Hepatol Int (2013) 7:468–476 DOI 10.1007/s12072-013-9422-7
ORIGINAL ARTICLE
Clinicopathologic study on complications of orthotopic liver transplantation in 54 patients with chronic hepatitis B viral infection Fei Pei • Kewei Shang • Bin Jiang • Hua Wang • Fang Mei • Yan Zhang • Juan Du • Haohao Zhong Jiangfeng You
•
Received: 21 July 2012 / Accepted: 6 January 2013 / Published online: 7 February 2013 Ó Asian Pacific Association for the Study of the Liver 2013
Abstract Objective To study the complication incidence of 54 patients with chronic HBV infection following orthotopic liver transplantation (OLT) and risk factors associated with HBV recurrence and hepatocellular carcinoma (HCC) recurrence or metastasis post-OLT. Methods The light-microscopic appearance of hepatic allograft biopsies in 54 patients with chronic HBV infection following OLT was examined. The related clinical data were analyzed. The incidence and occurrence time of post-OLT complications were studied. Furthermore, the relationship between hepatitis B virus recurrence and acute rejection and the relationship among HCC recurrence/ metastasis, acute rejection, tumor diameter, and portal vein invasion were particularly studied. Results Frequent complications of patients with chronic HBV infection following OLT were acute rejection [38 (70.4 %); occurrence time: 5–365 days], chronic rejection [1 (1.9 %); occurrence time: 10.7 months], bile duct complications [24 Kewei Shang contributed equally to the research. F. Pei (&) K. Shang H. Wang F. Mei Y. Zhang J. Du H. Zhong J. You Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xue Yuan Road, Haidian, Beijing 100191, People’s Republic of China e-mail: [email protected] K. Shang e-mail: [email protected] H. Wang e-mail: [email protected] F. Mei e-mail: [email protected] Y. Zhang e-mail: [email protected]
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(44.4 %);occurrence time: 7–940 days], HBV recurrence [7 (13.0 %); occurrence time: 1–540 days], HCV infection [3 (5.6 %); occurrence time: 60 days, 60 days, 33 months], CMV infection [8 (14.8 %); occurrence time: 67–90 days], and HCC recurrence or metastasis [17 (31.5 %); occurrence time: 2–41 months]. At the end of 1 year post-OLT, 95 % of patients with post-hepatitis B cirrhosis were alive. At the end of 3 years post-OLT, 85 % of patients with post-hepatitis B cirrhosis were alive. However, at the end of 1 year post-OLT, 67.6 % of patients with post-hepatitis B HCC were alive. At the end of 3 years post-OLT, 50 % of patients with post-hepatitis B HCC were alive. The number of acute rejection episodes in patients with recurrent HBV infection and in those without recurrent HBV infection was 0.86 ± 1.46 times/patient and 1.07 ± 0.90 times/patient, respectively (p [ 0.05); the number of moderate acute rejection episodes (RAI score C4) in patients with recurrent HBV infection and in those without recurrent HBV infection was 0.29 ± 0.49 times/patient and 0.50 ± 0.63 times/patient (p [ 0.05). Incidence of patients with C3 episodes of acute rejection in patient with recurrent HBV infection and in those without recurrent HBV infection was 14.3 and 10.6 % J. Du e-mail: [email protected] H. Zhong e-mail: [email protected] J. You e-mail: [email protected] B. Jiang Orthotopic Liver Transplantation Center from the Department of General Surgery, Peking University Third Hospital, Beijing 100191, People’s Republic of China e-mail: [email protected]
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(p [ 0.05). Furthermore, the number of acute rejection episodes in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 1.12 ± 0.93 times/ patient and 1.06 ± 1.39 times/patient, respectively (p [ 0.05). The number of moderate acute rejection episodes (RAI score C4) in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 0.65 ± 0.79 times/ patient and 0.65 ± 1.06 times/patient, respectively (p [ 0.05). Incidence of patients with C3 episodes of acute rejection in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 5.9 and 17.6 %, respectively (p [ 0.05). The tumor diameter in patients with HCC recurrence or metastasis was 6.72 ± 3.40 cm; however, that in patients without HCC recurrence or metastasis was 3.55 ± 2.17 cm (p = 0.0047). The incidence of portal vein invasion in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 68.75 and 33.3 %, respectively (p = 0.006). Conclusion There was no significant difference between HBV recurrence and acute rejection post-liver transplantation in patients with chronic HBV infection. There was no significant difference between HCC recurrence and acute rejection. The tumor diameter in patients with HCC recurrence or metastasis was significantly greater than that in patients with no HCC recurrence or metastasis. Portal vein invasion was significantly more frequent in patients with HCC recurrence or metastasis than in those with no HCC recurrence or metastasis. Keywords Hepatitis B Liver transplantation Postoperative complications Graft rejection Liver neoplasms
Introduction Since liver transplantation was first carried out by Starzl, an American, on 1 March 1963, orthotopic liver transplantation(OLT) has been widely used worldwide as the only effective treatment of end-stage liver disease. In China, liver transplantation started in 1977 and has made great progress with technological advances and improvement of living standards in recent years. Nearly 3,000 liver transplants per annum are currently performed in China, which ranks second, only behind the USA, among countries carrying out the largest numbers of liver transplants [1]. As China has a high incidence of hepatitis B, with an infection rate of 1/10, 90 % of patients undergoing liver transplantation are hepatitis B virus (HBV)-infected young adults with liver cirrhosis or hepatocellular carcinoma (HCC). Little research has been done on complications and risk factors associated with HBV recurrence and
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hepatocellular carcinoma (HCC) recurrence or metastasis post-OLT.
Materials and methods Basic data The hepatic allograft biopsies of 54 patients at Peking University Third Hospital with chronic HBV infection following OLT were analyzed retrospectively. The patients were operated on in August 2000–2004. Liver lesions of these patients were all caused by HBV infection before OLT. Related clinical data were analyzed thoroughly. The incidence and occurrence time of post-OLT complications were studied. Furthermore, the relationship between recurrent type B viral hepatitis and acute rejection and the relationship among HCC recurrence/metastasis, acute rejection, tumor diameter, and the existence of portal vein invasion were particularly studied. Pathologic diagnosis Pathologic diagnosis of acute rejection Pathologic diagnosis of acute rejection included: (1) mixed inflammatory cell infiltration in portal tracts of activated lymphocytes (lymphoblasts), lymphocytes(mainly T cells), macrophages, plasma cells, neutrophils and eosinophils, etc.; (2) biliary inflammation and bile duct damage characterized by inflammatory cell infiltration around the bile duct, biliary epithelial injury, as well as formation of vacuoles in the cytoplasm, size and shape change of the nucleus, an increased nuclear:cytoplasmic ratio, and apoptosis of biliary epithelial cells; (3) subendothelial inflammation of the interlobular veins and central veins, recognized by inflammatory cell infiltration of the venule walls, attachment of lymphocytes to epithelial cells, and swelling and shedding of epithelial cells. A semiquantitative approach to grade the severity of acute rejection, the Banff schema [2], was adopted: rejection activity index (RAI) = score 0–2: not diagnosed as acute rejection; RAI = score 3: suspected acute rejection; RAI = score 4–5: mild acute rejection, with \50 % portal tracts infiltrated by inflammatory cells, inflammatory cells restricted in the portal tracts, and occasional mixed inflammatory infiltration; RAI = score 6–7: moderate acute rejection with C50 % portal tracts infiltrated by inflammatory cells, moderate mixed inflammatory infiltration including a moderate number of eosinophils, interlobular bile duct inflammation, and venous subendothelial inflammation, and the junction of the portal tracts and hepatic lobule is still regular and smooth; RAI = score 8–9: severe acute
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rejection, characterized by C50 % portal tracts infiltrated by inflammatory cells with hepatic lobules involved, mixed inflammatory infiltration densely observed in the portal tracts with hepatic lobules affected, giving the inter-surface between the portal tracts and hepatic lobule an irregular appearance, obvious bile duct damage and venous subendothelial inflammation, and spotty necrosis of hepatocytes in the hepatic lobules (Fig. 1). Pathologic diagnosis of chronic rejection The pathologic diagnosis of chronic rejection includes: (1) more than 50 % of bile ducts within the portal tracts are lost (a firm diagnosis requires at least 20 portal tracts); (2) arteriopathic changes with foam cells within the intima of large arterial walls in the portal tracts and intimal thickening. Foam cells accumulate in the arterial lumens (Fig. 2). Pathologic diagnosis of HBV recurrence post-OLT Pathologic diagnosis of HBV recurrence post-OLT includes: (1) obvious mononuclear cell infiltration within portal tracts; (2) damage of hepatic plates (Fig. 3a); (3) local hepatic fibrosis; (4) necrosis of hepatocytes in the hepatic lobules with mononuclear cell infiltration; (5) appearance of ground-glass hepatocytes and sand-like hepatocyte nuclei caused by the accumulation of HBsAg in sER and hepatocyte nuclei; (6) immunohistochemistry can be used to identify HBsAg in the cytoplasm and cytomembrane of hepatocytes, which is positively correlated with the severity of hepatitis (Fig. 3b).
Fig. 2 Chronic rejection. The obliterative arteriopathy in a hilar artery is characterized by luminal narrowing because of intimal thickening. The intimal thickening is related to deposition of foamy macrophages and proliferation of myofibroblasts (H&E 910)
hepatocytes: among the viral hepatitises, Mallory bodies are only seen in case of chronic HCV infection; (3) the formation of hepatocyte apoptotic bodies in the hepatic lobules; (4) proliferation of Kupffer cells within the hepatic lobules; (5) accumulation of lymphocytes and formation of lymphoid follicles in the portal tracts as well as damage of interlobular bile ducts; (6) HCV RNA can be detected in the cytoplasm of hepatocytes using in situ hybridization (Fig. 4b). Clinical medicine and laboratory tests
Pathologic diagnosis of hepatitis C virus (HCV) infection includes: (1) mild to moderate macrovesicular hepatic steatosis (Fig. 4a); (2) Mallory bodies appear in the damaged
These patients were all HBsAg-positive before surgery and started using lamivuding 100 mg/day 1–12 months before transplantation. Furthermore, these patients received intravenous HBIG 10,000 during the anhepatic phase, then 2,000 IU daily for the next 7 days, then 2,000 IU monthly. At the same time, the patients received lamivudine 100 mg/day immediately after transplantation and remained on the drug. Cefotaxime (2 g every 8 h, intravenous drip) and ampicillin (1 g every 6 h, intravenous drip) were used as
Fig. 1 Acute rejection. a A male patient, 56 years old, day 30 after OLT. Allograft biopsy shows mild acute rejection (RAI: score 4). b A male patient, 56 years old, day 9 after OLT. Allograft biopsy shows
moderate acute rejection (RAI: score 6). c A female patient, 45 years old, day 90 after OLT. Allograft biopsy shows severe acute rejection (RAI: score 9) (H&E 910)
Pathologic diagnosis of hepatitis C virus (HCV) infection
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Fig. 3 Recurrent chronic hepatitis B. a Portal inflammation, interface hepatitis, and focal fibrosis (H&E 910). b Positive membrane and cytoplasmic staining for hepatitis B surface antigen (immunoperoxidase stain, IHC 920)
Fig. 4 Chronic hepatitis C. a Macrovesicular hepatic steatosis, acidophilic body with partially extruded nucleus, mononuclear portal inflammation, and lymphoid aggregates with terminal centers (H&E 910). b Positive cytoplasmic staining for HCV RNA (in situ hybridization, 920)
perioperative prophylactic antibiotics. If there were no signs or symptoms of infection, these agents were discontinued after 5 days. No prophylactic medication for fungal or viral infections was given. All post-OLT patients were treated conventionally with immunosuppressive combination therapy of tacrolimus (FK506) ? methylprednisolone ? mycophenolate mofetil (MMF) to suppress rejection. Intravenous methylprednisolone (intravenous dose of 1,000 mg of methylprednisolone followed by a 6-day taper from 200 to 20 mg/day) is used in our liver transplant center as first-line therapy for acute hepatic cellular rejection in patients who undergo liver transplants. Most take lamivudine orally and human hepatitis B immunoglobulin (HBIG) is injected to prevent the recurrence of HBV. All hepatitis B recurrences postOLT were confirmed by viral serological examination, fluorescence quantitative PCR of serum HBV DNA, liver biopsy, and immunochemical staining of HBsAg. PCR restriction fragment length polymorphism (RFLP) was adopted to determine whether the HBV DNA polymerase gene had a YMDD mutation [3]. All hepatitis C (HC) occurrences post-OLT were confirmed by viral serological examination, fluorescence quantitative PCR of serum HCV DNA, liver biopsy, and HCV RNA in situ hybridization [4].
All cytomegalovirus (CMV) infections post-OLT were identified by serological examination of CMV IgM and fluorescence quantitative PCR of serum CMV DNA [5]. Statistical methods Results were expressed as the mean and standard deviation. Survival curves were analyzed using SPSS 13.0 statistical software. Differences between groups were assessed by chi-square or Student’s t test, with p values \0.05 considered significant.
Results General All 54 patients with chronic HBV infection pre-OLT survived for more than 2 months post-OLT. Their average age was 48.11(±9.59) years (24–67); 49 were male (90.7 %) and 5 female (9.3 %). Follow-up post-OLT lasted until August 2007. Follow-up of 20 patients with post-hepatitis B cirrhosis required at least 3 years. Among them, 17 patients survived, and the longest follow-up was 74 months. Only
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three patients died, one of biliary complications (cholestatic cirrhosis) 36 months post-OLT, one of gastrointestinal bleeding and hepatic encephalopathy due to hepatitis B recurrence and hepatic cirrhosis 24 months post-OLT, and one of septicemia and multiple organ failure 2 months post-OLT. The 1-year post-OLT survival rate was 95 % (19/20), 2-year survival was 90 % (18/20), and 3-year survival was 85 % (17/20). Survival curves are shown in Fig. 5. Follow-up of 34 patients with post-hepatitis B hepatocellular carcinoma (HCC) required as least 3 years. Of the patients, 15 survived, and the longest follow-up was 84 months. Nineteen patients died. The 1-year survival rate post-OLT was 67.6 % (23/34), 2-year survival 58.1 % (18/ 34), and 3-year survival 50 % (17/34). Causes of deaths were as follows: HCC recurrence/metastasis in 13 patients (68.4 % of all deaths), biliary complications in 4 patients (21.1 % of all deaths), and infection in 2 patients (10.5 % of all deaths). Survival curves are shown in Fig. 5. Complications post-OLT (Table 1) Acute rejection Among 54 patients post-OLT, 38 experienced acute rejection, accounting for 70.4 %. Twenty-nine patients had acute rejection once (53.7 %); three had acute rejection twice (5.6 %); three had acute rejection three times (5.6 %); two had acute rejection four times (3.7 %); one had acute rejection five times (1.9 %). The number of patients with RAI scores C4 who had acute rejection was 21 (38.9 %). The first occurrence time of acute rejection for 38 patients was 64.67 (±98.62) days (5–365 days). The number of patients with first occurrence time B30 days was 25 (65.8 %), from 30 to 60 days was 4 (10.5 %), from 60 days to 6 months was 4 (10.5 %), and from 6 months to 1 year was 5 (13.2 %). Fig. 5 Survival curves of 54 patients with chronic HBV infection following orthotopic liver transplantation
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Chronic rejection Among 54 patients, only 1 had chronic rejection (1.9 %) 10.7 months post-OLT, complicated by miliary hepatic tuberculosis and chronic cavitary pulmonary tuberculosis. Biliary complications Twenty-four of 54 patients had biliary complications (44.4 %), which mainly included biliary stricture and obstruction. The occurrence time was 168.4 (±231.7) days (7–940 days) post-OLT. The patients with biliary complications underwent endoscopic biliary stricture dilatation and stent placement. Hepatitis B recurrence Seven of 54 patients experienced hepatitis B recurrence (13.0 %), the occurrence time of which was 344 (±206) days (1–540 days) post-OLT. Six patients had YMDD wild-type HBV, and one had HBV DNA polymerase gene YIDD mutation. Causes of hepatitis B recurrence were that three patients did not take or self-withdraw lamivudine for economic reasons; two adhered to lamivudine, but did not receive intramuscular HBIG injections; one was not responsive to lamivudine because of HBV DNA polymerase gene mutation; one had high-copy HBV DNA in serum and was controlled ineffectively. HCV infection Three of 54 patients were infected with HCV (5.6 %). The occurrence times were 60 days, 60 days, and 33 months, respectively. There was no hepatitis C viral coinfection in patients with chronic hepatitis B virus infection before or after liver transplantation, and the hepatitis C infection was diagnosed after OLT perhaps
Hepatol Int (2013) 7:468–476 Table 1 Post-OLT outcomes of 54 patients with chronic HBV infection
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Complications
Incidence (%)
Acute rejection
70.4
Chronic rejection
1.9
Bile duct complications
44.4
HBV recurrence
13.0
HCV infection
5.6
Occurrence time (days) 64.67 ± 98.62 days (5–365) 10.7 M 168.4 ± 231.7 days (7–940) 344 ± 206 days (1–540) 60 days, 60 days, 33 months
CMV infection
14.8
77.6 ± 10.4 days (67–90)
Bacterial and fungal infection
24.1
15 ± 45 days (5–60)
Vascular complications
14.8
56 ± 66 days (1–180)
Recurrent hepatocellular carcinoma
31.5
9.6 ± 10.4 months (2–41 months)
because of blood transfusions. Furthermore, the patients with hepatitis C infection underwent interferon alpha-2b (IFN-a) therapy after OLT. CMV infection Eight of 54 patients were infected with CMV (14.8 %). The occurrence time was 77.6 (±10.4) days (67–90 days) post-OLT. The patients did not undergo a prophylactic or preemptive therapy in the perioperative phase. It was not clear whether the patients who experienced a CMV infection belonged to risk groups (donor? or recipient?) because no serological examination for CMV was carried out before OLT. Furthermore, after OLT the CMV infection patients were treated with valganciclovir (900 mg twice daily) until three consecutive PCRs were negative. PCR was measured every 2 weeks during treatment. All patients had a quick recovery, and recurrent viremia was not observed. Bacterial and fungal infections Thirteen of 54 patients were infected post-OLT(24.1 %), among whom 2 were infected with fungi (3.7 %), 10 with common bacteria (18.5 %), and 1 with mycobacterium tuberculosis (hepatic tuberculosis) (1.9 %) who had a thick wall cavity in the lung pre-OLT. The diagnosis of bacterial or fungal infection was usually made in the first 2 months at a mean time of 15 days after OLT. The occurrence time was 15 ± 45 days (5–60 days). Two patients died within 6 months after transplantation because of Staphylococcus aureus infection or disseminated cryptococcosis. One patient with mycobacterium tuberculosis received a second OLT and died 20 months after the first transplantation. Portal vein or hepatic artery thrombosis, occlusion, or stenosis Eight of 54 patients experienced vascular complications (14.8 %), the occurrence time of which was 56
(± 66) days (1–180 days) post-OLT. The clinical presentation of vascular complications included elevated transaminases in all patients, biliary complications in two patients, portal hypertension with gastrointestinal bleeding in one patient, fever and sepsis in one patient, and graft dysfunction or failure in two patients. We observed two cases (3.7 %) of acute early hepatic artery thrombosis; one qualifed immediately for liver retransplantation, and the other underwent endovascular therapies, including intraarterial heparin infusion and percutaneous transluminal angioplasty with stent placement. The patients survived. Portal venous complications were seen in six patients (11.1 %), and they received anticoagulation without surgical therapy. HCC recurrence/metastasis post-OLT Seventeen of 34 patients with post-hepatitis B hepatocellular carcinoma had HCC recurrence/metastasis (50 %). The occurrence time was 9.6 (±10.4) months (2–41 months) post-OLT. Relationship between hepatitis B recurrence and acute rejection of patients with chronic HBV infection (Table 2) Among seven patients with hepatitis B recurrence, three experienced acute rejection. The number of acute rejection episodes was 0.86 (±1.46) times/patient. One patient had C3 episodes of acute rejection. The number of moderate acute rejections (RAI score C4) was 0.29 (±0.49) times/ patient. Among 47 patients without hepatitis B recurrence, 35 experienced acute rejection. The number of acute rejection episodes was 1.07 (±0.90) times/patient. Five patients had C3 episodes of acute rejection. The number of moderate acute rejections (RAI score C4) was 0.5 (±0.63) times/patient. The difference was not statistically significant (p [ 0.05). It was indicated that hepatitis B recurrence and acute rejection in patients with chronic HBV infection were not related.
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Factors associated with HCC recurrence/metastasis post-OLT (Table 3) Thirteen of 17 patients with HCC recurrence/metastasis post-OLT experienced acute rejection (76.5 %). The number of acute rejection episodes was 1.12 (±0.93) times/patients. One patient had C3 episodes of acute rejection (5.9 %). The number of moderate acute rejections (RAI score C4) was 0.65 (±0.79) times/patient. Eleven of 17 patients without HCC recurrence/metastasis post-OLT experienced acute rejection (64.7 %). The number of acute rejection episodes was 1.06 (±1.39) times/patients. Three patients had acute rejection C3 episodes (17.6 %). The number of moderate acute rejections (RAI score C4) was 0.65 (±1.06) times/ patient. The difference was not statistically significant (p [ 0.05). Indications showed HCC recurrence/metastasis post-OLT and acute rejection of patients with post-hepatitis B HCC were not related and that HCC recurrence/metastasis post-OLT of patients with post-hepatitis B HCC was not related to the existence of hepatic satellite nodules. Research has discovered that post-OLT HCC recurrence/metastasis in patients with post-hepatitis B HCC is not related to HCC differentiation (p [ 0.05). The difference in HCC diameter pre-OLT between that of patients with and without HCC recurrence/metastasis was statistically significant (6.72 ± 3.40 cm vs. 3.55 ± 2.17 cm; p = 0.0047). The difference in the occurrence rate of portal vein invasion was also statistically significant (68.75 % vs. 33.3 %, p = 0.006). It is suggested that post-OLT HCC recurrence/metastasis of patients with post-hepatitis B HCC is directly linked to the existence of large tumors and portal vein invasion. In addition, two patients with portal vein invasion and large HCCs underwent hepatic artery thrombosis and radiofrequency ablation therapy pre-OLT. HCC recurrence/metastasis did not occur after OLT. Both patients survived, and follow-ups lasted 60 and 62 months, respectively.
Discussion Complications in patients with chronic HBV infection post-OLT are acute rejection, chronic rejection, biliary
complications, HBV recurrence, HCV infection, CMV infection, and HCC recurrence/metastasis. The present study, the number of patients who experienced acute rejection post-OLT was 38, accounting for 70.4 % of the 54 patients with chronic HBV infection. The first occurrence time was 64.67 (±98.62) days. Acute rejection is one of the most common complications at the early stage following OLT, which often occurs 5–30 days post-OLT, especially at 7–14 days, but can also occur any time post-OLT and is usually related to viral infection or inadequate doses of immunosuppressive agents [6]. Acute rejection is mediated by cellular immunity. Antigen-presenting cells, dendric cells in the transplanted liver, activate T lymphocytes in recipients. Forty-eight hours after OLT, T lymphocytes can be observed in the transplanted liver clustering around the dendric cells, transforming to lymphoblasts, and secreting cytokines (like IL-2 and IL-5, etc.), which promote T lymphocyte division and proliferation, attracting immune effector cells (including eosinophils, neutrophils, macrophages, plasmocytes, etc.) to accumulate locally, proliferate, and differentiate, thus playing their biological roles. The present study, only 1 (1.9 %) of the 54 patients experienced chronic rejection, which occurred 10.7 months post-OLT. The patient also had complications of miliary hepatic tuberculosis and chronic cavitary pulmonary tuberculosis. Chronic rejection might be linked to the patient’s tuberculosis. Infection may stimulate autoimmune reaction. Anti-tuberculosis drugs can induce the liver to produce microsomal enzymes, accelerate hormone catabolism, and at the same time enhance the activity of P450 enzymes and speed up the catabolism of anti-rejection drugs; as a consequence, the plasma concentration is reduced, which may induce repeated occurrence of acute rejection and lead to chronic rejection in the end [7] . Of 54 patients, 24 (44.4 %) had biliary complications, the causes of which were as follows: (1) organic obstruction due to technical problems with the extrahepatic bile duct anastomosis; (2) intrahepatic bile duct stricture caused by technical problems with the hepatic artery anastomosis or thrombosis, and intrahepatic bile duct ischemia and hypoxia.
Table 2 Factors associated with HBV recurrence post-liver transplantation in patients with chronic HBV infection Evident HBV recurrence (n = 7)
No HBV recurrence (n = 47)
p value
Number of patients with rejection episodes
3 (42.9 %)
35 (74.5 %)
[0.05
Number of rejection episodes/patient
0.86 ± 1.46
1.07 ± 0.90
[0.05
Number of patients with C3 episodes of rejection
1 (14.3 %)
5 (10.6 %)
[0.05
Number of moderate rejection episodes/patient(RAI score C4)
0.29 ± 0.49
0.5 ± 0.63
0.32
There was no significant difference among HBV recurrence, acute rejection, and CMV infection post-liver transplantation in patients with chronic HBV infection (p [ 0.05)
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Table 3 Factors associated with hepatocellular carcinoma recurrence post-liver transplantation in patients with chronic HBV infection HCC recurrence or metastasis (n = 17)
No HCC recurrence or metastasis (n = 17)
p value
13 (76.5 %)
11 (64.7 %)
[0.05
Number of rejection episodes/patient
1.12 ± 0.93
1.06 ± 1.39
0.83
Number of patients with C3 episodes of rejection
1 (5.9 %)
3 (17.6 %)
0.20
Number of moderate rejection episodes/patient (RAI score C4)
0.65 ± 0.79
0.65 ± 1.06
1
Tumor diameter(cm)
6.72 ± 3.40
3.554 ± 2.17a
0.0047
Portal vein invasion
68.75 %
33.3 %b
0.006
Satellite tumor nodules Histologic grading of HCC
68.75 % Low grade: 6.25 %
47.1 % Low grade: 5.88 %
0.088 0.05
Moderate grade: 87.5 %
Moderate grade: 82.36 %
High grade: 6.25 %
High grade: 11.76 %
Number of patients with rejection episodes
a
The tumor diameter in patients with HCC recurrence or metastasis was significantly greater than in those with no HCC recurrence or metastasis (p = 0.0047)
b
The portal vein invasion in patients with HCC recurrence or metastasis was significantly more frequent than in those with no HCC recurrence or metastasis (p = 0.006) There was no significant difference among HCC recurrence, acute rejection, satellite tumor nodules, and histologic grading of HCC (p [ 0.05)
Three patients (5.6 %) were infected with HCV, which might have been caused by the massive blood transfusions during OLT. CMV infection in eight patients could be due to the risk constellation (donor? or -, recipient? or -) because of having no CMV serological examination before OLT. Seven cases (13.0 %) of HBV recurrence were directly related to the failure of combining lamivudine and HBIG therapy. The present study has shown that if no preventive measures are taken, HBV recurrence post-OLT in patients with chronic HBV infection will exceed 80 % [8, 9]. To prevent HBV recurrence post-OLT in patients with chronic HBV infection, combination therapy of HBIG and lamivudine is better than using lamivudine alone [10] and can reduce the recurrence of HBV infection post-OLT to 20 to 30 % [11]. The present study has also demonstrated that using drugs to minimize the serum HBV DNA concentration pre-OLT will reduce the recurrence rate of HBV infection post-OLT. As HBV infection frequently reccurs post-OLT in patients with chronic HBV infection, many liver transplant centers consider chronic HBV infection to be a contraindication to OLT. Our experience confirms that conventional combination therapy of HBIG and lamivudine can effectively prevent the recurrence of HBV infection post-OLT in patients with chronic HBV infection as well as increase patients’ survival rate and quality of life. The present study indicated that HBV recurrence is not related to acute rejection in patients with chronic HBV infection post-OLT and is linked to HBV virus replication in patients. There are no similar reports in China or abroad. However, it was reported abroad that acute rejection and/or treatment accelerates HCV infection in patients post-OLT
[12]. This difference requires more case studies for confirmation. In addition, HCC recurrence/metastasis and acute rejection are not related in patients with hepatitis B-related HCC post-OLT. Therefore, clinicians would not need to be concerned when applying immunosuppressive agents to treat acute rejection, as immunosuppressive agents would not increase the recurrence and metastasis rate of HCC. The conclusion lacks evidence, which may be explained by that fact that excessive autoimmune activation in patients with acute rejection has great suppressive effects on hepatocarcinoma cells and thereby sets off the negative effects of immunosuppressive agents. The present study also discovered that the immunosuppressive agent rapamycin can significantly reduce HCC recurrence postOLT in patients with HCC by suppressing tumor angiogenesis [13, 14]. However, the opposite point of view was raised in some studies from abroad. Studies by Vivarelli et al.[15] found that the doses of immunosuppressive agents were closely related to HCC recurrence 1 year postOLT in HCC patients and that high doses of cyclosporin 3–12 months post-OLT could significantly reduce the survival rate in patients without HCC. As a consequence, further studies are needed to determine the appropriate immunosuppressive agent doses and develop new immunosuppressive agents to reduce the HCC recurrence/ metastasis rate. Furthermore, the present study also revealed that HCC recurrence/metastasis post-OLT in patients with post-hepatitis B HCC is directly linked to the existence of large tumors and portal vein invasion pre-OLT. Pre-OLT HCC diameters in patients with and without HCC recurrence/ metastasis were 6.72 ± 3.40 cm and 3.55 ± 2.17 cm,
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respectively (p = 0.0047). Occurrence rates of portal vein invasion were 68.75 and 33.30 %, respectively (p = 0.006); the difference is statistically significant. This is consistent with the Shanghai Fudan Standard and the Milan criteria, formulated to screen OLT patients [1]. The latter requires that patients qualified for OLT must meet the following criteria: (1) the diameter of a single tumor is B9 cm, or B3 multiple tumors and the diameter of the largest B5 cm, total diameter of all tumors B9 cm; (2) no major vascular invasion (including the trunk and large branches of the portal vein, hepatic vein, and inferior vena cava); (3) no lymphatic and extrahepatic metastases. Two patients with portal vein invasion and large HCC underwent hepatic artery thrombosis and radiofrequency ablation therapy pre-OLT. HCC recurrence/metastasis did not occur after OLT. Both patients survived, and followups lasted 60 and 62 months, respectively. Indications show that pre-OLT cytoreductive therapy may reduce the HCC recurrence/metastasis rate in patients with large HCCs and portal vein invasion, prolong survival of patients, and expand the indications of OLT for HCC patients. This is consistent with studies elsewhere [16, 17]. In particular, studies showed transarterial chemoembolization (TACE) before OLT was safe and reduced the dropout rate from the waiting list [16, 17]. Furthermore, it induced significant downstaging (50 % reduction of the maximum diameter of the tumor) of tumors[3 cm in 52 % and complete tumour necrosis in 26 %, as assessed at explant. Therefore, these authors suggested that TACE before liver transplantation should be used in patients with large HCCs ([3 cm), and response to treatment should be regarded as a strong argument to proceed to liver transplantation. In summary, post-OLT complications in patients with chronic HBV infection are mainly acute rejection, biliary complications, HBV recurrence, and HCC recurrence/ metastasis post-OLT. HBV recurrence and HCC recurrence/metastasis are not related to acute rejection postOLT. HCC recurrence/metastasis post-OLT of patients with post-hepatitis B HCC is unrelated to the existence of hepatic satellite nodules and HCC differentiation, but is directly linked to large HCCs and portal vein invasion preOLT. Acknowledgments We thank all the staff of the Orthotopic Liver Transplantation Group of the Department of General Surgery, Peking University Third Hospital, for their support; Jingping Yang, Yuping Wang, Zhihui Han, Xishun Zhao, Wei Zhang, Xiaolong Ma, Wanjie Heng, and Yan Li from the Department of Pathology, Peking University School of Basic Medical Sciences, for their contributions; Prof. Zou Wanzhong especially for his attentive modifications and recommendations. The article was supported by the National Sciences Foundation of China (30971142).
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References 1. Fan J, Xu Y, Zhou J. What are the indications of liver transplantion in hepatic cancer patients? Natl Med J China. 2005; 85:1662–1664 2. International Working Party. Terminology for hepatic allograph rejection. Hepatology. 1995;22:648–654 3. Pei F, Zheng J, Ning J, et al. YMDD variants of HBV DNA polymerase gene: rapid detection and clinicopathological analysis with long-term lamivudine therapy after liver transplantation. World J Gastroenterol. 2005;11(18):2714–2719 4. Alzahrani AJ, Vallely PJ, McMahon RFT. Development of a novel nested in situ PCR-ISH method for detection of hepatitis C virus RNA in liver tissue. J Virol Methods. 2002;99:53–61 5. Perrella A, Esposito C, Ioia G, Campanella L, Taglialatela D, Cuomo O. Cytomegalovirus infection after liver transplantation: prophylaxis and preemptive treatment. A-single-center experience. Transpl Proc. 2010;42:1226–1228 6. Wiesner RA, Demetris AJ, Belle SH, et al. Acute hepatic allograft rejection: incidence, risk factors, and impact on outcome. Hepatology. 1998;28:638–645 7. Aguado JM, Herrero JA, Gavalda J, et al. Clinical presentation and outcome of tuberculosis in kidney, liver, and heart transplant recipients in Spain. Spanish transplantation infection study group, GE SITRA. Transplantation. 1997;63:1278 8. Rizzeffo M, Recchia S, Salizzoni M. Liver transplantation in carriers of the HBsAg. J Hepatol. 1991;13:5–7 9. Francesco PR, Marco B, Marco S, et al. Functional and morphological graft monitoring after liver transplantation. ClinChimActa. 2001;310:17–23 10. Gane EJ, Angus PW, Strasser S, Crawford DH, Ring J, Jeffrey GP, McCaughan GW. Lamivudine plus low-dose hepatitis B immunoglobulin to prevent recurrent hepatitis B following liver transplantation. Gastroenterology. 2007;132(3):931–937 11. Samuel D. Liver transplantation and hepatitis B virus infection: the situation seems to be under control, but the virus is still there. J Hepatol. 2001;34:943–945 12. Berenguer M, Prieto M, Cordoba J, et al. Early development of chronic active hepatitis in recurrent hepatitis C virus infection after liver transplantation: association with treatment of rejection. J Hepatol. 1998;28:756–763 13. Kneteman NM, Oberholzer J, AL Saghier M, et al. Sirolimusbased immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma. Liver Transp, J. 2004;10:1301–1311 14. Janus A, Robak T, Smolewski P, et al. The mammalian target of rapamycin (mToR) kinase pathway: its role in tumoringenesis and targeted entitum our therapy. Cell Mol Bio Lett. 2005;10: 479–498 15. Vivarelli M, Bellusci R, Cucchetti A, et al. Low recurrence rate of hepatocellular carcinoma after liver transplantation: better patient selection or low erimmunosuppression? Transplantation. 2002;74:1746–1751 16. Duffy JP, Vardanian A, Benjamin E, Watson M, Farmer DG, Ghobrial RM, Lipshutz G, Yersiz H, Lu DS, Lassman C, Tong MJ, Hiatt JR, Busuttil RW. Liver transplantation criteria for hepatocellular carcinoma should be expanded a 22-year experience with 467 patients at UCLA. Ann Surg. 2007;246:502–511 17. Marelli L, Stigliano R, Triantos C, Senzolo M, Cholongitas E, Davies N, Yu D, Meyer T, Patch DW, Burroughs AK. Treatment outcomes for hepatocellular carcinoma using chemoembolization in combination with other therapies. Cancer Treat Rev. 2006;32: 594–606
ORIGINAL ARTICLE
Clinicopathologic study on complications of orthotopic liver transplantation in 54 patients with chronic hepatitis B viral infection Fei Pei • Kewei Shang • Bin Jiang • Hua Wang • Fang Mei • Yan Zhang • Juan Du • Haohao Zhong Jiangfeng You
•
Received: 21 July 2012 / Accepted: 6 January 2013 / Published online: 7 February 2013 Ó Asian Pacific Association for the Study of the Liver 2013
Abstract Objective To study the complication incidence of 54 patients with chronic HBV infection following orthotopic liver transplantation (OLT) and risk factors associated with HBV recurrence and hepatocellular carcinoma (HCC) recurrence or metastasis post-OLT. Methods The light-microscopic appearance of hepatic allograft biopsies in 54 patients with chronic HBV infection following OLT was examined. The related clinical data were analyzed. The incidence and occurrence time of post-OLT complications were studied. Furthermore, the relationship between hepatitis B virus recurrence and acute rejection and the relationship among HCC recurrence/ metastasis, acute rejection, tumor diameter, and portal vein invasion were particularly studied. Results Frequent complications of patients with chronic HBV infection following OLT were acute rejection [38 (70.4 %); occurrence time: 5–365 days], chronic rejection [1 (1.9 %); occurrence time: 10.7 months], bile duct complications [24 Kewei Shang contributed equally to the research. F. Pei (&) K. Shang H. Wang F. Mei Y. Zhang J. Du H. Zhong J. You Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xue Yuan Road, Haidian, Beijing 100191, People’s Republic of China e-mail: [email protected] K. Shang e-mail: [email protected] H. Wang e-mail: [email protected] F. Mei e-mail: [email protected] Y. Zhang e-mail: [email protected]
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(44.4 %);occurrence time: 7–940 days], HBV recurrence [7 (13.0 %); occurrence time: 1–540 days], HCV infection [3 (5.6 %); occurrence time: 60 days, 60 days, 33 months], CMV infection [8 (14.8 %); occurrence time: 67–90 days], and HCC recurrence or metastasis [17 (31.5 %); occurrence time: 2–41 months]. At the end of 1 year post-OLT, 95 % of patients with post-hepatitis B cirrhosis were alive. At the end of 3 years post-OLT, 85 % of patients with post-hepatitis B cirrhosis were alive. However, at the end of 1 year post-OLT, 67.6 % of patients with post-hepatitis B HCC were alive. At the end of 3 years post-OLT, 50 % of patients with post-hepatitis B HCC were alive. The number of acute rejection episodes in patients with recurrent HBV infection and in those without recurrent HBV infection was 0.86 ± 1.46 times/patient and 1.07 ± 0.90 times/patient, respectively (p [ 0.05); the number of moderate acute rejection episodes (RAI score C4) in patients with recurrent HBV infection and in those without recurrent HBV infection was 0.29 ± 0.49 times/patient and 0.50 ± 0.63 times/patient (p [ 0.05). Incidence of patients with C3 episodes of acute rejection in patient with recurrent HBV infection and in those without recurrent HBV infection was 14.3 and 10.6 % J. Du e-mail: [email protected] H. Zhong e-mail: [email protected] J. You e-mail: [email protected] B. Jiang Orthotopic Liver Transplantation Center from the Department of General Surgery, Peking University Third Hospital, Beijing 100191, People’s Republic of China e-mail: [email protected]
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(p [ 0.05). Furthermore, the number of acute rejection episodes in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 1.12 ± 0.93 times/ patient and 1.06 ± 1.39 times/patient, respectively (p [ 0.05). The number of moderate acute rejection episodes (RAI score C4) in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 0.65 ± 0.79 times/ patient and 0.65 ± 1.06 times/patient, respectively (p [ 0.05). Incidence of patients with C3 episodes of acute rejection in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 5.9 and 17.6 %, respectively (p [ 0.05). The tumor diameter in patients with HCC recurrence or metastasis was 6.72 ± 3.40 cm; however, that in patients without HCC recurrence or metastasis was 3.55 ± 2.17 cm (p = 0.0047). The incidence of portal vein invasion in patients with HCC recurrence or metastasis and in those without HCC recurrence or metastasis was 68.75 and 33.3 %, respectively (p = 0.006). Conclusion There was no significant difference between HBV recurrence and acute rejection post-liver transplantation in patients with chronic HBV infection. There was no significant difference between HCC recurrence and acute rejection. The tumor diameter in patients with HCC recurrence or metastasis was significantly greater than that in patients with no HCC recurrence or metastasis. Portal vein invasion was significantly more frequent in patients with HCC recurrence or metastasis than in those with no HCC recurrence or metastasis. Keywords Hepatitis B Liver transplantation Postoperative complications Graft rejection Liver neoplasms
Introduction Since liver transplantation was first carried out by Starzl, an American, on 1 March 1963, orthotopic liver transplantation(OLT) has been widely used worldwide as the only effective treatment of end-stage liver disease. In China, liver transplantation started in 1977 and has made great progress with technological advances and improvement of living standards in recent years. Nearly 3,000 liver transplants per annum are currently performed in China, which ranks second, only behind the USA, among countries carrying out the largest numbers of liver transplants [1]. As China has a high incidence of hepatitis B, with an infection rate of 1/10, 90 % of patients undergoing liver transplantation are hepatitis B virus (HBV)-infected young adults with liver cirrhosis or hepatocellular carcinoma (HCC). Little research has been done on complications and risk factors associated with HBV recurrence and
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hepatocellular carcinoma (HCC) recurrence or metastasis post-OLT.
Materials and methods Basic data The hepatic allograft biopsies of 54 patients at Peking University Third Hospital with chronic HBV infection following OLT were analyzed retrospectively. The patients were operated on in August 2000–2004. Liver lesions of these patients were all caused by HBV infection before OLT. Related clinical data were analyzed thoroughly. The incidence and occurrence time of post-OLT complications were studied. Furthermore, the relationship between recurrent type B viral hepatitis and acute rejection and the relationship among HCC recurrence/metastasis, acute rejection, tumor diameter, and the existence of portal vein invasion were particularly studied. Pathologic diagnosis Pathologic diagnosis of acute rejection Pathologic diagnosis of acute rejection included: (1) mixed inflammatory cell infiltration in portal tracts of activated lymphocytes (lymphoblasts), lymphocytes(mainly T cells), macrophages, plasma cells, neutrophils and eosinophils, etc.; (2) biliary inflammation and bile duct damage characterized by inflammatory cell infiltration around the bile duct, biliary epithelial injury, as well as formation of vacuoles in the cytoplasm, size and shape change of the nucleus, an increased nuclear:cytoplasmic ratio, and apoptosis of biliary epithelial cells; (3) subendothelial inflammation of the interlobular veins and central veins, recognized by inflammatory cell infiltration of the venule walls, attachment of lymphocytes to epithelial cells, and swelling and shedding of epithelial cells. A semiquantitative approach to grade the severity of acute rejection, the Banff schema [2], was adopted: rejection activity index (RAI) = score 0–2: not diagnosed as acute rejection; RAI = score 3: suspected acute rejection; RAI = score 4–5: mild acute rejection, with \50 % portal tracts infiltrated by inflammatory cells, inflammatory cells restricted in the portal tracts, and occasional mixed inflammatory infiltration; RAI = score 6–7: moderate acute rejection with C50 % portal tracts infiltrated by inflammatory cells, moderate mixed inflammatory infiltration including a moderate number of eosinophils, interlobular bile duct inflammation, and venous subendothelial inflammation, and the junction of the portal tracts and hepatic lobule is still regular and smooth; RAI = score 8–9: severe acute
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rejection, characterized by C50 % portal tracts infiltrated by inflammatory cells with hepatic lobules involved, mixed inflammatory infiltration densely observed in the portal tracts with hepatic lobules affected, giving the inter-surface between the portal tracts and hepatic lobule an irregular appearance, obvious bile duct damage and venous subendothelial inflammation, and spotty necrosis of hepatocytes in the hepatic lobules (Fig. 1). Pathologic diagnosis of chronic rejection The pathologic diagnosis of chronic rejection includes: (1) more than 50 % of bile ducts within the portal tracts are lost (a firm diagnosis requires at least 20 portal tracts); (2) arteriopathic changes with foam cells within the intima of large arterial walls in the portal tracts and intimal thickening. Foam cells accumulate in the arterial lumens (Fig. 2). Pathologic diagnosis of HBV recurrence post-OLT Pathologic diagnosis of HBV recurrence post-OLT includes: (1) obvious mononuclear cell infiltration within portal tracts; (2) damage of hepatic plates (Fig. 3a); (3) local hepatic fibrosis; (4) necrosis of hepatocytes in the hepatic lobules with mononuclear cell infiltration; (5) appearance of ground-glass hepatocytes and sand-like hepatocyte nuclei caused by the accumulation of HBsAg in sER and hepatocyte nuclei; (6) immunohistochemistry can be used to identify HBsAg in the cytoplasm and cytomembrane of hepatocytes, which is positively correlated with the severity of hepatitis (Fig. 3b).
Fig. 2 Chronic rejection. The obliterative arteriopathy in a hilar artery is characterized by luminal narrowing because of intimal thickening. The intimal thickening is related to deposition of foamy macrophages and proliferation of myofibroblasts (H&E 910)
hepatocytes: among the viral hepatitises, Mallory bodies are only seen in case of chronic HCV infection; (3) the formation of hepatocyte apoptotic bodies in the hepatic lobules; (4) proliferation of Kupffer cells within the hepatic lobules; (5) accumulation of lymphocytes and formation of lymphoid follicles in the portal tracts as well as damage of interlobular bile ducts; (6) HCV RNA can be detected in the cytoplasm of hepatocytes using in situ hybridization (Fig. 4b). Clinical medicine and laboratory tests
Pathologic diagnosis of hepatitis C virus (HCV) infection includes: (1) mild to moderate macrovesicular hepatic steatosis (Fig. 4a); (2) Mallory bodies appear in the damaged
These patients were all HBsAg-positive before surgery and started using lamivuding 100 mg/day 1–12 months before transplantation. Furthermore, these patients received intravenous HBIG 10,000 during the anhepatic phase, then 2,000 IU daily for the next 7 days, then 2,000 IU monthly. At the same time, the patients received lamivudine 100 mg/day immediately after transplantation and remained on the drug. Cefotaxime (2 g every 8 h, intravenous drip) and ampicillin (1 g every 6 h, intravenous drip) were used as
Fig. 1 Acute rejection. a A male patient, 56 years old, day 30 after OLT. Allograft biopsy shows mild acute rejection (RAI: score 4). b A male patient, 56 years old, day 9 after OLT. Allograft biopsy shows
moderate acute rejection (RAI: score 6). c A female patient, 45 years old, day 90 after OLT. Allograft biopsy shows severe acute rejection (RAI: score 9) (H&E 910)
Pathologic diagnosis of hepatitis C virus (HCV) infection
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Fig. 3 Recurrent chronic hepatitis B. a Portal inflammation, interface hepatitis, and focal fibrosis (H&E 910). b Positive membrane and cytoplasmic staining for hepatitis B surface antigen (immunoperoxidase stain, IHC 920)
Fig. 4 Chronic hepatitis C. a Macrovesicular hepatic steatosis, acidophilic body with partially extruded nucleus, mononuclear portal inflammation, and lymphoid aggregates with terminal centers (H&E 910). b Positive cytoplasmic staining for HCV RNA (in situ hybridization, 920)
perioperative prophylactic antibiotics. If there were no signs or symptoms of infection, these agents were discontinued after 5 days. No prophylactic medication for fungal or viral infections was given. All post-OLT patients were treated conventionally with immunosuppressive combination therapy of tacrolimus (FK506) ? methylprednisolone ? mycophenolate mofetil (MMF) to suppress rejection. Intravenous methylprednisolone (intravenous dose of 1,000 mg of methylprednisolone followed by a 6-day taper from 200 to 20 mg/day) is used in our liver transplant center as first-line therapy for acute hepatic cellular rejection in patients who undergo liver transplants. Most take lamivudine orally and human hepatitis B immunoglobulin (HBIG) is injected to prevent the recurrence of HBV. All hepatitis B recurrences postOLT were confirmed by viral serological examination, fluorescence quantitative PCR of serum HBV DNA, liver biopsy, and immunochemical staining of HBsAg. PCR restriction fragment length polymorphism (RFLP) was adopted to determine whether the HBV DNA polymerase gene had a YMDD mutation [3]. All hepatitis C (HC) occurrences post-OLT were confirmed by viral serological examination, fluorescence quantitative PCR of serum HCV DNA, liver biopsy, and HCV RNA in situ hybridization [4].
All cytomegalovirus (CMV) infections post-OLT were identified by serological examination of CMV IgM and fluorescence quantitative PCR of serum CMV DNA [5]. Statistical methods Results were expressed as the mean and standard deviation. Survival curves were analyzed using SPSS 13.0 statistical software. Differences between groups were assessed by chi-square or Student’s t test, with p values \0.05 considered significant.
Results General All 54 patients with chronic HBV infection pre-OLT survived for more than 2 months post-OLT. Their average age was 48.11(±9.59) years (24–67); 49 were male (90.7 %) and 5 female (9.3 %). Follow-up post-OLT lasted until August 2007. Follow-up of 20 patients with post-hepatitis B cirrhosis required at least 3 years. Among them, 17 patients survived, and the longest follow-up was 74 months. Only
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three patients died, one of biliary complications (cholestatic cirrhosis) 36 months post-OLT, one of gastrointestinal bleeding and hepatic encephalopathy due to hepatitis B recurrence and hepatic cirrhosis 24 months post-OLT, and one of septicemia and multiple organ failure 2 months post-OLT. The 1-year post-OLT survival rate was 95 % (19/20), 2-year survival was 90 % (18/20), and 3-year survival was 85 % (17/20). Survival curves are shown in Fig. 5. Follow-up of 34 patients with post-hepatitis B hepatocellular carcinoma (HCC) required as least 3 years. Of the patients, 15 survived, and the longest follow-up was 84 months. Nineteen patients died. The 1-year survival rate post-OLT was 67.6 % (23/34), 2-year survival 58.1 % (18/ 34), and 3-year survival 50 % (17/34). Causes of deaths were as follows: HCC recurrence/metastasis in 13 patients (68.4 % of all deaths), biliary complications in 4 patients (21.1 % of all deaths), and infection in 2 patients (10.5 % of all deaths). Survival curves are shown in Fig. 5. Complications post-OLT (Table 1) Acute rejection Among 54 patients post-OLT, 38 experienced acute rejection, accounting for 70.4 %. Twenty-nine patients had acute rejection once (53.7 %); three had acute rejection twice (5.6 %); three had acute rejection three times (5.6 %); two had acute rejection four times (3.7 %); one had acute rejection five times (1.9 %). The number of patients with RAI scores C4 who had acute rejection was 21 (38.9 %). The first occurrence time of acute rejection for 38 patients was 64.67 (±98.62) days (5–365 days). The number of patients with first occurrence time B30 days was 25 (65.8 %), from 30 to 60 days was 4 (10.5 %), from 60 days to 6 months was 4 (10.5 %), and from 6 months to 1 year was 5 (13.2 %). Fig. 5 Survival curves of 54 patients with chronic HBV infection following orthotopic liver transplantation
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Chronic rejection Among 54 patients, only 1 had chronic rejection (1.9 %) 10.7 months post-OLT, complicated by miliary hepatic tuberculosis and chronic cavitary pulmonary tuberculosis. Biliary complications Twenty-four of 54 patients had biliary complications (44.4 %), which mainly included biliary stricture and obstruction. The occurrence time was 168.4 (±231.7) days (7–940 days) post-OLT. The patients with biliary complications underwent endoscopic biliary stricture dilatation and stent placement. Hepatitis B recurrence Seven of 54 patients experienced hepatitis B recurrence (13.0 %), the occurrence time of which was 344 (±206) days (1–540 days) post-OLT. Six patients had YMDD wild-type HBV, and one had HBV DNA polymerase gene YIDD mutation. Causes of hepatitis B recurrence were that three patients did not take or self-withdraw lamivudine for economic reasons; two adhered to lamivudine, but did not receive intramuscular HBIG injections; one was not responsive to lamivudine because of HBV DNA polymerase gene mutation; one had high-copy HBV DNA in serum and was controlled ineffectively. HCV infection Three of 54 patients were infected with HCV (5.6 %). The occurrence times were 60 days, 60 days, and 33 months, respectively. There was no hepatitis C viral coinfection in patients with chronic hepatitis B virus infection before or after liver transplantation, and the hepatitis C infection was diagnosed after OLT perhaps
Hepatol Int (2013) 7:468–476 Table 1 Post-OLT outcomes of 54 patients with chronic HBV infection
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Complications
Incidence (%)
Acute rejection
70.4
Chronic rejection
1.9
Bile duct complications
44.4
HBV recurrence
13.0
HCV infection
5.6
Occurrence time (days) 64.67 ± 98.62 days (5–365) 10.7 M 168.4 ± 231.7 days (7–940) 344 ± 206 days (1–540) 60 days, 60 days, 33 months
CMV infection
14.8
77.6 ± 10.4 days (67–90)
Bacterial and fungal infection
24.1
15 ± 45 days (5–60)
Vascular complications
14.8
56 ± 66 days (1–180)
Recurrent hepatocellular carcinoma
31.5
9.6 ± 10.4 months (2–41 months)
because of blood transfusions. Furthermore, the patients with hepatitis C infection underwent interferon alpha-2b (IFN-a) therapy after OLT. CMV infection Eight of 54 patients were infected with CMV (14.8 %). The occurrence time was 77.6 (±10.4) days (67–90 days) post-OLT. The patients did not undergo a prophylactic or preemptive therapy in the perioperative phase. It was not clear whether the patients who experienced a CMV infection belonged to risk groups (donor? or recipient?) because no serological examination for CMV was carried out before OLT. Furthermore, after OLT the CMV infection patients were treated with valganciclovir (900 mg twice daily) until three consecutive PCRs were negative. PCR was measured every 2 weeks during treatment. All patients had a quick recovery, and recurrent viremia was not observed. Bacterial and fungal infections Thirteen of 54 patients were infected post-OLT(24.1 %), among whom 2 were infected with fungi (3.7 %), 10 with common bacteria (18.5 %), and 1 with mycobacterium tuberculosis (hepatic tuberculosis) (1.9 %) who had a thick wall cavity in the lung pre-OLT. The diagnosis of bacterial or fungal infection was usually made in the first 2 months at a mean time of 15 days after OLT. The occurrence time was 15 ± 45 days (5–60 days). Two patients died within 6 months after transplantation because of Staphylococcus aureus infection or disseminated cryptococcosis. One patient with mycobacterium tuberculosis received a second OLT and died 20 months after the first transplantation. Portal vein or hepatic artery thrombosis, occlusion, or stenosis Eight of 54 patients experienced vascular complications (14.8 %), the occurrence time of which was 56
(± 66) days (1–180 days) post-OLT. The clinical presentation of vascular complications included elevated transaminases in all patients, biliary complications in two patients, portal hypertension with gastrointestinal bleeding in one patient, fever and sepsis in one patient, and graft dysfunction or failure in two patients. We observed two cases (3.7 %) of acute early hepatic artery thrombosis; one qualifed immediately for liver retransplantation, and the other underwent endovascular therapies, including intraarterial heparin infusion and percutaneous transluminal angioplasty with stent placement. The patients survived. Portal venous complications were seen in six patients (11.1 %), and they received anticoagulation without surgical therapy. HCC recurrence/metastasis post-OLT Seventeen of 34 patients with post-hepatitis B hepatocellular carcinoma had HCC recurrence/metastasis (50 %). The occurrence time was 9.6 (±10.4) months (2–41 months) post-OLT. Relationship between hepatitis B recurrence and acute rejection of patients with chronic HBV infection (Table 2) Among seven patients with hepatitis B recurrence, three experienced acute rejection. The number of acute rejection episodes was 0.86 (±1.46) times/patient. One patient had C3 episodes of acute rejection. The number of moderate acute rejections (RAI score C4) was 0.29 (±0.49) times/ patient. Among 47 patients without hepatitis B recurrence, 35 experienced acute rejection. The number of acute rejection episodes was 1.07 (±0.90) times/patient. Five patients had C3 episodes of acute rejection. The number of moderate acute rejections (RAI score C4) was 0.5 (±0.63) times/patient. The difference was not statistically significant (p [ 0.05). It was indicated that hepatitis B recurrence and acute rejection in patients with chronic HBV infection were not related.
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Factors associated with HCC recurrence/metastasis post-OLT (Table 3) Thirteen of 17 patients with HCC recurrence/metastasis post-OLT experienced acute rejection (76.5 %). The number of acute rejection episodes was 1.12 (±0.93) times/patients. One patient had C3 episodes of acute rejection (5.9 %). The number of moderate acute rejections (RAI score C4) was 0.65 (±0.79) times/patient. Eleven of 17 patients without HCC recurrence/metastasis post-OLT experienced acute rejection (64.7 %). The number of acute rejection episodes was 1.06 (±1.39) times/patients. Three patients had acute rejection C3 episodes (17.6 %). The number of moderate acute rejections (RAI score C4) was 0.65 (±1.06) times/ patient. The difference was not statistically significant (p [ 0.05). Indications showed HCC recurrence/metastasis post-OLT and acute rejection of patients with post-hepatitis B HCC were not related and that HCC recurrence/metastasis post-OLT of patients with post-hepatitis B HCC was not related to the existence of hepatic satellite nodules. Research has discovered that post-OLT HCC recurrence/metastasis in patients with post-hepatitis B HCC is not related to HCC differentiation (p [ 0.05). The difference in HCC diameter pre-OLT between that of patients with and without HCC recurrence/metastasis was statistically significant (6.72 ± 3.40 cm vs. 3.55 ± 2.17 cm; p = 0.0047). The difference in the occurrence rate of portal vein invasion was also statistically significant (68.75 % vs. 33.3 %, p = 0.006). It is suggested that post-OLT HCC recurrence/metastasis of patients with post-hepatitis B HCC is directly linked to the existence of large tumors and portal vein invasion. In addition, two patients with portal vein invasion and large HCCs underwent hepatic artery thrombosis and radiofrequency ablation therapy pre-OLT. HCC recurrence/metastasis did not occur after OLT. Both patients survived, and follow-ups lasted 60 and 62 months, respectively.
Discussion Complications in patients with chronic HBV infection post-OLT are acute rejection, chronic rejection, biliary
complications, HBV recurrence, HCV infection, CMV infection, and HCC recurrence/metastasis. The present study, the number of patients who experienced acute rejection post-OLT was 38, accounting for 70.4 % of the 54 patients with chronic HBV infection. The first occurrence time was 64.67 (±98.62) days. Acute rejection is one of the most common complications at the early stage following OLT, which often occurs 5–30 days post-OLT, especially at 7–14 days, but can also occur any time post-OLT and is usually related to viral infection or inadequate doses of immunosuppressive agents [6]. Acute rejection is mediated by cellular immunity. Antigen-presenting cells, dendric cells in the transplanted liver, activate T lymphocytes in recipients. Forty-eight hours after OLT, T lymphocytes can be observed in the transplanted liver clustering around the dendric cells, transforming to lymphoblasts, and secreting cytokines (like IL-2 and IL-5, etc.), which promote T lymphocyte division and proliferation, attracting immune effector cells (including eosinophils, neutrophils, macrophages, plasmocytes, etc.) to accumulate locally, proliferate, and differentiate, thus playing their biological roles. The present study, only 1 (1.9 %) of the 54 patients experienced chronic rejection, which occurred 10.7 months post-OLT. The patient also had complications of miliary hepatic tuberculosis and chronic cavitary pulmonary tuberculosis. Chronic rejection might be linked to the patient’s tuberculosis. Infection may stimulate autoimmune reaction. Anti-tuberculosis drugs can induce the liver to produce microsomal enzymes, accelerate hormone catabolism, and at the same time enhance the activity of P450 enzymes and speed up the catabolism of anti-rejection drugs; as a consequence, the plasma concentration is reduced, which may induce repeated occurrence of acute rejection and lead to chronic rejection in the end [7] . Of 54 patients, 24 (44.4 %) had biliary complications, the causes of which were as follows: (1) organic obstruction due to technical problems with the extrahepatic bile duct anastomosis; (2) intrahepatic bile duct stricture caused by technical problems with the hepatic artery anastomosis or thrombosis, and intrahepatic bile duct ischemia and hypoxia.
Table 2 Factors associated with HBV recurrence post-liver transplantation in patients with chronic HBV infection Evident HBV recurrence (n = 7)
No HBV recurrence (n = 47)
p value
Number of patients with rejection episodes
3 (42.9 %)
35 (74.5 %)
[0.05
Number of rejection episodes/patient
0.86 ± 1.46
1.07 ± 0.90
[0.05
Number of patients with C3 episodes of rejection
1 (14.3 %)
5 (10.6 %)
[0.05
Number of moderate rejection episodes/patient(RAI score C4)
0.29 ± 0.49
0.5 ± 0.63
0.32
There was no significant difference among HBV recurrence, acute rejection, and CMV infection post-liver transplantation in patients with chronic HBV infection (p [ 0.05)
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Table 3 Factors associated with hepatocellular carcinoma recurrence post-liver transplantation in patients with chronic HBV infection HCC recurrence or metastasis (n = 17)
No HCC recurrence or metastasis (n = 17)
p value
13 (76.5 %)
11 (64.7 %)
[0.05
Number of rejection episodes/patient
1.12 ± 0.93
1.06 ± 1.39
0.83
Number of patients with C3 episodes of rejection
1 (5.9 %)
3 (17.6 %)
0.20
Number of moderate rejection episodes/patient (RAI score C4)
0.65 ± 0.79
0.65 ± 1.06
1
Tumor diameter(cm)
6.72 ± 3.40
3.554 ± 2.17a
0.0047
Portal vein invasion
68.75 %
33.3 %b
0.006
Satellite tumor nodules Histologic grading of HCC
68.75 % Low grade: 6.25 %
47.1 % Low grade: 5.88 %
0.088 0.05
Moderate grade: 87.5 %
Moderate grade: 82.36 %
High grade: 6.25 %
High grade: 11.76 %
Number of patients with rejection episodes
a
The tumor diameter in patients with HCC recurrence or metastasis was significantly greater than in those with no HCC recurrence or metastasis (p = 0.0047)
b
The portal vein invasion in patients with HCC recurrence or metastasis was significantly more frequent than in those with no HCC recurrence or metastasis (p = 0.006) There was no significant difference among HCC recurrence, acute rejection, satellite tumor nodules, and histologic grading of HCC (p [ 0.05)
Three patients (5.6 %) were infected with HCV, which might have been caused by the massive blood transfusions during OLT. CMV infection in eight patients could be due to the risk constellation (donor? or -, recipient? or -) because of having no CMV serological examination before OLT. Seven cases (13.0 %) of HBV recurrence were directly related to the failure of combining lamivudine and HBIG therapy. The present study has shown that if no preventive measures are taken, HBV recurrence post-OLT in patients with chronic HBV infection will exceed 80 % [8, 9]. To prevent HBV recurrence post-OLT in patients with chronic HBV infection, combination therapy of HBIG and lamivudine is better than using lamivudine alone [10] and can reduce the recurrence of HBV infection post-OLT to 20 to 30 % [11]. The present study has also demonstrated that using drugs to minimize the serum HBV DNA concentration pre-OLT will reduce the recurrence rate of HBV infection post-OLT. As HBV infection frequently reccurs post-OLT in patients with chronic HBV infection, many liver transplant centers consider chronic HBV infection to be a contraindication to OLT. Our experience confirms that conventional combination therapy of HBIG and lamivudine can effectively prevent the recurrence of HBV infection post-OLT in patients with chronic HBV infection as well as increase patients’ survival rate and quality of life. The present study indicated that HBV recurrence is not related to acute rejection in patients with chronic HBV infection post-OLT and is linked to HBV virus replication in patients. There are no similar reports in China or abroad. However, it was reported abroad that acute rejection and/or treatment accelerates HCV infection in patients post-OLT
[12]. This difference requires more case studies for confirmation. In addition, HCC recurrence/metastasis and acute rejection are not related in patients with hepatitis B-related HCC post-OLT. Therefore, clinicians would not need to be concerned when applying immunosuppressive agents to treat acute rejection, as immunosuppressive agents would not increase the recurrence and metastasis rate of HCC. The conclusion lacks evidence, which may be explained by that fact that excessive autoimmune activation in patients with acute rejection has great suppressive effects on hepatocarcinoma cells and thereby sets off the negative effects of immunosuppressive agents. The present study also discovered that the immunosuppressive agent rapamycin can significantly reduce HCC recurrence postOLT in patients with HCC by suppressing tumor angiogenesis [13, 14]. However, the opposite point of view was raised in some studies from abroad. Studies by Vivarelli et al.[15] found that the doses of immunosuppressive agents were closely related to HCC recurrence 1 year postOLT in HCC patients and that high doses of cyclosporin 3–12 months post-OLT could significantly reduce the survival rate in patients without HCC. As a consequence, further studies are needed to determine the appropriate immunosuppressive agent doses and develop new immunosuppressive agents to reduce the HCC recurrence/ metastasis rate. Furthermore, the present study also revealed that HCC recurrence/metastasis post-OLT in patients with post-hepatitis B HCC is directly linked to the existence of large tumors and portal vein invasion pre-OLT. Pre-OLT HCC diameters in patients with and without HCC recurrence/ metastasis were 6.72 ± 3.40 cm and 3.55 ± 2.17 cm,
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respectively (p = 0.0047). Occurrence rates of portal vein invasion were 68.75 and 33.30 %, respectively (p = 0.006); the difference is statistically significant. This is consistent with the Shanghai Fudan Standard and the Milan criteria, formulated to screen OLT patients [1]. The latter requires that patients qualified for OLT must meet the following criteria: (1) the diameter of a single tumor is B9 cm, or B3 multiple tumors and the diameter of the largest B5 cm, total diameter of all tumors B9 cm; (2) no major vascular invasion (including the trunk and large branches of the portal vein, hepatic vein, and inferior vena cava); (3) no lymphatic and extrahepatic metastases. Two patients with portal vein invasion and large HCC underwent hepatic artery thrombosis and radiofrequency ablation therapy pre-OLT. HCC recurrence/metastasis did not occur after OLT. Both patients survived, and followups lasted 60 and 62 months, respectively. Indications show that pre-OLT cytoreductive therapy may reduce the HCC recurrence/metastasis rate in patients with large HCCs and portal vein invasion, prolong survival of patients, and expand the indications of OLT for HCC patients. This is consistent with studies elsewhere [16, 17]. In particular, studies showed transarterial chemoembolization (TACE) before OLT was safe and reduced the dropout rate from the waiting list [16, 17]. Furthermore, it induced significant downstaging (50 % reduction of the maximum diameter of the tumor) of tumors[3 cm in 52 % and complete tumour necrosis in 26 %, as assessed at explant. Therefore, these authors suggested that TACE before liver transplantation should be used in patients with large HCCs ([3 cm), and response to treatment should be regarded as a strong argument to proceed to liver transplantation. In summary, post-OLT complications in patients with chronic HBV infection are mainly acute rejection, biliary complications, HBV recurrence, and HCC recurrence/ metastasis post-OLT. HBV recurrence and HCC recurrence/metastasis are not related to acute rejection postOLT. HCC recurrence/metastasis post-OLT of patients with post-hepatitis B HCC is unrelated to the existence of hepatic satellite nodules and HCC differentiation, but is directly linked to large HCCs and portal vein invasion preOLT. Acknowledgments We thank all the staff of the Orthotopic Liver Transplantation Group of the Department of General Surgery, Peking University Third Hospital, for their support; Jingping Yang, Yuping Wang, Zhihui Han, Xishun Zhao, Wei Zhang, Xiaolong Ma, Wanjie Heng, and Yan Li from the Department of Pathology, Peking University School of Basic Medical Sciences, for their contributions; Prof. Zou Wanzhong especially for his attentive modifications and recommendations. The article was supported by the National Sciences Foundation of China (30971142).
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References 1. Fan J, Xu Y, Zhou J. What are the indications of liver transplantion in hepatic cancer patients? Natl Med J China. 2005; 85:1662–1664 2. International Working Party. Terminology for hepatic allograph rejection. Hepatology. 1995;22:648–654 3. Pei F, Zheng J, Ning J, et al. YMDD variants of HBV DNA polymerase gene: rapid detection and clinicopathological analysis with long-term lamivudine therapy after liver transplantation. World J Gastroenterol. 2005;11(18):2714–2719 4. Alzahrani AJ, Vallely PJ, McMahon RFT. Development of a novel nested in situ PCR-ISH method for detection of hepatitis C virus RNA in liver tissue. J Virol Methods. 2002;99:53–61 5. Perrella A, Esposito C, Ioia G, Campanella L, Taglialatela D, Cuomo O. Cytomegalovirus infection after liver transplantation: prophylaxis and preemptive treatment. A-single-center experience. Transpl Proc. 2010;42:1226–1228 6. Wiesner RA, Demetris AJ, Belle SH, et al. Acute hepatic allograft rejection: incidence, risk factors, and impact on outcome. Hepatology. 1998;28:638–645 7. Aguado JM, Herrero JA, Gavalda J, et al. Clinical presentation and outcome of tuberculosis in kidney, liver, and heart transplant recipients in Spain. Spanish transplantation infection study group, GE SITRA. Transplantation. 1997;63:1278 8. Rizzeffo M, Recchia S, Salizzoni M. Liver transplantation in carriers of the HBsAg. J Hepatol. 1991;13:5–7 9. Francesco PR, Marco B, Marco S, et al. Functional and morphological graft monitoring after liver transplantation. ClinChimActa. 2001;310:17–23 10. Gane EJ, Angus PW, Strasser S, Crawford DH, Ring J, Jeffrey GP, McCaughan GW. Lamivudine plus low-dose hepatitis B immunoglobulin to prevent recurrent hepatitis B following liver transplantation. Gastroenterology. 2007;132(3):931–937 11. Samuel D. Liver transplantation and hepatitis B virus infection: the situation seems to be under control, but the virus is still there. J Hepatol. 2001;34:943–945 12. Berenguer M, Prieto M, Cordoba J, et al. Early development of chronic active hepatitis in recurrent hepatitis C virus infection after liver transplantation: association with treatment of rejection. J Hepatol. 1998;28:756–763 13. Kneteman NM, Oberholzer J, AL Saghier M, et al. Sirolimusbased immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma. Liver Transp, J. 2004;10:1301–1311 14. Janus A, Robak T, Smolewski P, et al. The mammalian target of rapamycin (mToR) kinase pathway: its role in tumoringenesis and targeted entitum our therapy. Cell Mol Bio Lett. 2005;10: 479–498 15. Vivarelli M, Bellusci R, Cucchetti A, et al. Low recurrence rate of hepatocellular carcinoma after liver transplantation: better patient selection or low erimmunosuppression? Transplantation. 2002;74:1746–1751 16. Duffy JP, Vardanian A, Benjamin E, Watson M, Farmer DG, Ghobrial RM, Lipshutz G, Yersiz H, Lu DS, Lassman C, Tong MJ, Hiatt JR, Busuttil RW. Liver transplantation criteria for hepatocellular carcinoma should be expanded a 22-year experience with 467 patients at UCLA. Ann Surg. 2007;246:502–511 17. Marelli L, Stigliano R, Triantos C, Senzolo M, Cholongitas E, Davies N, Yu D, Meyer T, Patch DW, Burroughs AK. Treatment outcomes for hepatocellular carcinoma using chemoembolization in combination with other therapies. Cancer Treat Rev. 2006;32: 594–606
