Host conditioning and rejection monitoring in hepatocyte transplantation in humans

Abstract

Background & aims: Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Here we aimed to use preparative liver-directed radiation therapy, and continuous monitoring for possible rejection in an attempt to overcome these limitations.

Methods: Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression.

Results: Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67⁺ hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343±48μM (normal 30-119μM) to 854±25μM (treatment goal ≤360μM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900μM during supervised follow-up, but graft loss occurred after follow-up became inconsistent.

Conclusions: Radiation preconditioning and serial rejection risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure.

Lay summary: Hepatocyte transplantation can potentially be used to treat genetic liver disorders but its application in clinical practice has been impeded by inefficient hepatocyte engraftment and the inability to monitor rejection of transplanted liver cells. In this study, we first show in non-human primates that pretreatment of the host liver with radiation improves the engraftment of transplanted liver cells. We then used this knowledge in a series of clinical hepatocyte transplants in patients with genetic liver disorders to show that radiation pretreatment and rejection risk monitoring are safe and, if optimized, could improve engraftment and long-term survival of transplanted hepatocytes in patients.

Keywords: Hepatocyte transplantation; Hepatocyte xenotransplantation; Liver-based metabolic disease; Liver-directed radiation therapy; Phenylketonuria; Rejection risk monitoring.

Figure 1. Treatment planning for radiation therapy of the patient with classical PKU

Dose distributions along Axial (A), Sagittal (B), and (C) Coronal planes. The patient received a 10Gy dose in a single fraction with 7 fields gated IMRT. The portion of the irradiated right lobe is depicted in red. Light blue, green and deep blue colored isodose lines represent 5Gy, 8Gy, and 10Gy respectively. (D) Dose volume histogram for the patient.

Figure 2. Portal vein access for hepatocyte transplantation in the patient with classical PKU

(A) Schematic of hepatocyte infusion into the liver through the portal vein using the recanalized umbilical vein. (B) Photograph of catheter placement into the umbilical vein through a small peri-umbilical incision. (C) Contrast injection demonstrating the occlusion catheter going through the umbilical vein and the left portal vein, which is then directed into the right portal vein. (D) Balloon occlusion and contrast directed into right portal vein. (E) Contrast injection showing “pruning” of the portal venous system following hepatocyte infusion.

Figure 3. Engraftment and proliferation of porcine hepatocyte xenografts in NHPs

(A) Immunofluorescence staining for porcine albumin and β-Actin in the indicated liver tissue specimens. (B) Low power image of porcine albumin stained liver tissues showing large clusters of porcine albumin+ cells proximal to the blood vessels in the right lobe of transplant recipient liver. (C) Quantification of porcine albumin positivity in the left and right liver lobes of each animal. (D) Quantification of porcine albumin positivity in the left and right lobes relative to the whole liver. Values are presented as mean ± SEM.

Figure 4. Assessment of characteristics of isolated human hepatocytes

Measurement of the viability (A), apoptosis (B), and cytochrome P450 activity and ammonia metabolism (C) of hepatocytes right after isolation and during each round of hepatocyte transplantation.

Figure 5. Schematic presentation of pertinent laboratory investigations in infants that underwent hepatocyte transplants for UCD

(A) In the patient with CPS1 deficiency, elevated baseline IR at the time of transplant and increases in ammonia levels associated with minor increases in protein intake suggests poor graft function or graft failure. (B) In the patient with OTC deficiency, the increase in IR to a level above threshold at day 75 corresponding to an episode of severe ammonia level elevation suggests poor graft function or graft failure.

Figure 6. Histologic findings 6 months post-transplantation in the patient with classical PKU

FISH for the X and Y chromosomes (A) and immunocytochemistry for the PAH enzyme (B, C) confirming presence of small clusters of donor hepatocytes in the recipient liver. Histology showing mitoses (D) and Ki67+ cells (E) demonstrating proliferation of donor or host hepatocytes. (F) High resolution melt profile indicating the presence of ~6% donor DNA, that when corrected for salt concentration is estimated to be 3%.

Figure 7. Changes in peripheral PHE levels and IR levels following hepatocyte transplantation in a patient with PKU

(A) Comparison of PHE levels before and after transplantation showing a drop in PHE level to 854 ± 25μM (normal 30-120μM; treatment goal ≤360μM) in the PKU patient on an unrestricted diet after transplant. The decrease in PHE level by 36% compared to pre-transplant levels was statistically significant. Values are presented as mean ± SEM and significance was calculated using an unpaired one-tailed t test (**p<0.0001), followed by an F test to compare variances (ns). (B) PHE and IR levels measured at various time points post-transplant. Spikes in IR levels at 18 and 57 days post-transplant suggested increased risk of rejection but appeared to be transient as subsequent measurements were lower. At 219 days post-transplant, the IR level on three consecutive measurements indicated increased risk of rejection that coincided with an abrupt decrease in the patient’s PHE tolerance to pre-transplant baseline, suggesting that the allograft was failing due to rejection. Treatment of the patient with steroids improved PHE tolerance.

Figure 8. Evaluation of cellular graft function by whole body PHE oxidation

Serum PHE levels were intermittently elevated in the PKU patient after more than a year post-transplantation. In order to assess the status of donor hepatocytes, and quantitate the function of transplanted hepatocytes, whole body turnover analysis of 1-¹³C-Phe was performed in lieu of a liver biopsy. As measurement of whole body PHE oxidation was not available prior to transplant, the patient’s similarly affected brother was studied as a control. PHE oxidation (presented as a % of the dose of 1-¹³C-Phe oxidized) in the PKU patient (A) and her brother (B) were both low (~0.5-0.6%) and not different. (C) PHE oxidation in a normal control (Note the 50-fold change in scale).