Liver Transplantation and Immunologic Tolerance

1. Liver Transplantation and Immunologic Tolerance C. Andrew Bonham, M.D. Associate Professor of Surgery Stanford University Medical School

2. First Documented Liver Surgery

3. First liver gene therapy experiment First liver xenotransplant First split liver transplant First reduced size liver transplant First auxiliary liver transplant First human liver transplant First domino liver transplant Viaspan solution First large animal liver transplant Collins solution First adult-to- child transplant First adult-to-adult liver transplant Veno-venous bypass 1950 1960 1970 1980 1990 2000 2010 Azathioprine IL-2Ra RAPA OKT3 Antilymphocyte antisera Tacrolimus Cyclosporin Mycophenolate Mofetil Steroids

4. Patients Awaiting Organ Transplantation • Waiting = 87,700 (as of March 11, 2005) Kidney 61,067 Pancreas 1,683 Kidney/Pancreas 2437 Liver 17,263* Intestine 186 Heart 3199 Lung 3779 Heart/Lung 169 *1/5 dies while waiting.

5. Argentina Australia Austria Belgium Brazil Canada Chile China Columbia Croatia Czech Republic England Egypt Finland France Germany Greece Hong Kong India Iran Ireland Israel Italy Japan Korea Malaysia Mexico Netherlands New Zealand Norway Peru Philippines Countries Performing Liver Transplantation • Poland • Portugal • Russia • Saudi Arabia • Singapore • South Africa • Spain • Sweden • Switzerland • Taiwan • Turkey • Ukraine • United States • Venezuela

8. 4000 Consecutive Liver Transplants Patient Survival by Era Log Rank: p = 0.0001 Years Post Transplantation

9. 4000 Consecutive Liver Transplants Most Common Causes of Death ( n = 1633)

10. Infection Lymphoma Hypertension Renal failure Ulcers Seizures Diabetes Osteopenia Hirsutism Impaired healing Electrolyte abnormalities Accelerated disease recurrence Complications of Immunosuppression

11. Evolution of Immunosuppression • 1959 - First successful kidney transplants using TBI • Functioned 20 and 26 years without immunosuppression • Clusters of patients treated with azathioprine became drug-free after treatment of rejection • 1963 – Switch to heavy prophylactic immunosuppression with steroids prompted by 20-35% graft loss rate from rejection • No clusters of drug-free kidney recipients were ever described again • 1965 – Liver recognized as more tolerogenic

12. Evolution of Immunosuppression • 1966 – Spontaneous liver engraftment described • Tolerance to donor-derived skin or kidney grafts • 1990s – Role of chimerism in solid-organ transplantation re-examined • Owen’s freemartin cattle • Microchimerism in recipients of liver or kidney from opposite sex donor detected by PCR • Role of residual antigen to maintain immunity or tolerance (Zinkernagel and Starzl)

15. Evolution of Immunosuppression • Heavy multi-drug immunosuppression may erode the tolerance mechanism of clonal deletion/exhaustion • Long-term immunosuppression required to prevent emergence of reactive clones • Pretreatment and minimal post-transplant immunosuppression may be conducive to tolerance

16. WEANING OF IMMUNOSUPPRESSION IN LIVER TRANSPLANT RECIPIENTS Mazariegos, George V.3; Reyes, Jorge; Marino, Ignazio R.; Demetris, Anthony J.; Flynn, Bridget; Irish, William; McMichael, John; Fung, John J.; Starzl, Thomas E 95 patients weaned from immunosuppression 19% drug free for 10 months to 5 years

17. Pittsburgh Tolerance Protocol • Pretreatment with ATG • Post-transplant monotherapy immunosuppression • Dose weaning at 6 months

18. Current immunosuppression of 38 patients pretreated with ALG before liver transplantation between July 2001 and March 2002 *In most cases, tacrolimus PNAS, Oct. 2004, Starzl.

19. Total Lymphoid Irradiation • Is TLI conducive to tolerance induction? • Preconditioning regimen depletes immunoreactive clones • Antigen presentation and immune activation impaired

21. Outcome of ACI to Lewis Liver Transplants with or without TLI +/- Donor cell Infusion.

22. Cadaveric Liver Transplantation Protocol rATG (2.0 mg/kg) TLI Day 0 Liver Transplant Tacrolimus wean month 3-6 Withdraw immunosuppression if: -no evidence of rejection -no GVHD

23. Future Directions • Xenotransplantation • Artificial liver • Stem-cells and liver regeneration • Treatment and prevention of hepatitis

26. This three-year-old boy was born with biliary atresia and had been explored previously. The child was anesthetized with flouothane anesthesia and a large bore needle placed in a vein of the arm. A thoraco-abdominal incision was then made through the eighth right intercostals space and extending transversely down across the abdomen and then back up to the left costal margin. The operative wound was exceedingly difficult to keep dry during the deepening of the incision because of numerous collateral veins secondary to the portal hypertension. At this time, word was passed from the donor room that the pumping system for perfusion of the liver had failed and that it would be necessary to act with the utmost expediency to remove the child’s liver. The triangular ligaments were then cut and the liver removed with considerably less than pains-taking care for hemostasis. It should be mentioned that just before removing the liver, it was noticed that the needle had become disengaged from its position in the arm, and that the transfusions which had been given up to this point had all been passing out upon the table. Upon release of the clamps, the liver immediately assumed a good color, and despite the hectic circumstances of the transplant, it was initially thought that a good result would be obtained. However, after about 15 or 20 minutes, it was noticed that all raw surfaces were profusely bleeding. A sample was sent and the report came back that there were extremely high titres of fibrinolysin and low titres of fibrinogen in the submitted blood. The next 5 or 6 hours were spent in a desperate effort to control hemorrhage, using literally thousands of sutures and ties, but the hemorrhagic diathesis could never be satisfactorily controlled.

27. Excerpts from the autopsy report: Discussion: This three year old boy was first noted to be jaundiced at age one day. At the age of five months, an exploratory laparotomy revealed the absence of an extrahepatic biliary system and a liver biopsy was obtained. This showed the presence of early biliary cirrhosis. During the next two years the child was hospitalized several times for various infections. His weight gain was poor but growth in height was normal. He was admitted at his mother’s request so that some research procedure might be carried out in an effort to prevent this illness in other children. Autopsy examination showed that patent anastomoses of hepatic artery, portal vein and hepatic vein had been surgically effected. …The most significant findings were those of small hemorrhages in the adrenal cortex and lung and advanced, near total necrosis or autolysis of liver parenchyma. A review of current literature does not reveal a report of attempted liver transplant in humans, although there is great activity with experimental animals. Moore, Wheeler, et al, carried out well controlled series of liver and spleen transplants on dogs. They did not attempt to block the immunological response, but only to delineate the technical and mechanical problems. One of 31 homotransplants survived 12 days but more distressing is that none of 21 autotransplants survived more than 14 days. The causes of death in the two groups were similar and mostly unassociated with liver failure. Also five of thirteen “sham” dissections failed to survive. (The entire procedure except removal and replacement of liver was effected.) These two latter groups would seem to point out the tremendous technical difficulties attending this procedure above and beyond the immunological problems involved. These authors remark “If the transfusion volume Is large (during surgery), the dog will be found to have a coagulation defect that makes hemostasis difficult.” This observation may be of some significance in this case.

28. UNOS Regions

29. Indications for liver transplantation in adult and pediatric patients Biliary diseasesParenchymal diseases Primary biliary cirrhosis Viral hepatitis Secondary biliary cirrhosis Alcoholic cirrhosis Sclerosing cholangitis Neonatal hepatitis Biliary atresia Congenital Hepatic Fibrosis Autoimmune hepatitis Inborn errors of metabolismAcute liver failure Alpha 1-antitrypsin deficiency Severe hepatic trauma Hemochromatosis Byler’s disease Hepatic tumors Familial cholestasis Benign hepatic tumors Type I glycogen storage disease Primary hepatic malignancy Hemophilia Cystic fibrosis

30. Contraindications for liver transplantation Absolute • Advanced HCC - Stage IV • Liver metastases - except neuroendocrine tumors • Cholangiocarcinoma - except for in situ lesions • Active alcoholism • Active drug abuse • Active tuberculosis • AIDS • Refractory extra-hepatic sepsis • Extensive mesenteric venous thrombosis • Severe cardiopulmonary disease

31. Contraindications for liver transplantation Relative • HCC - Stage III • Alcohol abstinence < 6 months • Severe debilitation • Metastatic neuroendocrine tumors • HIV positive

32. Liver Transplantation: Pittsburgh Experience Total Transplant Activity (including re-transplants) Adults Children Total Liver Transplant 4426 1163 5589 Liver + Intestine 108 83 191 Liver + Kidney 46 9 55 Liver + Pancreas 29 1 30 Liver + Islets 14 2 16 Liver + Heart 5 3 8

33. 4000 Consecutive Liver Transplants Demographics 4000 primary liver transplants were performed between February 1981-April 1998 Follow-up to March 2000 Mean follow-up was 9.4 + 3.8 years (median 9.6 years, range 2-18 years)

34. 4000 Consecutive Liver Transplants Demographics EraTime FrameNumber of Patients A (Cyclosporine) 1981 – 1985 478 B (UW; OKT3) 1986 – 1990 1382 C (FK506) 1991 – 1998 2140

35. N (%) HCV/NANB 680 (21.3) Alcoholic 567 (17.8) PBC 409 (12.8) Cryptogenic 279 (8.7) PSC 253 (7.9) Malignancy 234 (7.3) HBV 217 (6.8) Autoimmune 147 (4.6) N (%) Metabolic Disease 100 (3.1) Acute Liver Failure 76 (2.4) Budd-Chiari 39 (1.2) SBC 38 (1.2) Carcinoid 22 (0.7) Cystic fibrosis 11 (0.3) Other 120 (3.8) Total 3192 4000 Consecutive Liver Transplants Indications -- Adults

36. 4000 Consecutive Liver Transplants

37. N (%) Biliary Atresia 416 (51.4) Metabolic Disorders 109 (13.5) A-1-A 62 (7.6) Wilson’s Disease 16 (2.0) Tyrosinemia 13 (1.6) GSD 13 (1.6) Hemochromatosis 2 (0.2) Other 3 (0.4) Acute Liver Failure 48 (5.9) N (%) Cryptogenic 30 (3.7) NANB/HCV 35 (4.3) Familial Cholestasis 27 (3.3) Malignancy 23 (2.8) Neonatal hepatitis 19 (2.3) SBC 17 (2.1) Hepatic Fibrosis 15 (1.8) Autoimmune 8 (1.0) Other 61 (7.5) Total 808 4000 Consecutive Liver Transplants Indications -- Children

38. 4000 Consecutive Liver Transplants

39. 4000 Consecutive Liver Transplants Overall Patient Survival Years Post Transplantation

40. 4000 Consecutive Liver Transplants Patient Survival by Age Log Rank: p = 0.0001 Years Post Transplantation

41. 4000 Consecutive Liver Transplants Survival Attrition % Years Post Transplantation

42. 4000 Consecutive Liver Transplants Overall Graft Survival Years Post Transplantation

43. 4000 Consecutive Liver Transplants Graft Survival by Era Log Rank: p = 0.0001 Years Post Transplantation

44. 4000 Consecutive Liver Transplants Re-Transplant NumberN% 0 3226 80.6 1 774 19.4 2 148 3.7 3 20 0.5 >3 5 0.13

45. 4000 Consecutive Liver Transplants Patient Survival Following 1, 2, 3 and >3 LTx Years Post Retransplantation

46. 4000 Consecutive Liver Transplants Patient Survival by Diagnosis Years Post Retransplantation

47. 4000 Consecutive Liver Transplants Causes of Retransplantation

48. Organ Shortage

49. Liver Transplant Status In the United States

50. Operative Technique