Liver transplantation (LT) is the preferred therapeutic option for selected patients with cirrhosis and hepatocellular carcinoma (HCC). After LT, patients within Milan criteria (a single nodule <5 cm or up to 3 nodules <3 cm each, without macrovascular invasion or extrahepatic spreading) show survival rates around 70% at 5 years, which are superior than those offered by any other surgical or ablative technique.1 However, tumor recurrence rates range between 10% and 20%, being even higher for patients with microvascular invasion (mVI).2,3 The only immunosuppression strategy able to reduce the risk of HCC recurrence is minimization of calcineurin inhibitors (CNI),4,5 which is particularly effective when implemented early after LT.6 In clinical practice, the potential risk of rejection associated with minimization is counteracted by combining CNIs with additional immunosuppressants, mainly antimetabolites, or mTOR inhibitors. In patients transplanted with HCC, mTOR inhibitors are particularly attractive because they allow for a marked reduction of CNI exposure while providing a theoretical antiproliferative effect. Indeed, the mTOR pathway expression is increased in HCC histological samples7 and its inhibition by using rapamycin results in tumor regression in animal models.8
The role of mTOR inhibitors in preventing HCC recurrence after LT is still under debate. In several observational retrospective series further meta-analyzed, a protective role of sirolimus was suggested,9 but the only randomized trial published hitherto found no clear benefit in avoiding tumor recurrence.10 A recent network meta-analysis of randomized trials showed that sirolimus containing protocols were associated with increased risk of graft loss and death in LT patients,11 and therefore prescribing sirolimus systematically in patients with HCC may not be justified. Everolimus is a more recently developed mTOR inhibitor which claims for an increased potency and improved safety profile as compared with sirolimus.12-14 In absence of supporting scientific evidence, many institutions have implemented everolimus-based immunosuppression for HCC-transplanted patients as part of routine clinical practice.15,16 A randomized trial is warranted but such design is precluded by its complexity, costs, increased sample size, and prolonged follow-up required. In this context, the aims of the present prospective study were to evaluate the impact of early introduced everolimus on HCC recurrence after LT and to identify clinical predictors of favorable response to everolimus.
MATERIALS AND METHODS
Study Design and Patients
A study design overview is shown in Figure S1 (SDC, http://links.lww.com/TP/B575). This is a prospective observational study designed in March 2011. A consecutive cohort of patients with HCC who underwent LT from June 2012 to December 2015 and survived longer than 1 month was included. Patients were initially recruited at a single center but, given the slow accrual, a second transplant institution was invited to participate from November 2013 onward. Exclusion criteria were as follows: younger than 18 years, HIV-positive, retransplantation, or combined organ transplantation. To enter the study, all patients were required to sign an informed consent document. The present clinical investigation was conducted according to the principles contained in the Declaration of Helsinki. This project is part of a broader research initiative (PI11/02867 and PI14/01469), which was approved by the local ethics committee.
Both recruiting institutions are located in Andalusia, southern Spain, and shared criteria for including and prioritizing patients within the waiting list. Only patients within Milan criteria in the pretransplant imaging assessment were accepted as candidates for LT. An empirical MELD score of 15 points was given at listing. An additional MELD point was assigned monthly within the waiting list until transplantation (a maximum of 23 MELD points were allowed for HCC patients). Locoregional ablative therapies (preferably transarterial chemoembolization) were used to prevent drop-out in patients with multinodular disease or with a single nodule larger than 20 mm unless technically unfeasible. The primary immunosuppression protocol consisted in tacrolimus, early introduction of everolimus and tapering corticosteroids. Dosage of tacrolimus was adjusted to keep trough concentrations between 6 and 10 ng/mL within the first month after LT, with a progressive reduction thereafter to achieve trough levels of 4 ng/mL in the long term.17 Everolimus was introduced at postoperative days 15 to 21 at an initial dose of 0.5 to 1 mg bid, which was tittered to maintain trough concentrations between 3 and 8 ng/mL. This early introduction of everolimus has proved its safety in observational series.18,19 Induction therapy with basiliximab was allowed in selected patients with pretransplant renal impairment or hepatic encephalopathy to delay the introduction of tacrolimus until day 7. Corticosteroids were progressively withdrawn within the first 6 months after LT.
Patients were followed up until July 2017 to ensure a minimum surveillance length of 18 months after LT for the most recently included patient. Median follow-up was 34 months. Clinical visits and determinations of trough concentrations of tacrolimus and everolimus were performed at least every other day until discharge, weekly until day 30, monthly until day 90, every 3 months within the first year and every 6 months thereafter.17 Additional visits and measurements of trough concentrations were performed whenever clinically indicated. Hepatocellular carcinoma recurrence was depicted by using abdominal ultrasound every 3 months within the first year and every 6 months thereafter. Any suspicious liver nodule detected in the ultrasound or rising alpha-fetoprotein otherwise unexplained motivated the indication of liver magnetic resonance or computed tomography. Imaging and histological criteria to diagnose HCC mirrored current international guidelines.20
The above described patients were compared with an historical cohort with identical inclusion and exclusion criteria. Historical controls were transplanted between January 2000 and June 2012. A propensity score matching strategy was performed to ensure comparability. The variables used to derive propensity scores were obtained from the histological analysis of the explanted liver as follows: (a) number of HCC nodules; (b) diameter of the largest nodule; (c) total tumor diameter; (d) Milan criteria fulfillment; (e) tumor differentiation according to Edmonson scale21; (f) mVI. The selection of historical controls was carried out by a study investigator who was blinded for patient outcomes, starting from the most recently transplanted patient and continuing backward. The nearest neighbor match was selected (1:2 ratio). None of the historical controls had received mTOR inhibitors.
The main outcome of the study was HCC recurrence. Secondary outcomes were recurrence-free survival and overall survival after LT. Those adverse events leading to greater than 50% dose reduction of everolimus or discontinuation were also recorded. The histological evaluation of the explanted liver was systematically performed to determine number of HCC nodules, diameter of the largest nodule, total tumor diameter (sum of diameter of each recorded nodule), histological differentiation according to Edmonson scale,21 macrovascular invasion and mVI (defined either as a tumor emboli within a peritumoral vessel, or as a satellite nodule surrounded by endothelium3). The primary immunosuppression protocol was recorded, as well as tacrolimus and everolimus trough concentrations and serum creatinine during follow-up. Vascular and biliary complications, biopsy-proven acute cellular rejection, wound healing complications, and need for retransplantation were registered.
Variables were displayed in frequency tables or expressed as means and standard deviations, except for those with asymmetric distribution in which median and interquartile range (IQR) were used. Kaplan-Meier curves (Log rank) and Multivariate Cox regression were used to analyze the impact of early introduced everolimus on HCC recurrence and other time-dependent outcomes, while controlling for potential confounders. Those variables with P values of 0.25 or less in the univariate analysis entered the initial multivariate model. Covariates were removed from the model in a backward stepwise process. All possible interactions among covariates were tested. Variables with a P value between 0.05 and 0.20 were screened to identify potential confounding factors, and further removed from the model if they did not behave as such. A confounding factor was confirmed when its removal from the model motivated greater than 15% variation in the β coefficients of the remaining covariables. Cumulative incidence of HCC recurrence after LT was adjusted for the risk of death by using competing risks analysis. Every hypothesis tested was 2 tailed and considered significant if P value is less than 0.05. Statistical analysis was performed using SPSS 20.0 (IBM, Chicago, IL).
Sample Size Calculation
EPIDAT 3.1 (Xunta de Galicia) was used for sample size calculation. The study was powered to detect a clinically significant reduction in HCC recurrence rates at maximal follow-up after LT (median 34 months). The following assumptions were made based on the best evidence available during the study design (ie, March 2011)22: (a) expected HCC recurrence rates in the prospective cohort (ie, everolimus cohort): 5%; (b) expected HCC recurrence rates in the historical cohort (ie, without everolimus): 20%; (c) statistical power: 80%; (d) alpha error: 5%; (e) ratio cases/controls: 1/2. After applying Yates correction, the sample size required under these premises was 186 patients (including 62 patients in the prospective cohort and 124 matched historical controls).
A total of 192 patients were included, comprising 64 prospectively enrolled patients who received early initiated everolimus and 128 matched historical controls without everolimus. One hundred forty-seven (76.6%) patients were recruited at the Reina Sofía University Hospital (ie, 49 cases and 98 controls), whereas the remaining 45 (23.4%) patients were enrolled at the Hospital Virgen del Rocío (ie, 15 cases and 30 controls). In the overall cohort, mean recipient age was 55.9 ± 7.2 years, and there was a male predominance (n = 170; 88.5%). The main etiologies of liver disease before LT were chronic hepatitis C (57.8%), alcoholic cirrhosis (49.5%), and chronic hepatitis B (14.1%). The uncorrected MELD and Child-Pugh scores at transplantation were 12.6 ± 4.6 points and 7.3 ± 2 points, respectively. Clinical features of portal hypertension, including derived liver decompensations or compatible endoscopic/ultrasonographic findings, were present in 168 (87.5%) patients. In the pretransplant radiological assessment, the number of nodules was 1.46 ± 0.7 (29.6% of patients had multinodular disease), and the diameter of the largest nodule was 30.7 ± 12 mm. Median pretransplant alpha-fetoprotein was 6.86 ng/dL (IQR, 3.1-31). Bridging locoregional therapies of HCC were used in 112 (58.3%) patients, including transarterial chemoembolization in 68 (60.8%) patients, radiofrequency ablation in 26 (23.2%) patients, combination of both in 13 (11.6%) patients, and surgical resection in 5 (4.4%) patients. Demographics, clinical characteristics, and HCC histological features of prospectively enrolled patients receiving everolimus and matched historical controls (without everolimus) are shown in Table 1. Patients with and without everolimus were balanced in terms of age, sex, presence of portal hypertension, and liver function according to MELD and Child-Pugh scores. There was a predominance of chronic hepatitis C in patients receiving everolimus (68.8% vs 52.3%; P = 0.03), whereas chronic hepatitis B was more frequent among historical controls (18.8% vs 4.7%; P = 0.008). Serum alpha-fetoprotein was similar between groups (P = 0.67). In the pretransplant radiological assessment, prospectively enrolled patients receiving everolimus had an increased proportion of multinodular disease (49.1% vs 27.6%; P = 0.006). However, the diameter of the main nodule was greater in historical controls (33.4 ± 12.4 mm vs 25.7 ± 9.3 mm; P < 0.001). Regarding HCC histological features, propensity score matching allowed for an adequate balance in terms of number of nodules (P = 0.37), diameter of the main nodule (P = 0.68), total tumor diameter (P = 0.44), tumor differentiation (P = 0.61), and Milan criteria fulfillment (P = 0.56). However, there were increased mVI rates in patients receiving everolimus (26.5%) as compared with historical controls (13.3%) (P = 0.026).
Impact of Early Everolimus on HCC Recurrence and Overall Survival
Hepatocellular carcinoma recurrence occurred in 8 patients within the group of early everolimus (12.5%), and in 16 historical controls (12.5%). Median follow-up after LT was shorter in the early everolimus group (34 months vs 60.7 months). Hepatocellular carcinoma recurrence was extrahepatic in 50% of recurrent patients with everolimus as compared with 66.8% of recurrent historical controls (P = 0.41). Actuarial HCC recurrence rates were not significantly different between groups (Figure 1): 9.3% and 10.9% at 24 and 36 months, respectively, in patients receiving everolimus, as compared with 6.1% and 9.9% at 24 and 36 months, respectively, in historical controls (P = 0.18). These results remained statistically not significant irrespective of the mVI status (Figure S2, SDC,http://links.lww.com/TP/B575). In the subgroup of patients who trespassed Milan criteria in the explanted liver (n = 50), the addition of everolimus did not impact on tumor recurrence rates (P = 0.79). Subgroups of patients with at least 1 histological risk factor (trespassing Milan criteria and/or with mVI and/or poor tumor differentiation), and without any of these risk factors were also analyzed, but no statistical significance was found (P = 0.76 and P = 0.90 respectively). Hepatocellular carcinoma recurrence-free survival rates were not significantly different between groups (Figure 2A): 80.9% and 76.4% at 24 and 36 months, respectively, in patients receiving everolimus, as compared with 78.1% and 71.1% at 24 and 36 months, respectively, in historical controls (P = 0.93). Again, subgroup analyses showed no significant influence of everolimus on recurrence-free survival depending on mVI status (Figure S2, SDC,http://links.lww.com/TP/B575). Overall survival rates were not influenced by the use of early everolimus (Figure 2B): 83.9% and 78.7% at 24 and 36 months, respectively, in patients receiving everolimus, as compared with 78.9% and 72.7% at 24 and 36 months, respectively, in historical controls (P = 0.66). The competing risks analysis for HCC recurrence and death confirmed the negligible impact of everolimus (relative risk [RR], 1.02; 95% confidence interval [CI], 0.57-1.84]; P = 0.94).
The univariate and multivariate analyses evaluating potential predictors of HCC recurrence are shown in Table 2. Patients with tumor recurrence were characterized by increased number of HCC nodules and total tumor diameter, both in the pretransplant radiological assessment and in the histological examination of the explanted liver. Indeed, the number of nodules detected in the pretransplant radiological examination was 1.81 ± 0.81 in patients who experienced tumor recurrence, as compared with 1.41 ± 0.67 in patients without tumor recurrence (P = 0.015). Total tumor diameter in patients with and without tumor recurrence was 46.1 ± 23 mm and 36.2 ± 15 mm, respectively (P = 0.01). In the explanted liver, patients with HCC recurrence had increased total tumor diameter (49.2 ± 31.3 mm vs 37.9 ± 21.7 mm; P = 0.029), more frequent mVI (45.8% vs 13.9%; P < 0.001), and a trend toward increased number of HCC nodules (1.95 ± 1.11 vs 1.59 ± 0.77; P = 0.056). In the multivariate Cox's regression analysis, the presence of mVI (RR, 3.37; 95% CI, 1.18-9.62; P = 0.023) and the number of nodules in the explanted liver (RR, 1.71; 95% CI, 1.03-2.84; P = 0.036) were independent predictors of HCC recurrence, whereas the use of everolimus was not (RR, 3.23; 95% CI, 0.83-12.5; P = 0.09). Although not statistically significant, mean tacrolimus trough concentrations within the first month were kept in the final model to be controlled as a confounding factor.
Drug Exposure, Renal Function, and Adverse Events
Trough concentrations of everolimus at day 30 post-LT were 2.2 ± 1.2 ng/mL, but they were raised and kept within the target range, thereafter: 3.31 ± 1.45 ng/mL at 3 months, 3.72 ± 1.68 ng/mL at 6 months, 4.28 ± 2.43 ng/mL at 12 months, 4.44 ± 1.41 ng/mL at 18 months, and 3.69 ± 1.76 ng/mL at 24 months. Fifteen (23.4%) patients had significant adverse events which motivated greater than 50% dose reduction of everolimus or complete drug withdrawal during follow-up. It is noteworthy that only one of these patients experienced HCC recurrence. In other words, all patients showing tumor recurrence but one was on active therapy with everolimus. Adverse events leading to greater than 50% dose reduction or discontinuation of everolimus were the following: leucopenia (n = 4), dyslipidemia (n = 3), peripheral edema (n = 2), stomatitis (n = 2), fatigue (n = 2), proteinuria (n = 1), and diarrhea (n = 1). All these events were resolved or improved significantly and did not require hospital admission.
Mean trough concentrations of tacrolimus within the first month after LT were lower in combination with everolimus (6.67 ± 2.1 ng/mL), as compared with combinations with mycophenolate (9.23 ± 3.3 ng/mL; P < 0.001) and tacrolimus in monotherapy (9.49 ± 2.9 ng/mL; P < 0.001). During follow-up, trough concentrations of tacrolimus remained significantly reduced in patients receiving everolimus (Figure 3). At 24 months post-LT, differences in tacrolimus trough concentrations were less pronounced and became not significant between combinations with everolimus and mycophenolate (4.51 ± 1.9 ng/mL vs 5.52 ± 2.5 ng/mL, respectively; P = 0.12). Despite a similar pretransplant renal function, patients with everolimus had a significantly lower serum creatinine during follow-up as compared with patients receiving tacrolimus monotherapy or tacrolimus with mycophenolate (Figure 4). There were no differences in the proportion of patients with histologically proven acute cellular rejection, which was 7.8% in everolimus treated patients, 12.9% in patients who received mycophenolate, and 9.6% in patients with tacrolimus monotherapy (P = 0.62).
The antiproliferative potential of mTOR inhibitors, which has been demonstrated in preclinical studies,14,23,24 makes them an attractive therapeutic option for LT patients with HCC. Indeed, an mTOR-based immunosuppression protocol would allow for a minimization of tacrolimus, which is prooncogenic in a dose-dependent manner,14 whereas hampering the proliferation of remnant HCC cells after surgery. Several retrospective case series of HCC transplanted patients who received sirolimus, further meta-analyzed, provided encouraging results with a reduction of HCC rates from 13.8% for CNI-based immunosuppression, to 8% for sirolimus-based immunosuppression (P < 0.001).9 However, the only randomized trial published hitherto, which was sufficiently powered and had a prolonged surveillance, did not confirm a clear benefit.10 This uncertainty has led some authors to advise the use of mTOR inhibitors in HCC transplanted patients, particularly when histological features of increased aggressiveness are found,16 whereas other authors argue against this practice.25 Further prospective studies are needed, particularly evaluating everolimus with which no randomized trial aiming at HCC recurrence has been published or even designed hitherto. Here, we present the first prospective study with a propensity score-matched historical cohort evaluating the role of early introduced everolimus on HCC recurrence after LT. No significant benefit was demonstrated in terms of prevention of tumor recurrence by implementing this strategy, and therefore, while awaiting randomized trials, a universal prescription of everolimus in HCC transplanted patients may not be justified.
The histological findings of HCC in the explanted liver, including number of nodules, diameter of the main nodule, tumor differentiation, and mVI are the main predictors of tumor recurrence after LT,26 and they had to be controlled as potential confounding factors given the lack of randomization. A double control mechanism was implemented including propensity score matching between prospective and historical cohorts according to the previously referred histological features, and multivariate Cox's regression. The matching strategy resulted in an adequate balance in terms of tumor burden and histological differentiation. However, patients receiving everolimus had unexpectedly high mVI rates which could not be counteracted in the historical cohort. The multivariate Cox's regression was able to control for mVI, which behaved as an independent predictor of HCC recurrence, without producing a dramatic change in the β coefficient of everolimus. The use of early everolimus remained statistically not significant in the univariate, multivariate, and competing risks analyses. Thus, we can conclude that the results of the present study were unlikely influenced by the baseline HCC aggressiveness.
Although we failed to prove an advantage of early everolimus in subgroups with a priori increased risk of recurrence such as patients above Milan criteria or showing mVI in the explanted liver, the present study was not sufficiently powered to extract solid conclusions from such small subgroups. Future randomized studies should be focused preferably on subpopulations at high risk of tumor recurrence and will have to face significant caveats including increased sample size, multicenter involvement, and prolonged follow-up. The assessment of the mTOR pathway activation in histological samples from the explanted liver would be a more rational approach to select candidates to receive everolimus after LT, and this hypothesis deserves further investigation.
The use of mTOR inhibitors is associated with adverse events in some LT patients. A recent network meta-analysis of randomized trials found that sirolimus-containing regimes are associated with shorter survival.11 Everolimus has an improved safety profile as compared with sirolimus, but still the risk of side effects is not negligible. In the H2024 study, which is the largest randomized trial performed in LT, the incidence of adverse events leading to study drug discontinuation was 25.7% in patients receiving everolimus.27 Similar findings were obtained in the present study, where 23.4% of patients receiving everolimus required greater than 50% dose reduction or complete withdrawal of everolimus due to adverse events. For outcome analysis, patients with complete withdrawal of everolimus during follow-up were analyzed in their initial group to preserve the intention-to-treat principle. However, we believe that withdrawal of everolimus had little impact on outcome analysis because nearly all patients who experienced HCC recurrence were on active therapy with everolimus (7 of 8). None of the adverse events associated with everolimus were linked to an earlier introduction of the drug (ie, before postoperative day 30), thus confirming previous observations.18,19 Trough concentrations of everolimus were within the recommended therapeutic range for LT patients, but close to the lower threshold during follow-up. Whether an increased exposure to everolimus would result into a more effective prevention of tumor recurrence remains to be determined. If future randomized studies are designed to target the upper range of everolimus trough concentrations (ie, 8 ng/mL), a close surveillance to detect and manage potential adverse events would be warranted. Until other evidence proves otherwise, the prescription of everolimus after LT should be aimed preferably to preserve renal function where the expected benefits would overcome safety issues in most patients.27,28
Everolimus allowed for a significant reduction in tacrolimus trough concentrations during follow-up which resulted in a more effective preservation of renal function after LT. These findings have been consistently reported in previous randomized trials.27,28 Tacrolimus sparing was more pronounced with everolimus as compared with mycophenolate within the first 18 months after LT, which may be explained by the widely spread belief that everolimus is a more potent immunosuppressant. In previous observational studies, reduced tacrolimus trough concentrations had a protective effect on HCC recurrence,5 particularly when implemented early after LT.6 It is noteworthy that in these studies, HCC recurrence rates were significantly increased when mean tacrolimus trough concentrations exceeded 10 ng/mL. This is a high threshold which is not usually targeted nowadays and indeed neither everolimus-treated patients, nor historical controls have overtook such levels in the present study, thus explaining why the successful tacrolimus sparing obtained with everolimus was not translated into reduced HCC recurrence rates in the multivariate Cox regression analysis.
The main limitation of the present study is the lack of randomization. However, the implementation of propensity score matching and the use of multivariate Cox regression and competing risks analyses have minimized the risk of bias. The relatively reduced sample size may have weakened some conclusions. In addition, adverse events could not be systematically evaluated in the retrospective cohort and no solid conclusion could be drawn regarding drug safety. The study strengths are also to be noted including the involvement of 2 transplant institutions sharing a common transplant waiting list, a prospective recruitment of everolimus-treated patients and a protocolized surveillance after LT, which allowed recording high-quality data on patient outcomes.
In conclusion, an early introduction of everolimus after LT was able to reduce the exposure to tacrolimus and to preserve renal function, but it had no significant impact on HCC recurrence rates. We failed to identify a subpopulation potentially benefited by an early introduction of everolimus. In absence of biological markers related with increased efficacy of everolimus, we believe that future randomized trials may be focused in patients with histological features associated with increased risk of tumor recurrence such as mVI or exceeding Milan criteria.
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