Wilson’s disease (WD), described for the first time by Kinnear Wilson in 1912, is a rare autosomal recessive disorder, characterized by an abnormal accumulation of copper in the body with a prevalence of about one in 30 000 individuals 1,2. It is characterized by a decreased biliary copper excretion and a defective incorporation of copper into caeruloplasmin, leading to copper accumulation in the liver, brain, and kidneys 3. Most patients with WD, whatever their clinical presentation or presymptomatic status, have some degree of liver disease. The most common age of hepatic manifestation is between 8 and 18 years; however, cirrhosis may be present in children younger than 5 years of age, or may be diagnosed in patients presenting with advanced chronic liver disease in their 50s or 60s, without neurological symptoms and without Kayser–Fleischer rings 4,5. Liver disease may mimic all forms of common liver conditions, including asymptomatic transaminasemia, acute or chronic hepatitis, fulminant hepatic failure, and cirrhosis. With the progression of parenchymal damage and inflammation, fibrosis, and subsequently cirrhosis, invariably develop 6. Cirrhosis can have either a micronodular or a mixed macro–micronodular pattern, and it is rarely complicated by hepatocellular carcinoma or cholangiocarcinoma 7. In terms of the timing of disease progression, cirrhosis is often diagnosed by the second decade, but there are some individuals who do not develop cirrhosis even after the fourth decade of life. The diagnosis of WD is made on the basis of the results of multiple clinical, lab, and genetic studies. Each diagnostic test has its limitations, and a combination of all of them allows a definite diagnosis of WD 8. The aim of medical treatment for WD is to remove the toxic deposit of copper from the body to produce a negative copper balance, and to prevent its reaccumulation. Successful therapy is measured in terms of a restoration of normal levels of free serum copper and its excretion in the urine. The current treatment is based on the use of chelating agents such as penicillamine and trientine hydrochloride and zinc to prevent copper intestinal absorption. In patients with decompensated liver cirrhosis or with neurological disease in early stages, a liver transplant is required 9–12. As data are rare on larger cohorts with WD, we analyzed 60 patients with WD for their initial clinical presentation and diagnostic findings, including laboratory results, liver histology, and mutational analysis. The aim of this study was to determine the clinical presentation and long-term outcome, and to investigate the progression of hepatic histopathology in serial liver biopsies from patients with WD.
Materials and methods
Patients and treatment
A cohort of 60 WD patients has been followed over the past three decades at the Hepato-Gastroenterology Service of Policlinico Universitario di Monserrato, Cagliari, Italy. A combination of clinical symptoms, laboratory tests, and the results of mutation analysis were used to establish a diagnosis in each patient. Confirmation of clinical diagnosis included a decreased serum ceruloplasmin concentration, a high serum non-ceruloplasmin-bound copper concentration, a low serum copper concentration, an elevated 24-h urinary copper excretion, an elevated hepatic copper content of more than 250 µg/g dry weight, the presence of a Kayser–Fleischer ring by slit lamp examination, and an ATP7B mutation. Age, symptoms, and laboratory tests at diagnosis are baseline characteristics for each patient. The methods used for the determination of hepatic function indices and copper metabolism parameters collected over different years were the same throughout the study and no standardization was necessary. Patients were categorized as hepatic when presenting signs of acute or chronic liver disease, without any extrapyramidal symptoms, and/or neurologic when presenting psychiatric and/or neurologic symptoms such as dysartria, involuntary movements, tremors, and rigidity.
The approach to treatment included restriction of dietary copper intake, lifelong medical therapy, or orthotopic liver transplantation. The treatment lasted from 3 to 25 years with a standard regime of penicillamine, trientine, and/or zinc sulfate. The treatment consisted of the introduction phase with an initial dose of penicillamine or trientine at 600 mg/day and a gradual increase to a daily dose at 1200 mg/day thereafter for the first year. A variable dosage reduction was administered for maintenance therapy according to serum nonceruloplasmin-bound copper levels (<15 µg/dl) and 24-h urinary copper excretion (250–500 mg/dl).
Regular supplementation with pyridoxine is required at a dose of 25 to 50 mg daily (30 mg/day). Elemental zinc was administered at 150–300 mg/day.
All patients were followed up periodically for the assessment of compliance, adverse effects, and response to treatment and underwent laboratory measurements of full blood count, liver function tests, copper indices, and liver biopsy.
In 40 patients who consented to the histological study, liver biopsies were obtained by the Menghini technique under ultrasound guidance. For histological examination, paraffin-embedded cores more than 2.5 cm in length and containing 11 or more complete portal areas were stained with hematoxylin and eosin, rhodanine, natural orceine, Timm’s reagent, and Perls’ Prussian blue. Liver copper concentrations in dried liver tissue were measured using flame atomic absorption spectrophotometry. Liver histology was evaluated in a blinded manner and staged according to the Desmet scoring system 13. Fibrosis was staged on a 0–4 scale as follows: F0 – no fibrosis; F1 – portal fibrosis without septa; F2 – portal fibrosis and few septa; F3 – numerous septa without cirrhosis; and F4 – cirrhosis.
Patients divided separated into two groups. We considered as progressors the patients who presented worsening of at least one unit of fibrosis. Nonprogressors presented stable or improved fibrosis scores. The aim of this study was to assess the effectiveness of therapy through the clinical course and histopathology pattern during follow-up of patients with WD.
All analyses were carried out using the SPSS software package (version 17.0; Chicago, Illinois, USA). Continuous variables were expressed as mean±SEM and differences between groups were compared using the unpaired t-test, the Mann–Whitney U-test, and one-way analysis of variance, followed by the Bonferroni test. Categorical variables were compared using the χ 2-test. Significance criteria of P value less than 0.05 were used for all inferences.
This study group included 60 patients with WD, followed for an average of 25±9 years, with a predominance of women over men.
Tables 1 and 2 show the epidemiological and clinical characteristics, and the baseline laboratory findings of 60 patients. The most frequent symptoms or signs in our WD patient population were of hepatic origin: 11/60 (19%) patients with cirrhosis and 49/60 (81%) with chronic hepatitis. Neurological manifestations were observed in 16/60 patients (26.7%) and psychiatric manifestations were observed in 7/60 patients (11.7%). There was no case of clinical presentation of acute liver failure. No patient had a clinical presentation that required a liver transplant.
All patients with WD had low serum ceruloplasmin (mean value 8.1±1.8 mg/dl) and low serum copper (32.6±8 mg/dl), elevated 24 h urinary copper excretion (359±66 mg/dl), and elevated nonceruloplasmin-bound serum copper levels (25±6 μg/l).
In the 40/60 patients in whom a liver biopsy was performed, we found values of hepatic copper in a range between 113 and 1670 mcg/ (717±70 mcg/dry weight).
We analyzed the different biochemical liver tests and copper metabolism according to the clinical presentation. No statistically significant difference was observed between the two groups. Only alanine aminotransferase (ALT) was significantly higher in patients with a hepatic manifestation at the time of diagnosis (87±15 vs. 40±7, P=0.001).
All patients were treated with penicillamine and/or trientine alone or in combination with zinc (acetate or sulfate). In patients on treatment with decoppering agents, compliance was tested by repeated measurements of the 24-h urinary copper excretion.
In all patients treated, five developed penicillamine-related side-effects, but none had a severe event. Four patients had skin rash and one patient had epigastric discomfort. In these patients, changes in the treatment regimen were necessary. The treatment was changed once in all patients to trientine. In all these patients, penicillamine-related side-effects improved after changing the treatment.
After a median follow-up period of 25 years, we observed a statistically significant difference in the ALT values (P=0.01), serum free copper (P=0.03), and 24-h urinary copper (P=0.01), whereas no statistically significant difference was observed in serum ceruloplasmin levels and the liver copper content (Table 3).
The availability of liver tissue by liver biopsy for histological study was possible in 40/60 patients. None of the patients was on anticopper treatment at the time of the first liver biopsy. The follow-up liver biopsies were carried out to monitor the potential disease progression.
In Table 4, the clinical characteristics and treatment regimen of 40 patients who underwent at least two liver biopsies are presented. The mean interval between the first and the last biopsy was 11±1.1 years.
In terms of the stage of fibrosis, at baseline, 14/40 had stage 1, 7/40 had stage 2, 13/40 had stage 3, and 6/40 had stage 4 fibrosis. No patient had a histological study within normal limits. According to the histological evolution, we identified two groups, respectively: group A: 7/40 patients (17%) who had worsening histology, named progressors (Table 5); group B: 33/40 patients (83%) who showed a stabilization or a regression of fibrosis, named nonprogressors. Between the patients who manifested histological progression, six had received combination therapy and one patient was on treatment with penicillamine in monotherapy. Four patients, progressors, had a mixed phenotype, whereas the other three patients showed a pure hepatic phenotype.
Of the 33 patients who were nonprogressors, 22/33 showed a stable hepatic condition and 11/33 patients showed improvement in their liver histology under treatment. Three of 33 patients had received monotherapy with zinc salts, 14/33 patients had received monotherapy with penicillamine, and 16/33 patients had received penicillamine/zinc salts combination. 9/33 Patients showed a mixed phenotype; 24/33 patients had a pure hepatic phenotype.
We compared the baseline clinical characteristics of the two groups and we did not find any statistically significant difference in sex, age of disease onset, and phenotypic manifestations. The time of therapy showed a statistically significant difference between two groups: histological progression correlates with a median time of therapy of 12 years, whereas no progressors showed a median time of therapy of 22 years (P=0.01). Also, the histopathologic evolution did not correlate with different treatment regimens.
Laboratory data did not show a statistically significant difference between the two groups. (Table 6).
Hepatic copper concentration and ALT were higher in the progressors group than the nonprogressors, but the results were not statistically different because of the small sample.
Finally, we compared the biochemical parameters at the end of follow-up between the two groups. The aminotransferase levels, 24 h urinary copper excretion, and liver copper concentrations were lower in nonprogressors than in progressors, but the results were not statistically significant.
In our study, we aimed to evaluate the clinical presentation and long-term outcome, and to examine the progression of hepatic histopathology in serial liver biopsies from patients with WD. We attempted to identify possible correlations between the histological progression of the disease and different clinical and epidemiological parameters of WD. WD is a curable disease, but early diagnosis is essential to stop the progression to cirrhosis or worsening of the neurological and psychiatric conditions.
In fact, in terms of the rate of progression of the disease, cirrhosis is usually diagnosed in the second decade of life, although some individuals do not develop cirrhosis, even after the fourth decade of life. In our series of patients, two patients presented a delayed clinical onset, 50 and 51 years (called ‘late onset’), with an exclusive hepatic involvement.
The aim of therapy is to reduce copper accumulation by increasing its excretion with the use of chelating agents and/or by reducing the intestinal absorption of zinc salts.
In this study, therapy was well tolerated, and only five patients required the use of trientine because of the occurrence of penicillamine-related adverse events, and none developed severe side-effects. Because of the rarity of the disease, and the fact that liver biopsies in many centers are not part of the follow-up routine, the progression of liver disease and its correlation with treatment and other clinical parameters are poorly characterized.
Clinical data in our patients highlighted a predominant hepatic involvement and only a small minority of patients presented psychiatric–neurological involvement. An important aspect of our analysis was the long-term follow-up (median age 25 year) with clinical and histopathological observations of liver disease.
Not many studies have been published on serial liver biopsies and the correlation between liver histology and clinical parameters.
Some authors have observed improvements in the degree of steatosis and liver inflammation on long-term therapy 14; others have reported improvements in the stage of fibrosis during the long-term follow-up 15,16.
The effect of penicillamine on the progression of long-term damage has been described in the literature in two small series of patients: four pediatric patients showed improvement in or stabilization of hepatic fibrosis after 2–7 years of treatment 17 and seven adult patients showed a marked improvement in or regression of steatosis, and improved mitochondrial morphological changes after 3–5 years of treatment with penicillamine 18.
A majority of our patients were treated with penicillamine, and later, a change to trientine was required in five patients because of side-effects, a percentage significantly lower than that reported in other series. In our study, treatment with penicillamine, trientine, and/or zinc salts was effective in 87% of patients. In these patients, histological stage had not progressed, remaining stable or improving compared with the baseline data, whereas only 13% of the patients showed progression.
Compared with the few data reported in the literature, our result (87%) is significant and shows the remarkable effectiveness of chelation therapy alone or in combination with zinc salts.
Linn and colleagues have described 17 patients who underwent follow-up for an average of 14 years, and who were treated with zinc. In two cases, a second liver biopsy, performed at the baseline, showed the resolution of a picture of moderate fibrosis, whereas in another case, we observed the development of cirrhosis. In this small cohort, although the data are heterogeneous and the progression of the histopathologic features have been described according to different criteria, it seems that there has been an improvement in the histology in most of the patients described (60%) 17.
In a recent paper, Cope-Yokoyama et al. 19 observed an improvement or stabilization in only 50% of cases, but perhaps this difference may be attributed to the fact that in this case, the biopsies were carried out only in the presence of a clinical indication; that is, if there is a lack of response to medical therapy, which led to recruit only patients in the worse clinical condition.
We carried out a histologic follow-up in all patients with WD, irrespective of clinical indications. The need to carry out a follow-up of the histology features is further supported by the fact that in our study, the clinical course and histopathologic evolution of liver disease did not correlate with the laboratory data examined. We compared the demographic characteristics of the two groups and we did not observe any statistically significant difference in sex, age of onset of disease, and phenotypic manifestations between progressors and nonprogressors. Also, laboratory data did not show a statistically significant difference between the two groups (Table 7).
Hepatic copper concentration and ALT were higher in the progressors group than in the nonprogressors group, but the results were not statistically different; this may have been because of the small sample. This is why biochemical parameters are not sufficient to assess the effectiveness of medical therapy on the evolution of liver disease, and we suggest the need to carry out a clinical follow-up and periodic histologic evaluation. Alternatively, as observed in our recent paper, transient elastography could be useful as a noninvasive method to evaluate liver fibrosis, to monitor liver disease progression, and to aid decision-making in daily clinical practice.
One aspect of our analysis is the clinical and histologic evolution of cirrhotic patients and the effectiveness of maintenance treatment. WD often has a high rate of progression with an age at onset in the second decade of life. It is essential to understand whether the therapy can stop the evolution of chronic hepatitis to cirrhosis or whether it is able to determine a regression in patients with advanced liver disease.
Askari and colleagues have observed different degrees of improvement of fibrosis in three patients with WD with cirrhosis initially treated with zinc and trientine, and only secondarily with zinc as maintenance treatment. One patient showed persistence of stage III–IV fibrosis, a second patient had stage II–III fibrosis, and only one patient had stage I fibrosis 16.
Our data indicated higher effectiveness in cirrhotic patients with WD. In the group of nonprogressors, six patients showed a histological picture of stage IV fibrosis, one of six patients showed no significant change in histology over 13 years of therapy, and five of six patients showed a histologic improvement in the last biopsy. Of these five patients, four showed a regression to stage I fibrosis and one patient showed histologic improvement to stage II fibrosis (Fig. 1). Among the seven patients who showed histological progression of fibrosis, two had worsening to stage IV.
Fig.1. Liver histolo...Image Tools
Interestingly, in both groups, the series of biopsies performed during the long-term follow-up showed a variable histologic evolution and the trends are not linear.
It is difficult to assess the clinical or laboratory factors that could influence the histologic evolution. Clinical phenotype, sex, age of onset, and type of therapy did not show a variable distribution between progressors and nonprogressors. Twenty-four hour urinary copper excretion and hepatic copper concentration were not different between the two groups. The role of hepatic copper content in fibrosis progression has often been reported in the literature, sometimes in a contradictory manner. Goldfischer and Sternlieb 20 observed a higher concentration of hepatic copper content in patients who presented worsening of liver fibrosis compared with patients with improved or stable fibrosis, and this may indicate the importance of intrahepatic copper dosage during follow-up.
Marcellin has described a population of 22 pediatric patients undergoing liver biopsy after 10 years of diagnosis, and all had been receiving treatment with zinc sulfate. The authors observed an improvement in all parameters of histological damage (inflammation, steatosis, and fibrosis) and a reduction in the concentration of hepatic copper, but these concentrations remained higher than those in normal controls 15. Our results evidenced stable hepatic copper concentration in progressors and a marked reduction in nonprogressors during follow-up.
Finally, we evaluated the role of different treatment regimens in histologic evolution and we did not observe a statistically significant difference, as reported in the literature 19.
We compared the time of therapy with the histological changes, and we observed a statistically significant difference with stabilization or improvement in histology: in fact, patients who showed a stabilization or an improvement in liver fibrosis had a mean time of therapy of 22 years and patients who showed a progression of liver fibrosis had a mean time of follow-up of 12 years (P=0.01).
These data indicate that patients with WD require a very long follow-up period and that patients who apparently are progressing maybe are only apparently treatment failure and need a longer time of treatment.
Our data showed that good adherence to therapy is essential, both in the initial approach with higher doses and during the period of maintenance over the years.
These data confirm at least two important concepts for the regression of the disease or its stabilization:
compliance to therapy with the individual regimen; and
long duration of therapy.
We can assume that those patients who still have not achieved substantial improvements require further optimized therapy or require more time to see a satisfactory answer.
We have not identified any factors predictive of response and further studies are required to assess the impact of therapeutic response and consequently the histological progression of various clinical parameters and especially the role of the genotype in the phenotype of the disease.
Conflicts of interest
There are no conflicts of interest.
© 2013 Lippincott Williams & Wilkins, Inc.