A. Joseph Tector

Stereotactic body radiotherapy for primary hepatocellular carcinoma.

PURPOSE To evaluate the safety and efficacy of stereotactic body radiotherapy (SBRT) for the treatment of primary hepatocellular carcinoma (HCC). METHODS AND MATERIALS From 2005 to 2009, 60 patients with liver-confined HCC were treated with SBRT at the Indiana University Simon Cancer Center: 36 Child-Turcotte-Pugh (CTP) Class A and 24 CTP Class B. The median number of fractions, dose per fraction, and total dose, was 3, 14 Gy, and 44 Gy, respectively, for those with CTP Class A cirrhosis and 5, 8 Gy, and 40 Gy, respectively, for those with CTP Class B. Treatment was delivered via 6 to 12 beams and in nearly all cases was prescribed to the 80% isodose line. The records of all patients were reviewed, and treatment response was scored according to Response Evaluation Criteria in Solid Tumors v1.1. Toxicity was graded according to the Common Terminology Criteria for Adverse Events v4.0. Local control (LC), time to progression (TTP), progression-free survival (PFS), and overall survival (OS) were calculated according to the method of Kaplan and Meier. RESULTS The median follow-up time was 27 months, and the median tumor diameter was 3.2 cm. The 2-year LC, PFS, and OS were 90%, 48%, and 67%, respectively, with median TTP of 47.8 months. Subsequently, 23 patients underwent transplant, with a median time to transplant of 7 months. There were no ≥Grade 3 nonhematologic toxicities. Thirteen percent of patients experienced an increase in hematologic/hepatic dysfunction greater than 1 grade, and 20% experienced progression in CTP class within 3 months of treatment. CONCLUSIONS SBRT is a safe, effective, noninvasive option for patients with HCC ≤6 cm. As such, SBRT should be considered when bridging to transplant or as definitive therapy for those ineligible for transplant.

Use of extended criteria livers decreases wait time for liver transplantation without adversely impacting posttransplant survival.

Introduction:The use of extended criteria donors (ECDs) could minimize shortage of suitable donor livers for transplantation. In 3 years, the aggressive use of ECD livers has reduced the wait list at our center from 257 to 30 patients with a median wait time of 18 days without using living donors. This study compares the graft/patient survival from standard (SD) and ECD for our transplant population between 2001 and 2005. Methods:Records of all adult liver transplant recipients over 4 years were reviewed (n = 571). ECD criteria included: age >59 years, BMI >34.9, maximum AST/ALT >500, maximum bilirubin >2.0, peak serum sodium >170, HBV/HCV/HTLV reactive, donation after cardiac death, cold ischemia time >12 hours, ICU stay >5 days, 3 or more pressors simultaneously, extensive alcohol abuse, cancer history (nonskin), active meningitis/bacteremia, or significant donor liver trauma. Outcomes included graft and patient survival at 90 days, 1 year, and 2 years. Results:Sixty-eight percent of recipients (n = 388) received ECD livers. Primary factors accounting for ECD-liver status included: elevated liver function tests (20%), hypernatremia (12.6%), and extensive alcohol abuse (11.4%). Graft survival was (SD, ECD): 90-day 91%, 88%; 1-year 84%, 80%; 2-year 78%, 77%; patient survival was: 90-day 93%, 90%; 1-year 87%, 82%; 2-year 83%, 79%. Kaplan-Meier survival analysis failed to demonstrate an overall difference in graft or patient survival at any time point. Only donor age >60 years was associated with decreased graft and patient survival. Conclusions:Liver grafts from ECD can be used to dramatically reduce wait list time with outcomes comparable to those for SD without resorting to living donor liver transplantation.

Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose α-1,3-Galactose reduce the humoral barrier to xenotransplantation

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα‐1,3‐Gal (Gal) and N‐glycolylneuraminic acid (Neu5Gc) are two known xenoantigens.

Comparison of histidine‐tryptophan‐ketoglutarate solution (HTK) and University of Wisconsin solution (UW) in adult liver transplantation

Histidine‐tryptophan‐ketoglutarate solution (HTK) and University of Wisconsin solution (UW) have been shown to have similar outcomes in cadaveric kidney, pancreas, and liver transplantation. Our institution changed from UW to HTK as the primary preservation solution for liver, kidney and pancreas transplantation. This study compares the perioperative and first year outcomes of liver transplantation using UW or HTK. Primary use of HTK began on May 1, 2003. We reviewed the records of all adult liver transplant recipients from July 1, 2002 to December 31, 2004. Recipients were compared based on organ preservation solution (UW n=204, HTK n=174). Outcomes included 1‐, 6‐ and 12‐month graft and patient survival and 1‐, 7‐, 14‐, and 30‐day liver function and serum creatinine. During the entire study period, the two groups were managed similarly in operative technique, immunosuppressive regimens, and donor liver criteria. Over 30 months, 378 adult patients underwent liver transplantation. There were no significant differences between UW and HTK in 1‐, 6‐, or 12‐month graft or patient survival. The HTK group had a higher day 1 median AST, ALT, and total bilirubin, but the two groups were similar thereafter. An anticipated difference in infused volume between UW and HTK was demonstrated. In conclusion, to our knowledge, this is the first reported large case series from North America comparing HTK and UW in liver transplantation with 2‐ to 12‐month follow‐up. There were no significant differences between HTK and UW in this population when comparing 1 month graft function and first‐year graft and patient survival. Liver Transpl 12:226–230, 2006.

Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection.

Manipulating the pig genome to increase compatibility with human biology may facilitate the clinical application of xenotransplantation. Genetic modifications to pig cells have been made by sequential recombination in fetal fibroblasts and liver‐derived cells followed by cross‐breeding or somatic cell nuclear transfer. The generation of pigs for research or organ donation by these methods is slow, expensive and requires technical expertise. A novel system incorporating the bacterial nuclease Cas9 and single‐guide RNA targeting a 20 nucleotide site within a gene can be expressed from a single plasmid leading to a double‐strand break and gene disruption. Coexpression of multiple unique single‐guide RNA can modify several genetic loci in a single step. We describe a process for increasing the efficiency of selecting cells with multiple genetic modifications.

Pre‐transplant antibody screening and anti‐CD154 costimulation blockade promote long‐term xenograft survival in a pig‐to‐primate kidney transplant model

Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end‐stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig‐to‐primate kidney xenotransplantation has met limited success. Preformed anti‐pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti‐pig antibody levels. We then selected animals with both low and high titers of anti‐pig antibodies to proceed with kidney transplant from galactose‐α1,3‐galactose knockout/CD55 transgenic pig donors. All animals received T‐cell depletion followed by maintenance therapy with costimulation blockade (either anti‐CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti‐pig antibody rejected the kidney xenograft within the first week. Low‐titer animals treated with anti‐CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long‐term surviving animals treated with the anti‐CD154‐based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig‐to‐non‐human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.

Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution in extended criteria liver donors

Liver, pancreas, and kidney allografts preserved in histidine‐tryptophan‐ketoglutarate (HTK) and University of Wisconsin (UW) solutions have similar clinical outcomes. This study compares HTK and UW in a large number of standard criteria donor (SCD) and extended criteria donor (ECD) livers at a single center over 5 years. All adult, cadaveric liver and liver‐kidney transplants performed between July 1, 2001 and June 30, 2006 were reviewed (n = 698). There were 435 livers (62%) categorized as ECD for severe physiologic stress and 70 (10%) because of old age. Recipient outcomes included perioperative death or graft loss and overall survival. Liver enzymes were analyzed for the first month post‐transplant. Biliary complications were assessed through chart review. Overall, 371 donor livers were preserved in HTK (53%), and 327 were preserved in UW (47%). There were no statistically significant differences in any of the primary outcome measures comparing HTK and UW. The HTK group overall had a higher day 1 median aspartate aminotransferase and alanine aminotransferase, but the two groups were similar in function thereafter. HTK was superior to UW in protection against biliary complications. Kaplan‐Meier graft survival curves failed to demonstrate a significant difference in SCD or ECD livers. In conclusion, HTK and UW are not clinically distinguishable in this large sample of liver transplants, although HTK may be protective against biliary complications when compared to UW. These findings persisted for both SCD and ECD livers. Given the lower cost per donor for HTK, this preservation solution may be preferable for general use. Liver Transpl 14:365–373, 2008.

Creating Class I MHC–Null Pigs Using Guide RNA and the Cas9 Endonuclease

Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC− cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4−CD8+ T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.

Promising early results with immunosuppression using rabbit anti-thymocyte globulin and steroids with delayed introduction of tacrolimus in adult liver transplant recipients

Induction therapy with T‐cell depleting drugs in liver transplantation is controversial. This study examined the use of rabbit antithymocyte globulin (RATG) with delayed introduction of tacrolimus in liver transplant recipients. Additional subgroup analysis compared patients with or without hepatitis C (HCV) cirrhosis. Over 17 months, 116 adults received 120 liver allografts. Four patients who died before receiving RATG were excluded. Immunosuppression included steroids, 3 doses of RATG (2 mg/kg), and tacrolimus started on postoperative day 3 to 4. Ninety‐six percent of patients were alive with a mean follow‐up of 12.9±4.5 months. No graft was lost to rejection. Two patients developed hepatic artery thrombosis. Six percent of patients had acute rejection. No patient had steroid resistant or recurrent rejection. RATG related drug events were limited to fever, chills, tachycardia, and oxygen desaturation. There were no cases of lymphoproliferative disease. Forty‐two percent of patients were transplanted for HCV. Thirty‐two percent of HCV‐patients had biopsy proven hepatitis C recurrence occurring at 4 weeks to 10 months posttransplant. RATG induction therapy is associated with good patient and graft survival, a low incidence of rejection, and minimal side effects. In addition, RATG induction is safe in patients transplanted for HCV. (Liver Transpl 2004;10:404–407.)

Pig liver xenotransplantation as a bridge to allotransplantation: Which patients might benefit?

Acute liver failure is a potentially devastating clinical syndrome that, without liver transplantation (Tx), is associated with high mortality. Rapid deterioration in clinical status and a shortage of deceased human organs prohibits liver Tx in many patients. Bridging to liver Tx has been attempted by various approaches, for example, bioartificial liver support, extracorporeal pig liver perfusion, and hepatocyte Tx, but none of these approaches has convincingly improved patient survival. The orthotopic Tx of a genetically engineered pig liver could theoretically provide successful bridging. Immediate availability, perfect metabolic condition, adequate size-match and hepatocyte mass, and freedom from potentially pathogenic microorganisms could be assured. The advantages and disadvantages of bridging by pig liver Tx compared with other approaches are discussed. The selection of patients for an initial clinical trial of pig liver Tx would be similar to that of various prior trials in patients experiencing rapid and severe deterioration in liver function. The ability to give truly informed consent for a pig bridging procedure at the time of listing for liver Tx renders the patient with acute-on-chronic liver failure or primary allograft failure is a preferable candidate for this procedure than a patient who is admitted urgently with acute (fulminant) liver failure in whom consent may not be possible. Although several barriers to successful pig organ xenoTx remain, for example, coagulation dysfunction between pig and primate, if these can be resolved by further genetic engineering of the organ-source pigs, a pig liver may prove life saving to patients dying rapidly of liver failure.