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Clinical Cancer Research Vol. 9, 1773-1778, May 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Promoter Methylation of INK4a/ARF As Detected in Bile-Significance for the Differential Diagnosis in Biliary Disease1

Bodo Klump,2,3, Chih-Jen Hsieh3, Stephan Dette, Karlheinz Holzmann, Ralf Kießlich, Michael Jung, Udo Sinn, Marianne Ortner, Rainer Porschen and Michael Gregor

Department of Internal Medicine I, University Hospital of Tübingen, D-72076 Tübingen [B. K., C-J. H., S. D., K. H., M. G.]; Department of Internal Medicine, St. Hildegardis Hospital Mainz [R. K., M. J.]; Department of Internal Medicine, Hospital Bad Cannstatt [U. S.]; Department of Internal Medicine, Campus Charite Berlin [M. O.]; Department of Internal Medicine, Central Hospital Bremen East [R. P.], Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: There is a need to enhance endobiliary cytotechniques by molecular marker lesions. This is of special significance for patients with primary sclerosing cholangitis, a disease predisposing for the development of cholangiocarcinoma. The INK4a/ADP ribosylation factor (ARF) locus encodes two tumor suppressor genes: p16INK4a and p14ARF. p16INK4a has been shown to be of major significance in cholangiocarcinoma.

Experimental Design: In an effort to evaluate the potential diagnostic role of p16INK4a and p14ARF promoter methylation in biliary disease, endoscopical obtained bile specimens of 71 patients were analyzed (26 choledocholithiasis, 6 with normal results, 23 bile duct carcinoma, 5 gall bladder carcinoma). Eleven patients with primary sclerosing cholangitis were enrolled.

Results: Merely 6% of specimens (2 of 32) obtained from patients without evidence for malignant biliary disease but 53.5% of malignancies (15 of 28) showed p16 promoter methylation (p14: 3 and 46.2%, respectively). The concordance of methylation rates detected in either bile or tissue specimens was high. In primary sclerosing cholangitis, a similar prevalence of methylation was detected as in malignant disease.

Conclusions: This study demonstrates: (a) a high frequency and specificity of INK4a/ARF methylation in malignant biliary disease compared with mere cholangitis; and (b) the capability to detect these alterations reliably in endoscopically obtained bile. Thus, INK4a/ARF’s promoter methylation status represents a candidate marker for the endoscopic diagnosis of biliary disease.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Approximately 5000 new cases of cholangiocarcinoma have been diagnosed in the United States in 1996. Unfortunately, cholangiocarcinomas are rarely diagnosed in time for cure with most patients dying within 3–6 months after diagnosis (1) . An improved management of cholangiocarcinoma would include the earlier identification of patients and a more efficient diagnosis of unexplained biliary stricture. Endobiliary cytotechniques, conveniently applied during endoscopic retrograde cholangiopancreatography, for the first time offered the opportunity to obtain tissue diagnosis in pancreaticobiliary disease without surgery (2) . However, in terms of clinical management, the sensitivity of bile cytology proved to be low because of an often low cell count or disintegration (3) . Hence, there is a considerable interest in molecular markers that might complement conventional endobiliary cytotechniques. This would be of even greater significance in PSC,4 a chronic cholestatic disease that is supposed to be of autoimmune etiology. Inflammation and obliterative fibrosis affect intra-, as well as extrahepatic, bile ducts (4) . Although a majority of patients develops end stage liver disease, the occurrence of cholangiocarcinoma is another severe complication. Unfortunately, often even the most sophisticated imaging procedures or biochemical methods fail to detect neoplastic alterations within PSC. Although the time period from the diagnosis of PSC to the clinical occurrence of cholangiocarcinoma shows a broad variability, once cholangiocarcinoma is clinically apparent, the prognosis is as grim as in sporadic cholagiocarcinoma with reported survival times of ~6 months (5, 6, 7, 8) . The detection of clinically unapparent cholangiocarcinoma through the pathologist in 10–36% of explanted livers from PSC patients reflects the current shortcomings in the clinical management and surveillance of patients with PSC (9, 10, 11, 12, 13) .

Most human cancer cells are characterized by the functional disruption of p53 and retinoblastoma tumor suppressive pathways (14) . Herein, the deregulation of genes controlling the phosphorylation status of retinoblastoma represents a major inactivating mechanism. These alterations include cyclin D1 amplification, CDK4-activating mutations, and inactivation of CDK4 inhibitors (14 , 15) . The p16INK4a gene preferentially displays its tumor suppressive function through binding and inactivating CDKs 4 and 6. Thus, impaired function of p16INK4a may lead to an accelerated cell cycle progression and uncontrolled cell growth (14 , 16 , 17) . The p16INK4a gene has a common exon with murine p19ARF or human p14ARF. p14ARF’s unique first exon (1ß) is located ~20 kb centromeric to p16INK4a’s first exon (1{alpha}), and both genes are controlled by their own specific promoters. Exon 1ß is spliced into exon 2 of INK4a in an alternative reading frame, resulting in a protein that shows near to no homology with the p16INK4a transcript. p19ARF is capable to induce G1-G2 arrest. It has been suggested that p19/p14ARF binds to MDM2, thus neutralizing MDM2-mediated degradation of p53 (15 , 18 , 19) . Although alterations of the p16INK4a tumor suppressor gene include mutation, deletion, or promoter methylation and are only second to p53 alterations in human malignancies (20 , 21) , little is known about the tumor suppressive function of p14ARF in humans. Recent reports suggested aberrant promoter methylation as an inactivating mechanism also of p14ARF in colorectal cancer (22 , 23) .

We and others have shown that de novo methylation of the p16INK4a promoter represents a frequent, early, and specific alteration during neoplastic transformation in precancerous conditions of the gastrointestinal tract (24, 25, 26) . A recent study has suggested promoter methylation as a major inactivating mechanism of p16INK4a in intrahepatic cholangiocarcinoma (27) . Hence, we speculated whether the methylation pattern of this locus would confer diagnostic information also for the endoscopic differentiation of malignant and benign biliary disease. Furthermore, we sought to evaluate the methylation pattern of INK4a/ARF’s promoter in clinical precancerous PSC to get first insights into this locus’ role during neoplastic transformation in PSC patients.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Specimens.
Seventy-one patients in four endoscopic units, who were routinely investigated by endoscopic retrograde cholangiopancreatography, were enrolled into this study (26 CDL, 11 PSC, 23 bile duct carcinoma, 5 gall bladder carcinoma, and 6 NAD). Because of their different biological nature, intrahepatic tumors or neoplasms of the Papilla Vateri were not included. The study was approved by the local ethical committees. The individual diagnosis of biliary disease was based on unequivocal histological, biochemical, and radiological results and the immediate clinical course. Because of the often complex differential diagnosis of biliary obstruction and a lack of tissue diagnosis in mere palliative clinical management, bile specimens of patients in whom malignant diseases other than extrahepatic cholangiocarcinoma could not definitely be ruled out were excluded from subsequent molecular analysis. The diagnosis was accomplished by histopathology in 14 of 28 patients with malignant disease. Additional characteristics of patients with PSC are given in Table 1Citation .


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  		Table 1 Characteristics of patients with PSC

 
To get some insight into the concordance of bile and tissue data in those patients, in whom tissue specimens were available, both bile and tissue specimens were subjected for further molecular analysis (n = 7). To enlarge the sample of tissue specimens, five additional archival paraffin blocks from patients in whom no bile specimens had been obtained were included.

After cannulation of the common bile duct, between 1 and 10 ml of bile were aspirated. No irrigation maneuvers were performed. No standardized strategy with regard to the exact localization of the catheter tip was followed. Immediately after aspiration, obtained specimens were stored without any further processing at -20°C.

DNA Extraction.
Genomic DNA was extracted from thawed bile specimens. A measurement of 2 ml of bile was digested for 3 days at 55°C using a proteinase K/SDS solution. DNA was extracted using the conventional phenol/chloroform method. In those patients in whom diagnosis of malignancy was accomplished by histopathology and archival tissue specimens were retrievable, DNA was extracted from paraffin-embedded tissue slices.

Methylation-specific PCR Amplification.
A slight modification of the protocol suggested by Herman et al. (28) has been implemented. In brief, DNA modification by bisulfite converts exclusively unmethylated cytosines to uracil. Subsequent PCR amplification with primers specific for unmethylated versus methylated DNA reveals the methylation status of investigated DNA sections. Initially, 1 µg of DNA was denatured in a volume of 50 µl (final NaOH concentration of 0.2 M) for 20 min at 37°C. A measurement of 30 µl of hydroquinone (10 mM Deisenhofen; Sigma) and 520 µl of 3M sodium bisulfite (Deisenhofen; Sigma) at pH 5.5 were added and mixed. Samples were incubated at 55°C for 21 h. Modified DNA was purified using a commercially available PCR-Purification Kit according to the manufacturer’s recommendations (Qiagen, Hilden, Germany). Finally, a second NaOH treatment was performed (20 min at room temperature and a final concentration of 0.3 M). Modified and purified DNA were precipitated by ethanol for 12 h and resuspended in 100 µl of water. Primer pairs for PCR amplification are given in Table 2Citation and were purchased (MWG-Biotech, Ebersberg, Germany). A volume of 100 µl of PCR mixtures contained 10 µl of buffer (10 mM Tris-HCl, 50 mM KCl, and 0.1% Triton X-100), 1 µl of MgCl2, 1.5 µl of deoxynucleotide triphosphates (1.25 mM), primers, two units of Taq polymerase (PAN-Systems, Aidenbach, Germany), and 0.1 µg of DNA. Amplification was performed in a thermal cycler (Biometra, Göttingen, Germany) for 35 cycles (95°C/5 min, annealing temperature/90 s, 72°C/60 s) and concluded by a final 8-min extension at 72°C. A control without the addition of DNA was performed for each PCR set. A measurement of 20 µl of PCR product was loaded onto nondenaturing polyacrylamide gels (8%) and visualized by silver staining. A methylation was confirmed if at least two experiments had demonstrated an unequivocal amplification product of methylation-specific PCR.


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  		Table 2 Primer pairsa

 
Around 10 consecutive specimens were analyzed as common slots without clinical information on singular specimens.

Statistical Analysis.
The association of p16INK4a and p14ARF promoter methylation in malignant versus nonmalignant biliary alterations and PSC was tested by {chi}2 test and Yates’ correction.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
p16 Promoter Methylation.
More than half (52.2%) of the specimens obtained from patients with bile duct carcinoma revealed p16 promoter methylation (12 of 23). The percentage for gall bladder carcinoma was 60%. Conversely, merely 2 of 32 specimens obtained from patients with biliary stone disease or missing alterations showed p16 promoter methylation (6.3%; Figs. 1Citation and 2Citation ; Tables 3Citation and 4Citation ). Both specimens were obtained from patients with CDL and the clinical diagnosis of cholangitis (Table 4)Citation . The sensitivity found for p16 promoter methylation in regard to the detection of malignancy was 53.6%, the specificity 93.8%.



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  		Fig. 1. Gel with exemplary products of PCR specific for either an unmethylated (U) or methylated (M) promoter region. White arrows, detected promoter methylation (CA, carcinoma).

 


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  		Fig. 2. Percentage of specimens showing methylation of the p16 promoter (p16M), the p14 promoter (p14), or both promoters (p14M/p16M) in patients with gall bladder/bile duct carcinoma (CA), PSC, or CDL/NAD.

 

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  		Table 3 Methylation data within tissue specimens and/or bile secretions (only patients with malignancy or PSC displayed)

 

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  		Table 4 Methylation analysis (p14M, p16M) within bile fluid for patients without clinical signs of biliary disease (NAD), CDL, PSC, bile duct carcinoma (BDC), or gall bladder carcinoma (GBCA)

 
p16 promoter methylation was detected in 45.6% of patients with PSC and missing clinical signs of malignancy (Fig. 2Citation ; Table 3Citation ).

In 6 of 7 patients in whom both bile and tissue specimens were analyzed, concordant results were found (85.7%; Table 3Citation ).

p14 Promoter Methylation.
Similarly, 47.6% of specimens of patients with bile duct carcinoma showed p14 promoter methylation (10 of 21); the percentage for gall bladder carcinoma was 40%. A mere 3.1% of methylation-positive specimens was found in patients without biliary disease or CDL (1 of 32; Tables 3Citation and 4Citation ; Figs. 1Citation and 2Citation ). The sensitivity found for p14 promoter methylation in regard to the detection of malignancy was 46.2%, the specificity 96.9%.

Remarkably, 60% of samples obtained from patients with PSC revealed a methylated p14 promoter (Fig. 2Citation ; Tables 3Citation and 4Citation ). In 4 of 7 patients, tissue and bile specimens revealed the same methylation status (Table 3)Citation .

Relation of p14 and p16 Promoter Methylation.
Although a simultaneous promoter methylation of p14 and p16 was seen in none of the patients with either missing alterations or CDL (Fig. 2)Citation , a substantial percentage of specimens obtained from patients with either PSC or carcinoma showed simultaneous p14 and p16 promoter methylation (Table 3Citation ; Fig. 2Citation ). In bile duct and gall bladder carcinoma, 42.9% of specimens with p16 promoter methylation showed a simultaneous methylation of the adjacent p14 promoter (6 of 14). An exclusive methylation of either the p16 or p14 promoter region was observed in 30.8% (8 of 26) and 23.1% of carcinoma specimens, respectively (6 of 26; Table 3Citation ).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates the diagnostic potential of INK4a/ARF promoter methylation in bile samples of patients with biliary disease. Methylation status of both the p14ARF and p16INK4a promoters was successfully assessed routinely during endoscopic retrograde cholangiography-obtained bile specimens. Although the outcome of detected promoter methylation in terms of protein expression or functional end points (e.g., proliferation rate, apoptotic cell death, etc.) has not been addressed within this study, promoter methylation of suggested tumor suppressor genes p14ARF and p16INK4a was shown to be highly specific when cholangiocarcinoma of the bile duct and gallbladder was compared with cholangitis or patients with a normal cholangiogram.

p14ARF and p16INK4a promoter methylation were detected simultaneously in a substantial percentage of cases; however, in some specimens, either p14ARF or p16INK4a methylation was found alone. The concordance of methylation data determined in either bile or tissue specimens was rather high. Surprisingly, in PSC, a similar prevalence of INK4a/ARF methylation as in frank biliary tract cancer was observed.

Molecular approaches keep the promise to complement endobiliary cytotechniques in the noninvasive diagnosis of biliary disease. The potential sensitivity of any molecular alteration, however, is defined by its prevalence in biliary tract cancer and the technical options to detect it reliably. The rate of p16INK4a promoter methylation found in this study corresponds with the data reported by Tannapfel et al. (27) who found p16INK4a promoter methylation in 83% of 41 fresh-frozen tissue specimens of cholangiocarcinoma. We speculate that not only the prevalence of p16INK4a promoter methylation in biliary tract cancer but also the capability of the tumor to reject cells into the bile fluid and the localization of the catheter tip, as well as irrigation maneuvers, contribute to the somewhat lower rate of detected methylation in our study. However, it is to be taken into consideration that p14 and p16 methylation, respectively, was detected in only 25 and 33% of archival tissue specimens included in this study. In our hands, it was rather obvious that methylation analysis was more stable and reproducible in bile specimens than in tissue slices obtained from archival paraffin blocks (data not shown).

As far as we know, no data have been reported regarding the prevalence of p14ARF alterations in biliary tract cancer. Although no expression data have been generated within the current study and the association of p14ARF promoter methylation and tumor suppression remains to be elucidated, the frequency of detected p14 promoter methylation in this study suggests further work focusing on the role of p14ARF in tumorigenesis of the biliary tract.

Furthermore, the value of molecular alterations as diagnostic tools is essentially connected to the specificity of alterations under investigation. Although the prevalence of K-ras mutations has been shown to be <=90% in pancreatic adenocarcinoma, the occurrence in benign ductal alterations associated to chronic inflammatory processes seriously limits the use of K-ras mutations in the clinical management of pancreatic disease (29 , 30) . Considering benign biliary disease and biliary tract carcinoma, p16INK4a, as well as p14ARF, methylation revealed to be highly specific for clinical malignant disease in our study when cholangiocarcinoma was compared with mere cholangitis. The detection of p16INK4a promoter methylation in 2 patients and p14ARF promoter methylation in 1 patient, respectively, without clinical features of malignant disease or PSC remains unclear. Up to now, p16INK4a methylation has not been described in merely inflammatory or hyperproliferative conditions. However, aberrant methylation not only is a gradual process but also has been reported in precancerous lesions in the lung, colon, and esophagus (25 , 26 , 31) . Meticulous follow-up hopefully will show whether possibly a preneoplastic alteration had been the reason for these results.

Although this study has targeted exclusively promoter methylation, which has been shown to be associated to gene silencing, the role of aberrant methylation in carcinogenesis within the biliary tract as a whole remains to be thoroughly investigated. In this context, issues such as the extension and localization of methylation, as well as allele-specific methylation, require further analysis.

Similarly, the high prevalence of INK4a/ARF promoter methylation in patients with PSC without clinical signs of cholangiocarcinoma raises additional questions; although pathologists have detected clinically unapparent cholangiocarcinoma in <=36% of explanted livers of patients with PSC (9, 10, 11, 12, 13) , it is intriguing to speculate that INK4a/ARF methylation as detected within bile specimens from the common bile duct (reflecting the situation of the entire bile duct system) would indicate those PSC patients with either already existing or developing cholangiocarcinoma. Moreover, the frequent and early involvement of INK4a methylation during the multistep process of carcinogenesis in the gastrointestinal tract in mind, the occurrence of INK4a methylation in PSC could reflect the existence of precancerous or preinvasive dysplastic alterations in PSC. A recent study of Kersting et al. (31) reported the detection of p16 promoter methylation in the sputum of smokers, some of whom developed lung cancer during follow-up. Another explanation would be that methylation of tumor suppressor genes is not necessarily specific for neoplastic transformation and would be detected in (chronic) inflammatory conditions as well. However, this has not been reported for p14ARF or p16INK4a in any inflammation or hyperproliferative situation thus far, and in this study, almost no aberrant promoter methylation was detected in cholangitis because of biliary stone disease. Prospective studies, including clinical follow-up of patients, as well as thorough histopathological work up of explanted livers, will further determine the diagnostic and predictive roles of INK4a methylation in PSC.

Taken together, our study demonstrates the technical feasibility to detect INK4a/ARF’s methylation status in endoscopically obtained bile specimens. Published data on the high prevalence of p16INK4a promoter methylation in biliary carcinoma tissues are confirmed by our findings, and in addition, p14ARF is suggested as another diagnostic candidate marker in biliary cancer. Moreover, our data lead us to further investigate whether INK4a/ARF promoter methylation is a marker of preinvasive or clinically unapparent neoplastic alterations in patients with PSC.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant IZKF IIIB2 of the Interdisciplinary Clinical Research Center Tübingen and the German Competence Network for Inflammatory Bowel Disease. B. K. is supported by the Deutsche Krebshilfe (70-2601). Back

2 To whom requests for reprints should be addressed, at Department of Internal Medicine I, University Hospital of Tübingen, Otfried-Müller-Street 10, D-72076 Tübingen, Germany. Phone: 49-7071-2982711; Fax: 49-7071-295754; E-mail: bodo.klump{at}med.uni-tuebingen.de Back

3 B. K. and C-J. H. contributed equally to this work. Back

4 The abbreviations used are: PSC, primary sclerosing cholangitis; CDK, cyclin-dependent kinase; CDL, choledocholithiasis; NAD, nothing abnormal detected; ARF, ADP ribosylation factor. Back

Received 7/ 2/02; revised 11/ 4/02; accepted 11/ 6/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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