Liver International ISSN 1478-3223
GENETIC LIVER DISEASE
Hepatobiliary malignancies in Wilson disease Jan Pfeiffenberger1,2, Carolin Mogler2,3, Daniel N. Gotthardt1,2, Henning Schulze-Bergkamen2,4, Thomasz Litwin5, Ulrike Reuner6, Harald Hefter7, Dominik Huster8, Peter Schemmer2,9, Anna Członkowska5,10, Peter Schirmacher2,3, Wolfgang Stremmel1,2, David Cassiman11 and Karl Heinz Weiss1,2 1 Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany 2 Liver Cancer Center Heidelberg, University Hospital Heidelberg, Heidelberg, Germany 3 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany 4 National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany 5 Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland 6 Department of Neurology, University Hospital Dresden, Dresden, Germany 7 Department of Neurology, University Hospital Duesseldorf, Duesseldorf, Germany 8 Department of Gastroenterology and Oncology, Deaconess Hospital Leipzig, Leipzig, Germany 9 Department of General and Transplant Surgery, University Hospital Heidelberg, Heidelberg, Germany 10 Department of Clinical and Experimental Pharmacology, Medical University of Warsaw, Warsaw, Poland 11 Department of Hepatology and Metabolic Center, University of Leuven, Leuven, Belgium
Keywords hepatobiliary malignancies – liver cancer – Wilson’s disease – ATP7B Abbreviations DPA, D-penicillamine; FFPE, formalin fixed paraffin embedded; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinomas; KFR, Kayser–Fleischer rings; WD, Wilson disease. Correspondence Karl Heinz Weiss, MD, Department of Gastroenterology and Hepatology, University Hospital of Heidelberg, INF 410, Heidelberg D-69120, Germany Tel: +49 6221 56 37911 Fax: +49 6221 56 5255 e-mail: [email protected] berg.de Received 10 September 2014 Accepted 30 October 2014
Abstract Backgrounds & Aims: Reports of hepatobiliary malignancies in Wilson disease are sparse. The aim of this study was to evaluate hepatobiliary malignancies in Wilson disease patients concerning the clinical course of tumour disease and pathological analysis of tumour tissue. Methods: Multicenter cohort study of patients with confirmed diagnosis of Wilson disease treated at the Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland, the university hospitals Heidelberg, Duesseldorf and Dresden, Germany, and the Department of Hepatology, University Leuven, Belgium. Occurrence, treatment and outcome of hepatobiliary tumours were analysed retrospectively. Results: Of a total of 1186 patients, fourteen developed hepatobiliary malignancies. Eight were hepatocellular carcinomas (HCC) and six were intrahepatic cholangiocellular carcinomas (ICC). The prevalence of hepatobiliary malignancies in the cohort was 1.2% and the incidence was 0.28 per 1000 person years. Pathological analysis of tumour material showed no abnormal copper concentration. Conclusions: The rate of hepatobiliary malignancies in Wilson disease is very low, even in cirrhotic patients. As a result of the relevant number of ICC in addition to HCC histological analysis through surgical resection or biopsy should be mandatory when a suspect liver lesion is detected. The influence of copper depletion from Wilson disease-specific medical treatment on tumour activity remains to be elucidated.
DOI:10.1111/liv.12727 Liver Int. 2015; 35: 1615–1622
Wilson disease (WD) is a rare, autosomal recessive inherited disorder of human copper metabolism characterized by an impaired excretion of copper into the bile. Toxic copper accumulates especially in the liver and the brain. This causes progressive liver damage and neurological impairment (1, 2). The spectrum of clinical manifestations of hepatic disease is highly variable, ranging from asymptomatic patients to acute liver failure (3–6). The underlying molecular defect affects the coppertransporting P-Type ATPase ATP7B (7). In the presence Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
of elevated hepatocellular copper levels ATP7B facilitates biliary copper excretion. Under steady state conditions, ATP7B resides in the trans Golgi network (TGN) and transports copper ions across the TGN membrane for incorporation of copper into cuproenzymes (8). The occurrence of hepatobiliary malignancies is a common event in chronic liver diseases. In patients with compensated cirrhosis because of chronic hepatitis C, the incidence of hepatocellular carcinoma (HCC) is approximately 4% per year (9). In patients suffering
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Key Points
• The rate of hepatobiliary malignancies in patients suffering from Wilson disease is low, even in cirrhotic patients. • Our collective shows an above average rate of intrahepatic cholangiocellular. • Therefore, we recommend histological analysis by use of complete surgical resection or fine needle biopsy in every case. • The role of Wilson disease-specific medical treatment on tumour occurrence remains to be elucidated. from primary sclerosing cholangitis (PSC), the lifetime risk for developing intrahepatic cholangiocarcinoma (ICC) is 5–10% (10). Although data are sparse, the occurrence of hepatobiliary malignancies seems to be an uncommon event in WD (11, 12). The reasons for this finding are unclear and the discussion seems contradictory: Copper in the cell nucleus at physiological concentrations seems to play a role in stabilizing the chromatin by direct binding to the DNA (13, 14). Moreover, excess copper intake can protect against chemically induced carcinogenesis in rats (15). On the other hand, elevated copper concentration is known to induce DNA damage (16). Copper deficiency could likewise be a factor in WD. WD patients are in many ways functionally copper deficient, as copper is not properly handled and is mislocated in the cell so that cupro enzymes are not properly loaded with copper in the TGN. This could even be aggravated by the copper depletion induced by WD-specific medical anticopper therapy. Such copper deficiency could lead to diminished angiogenesis and reduced tumour growth (17). For this reason, decoppering therapies have been evaluated in various tumour entities without significant adverse effects but only a modest clinical benefit (18, 19). The aim of this study was to analyse the risk, prevalence and type of hepatobiliary malignancies in WD. This included the clinical course of these patients as well as pathological analysis of available liver specimens with regard to histology and histochemically detectable copper. Material and methods Patients
We retrospectively reviewed cases of WD patients at the Second Department of Neurology, Institute of Psychiatry and Neurology (n = 753), Warsaw, Poland, at the university hospitals in Heidelberg (n = 327), Duesseldorf (n = 49), and Dresden (n = 32), Germany and at the university hospital Leuven (n = 25), Belgium. Only cases with confirmed diagnosis of WD, defined as a
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Leipzig score >3, were included in this study, as described previously (7). Written consent was obtained from the patients and the study protocol conformed to the ethical guidelines of the Declaration of Helsinki as reflected by an a priori approval by the ethical committee of the Medical University of Heidelberg. Presence of cirrhosis was assessed by liver histology or – when a liver biopsy was not available – by imaging studies with clear signs of cirrhosis and signs of portal hypertension (splenomegaly, ascites or oesophageal varices). Cases with hepatobiliary malignancies were identified and analysed for age at initial diagnosis of WD, age at malignancy diagnosis, initial presentation, occurrence of Kayser–Fleischer ring (KFR), presence of cirrhosis and medical treatment for WD. Other concomitant liver diseases were ruled out by screening for viral hepatitis (hepatitis B/D, C), evaluation of metabolic parameters (ferritin, transferrin saturation, a1-antitrypsin and assessment of autoimmune markers. Ultrasound of the liver was performed in all patients. In cases with unclear results or suspicious findings MR-imaging was performed. The type of tumour, tumour treatment and outcome were also studied. Approach to monitoring and medical therapy
Patients with a stable course of WD were seen in the tertiary centres approximately once a year. The overall approach to medical treatment was based on current guidelines (20), however, the choice of medication remained an individual decision of the treating physician. Patients were preferably started on a chelator treatment when symptomatic and most patients received DPA as initial therapy. The use of trientine was limited in some centres because of national regulations (reimbursement, availability), while DPA and zinc treatment was commonly available. Screening for intrahepatic lesions by abdominal ultrasound was performed every 6 in patients with hepatic symptoms or suspected or confirmed cirrhosis. Patients without cirrhosis did undergo routine ultrasound examinations in more widely spaced intervals, but received imaging in case of clinical or laboratory abnormalities. In the event of hepatobiliary malignancies, each case was discussed by interdisciplinary tumour boards and treatment was based on current guidelines (21). Pathological analysis
Liver samples from five patients were available (patients 1, 2, 3, 5 and 13). Histological analysis of formalin fixed paraffin embedded (FFPE) liver tissue was performed by experienced liver pathologist with special regards to tumour type and surrounding non-tumourous liver tissue. Potential cooper accumulation in the tumour and non-tumourous liver tissue was investigated using rhodanine staining. Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pfeiffenberger et al.
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Yes Yes No Yes Yes Yes No Yes No Yes Yes N/A Yes Yes Hep + neurol Hep Hep + neurol Neurol Neurol Hep Neurol Hep + neurol Hep Hep + neurol Hep Hep Hep + neurol Hep + neurol M M F F M M F M M M F M M F 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Survival, time between tumour diagnosis and death; M, Male; F, Female; Hep, Hepatic; Neurol, Neurological; OLT, orthotopic liver transplantation; FU, follow-up; TACE, transarterial chemo embolisation; TAC, transarterial chemotherapy; Gem, gemcitabine; Cis, cisplatin; Dox, doxorubicin; Sor, sorafenib, Erl, erlotinib; Bev, Bevacicumab.
OLT, adj gem/cis Gem Cis/gem Surgical resection Dox/sor/erl/bev None TAC TACE/microwave ablation TACE Surgical resection OLT TACE, OLT RFA, cis/gem Surgical resection ICC ICC ICC HCC HCC HCC HCC HCC HCC ICC HCC HCC ICC ICC
KFR Age at WD diagnosis Sex Patient no.
The histological analysis of tumour tissue of patients 1, 2, 3 and 13 revealed moderately differentiated ICC. Liver cirrhosis was present in the tumour adjacent tissue in all cases. Rhodanine staining of tumour samples showed no significant copper accumulation, in contrast to the surrounding liver tissue. In patient 5, histological analysis showed macrotrabecular poorly differentiated HCC with cirrhosis in the surrounding tissue. Rhodanine staining showed no abnormal copper accumulation of tumour tissue in contrast to tumour adjacent tissue (see Fig. 1).
Table 1. Patients’ characteristics
Histopathology
Initial presentation
Liver cirrhosis
WD therapy
Age at tumour diagnosis
A total of 1186 patients with a confirmed diagnosis of WD were analysed. Fourteen cases (1.2%) of histologically confirmed hepatobiliary malignancies were detected (for details see Data S1). Nine patients were male and five patients were female. The median age at time of initial diagnosis of WD was 29 (range 20–49) years. Five patients presented with hepatic manifestations only at the time of WD diagnosis, three patients presented with neurological manifestations and six patients had combined hepatic and neurological symptoms at the time of diagnosis. KFR were present in ten cases. Liver cirrhosis was evident in eleven cases at the time of WD diagnosis. Treatment of WD consisted of DPA in eight cases, zinc in one case and trientine in two cases. Three patients who initially were on DPA were switched to trientine due to intolerance to penicillamine. Two patients (patient 1 and 5) reported daily alcohol consumption of 2–3 units/day. No patient presented with concomitant liver diseases. The median age at time of cancer diagnosis was 53 (range 33–72) years. The median time between diagnosis of WD and tumour diagnosis was 20.5 (range 2–41) years. In eight patients histopathology revealed HCC, while ICC was diagnosed in six patients (see Table 1). The incidence of hepatobiliary malignancies in this cohort was 0.28 per 1000 person years (see Table 2).
years years years years years years years years years years years years years years
Patient characteristics
53 43 33 42 43 55 40 58 57 72 44 53 56 70
Histology
Results
years years years years years years years years years years years years years years
Calculations were performed using SPSS for Windows software version 18.0 (IBM, Chicago, IL, USA).
31 24 27 20 41 22 31 23 22 31 29 47 49 29
Statistical analysis
DPA Zinc Trientine DPA/trientine Trientine DPA DPA DPA/trientine DPA Trientine/DPA DPA DPA DPA DPA
Tumour therapy
We performed a MEDLINE search (keywords: Wilson’s disease, hepatobiliary malignancies, liver cancer) regarding other cases of WD patients who suffered from hepatobiliary malignancies.
Yes Yes Yes Yes Yes Yes No No Yes No Yes Yes Yes Yes
Survival
Review of literature
26 months 12 months Lost for FU Alive Dead 1 month 1 month Alive 14 months Alive Alive 11 months 12 months Alive
Hepatobiliary malignancies in Wilson disease
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Table 2. Wilson’s disease cohorts of participating centres Centre
Warsaw
Heidelberg
Duesseldorf
Dresden
Leuven
Total
Total number of WD patients Female [n] (%) Patients with hepatic symptoms at diagnosis [n] (%) Patients with neurological symptoms at diagnosis [n] (%) Total patient years under surveillance since diagnosis of WD (y) Total patient years since birth (y) Number of HCC cases Number of ICC cases
753 363 (48%) 446 (59%)
327 198 (61%) 229 (70%)
49 29 (59%) 12 (25%)
32 17 (53%) 12 (38%)
25 11 (44%) 17 (68%)
1186 618 (52%) 716 (60%)
400 (53%)
96 (29%)
16 (33%)
17 (53%)
6 (24%)
535 (45%)
12 941
6392.41
1844.90
1013.08
391
22 582.39
32 442 2 (pat. 6; 7) –
12 880.49 2 (pat. 4; 9) 4 (pat 1; 2; 3; 10)
2120.78 1 (pat. 5) –
1611.84 1 (pat. 8) –
1260 2 (pat. 11; 12) 2 (pat. 13; 14)
50 315.11 8 6
(A)
(B)
(C)
(D)
Fig. 1. (A and B) Pathology specimen from patient 1 (moderately differentiated ICC). (C and D) Pathology specimen from patient 5 (poorly differentiated HCC). Left: Haematoxylin staining. Right: Rhodanine staining. Arrows: Tumour, NL: Normal liver tissue
Discussion
With a prevalence of 1.2% in our study, the rate of hepatic malignancies in WD is low and not comparable to other chronic liver diseases. This is consistent with the sparse other reports of liver cancer in WD (11, 12, 14, 22–39) that are summarized in Table 3. This finding is surprising for a disease characterized by disturbed copper homoeostasis. Under physiological conditions, a significant proportion of cellular copper is located in the cell nucleus (40). There it seems to stabilize the chromatin by directly binding to DNA under physiological conditions (41, 42). However, there is also clear evidence that excess copper – like observed in WD – can induce DNA damage through reactive oxygen species, which leads to oxidation or double and single strand breaks of DNA (16).
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Analysis of human HCCs (with other underlying liver disease than WD) regarding copper content has shown an increased copper concentration in comparison to the surrounding liver tissue in relatively small HCCs, while this finding is absent in larger HCCs (43, 44). In patients of our study, the copper concentration did not differ between the tumours and the surrounding liver tissue regardless of tumour size. This was true for HCC as well as ICC. This finding could be because of the fact that all of our patients had already been on decoppering therapy for several years when the malignancies were diagnosed, therefore masking the ‘natural’ distribution of copper in these tumours. Following this line of reasoning, one might speculate that it is not copper itself, but its depletion that has a protective effect and thus might be responsible for the very low incidence of malignancies in WD. Evidence
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Table 3. Overview of published cases of hepatobiliary malignancies in WD WD treatment
Tumour histology
Age at tumour diagnosis (y)
DPA, trientine DPA, trientine DPA DPA DPA None, diagnosis after LT DPA
ICC ICC HCC HCC ICC HCC
28 40 46 42 85 29
HCC
References
Sex
Age at WD diagnosis
Walshe JM, QJM 2003 (9)
F F M M F M
13 16 8 11 63 29
M
6
M
60
M
17
None until tumour diagnosis DPA
M
28
M
Xu R, Semin Liver Dis 2007 (37) Savas N, Pediatric transplant 2006 (36) Kumagi T, Intern Med 2005 (30) Iwadate H, Intern Med 2004 (28) Ikegawa S, Intern Med 2011 (27) Aydinli M, J Natl Med Assoc 2006 (21) Cheng WS, Liver 1992 (23) Polio J, J Clin Gastroenterology 1989 (34) Guan R, Postgrad Med J 1985 (26) Wilkinson ML, Gut 1983 (12) Thattil R, WJG 2013 (10) Girard PF, Lyon Med 1968 (25) Kamakura K, Nihon Naika Gakkai Zasshi, 1975 (29) Buffet C, Gastroenterol Clin Biol 1984 (22) Madden JW, QJM 1985 (32) Agret F, Gastroenterol Clin Biol 2003 (20) Reyes CV, Gastroenterol Hepatol 2008 (35) Emlakcioglu E, Acta Rheumatol Port 2009 (24)
Tumour treatment
Outcome N.A. N.A. N.A. N.A. N.A. Alive
12
N.A. N.A. Liver transplantation N.A. N.A. TACE, liver transplantation Liver transplantation
HCC
66
TACE, PEI
Dead
HCC
23
Best supportive care
Dead
DPA
HCC
37
RFA
Alive
22
DPA, zinc
HCC
22
RFA, liver transplantation
Alive
F
39
DPA
HCC
72
None
Dead
M
32
DPA
HCC
33
Chemotherapy
Dead
F
27
DPA
HCC
30
Resection
Alive
M
31
DPA
HCC
40
Dead
M
58
DPA
HCC
61
None, postmortem diagnosis Resection
Alive
M
22
DPA
HCC
41
None
Dead
M
26
DPA
HCC
32
None
Dead
M
45
DPA
HCC
57
None
Dead
M
61
DPA
HCC
61
Dead
M
73
None
HCC
73
None, post-mortem diagnosis None
Dead
M
59
None
HCC
59
None
Dead
DPA
HCC
50
TACE
Alive
F
30
from animal models show that copper depletion by chelating agents (trientine) suppressed hepatocellular tumour development in a murine model (17), while the untreated Long Evans cinnamon rat (LEC), the animal Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Alive
model for WD, frequently develops malignant liver tumours (45, 46). The underlying pathophysiological protective mechanism of copper depletion seems to be an alteration
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(inhibition) of angiogenesis, which is often copperdependent (47). This potentially protective effect of copper depletion has been already assessed in clinical studies in non-WD patients with solid cancers. Pass and colleagues demonstrated that treatment with the copper chelating agent tetrathiomolybdate (TM) could decrease tumour growth after surgery for malignant mesothelioma (18). In patients with advanced renal cancer, treatment with TM showed a modest disease stabilizing effect (19). However, these encouraging results were achieved in patients without an impaired copper metabolism and in these patients the TM-induced state of copper deficiency was most likely more extensive compared to steady state treatment conditions in WD patients. Thus, the anti-angiogenetic effect of any chelator therapy in WD patients might be limited as the overall goal of anticopper therapy in WD is to maintain copper homeostasis in terms of lowering the copper burden from the elevated starting point to normal levels in most cases. Thus, the decoppering therapy cannot fully explain the rare prevalence of hepatobiliary malignancies in WD, but might have a role in the reduction in elevated reactive copper. Additional risk factors for HCC formation were only evident in two patients, who reported significant alcohol consumption. In our study, six out of fourteen patients developed cholangiocellular carcinomas. This is the second report of this tumour entity in WD. Walshe and colleagues also reported three cases of ICC in their WD collective (11). This underlines the importance of histological analysis when a suspect liver lesion is diagnosed in WD patients. Overall, this finding of a relatively high number of tumours with cholangiocellular differentiation is in line with reports from WD animal models: both rodent models of WD, the LEC rats and Atp7b / mice, display inflammation and cholangiofibrosis, a condition preceding development of cholangiocarcinoma in these models (48). This observation of a frequent ICC development in WD patients is of relevance for the discussion on copper-mediated carcinogenesis in chronic cholestatic diseases. The only chronic liver disease leading to consistent and frequent formation of ICC is primary sclerosing cholangitis (PSC). Ferenci and colleagues showed a significant concentration of copper in the hepatocytes of patients with PSC (49). One could speculate on analogous effects because of elevated hepatocellular copper levels or because of altered hepatocellular copper compartimentation. However, a remarkable difference between tumour formation in PSC and WD is the later onset of tumour diagnosis in WD in our cohort. Our study has several limitations, which partially relate to the retrospective study design (e.g. patients who were lost for follow-up might have developed hepatobiliary malignancies or patients who died of natural causes might have suffered from undiagnosed
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hepatobiliary malignancies) and non-uniform ultrasound screening for liver lesions. In summary, the formation of hepatobiliary malignancies in WD is a rare event and the specific pathophysiology under which these tumours develop is not yet understood. Concomitant risk factors like alcohol should be avoided following the recommendations developed for other chronic liver diseases (50). However, the late onset and the occurrence of liver tumours even after years of decoppering therapy suggest a continuous tumour surveillance strategy in WD even in the absence of additional risk factors. In our opinion, screening ultrasound examinations every 6 months, comparable to HCC screening for other cirrhotic liver diseases, are justified besides the low prevalence of liver tumours in WD. Such procedure may be justified as in general in treated WD patients – even with cirrhosis – liver function might show a stable course over decades under anticopper treatment. Thus, early detection of HCC or ICC might benefit patients, leading to improved long term survival in this rare subgroup of WD patients, as these can be considered ideal candidates for curative treatment options as resection and local ablation – when the tumour is diagnosed early enough. In cases not eligible for resection or local ablation, liver transplantation is likewise a curative option for HCC cases who fulfil transplantation criteria (51). But in any case, a histological analysis of suspicious lesions – through complete surgical resection or fine needle biopsy – should be performed because of the balanced incidence of tumours with hepatocellular or cholangiocellular differentiation. This is not only important for the evaluation of liver transplantation, as national transplant guidelines might restrict this treatment option to patients with hepatocellular carcinoma. In the absence of curative treatments, the histological subtype of the lesion has implication for the choice of systemic oncologic treatment options. Acknowledgements
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44. Jie J, Hao S, Hongxiu Y, et al. Evaluation of Cu in hepatocellular carcinoma by particle induced X-ray emission. J Trace Elem Med Biol 2007; 21: 255–60. 45. Mori M, Hattori A, Sawaki M, et al. The LEC rat: a model for human hepatitis, liver cancer, and much more. Am J Pathol 1994; 144: 200–4. 46. Masaki T, Shiratori Y, Rengifo W, et al. Hepatocellular carcinoma cell cycle: study of Long-Evans cinnamon rats. Hepatology 2000; 32: 711–20. 47. Raju KS, Alessandri G, Ziche M, Gullino PM. Ceruloplasmin, copper ions, and angiogenesis. J Natl Cancer Inst 1982; 69: 1183–8. 48. Huster D, Finegold MJ, Morgan CT, et al. Consequences of copper accumulation in the livers of the Atp7b-/- (Wilson disease gene) knockout mice. Am J Pathol 2006; 168: 423–34. 49. Ferenci P, Steindl-Munda P, Vogel W, et al. Diagnostic value of quantitative hepatic copper determination in
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patients with Wilson’s Disease. Clin Gastroenterol Hepatol 2005; 3: 811–8. 50. Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology 2011; 53: 1020–2. 51. Weiss KH, Schafer M, Gotthardt DN, et al. Outcome and development of symptoms after orthotopic liver transplantation for Wilson disease. Clin Transplant 2013; 27: 914–22.
Supporting information
Additional Supporting Information may be found in the online version of this article: Data S1. Patient characteristics with individual courses of disease.
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GENETIC LIVER DISEASE
Hepatobiliary malignancies in Wilson disease Jan Pfeiffenberger1,2, Carolin Mogler2,3, Daniel N. Gotthardt1,2, Henning Schulze-Bergkamen2,4, Thomasz Litwin5, Ulrike Reuner6, Harald Hefter7, Dominik Huster8, Peter Schemmer2,9, Anna Członkowska5,10, Peter Schirmacher2,3, Wolfgang Stremmel1,2, David Cassiman11 and Karl Heinz Weiss1,2 1 Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany 2 Liver Cancer Center Heidelberg, University Hospital Heidelberg, Heidelberg, Germany 3 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany 4 National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany 5 Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland 6 Department of Neurology, University Hospital Dresden, Dresden, Germany 7 Department of Neurology, University Hospital Duesseldorf, Duesseldorf, Germany 8 Department of Gastroenterology and Oncology, Deaconess Hospital Leipzig, Leipzig, Germany 9 Department of General and Transplant Surgery, University Hospital Heidelberg, Heidelberg, Germany 10 Department of Clinical and Experimental Pharmacology, Medical University of Warsaw, Warsaw, Poland 11 Department of Hepatology and Metabolic Center, University of Leuven, Leuven, Belgium
Keywords hepatobiliary malignancies – liver cancer – Wilson’s disease – ATP7B Abbreviations DPA, D-penicillamine; FFPE, formalin fixed paraffin embedded; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinomas; KFR, Kayser–Fleischer rings; WD, Wilson disease. Correspondence Karl Heinz Weiss, MD, Department of Gastroenterology and Hepatology, University Hospital of Heidelberg, INF 410, Heidelberg D-69120, Germany Tel: +49 6221 56 37911 Fax: +49 6221 56 5255 e-mail: [email protected] berg.de Received 10 September 2014 Accepted 30 October 2014
Abstract Backgrounds & Aims: Reports of hepatobiliary malignancies in Wilson disease are sparse. The aim of this study was to evaluate hepatobiliary malignancies in Wilson disease patients concerning the clinical course of tumour disease and pathological analysis of tumour tissue. Methods: Multicenter cohort study of patients with confirmed diagnosis of Wilson disease treated at the Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland, the university hospitals Heidelberg, Duesseldorf and Dresden, Germany, and the Department of Hepatology, University Leuven, Belgium. Occurrence, treatment and outcome of hepatobiliary tumours were analysed retrospectively. Results: Of a total of 1186 patients, fourteen developed hepatobiliary malignancies. Eight were hepatocellular carcinomas (HCC) and six were intrahepatic cholangiocellular carcinomas (ICC). The prevalence of hepatobiliary malignancies in the cohort was 1.2% and the incidence was 0.28 per 1000 person years. Pathological analysis of tumour material showed no abnormal copper concentration. Conclusions: The rate of hepatobiliary malignancies in Wilson disease is very low, even in cirrhotic patients. As a result of the relevant number of ICC in addition to HCC histological analysis through surgical resection or biopsy should be mandatory when a suspect liver lesion is detected. The influence of copper depletion from Wilson disease-specific medical treatment on tumour activity remains to be elucidated.
DOI:10.1111/liv.12727 Liver Int. 2015; 35: 1615–1622
Wilson disease (WD) is a rare, autosomal recessive inherited disorder of human copper metabolism characterized by an impaired excretion of copper into the bile. Toxic copper accumulates especially in the liver and the brain. This causes progressive liver damage and neurological impairment (1, 2). The spectrum of clinical manifestations of hepatic disease is highly variable, ranging from asymptomatic patients to acute liver failure (3–6). The underlying molecular defect affects the coppertransporting P-Type ATPase ATP7B (7). In the presence Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
of elevated hepatocellular copper levels ATP7B facilitates biliary copper excretion. Under steady state conditions, ATP7B resides in the trans Golgi network (TGN) and transports copper ions across the TGN membrane for incorporation of copper into cuproenzymes (8). The occurrence of hepatobiliary malignancies is a common event in chronic liver diseases. In patients with compensated cirrhosis because of chronic hepatitis C, the incidence of hepatocellular carcinoma (HCC) is approximately 4% per year (9). In patients suffering
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Key Points
• The rate of hepatobiliary malignancies in patients suffering from Wilson disease is low, even in cirrhotic patients. • Our collective shows an above average rate of intrahepatic cholangiocellular. • Therefore, we recommend histological analysis by use of complete surgical resection or fine needle biopsy in every case. • The role of Wilson disease-specific medical treatment on tumour occurrence remains to be elucidated. from primary sclerosing cholangitis (PSC), the lifetime risk for developing intrahepatic cholangiocarcinoma (ICC) is 5–10% (10). Although data are sparse, the occurrence of hepatobiliary malignancies seems to be an uncommon event in WD (11, 12). The reasons for this finding are unclear and the discussion seems contradictory: Copper in the cell nucleus at physiological concentrations seems to play a role in stabilizing the chromatin by direct binding to the DNA (13, 14). Moreover, excess copper intake can protect against chemically induced carcinogenesis in rats (15). On the other hand, elevated copper concentration is known to induce DNA damage (16). Copper deficiency could likewise be a factor in WD. WD patients are in many ways functionally copper deficient, as copper is not properly handled and is mislocated in the cell so that cupro enzymes are not properly loaded with copper in the TGN. This could even be aggravated by the copper depletion induced by WD-specific medical anticopper therapy. Such copper deficiency could lead to diminished angiogenesis and reduced tumour growth (17). For this reason, decoppering therapies have been evaluated in various tumour entities without significant adverse effects but only a modest clinical benefit (18, 19). The aim of this study was to analyse the risk, prevalence and type of hepatobiliary malignancies in WD. This included the clinical course of these patients as well as pathological analysis of available liver specimens with regard to histology and histochemically detectable copper. Material and methods Patients
We retrospectively reviewed cases of WD patients at the Second Department of Neurology, Institute of Psychiatry and Neurology (n = 753), Warsaw, Poland, at the university hospitals in Heidelberg (n = 327), Duesseldorf (n = 49), and Dresden (n = 32), Germany and at the university hospital Leuven (n = 25), Belgium. Only cases with confirmed diagnosis of WD, defined as a
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Leipzig score >3, were included in this study, as described previously (7). Written consent was obtained from the patients and the study protocol conformed to the ethical guidelines of the Declaration of Helsinki as reflected by an a priori approval by the ethical committee of the Medical University of Heidelberg. Presence of cirrhosis was assessed by liver histology or – when a liver biopsy was not available – by imaging studies with clear signs of cirrhosis and signs of portal hypertension (splenomegaly, ascites or oesophageal varices). Cases with hepatobiliary malignancies were identified and analysed for age at initial diagnosis of WD, age at malignancy diagnosis, initial presentation, occurrence of Kayser–Fleischer ring (KFR), presence of cirrhosis and medical treatment for WD. Other concomitant liver diseases were ruled out by screening for viral hepatitis (hepatitis B/D, C), evaluation of metabolic parameters (ferritin, transferrin saturation, a1-antitrypsin and assessment of autoimmune markers. Ultrasound of the liver was performed in all patients. In cases with unclear results or suspicious findings MR-imaging was performed. The type of tumour, tumour treatment and outcome were also studied. Approach to monitoring and medical therapy
Patients with a stable course of WD were seen in the tertiary centres approximately once a year. The overall approach to medical treatment was based on current guidelines (20), however, the choice of medication remained an individual decision of the treating physician. Patients were preferably started on a chelator treatment when symptomatic and most patients received DPA as initial therapy. The use of trientine was limited in some centres because of national regulations (reimbursement, availability), while DPA and zinc treatment was commonly available. Screening for intrahepatic lesions by abdominal ultrasound was performed every 6 in patients with hepatic symptoms or suspected or confirmed cirrhosis. Patients without cirrhosis did undergo routine ultrasound examinations in more widely spaced intervals, but received imaging in case of clinical or laboratory abnormalities. In the event of hepatobiliary malignancies, each case was discussed by interdisciplinary tumour boards and treatment was based on current guidelines (21). Pathological analysis
Liver samples from five patients were available (patients 1, 2, 3, 5 and 13). Histological analysis of formalin fixed paraffin embedded (FFPE) liver tissue was performed by experienced liver pathologist with special regards to tumour type and surrounding non-tumourous liver tissue. Potential cooper accumulation in the tumour and non-tumourous liver tissue was investigated using rhodanine staining. Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pfeiffenberger et al.
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Yes Yes No Yes Yes Yes No Yes No Yes Yes N/A Yes Yes Hep + neurol Hep Hep + neurol Neurol Neurol Hep Neurol Hep + neurol Hep Hep + neurol Hep Hep Hep + neurol Hep + neurol M M F F M M F M M M F M M F 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Survival, time between tumour diagnosis and death; M, Male; F, Female; Hep, Hepatic; Neurol, Neurological; OLT, orthotopic liver transplantation; FU, follow-up; TACE, transarterial chemo embolisation; TAC, transarterial chemotherapy; Gem, gemcitabine; Cis, cisplatin; Dox, doxorubicin; Sor, sorafenib, Erl, erlotinib; Bev, Bevacicumab.
OLT, adj gem/cis Gem Cis/gem Surgical resection Dox/sor/erl/bev None TAC TACE/microwave ablation TACE Surgical resection OLT TACE, OLT RFA, cis/gem Surgical resection ICC ICC ICC HCC HCC HCC HCC HCC HCC ICC HCC HCC ICC ICC
KFR Age at WD diagnosis Sex Patient no.
The histological analysis of tumour tissue of patients 1, 2, 3 and 13 revealed moderately differentiated ICC. Liver cirrhosis was present in the tumour adjacent tissue in all cases. Rhodanine staining of tumour samples showed no significant copper accumulation, in contrast to the surrounding liver tissue. In patient 5, histological analysis showed macrotrabecular poorly differentiated HCC with cirrhosis in the surrounding tissue. Rhodanine staining showed no abnormal copper accumulation of tumour tissue in contrast to tumour adjacent tissue (see Fig. 1).
Table 1. Patients’ characteristics
Histopathology
Initial presentation
Liver cirrhosis
WD therapy
Age at tumour diagnosis
A total of 1186 patients with a confirmed diagnosis of WD were analysed. Fourteen cases (1.2%) of histologically confirmed hepatobiliary malignancies were detected (for details see Data S1). Nine patients were male and five patients were female. The median age at time of initial diagnosis of WD was 29 (range 20–49) years. Five patients presented with hepatic manifestations only at the time of WD diagnosis, three patients presented with neurological manifestations and six patients had combined hepatic and neurological symptoms at the time of diagnosis. KFR were present in ten cases. Liver cirrhosis was evident in eleven cases at the time of WD diagnosis. Treatment of WD consisted of DPA in eight cases, zinc in one case and trientine in two cases. Three patients who initially were on DPA were switched to trientine due to intolerance to penicillamine. Two patients (patient 1 and 5) reported daily alcohol consumption of 2–3 units/day. No patient presented with concomitant liver diseases. The median age at time of cancer diagnosis was 53 (range 33–72) years. The median time between diagnosis of WD and tumour diagnosis was 20.5 (range 2–41) years. In eight patients histopathology revealed HCC, while ICC was diagnosed in six patients (see Table 1). The incidence of hepatobiliary malignancies in this cohort was 0.28 per 1000 person years (see Table 2).
years years years years years years years years years years years years years years
Patient characteristics
53 43 33 42 43 55 40 58 57 72 44 53 56 70
Histology
Results
years years years years years years years years years years years years years years
Calculations were performed using SPSS for Windows software version 18.0 (IBM, Chicago, IL, USA).
31 24 27 20 41 22 31 23 22 31 29 47 49 29
Statistical analysis
DPA Zinc Trientine DPA/trientine Trientine DPA DPA DPA/trientine DPA Trientine/DPA DPA DPA DPA DPA
Tumour therapy
We performed a MEDLINE search (keywords: Wilson’s disease, hepatobiliary malignancies, liver cancer) regarding other cases of WD patients who suffered from hepatobiliary malignancies.
Yes Yes Yes Yes Yes Yes No No Yes No Yes Yes Yes Yes
Survival
Review of literature
26 months 12 months Lost for FU Alive Dead 1 month 1 month Alive 14 months Alive Alive 11 months 12 months Alive
Hepatobiliary malignancies in Wilson disease
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Table 2. Wilson’s disease cohorts of participating centres Centre
Warsaw
Heidelberg
Duesseldorf
Dresden
Leuven
Total
Total number of WD patients Female [n] (%) Patients with hepatic symptoms at diagnosis [n] (%) Patients with neurological symptoms at diagnosis [n] (%) Total patient years under surveillance since diagnosis of WD (y) Total patient years since birth (y) Number of HCC cases Number of ICC cases
753 363 (48%) 446 (59%)
327 198 (61%) 229 (70%)
49 29 (59%) 12 (25%)
32 17 (53%) 12 (38%)
25 11 (44%) 17 (68%)
1186 618 (52%) 716 (60%)
400 (53%)
96 (29%)
16 (33%)
17 (53%)
6 (24%)
535 (45%)
12 941
6392.41
1844.90
1013.08
391
22 582.39
32 442 2 (pat. 6; 7) –
12 880.49 2 (pat. 4; 9) 4 (pat 1; 2; 3; 10)
2120.78 1 (pat. 5) –
1611.84 1 (pat. 8) –
1260 2 (pat. 11; 12) 2 (pat. 13; 14)
50 315.11 8 6
(A)
(B)
(C)
(D)
Fig. 1. (A and B) Pathology specimen from patient 1 (moderately differentiated ICC). (C and D) Pathology specimen from patient 5 (poorly differentiated HCC). Left: Haematoxylin staining. Right: Rhodanine staining. Arrows: Tumour, NL: Normal liver tissue
Discussion
With a prevalence of 1.2% in our study, the rate of hepatic malignancies in WD is low and not comparable to other chronic liver diseases. This is consistent with the sparse other reports of liver cancer in WD (11, 12, 14, 22–39) that are summarized in Table 3. This finding is surprising for a disease characterized by disturbed copper homoeostasis. Under physiological conditions, a significant proportion of cellular copper is located in the cell nucleus (40). There it seems to stabilize the chromatin by directly binding to DNA under physiological conditions (41, 42). However, there is also clear evidence that excess copper – like observed in WD – can induce DNA damage through reactive oxygen species, which leads to oxidation or double and single strand breaks of DNA (16).
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Analysis of human HCCs (with other underlying liver disease than WD) regarding copper content has shown an increased copper concentration in comparison to the surrounding liver tissue in relatively small HCCs, while this finding is absent in larger HCCs (43, 44). In patients of our study, the copper concentration did not differ between the tumours and the surrounding liver tissue regardless of tumour size. This was true for HCC as well as ICC. This finding could be because of the fact that all of our patients had already been on decoppering therapy for several years when the malignancies were diagnosed, therefore masking the ‘natural’ distribution of copper in these tumours. Following this line of reasoning, one might speculate that it is not copper itself, but its depletion that has a protective effect and thus might be responsible for the very low incidence of malignancies in WD. Evidence
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Table 3. Overview of published cases of hepatobiliary malignancies in WD WD treatment
Tumour histology
Age at tumour diagnosis (y)
DPA, trientine DPA, trientine DPA DPA DPA None, diagnosis after LT DPA
ICC ICC HCC HCC ICC HCC
28 40 46 42 85 29
HCC
References
Sex
Age at WD diagnosis
Walshe JM, QJM 2003 (9)
F F M M F M
13 16 8 11 63 29
M
6
M
60
M
17
None until tumour diagnosis DPA
M
28
M
Xu R, Semin Liver Dis 2007 (37) Savas N, Pediatric transplant 2006 (36) Kumagi T, Intern Med 2005 (30) Iwadate H, Intern Med 2004 (28) Ikegawa S, Intern Med 2011 (27) Aydinli M, J Natl Med Assoc 2006 (21) Cheng WS, Liver 1992 (23) Polio J, J Clin Gastroenterology 1989 (34) Guan R, Postgrad Med J 1985 (26) Wilkinson ML, Gut 1983 (12) Thattil R, WJG 2013 (10) Girard PF, Lyon Med 1968 (25) Kamakura K, Nihon Naika Gakkai Zasshi, 1975 (29) Buffet C, Gastroenterol Clin Biol 1984 (22) Madden JW, QJM 1985 (32) Agret F, Gastroenterol Clin Biol 2003 (20) Reyes CV, Gastroenterol Hepatol 2008 (35) Emlakcioglu E, Acta Rheumatol Port 2009 (24)
Tumour treatment
Outcome N.A. N.A. N.A. N.A. N.A. Alive
12
N.A. N.A. Liver transplantation N.A. N.A. TACE, liver transplantation Liver transplantation
HCC
66
TACE, PEI
Dead
HCC
23
Best supportive care
Dead
DPA
HCC
37
RFA
Alive
22
DPA, zinc
HCC
22
RFA, liver transplantation
Alive
F
39
DPA
HCC
72
None
Dead
M
32
DPA
HCC
33
Chemotherapy
Dead
F
27
DPA
HCC
30
Resection
Alive
M
31
DPA
HCC
40
Dead
M
58
DPA
HCC
61
None, postmortem diagnosis Resection
Alive
M
22
DPA
HCC
41
None
Dead
M
26
DPA
HCC
32
None
Dead
M
45
DPA
HCC
57
None
Dead
M
61
DPA
HCC
61
Dead
M
73
None
HCC
73
None, post-mortem diagnosis None
Dead
M
59
None
HCC
59
None
Dead
DPA
HCC
50
TACE
Alive
F
30
from animal models show that copper depletion by chelating agents (trientine) suppressed hepatocellular tumour development in a murine model (17), while the untreated Long Evans cinnamon rat (LEC), the animal Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Alive
model for WD, frequently develops malignant liver tumours (45, 46). The underlying pathophysiological protective mechanism of copper depletion seems to be an alteration
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(inhibition) of angiogenesis, which is often copperdependent (47). This potentially protective effect of copper depletion has been already assessed in clinical studies in non-WD patients with solid cancers. Pass and colleagues demonstrated that treatment with the copper chelating agent tetrathiomolybdate (TM) could decrease tumour growth after surgery for malignant mesothelioma (18). In patients with advanced renal cancer, treatment with TM showed a modest disease stabilizing effect (19). However, these encouraging results were achieved in patients without an impaired copper metabolism and in these patients the TM-induced state of copper deficiency was most likely more extensive compared to steady state treatment conditions in WD patients. Thus, the anti-angiogenetic effect of any chelator therapy in WD patients might be limited as the overall goal of anticopper therapy in WD is to maintain copper homeostasis in terms of lowering the copper burden from the elevated starting point to normal levels in most cases. Thus, the decoppering therapy cannot fully explain the rare prevalence of hepatobiliary malignancies in WD, but might have a role in the reduction in elevated reactive copper. Additional risk factors for HCC formation were only evident in two patients, who reported significant alcohol consumption. In our study, six out of fourteen patients developed cholangiocellular carcinomas. This is the second report of this tumour entity in WD. Walshe and colleagues also reported three cases of ICC in their WD collective (11). This underlines the importance of histological analysis when a suspect liver lesion is diagnosed in WD patients. Overall, this finding of a relatively high number of tumours with cholangiocellular differentiation is in line with reports from WD animal models: both rodent models of WD, the LEC rats and Atp7b / mice, display inflammation and cholangiofibrosis, a condition preceding development of cholangiocarcinoma in these models (48). This observation of a frequent ICC development in WD patients is of relevance for the discussion on copper-mediated carcinogenesis in chronic cholestatic diseases. The only chronic liver disease leading to consistent and frequent formation of ICC is primary sclerosing cholangitis (PSC). Ferenci and colleagues showed a significant concentration of copper in the hepatocytes of patients with PSC (49). One could speculate on analogous effects because of elevated hepatocellular copper levels or because of altered hepatocellular copper compartimentation. However, a remarkable difference between tumour formation in PSC and WD is the later onset of tumour diagnosis in WD in our cohort. Our study has several limitations, which partially relate to the retrospective study design (e.g. patients who were lost for follow-up might have developed hepatobiliary malignancies or patients who died of natural causes might have suffered from undiagnosed
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hepatobiliary malignancies) and non-uniform ultrasound screening for liver lesions. In summary, the formation of hepatobiliary malignancies in WD is a rare event and the specific pathophysiology under which these tumours develop is not yet understood. Concomitant risk factors like alcohol should be avoided following the recommendations developed for other chronic liver diseases (50). However, the late onset and the occurrence of liver tumours even after years of decoppering therapy suggest a continuous tumour surveillance strategy in WD even in the absence of additional risk factors. In our opinion, screening ultrasound examinations every 6 months, comparable to HCC screening for other cirrhotic liver diseases, are justified besides the low prevalence of liver tumours in WD. Such procedure may be justified as in general in treated WD patients – even with cirrhosis – liver function might show a stable course over decades under anticopper treatment. Thus, early detection of HCC or ICC might benefit patients, leading to improved long term survival in this rare subgroup of WD patients, as these can be considered ideal candidates for curative treatment options as resection and local ablation – when the tumour is diagnosed early enough. In cases not eligible for resection or local ablation, liver transplantation is likewise a curative option for HCC cases who fulfil transplantation criteria (51). But in any case, a histological analysis of suspicious lesions – through complete surgical resection or fine needle biopsy – should be performed because of the balanced incidence of tumours with hepatocellular or cholangiocellular differentiation. This is not only important for the evaluation of liver transplantation, as national transplant guidelines might restrict this treatment option to patients with hepatocellular carcinoma. In the absence of curative treatments, the histological subtype of the lesion has implication for the choice of systemic oncologic treatment options. Acknowledgements
Financial support: This study was conducted without any financial support. Conflicts of interests: The authors do not have any disclosures to report. References 1. Stremmel W, Meyerrose KW, Niederau C, et al. Wilson disease: clinical presentation, treatment, and survival. Ann Intern Med 1991; 115: 720–6. 2. Liver E.A.f.S.o. EASL Clinical Practice Guidelines: Wilson’s disease. J Hepatol 2012; 56: 671–85. 3. Schilsky ML, Scheinberg IH, Sternlieb I. Liver transplantation for Wilson’s disease: indications and outcome. Hepatology 1994; 19: 583–7. 4. Weiss KH, Gotthardt D, Schmidt J, et al. , Liver transplantation for metabolic liver diseases in adults: indications
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Hepatobiliary malignancies in Wilson disease
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Supporting information
Additional Supporting Information may be found in the online version of this article: Data S1. Patient characteristics with individual courses of disease.
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