

men

tion

the

met

hodo

logy

used

.



Table 2: Quality assessment of randomized controlled trials comparing conventional (>10 ng/mL) and low (<10 ng/mL) trough concen-trations of tacrolimus in opposite arms within the first month after liver transplantation

AuthorSequencegeneration

Allocationconcealment Blinding

Incompleteoutcome data

Selectiveoutcomereporting

Other sources ofbias

Boudjema (28) Yes Yes Yes∗ Yes Yes Yes“Randomization

was based oncomputer-generatedlists”

“Randomizationlists were kept bythe pharmacist ofthe coordinatingcenter”.

Not blinded butoutcome isunlikely to beaffected

Missing databalancedacross groups

Benıtez(13) Yes Yes Yes∗ Yes Yes No“The randomization

sequence wascomputed-generated”

“The randomizationsequence waskept in opaquesealedenvelopes”


Causes ofprotocol dis-continuationwere notrelated withprimaryoutcome(rejection)

The study wasprematurelystopped dueto imbalancein outcome

Nashan (32) Unclear Unclear Yes∗ No Yes YesThe authors did

randomization butthe method usedis not described

Information aboutallocationconcealment isnot provided


“Other reasonsfor withdrawalin thetacrolimusstandard-dosegroup wereinsufficienttherapeuticresponse (2patients)”. Thiscould berelated tooutcome

Neuberger (37) Yes Yes Yes∗ Yes Yes Yes“using a

randomization listwith blocks of sixpatients”

“Patients wererandomizedcentrally”


36% of patientsdiscontinuedthe studyprematurelybut thereasons wereunlikelyrelated tooutcome

Yoshida (38) Unclear Unclear Yes∗ Yes Yes YesThe authors did

randomization butthe method usedis not described

Information aboutallocationconcealment isnot provided


Case analysisavailable

∗Measured outcomes were objectively assessed (serum creatinine for renal impairment and Banff grading for histological acute rejection);in the authors’ opinion these are unlikely to be influenced by the lack of blinding and do not influence the result of the meta-analysis.


Tacrolimus Trough Levels, Rejection and Renal Impairment in Liver Transplantation: A Systematic...

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