Hepatocellular Carcinoma that Relapsed 54 Months after Living Donor Liver Transplantation

We report the case of a 41-year-old woman with hepatocellular carcinoma (HCC). She received living donor liver transplantation (LDLT) from her husband for HCC at 36 years of age. She had few risk factors for HCC recurrence, such as elevated alpha-fetoprotein (AFP), protein induced by vitamin K absence (PIVKA) II, vascular invasion, and number, size of tumors. However, recurrent tumors were found in the graft at 54 months after LDLT. Although we examined the sex chromosomes of the HCC by fluorescence in situ hybridization (FISH) methods, the origin of the HCC was unclear. This is a very rare case of recurrent HCC appearing more than 4 years after LDLT in the absence of risk factors for recurrence.


Introduction
In recent years, liver transplantation (LT) has become a widely performed treatment option for hepatocellular carcinoma (HCC).The Milan criteria (a single tumor <5 cm or 2 to 3 tumors all <3 cm, no extrahepatic manifestations and no vascular invasion) are widely accepted for candidate selection of LT.Yet, the recurrence of HCC after LT is an unresolved issue.There are many reports on the risk factors of HCC recurrence after LT (1)(2)(3)(4)(5)(6)(7)(8).
Tumor stage, age, AFP, PIVKA II and the pathological characteristics of tumors are reportedly closely associated with HCC recurrence.In previous studies in which the Milan criteria were applied, the tumor recurrence rate was 8% and tumor-free patient survival at 4 years was 83%, while 5-year survival rates were between 60% and 80% (9)(10)(11).In light of this, LT is only performed for recipients who do not exhibit many risks factors for recurrence.We describe herein a patient who developed HCC more than four years after living donor liver transplantation (LDLT).

Case Report
A 41-year-old woman was admitted to our department in September 2007 for close examination of a liver tumor.She had a past history of splenectomy for hypersplenism at 6 years of age.At that time, her liver was cirrhotic, although the cause of cirrhosis was uncertain because detailed examination was not performed.Endoscopic injection sclerotherapy for esophageal varices was performed at 26 years of age.She developed HCC for the first time at 30 years of age for which she underwent percutaneous ethanol injection therapy (PEIT) and trans-catheter arterial chemoembolization (TACE).At that time, her body mass index (BMI) was 23.2 and she had no other positive medical history, such as diabetes mellitus.
The HCC, however, recurred the following year.Her laboratory findings at that time were as shown in Table .We could not rule out HBV infection as the cause of cirrhosis and HCC because she was seropositive for HBc antibodies.Since the HCC was within the Milan criteria on computed  tomography (CT) and all tumor markers tested showed normal values, LDLT from her husband was performed in January 2003.Examination of the explanted liver showed that the HCC was composed of two small lesions, one of approximately 15 mm in diameter in S3 (Fig. 1A), and the other of approximately 10 mm in diameter in S8 of the liver (Fig. 1B), with no vascular invasion or metastasis.Pathologically, the tumors were moderately (S3) and well (S8) differentiated HCC, respectively.The remaining non-cancerous liver tissue was cirrhotic.
Fifty-four months after LDLT, abdominal CT showed a recurrent tumor in the liver.The following month, the patient was admitted to our department for detailed examination.She had no history of habitual alcohol consumption and no family history of liver disease, including hepatitis B viral infection.On admission, she was anicteric and her heart and respiratory sounds were normal.The liver, spleen and tumor were not palpable.Peripheral blood and biochemical tests on admission were negative for inflammation and abnormal liver function.Her liver function had been normal after LDLT.Moreover, HBV DNA was negative and HBV markers did not change before and after LDLT, although she was treated with immunosuppressive drugs after LDLT.AFP levels were within normal limits, while levels of PIVKA-II were elevated (136 mAu/mL, criterion, <40).Abdominal ultrasonography (US) and enhanced CT showed a tumor approximately 10 mm in diameter in the liver (Fig. 2A, 2B).Angiography demonstrated that the increased tumor vascularity originated from the right hepatic artery.Based on elevation of PIVKA-II and the imaging results, the patient was diagnosed with HCC and right hepatectomy was performed in October 2007.An 11-mm tumor was located in the anterior segment of the right hepatic lobe.The tumor had a fibrous capsule and its cut surface was slightly yellowish (Fig. 3A).Pathologically, the tumor was a moderately differentiated HCC (Fig. 3B), while the non-cancerous liver tissue showed mild chronic hepatitis (Fig. 3C).Fluorescence in situ hybridization (FISH) was performed to evaluate the origin of the cancer; 82.6% of the sex chromosomes of the cancer cells were X, 5.0% were XX and 12.4% were XY.In the non-cancerous areas, 0.2% of the sex chromosomes were XX, 99.4% were XY and 0.4% were YY (Fig. 4A, 4B).These findings showed that while the origin of the non-cancerous lesion was donor liver, the origin of the cancer was unclear.
Twelve months after the right hepatectomy, the tumors recurred once again.Despite various therapies, such as TACE, PEIT and medication with sorafenib tosilate, the tumors spread uncontrollably, and the patient died 99 months after LDLT, 45 months after the post-LDLT recurrence.

Discussion
A large survey conducted in Japan of 653 patients with HCC who received LDLT (3) demonstrated that 92 patients (14.1%) developed HCC recurrence after LDLT.By multivariate analysis, AFP, PIVKA II, vascular invasion, and number, distribution, and size of tumors were found to be independent risk factors for recurrence.The present patient  (8,12,13).In the above-mentioned survey, the cumulative recurrence rate after LDLT in Japan was 9.2% at 1 year, 19.9% at 3 years and 21.6% at 5 years (3).As a result, the HCC recurrence rate from 3 to 5 years after LDLT was 1.7%.The present patient developed HCC recurrence at 54 months after LDLT, which is very rare, the slowness of recurrence probably being related to her lack of risk factors for recurrence.
Recently, the existence of cancer stem cells in HCC has been proposed (14), although the details regarding these cells, such as their precise cellular origin and molecular genetics, are poorly understood.If the gender of the donor and recipient is different, it may be possible to elucidate the origin of the recurrent tumor by FISH methods (15).Although we examined the origin of HCC by FISH methods, the origin of our patient's HCC recurrence was unclear.In our patient, FISH results demonstrated that 82.6% of the sex chromosomes of the cancer cells were X, indicating that the origin of the HCC cells was abnormal tissue of indeterminate gender.Her husband, who was the donor in her case, had no risk of HCC, including no history of hepatitis B or C infection or alcohol consumption.
Although many reports have evaluated HCC patients to determine the optimal candidates for LT (1-8), HCC recur-

Figure 1 .
Figure 1.At the time of live donor liver transplantation, there were two HCC lesions in the isolated liver.One was a confluent multinodular type tumor with the histopathological diagnosis of moderately differentiated hepatocellular carcinoma (A).The other was a simple nodular type tumor with extranodular growth, histopathologically a well-differentiated hepatocellular carcinoma (B).