Donor and recipient genetics: Implications for the development of posttransplant diabetes mellitus.

Posttransplant diabetes mellitus (PTDM) is a prevalent complication of liver transplantation and is associated with cardiometabolic complications. We studied the consequences of genetic effects of liver donors and recipients on PTDM outcomes, focusing on the diverse genetic pathways related to insulin that play a role in the development of PTDM. One thousand one hundred fifteen liver transplant recipients without a pretransplant diagnosis of type 2 diabetes mellitus (T2D) and their paired donors recruited from 2 transplant centers had polygenic risk scores (PRS) for T2D, insulin secretion, and insulin sensitivity calculated. Among recipients in the highest T2D-PRS quintile, donor T2D-PRS did not contribute significantly to PTDM. However, in recipients with the lowest T2D genetic risk, donor livers with the highest T2D-PRS contributed to the development of PTDM (OR [95% CI] = 3.79 [1.10-13.1], P = .035). Recipient risk was linked to factors associated with insulin secretion (OR [95% CI] = 0.85 [0.74-0.98], P = .02), while donor livers contributed to PTDM via gene pathways involved in insulin sensitivity (OR [95% CI] = 0.86 [0.75-0.99], P = .03). Recipient and donor PRS independently and collectively serve as predictors of PTDM onset. The genetically influenced biological pathways in recipients primarily pertain to insulin secretion, whereas the genetic makeup of donors exerts an influence on insulin sensitivity.

Whole-exome sequencing analyses in a Saudi Ischemic Stroke Cohort reveal association signals, and shows polygenic risk scores are related to Modified Rankin Scale Risk.

Ischemic stroke represents a significant societal burden across the globe. Rare high penetrant monogenic variants and less pathogenic common single nucleotide polymorphisms (SNPs) have been described as being associated with risk of diseases. Genetic studies in Saudi Arabian patients offer a greater opportunity to detect rare high penetrant mutations enriched in these consanguineous populations. We performed whole exome sequencing on 387 ischemic stroke subjects from Saudi Arabian hospital networks with up to 20,230 controls from the Saudi Human Genome Project and performed gene burden analyses of variants in 177 a priori loci derived from knowledge-driven curation of monogenic and genome-wide association studies of stroke. Using gene-burden analyses, we observed significant associations in numerous loci under autosomal dominant and/or recessive modelling. Stroke subjects with modified Rankin Scale (mRSs) above 3 were found to carry greater cumulative polygenic risk score (PRS) from rare variants in stroke genes (standardized PRS mean > 0) compared to the population average (standardized PRS mean = 0). However, patients with mRS of 3 or lower had lower cumulative genetic risk from rare variants in stroke genes (OR (95%CI) = 1.79 (1.29-2.49), p = 0.0005), with the means of standardized PRS at or lower than 0. In conclusion, gene burden testing in Saudi stroke populations reveals a number of statistically significant signals under different disease inheritance models. However, interestingly, stroke subjects with mRS of 3 or lower had lower cumulative genetic risk from rare variants in stroke genes and therefore, determining the potential mRS cutoffs to use for clinical significance may allow risk stratification of this population.

Joint testing of donor and recipient genetic matching scores and recipient genotype has robust power for finding genes associated with transplant outcomes.

Genetic matching between transplant donor and recipient pairs has traditionally focused on the human leukocyte antigen (HLA) regions of the genome, but recent studies suggest that matching for non-HLA regions may be important as well. We assess four genetic matching scores for use in association analyses of transplant outcomes. These scores describe genetic ancestry distance using identity-by-state, or genetic incompatibility or mismatch of the two genomes and therefore may reflect different underlying biological mechanisms for donor and recipient genes to influence transplant outcomes. Our simulation studies show that jointly testing these scores with the recipient genotype is a powerful method for preliminary screening and discovery of transplant outcome related single nucleotide polymorphisms (SNPs) and gene regions. Following these joint tests with marginal testing of the recipient genotype and matching score separately can lead to further understanding of the biological mechanisms behind transplant outcomes. In addition, we present results of a liver transplant data analysis that shows joint testing can detect SNPs significantly associated with acute rejection in liver transplant.

Genome-wide non-HLA donor-recipient genetic differences influence renal allograft survival via early allograft fibrosis.

Donor-recipient (D-R) differences at human leukocyte antigen (HLA) loci are currently incorporated into organ sharing, allocation and immunosuppression decisions. However, while acute rejection episodes have substantially diminished, progressive histologic damage occurs in allografts and improved long-term survival remains an unrealized goal among kidney recipients. Here we tested the hypothesis that non-HLA dependent, genome-wide D-R genetic differences could contribute to unchecked alloimmunity with histologic and functional consequences, culminating in long-term allograft failure. Genome-wide single nucleotide polymorphism (SNP) array data, excluding the HLA region, was utilized from 385 transplants to study the role of D-R differences upon serial histology and allograft survival. ADMIXTURE analysis was performed to quantitatively estimate ancestry in each D-R pair and PLINK was used to estimate the proportion of genome-shared identity-by-descent (pIBD) between D-R pairs. Subsequently, quantitative measures of recipient ancestry based on non-HLA SNPs was associated with death-censored allograft survival in adjusted Cox models. In D-R pairs of similar ancestry, pIBD was significantly associated with allograft survival independent of HLA mismatches in 224 transplants. Surprisingly, pIBD and recipient ancestry were not associated with clinical or subclinical rejection at any time post-transplant. Significantly, in multivariable analysis, pIBD inversely correlated with vascular intimal fibrosis in 160 biopsies obtained less than one year which in turn was significantly associated with allograft survival. Thus, our novel data show that non-HLA D-R differences associate with early vascular intimal fibrosis and allograft survival.

Contribution of non-HLA incompatibility between donor and recipient to kidney allograft survival: genome-wide analysis in a prospective cohort.

BACKGROUND: The introduction of HLA matching of donors and recipients was a breakthrough in kidney transplantation. However, half of all transplanted kidneys still fail within 15 years after transplantation. Epidemiological data suggest a fundamental role of non-HLA alloimmunity. METHODS: We genotyped 477 pairs of deceased donors and first kidney transplant recipients with stable graft function at three months that were transplanted between Dec 1, 2005, and April 30, 2015. Genome-wide genetic mismatches in non-synonymous single nucleotide polymorphisms (nsSNPs) were calculated to identify incompatibilities in transmembrane and secreted proteins. We estimated the association between nsSNP mismatch and graft loss in a Cox proportional hazard model, adjusting for HLA mismatch and clinical covariates. Customised peptide arrays were generated to screen for antibodies against genotype-derived mismatched epitopes in 25 patients with biopsy-confirmed chronic antibody-mediated rejection. FINDINGS: 59 268 nsSNPs affecting a transmembrane or secreted protein were analysed. The median number of nsSNP mismatches in immune-accessible transmembrane and secreted proteins between donors and recipients was 1892 (IQR 1850-1936). The degree of nsSNP mismatch was independently associated with graft loss in a multivariable model adjusted for HLA eplet mismatch (HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR). Each increase by a unit of one IQR had an HR of 1·68 (95% CI 1·17-2·41, p=0·005). 5-year death censored graft survival was 98% in the quartile with the lowest mismatch, 91% in the second quartile, 89% in the third quartile, and 82% in the highest quartile (p=0·003, log-rank test). Customised peptide arrays verified a donor-specific alloimmune response to genetically predicted mismatched epitopes. INTERPRETATION: Genetic mismatch of non-HLA haplotypes coding for transmembrane or secreted proteins is associated with an increased risk of functional graft loss independently of HLA incompatibility. As in HLA alloimmunity, donor-specific alloantibodies can be identified against genotype derived non-HLA epitopes. FUNDING: Austrian Science Fund, WWTF (Vienna Science and Technology Fund), and Ministry of Health of the Czech Republic.

High sustained virologic response in genotypes 3 and 6 with generic NS5A inhibitor and sofosbuvir regimens in chronic HCV in myanmar.

Exclusive HCV therapy clinical trials with genotype 6 patients in high prevalence areas have been sparse. We analysed the safety and efficacy of two generic, pangenotypic NS5A/NS5B combination oral DAA regimens, primarily in genotypes 3 and 6, in a real-world setting: (a) daclatasvir/sofosbuvir (DCV/SOF) ± ribavirin (RBV) and (b) Velpatasvir/sofosbuvir (VEL/SOF ± RBV). Between December 2015 and November 2017, data from 522 patients were analysed, 311 of whom were treated with DCV/SOF ± RBV for 12/24 weeks (genotype 3: n = 193, genotype 6: n = 89) and 211 were treated with VEL/SOF ± RBV for 12/24 weeks (genotype 3: n = 83, genotype 6: n = 77). Overall SVR rates were high for both DCV/SOF ± RBV (96.1%, n = 299/311) and VEL/SOF ± RBV (95.3%, n = 201/211), and there was a good adverse event profile. Treatment naïve status and inclusion of RBV (in advanced fibrosis/cirrhosis) were significant independent predictors of achieving SVR12, while type of DAA regimen was not predictive. In this large cohort of genotypes 3 (n = 276) and 6 (n = 166; n = 127 unique subtype of 6c-l), high SVR rates of 94.9% (n = 262/276) and 95.2% (n = 158/166), respectively, were noted. In conclusion, generic and pangenotypic DCV/SOF and VEL/SOF ± RBV regimens were highly effective and safe, in genotypes 3 and 6 chronic HCV in Myanmar. These efficacious pangenotypic regimens suggest that baseline genotype testing can be eliminated moving forward. While RBV may still be needed for those with advanced fibrosis/cirrhosis, in a global elimination strategy it would not be practical even if it does compromise SVR in a minority with difficult to treat characteristics.

Assessing the Utility of a Genotype-Guided Tacrolimus Equation in African American Kidney Transplant Recipients: A Single Institution Retrospective Study.

Tacrolimus metabolism is heavily influenced by the CYP3A5 genotype, which varies widely among African Americans (AA). We aimed to assess the performance of a published genotype-informed tacrolimus dosing model in an independent set of adult AA kidney transplant (KTx) recipients. CYP3A5 genotypes were obtained for all AA KTx recipients (n = 232) from 2010 to 2019 who met inclusion criteria at a single transplant center in Philadelphia, Pennsylvania, USA. Medical record data were used to calculate predicted tacrolimus clearance using the published AA KTx dosing equation and two modified iterations. Observed and model-predicted trough levels were compared at 3 days, 3 months, and 6 months post-transplant. The mean prediction error at day 3 post-transplant was 3.05 ng/mL, indicating that the model tended to overpredict the tacrolimus trough. This bias improved over time to 1.36 and 0.78 ng/mL at 3 and 6 months post-transplant, respectively. Mean absolute prediction error-a marker of model precision-improved with time to 2.33 ng/mL at 6 months. Limiting genotype data in the model decreased bias and improved precision. The bias and precision of the published model improved over time and were comparable to studies in previous cohorts. The overprediction observed by the published model may represent overfitting to the initial cohort, possibly limiting generalizability.

Dietary assessment of type 2 diabetic patients using healthful plant-based diet score in the Eastern Province of Saudi Arabia.

BACKGROUND: Diabetes mellitus is a chronic disease characterized by a wide range of metabolic problems. The current study sought to assess nutritional habits of Saudi patients with type 2 diabetes (T2D) and to propose recommendations to improve these patients' dietary habits and delay possible disease complications. METHODS: Over a period of three years, (2017-2019) 577 patients with T2D attending the outpatient's diabetic clinics at King Fahd Hospital of the University, Al Khobar, Saudi Arabia were invited to participate in this study. Data of dietary intake were collected by trained nurses using a pretested structured validated semi quantitative food frequency questionnaire. The dietary data were collected using 7-day dietary recall questionnaire. A modified score system that associates dietary habits with glycemic control and lipid profile was used. RESULTS: Overall, a high healthful plant-based diet score was associated with a significant (P = 0.018) reduction in triglycerides (TG) level (mean difference - 3.78%; 95% CI, -0.65% to -6.81%) and a statistically non-significant (P = 0.06) increase in high density lipoprotein (HDL) levels (mean difference 1.87%; 95% CI -0.06-3.84%) in T2D patients from the Eastern Province of Saudi Arabia. Additionally, in our patient group, the prevalence of coronary artery disease, stroke, peripheral artery disease, and chronic kidney disease in T2D patients was 11.3%, 6.2%, 3.3%, and 8.4%, respectively and were higher when compared to the prevalence in the general population. CONCLUSION: The present study showed that adherence to a healthful plant-based diet, when compared to high glycemic index diet, is associated with a favorable outcome in glycemic control and lipid profile in T2D patients. Prior assessment of total diet quality may be beneficial when giving nutritional advice to T2D patients with the possibility of improving glycemic control and lipid profile.

Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

LoFTK: a framework for fully automated calculation of predicted Loss-of-Function variants and genes.

BACKGROUND: Loss-of-Function (LoF) variants in human genes are important due to their impact on clinical phenotypes and frequent occurrence in the genomes of healthy individuals. The association of LoF variants with complex diseases and traits may lead to the discovery and validation of novel therapeutic targets. Current approaches predict high-confidence LoF variants without identifying the specific genes or the number of copies they affect. Moreover, there is a lack of methods for detecting knockout genes caused by compound heterozygous (CH) LoF variants. RESULTS: We have developed the Loss-of-Function ToolKit (LoFTK), which allows efficient and automated prediction of LoF variants from genotyped, imputed and sequenced genomes. LoFTK enables the identification of genes that are inactive in one or two copies and provides summary statistics for downstream analyses. LoFTK can identify CH LoF variants, which result in LoF genes with two copies lost. Using data from parents and offspring we show that 96% of CH LoF genes predicted by LoFTK in the offspring have the respective alleles donated by each parent. CONCLUSIONS: LoFTK is a command-line based tool that provides a reliable computational workflow for predicting LoF variants from genotyped and sequenced genomes, identifying genes that are inactive in 1 or 2 copies. LoFTK is an open software and is freely available to non-commercial users at https://github.com/CirculatoryHealth/LoFTK .

The Course of LIRADS 3 and 4 Hepatic Abnormalities as Correlated With Explant Pathology: A Single Center Experience.

Background and aims: The Liver Reporting and Data System (LI-RADS) is the standard classification of imaging findings of hepatic abnormalities for hepatocellular carcinoma (HCC) surveillance. We aimed to study the course of LI-RADS 3 and 4 (LR-3 and LR-4) abnormalities through correlations with explant pathology. Methods: A single center retrospective study of liver transplant recipients between January 2016 and September 2019 with HCC on explant pathology was conducted. Eligible patients were divided into three subgroups based on their LI-RADS classification: LR-3/4, LR-5 only, and combination of LR-3/4/5. Results: There were 116 eligible patients with 99 LR-3/4 observations (60 LR-3 and 39 LR-4); the rest had LR-5 lesions. LR-4 more often than LR-3 observations progressed to LR-5 (36% vs 12%) and with shorter duration during follow-up (median 175 days and 196 days). Mean size growth of LR-3 and LR-4 abnormalities were 2.6 and 3.8 mm; median growth rates were 0.2 and 0.4 mm/month, respectively. Numbers of HCC lesions per explant, largest HCC lesion size, and cumulative size were higher in LR-3/4/5 subgroup than LR-5 subgroup (P = 0.007, 0.007 and 0.006, respectively); 68% of LR-3 and 82% of LR-4 abnormalities were confirmed HCC on explant (P = 0.09). Conclusion: Compared to LR-3, more LR-4 abnormalities progressed to LR-5 (12% and 36%, respectively) in a shorter time and with faster growth rate. A high proportion of LR-3 and LR-4 lesions (68% and 82%, respectively) were confirmed HCC on explant, raising the question of whether excluding HCC based on radiologic criteria alone is adequate in those with LR-3/4 abnormalities.

Impact of Donor and Recipient Clinical Characteristics and Hepatic Histology on Steatosis/Fibrosis Following Liver Transplantation.

BACKGROUND: Deceased donor and recipient predictors of posttransplant steatosis/steatohepatitis and fibrosis are not well known. Our aim was to evaluate the prevalence and assess donor and recipient predictors of steatosis, steatohepatitis, and fibrosis in liver transplantation recipients. METHODS: Using the immune tolerance network A-WISH multicenter study (NCT00135694), donor and recipient demographic and clinical features were collected. Liver biopsies were taken from the donor liver at transplant, and from recipients per protocol and for-cause (ie, abnormal transaminases and to rule out rejection) and were interpreted by a central pathologist. RESULTS: One hundred eighty-three paired donor/recipients liver biopsies at the time of transplant and posttransplant follow-up (median time 582 d; average time to last biopsies was 704 d [SD ± 402 d]) were analyzed. Donor steatosis did not influence recipient steatosis or fibrosis. Ten of 183 recipients had steatohepatitis on the last biopsy. Recipient body mass index at the time of liver biopsy was the most influential factor associated with posttransplant steatosis. Both donor and recipient metabolic syndrome features were not associated with graft steatosis. Untreated hepatitis C viral (HCV) infection was the most influential factor associated with the development of allograft fibrosis. CONCLUSIONS: In a large experience evaluating paired donor and recipient characteristics, recipient body mass index at the time of liver biopsy was most significantly associated with posttransplant steatosis. Untreated HCV etiology influenced graft fibrosis. Thus relative to untreated HCV, hepatic fibrosis in those with steatosis/steatohepatitis is less common though long-term follow-up is needed to determine the course of posttransplant fibrosis. Emphasis on recipient weight control is essential.

Donor and recipient polygenic risk scores influence the risk of post-transplant diabetes.

Post-transplant diabetes mellitus (PTDM) reduces allograft and recipient life span. Polygenic risk scores (PRSs) show robust association with greater risk of developing type 2 diabetes (T2D). We examined the association of PTDM with T2D PRS in liver recipients (n = 1,581) and their donors (n = 1,555), and kidney recipients (n = 2,062) and their donors (n = 533). Recipient T2D PRS was associated with pre-transplant T2D and the development of PTDM. T2D PRS in liver donors, but not in kidney donors, was an independent risk factor for PTDM development. The inclusion of a combined liver donor and recipient T2D PRS significantly improved PTDM prediction compared with a model that included only clinical characteristics: the area under the curve (AUC) was 67.6% (95% confidence interval (CI) 64.1-71.1%) for the combined T2D PRS versus 62.3% (95% CI 58.8-65.8%) for the clinical characteristics model (P = 0.0001). Liver recipients in the highest quintile of combined donor and recipient T2D PRS had the greatest risk of PTDM, with an odds ratio of 3.22 (95% CI 2.07-5.00) (P = 1.92 × 10-7) compared with those in the lowest quintile. In conclusion, T2D PRS identifies transplant candidates with high risk of PTDM for which pre-emptive diabetes management and donor selection may be warranted.

Whole transcriptome profiling of prospective endomyocardial biopsies reveals prognostic and diagnostic signatures of cardiac allograft rejection.

BACKGROUND: Heart transplantation provides a significant improvement in survival and quality of life for patients with end-stage heart disease, however many recipients experience different levels of graft rejection that can be associated with significant morbidities and mortality. Current clinical standard-of-care for the evaluation of heart transplant acute rejection (AR) consists of routine endomyocardial biopsy (EMB) followed by visual assessment by histopathology for immune infiltration and cardiomyocyte damage. We assessed whether the sensitivity and/or specificity of this process could be improved upon by adding RNA sequencing (RNA-seq) of EMBs coupled with histopathological interpretation. METHODS: Up to 6 standard-of-care, or for-cause EMBs, were collected from 26 heart transplant recipients from the prospective observational Clinical Trials of Transplantation (CTOT)-03 study, during the first 12-months post-transplant and subjected to RNA-seq (n = 125 EMBs total). Differential expression and random-forest-based machine learning were applied to develop signatures for classification and prognostication. RESULTS: Leveraging the unique longitudinal nature of this study, we show that transcriptional hallmarks for significant rejection events occur months before the actual event and are not visible using traditional histopathology. Using this information, we identified a prognostic signature for 0R/1R biopsies that with 90% accuracy can predict whether the next biopsy will be 2R/3R. CONCLUSIONS: RNA-seq-based molecular characterization of EMBs shows significant promise for the early detection of cardiac allograft rejection.

Bedside testing of CYP2C19 vs. conventional clopidogrel treatment to guide antiplatelet therapy in ST-segment elevation myocardial infarction patients.

BACKGROUND: ST-segment elevation myocardial infarction (STEMI) patients are treated with dual antiplatelet therapy comprising aspirin and a P2Y12 inhibitor. Clopidogrel is widely used in these patients in several areas worldwide, such as Middle East, but is associated to sub-optimal platelet inhibition in up to 1/3 of treated patients. We investigated a CYP2C19 genotype-guided strategy to select the optimal P2Y12 inhibitor. METHODS: This prospective randomized clinical trial included STEMI patients. The standard-treatment group received clopidogrel, while the genotype-guided group were genotyped for CYP2C19 loss-of-function alleles and carriers were prescribed ticagrelor and noncarriers were prescribed clopidogrel. Primary outcome was a combined ischemic and bleeding outcome, comprising myocardial infarction, non-fatal stroke, cardiovascular death, or Platelet Inhibition and Patient Outcomes major bleeding one year after STEMI. RESULTS: STEMI patients (755) were randomized into a genotype-guided- (383) and standard-treatment group (372). In the genotype-guided group, 31 patients carrying a loss-of-function allele were treated with ticagrelor, while all other patients in both groups were treated with clopidogrel. Patients in the genotype-guided group had a significantly lower risk of primary outcome (odds ratio (OR) 0.34, 95% confidence interval (CI) 0.20-0.59,), recurrent myocardial infarction (OR 0.25, 95%CI 0.11-0.53), cardiovascular death (OR 0.16, 95%CI0.06-0.42) and major bleeding (OR 0.49, 95%CI 0.32-0.74). There was no significant difference in the rate of stent thrombosis (OR 0.85, 95%CI 0.43-1.71). CONCLUSION: A genotype-guided escalation of P2Y12 inhibitor strategy is feasible in STEMI patients treated with clopidogrel and undergoing PCI and is associated with a reduction of primary outcomes compared to conventional antiplatelet therapy.

Predictors, Presentation, and Treatment Outcomes of Recurrent Hepatocellular Carcinoma After Liver Transplantation: A Large Single Center Experience.

BACKGROUND: Liver transplantation (LT) is an accepted therapeutic option for hepatocellular carcinoma (HCC) in patients with cirrhosis. Despite careful candidate selection, HCC recurrence occurs. We aimed to describe the predictors of recurrence, clinical presentation, and predictors of survival after HCC recurrence post-LT. METHODS: Patients with recurrent HCC after LT between January 1996 and December 2017 were retrospectively reviewed. RESULTS: Of 711 patients, 96 (13.5%) patients had post-LT HCC recurrence. The median time to recurrence was 17.1 months, and the median survival was 10.1 months. Initial recurrence was more often in the graft (34.4%), and most (60.4%) had multiple recurrent lesions, and 26% were in multiple sites. In multivariate analysis, factors associated with shorter survival were poorly differentiated histology in explant (Hazard ratio [HR] = 1.96; p = 0.027), bilirubin ≥1.2 mg/dL (HR = 2.47; p = 0.025), and albumin <3.5 mg/dL (HR = 2.13; p = 0.014) at recurrence, alpha-fetoprotein at recurrence ≥ 1000 ng/mL (HR = 2.96; p = 0.005), and peritoneal disease (HR = 3.20; p = 0.022). There was an increased survival in patients exposed to sirolimus (HR = 0.32; p < 0.0001). CONCLUSIONS: Recurrent HCC after LT is often in extrahepatic sites with a decreased survival in those with poorly differentiated explant pathology, high bilirubin, low albumin, marked elevation of alpha-fetoprotein at recurrence, and peritoneal recurrence. Sirolimus-based immunosuppression may provide benefit.

Monitoring liver damage using hepatocyte-specific methylation markers in cell-free circulating DNA.

Liver damage is typically inferred from serum measurements of cytoplasmic liver enzymes. DNA molecules released from dying hepatocytes are an alternative biomarker, unexplored so far, potentially allowing for quantitative assessment of liver cell death. Here we describe a method for detecting acute hepatocyte death, based on quantification of circulating, cell-free DNA (cfDNA) fragments carrying hepatocyte-specific methylation patterns. We identified 3 genomic loci that are unmethylated specifically in hepatocytes, and used bisulfite conversion, PCR, and massively parallel sequencing to quantify the concentration of hepatocyte-derived DNA in mixed samples. Healthy donors had, on average, 30 hepatocyte genomes/ml plasma, reflective of basal cell turnover in the liver. We identified elevations of hepatocyte cfDNA in patients shortly after liver transplantation, during acute rejection of an established liver transplant, and also in healthy individuals after partial hepatectomy. Furthermore, patients with sepsis had high levels of hepatocyte cfDNA, which correlated with levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Duchenne muscular dystrophy patients, in which elevated AST and ALT derive from damaged muscle rather than liver, did not have elevated hepatocyte cfDNA. We conclude that measurements of hepatocyte-derived cfDNA can provide specific and sensitive information on hepatocyte death, for monitoring human liver dynamics, disease, and toxicity.