Everolimus-Based Immunosuppression in Patients With Hepatocellular Carcinoma at High Risk of Recurrence After Liver Transplantation: A Case Series A.O. Ferreiroa, M.A. Vazquez-Millána,*, F.S. Lópeza, M.G. Gutiérreza, S.P. Diazb, and M.J.L. Patiñoc a
Liver Transplant Unit, bClinical Epidemiology and Biostatistics Unit, and cPathology Service, A Coruña Hospital, A Coruña, Spain
ABSTRACT Background. Liver transplantation offers the most effective treatment in patients with hepatocellular carcinoma (HCC). However, transplant patients outside the Milan criteria have a high risk of tumor recurrence, which has been linked to standard immunosuppression regimens. Everolimus is a mammalian target of rapamycin inhibitor that has been used for immunosuppression, but its effect on recurrence and survival in HCC patients with a high risk of tumor recurrence has not been examined. We compared long-term survival and cumulative recurrence in high-risk patients receiving everolimus-based immunosuppression after liver transplantation for HCC with an historic control group. Methods. The everolimus group comprised 21 patients receiving a liver transplant at our center from February 2005 to December 2010. The control group comprised 31 patients receiving a liver transplant from May 1994 to January 2005. All patients received cyclosporine or tacrolimus as initial post-transplant immunosuppression. Patients in the everolimus group switched to everolimus 2 weeks later. Results. There were no differences between the two groups in number of rejection episodes or of infectious or surgical complications. Five-year survival was 60.2% in the everolimus group and 32.3% in the control group (P ¼ .05). Five-year cumulative recurrence rate was 61.3% in the control group and 41.3% in the everolimus group. Treatment with everolimus was identified as an independent predictor of longer survival (hazard ratio ¼ 0.34; P ¼ .02). Conclusions. Patients receiving liver transplantation for HCC with a high risk of tumor recurrence may well benefit from everolimus-based immunosuppression, with no added risks of rejection or other post-transplant complications.
H
EPATOCELLULAR CARCINOMA (HCC) is the most common primary liver tumor and is now the third cause of cancer-related deaths [1,2]. Liver transplantation offers the best long-term effective treatment for patients with HCC [3], and patients who meet the Milan criteria (single tumor 5 cm; two or three tumors, none >3 cm; no gross vascular invasion) can attain excellent longterm survival and low rates of tumor recurrence (5-year survival >70%) [4,5]. However, despite the adoption of the restrictive Milan criteria, tumor recurrence occurs in 10% to 30% of patients after transplantation, and their prognosis is dismal. Moreover, 5-year survival falls to approximately 50% in patients with advanced HCC who receive liver transplants under extended criteria and who
can be considered to have a high risk of tumor recurrence after transplantation [4,6]. Although there is no clear consensus on how to prevent tumor recurrence in these patients, it is known that the pharmacologic immunosuppression required after transplantation can accelerate tumor growth. Vivarelli et al [7] reported that a high level of cyclosporine exposure was an independent marker of tumor recurrence. The mammalian target of rapamycin inhibitors, such as everolimus and sirolimus, can prevent angiogenesis by interfering with *Address correspondence to Maria Angeles Vazquez-Millán, MD, Xubias de Arriba n 84, CP 15006 A Coruña, Spain. E-mail: [email protected]
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.08.045
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3496
Transplantation Proceedings, 46, 3496e3501 (2014)
EVEROLIMUS-BASED IMMUNOSUPPRESSION
vascular endothelial growth factoremediated pathways in endothelial cells, limiting the growth of several tumors [8]. Immunosuppression with tacrolimus and sirolimus after orthotopic liver transplantation was shown to be safe, well tolerated, and effective [9]. Rapamycin inhibited the growth of tumor cells in vitro [10], but it is unclear whether it can confer any benefit in HCC patients after liver transplantation. Moreover, sirolimus has been shown both to inhibit the growth of human hepatoma cells in vitro [11] and also to reduce HCC recurrence and improve patient survival when used as immunosuppression after liver transplantation [12e16]. Recent meta-analyses have also shown a survival benefit in patients with pre-transplant HCC receiving sirolimus after liver transplantation [17,18]. However, the majority of these studies included only patients who met the Milan criteria, and little is known of the effect of sirolimus on HCC patients at high risk of recurrence after transplantation. Moreover, although several studies have evaluated the efficacy of everolimus-based immunosuppression [19,20], to the best of our knowledge, none has examined its effect specifically on tumor recurrence and survival in high-risk HCC patients. We report our center’s experience with everolimus-based immunosuppression after liver transplantation for end-stage liver disease (ESLD) and HCC in patients with a high risk of post-transplant recurrence. Long-term survival and tumor recurrence in these patients were compared with those in a historic control group treated with standard immunosuppression regimens.
METHODS Patient Populations and Study Design The study compared two groups of patients receiving liver transplants for ESLD and HCC at A Coruña Hospital (A Coruña, Spain) who were deemed to have a high risk of post-transplant recurrence according to histological features. Patients were considered highrisk if they exceeded the Milan criteria or if they had macrovascular or microvascular invasion or poorly differentiated tumors in the explanted liver. The everolimus group comprised 21 patients selected from a total of 72 patients receiving liver transplantation for ESLD and hepatocellular carcinoma from February 2005 to December 2010. The control group comprised 31 patients, selected from a historic control group of 120 patients with HCC receiving liver transplantation from May 1994 to January 2005. The study was approved by the Ethics Committee of A Coruña Hospital and was carried out in accordance with the Helsinki Declaration of 1975. All patients gave their signed informed consent. The patients in the everolimus group initially received calcineurin inhibitors (CNI), either cyclosporine (Neoral, Novartis, Barcelona, Spain) 5 mg/kg orally twice daily (target C2 levels of 800e1200 ng/L) or tacrolimus (Prograf, Astellas, Madrid, Spain) 0.05 mg/kg orally twice daily (target trough levels of 5e10 ng/L). They also received steroids (500 mg intraoperative methylprednisolone, tapering to 20 mg/day at day 5 and to suppression at month 3) and mycophenolate mofetil (MMF) (Cellcept, Roche, Madrid, Spain) 1gr orally twice daily. Results from the post-transplant pathology review of the explanted liver were available approximately 2 weeks after transplant, at which time MMF was replaced by
3497 everolimus (Certican, Novartis, Barcelona, Spain) 0.5 to 1.5 mg twice daily (target trough levels of 3e8 ng/mL), and CNI doses were progressively reduced. The patients in the control group received CNI, either cyclosporine (Neoral, Novartis) 5 mg/kg orally twice daily (target C2 levels of 800e1200 ng/L) or tacrolimus (Prograf, Astellas) 0.05 mg/ kg orally twice daily (target trough levels of 5e10 ng/L). They also received steroids (500 mg intraoperative methylprednisolone, tapering to 20 mg/day at day 5 and to suppression at month 3). From May 1994 to June 2000, these patients also received azathioprine 100 mg daily; after June 2000, patients received MMF (CellCept, Roche) 1gr orally twice daily. Data on demographic factors and tumor characteristics (size, number of nodules, histological grade, macrovascular or microvascular invasion, and pre-transplant alpha-fetoprotein [AFP] levels) were recorded for all patients. Follow-up included an AFP blood test every 3 months and abdominal ultrasound or CT scanning every 6 months during the first 5 years after transplantation.
Statistical Analyses The sample size was calculated to detect a significant hazard ratio (HR) 0.36 associated with everolimus immunosuppression in a Table 1. Baseline Patient Characteristics for the Everolimus and Control Groups
Age, years Mean SD Median Sex Male Female Disease etiology Alcohol Hepatitis C virus Hepatitis B virus Other Number of nodules 3 >3 Differentiation Well/moderately differentiated Poorly differentiated Vascular invasion Yes No Alpha-fetoprotein levels Mean SD Median Tumour size Mean SD Median Within Milan criteria† Within UCSF criteria‡
Everolimus Group (n ¼ 21) n (%)
Control Group (n ¼ 31) P (%)
56.8 7.0 56.0
58.1 6.5 59.0
20 (95.2) 1 (4.8)
28 (90.3) 3 (9.7)
8 10 2 1
11 17 1 2
P
.47
.46
NC* (38.1) (47.6) (9.5) (4.8)
(35.5) (54.8) (3.2) (6.5) .45
10 (47.6) 11 (52.4)
13 (41.9) 18 (58.1)
10 (47.6) 11 (52.4)
22 (71.0) 9 (29.0)
17 (80.9) 4 (19.1)
23 (74.2) 8 (25.8)
60.8 113 10.0
186 619 20.5
3.8 2.4 3.0 1 (4.8) 2 (9.5)
3.58 1.8 3.0 9 (29.0) 11 (35.5)
.08
.41
.31
.96
.03 .03
Abbreviations: SD, standard deviation; UCSF, University of California at San Francisco. *NC: Not calculated because of the small sample size in subgroups. † Milan criteria: 1 lesion 5 cm or up to 3 lesions 3 cm. ‡ UCSF criteria: 1 nodule 6.5 cm or 2 to 3 nodules 4.5 cm and total tumor diameter 8 cm.
3498 Cox regression model (censored data percentage, 40%; security, 95%; power, 80%). Overall survival probability was estimated by use of the Kaplan-Meier method, and the Cox proportional hazards regression model was used to identify the HRs for risk of death with their 95% confidence intervals (CIs). The cumulative recurrence rate was calculated by treating deaths as competing risks, with the use of Kalbfleisch and Prentice’s [21] cumulative incidence technique, in which the events of cancer recurrence and of death, whichever occurs first, are considered as competing events, censoring for end of follow-up. Failure to take competing risks into account could result in biased estimates of the risk of recurrence. Cumulative recurrence in patient subgroups classified according to clinical and pathological factors was compared with the use of Gray’s K-sample test [22]. A multivariate analysis with the use of Fine and Gray’s [23] survival model was performed to identify the HRs, with their 95% CIs for risk of recurrence. All statistical tests were 2-sided, and significance was set at P < .05. All analyses were performed with the use of IBM SPSS Statistics for Windows version 19.0 and R 2.12.2 (R Foundation for Statistical Computing, Vienna, Austria) with the cmprsk package.
RESULTS Patient Characteristics
Twenty-one patients at high risk of HCC recurrence were included in the everolimus group, and 31 patients at high risk of recurrence were included in the historical control group. Table 1 displays the characteristics of the two groups of patients. The histological analyses of the explanted livers showed that one patient in the everolimus group (4.8%) and nine (29%) in the control group met the Milan criteria (P ¼ .03), and two patients (9.5%) in the everolimus group and 11 in the control group met the University of California at San Francisco criteria (P ¼ .03). No other significant differences were observed between the two groups.
FERREIRO, VAZQUEZ-MILLÁN, LÓPEZ ET AL
Survival and HCC Recurrence
Mean follow-up was 41 (3.9e94) months in the everolimus group and 64 (12.7e212) months in the control group. Figure 1 shows overall survival for the two groups. Two-year survival in the everolimus group was 85.7%, compared with 64.5% in the control group. Five-year survival was 60.2% and 32.3%, respectively (P ¼ .05). A trend toward longer survival was observed in patients with well or moderately differentiated tumors (55% at 5 years) compared with those with poorly differentiated tumors (20.3% at 5 years) (P ¼ .07). Two- and five-year survival rates were higher for patients with 3 nodules and for those without vascular invasion, but differences did not reach statistical significance (Table 2). The 2- and 5-year cumulative recurrence rates were 23.8% and 41.3% in the everolimus group, compared with 45.2% and 61.3%, respectively, in the control group (P ¼ .17) (Fig 2). A lower recurrence rate was observed in patients with 3 nodules, in those with well or moderately differentiated HCC, and in those without vascular invasion, but differences were not significant (Table 2). Thirty-two patients had well or moderately differentiated HCC (10 in the everolimus group and 22 in the control group). In this subgroup of patients, 2-year survival rates were 100% and 68.2%, respectively, and 5-year survival rates were 88.9% and 40.9%, respectively (P ¼ .02), but no significant differences were observed in cumulative recurrence rates between the two groups (P ¼ .22) (Table 3, Fig 3). Among the 20 patients with poorly differentiated HCC (11 in the everolimus group and nine in the control group), there were no significant differences in survival (P ¼ .32) or cumulative recurrence rates (P ¼ .25) (Table 3, Fig 3). Twenty-three patients had 3 nodules (10 in the everolimus group and 13 in the control group). No differences in survival (P ¼ .17) or cumulative recurrence rates (P ¼ .57) were observed between the two groups. Among the 29 patients with >3 nodules (11 in the everolimus group and 18 Table 2. Overall Survival and Cumulative Recurrence in Patients With a High Risk of Recurrence After Liver Transplantation for Hepatocellular Carcinoma Overall Survival (%) 1-Year 2-Year 5-Year
Fig 1. Overall survival by immunosuppression regimen in patients with a high risk of recurrence after liver transplantation for hepatocellular carcinoma.
Immunosuppression Everolimus Control Number of nodules 3 nodules >3 nodules Tumor differentiation Well/moderately differentiated Poorly differentiated Vascular invasion No Yes
Cumulative Recurrence (%) P
1-Year 2-Year 5-Year
.05 95.2 100
85.7 64.5
60.2 32.3
100 96.6
78.3 69.0
57.9 31.2
100
78.1
95.0
100 97.5
P .17
9.5 22.6
23.8 45.2
41.3 61.3
17.4 17.2
30.4 41.4
40.5 61.0
55.0
12.5
31.2
49.3
65.0
20.3
25.0
45.0
67.9
83.3 70.0
45.7 41.4
8.3 20.0
16.7 42.5
36.4 58.4
.15
.18
.07
.21
.51
.13
EVEROLIMUS-BASED IMMUNOSUPPRESSION
3499
0.34; 95% CI, 0.14e0.83; P ¼ .02), whereas poorly differentiated tumors (HR ¼ 2.77; 95% CI, 1.24e6.15; P ¼ .01) and >3 nodules (HR ¼ 2.37; 95% CI, 1.07e5.26; P ¼ .03) were markers of shorter survival. No factor was identified as an independent marker of risk of recurrence (Table 4). Rejection and Everolimus-Associated Side Effects
Three patients (14.3%) had an episode of acute rejection in the everolimus group, all of whom received cyclosporine as a concomitant immunosuppressor, compared with seven patients (22.6%) in the control group (P ¼ .35). Side effects associated with everolimus treatment were anaemia (19%), oral ulcers (9.5%) and wound dehiscence (4.8%). DISCUSSION
Fig 2. Cumulative recurrence by immunosuppression regimen in patients with a high risk of recurrence after liver transplantation for hepatocellular carcinoma.
in the control group), there were also no significant differences in survival (P ¼ .16) or cumulative recurrence rates (P ¼ .12) (Table 3). Vascular invasion was present in 40 patients (17 in the everolimus group and 23 in the control group). Among these patients, 5-year survival was 58.8% in the everolimus group, compared with 30.4% in the control group (P ¼ .31), and the 5-year cumulative recurrence rates were 50.7% and 65.2%, respectively (P ¼ .33). Among the 12 patients without vascular invasion (four in the everolimus group and eight in the control group), 2-year survival rates were 100% for the everolimus group and 75% for the control group, and the 5-year survival rates were 66.7% and 37.5%, respectively (P ¼ .09). Cumulative recurrence rates were 0% and 25% at 2 years and 0% and 50% at 5 years, respectively (P < .01) (Table 3). In the multivariate analysis, everolimus treatment surfaced as an independent marker of longer survival (HR ¼
Liver transplantation is the only curative treatment for patients with ESLD and HCC, with the most benefit attained in patients meeting the Milan criteria [4,6]. Several prognostic factors, including vascular invasion and poor tumor differentiation, can predict HCC recurrence after liver transplantation [24], and CNIs have also been linked to HCC recurrence [7,25]. Despite an increasing interest in the development of drugs to improve post-transplant outcome, there is as yet no consensus on the pharmacological management of HCC patients after liver transplantation. Several studies have reported lower recurrence rates and longer survival with sirolimus [14e17,26]; however, to the best of our knowledge, the present study is the first to examine the effect of everolimus-based immunosuppression on recurrence and survival of HCC patients with a high risk of recurrence. We have observed a strong but non-significant trend toward longer survival and lower recurrence in patients treated with everolimus. Treatment with everolimus was also identified as an independent predictor of longer posttransplant survival (HR ¼ 0.34; P ¼ .02). Moreover, 52.4% of the patients receiving everolimus had poorly differentiated tumors and 81% had vascular invasion, which was found to double the risk of death in a retrospective analysis of more than 1500 patients who received liver
Table 3. Overall Survival and Cumulative Recurrence in the Subgroups of Patients Classified According to Number of Nodules, Tumor Differentiation, and Presence of Vascular Invasion Overall Survival (%) 2-Year Everolimus
Number of nodules 3 nodules >3 nodules Tumor differentiation Well/moderately differentiated Poorly differentiated Vascular invasion No Yes
Cumulative Recurrence (%)
5-Year
2-Year
5-Year
Control
Everolimus
Control
P
Everolimus
Control
Everolimus
Control
P
90.0 81.8
69.2 61.1
77.1 46.8
46.2 22.2
.17 .16
20.0 27.3
38.5 50.0
20.0 50.6
53.8 66.7
.57 .12
100 72.7
68.2 55.6
88.9 31.2
40.9 11.1
.02 .32
10.0 36.4
40.9 55.6
40.9 36.4
54.5 77.8
.22 .25
100 82.4
75.0 60.9
66.7 58.8
37.5 30.4
.09 .31
0 29.4
25.0 52.2
0 50.7
50 65.2
3 Everolimus-based immunosuppression
Recurrence
HR
95% CI
P
HR
95% CI
P
0.94 2.77 1.45 2.37 0.34
0.94e1.05 1.24e6.15 0.55e3.83 1.07e5.26 0.14e0.83
.83 .01 .45 .03 .02
1.01 1.87 2.49 2.25 0.49
0.94e1.08 0.80e4.35 0.83e7.46 0.95e5.32 0.21e1.12
.80 .15 .10 .06 .09
Abbreviations: HR, hazard ratio; CI, confidence interval.
these studies included only patients within the Milan criteria. In contrast, the present study included only patients at high risk of HCC recurrence, and the 2-year (87.7%) and 5-year (60.2%) survival rates attained with everolimusbased immunosuppression are promising and compare favourably with the 53.6% 5-year survival rate reported for patients outside the Milan criteria [6]. The beneficial effect of everolimus observed in our patients could be a direct result of the anti-proliferative properties of the drug itself or, as suggested by Vivarelli [7], an indirect result of reduced exposure to CNIs. Further studies could help to clarify this point. Our report has several limitations, including the relatively small number of patients and the non-randomized design. In addition, the comparison with a historical control group could be the source of bias, because the better survival rates attained with everolimus may be partly the result of general improvements in post-transplant care in recent years. In conclusion, our findings indicate that patients receiving liver transplantation for ESLD and HCC who have a high risk of tumor recurrence may well benefit from everolimusbased immunosuppression, with no added risks of rejection or other post-transplant complications. Prospective studies with a larger number of patients are warranted to further examine the role of everolimus in reducing post-transplant HCC recurrence. REFERENCES [1] Parkin DM, Bray F, Ferlay J, et al. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001;94:153e6. [2] El-Serag HB, Davila JA, Petersen NJ, Mcglynn KA. The continuing increase in the incidence of hepatocellular carcinoma
EVEROLIMUS-BASED IMMUNOSUPPRESSION in the United States: an update. Ann Intern Med 2003;139: 817e23. [3] Jelic S, Sotiropoulos GC. Hepatocellular carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010;21(Suppl 5):v59e64. [4] Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med 1996;334:693e9. [5] Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology 2011;53:1020e2. [6] Mazzaferro V, Llovet JM, Miceli R, et al. Predicting survival after liver transplantation in patients with hepatocellular carcinoma beyond the Milan criteria: a retrospective, exploratory analysis. Lancet Oncol 2009;10:35e43. [7] Vivarelli M, Cucchetti A, Piscaglia F, et al. Analysis of risk factors for tumor recurrence after liver transplantation for hepatocellular carcinoma: key role of immunosuppression. Liver Transpl 2005;11:497e503. [8] Guba M, Von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002;8: 128e35. [9] Mcalister VC, Peltekian KM, Malatjalian DA, et al. Orthotopic liver transplantation using low-dose tacrolimus and sirolimus. Liver Transpl 2001;7:701e8. [10] Casadio F, Croci S, D’errico Grigioni A, et al. Toward the definition of immunosuppressive regimens with antitumor activity. Transplant Proc 2005;37:2144e7. [11] Schumacher G, Oidtmann M, Rueggeberg A, et al. Sirolimus inhibits growth of human hepatoma cells alone or combined with tacrolimus, while tacrolimus promotes cell growth. World J Gastroenterol 2005;11:1420e5. [12] Trotter JF, Wachs M, Bak T, et al. Liver transplantation using sirolimus and minimal corticosteroids (3-day taper). Liver Transpl 2001;7:343e51. [13] Zhou J, Fan J, Wang Z, et al. Conversion to sirolimus immunosuppression in liver transplantation recipients with hepatocellular carcinoma: report of an initial experience. World J Gastroenterol 2006;12:3114e8. [14] Toso C, Meeberg GA, Bigam DL, et al. De novo sirolimus-based immunosuppression after liver transplantation
3501 for hepatocellular carcinoma: long-term outcomes and side effects. Transplantation 2007;83:1162e8. [15] Zimmerman MA, Trotter JF, Wachs M, et al. Sirolimusbased immunosuppression following liver transplantation for hepatocellular carcinoma. Liver Transpl 2008;14:633e8. [16] Chinnakotla S, Davis GL, Vasani S, et al. Impact of sirolimus on the recurrence of hepatocellular carcinoma after liver transplantation. Liver Transpl 2009;15:1834e42. [17] Liang W, Wang D, Ling X, et al. Sirolimus-based immunosuppression in liver transplantation for hepatocellular carcinoma: a meta-analysis. Liver Transpl 2012;18:62e9. [18] Menon KV, Hakeem AR, Heaton ND. Meta-analysis: recurrence and survival following the use of sirolimus in liver transplantation for hepatocellular carcinoma. Aliment Pharmacol Ther 2013;37:411e9. [19] Saliba F, De Simone P, Nevens F, et al. Renal function at two years in liver transplant patients receiving everolimus: results of a randomized, multicenter study. Am J Transplant 2013;13: 1734e45. [20] De Simone P, Nevens F, De Carlis L, et al. Everolimus with reduced tacrolimus improves renal function in de novo liver transplant recipients: a randomized controlled trial. Am J Transplant 2012;12:3008e20. [21] Kalbfleisch J, Prentic R. The Statistical Analysis of Failure Time Data. New York: John Wiley and Sons; 1980. [22] Gray R. A class of K-sample tests for comparing the cumulative incidence of a competing risk. Ann Stat 1988;16:1141e54. [23] Fine J, Gray R. A proportional hazards model for the subdistribution of a competing risk. JASA 1999;94:496e509. [24] Cescon M, Ravaioli M, Grazi GL, et al. Prognostic factors for tumor recurrence after a 12-year, single-center experience of liver transplantations in patients with hepatocellular carcinoma. J Transplant 2010;2010. doi: 10.1155/2010/904152. Epub 2010 Aug 25. [25] Vivarelli M, Dazzi A, Zanello M, et al. Effect of different immunosuppressive schedules on recurrence-free survival after liver transplantation for hepatocellular carcinoma. Transplantation 2010;89:227e31. [26] Gomez-Martin C, Bustamante J, Castroagudin JF, et al. Efficacy and safety of sorafenib in combination with mammalian target of rapamycin inhibitors for recurrent hepatocellular carcinoma after liver transplantation. Liver Transpl 2012;18:45e52.
Liver Transplant Unit, bClinical Epidemiology and Biostatistics Unit, and cPathology Service, A Coruña Hospital, A Coruña, Spain
ABSTRACT Background. Liver transplantation offers the most effective treatment in patients with hepatocellular carcinoma (HCC). However, transplant patients outside the Milan criteria have a high risk of tumor recurrence, which has been linked to standard immunosuppression regimens. Everolimus is a mammalian target of rapamycin inhibitor that has been used for immunosuppression, but its effect on recurrence and survival in HCC patients with a high risk of tumor recurrence has not been examined. We compared long-term survival and cumulative recurrence in high-risk patients receiving everolimus-based immunosuppression after liver transplantation for HCC with an historic control group. Methods. The everolimus group comprised 21 patients receiving a liver transplant at our center from February 2005 to December 2010. The control group comprised 31 patients receiving a liver transplant from May 1994 to January 2005. All patients received cyclosporine or tacrolimus as initial post-transplant immunosuppression. Patients in the everolimus group switched to everolimus 2 weeks later. Results. There were no differences between the two groups in number of rejection episodes or of infectious or surgical complications. Five-year survival was 60.2% in the everolimus group and 32.3% in the control group (P ¼ .05). Five-year cumulative recurrence rate was 61.3% in the control group and 41.3% in the everolimus group. Treatment with everolimus was identified as an independent predictor of longer survival (hazard ratio ¼ 0.34; P ¼ .02). Conclusions. Patients receiving liver transplantation for HCC with a high risk of tumor recurrence may well benefit from everolimus-based immunosuppression, with no added risks of rejection or other post-transplant complications.
H
EPATOCELLULAR CARCINOMA (HCC) is the most common primary liver tumor and is now the third cause of cancer-related deaths [1,2]. Liver transplantation offers the best long-term effective treatment for patients with HCC [3], and patients who meet the Milan criteria (single tumor 5 cm; two or three tumors, none >3 cm; no gross vascular invasion) can attain excellent longterm survival and low rates of tumor recurrence (5-year survival >70%) [4,5]. However, despite the adoption of the restrictive Milan criteria, tumor recurrence occurs in 10% to 30% of patients after transplantation, and their prognosis is dismal. Moreover, 5-year survival falls to approximately 50% in patients with advanced HCC who receive liver transplants under extended criteria and who
can be considered to have a high risk of tumor recurrence after transplantation [4,6]. Although there is no clear consensus on how to prevent tumor recurrence in these patients, it is known that the pharmacologic immunosuppression required after transplantation can accelerate tumor growth. Vivarelli et al [7] reported that a high level of cyclosporine exposure was an independent marker of tumor recurrence. The mammalian target of rapamycin inhibitors, such as everolimus and sirolimus, can prevent angiogenesis by interfering with *Address correspondence to Maria Angeles Vazquez-Millán, MD, Xubias de Arriba n 84, CP 15006 A Coruña, Spain. E-mail: [email protected]
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.08.045
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3496
Transplantation Proceedings, 46, 3496e3501 (2014)
EVEROLIMUS-BASED IMMUNOSUPPRESSION
vascular endothelial growth factoremediated pathways in endothelial cells, limiting the growth of several tumors [8]. Immunosuppression with tacrolimus and sirolimus after orthotopic liver transplantation was shown to be safe, well tolerated, and effective [9]. Rapamycin inhibited the growth of tumor cells in vitro [10], but it is unclear whether it can confer any benefit in HCC patients after liver transplantation. Moreover, sirolimus has been shown both to inhibit the growth of human hepatoma cells in vitro [11] and also to reduce HCC recurrence and improve patient survival when used as immunosuppression after liver transplantation [12e16]. Recent meta-analyses have also shown a survival benefit in patients with pre-transplant HCC receiving sirolimus after liver transplantation [17,18]. However, the majority of these studies included only patients who met the Milan criteria, and little is known of the effect of sirolimus on HCC patients at high risk of recurrence after transplantation. Moreover, although several studies have evaluated the efficacy of everolimus-based immunosuppression [19,20], to the best of our knowledge, none has examined its effect specifically on tumor recurrence and survival in high-risk HCC patients. We report our center’s experience with everolimus-based immunosuppression after liver transplantation for end-stage liver disease (ESLD) and HCC in patients with a high risk of post-transplant recurrence. Long-term survival and tumor recurrence in these patients were compared with those in a historic control group treated with standard immunosuppression regimens.
METHODS Patient Populations and Study Design The study compared two groups of patients receiving liver transplants for ESLD and HCC at A Coruña Hospital (A Coruña, Spain) who were deemed to have a high risk of post-transplant recurrence according to histological features. Patients were considered highrisk if they exceeded the Milan criteria or if they had macrovascular or microvascular invasion or poorly differentiated tumors in the explanted liver. The everolimus group comprised 21 patients selected from a total of 72 patients receiving liver transplantation for ESLD and hepatocellular carcinoma from February 2005 to December 2010. The control group comprised 31 patients, selected from a historic control group of 120 patients with HCC receiving liver transplantation from May 1994 to January 2005. The study was approved by the Ethics Committee of A Coruña Hospital and was carried out in accordance with the Helsinki Declaration of 1975. All patients gave their signed informed consent. The patients in the everolimus group initially received calcineurin inhibitors (CNI), either cyclosporine (Neoral, Novartis, Barcelona, Spain) 5 mg/kg orally twice daily (target C2 levels of 800e1200 ng/L) or tacrolimus (Prograf, Astellas, Madrid, Spain) 0.05 mg/kg orally twice daily (target trough levels of 5e10 ng/L). They also received steroids (500 mg intraoperative methylprednisolone, tapering to 20 mg/day at day 5 and to suppression at month 3) and mycophenolate mofetil (MMF) (Cellcept, Roche, Madrid, Spain) 1gr orally twice daily. Results from the post-transplant pathology review of the explanted liver were available approximately 2 weeks after transplant, at which time MMF was replaced by
3497 everolimus (Certican, Novartis, Barcelona, Spain) 0.5 to 1.5 mg twice daily (target trough levels of 3e8 ng/mL), and CNI doses were progressively reduced. The patients in the control group received CNI, either cyclosporine (Neoral, Novartis) 5 mg/kg orally twice daily (target C2 levels of 800e1200 ng/L) or tacrolimus (Prograf, Astellas) 0.05 mg/ kg orally twice daily (target trough levels of 5e10 ng/L). They also received steroids (500 mg intraoperative methylprednisolone, tapering to 20 mg/day at day 5 and to suppression at month 3). From May 1994 to June 2000, these patients also received azathioprine 100 mg daily; after June 2000, patients received MMF (CellCept, Roche) 1gr orally twice daily. Data on demographic factors and tumor characteristics (size, number of nodules, histological grade, macrovascular or microvascular invasion, and pre-transplant alpha-fetoprotein [AFP] levels) were recorded for all patients. Follow-up included an AFP blood test every 3 months and abdominal ultrasound or CT scanning every 6 months during the first 5 years after transplantation.
Statistical Analyses The sample size was calculated to detect a significant hazard ratio (HR) 0.36 associated with everolimus immunosuppression in a Table 1. Baseline Patient Characteristics for the Everolimus and Control Groups
Age, years Mean SD Median Sex Male Female Disease etiology Alcohol Hepatitis C virus Hepatitis B virus Other Number of nodules 3 >3 Differentiation Well/moderately differentiated Poorly differentiated Vascular invasion Yes No Alpha-fetoprotein levels Mean SD Median Tumour size Mean SD Median Within Milan criteria† Within UCSF criteria‡
Everolimus Group (n ¼ 21) n (%)
Control Group (n ¼ 31) P (%)
56.8 7.0 56.0
58.1 6.5 59.0
20 (95.2) 1 (4.8)
28 (90.3) 3 (9.7)
8 10 2 1
11 17 1 2
P
.47
.46
NC* (38.1) (47.6) (9.5) (4.8)
(35.5) (54.8) (3.2) (6.5) .45
10 (47.6) 11 (52.4)
13 (41.9) 18 (58.1)
10 (47.6) 11 (52.4)
22 (71.0) 9 (29.0)
17 (80.9) 4 (19.1)
23 (74.2) 8 (25.8)
60.8 113 10.0
186 619 20.5
3.8 2.4 3.0 1 (4.8) 2 (9.5)
3.58 1.8 3.0 9 (29.0) 11 (35.5)
.08
.41
.31
.96
.03 .03
Abbreviations: SD, standard deviation; UCSF, University of California at San Francisco. *NC: Not calculated because of the small sample size in subgroups. † Milan criteria: 1 lesion 5 cm or up to 3 lesions 3 cm. ‡ UCSF criteria: 1 nodule 6.5 cm or 2 to 3 nodules 4.5 cm and total tumor diameter 8 cm.
3498 Cox regression model (censored data percentage, 40%; security, 95%; power, 80%). Overall survival probability was estimated by use of the Kaplan-Meier method, and the Cox proportional hazards regression model was used to identify the HRs for risk of death with their 95% confidence intervals (CIs). The cumulative recurrence rate was calculated by treating deaths as competing risks, with the use of Kalbfleisch and Prentice’s [21] cumulative incidence technique, in which the events of cancer recurrence and of death, whichever occurs first, are considered as competing events, censoring for end of follow-up. Failure to take competing risks into account could result in biased estimates of the risk of recurrence. Cumulative recurrence in patient subgroups classified according to clinical and pathological factors was compared with the use of Gray’s K-sample test [22]. A multivariate analysis with the use of Fine and Gray’s [23] survival model was performed to identify the HRs, with their 95% CIs for risk of recurrence. All statistical tests were 2-sided, and significance was set at P < .05. All analyses were performed with the use of IBM SPSS Statistics for Windows version 19.0 and R 2.12.2 (R Foundation for Statistical Computing, Vienna, Austria) with the cmprsk package.
RESULTS Patient Characteristics
Twenty-one patients at high risk of HCC recurrence were included in the everolimus group, and 31 patients at high risk of recurrence were included in the historical control group. Table 1 displays the characteristics of the two groups of patients. The histological analyses of the explanted livers showed that one patient in the everolimus group (4.8%) and nine (29%) in the control group met the Milan criteria (P ¼ .03), and two patients (9.5%) in the everolimus group and 11 in the control group met the University of California at San Francisco criteria (P ¼ .03). No other significant differences were observed between the two groups.
FERREIRO, VAZQUEZ-MILLÁN, LÓPEZ ET AL
Survival and HCC Recurrence
Mean follow-up was 41 (3.9e94) months in the everolimus group and 64 (12.7e212) months in the control group. Figure 1 shows overall survival for the two groups. Two-year survival in the everolimus group was 85.7%, compared with 64.5% in the control group. Five-year survival was 60.2% and 32.3%, respectively (P ¼ .05). A trend toward longer survival was observed in patients with well or moderately differentiated tumors (55% at 5 years) compared with those with poorly differentiated tumors (20.3% at 5 years) (P ¼ .07). Two- and five-year survival rates were higher for patients with 3 nodules and for those without vascular invasion, but differences did not reach statistical significance (Table 2). The 2- and 5-year cumulative recurrence rates were 23.8% and 41.3% in the everolimus group, compared with 45.2% and 61.3%, respectively, in the control group (P ¼ .17) (Fig 2). A lower recurrence rate was observed in patients with 3 nodules, in those with well or moderately differentiated HCC, and in those without vascular invasion, but differences were not significant (Table 2). Thirty-two patients had well or moderately differentiated HCC (10 in the everolimus group and 22 in the control group). In this subgroup of patients, 2-year survival rates were 100% and 68.2%, respectively, and 5-year survival rates were 88.9% and 40.9%, respectively (P ¼ .02), but no significant differences were observed in cumulative recurrence rates between the two groups (P ¼ .22) (Table 3, Fig 3). Among the 20 patients with poorly differentiated HCC (11 in the everolimus group and nine in the control group), there were no significant differences in survival (P ¼ .32) or cumulative recurrence rates (P ¼ .25) (Table 3, Fig 3). Twenty-three patients had 3 nodules (10 in the everolimus group and 13 in the control group). No differences in survival (P ¼ .17) or cumulative recurrence rates (P ¼ .57) were observed between the two groups. Among the 29 patients with >3 nodules (11 in the everolimus group and 18 Table 2. Overall Survival and Cumulative Recurrence in Patients With a High Risk of Recurrence After Liver Transplantation for Hepatocellular Carcinoma Overall Survival (%) 1-Year 2-Year 5-Year
Fig 1. Overall survival by immunosuppression regimen in patients with a high risk of recurrence after liver transplantation for hepatocellular carcinoma.
Immunosuppression Everolimus Control Number of nodules 3 nodules >3 nodules Tumor differentiation Well/moderately differentiated Poorly differentiated Vascular invasion No Yes
Cumulative Recurrence (%) P
1-Year 2-Year 5-Year
.05 95.2 100
85.7 64.5
60.2 32.3
100 96.6
78.3 69.0
57.9 31.2
100
78.1
95.0
100 97.5
P .17
9.5 22.6
23.8 45.2
41.3 61.3
17.4 17.2
30.4 41.4
40.5 61.0
55.0
12.5
31.2
49.3
65.0
20.3
25.0
45.0
67.9
83.3 70.0
45.7 41.4
8.3 20.0
16.7 42.5
36.4 58.4
.15
.18
.07
.21
.51
.13
EVEROLIMUS-BASED IMMUNOSUPPRESSION
3499
0.34; 95% CI, 0.14e0.83; P ¼ .02), whereas poorly differentiated tumors (HR ¼ 2.77; 95% CI, 1.24e6.15; P ¼ .01) and >3 nodules (HR ¼ 2.37; 95% CI, 1.07e5.26; P ¼ .03) were markers of shorter survival. No factor was identified as an independent marker of risk of recurrence (Table 4). Rejection and Everolimus-Associated Side Effects
Three patients (14.3%) had an episode of acute rejection in the everolimus group, all of whom received cyclosporine as a concomitant immunosuppressor, compared with seven patients (22.6%) in the control group (P ¼ .35). Side effects associated with everolimus treatment were anaemia (19%), oral ulcers (9.5%) and wound dehiscence (4.8%). DISCUSSION
Fig 2. Cumulative recurrence by immunosuppression regimen in patients with a high risk of recurrence after liver transplantation for hepatocellular carcinoma.
in the control group), there were also no significant differences in survival (P ¼ .16) or cumulative recurrence rates (P ¼ .12) (Table 3). Vascular invasion was present in 40 patients (17 in the everolimus group and 23 in the control group). Among these patients, 5-year survival was 58.8% in the everolimus group, compared with 30.4% in the control group (P ¼ .31), and the 5-year cumulative recurrence rates were 50.7% and 65.2%, respectively (P ¼ .33). Among the 12 patients without vascular invasion (four in the everolimus group and eight in the control group), 2-year survival rates were 100% for the everolimus group and 75% for the control group, and the 5-year survival rates were 66.7% and 37.5%, respectively (P ¼ .09). Cumulative recurrence rates were 0% and 25% at 2 years and 0% and 50% at 5 years, respectively (P < .01) (Table 3). In the multivariate analysis, everolimus treatment surfaced as an independent marker of longer survival (HR ¼
Liver transplantation is the only curative treatment for patients with ESLD and HCC, with the most benefit attained in patients meeting the Milan criteria [4,6]. Several prognostic factors, including vascular invasion and poor tumor differentiation, can predict HCC recurrence after liver transplantation [24], and CNIs have also been linked to HCC recurrence [7,25]. Despite an increasing interest in the development of drugs to improve post-transplant outcome, there is as yet no consensus on the pharmacological management of HCC patients after liver transplantation. Several studies have reported lower recurrence rates and longer survival with sirolimus [14e17,26]; however, to the best of our knowledge, the present study is the first to examine the effect of everolimus-based immunosuppression on recurrence and survival of HCC patients with a high risk of recurrence. We have observed a strong but non-significant trend toward longer survival and lower recurrence in patients treated with everolimus. Treatment with everolimus was also identified as an independent predictor of longer posttransplant survival (HR ¼ 0.34; P ¼ .02). Moreover, 52.4% of the patients receiving everolimus had poorly differentiated tumors and 81% had vascular invasion, which was found to double the risk of death in a retrospective analysis of more than 1500 patients who received liver
Table 3. Overall Survival and Cumulative Recurrence in the Subgroups of Patients Classified According to Number of Nodules, Tumor Differentiation, and Presence of Vascular Invasion Overall Survival (%) 2-Year Everolimus
Number of nodules 3 nodules >3 nodules Tumor differentiation Well/moderately differentiated Poorly differentiated Vascular invasion No Yes
Cumulative Recurrence (%)
5-Year
2-Year
5-Year
Control
Everolimus
Control
P
Everolimus
Control
Everolimus
Control
P
90.0 81.8
69.2 61.1
77.1 46.8
46.2 22.2
.17 .16
20.0 27.3
38.5 50.0
20.0 50.6
53.8 66.7
.57 .12
100 72.7
68.2 55.6
88.9 31.2
40.9 11.1
.02 .32
10.0 36.4
40.9 55.6
40.9 36.4
54.5 77.8
.22 .25
100 82.4
75.0 60.9
66.7 58.8
37.5 30.4
.09 .31
0 29.4
25.0 52.2
0 50.7
50 65.2
3 Everolimus-based immunosuppression
Recurrence
HR
95% CI
P
HR
95% CI
P
0.94 2.77 1.45 2.37 0.34
0.94e1.05 1.24e6.15 0.55e3.83 1.07e5.26 0.14e0.83
.83 .01 .45 .03 .02
1.01 1.87 2.49 2.25 0.49
0.94e1.08 0.80e4.35 0.83e7.46 0.95e5.32 0.21e1.12
.80 .15 .10 .06 .09
Abbreviations: HR, hazard ratio; CI, confidence interval.
these studies included only patients within the Milan criteria. In contrast, the present study included only patients at high risk of HCC recurrence, and the 2-year (87.7%) and 5-year (60.2%) survival rates attained with everolimusbased immunosuppression are promising and compare favourably with the 53.6% 5-year survival rate reported for patients outside the Milan criteria [6]. The beneficial effect of everolimus observed in our patients could be a direct result of the anti-proliferative properties of the drug itself or, as suggested by Vivarelli [7], an indirect result of reduced exposure to CNIs. Further studies could help to clarify this point. Our report has several limitations, including the relatively small number of patients and the non-randomized design. In addition, the comparison with a historical control group could be the source of bias, because the better survival rates attained with everolimus may be partly the result of general improvements in post-transplant care in recent years. In conclusion, our findings indicate that patients receiving liver transplantation for ESLD and HCC who have a high risk of tumor recurrence may well benefit from everolimusbased immunosuppression, with no added risks of rejection or other post-transplant complications. Prospective studies with a larger number of patients are warranted to further examine the role of everolimus in reducing post-transplant HCC recurrence. REFERENCES [1] Parkin DM, Bray F, Ferlay J, et al. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001;94:153e6. [2] El-Serag HB, Davila JA, Petersen NJ, Mcglynn KA. The continuing increase in the incidence of hepatocellular carcinoma
EVEROLIMUS-BASED IMMUNOSUPPRESSION in the United States: an update. Ann Intern Med 2003;139: 817e23. [3] Jelic S, Sotiropoulos GC. Hepatocellular carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010;21(Suppl 5):v59e64. [4] Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med 1996;334:693e9. [5] Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology 2011;53:1020e2. [6] Mazzaferro V, Llovet JM, Miceli R, et al. Predicting survival after liver transplantation in patients with hepatocellular carcinoma beyond the Milan criteria: a retrospective, exploratory analysis. Lancet Oncol 2009;10:35e43. [7] Vivarelli M, Cucchetti A, Piscaglia F, et al. Analysis of risk factors for tumor recurrence after liver transplantation for hepatocellular carcinoma: key role of immunosuppression. Liver Transpl 2005;11:497e503. [8] Guba M, Von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002;8: 128e35. [9] Mcalister VC, Peltekian KM, Malatjalian DA, et al. Orthotopic liver transplantation using low-dose tacrolimus and sirolimus. Liver Transpl 2001;7:701e8. [10] Casadio F, Croci S, D’errico Grigioni A, et al. Toward the definition of immunosuppressive regimens with antitumor activity. Transplant Proc 2005;37:2144e7. [11] Schumacher G, Oidtmann M, Rueggeberg A, et al. Sirolimus inhibits growth of human hepatoma cells alone or combined with tacrolimus, while tacrolimus promotes cell growth. World J Gastroenterol 2005;11:1420e5. [12] Trotter JF, Wachs M, Bak T, et al. Liver transplantation using sirolimus and minimal corticosteroids (3-day taper). Liver Transpl 2001;7:343e51. [13] Zhou J, Fan J, Wang Z, et al. Conversion to sirolimus immunosuppression in liver transplantation recipients with hepatocellular carcinoma: report of an initial experience. World J Gastroenterol 2006;12:3114e8. [14] Toso C, Meeberg GA, Bigam DL, et al. De novo sirolimus-based immunosuppression after liver transplantation
3501 for hepatocellular carcinoma: long-term outcomes and side effects. Transplantation 2007;83:1162e8. [15] Zimmerman MA, Trotter JF, Wachs M, et al. Sirolimusbased immunosuppression following liver transplantation for hepatocellular carcinoma. Liver Transpl 2008;14:633e8. [16] Chinnakotla S, Davis GL, Vasani S, et al. Impact of sirolimus on the recurrence of hepatocellular carcinoma after liver transplantation. Liver Transpl 2009;15:1834e42. [17] Liang W, Wang D, Ling X, et al. Sirolimus-based immunosuppression in liver transplantation for hepatocellular carcinoma: a meta-analysis. Liver Transpl 2012;18:62e9. [18] Menon KV, Hakeem AR, Heaton ND. Meta-analysis: recurrence and survival following the use of sirolimus in liver transplantation for hepatocellular carcinoma. Aliment Pharmacol Ther 2013;37:411e9. [19] Saliba F, De Simone P, Nevens F, et al. Renal function at two years in liver transplant patients receiving everolimus: results of a randomized, multicenter study. Am J Transplant 2013;13: 1734e45. [20] De Simone P, Nevens F, De Carlis L, et al. Everolimus with reduced tacrolimus improves renal function in de novo liver transplant recipients: a randomized controlled trial. Am J Transplant 2012;12:3008e20. [21] Kalbfleisch J, Prentic R. The Statistical Analysis of Failure Time Data. New York: John Wiley and Sons; 1980. [22] Gray R. A class of K-sample tests for comparing the cumulative incidence of a competing risk. Ann Stat 1988;16:1141e54. [23] Fine J, Gray R. A proportional hazards model for the subdistribution of a competing risk. JASA 1999;94:496e509. [24] Cescon M, Ravaioli M, Grazi GL, et al. Prognostic factors for tumor recurrence after a 12-year, single-center experience of liver transplantations in patients with hepatocellular carcinoma. J Transplant 2010;2010. doi: 10.1155/2010/904152. Epub 2010 Aug 25. [25] Vivarelli M, Dazzi A, Zanello M, et al. Effect of different immunosuppressive schedules on recurrence-free survival after liver transplantation for hepatocellular carcinoma. Transplantation 2010;89:227e31. [26] Gomez-Martin C, Bustamante J, Castroagudin JF, et al. Efficacy and safety of sorafenib in combination with mammalian target of rapamycin inhibitors for recurrent hepatocellular carcinoma after liver transplantation. Liver Transpl 2012;18:45e52.
