Metallothionein: a game changer in histopathological diagnosis of Wilson disease.

AIMS: Wilson disease (WD) is a genetic disorder of copper metabolism caused by mutations in the ATP7B gene. Toxic copper accumulation leads to hepatic, neurologic, and psychiatric disorders with variable presentation. Metallothionein (MT) immunohistochemistry was proposed as a diagnostic marker. METHODS: MT immunohistochemistry was performed on liver specimens of WD patients (n = 64) and control cases (n = 160) including acute liver failure, steatotic liver disease, autoimmune hepatitis, normal liver, primary biliary cholangitis, primary and secondary sclerosing cholangitis, and progressive familial intrahepatic cholestasis. The optimal cutoff for detection of WD was determined by receiver operating characteristic (ROC) analysis. RESULTS: At least moderate staining in >50% of hepatocytes was observed in 81% of analysed liver specimens (n = 56/69) of WD patients, while only five control cases showed this staining pattern. The sensitivity, specificity, and accuracy for a new diagnosis of WD were 85.7%, 96.9%, and 94.9%, respectively. Sensitivity in nonfibrotic patients was 70.6% and this MT pattern was robust in small biopsies. The hepatic copper concentration was similar between MT-positive and MT-negative liver samples (P > 0.05). Zinc treatment may induce hepatocellular MT expression. Kayser-Fleischer rings (50% versus 15%) and neurologic disorders (50% versus 13%) were significantly more prevalent in MT-negative compared to MT-positive WD patients, respectively. CONCLUSION: MT immunostaining is an excellent biomarker for histological diagnosis of WD, should be incorporated in the diagnostic work-up of patients with potential WD, and is useful in a modified Leipzig score.

A fluorometric assay to determine labile copper(II) ions in serum.

Labile copper(II) ions (Cu2+) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu2+ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu2+ subset. This study introduces a fluorometric high throughput assay using the novel Cu2+ binding fluoresceine-peptide sensor FP4 (Kd of the Cu2+-FP4-complex 0.38 pM) to determine labile Cu2+ in human and rat serum. Using 96 human serum samples, labile Cu2+was measured to be 0.14 ± 0.05 pM, showing no correlation with age or other serum trace elements. No sex-specific differences in labile Cu2+ concentrations were noted, in contrast to the total copper levels in serum. Analysis of the effect of drug therapy on labile Cu2+ in the sera of 19 patients with WD showed a significant decrease in labile Cu2+ following copper chelation therapy, suggesting that labile Cu2+ may be a specific marker of disease status and that the assay could be suitable for monitoring treatment progress.

Excessive copper impairs intrahepatocyte trafficking and secretion of selenoprotein P.

Selenium homeostasis depends on hepatic biosynthesis of selenoprotein P (SELENOP) and SELENOP-mediated transport from the liver to e.g. the brain. In addition, the liver maintains copper homeostasis. Selenium and copper metabolism are inversely regulated, as increasing copper and decreasing selenium levels are observed in blood during aging and inflammation. Here we show that copper treatment increased intracellular selenium and SELENOP in hepatocytes and decreased extracellular SELENOP levels. Hepatic accumulation of copper is a characteristic of Wilson's disease. Accordingly, SELENOP levels were low in serum of Wilson's disease patients and Wilson's rats. Mechanistically, drugs targeting protein transport in the Golgi complex mimicked some of the effects observed, indicating a disrupting effect of excessive copper on intracellular SELENOP transport resulting in its accumulation in the late Golgi. Our data suggest that hepatic copper levels determine SELENOP release from the liver and may affect selenium transport to peripheral organs such as the brain.

[Hereditary Liver Diseases: Wilson's Disease and Hemochromatosis]. / Hereditäre Lebererkrankungen: Morbus Wilson und Hämochromatose.

Wilson's disease and HFE-hemochromatosis are autosomal-recessively inherited metabolic diseases of the liver. Copper overload in case of Wilson's disease and iron overload in case of hemochromatosis lead to organ damage of the liver and other organs. In order to diagnose these diseases at an early stage and introduce therapy, knowledge of the symptoms and diagnostic criteria of these diseases is important. Iron overload in hemochromatosis patients is treated with phlebotomies and copper overload in Wilson's disease patients with either chelating medications (D-penicillamine or trientine) or zinc salts. After introduction of lifelong therapy both diseases typically have a favorable disease course and further development of organ-damage can be prevented, especially with respect to liver-damage.

Medical care of patients with Wilson disease in Germany: a multidisciplinary survey among university centers.

BACKGROUND: Wilson disease (WD) is a rare, hereditary disorder of copper metabolism. Due to its variable symptoms and manifestations, diagnosis remains challenging. Affected patients must obtain lifelong medical treatment, as the disease is fatal if untreated. Patients require continuous monitoring, but little is known about the care of these patients in Germany. Therefore, we analyzed the medical care of WD patients at German university centers. We sent a questionnaire containing 20 questions to a total of 108 departments of pediatrics, neurology and gastroenterology in 36 university hospitals. Our questions referred to the characteristics of WD patients at the different sites and internal procedures regarding diagnosis, therapy and follow-up. A descriptive statistical analysis was performed. RESULTS: Sixty-three departments (58%) returned our questionnaire. In total, approximately one-third of the estimated WD patients in Germany are seen annually in the outpatient clinics of these departments (approx. 950 patients). There are only a few departments which treat patients in a multidisciplinary setting (12%). Our survey revealed that for diagnosis, 51% of all departments used an algorithm based on the Leipzig score as recommended by international guidelines. Most departments apply essential parameters recommended by WD guidelines. Routine monitoring is performed at least biannually by 84% of the departments, and standard investigations for monitoring are regularly applied. A routine family screening is performed by 84% of all departments. A reduction in medical therapy during pregnancy is recommended by 46% of the departments. Only 14% suggested that WD patients should not breastfeed. Liver transplantation (LT) due to WD is a rare but repeatedly occurring event. Most departments of gastroenterology (72%) reported at least one patient with LT within the last decade. CONCLUSIONS: Medical care of WD patients at German university centers follows the recommendations set forth by international guidelines, but only a few centers treat significant numbers of patients. The surveillance of patients does not follow specified standards, but most departments adhere to the accepted guidelines. The formation of central units and networks in a multidisciplinary setting should be evaluated to improve the care of WD patients.

Neurological worsening in Wilson disease - clinical classification and outcome.

BACKGROUND & AIMS: Prevention of neurological worsening (NW) under therapy is an unmet need in the management of Wilson disease (WD). In this study, we aimed to characterize the occurrence, associated outcomes and potential reversibility of NW in WD. METHODS: From a total cohort of 457 patients with WD, 128 patients with WD and neurological features at any time point (all Caucasian, 63 females, median age at diagnosis 22 years) were identified by chart review at University Hospital Heidelberg and grouped according to initial presentation. The timing and occurrence of NW was assessed following a structured clinical examination during clinical visits. RESULTS: Early NW (within the first 3 months of therapy) was observed in 30 out of 115 (26.1%) patients with neurological or mixed presentation and never in patients with a purely hepatic or asymptomatic presentation (0%). Late NW (after >12 months) was seen in a further 23 (20%) with neurological or mixed presentation and in 13 out of 294 (4.4%) patients with a hepatic or asymptomatic presentation. The median time from start of treatment to late NW was 20 months. Only three patients experienced NW between 3 and 12 months. NW was observed with D-penicillamine, trientine and zinc therapy and was reversible in 15/30 (50%) with early NW and in 29/36 (81%) with late NW. CONCLUSIONS: In this study, we identified two peaks in NW: an early (≤3 months) treatment-associated peak and a late (>12 months of treatment) adherence-associated peak. Early paradoxical NW was attributed to treatment initiation and pre-existing neurological damage, and was not observed in those with a hepatic or asymptomatic presentation. Late NW is likely to be associated with non-adherence. IMPACT AND IMPLICATIONS: In patients with Wilson disease, defined as an excess accumulation of copper which can damage the liver, brain and other vital organs, neurological worsening can occur despite chelation therapy. The study identifies different patterns of 'early' (<3 months) vs. 'late' (>12 months) neurological worsening in relation to initiation of chelation therapy and establishes possible causes and the potential for reversibility. These data should be useful for counseling patients and for guiding the optimal management of chelation therapy.

Experience on switching trientine formulations in Wilson disease: Efficacy and safety after initiation of TETA 4HCl as substitute for TETA 2HCl.

BACKGROUND AND AIM: This retrospective, multicenter study aims to assess the efficacy and safety in Wilson disease (WD) patients treated with trientine tetrahydrochloride (TETA 4HCl) after switch from trientine dihydrochloride (TETA 2HCl). METHODS: In total, 68 WD patients with stable copper metabolism were identified to receive TETA 4HCl (Cuprior™) after previous treatment with TETA 2HCl. We analyzed biochemical markers such as urinary copper, serum copper, non-coeruloplasmin bound copper (NCC), and transaminases as well as clinical scores (APRI; FIB-4 score) at baseline with a follow-up (FU) of 12 months. Safety of TETA 4HCl treatment was based on reported adverse events (AEs). RESULTS: The study cohort reflects a common WD cohort with a mean age of 20.3 years at diagnosis and 38.3 years at baseline. There are no significant differences concerning serum copper, NCC, transaminases, APRI, and FIB-4 score in the 3-month FU. Six-month FU revealed a decreased AST (P = 0.008), APRI (P = 0.042), and FIB-4 score (P = 0.039). GGT varied only borderline significantly in the 3-month, but not in the 6-month FU. Comparison of urinary copper within the subsets did not reveal a difference to baseline in all FUs, suggesting stable control of copper metabolism. Few AEs during TETA 4HCl treatment were reported, most commonly gastrointestinal discomfort. Only three treatments with TETA 4HCl were discontinued. CONCLUSION: Copper parameters and liver function were stable after treatment switch to TETA 4HCl. Treatment with TETA 4HCl was generally well tolerated. This study indicates that the switch from TETA 2HCl to TETA 4HCl is safe and viable.

Clinical effects and safety of different transarterial chemoembolization methods for bridging and palliative treatments in hepatocellular carcinoma.

PURPOSE: We assessed and compared clinical effects and safety endpoints of three methods of transarterial chemoembolization (TACE), conventional (cTACE), with drug-eluting beads (DEB-TACE), and with degradable starch microspheres (DSM-TACE), used in patients with hepatocellular carcinoma (HCC) in the bridging to liver transplant (LT) and the palliative setting. METHODS: In our center, 148 patients with HCC underwent 492 completed TACE procedures between 2008 and 2017 (158 for bridging to LT; 334 for palliative treatment) which we analyzed retrospectively. Of these procedures, 348 were DEB-TACE, 60 cTACE, and 84 DSM-TACE. RESULTS: The cTACE procedure revealed a significantly longer period of hospitalization (p = 0.02), increased occurrence of nausea (p = 0.025), and rise in alanine transaminase (ALT) levels (p = 0.001), especially in the palliative setting. In the bridging to LT cohort, these clinical endpoints did not reach statistical significance. CONCLUSIONS: The clinical safety of different TACE methods for HCC in both the palliative and the bridging to LT setting was equivalent. In the palliative setting, the cTACE procedure revealed an increased risk for adverse clinical effects such as nausea, elevation of ALT, and a prolonged period of hospitalization what might either be related to the systemic effects of the chemotherapeutic agent or to the differences in both collectives. Thus, further studies must be conducted on a larger number of TACE procedures to effectively explore the clinical side effects of the various TACE variants.

The impact of Wilson disease on myocardial tissue and function: a cardiovascular magnetic resonance study.

BACKGROUND: Systemic effects of altered serum copper processing in Wilson Disease (WD) might induce myocardial copper deposition and consequently myocardial dysfunction and structural remodeling. This study sought to investigate the prevalence, manifestation and predictors of myocardial tissue abnormalities in WD patients. METHODS: We prospectively enrolled WD patients and an age-matched group of healthy individuals. We applied cardiovascular magnetic resonance (CMR) to analyze myocardial function, strain, and tissue characteristics. A subgroup analysis of WD patients with predominant neurological (WD-neuro+) or hepatic manifestation only (WD-neuro-) was performed. RESULTS: Seventy-six patients (37 years (27-49), 47% women) with known WD and 76 age-matched healthy control subjects were studied. The prevalence of atrial fibrillation in WD patients was 5% and the prevalence of symptomatic heart failure was 2.6%. Compared to healthy controls, patients with WD had a reduced left ventricular global circumferential strain (LV-GCS), and also showed abnormalities consistent with global and regional myocardial fibrosis. WD-neuro+ patients presented with more severe structural remodeling and functional impairment when compared to WD-neuro- patients. CONCLUSIONS: In a large cohort, WD was not linked to a distinct cardiac phenotype except CMR indexes of myocardial fibrosis. More research is warranted to assess the prognostic implications of these findings. TRIAL REGISTRATION: This trial is registered at the local institutional ethics committee (S-188/2018).

HBV-infection rate and long-term outcome after liver-transplantation of anti-HBc-positive liver-grafts to HBV-naïve recipients: A retrospective study.

BACKGROUND: Anti-HBc only positive liver grafts may be suitable for HBV-naive recipients insofar as an appropriate infection prophylaxis is performed. Therefore, we investigated the effect of prophylactic regimens on HBV infection prevention and long-term outcome of anti-HBc-positive graft recipients. PATIENTS AND METHODS: This retrospective monocenter study consisted of a cohort of 1912 patients who underwent deceased donor liver transplantation at our transplant center between June 1987 and July 2019. 81 HBV-naïve patients after reception of an anti-HBc-positive liver-graft and consecutive HBV prophylaxis were selected for further examination. HBV infection rate and host- and graft-survival rates were compared to a matched control group consisting of 162 HBV-naïve patients after reception of anti-HBc-negative grafts. Pharmaceutical HBV prophylaxis included: only HBIG, only NUCs, or combined HBIG and NUCs. RESULTS: Compared to control cases of HBV-naïve anti-HBc-negative graft recipients, no differences in host- and graft-survival rate were determined.13 of 81 anti-HBc-positive graft recipients (16%) developed HBV-infection after liver transplantation. No patient suffered from HBV infection after receiving modern NUCs. Survival analysis showed no statistical differences between patients with and without infection concerning host- and graft-survival. CONCLUSION: Especially in times of organ shortage, anti-HBc-positive liver grafts may be useful for liver transplantation in HBV-naïve recipients. Efficient prophylactic regimens can prevent HBV-infection.

Optimized Trientine-dihydrochloride Therapy in Pediatric Patients With Wilson Disease: Is Weight-based Dosing Justified?

OBJECTIVES: The aim of the study was to investigate the efficacy and safety of trientine-dihydrochloride (TD) in pediatric patients with Wilson disease (WD) and the effect of different weight-based dosages on their clinical and biochemical outcome. METHODS: We retrospectively reviewed the clinical data of 31 children with WD receiving TD therapy ages under 18 years at the time of diagnosis. Outcome measures included parameters of copper metabolism and liver function tests. To examine the impact of different weight-based dosages, 2 dosage subgroups were analyzed. Group 1 received less than 20 mg/kg TD per day, group 2 more than 20 mg ·â€Škg-1 ·â€Šday-1. RESULTS: Median follow-up was 60 (5-60) months in the total study group. During TD therapy, nonceruloplasmin-bound copper was reduced from mean 1.53 (0.01-6.95) at baseline to 0.62 (0.01-4.57) µmol/l. 24h-urinary copper excretion diminished to 1.85 (0.8-9.6) µmol/day approximating the therapeutic goal of 1.6 µmol/day. Seven of 31 patients (22.6%) required discontinuation of TD treatment, in 4 cases it was because of adverse events (ulcerative colitis, gingival and breast hypertrophy, hirsutism, elevation of transaminases).Investigations about weight-based dosage showed no significant difference of any laboratory parameter between the 2 cohorts. But in terms of clinical safety, adverse effects because of TD were only found in 6.7% of children in group 1 (<20 mg ·â€Škg-1 ·â€Šday-1, median follow-up 60 [9-60] months), whereas in group 2 (>20 mg ·â€Škg-1 ·â€Šday-1, median follow-up 60 [14-60] months), it was 63.6%. CONCLUSIONS: TD proves to be an efficacious alternative chelating agent for children with WD. Weight-based dosages above the recommended 20 mg ·â€Škg-1 ·â€Šday-1 may increase the rate of adverse effects in pediatric patients.

[Retrospective cross-sectional study based on nationwide data of drug prescriptions and contractional data of outpatient offices regarding Morbus Wilson's disease]. / Retrospektive Querschnittsstudie von bundesweiten Arzneiverordnungsdaten und vertragsärztlichen Abrechnungsdaten von Morbus Wilson.

INTRODUCTION: In this article, the prevalence of the Morbus Wilson disease in Germany is determined. This is based on nationwide data of drug prescriptions and contractional data of outpatient offices. The prevalence is set in ratio to the found prevalence of prescriptions in Germany. METHOD: The descriptive evaluation is based on the database of the Central Research Institute of Ambulatory Health Care (Zi) in Germany. Additionally, data of the Federal Office of Statistics regarding inpatient treatment are available. RESULTS: It can be seen that there is a notable difference between the prevalence of patients undergoing therapy and the patients with verified diagnoses. In total, prevalence is increasing. The incidence on hand and the given dynamic of the patient population could indicate that, possibly, there is an increased rate of misdiagnosis in the first year of diagnosis. According to data, the hepatic form is the more often diagnosed form. The human genetic diagnostic increases, on average, are most distinct. ATTRIBUTES: Wilson Disease, Prevalence, Incidence, Trientine, Trientintetrahydrochlorid, D-Penicillamin, Zinc acetat, Zinc.

Applicability of scoring systems predicting outcome of transarterial chemoembolization for hepatocellular carcinoma.

PURPOSE: Several scoring systems have been proposed to predict the outcome of transarterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC). However, the application of these scores to a bridging to transplant setting is poorly validated. Evaluation of the applicability of prognostic scores for patients undergoing TACE in palliative intention vs. bridging therapy to liver transplantation (LT) is necessary. METHODS: Between 2008 and 2017, 148 patients with HCC received 492 completed TACE procedures (158 for bridging to transplant; 334 TACE procedures in palliative treatment intention at our center and were analyzed retrospectively. Scores (ART, CLIP, ALBI, APRI, SNACOR, HAP, STATE score, Child-Pugh, MELD, Okuda and BCLC) were calculated and evaluated for prediction of overall survival. ROC analysis was performed to assess prediction of 3-year survival and treatment discontinuation. RESULTS: In patients receiving TACE in palliative intention most scores predicted OS in univariate analysis but only mSNACOR score (p = 0.006), State score (p < 0.001) and Child-Pugh score (p < 0.001) revealed statistical significance in the multivariate analysis. In the bridging to LT cohort only the BCLC score revealed statistical significance (p = 0.002). CONCLUSIONS: Clinical usability of suggested scoring systems for TACE might be limited depending on the individual patient cohorts and the indication. Especially in patients receiving TACE as bridging to LT none of the scores showed sufficiently applicability. In our study Child-Pugh score, STATE score and mSNACOR score showed the best performance assessing OS in patients with TACE as palliative therapy.

The MBOAT7 rs641738 variant is associated with an improved outcome in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease that causes liver cirrhosis, leading to liver failure. Additionally, PSC is a risk factor for cholangiocarcinoma. Its mechanism is unknown, and liver transplantation remains the sole curative option. The membrane bound O-acyltransferase domain containing 7 (MBOAT7) rs641738 and rs626283 variant alleles have been associated with both an accelerated progression of the disease and a higher risk for developing a more severe phenotype in many chronic hepatic diseases. Thus, we analysed their effect on long-term outcomes and laboratory parameters in PSC patients. METHODS: We determined MBOAT7 genotypes and estimated the actuarial survival rate free of liver transplantation, using the Kaplan-Meier estimator. The differences between the estimates were analysed using the log-rank test. Patient blood was drawn and analysed for different serum parameters including cholestatic markers. Additionally, MBOAT7 RNA expression in human hepatic cell lines MZCHA1 (a biliary adenocarcinoma cell line), HepG2 (a hepatocellular carcinoma cell line), LX-2 (hepatic stellate cell line) and H-69 (cholangiocyte cell line) was analysed. RESULTS: Transplant-free survival was significantly prolonged in carriers of two rs641738 variant alleles, which was referred to as the TT genotype (mean 19.6 years; 95% confidence interval [CI]: 16.3-22.9 years) compared to the CC (mean 15.4 years, 95% CI 12.8-18.0 years) and heterozygous genotypes (mean 13.2 years, 95% CI 11.4-15.0 years) (P=0.017). This effect was restricted to male patients. We confirmed the high expression of MBOAT7 in hepatic stellate cells and found that MBOAT7 is less expressed in biliary epithelial cell lines, compared to parenchymal hepatic cells. CONCLUSIONS: Unlike other chronic liver diseases, carrying two MBOAT7 variant alleles does not seem to affect PSC patients negatively, but seems to have a positive effect on transplant-free survival. This study could help improve individual prognosis in PSC patients and give some new perspective on the involvement of the immune system in PSC.

An ultra-sensitive UHPLC-MS/MS assay for the quantification of orally administered vancomycin in plasma.

Though marketed for over half a century, little is known about the pharmacokinetics of oral vancomycin except that its bioavailability is low, thus making accurate determination of plasma concentrations difficult. To quantify plasma concentrations of vancomycin after oral administration, we developed an ultra-sensitive UHPLC-MS/MS assay and validated it according to FDA´s and EMA´s pertinent guidelines. A fast and simple protein precipitation method followed by short UHPLC chromatography was developed for extraction and separation of vancomycin from plasma. Quantification was performed via heated positive electrospray tandem mass spectrometry with multiple reaction monitoring using deuterated internal standard. The assay was linear in the calibrated concentration range of 0.05-100 ng/mL showing correlation coefficients >0.997. Intraday and interday accuracy showed coefficients of variation <12% at the lower limit of quantification (LLOQ) of 0.05 ng/mL and <6% in the calibrated range while corresponding values for precision were <13% and <8%, respectively. With its high sensitivity, the assay allows for the accurate quantification of therapeutic plasma concentrations in the therapeutic range (up to 100 µg/mL) in 1000-fold diluted samples with a sample volume decreased down to 1 µL. The UHPLC-MS/MS assay was successfully used for the determination of trough plasma concentrations of two patients with Clostridium difficile infection receiving oral vancomycin therapy and its performance was compared to a commercial immunoassay for concentrations close to its LLOQ.

Biochemical Markers for the Diagnosis and Monitoring of Wilson Disease.

Wilson disease (WD) is an autosomal recessively-inherited disorder of copper metabolism and characterised by a pathological accumulation of copper. The ATP7B gene encodes for a transmembrane copper transporter essential for biliary copper excretion. Depending on time of diagnosis, severity of disease can vary widely. Almost all patients show evidence of progressive liver disease. Neurological impairments or psychiatric symptoms are common in WD patients not diagnosed during adolescence. WD is a treatable disorder, and early treatment can prevent the development of symptoms in patients diagnosed while still asymptomatic. This is why the early diagnosis of WD is crucial. The diagnosis is based on clinical symptoms, abnormal measures of copper metabolism and DNA analysis. Available treatment includes chelators and zinc salts which increase copper excretion and reduce copper uptake. In severe cases, liver transplantation is indicated and accomplishes a phenotypic correction of the hepatic gene defect. Recently, clinical development of the new copper modulating agent tetrathiomolybdate has started and direct genetic therapies are being tested in animal models. The following review focuses especially on biochemical markers and how they can be utilised in diagnosis and drug monitoring.