Objectives: To identify the predictors of overall survival and tumor-free survival of 88 hepatocellular carcinoma patients who were treated with orthotopic liver transplant at Shiraz Organ Transplant Center.

Metarials and Methods: We performed this retrospective study after reviewing the transplant database of all patients who underwent orthotopic liver transplant secondary to hepatocellular carcinoma and liver cirrhosis. Hepatocellular carcinoma was diagnosed in 70 patients before liver transplant and 18 patients on histologic examination of the explanted livers. Cox regression identified independent factors that affected post­transplant survival.

Results: The overall survival rate was 83% and the tumor-free survival rate was 79.5%. Independent factors for tumor recurrence were Milan criteria, alpha-fetoprotein level before operation ≥ 400 ng/mL, tumor grade, vascular invasion, and age. Vascular invasion (odds ratio, 5; 95% confidence interval, 1.1 to 25.496; P = .049) and tumor grade (odds ratio, 14.42; 95% confidence interval, 3.652 to 56.95; P < .001 were statistically significant.

Conclusions: Vascular invasion and tumor grade were predictive factors for tumor-free survival.

Key words : Cancer, End-stage liver disease, Outcome, Vascular

Introduction

Hepatocellular carcinoma (HCC), with an annual incidence of more than half a million cases, is the most common primary hepatic carcinoma and the fifth most common cause of cancer-related mortality globally. This neoplasm almost invariably arises in the setting of cirrhosis induced by hepatitis B virus (HBV) or hepatitis C virus (HCV) at a rate of 2.5% per year.^1,2

The incidence of HCC has doubled in the past 20 years, with an estimated 8500 to 11 000 new cases annually in the United States. The global burden of HCC is expected to double in the next few decades HCV accounted for this increase from the 1970s and 1980s. Overall, HCC is a lethal malignancy with poor outcomes regardless of treatment, with overall 5-year survival rate 20% to 40%. Moreover, survival without transplant in patients with HCC and end-stage cirrhosis is estimated at < 1 year. Chronic HCV hepatitis, chronic HBV infection, exposure to aflatoxins, tyrosinemia, exogenous hormone intake, and heavy alcohol use are important risk factors associated with the development of HCC.^3,4

Orthotopic liver transplant (OLT) remains the preferred curative modality for patients with cirrhosis and HCC because it removes the tumor and premalignant cirrhotic liver, which is the main risk factor for recurrence. Nonetheless, recurrence after OLT is occurring with growing evidence, and many reports suggest that the tumor biology of HCC (perhaps the most important predictor of HCC recurrence) can be highly variable. The HCV, as the most important risk factor for HCC, typically affects patients who are older, have more advanced liver disease, and have higher numbers of neoplastic lesions at diagnosis, and HCV leads to higher risk of HCC recurrence after resection.⁵

Various factors (such as size and number of lesions) should be identified at the time of patient selection to reduce recurrence of HCC after liver transplant. Based on this fact, several criteria have been introduced such as Milan criteria (single tumor diameter ≤ 5 cm or 3 tumors with diameter ≤ 3 cm) and University of California, San Francisco (UCSF) criteria (single tumor < 6.5 cm; maximum of 3 tumors with the largest < 4.5 cm; and cumulative tumor size < 8 cm).2 A study conducted by Yao et al. showed several important predictors of survival in univariate analysis such as α-fetoprotein (AFP) level > 1000 ng/mL, total tumor diameter > 8 cm, age ≥ 55 years, and poorly differentiated histologic grade. Only tumor stage and total tumor diameter remained statistically significant in multivariate analysis.⁶

Some studies showed that liver transplant offers better outcomes than other strategies, with expected 5-year survival 70% to 90%. Despite these good results, 10% patients experience posttransplant HCC recurrence, many ending with death.⁷

Although the true prevalence of HCC in Iran is unknown, it is not an uncommon malignancy; 80% HCC cases in Iran are positive for at least 1 of the markers of HBV, and this virus appears to be the most common cause of HCC in Iran.⁸

The primary aim of our study was to identify the potential predictors of survival and tumor-free survival in a cohort of 88 HCC patients who were treated with OLT at Shiraz Organ Transplant Center between 2008 and 2013.

Materials and Methods

Patients
We performed this retrospective study after reviewing the transplant database of all patients who underwent OLT secondary to HCC and liver cirrhosis at Nemazee Hospital, Shiraz, Iran, a referral organ transplant center. Between January 2008 and December 2013, approximately 1000 liver transplants were performed at Nemazee Hospital. To ensure adequate follow-up, only patients who received liver transplant before December 31, 2013 were enrolled in the study. We selected the patients according to Milan criteria. With some exceptions, patients meeting UCSF criteria by preoperative imaging were included in the study. If OLT was not feasible according to UCSF criteria, patients were considered for percutaneous ethanol injection, transarterial chemoembolization, and/or radiofrequency ablation. Patients who had successful down-staging and came within UCSF criteria were enrolled in the transplant list. In the 5 patients (5.7%) who had successful down-staging, 3 patients received transarterial chemo­embolization and 2 patients received radio­frequency ablation.

Tumor-related criteria for exclusion were gross hilar involvement and extrahepatic spread based on computed tomography (CT) or ultrasonography (US). Vascular involvement also was recognized after transplant by the pathologist.

Study design
According to the Child-Turcotte-Pugh classification of cirrhosis, patients with Child class A or B who had HCC as the primary indication for OLT were diagnosed based on histologic findings or imaging evidence of tumor formation in the liver (with arterial hypervascularization) on at least 2 imaging techniques, CT and US or magnetic resonance imaging. In patients with Child class C, the diagnosis of HCC was based on imaging evidence of a focal liver lesion on at least 2 imaging techniques. Vascular invasion was assessed by Doppler US and/or contrast-enhanced CT scan. To exclude extrahepatic metastasis before OLT, bone scintigraphy, brain CT, and thoracic CT were performed. Serum AFP levels were repeatedly measured in all cases before and after transplant. The explanted livers were fixed in formalin, cut into slices (thickness, 1 cm), and examined by an experienced pathologist. Parameters recorded included the number, site, and maximum diameter of all tumor nodules, capsule formation, presence of vascular invasion, and degree of differentiation (well, moderately, or poorly differentiated). Incidental (or undetected) HCCs were defined as carcinomas identified only on pathologic evaluation of the explanted liver in patients with negative pretransplant evaluation for focal liver lesions. Macroscopic vascular invasion was defined by gross involvement of the lobar or segmental branches of the portal or hepatic veins. Microscopic vascular invasion was defined by the presence of tumor emboli within the central hepatic vein, portal vein, or large capsular vessels.

Follow-up
The OLT recipients were screened for tumor recurrence every 3 months by US and measurement of AFP level and every 12 months with abdominal CT scan and chest radiography. Thoracic or cerebral CT scans and radionuclide bone scans were performed only when there was a suspicion or evidence of extrahepatic neoplastic disease.

Immunosuppression and antiviral protocols
Immunosuppressive therapy after OLT consisted of a triple drug regimen with calcineurin inhibitors (tacrolimus or cyclosporine), mycophenolate mofetil (MMF), and prednisolone which were administered to all patients. Prednisolone was administered as methylprednisolone (1 g/d for 3 days; maintenance dose, 20 mg per day for 1 week, then gradually tapered and discontinued in most patients during the next 6 months). Cyclosporine dose adjustments were based on blood concentrations, aiming at drug levels from 200 to 350 ng/mL during the first month and 150 to 250 ng/mL thereafter. For tacrolimus, the drug blood level was adjusted to 10 to 15 ng/mL in the first month and < 10 ng/mL thereafter. The MMF was started with 2 to 3 g/d and reduced according to the patient’s laboratory data. After the first post­transplant month, sirolimus (whole blood trough level aimed at 6 to 10 ng/mL) was added and the doses of prednisolone and calcineurin inhibitors were decreased. Acute rejection episodes were treated with 3 to 5 episodes of steroid pulses.

Hepatitis B immunoglobulin was administered to all HBV-infected patients. Antiviral drugs such as lamivudine and/or tenofovir were added to the treatment regimen of patients who were positive for hepatitis B surface antigen.

We assessed demographics, laboratory status, tumor characteristics, and transplant technique (piggy-back or standard technique) applied for 88 recipients in our study (Table 1). Patient survival from time of transplant to the time of HCC recurrence and death were selected as end points in our study.

Statistical analyses
Cox proportional hazards regression model was performed to identify independent factors that affected posttransplant survival. All analyses were performed using statistical software (SPSS, version 16.0, SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at P ≤ .05.

Results

Average age was 46.6 ± 18 years; 70 patients (79.5%) were men and 18 patients (20.5%) were women. Underlying liver disease was present in all patients and most commonly was caused by HBV. The presence of tumor was confirmed before transplant in 70 patients (79.5%) and incidentally at OLT in 18 patients (20.5%) on histologic examination of the explanted livers (incidental HCC) (Table 1). In the latter group of patients, imaging studies performed before OLT did not reveal the presence of HCC. In this study, 62 patients (70.5%) were within Milan criteria and 26 patients (29.5%) were beyond Milan criteria; 74 patients (84.1%) were within UCSF criteria and 14 patients (15.9%) exceeded UCSF criteria (Table 2). The median Model for End-Stage Liver Disease (MELD) score was 22 (mean, 20.8 ± 3.4). Serum AFP levels were obtained before transplant, with median value 124 ng/mL; 23 patients (32%) had AFP ≥ 400 ng/mL. The overall survival rate was 83% and tumor-free survival rate was 79.5%.

Recurrence characteristics
To understand the cause of recurrence, the profile of each patient with recurrence was assessed independently. In our entire cohort of 88 patients who underwent liver transplant, tumor recurrence was detected in 18 patients (20.5%). Recurrence appeared from 3 to 35 months after transplant (mean, 11.3 ± 9 mo).

In 18 patients with HCC recurrence, sites of recurrence were the liver (8 patients), pancreas (2 patients), adrenal (1 patient), pelvis (1 patient), scalp (1 patient), lung and liver (2 patients), skin and liver (1 patient), and liver and extrahepatic lymph nodes (2 patients). There were 8 deaths (44.4%) in 18 patients with HCC recurrence during follow-up.

There was no significant association between liver transplant technique (piggy-back vs standard technique), site of HCC in the liver (right, left, or bilobar), type of transplant (deceased- vs living donor liver transplant) and tumor recurrence (Table 1).

The overall survival rates of patients with incidental and nonincidental HCC were 94.4% and 80%. Tumor-free survival rates of patients with incidental and nonincidental HCC were 94.4% and 75.7%. Recurrence of HCC occurred in 8 patients beyond Milan criteria (30.8%) and 5 patients beyond UCSF criteria (35.7%), but there was no significant difference (Table 2).

Multivariate Cox regression analysis was performed with the following variables: Milan criteria, AFP levels before operation ≥ 400 ng/mL, tumor grade, age, and vascular invasion to predict tumor-free survival and overall survival. Multivariate analysis showed that vascular invasion (odds ratio [OR], 5; 95% confidence interval [CI]: 1.1 - 25.496; P= .049) and tumor grade (OR, 14.42; 95% CI: 3.652 - 56.95; P < .001) were independent predictors of tumor-free survival. Multivariate analysis also showed that vascular invasion (OR, 7.14; 95% CI: 1.781 - 28.623; P = .006) and age ≥ 60 years (OR, 3.25; 95% CI: 1.078 - 9.811; P = .036) were independent predictors of overall survival.

Discussion

Liver transplant is the best available option for the treatment of HCC, with > 70%, 5-year survival and low recurrence rates (< 10%). However, despite various patient selection criteria available, recurrence following transplant is inevitable and a major challenge. Moreover, there are few treatment strategies available for HCC recurrence and no absolute adjuvant therapy. Following the introduction of the Milan criteria in 1996, application of OLT in HCC patients has been limited by restrictive selection criteria. Proposals to expand the selection criteria from transplant centers include UCSF criteria, Hangzhou criteria, and up to 7 criteria, and these have made patients with larger tumors eligible for liver transplant.^9,10 The OLT also is limited by a shortage of donors, which facilitates tumor progression while waiting for transplant and might lead to poor patient survival.²

Although the expanded criteria (UCSF criteria) for liver transplant have provided satisfactory results in several consensus studies, several unclear issues remain for discussion. Tumor size is one of the most important parameters, and there is no clear boundary for tumor diameter with the lowest recurrence rate. Radiographic imaging under­estimates tumor size in 27% to 49% cases. However, there are other important factors in tumor pathology that could aid patient selection, such as DNA heterogeneity, tumor differentiation degree, and vascular invasion, but pretransplant biopsy is required for detection of these factors and as such risks tumor spread.¹¹

In the present study, the overall survival (83%) and tumor-free survival (79.5%) were within the expected range. However, we observed a higher rate of HCC recurrence (20.5%), with most (70.5%) within the Milan criteria at transplant time. This result is higher than reported in comparable studies from Switzerland (12.4%), Canada (13.1%), and the United States (18.3%).7,12 A study from Korea reported a higher rate of recurrence than our study (22.4%).¹³

Currently, HCC is one of the major indications for OLT, with expected recurrence rates between 15% to 20% and less than 10% in patients who meet the Milan criteria, and overall survival rate of patients within Milan criteria is over than 70% after liver transplant.^10,14 In patients in our study, the tumor-free survival rate for tumors within Milan criteria was 83.9% and beyond Milan criteria was 69.2%. However, in our study there was no significant difference in survival of patients who were within or beyond Milan criteria; this finding could be explained by small sample size. The successful outcome of OLT with HCC patients and also presence of several consensus studies demonstrated that patient survival was not worse following OLT in tumors exceeding the Milan criteria.¹⁵

When comparing risk factors for posttransplant recurrence, a study by Lai et al. identified 4 independent factors: microvascular invasion, poor tumor grading, diameter of the largest tumor, and previous liver resection.¹⁶ Another study using Cox multiple regression analysis revealed that only histologic grading could negatively affect recipient survival.¹⁷

Based on our analysis, histologic grade of differentiation and vascular invasion were strong independent predictors of tumor-free survival. Moreover, we observed that age and vascular invasion were predictive factors for overall survival.A meta-analysis of tumor recurrence of HCC after liver transplant reported that patients who had tumors within the Milan criteria had 8% recurrence and patients with tumor pathology outside these criteria had a much higher incidence of recurrence (50%). These results differed from our findings, suggesting that characteristics of the pathologic evaluation of the resected specimen should be used to stratify screening.¹⁸

There were 2 main limitations of our study, including the small number of cases of OLT due to HCC in our center and short follow-up because the liver transplant program for HCC patients was started only 5 years ago at Shiraz University Hospital.

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