Frieder Berr

Diagnosis and phenotypic classification of Wilson disease

Wilson disease is an inherited autosomal recessive disorder of hepatic copper metabolism leading to copper accumulation in hepatocytes and in extrahepatic organs such as the brain and the cornea. Originally Wilson disease was described as a neurodegerative disorder associated with cirrhosis of the liver. Later, Wilson disease was observed in children and adolescents presenting with acute or chronic liver disease without any neurologic symptoms. While diagnosis of neurologic Wilson disease is straightforward, it may be quite difficult in non‐neurologic cases. Up to now, no single diagnostic test can exclude or confirm Wilson disease with 100% certainty. In 1993, the gene responsible for Wilson disease was cloned and localized on chromosome 13q14.3 (MIM277900) ( 1,u20032 ). The Wilson disease gene ATP7B encodes a P‐type ATPase. More than 200 disease causing mutations of this gene have been described so far ( 3 ). Most of these mutations occur in single families, only a few are more frequent (like H1069Q, 3400delC and 2299insC in Caucasian ( 4–6 ) or R778L in Japanese ( 7 ), Chinese and Korean patients). Studies of phenotype‐genotype relations are hampered by the lack of standard diagnostic criteria and phenotypic classifications. To overcome this problem, a working party discussed these problems in depth at the 8th International Meeting on Wilson disease and Menkes disease in Leipzig/Germany (April 16–18,u20032001) 2 . After the meeting, a preliminary draft of a consensus report was mailed to all active participants and their comments were incorporated in the final text.

Surgical and palliative management and outcome in 184 patients with hilar cholangiocarcinoma: palliative photodynamic therapy plus stenting is comparable to r1/r2 resection.

Objective:First, to analyze the strategy for 184 patients with hilar cholangiocarcinoma seen and treated at a single interdisciplinary hepatobiliary center during a 10-year period. Second, to compare long-term outcome in patients undergoing surgical or palliative treatment, and third to evaluate the role of photodynamic therapy in this concept. Summary Background Data:Tumor resection is attainable in a minority of patients (<30%). When resection is not possible, radiotherapy and/or chemotherapy have been found to be an ineffective palliative option. Recently, photodynamic therapy (PDT) has been evaluated as a palliative and neoadjuvant modality. Methods:Treatment and outcome data of 184 patients with hilar cholangiocarcinoma were analyzed prospectively between 1994 and 2004. Sixty patients underwent resection (8 after neoadjuvant PDT); 68 had PDT in addition to stenting and 56 had stenting alone. Results:The 30-day death rate after resection was 8.3%. Major complications occurred in 52%. The overall 1-, 3-, and 5-year survival rates were 69%, 30%, and 22%, respectively. R0, R1, and R2 resection resulted in 5-year survival rates of 27%, 10%, and 0%, respectively. Multivariate analysis identified R0 resection (P < 0.01), grading (P < 0.05), and on the limit to significance venous invasion (P = 0.06) as independent prognostic factors for survival. PDT and stenting resulted in longer median survival (12 vs. 6.4 months, P < 0.01), lower serum bilirubin levels (P < 0.05), and higher Karnofsky performance status (P < 0.01) as compared with stenting alone. Median survival after PDT and stenting, but not after stenting alone, did not differ from that after both R1 and R2 resection. Conclusion:Only complete tumor resection, including hepatic resection, enables long-term survival for patients with hilar cholangiocarcinoma. Palliative PDT and subsequent stenting resulted in longer survival than stenting alone and has a similar survival time compared with incomplete R1 and R2 resection. However, these improvements in palliative treatment by PDT will not change the concept of an aggressive resectional approach.

High prevalence of the H1069Q mutation in East German patients with Wilson disease: rapid detection of mutations by limited sequencing and phenotype–genotype analysis

BACKGROUND/AIMSnWilson disease is caused by a large number of different mutations in the ATP7B gene. Wilson disease patients from a homogeneous ethnical background (Saxonia) were studied for distribution and phenotypes of ATP7B mutations.nnnMETHODSnEighty-two patients were analyzed. The H1069Q mutation was assayed by a polymerase chain reaction-based restriction fragment length polymorphism test. Exons 8 and 15 were sequenced in all, and the entire gene in 30, non-H1069Q-homozygotes.nnnRESULTSnFour novel and 12 known mutations were found. Thirty-two (39%) Wilson disease patients were homozygous and 39 (48%) heterozygous for the H1069Q mutation (allele frequency 63%). Together with sequence analysis of exons 8 and 15 mutations in both alleles were identified in 65% of patients. Only one patient had both mutations at other locations. In H1069Q homozygotes symptoms started later (21.3+/-7.2 years) than in H1069Q compound heterozygotes (14.6+/-5.8, P<0.001) or H1069Q negatives (10+/-4.4, P<0.001), and they had more frequently neurologic symptoms (93 vs. 47%, P<0.001) and Kayser-Fleischer rings (82 vs. 51%, P<0.001). Mutation status did not correlate with liver biopsy findings, serum ceruloplasmin levels or (64)Cu-assay results.nnnCONCLUSIONSnIn spite of many known ATP7B mutations, only few occur in this homogeneous population. Limited genetic testing is useful to confirm Wilson disease in this population.

Photodynamic therapy in patients with non-resectable hilar cholangiocarcinoma: 5-year follow-up of a prospective phase II study

BACKGROUNDnMedian survival of patients with non-resectable hilar cholangiocarcinoma is 3 to 6 months, even after biliary drainage. Therefore, a single-arm phase II study was conducted (July 1996 to October 1998) to investigate the effect of local photodynamic therapy; a significant improvement in survival (74%) was noted at 6 months. The present study is an analysis of the long-term follow-up for patients enrolled in that phase II study.nnnMETHODSnFive-year follow-up data for the 23 patients enrolled in the original prospective study were analyzed by using Kaplan-Meier log-rank analysis.nnnRESULTSnMedian survival after treatment was 11.2 months for patients without distant metastases (M0) and 9.3 months for all patients (M0+M1). The 1-year, 2-year, 3-year, and 4-year survival rates were estimated to be 47%, 21%, 11% and 5%, respectively, for patients with stage M0 cholangiocarcinoma, and 39%, 17%, 9%, and 4%, respectively, for patients with stages M0 and M1. Of the patients who died, 73.9% (n=17) were because of tumor progression; 26.1% (n=6) died as a result of cholangitis (n=4), septic shock (n=1), or appendicitis/peritonitis (n=1). For all patients, except one with diffuse liver metastases, there was improvement in cholestasis, performance, and quality of life, which was maintained for an extended period.nnnCONCLUSIONSnThis 5-year follow-up study confirms that photodynamic therapy is safe and effective for non-resectable hilar cholangiocarcinoma, although it does not prevent progression of the disease.

Comparative characterization of the efficiency and cellular pharmacokinetics of Foscan®- and Foslip®-based photodynamic treatment in human biliary tract cancer cell lines

Due to the poor prognosis and limited management options for perihilar cholangiocarcinoma (CC) the development of alternatives for treatment is an important topic. Photodynamic therapy (PDT) with porfimer as palliative or neoadjuvant endoscopic treatment of non-resectable perihilar CC has improved quality of life and survival time, but cannot eradicate the primary tumors because of inadequate tumoricidal depth (4 mm only around the tumor stenoses). The use of meta-tetrahydroxyphenyl chlorin (mTHPC) and photoactivation at higher wavelengths (650-660 nm) provides high tumoricidal depth (10 mm) for PDT of pancreatic cancer and should yield similar tumoricidal depth in CC. This study investigates the photodynamic characteristics of mTHPC in solvent-based formulation (Foscan) and in liposomal (water soluble) formulation (Foslip) in an in vitro model system consisting of two biliary cancer cell lines (GBC, gall bladder cancer and BDC, bile duct cancer cells). Dark toxicity, photodynamic efficiency, time-dependent uptake and retention and intracellular localization of Foscan and Foslip were studied. The results prove mTHPC as a potent photosensitizing agent with high phototoxic potential in biliary cancer cells as a concentration of 600 ng ml(-1) and irradiation with 1.5 J cm(-2) (660 +/- 10 nm) is sufficient for about 90% cell killing. Addition of foetal bovine serum (FBS) to the incubation medium and analysis of the uptake and phototoxic properties reveals that both photosensitizer formulations bind to serum protein fractions, i.e. no difference between Foscan and Foslip can be found in the presence of FBS. Laser scanning fluorescence microscopy indicates a similar pattern of perinuclear localization of both sensitizers. This study demonstrates the potential of mTHPC for treatment of bile duct malignancies and provides evidence that Foslip is an equivalent water-soluble formulation of mTHPC that should ease intravenous application and thus clinical use of mTHPC.

Endoscopic radiofrequency ablation for malignant biliary obstruction: a nationwide retrospective study of 84 consecutive applications

AbstractBackgroundnBiliary radiofrequency ablation (RFA) using the Habib™ EndoHBP catheter is a new endoscopic palliation therapy for malignant biliary obstruction. The aim of this study was to assess the feasibility and safety of this technique.MethodsIn this nationwide retrospective study of prospectively collected clinical data, all patients treated by biliary RFA in Austria between November 2010 and December 2012 were included. Procedure-related complications, adverse events within 30 days post-intervention, stent patency, and mortality rates were investigated.ResultsA total of 58 patients (31 male, 27 female, median age 75 years) underwent 84 RFA procedures at 11 Austrian referral centers for biliary endoscopy. The predominant underlying condition was Klatskin tumor (45 of 58 cases). All 84 RFA procedures were feasible without technical problems. A partial liver infarction was induced by RFA in a 49-year-old Klatskin tumor patient. During 30 days after each RFA procedure, five cases of cholangitis, three cases of hemobilia, two cases of cholangiosepsis, and one case each of gallbladder empyema, hepatic coma, and newly diagnosed left bundle branch block occurred. Median stent patency after last electively performed RFA was 170 days (95xa0% CI 63–277) and was almost significantly different between metal and plastic stenting (218 vs. 115 days; pxa0=xa00.051). Median survival was 10.6 months (95xa0% CI 6.9–14.4) from the time of the first RFA in each patient and 17.9 months (95xa0% CI 10.3–25.6) from the time of initial diagnosis.ConclusionsExcept for one severe interventional complication (hepatic infarct), RFA presented as a technically feasible and safe therapeutic option for the palliative treatment of malignant biliary obstruction. The good results of stent patency and survival in this study should be proven in prospective (controlled) trials to further quantify the efficacy of this promising new technique.

Diverse Functional Properties of Wilson Disease ATP7B Variants

BACKGROUND & AIMSnWilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease.nnnMETHODSnWe analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper ((64)Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy.nnnRESULTSnProperties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization.nnnCONCLUSIONSnVariants in ATP7B associated with Wilson disease disrupt the proteins transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype-phenotype correlation and mechanisms of disease pathogenesis.

Neoadjuvant photodynamic therapy before curative resection of proximal bile duct carcinoma

BACKGROUNDnHilar bile duct carcinoma has an 80% probability of local recurrence after curative resection, which might be reduced if neoadjuvant photodynamic therapy is feasible. CASE AND TREATMENT: After intravenous injection of sodium porfimer we treated an adenocarcinoma of the proximal common bile duct (T2 N0 M0, Bismuth type II) in a 72-year-old man with red laser light (applied from the lumen at a dose 250 Joules/cm2), and the adjacent right and left hepatic and common bile duct at a dose of 125 Joules/cm2. After 23 days the tumor was completely resected (adenocarcinoma pT2 pNO; G2).nnnRESULTSnIn the lumenal, 4-mm-thick layer the bile duct specimen exhibited complete tumor necrosis with pigmentation of photodegraded porfimer and no viable tumor cells, while in the outer layer of the wall (at 5-8-mm depth) viable cancer cell nests without degraded porfimer were seen. The bile duct tissue showed little damage. Eighteen months after surgery, neither tumor recurrence nor stricture formation was found at the pretreated bilioenteric anastomoses.nnnCONCLUSIONSna) Photodynamic therapy with sodium porfimer seems to be confined to the superficial 4-mm layer of bile duct cancer. b) Neoadjuvant photodynamic therapy is feasible for hilar bile duct carcinoma.

A CDE/CHR tandem element regulates cell cycle‐dependent repression of cyclin B2 transcription

Cyclin B is an important regulator of progression through the cell division cycle. The oscillating appearance of cyclin B1 and B2 proteins during the cell cycle is in part due to fluctuating mRNA levels. We had identified earlier a tandem promoter element named cell cycle‐dependent element (CDE) and cell cycle genes homology region (CHR) which regulates cell cycle‐dependent transcription of cdc25C, cyclin A and cdc2. Here we describe that cyclin B2 transcription is repressed through a novel CDE/CHR element in resting and G1 cells. By relief of this repression in S and G2 oscillating expression of cyclin B2 mRNA is achieved during the cell cycle.

Apoptosis and the expression of Fas and Fas ligand (FasL) antigen in rejection and reinfection in liver allograft specimens.

BACKGROUND AND METHODSnTo investigate the frequency of apoptosis and the expression of Fas/Fas ligand (FasL) in liver allografts, we examined 97 biopsy specimens from 62 patients after orthotopic liver transplantation. The results of the biopsies were as follows: acute allograft rejection (n=32); hepatitis C virus (HCV) reinfection (n=18); cytomegalovirus infection (n=5); acute rejection plus HCV reinfection (n=3); and stable graft function (n=30); and after treatment of acute rejection (n=9).nnnRESULTSnApoptotic cells were found in all cases examined, and their frequency increased significantly during acute rejection (0.17 vs. 9.0; P<0.05). The immunoreactivity of Fas and FasL antigen was higher in specimens with acute rejection than in those with stable graft function. Increased apoptosis, Fas, and FasL expression, however, were also seen in HCV reinfection.nnnCONCLUSIONnWe conclude that apoptosis plays an important role in the hepatocellular damage observed in acute rejection and also in HCV reinfection. However, these parameters are, taken by themselves, not useful as indicators of acute rejection or HCV reinfection.