Prognostic Abilities and Quality Assessment of Models for the Prediction of 90-Day Mortality in Liver Transplant Waiting List Patients.

BACKGROUND: Model of end-stage liver disease (MELD)-score and diverse variants are widely used for prognosis on liver transplant waiting-lists. METHODS: 818 consecutive patients on the liver transplant waiting-list included to calculate the MELD, MESO Index, MELD-Na, UKELD, iMELD, refitMELD, refitMELD-Na, upMELD and PELD-scores. Prognostic abilities for 90-day mortality were investigated applying Receiver-operating-characteristic-curve analysis. Independent risk factors for 90-day mortality were identified with multivariable binary logistic regression modelling. Methodological quality of the underlying development studies was assessed with a systematic assessment tool. RESULTS: 74 patients (9%) died on the liver transplant waiting list within 90 days after listing. All but one scores, refitMELD-Na, had acceptable prognostic performance with areas under the ROC-curves (AUROCs)>0.700. The iMELD performed best (AUROC = 0.798). In pediatric cases, the PELD-score just failed to reach the acceptable threshold with an AUROC = 0.699. All scores reached a mean quality score of 72.3%. Highest quality scores could be achieved by the UKELD and PELD-scores. Studies specifically lack statistical validity and model evaluation. CONCLUSIONS: Inferior quality assessment of prognostic models does not necessarily imply inferior prognostic abilities. The iMELD might be a more reliable tool representing urgency of transplantation than the MELD-score. PELD-score is assumedly not accurate enough to allow graft allocation decision in pediatric liver transplantation.

Independent Pre-Transplant Recipient Cancer Risk Factors after Kidney Transplantation and the Utility of G-Chart Analysis for Clinical Process Control.

BACKGROUND: The aim of this study is to identify independent pre-transplant cancer risk factors after kidney transplantation and to assess the utility of G-chart analysis for clinical process control. This may contribute to the improvement of cancer surveillance processes in individual transplant centers. PATIENTS AND METHODS: 1655 patients after kidney transplantation at our institution with a total of 9,425 person-years of follow-up were compared retrospectively to the general German population using site-specific standardized-incidence-ratios (SIRs) of observed malignancies. Risk-adjusted multivariable Cox regression was used to identify independent pre-transplant cancer risk factors. G-chart analysis was applied to determine relevant differences in the frequency of cancer occurrences. RESULTS: Cancer incidence rates were almost three times higher as compared to the matched general population (SIR = 2.75; 95%-CI: 2.33-3.21). Significantly increased SIRs were observed for renal cell carcinoma (SIR = 22.46), post-transplant lymphoproliferative disorder (SIR = 8.36), prostate cancer (SIR = 2.22), bladder cancer (SIR = 3.24), thyroid cancer (SIR = 10.13) and melanoma (SIR = 3.08). Independent pre-transplant risk factors for cancer-free survival were age <52.3 years (p = 0.007, Hazard ratio (HR): 0.82), age >62.6 years (p = 0.001, HR: 1.29), polycystic kidney disease other than autosomal dominant polycystic kidney disease (ADPKD) (p = 0.001, HR: 0.68), high body mass index in kg/m2 (p<0.001, HR: 1.04), ADPKD (p = 0.008, HR: 1.26) and diabetic nephropathy (p = 0.004, HR = 1.51). G-chart analysis identified relevant changes in the detection rates of cancer during aftercare with no significant relation to identified risk factors for cancer-free survival (p<0.05). CONCLUSIONS: Risk-adapted cancer surveillance combined with prospective G-chart analysis likely improves cancer surveillance schemes by adapting processes to identified risk factors and by using G-chart alarm signals to trigger Kaizen events and audits for root-cause analysis of relevant detection rate changes. Further, comparative G-chart analysis would enable benchmarking of cancer surveillance processes between centers.

Risk factors for short- and long-term mortality in liver transplant recipients with MELD score ≥30.

BACKGROUND: After introduction of MELD-based allocation in Germany, decreased waiting list mortality and increased mortality after transplantation have been reported. MATERIAL AND METHODS: This study compares relevant outcome parameters in patients with high MELD ≥30 versus lower MELD scores in a retrospective analysis including 454 consecutively performed liver transplantations in adults (age >16 years) at Hannover Medical School between 01/01/2007 and 31/12/2012 and a follow-up until 31/12/2013. Multivariable risk-adjusted models were applied to identify independent risk factors for 90-day and long-term mortality. RESULTS: MELD score ≥30 (n=117; 26.1%) was an independent risk factor for 90-day mortality (p=0.004, odds ratio: 3.045, 95% CI 1.439-6.498) and long-term mortality (p=0.016, hazard ratio: 1.620, 95% CI 1.095-2.396) and was associated with significantly longer hospital and intensive care unit stays (p<0.001), and death occurred in more cases earlier after transplantation (90-day mortality 21.6% vs. 13.0%; p=0.029). Portal vein thrombosis at transplantation was significantly associated with 90-day mortality after transplantation in patients with MELD scores ≥30 (p=0.041), but this was not the case for patients with MELD scores <30, although portal vein thrombosis was equally frequent in individuals of both groups (3.0% vs. 3.4%, p=0.824). CONCLUSIONS: Results of this study suggest that liver transplant recipients with portal vein thrombosis at transplantation should be transplanted before reaching a MELD score ≥30.

Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells.

BACKGROUND: The adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6. METHODS: To obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ(+) T cells exceeded 0.03% of CD3(+) lymphocytes as determined by IFN-γ cytokine secretion assay. RESULTS: Starting with low concentration of IFN-γ(+) T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ(+)CD3(+) T cells (1.42 × 10(6), 0.05 × 10(6), and 1.15 × 10(6)) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8(+) than CD4(+) memory T cells, both of which were effectively enriched to a total of 81.0% CD8(+)IFN-γ(+) and 38.4% CD4(+)IFN-γ(+) T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes. CONCLUSIONS: The generation of antiviral CD4(+) and CD8(+) T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.

Value and limitations of the BAR-score for donor allocation in liver transplantation.

PURPOSE: The MELD-score was shown to be able to predict 90-day mortality in most patients with end-stage liver disease prior to liver transplantation and is used as a widely accepted measure for transplantation urgency. Prognostic ability of the BAR-score to predict 90-day post-transplant mortality by detection of unfavourable pretransplant combinations of donor and recipient factors may help to better balance urgency versus utility. METHODS: Two German cohorts (Hannover, n=453; Kiel, n=234) were retrospectively analyzed using ROC-curve analysis, goodness-of-model-fit tests, summary measures and risk-adjusted multivariate binary regression. Included were all consecutive liver transplants performed in adult recipients (minimum age 18 years). Excluded were all combined transplants and living-related organ donor transplants. RESULTS: Risk-adjusted multivariate regression revealed that the BAR-score is an independent risk factor for 90-day mortality after transplantation in both cohorts from Hannover and Kiel combined (p<0.001, OR=1.017, 95% CI:1.031-1.113). The area under the ROC-curve (AUROC) for the prediction of 90-day mortality using the BAR-score was 0.662 (95% CI 0.624-0.699, power>95%). Measures for association between observed 90-day mortality and the predicted probabilities in the combined cohort were concordant in 63.5% with low summary measures (Somers' D test 0.32, Goodman-Kruskal Gamma test 0.34 and Kendall's Tau a test 0.07). CONCLUSIONS: The BAR-score performed below accepted thresholds for potentially useful clinical prognostic models. Prognostic models with better predictive ability with AUROCs>0.700, concordance>70% and larger summary measures are required for the prediction of 90-day post-transplant mortality to enable donor organ allocation with reliable weighing of urgency versus utility.

Liver cell transplantation: basic investigations for safe application in infants and small children.

Liver cell transplantation (LCT) is a very promising method for the use in pediatric patients. It is significantly less invasive than whole organ transplantation, but has the potential to cure or at least to substantially improve severe disorders like inborn errors of metabolism or acute liver failure. Prior to a widespread use of the technique in children, some important issues regarding safety and efficacy must be addressed. We developed a mathematical model to estimate total hepatocyte counts in relation to bodyweight to make possible more appropriate dose calculations. Different liver cell suspensions were studied at different flow rates and different catheter sizes to determine mechanical damage of cells by shear forces. At moderate flow rates, no significant loss of viability was observed even at a catheter diameter of 4.2F. Addition of heparin to the cell suspension is favored, which is in contrast to previous animal experiments. Mitochondrial function of the hepatocytes was determined with the WST-1 assay and was not substantially altered by cryopreservation. We conclude that especially with the use of small catheters, human LCT should be safe and efficient even in small infants and neonates.

Enzyme induction in cryopreserved human hepatocyte cultures.

Freshly isolated human hepatocytes are considered as the gold standard for in vitro testing of drug candidates. Meanwhile also cryopreserved human hepatocyte suspensions are available. However, a drawback of these cells is the incalculability of attachment to the culture dish. Therefore, we established a technique freezing hepatocytes cultured on a collagen gel. After thawing damaged cells were removed to a certain extent by gentle washing with culture medium prior to adding an upper gel layer. The morphology of the resulting hepatocyte cultures could not be distinguished from that of non-frozen cells. However, basal activities of cytochrome P450 isoforms decreased in cryopreserved compared to non-frozen hepatocytes, as evidenced by analysis of testosterone hydroxylation (OHT) in positions 6beta, 16alpha, 2beta and 6alpha. Nevertheless, enzyme induction factors caused by 24 h incubation with 50 microM rifampicin were similar in cryopreserved and non-frozen hepatocytes. In cryopreserved hepatocytes rifampicin caused an increase in mean values of 6beta-OHT formation from 57.2 to 157.7 pmol/well/min (2.8-fold), compared to an increase from 115.8 to 269.1 pmol/well/min (2.3-fold) in non-frozen cells. Similarly, 16alpha- and 2beta-OHT showed induction factors of 2.4- and 2.3-fold in cryopreserved compared to 1.6- and 2.4-fold in non-frozen hepatocytes, respectively. In conclusion, human hepatocytes cryopreserved on collagen gels show a clear induction of CYP3A4 by rifampicin, although the basal activities are reduced compared to non-frozen cells.

Large-scale isolation of human hepatocytes for therapeutic application.

During the last decade, hepatocyte transplantation has been suggested as a safe and potentially effective clinical option for the treatment of acute or decompensating chronic liver failure as well as for hereditary liver disease. Currently, one of the major limiting factors for clinical application is the insufficient access to suitable liver cell preparations. In cooperation with the German and Catalane organ procurement organizations, a routine procedure for the isolation of hepatocytes from donor organs rejected for transplantation (n = 117) has been established. The process is performed according to the current EC Guidelines for Good Manufacturing Practice (cGMP) and all corresponding national laws and regulations concerning donor organ and tissue procurement. In about 50% of the cases (n = 58) the three-step perfusion procedure has been completed with an average total cell yield of 5.9 x 10(9) cells per organ, the cell preparations displaying a mean viability of 64%. The mean specific yield was 3.6 x 10(6) total and 2.6 x 10(6) viable cells per gram liver tissue, respectively. Specific cell yields from three infantile donor livers were considerably higher. No correlation between isolation efficiency and cold ischemia time or donor age was found within the adult organ donors. In contrast, organs with a severe steatosis generally did not result in successful cell isolation. Results of sterility and endotoxin determination are also presented. In summary, a standardized and cGMP conform method of hepatocyte isolation from nontransplantable liver organs was established, which reproducibly yields large amounts of hepatocytes suitable for therapeutic application.