Impact of sirolimus on the recurrence of hepatocellular carcinoma after liver transplantation

ORIGINAL ARTICLE

Impact of Sirolimus on the Recurrence ofHepatocellular Carcinoma After LiverTransplantationSrinath Chinnakotla,1 Gary L. Davis,2 Sugam Vasani,1 Peter Kim,1 Koji Tomiyama,1 Edmund Sanchez,1

Nicholas Onaca,1 Robert Goldstein,1 Marlon Levy,1 and Goran B. Klintmalm1

1Department of Transplant Surgery, Baylor Regional Transplant Institute, and 2Department of Hepatology,Baylor University Medical Center, Dallas, TX

Tumor recurrence after liver transplantation for hepatocellular carcinoma is associated with a poor prognosis. Becauseimmunosuppression is a well-known risk factor for tumor growth, it is surprising that its possible role in the outcome of livertransplantation has been poorly evaluated. We performed a case-control review of prospectively collected data and compared2 groups of patients according to the type of immunosuppression after liver transplantation for hepatocellular carcinoma at asingle center. One hundred six patients received tacrolimus and mycophenolate mofetil, and 121 received sirolimus. Patientsin the sirolimus group had significantly higher recurrence-free survival rates than patients in the tacrolimus group (P � 0.0003).The sirolimus group also had significantly higher patient survival rates than the tacrolimus group at 1 year (94% versus 79%),3 years (85% versus 66%), and 5 years (80% versus 59%; P � 0.001). Sirolimus was well tolerated, and the patients in thisstudy did not have the increase in surgical complications noted by other investigators. Leukopenia was the most common sideeffect, but it typically resolved with dose reduction. Dyslipidemia and mouth ulcers were common but were easily controlled.In summary, the data suggest a beneficial effect of sirolimus immunosuppression on recurrence-free survival, which translatesinto patient survival benefits. Liver Transpl 15:1834-1842, 2009. © 2009 AASLD.

Received June 5, 2009; accepted September 14, 2009.

The incidence of hepatocellular carcinoma (HCC) is in-creasing in the United States and throughout the world;this trend is projected to continue for several years.1-4

Currently, HCC is the indication for approximately 750liver transplants in the United States each year. Earlyexperiences with transplantation in patients with HCCwere poor because of a high recurrence rate. However,since the implementation of the Milan criteria, patientswith HCC have been more likely to reach transplanta-tion without tumor progression.5,6 Nonetheless, tumorrecurrence after transplantation carries an ominousprognosis; therefore, considerable effort goes into se-lecting candidates for whom the likelihood of recur-rence is low.

Measures to prevent tumor recurrence have alwaysbeen the subject of debate. Several centers have usedpostoperative chemotherapy to prevent tumor recur-

rence post-transplant,7-9 although these studies sug-gested that the greatest impact was in patients withadvanced tumors who would generally not undergotransplantation today. Immunosuppression is a well-known risk factor for tumor growth, so it is surprisingthat its possible role in the outcome of liver transplan-tation has been poorly evaluated. Vivarelli et al.10 re-ported that a higher level of cyclosporine exposure wasan independent prognostic determinant of tumor recur-rence. Experience with sirolimus-based immunosup-pression following orthotopic liver transplantation israpidly accumulating. Sirolimus has been reported toinhibit the growth of human hepatoma cells in severalin vitro studies,11 and a few clinical case reports havedescribed the effectiveness of sirolimus in treating var-ious malignancies in patients after liver transplanta-tion.12-15

Abbreviations: CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; ICU, intensive care unit; MELD, Model forEnd-Stage Liver Disease; NS, not significant; WBC, white blood cell.Address reprint requests to Srinath Chinnakotla, M.B.B.S., M.Ch., Department of Transplant Surgery, Baylor University Medical Center, 3500Gaston Avenue, Dallas, TX 75246. Telephone: 214-820-2050; FAX: 214-820-4527; E-mail: [email protected]

DOI 10.1002/lt.21953Published online in Wiley InterScience (www.interscience.wiley.com).

LIVER TRANSPLANTATION 15:1834-1842, 2009

© 2009 American Association for the Study of Liver Diseases.

Despite all the in vitro evidence and sporadic casereports, it is unclear whether sirolimus has any directoncological benefits in patients transplanted for HCC.Our aim in the current report was to examine our cen-ter’s experience with sirolimus-based immunosuppres-sion in patients who underwent liver transplantationfor HCC to determine if the drug affected tumor recur-rence and was safe when combined with prophylacticchemotherapy.

PATIENTS AND METHODS

Study Group

The study group consisted of 227 patients who under-went primary liver transplantation for HCC at the Bay-lor Regional Transplant Institute (Dallas, TX) betweenJanuary 1995 and July 2006. Patients who exceededthe Milan criteria or had evidence of portal vein invasionby imaging were excluded. From 2000 onward, preop-erative tumor ablation with chemoembolization (a se-lective hepatic artery infusion of 50 mg of doxorubicin,300 mg of carbopentin, and 10 mg of mitomycin C) orradiofrequency thermal ablation was considered in allcases.

Patient data were collected prospectively and enteredinto an institutional transplant database. The BaylorHealth Care System Institutional Review Board ap-proved the review of these data.

Immunosuppression Protocols

One hundred six patients received tacrolimus and my-cophenolate mofetil immunosuppression, and 121 pa-tients received sirolimus-based immunosuppression.There were no consistent criteria for the selection of aspecific agent over time. Although sirolimus was thepreferred agent for HCC patients after 2000, many stillreceived tacrolimus because of participation in clinicaltrials, clinical circumstances, or surgeon preference.Both groups received posttransplant chemotherapy asdescribed later. The minimum follow-up for all patientswas 2 years to allow an adequate assessment of tumorrecurrence and a full evaluation of the safety profile ofsirolimus.

Sirolimus Group

On postoperative day 1, patients in the sirolimus groupbegan receiving the drug at a dose of 2 mg orally, oncedaily, along with cyclosporine (Neoral, Novartis Phar-maceuticals Corp., East Hanover, NJ) at a dose of 5mg/kg orally, twice daily. In patients with incidentaltumors, sirolimus was started as soon as the explantpathology was reported, usually on postoperative day 3.Sirolimus levels were maintained at 5 to 8 ng/mL forthe first 3 months and at 5 ng/mL thereafter. Cyclo-sporine levels were maintained at 200 ng/mL. Patientstransplanted between 1995 and 2005 also receivedmethylprednisolone; this began intraoperatively. Corti-costeroids were tapered and discontinued on postoper-ative day 60. Since 2005, we have used a completelysteroid-free protocol that includes induction with dacli-

zumab (Zenapax, Roche Pharmaceuticals, Nutley, NJ)at 2 mg/kg on days 0 and 3 and at 1 mg/kg on day 8.

Tacrolimus Group

Patients in the tacrolimus group started receiving thedrug at a dose of 0.05 mg/kg orally, twice daily, on day1, and the levels were maintained at 10 to 12 ng/mLduring the first 2 weeks, at 8 to 10 ng/mL during weeks3 to 12, and at 5 to 7 ng/mL thereafter. Mycophenolatemofetil was started at a dose of 1000 mg orally, twicedaily, on day 1. Patients transplanted between 1995and 2005 received the same corticosteroids as those inthe sirolimus group, and patients transplanted after2005 followed the corresponding steroid-free protocol.

Systemic Chemotherapy

Patients with tumors discovered before transplantationwere offered chemotherapy that began with 10 mg/m2

doxorubicin during the anhepatic phase of the trans-plant and continued postoperatively at weekly intervalsfor 20 weeks.7 Therapy was interrupted if patients de-veloped leukopenia [white blood cell (WBC) count �4000/�L] or thrombocytopenia (platelet count �30,000/�L). Patients with HCC discovered incidentallyin the explanted liver received postoperative chemo-therapy if the lesion met T2 criteria and had unfavor-able histological features, including vascular invasionor a poor grade, and if the patient agreed to treatment.

Surgical Technique

Liver transplantation was performed with the standardorthotopic technique.16 All patients except one receivedorgans from deceased donors. Extra care was taken toavoid unnecessary manipulation of the tumor at thetime of dissection for hepatectomy. A cell saver was notused in patients with known tumors. Liver transplan-tation was performed only when the tumor was con-fined to the liver and there was no evidence of extrahe-patic spread. A careful visual examination wasperformed at the time of transplant to exclude any ex-trahepatic malignancy. If lymphadenopathy or suspi-cious nodules were noted in the abdomen, biopsy wasperformed, and frozen sections were evaluated to ex-clude metastatic disease before the surgeon proceededwith the transplant.

Pathology

A single hepatopathologist examined all explanted liv-ers. The number of tumors, lobar distribution of thetumors, maximal tumor diameter, histological gradeaccording to the modified Edmondson criteria17 for thedegree of tumor differentiation (grades I-IV), and pres-ence or absence of macrovesicular and microvascularinvasion were recorded. The total tumor diameter forpatients with multiple tumor nodules was calculated.The new prognostic score predicting disease-free sur-vival post–liver transplantation for HCC, recently pro-posed by Marelli et al.18 and Decaens et al.,19 was

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LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

calculated for each patient. This score evaluates 3 vari-ables: the largest nodule diameter (�20 mm � 0 points,21-30 mm � 1 point, 31-50 mm � 2 points, �50 mm �5 points), the number of nodules (1 nodule � 0 points,2-3 nodules � 1 point, �4 nodules � 2 points), andtumor differentiation (well � 0 points, moderate � 1point, poor � 3 points). Projected 5-year survival rateswere estimated with Metroticket for subjects whose tu-mors exceeded the Milan criteria.20

Posttransplant Follow-Up

All patients were seen twice weekly in the posttrans-plant clinic for 3 months. Thereafter, their respectiveprimary care physicians provided follow-up. The pa-tients were seen monthly for the first 5 years post-transplant by the primary care physicians; laboratorytests results were faxed to Baylor University MedicalCenter and reviewed by an attending transplant sur-geon, who performed immunosuppression changes, ifthere were any. Patients with abnormal clinical findingsor liver dysfunction suggestive of acute cellular rejec-tion were seen at the posttransplant clinic, and liverbiopsy was performed when indicated. Rapamycin waswithheld transiently if a patient had wound complica-tions, leukopenia (WBC count � 4000/�L), or severediarrhea. Chemotherapy was initiated after the healingof wounds and was interrupted if the WBC count was�3000/�L or if the platelet count was �30,000/�L.Patients were evaluated by the transplant surgeon andoncologist for clinically apparent recurrence or other-wise routinely at 1, 2, 5, and 10 years post-transplant,at which time a clinical examination, laboratory testing(including the serum alpha fetoprotein level), an echo-cardiogram, a computed tomography scan of the abdo-men, and a bone scan were routinely performed to seekevidence of recurrence.

Statistical Analysis

Because this was not a prospective randomized study,great care was taken to evaluate the characteristics ofthe patients in the sirolimus group and the tacrolimusgroup to identify potential bias. Using all the possiblefactors known in the literature to affect recurrence, wefirst performed a univariate analysis. The studied vari-ables included demographic factors (age, gender, era oftransplant, and waiting time prior to transplant), etiol-ogy for liver cirrhosis (hepatitis C and hepatitis B), tu-mor characteristics (size, number of nodules, lobar dis-tribution, tumor grade, vascular invasion, and serumalpha fetoprotein levels), and treatment factors (use ofany ablative treatment prior to transplant, use of intra-operative and postoperative chemotherapy, and use ofsirolimus). Variables that were identified as potentiallydifferent by univariate analysis (P � 0.10) were includedin a multivariate stepwise Cox regression analysis fortime to recurrence and patient survival. Recurrence-free survival and patient survival were calculated withthe Kaplan-Meier method and compared with the log-rank test. Other comparisons of dichotomous variables

between the groups (demographic data, complications,and tumor characteristics) were made with Fisher’s ex-act test for 2 � 2 tables; continuous variables werecompared with the Wilcoxon 2-sample test and the like-lihood ratio chi-square test for larger tables, with P �0.05 considered significant. Analysis was performedwith SAS 9.1.3 (SAS Institute, Cary, NC).

RESULTS

Patients

The characteristics of the patients in the tacrolimusand sirolimus groups are shown in Table 1. The groupswere similar with respect to age, gender, and operativecharacteristics. Patients in the tacrolimus group had ahigher calculated Model for End-Stage Liver Diseasescore and were more likely to have been transplanted inthe pre–Model for End-Stage Liver Disease era becausesirolimus did not become available until 2000. Thewarm ischemia time was approximately 10 minuteslonger in the tacrolimus group. More patients in thesirolimus group received ablative treatment whileawaiting transplantation, as these cases occurred after2000 when locoregional ablation was more common atour center (Table 1). However, when we separated andcompared the patients who received ablative treatment(n � 99) and the patients who did not receive ablativetreatment (n � 110), there was no significant differencein recurrence-free survival or patient survival (P �0.1461). The median lengths of follow-up in the tacroli-mus and sirolimus groups were 45 and 50 months,respectively.

Tumor Characteristics and Tumor PrognosticScore

Characteristics of the tumors in the 2 groups are shownin Table 2. There were significantly more incidentaltumors in the tacrolimus group, and this may havebeen related to better imaging during later years whenmost sirolimus patients were enrolled. The probabilityof recurrence of incidental and known tumors was notdifferent (Fig. 1). There were otherwise no differencesbetween the groups in terms of tumor size, number ofnodules, proportion meeting or exceeding the Milan cri-teria, vascular invasion, Metroticket-estimated 5-yearsurvival (for those exceeding the Milan criteria), or tu-mor grade. The tumor prognostic score18 was not sta-tistically different between the sirolimus and tacroli-mus groups (Table 3).

Posttransplant Complications

The surgical complications for the 2 groups are listed inTable 4. Two patients in the sirolimus group developedarterial thrombosis versus none in the tacrolimusgroup, but this difference was not statistically signifi-cant. Both cases of arterial thrombosis were technical.No significant difference was found in the incidence ofportal vein complications, biliary complications, wound

1836 CHINNAKOTLA ET AL.


TABLE 1. Characteristics of the Patients According to the Type of Immunosuppression

Characteristic Tacrolimus Sirolimus P Value

Number 106 121 —Age (years) 53.9 � 7.4 53.9 � 7.8 0.7874Number of males 81 (76%) 91 (75%) 0.8774Etiology of cirrhosis

Hepatitis C 67 (63.2%) 82 (67.8%) 0.1937Hepatitis B 13 (12.3%) 12 (9.9%)Alcohol 12 (11.3%) 16 (13.2%)Cryptogenic 8 (7.6%) 6 (5%)Other 4 (3.8%) 4 (3.3%)No cirrhosis 2 (1.9%) 1 (0.8%)

Alpha fetoprotein � 200 ng/mL 12/93 (12.9%) 10/107 (9.3%) 0.4993Calculated MELD score 16.48 � 7.52 13.61 � 5.02 0.0060Era of transplant

Pre-MELD 63 (59.4%) 23 (19%) �0.001MELD 43 (40.6%) 98 (81%)

Days on waiting list 225 � 299 271 � 415 0.4113Pretransplant status ICU, 6 ICU, 3 0.1937

Hospital, 12 Hospital, 8Home, 88 Home, 110

Cold ischemia time (hours) 8.32 � 2.91 7.56 � 2.88 0.0616Warm ischemia time (hours) 0.98 � 0.42 0.81 � 0.25 0.0007Received preoperative ablation 32/95 (33.7%) 76/110 (69.1%) �0.001Received intraoperative and/or

postoperative chemotherapy70/96 (72.9%) 97/113 (85.8%) 0.0244

NOTE: Values are shown as means and standard deviations.Abbreviation: ICU, intensive care unit; MELD, Model for End-Stage Liver Disease.

TABLE 2. Characteristics of the Tumors in the Two Groups

Characteristic Tacrolimus Group (n � 106) Sirolimus Group (n � 121) P Value

Incidental tumor 44 (41.51%) 11 (9.57%) �0.0001Milan criteria

Solitary 45 68Diameter (cm) 2.30 � 1.15 2.57 � 1.16 0.2296

Multiple 18 20Number 2.27 � 0.46 2.35 � 0.49 0.6434Largest (cm) 2.02 � 0.66 2.00 � 0.59 0.8255

Vascular invasion 8 (13.1%) 5 (5.6%) 0.1453Exceeding the Milan criteria

Solitary 3 2Diameter (cm) 6.67 � 0.58 9.25 � 2.47 0.0756

Multiple 31 20Number* 2.75 � 0.85 2.62 � 0.96 0.6758Largest (cm) 4.21 � 2.08 3.78 � 1.42 0.4164

�5 lesions (% of cases outside Milan) 11 (32.4%) 7 (31.8%) 1.0000Vascular invasion 12 (35.3%) 6 (27.3%) 0.7691Metroticket (estimated 5-year survival) 52.29 � 9.79 52.89 � 11.33 0.3684

Edmondson gradeI (41) 22 (25.29%) 19 (21.11%) 0.7906II (75) 38 (43.68%) 37 (41.11%)III (57) 25 (28.74%) 32 (35.56%)IV (4) 2 (2.3%) 2 (2.22%)

NOTE: The columns may not add up to the same total because not all data were available for every case.*This does not include cases with �5 tumors (ie, “too numerous to count”).



infections, or acute rejection when the groups werecompared.

A higher incidence of pleural effusions occurred inthe sirolimus group than in the tacrolimus group(16.53% versus 10.38%, respectively), but the differ-ence was not statistically significant (Table 4). Siroli-mus-induced pneumonitis was not seen in any of thepatients.

The incidence of postoperative acute renal failure wassimilar for both groups. Longitudinal follow-up showed

that both groups had a 40% drop in the median glomer-ular filtration rate at 3 months. However, the sirolimusgroup had a better median glomerular filtration rate at5 years (Fig. 2).

Leukopenia (�4000/�L) was the most common sideeffect of sirolimus (45% of patients), and the WBCcounts remained lower in this group for 3 years post-transplant. Despite this fact, there was no significantdifference in the rate of infectious complications be-tween the 2 groups. The WBC count rose in the siroli-mus group after year 3, and there was no significantdifference between the groups at 5 years.

Thrombocytopenia was not a problem in either groupand usually normalized by 3 months after transplanta-tion, regardless of the immunosuppressive regimen orreceipt of chemotherapy. The median platelet counts(�103/�L) in the tacrolimus and sirolimus groups were72.5 and 73, respectively, before transplant; 148 and144, respectively, at 3 months post-transplant; 152and 155, respectively, at 1 year; 154 and 167, respec-tively, at 3 years; and 176 and 175, respectively, at 5years.

Thirteen patients (12.26%) in the tacrolimus groupdeveloped new-onset diabetes, whereas none in thesirolimus group did (P � 0.001). No significant differ-ences were found between the groups with respect tothe levels of total cholesterol, low-density lipoproteincholesterol, high-density lipoprotein cholesterol, ortriglycerides at 3 months, 1 year, 2 years, and 5 years.Although patients receiving sirolimus had elevated lipidlevels, treatment with antihyperlipidemic agents waseffective in reducing the levels to those of the tacrolimusgroup.

Predictors of HCC Recurrence and PatientSurvival

Multivariate stepwise Cox regression analysis for HCCtumor recurrence (death censored) found that vascularinvasion [hazard ratio (HR) � 6.6, 95% confidence in-terval (CI) � 2.65-16.44, P � 0.0001], serum alphafetoprotein � 200 ng/mL (HR � 4.9, 95% CI � 1.77-13.56, P � 0.0005), 4 or more tumor nodules (HR � 5.3,95% CI � 2.08-13.52, P � 0.001), and not using siroli-mus (HR � 2.62, 95% CI � 0.99-6.96, P � 0.0465) wereindependently associated with HCC recurrence. The in-cidental discovery of tumors and ablative treatmentprior to transplant did not influence HCC recurrence inthis analysis.

Three variables were independently associated withpatient death by multivariate stepwise Cox regressionanalysis. They included absence of sirolimus (HR � 3.0,95% CI � 1.6-5.6, P � 0.0002), 4 or more tumor nod-ules (HR � 3.8, 95% CI � 1.9-7.8, P � 0.0001), andpresence of vascular invasion (HR � 1.8, 95% CI �1.0-3.3, P � 0.0433).

Recurrence Probability and Patient Survival

Tumors recurred in 25 patients (11.0%) transplantedwith HCC, with most occurring within the first 2 years

Figure 1. Kaplan-Meier plot of patient survival according towhether the tumor was found before transplantation or dis-covered at the time of transplantation (incidental).

TABLE 3. Tumor Prognostic Score

Description

Tacrolimus

Group

Sirolimus

Group P Value

Prognostic score0 5 (5.88%) 5 (5.56%) 0.67271 12 (14.12%) 19 (21.11%)2 17 (20%) 11 (12.22%)2 15 (17.65%) 18 (20%)4 14 (16.47%) 16 (17.78%)5-10 22 (25.88%) 21 (23.33%)

Diameter score0 37 (37.6%) 42 (37.5%) 0.46681 22 (22.45%) 32 (28.57%)2 30 (30.61%) 33 (29.46%)5 9 (9.18%) 5 (4.46%)

Nodule score0 49 (50%) 71 (63.96%) 0.07081 33 (33.67%) 31 (27.93%)2 16 (16.33%) 9 (8.11%)

NOTE: The tumor prognostic score predictive disease-freesurvival for this table was calculated by methods describedby Marelli et al.18



(probability of recurrence: 5.2% at year 1, 9.2% at year2, 10.2% at year 3, and 11.0% at year 5). The patients inthe sirolimus group had a significantly lower rate oftumor recurrence (P � 0.0001) than the tacrolimusgroup (Fig. 3).

The sirolimus group also had significantly higher pa-tient survival rates than the tacrolimus group at 1 year(94% versus 79%), 3 years (85% versus 66%), and 5years (80% versus 59%, P � 0.0001; Fig. 4). Whenpatients with incidental tumors and those with knowntumors were compared, there was no significant differ-ence in recurrence-free survival or patient survival (P �0.3162; Fig. 5).

A significantly higher number of deaths were due torecurrent tumors in the tacrolimus group (n � 19) ver-sus the sirolimus group (n � 4). The remaining causes

of death in the tacrolimus group were recurrent hepa-titis in 5 patients, sepsis in 6, de novo cancer in 6,cardiac events in 2, stroke in 2, posttransplant lympho-proliferative disorder in 2, technical reasons in 3, graft-versus-host disease in 2, graft failure in 2, other causesin 2, and unknown causes in 2. The remaining causes

TABLE 4. Posttransplant Complications

Complication

Tacrolimus Group

(n � 106)

Sirolimus Group

(n � 121) P Value

Primary nonfunction 1 (0.94%) 0 0.4670Hepatic artery stenosis 3 (2.83) 6 (4.96%) 0.5079Hepatic artery thrombosis 0 2 (1.65%) 0.5000Portal vein stenosis 0 1 (0.83%) 1.0000Portal vein thrombosis 1 (0.94%) 2 (1.65%) 1.0000Wound infection 6 (5.66%) 12 (9.92%) 0.3257Wound dehiscence 2 (1.89%) 4 (3.31%) 0.6875Bile duct stricture 10 (9.43%) 16 (13.2%) 0.4097Bile leak 5 (4.72%) 6 (4.96%) 1.0000Neurological: seizures 3 (2.83%) 2 (1.65%) 0.6665Tacrolimus neurotoxicity 2 (1.89%) 2 (1.65%) 1.0000Cyclosporine neurotoxicity 1 (0.94%) 0 0.4670Gastrointestinal ulcers 8 (7.55%) 14 (11.57%) 0.3717Chronic diarrhea 17 (16.04%) 10 (8.26%) 0.0991Septic shock 10 (9.43%) 4 (3.31%) 0.0940Pneumonia 7 (6.60%) 7 (5.79%) 1.0000Pleural effusions 11 (10.38%) 20 (16.53%) 0.2449Acute renal failure 17 (16.04%) 17 (14.05%) 0.7121New-onset diabetes mellitus 13 (12.26%) 0 �0.001Acute cellular rejection 58 (54.72%) 76 (62.81%) NS

Abbreviation: NS, not significant.

Figure 2. Median glomerular filtration rate in the sirolimusand tacrolimus groups.

Figure 3. Kaplan-Meier plot of the time-to-recurrence prob-ability according to the type of immunosuppression. No indi-cates no sirolimus (the patients received tacrolimus/mycophe-nolate mofetil).



of death in the sirolimus group were recurrent hepatitisin 5 patients, sepsis in 5, de novo cancer in 1, post-transplant lymphoproliferative disorder in 1, technicalreasons in 1, and anoxic brain injury in 2.

DISCUSSION

Sirolimus has potent in vitro effects on tumorgrowth.11,21 These effects are related to the antiangio-genic properties of the drug, which are derived from theinhibition of vascular endothelial growth factor produc-tion and the stimulation of endothelial cells. Angiogen-esis, or the growth of new blood vessels, occurs primar-ily during embryonic development and, in adults, is

triggered locally in finely balanced processes such aswound healing.

Data from in vitro studies22 suggest that the effective-ness of sirolimus as an anticancer agent differs withdose, such that low nanogram per milliliter concentra-tions of the drug produce a potent antiangiogenic andpotential antiproliferative effect, whereas higher dosesmay actually have a direct cytotoxic effect on tumorcells. Even though the doses used in posttransplantimmunosuppression would likely be antiangiogenic,angiogenesis is only one of the mechanisms by whichtumors proliferate; therefore, an antiangiogenic ap-proach would likely be insufficient to eradicate all tu-mors or prevent tumor recurrence.23 Thus, combiningsirolimus with other agents, including traditional sys-temic chemotherapy, may be logical. One could specu-late that a combined approach including long-term an-tiangiogenic treatment with intermittent cytotoxic orother therapy might provide the best outcomes. Ourposttransplant protocol for HCC patients includesdoxorubicin for the first 20 postoperative weeks. Fur-thermore, cyclosporine is added to the immunosup-pressive regimen because we have previously shownthat pharmacological concentrations of cyclosporineenhance the in vitro doxorubicin cytotoxicity for multi-drug resistance protein 1–positive hepatoma cells bymodulating drug retention.24

Recurrence of tumors after liver transplantation forHCC is associated with a very poor prognosis: the me-dian survival is just 13 months.25 Despite the impor-tance of this problem, there is a paucity of clinicalexperience with different prophylactic regimens, in-cluding sirolimus, as strategies to reduce HCC recur-rence. Kneteman et al.26 reported a pilot study of 40patients with HCC who were treated with a sirolimus-based protocol post-transplant. The authors catego-rized patients within and beyond the Milan criteria, andthey concluded that both the 1- and 4-year survivalrates were not significantly different between thegroups. They were not, however, able to addresswhether sirolimus affected clinical outcome. Similarly,Tosco et al.27 reported on a cohort of 70 patients withHCC who received sirolimus and showed no differencein short-term tumor-free survival in patients within orbeyond the Milan criteria. Although cancer recurrencewas reasonably low (11%), nearly 50% of patients suf-fered from delayed wound healing or incisional hernia.

Zhou et al.28 described 18 patients with HCC recur-rence after liver transplantation who were switched tosirolimus-based immunosuppression from tacrolimus.Twelve of the 18 patients who had documented recur-rence remained alive after 10.4 months of follow-up,and the authors concluded that sirolimus may haveinhibited the progression of the recurrent tumor. How-ever, this study had no comparison group and did notlook prospectively at prevention of recurrence. Zimmer-man et al.29 compared 2 groups of patients who under-went liver transplantation for HCC: 45 patients receivedsirolimus and a calcineurin inhibitor post-transplant,and 52 patients received cyclosporine or tacrolimusplus mycophenolate mofetil and corticosteroids. The 1-

Figure 4. Kaplan-Meier plot of patient survival according tothe type of immunosuppression. No indicates no sirolimus(the patients received tacrolimus/mycophenolate mofetil).

Figure 5. Kaplan-Meier plot of the time-to-recurrence prob-ability according to whether the tumor was found before trans-plantation or discovered at the time of transplantation (inci-dental).



and 5-year survival rates for the sirolimus-treatedgroup were 95.5% and 78%, respectively, versus 83%and 62% for the nonsirolimus group. The investigatorsfound no significant difference in the incidence of majorcomplications, and this was similar to our large expe-rience.

Our present investigation is the first case-controlstudy to report the usefulness of sirolimus, in conjunc-tion with systemic chemotherapy, in preventing tumorrecurrence after liver transplantation for HCC. Becauseof its proven cytotoxic effects on hepatoma cells in vitro,we have used doxorubicin chemotherapy for all pa-tients undergoing liver transplantation for HCC at Bay-lor University Medical Center since 1987.7 To reap itsclear antiangiogenic benefits, we began to add siroli-mus to our tumor protocol on a limited basis in late2000, and we combined it with doxorubicin, a cytotoxicagent capable of directly killing the residual cancer cellsas they emerge from a dormant stage. We cautiouslychose a lower dosage of sirolimus to avoid the potentialside effects associated with a higher dosage; thus, ourpatients were able to receive sirolimus in the immediateposttransplant period and thereafter.

Our data suggest a beneficial effect of sirolimus im-munosuppression on recurrence-free survival, whichtranslates into patient survival benefits. At our institu-tion’s liver transplant program, we transplant patientswithin United Network for Organ Sharing criteria, andsince December 2000, we have employed ablative treat-ment when feasible in patients waiting for liver trans-plants. One could argue that the survival benefits in thesirolimus group could be attributed to the ablativetreatment that the patients received while awaitingtransplantation.30 Hence, we compared the patientswho received pretransplant ablative treatment withthose who did not, and there was no difference in sur-vival. We also performed a multivariate stepwise Coxregression analysis, and the ablative treatment did notinfluence HCC tumor recurrence. This finding suggeststhat the improved survival in the patients receivingsirolimus-based immunosuppression was not attribut-able to locoregional ablation.

The use of sirolimus during the early posttransplantperiod is currently challenged by the US Food and DrugAdministration’s black box recommendation againstimmunosuppression with sirolimus after liver trans-plantation. A recent clinical trial reported a trend to-ward an increased rate of graft loss and death (R.Wiesner, unpublished data, American Transplant Con-gress, Washington, DC, June 2003). However, siroli-mus was well tolerated by our patients and did not haveto be discontinued in any patient. We did not note theincrease in surgical complications (eg, wound dehis-cence, wound infections, and arterial thrombosis) thatothers have reported,27 and we suspect that our use ofa lower dose of sirolimus could explain this difference.The most common side effect was leukopenia in nearlyhalf of the patients, but it typically resolved with dosereduction. Infections were not more common in thesirolimus group. Dyslipidemia and mouth ulcers were

common, as others have reported, but these were easilycontrolled with local or drug therapy.

The limitation of our experience is that it is a retro-spective case-control study. As such, there is a poten-tial for selection bias in this study. This is particularlyimportant because sirolimus has been available foronly the last 10 years of the study period and becameour primary immunosuppressive drug of choice for tu-mor patients after 2000. However, the tumor charac-teristics in the tacrolimus and sirolimus groups werewell-matched, and there were few other differences be-tween the groups. Furthermore, multivariate analysisfound that only tumor extent (vascular invasion andnumber of lesions) and sirolimus influenced HCC re-currence and patient survival after liver transplanta-tion. Thus, we are confident that sirolimus was inde-pendently associated with lower HCC tumor recurrenceand improved patient survival. Our data should be con-firmed in a randomized prospective trial, and such atrial is currently beginning in Europe. However, thisstudy is still in the enrollment phase, and there are noresults published yet.31 Nonetheless, on the basis ofour data, we can conclude that sirolimus is well toler-ated at the studied doses as a treatment for patientstransplanted for HCC, and it was not associated withtoxicity beyond that from the systemic chemotherapyregimen. Patients who received sirolimus and post-transplant chemotherapy had better recurrence-freesurvival than patients who were treated with tacroli-mus and mycophenolate mofetil along with posttrans-plant chemotherapy.

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Impact of sirolimus on the recurrence of hepatocellular carcinoma after liver transplantation