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Frequent p15 Promoter Methylation in Tumor and Peripheral Blood from Hepatocellular Carcinoma Patients¹

Departments of Anatomical and Cellular Pathology [I. H. N. W.], Chemical Pathology [Y. M. D. L.], Surgery [W. Y. L.], and Clinical Oncology [W. Y., P. J. J.], The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR

	ABSTRACT

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We prospectively analyzed p15 methylation patterns in 25 surgically resected tumors and 130 plasma, serum, and buffy coat samples from hepatocellular carcinoma (HCC) patients, controls with chronic hepatitis/cirrhosis, and healthy subjects. Using methylation-specific PCR, we demonstrated for the first time p15 promoter methylation in 64% of tumors and 25% (4 of 16) of patients’ plasma and serum samples. Concurrent p15 and p16 methylation was shown in 48% of tumors, and p15/p16 methylation was detected in the plasma/serum of 92% (11 of 12) of patients. Of note, 75% of 12 patients with concurrent tumor methylation developed clinical metastasis/recurrence (P = 0.027). In buffy coat samples, p15 methylation was detected in all eight patients with tumor p15 methylation, suggesting the presence of circulating tumor cells. None of the control samples were methylation positive. Our data underscore the important role(s) of p15 and p16 methylation in hepatocarcinogenesis and tumor progression. Among 92% (23 of 25) of patients with tumor p15/p16 methylation, circulating tumor DNA and HCC cells were detected in the peripheral blood of 87% (20 of 23) of patients. The combination of these epigenetic markers may prove valuable for noninvasive HCC diagnosis and disease monitoring.

	INTRODUCTION

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HCC³ is one of the most rapidly fatal human neoplasms. Treatment outcome after surgery is generally poor because of the frequent presence of recurrence or metastasis, which is a major limitation of long-term survival among HCC patients. Hematogeneous dissemination is presumably the main route of metastasis; circulating tumor cells may persist for a long period of time before the formation of clinical metastasis or recurrence (1) . Chronic hepatitis infection and cirrhosis are well-documented risk factors for HCC (2 , 3) . However, patients with chronic hepatitis/cirrhosis may develop HCC only after many years. The long-sought goal is therefore the development of sensitive, specific, and noninvasive blood tests or early detection of HCC and disease monitoring.

Although the molecular mechanisms of hepatocarcinogenesis remain unclear, an emerging number of genetic lesions have been identified (2) . Different spectra of p53 and Rb alterations have been found in HCCs (4 , 5) . Hypermethylation of p16, a cyclin-dependent kinase inhibitor gene that regulates cell cycling, has been detected frequently in human cancers including HCC (6 , 12) . The p15 gene, another cyclin-dependent kinase inhibitor gene adjacent to p16 on chromosome 9p21, is also aberrantly methylated in several human neoplasms, especially among hematopoietic malignancies (7, 8, 9, 10) . p15 has been postulated to be a tumor suppressor gene modulating pRb phosphorylation (13) . Among solid and soft tissue tumors, p15 hypermethylation has occasionally been found in plasmacytoma, brain lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, and mantle cell lymphoma (7, 8, 9, 10) . To improve the understanding of the molecular and cell biology of HCC, we prospectively analyzed p15 promoter methylation profiles in tumor, plasma/serum, and buffy coat samples from HCC patients using MSP, which can detect methylated CpG sites critical for transcriptional silencing. To explore the potential clinical implications, we studied the association of p15 and p16 methylation with the development of recurrence or metastasis.

	MATERIALS AND METHODS

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Patients and Control Subjects.
With informed consent of patients and ethics board approval, we recruited 25 HCC patients (median follow-up time, 14 months postsurgery) from the Joint Hepatoma Clinic at Prince of Wales Hospital. In this prospective study, preoperative peripheral blood and surgically resected HCC specimen were collected from each HCC patient. The diagnosis of HCC was confirmed histologically in all cases. Eighty plasma/serum samples were obtained from 25 HCC patients, 35 non-HCC patients with chronic hepatitis/cirrhosis, and 20 healthy volunteers. Fifty buffy coat samples were collected from 15 HCC patients, 15 non-HCC patients with chronic hepatitis/cirrhosis, and 20 healthy volunteers.

DNA Extraction from Tumors, Plasma, Serum, and Buffy Coat Samples.
DNA was extracted from HCCs using the QIAamp Tissue Kit (Qiagen, Hilden, Germany). Peripheral blood samples were centrifuged at 3000 x g, and plasma and serum samples were carefully collected from the EDTAcontaining and plain tubes, respectively. DNA was extracted from 400 µl of plasma/serum using the QIAamp Blood Kit (Qiagen) following the blood and body fluid protocol. The buffy coat fraction was isolated from peripheral blood, and DNA was extracted using standard proteinase K treatment and phenol/chloroform/isoamyalcohol extraction.

Bisulfite Conversion of DNA.
Bisulfite modification and MSP were conducted based on the principle that bisulfite treatment of DNA would convert unmethylated cytosine residues into uracil, whereas methylated cytosine residues would remain unmodified (7 , 11 , 12) . Thus, after bisulfite conversion, methylated and unmethylated DNA sequences would be distinguishable by sequence-specific primers. Bisulfite treatment was conducted using the CpGenome DNA Modification Kit (Intergen, New York, NY). One µg of tumor/buffy coat DNA or extracted plasma/serum DNA was treated with sodium bisulfite following the manufacturer’s recommendations. Two hundred ng of tumor/buffy coat DNA or one-fourth of the extracted amount of plasma/serum DNA was subjected to MSP.

MSP and Southern Blot Analysis.
Bisulfite-modified DNA was amplified using primers specific for the methylated p15 or p16 sequence (7 , 11 , 12 , 14) . The sense and antisense primers for the methylated p15 sequence were 5'-GCGTTCGTATTTTGCGGTT-3' and 5'-CGTACAATAACCGAACGACCGA-3'. The sense and antisense primers for the methylated p16 sequence were 5'-TTATTAGAGGGTGGGGCGGATCGC-3' and 5'-GACCCCGAACCGCGACCGTAA-3'. All bisulfite-converted DNA samples were also amplified using primers specific for the unmethylated p15 or p16 sequence. The sense and antisense primers for the unmethylated p15 sequence were 5'-TGTGATGTGTTTGTATTTTGTGGTT-3' and 5'-CCATACAATAACCAAACAACCAA-3'. The sense and antisense primers for the unmethylated p16 sequence were 5'-TTATTAGAGGGTGGGGTGGATTGT-3' and 5'-CAACCCCAAACCACAACCATAA-3'.

PCR was conducted using the GeneAmp DNA Amplification Kit and AmpliTaq Gold polymerase (Perkin Elmer, Foster City, CA). Thirty-five and fifty-five PCR cycles were used for tumor/buffy coat DNA and plasma/serum DNA, respectively. The thermal profile consisted of an initial denaturation step of 95°C for 12 min followed by repetitions of 95°C for 45 s, 60°C for 45 s, and 72°C for 60 s, with a final extension step of 72°C for 10 min. PCR products were loaded onto 2% agarose gels and stained with ethidium bromide. Each sample was analyzed in duplicate in parallel with a methylated cell line control, unmethylated normal controls, and multiple negative water blanks.

The identity of the PCR product for the methylated p15 or p16 sequence was confirmed by nonradioactive Southern blot analysis (15) . The probe designed to hybridize to the methylated p15 sequence was 5'-TAGGC/TGTTTTTTTTTAGAAGTAATTTAGG-3'. The probe designed to hybridize to the methylated p16 sequence was 5'-GAGTAGTATGGAGTTTTCGGTTGATTGGTTG-3'.

Human plasmacytoma cell line HS-Sultan (American Type Culture Collection CRL-1484), which was previously shown to have p15 and p16 methylation by Southern blot analysis using methylation-sensitive restriction enzymes (10) , was used as a methylated control for MSP. To determine the sensitivity of MSP, HS-Sultan DNA was serially diluted in water, mixed with normal peripheral blood cell DNA, bisulfite converted, and then amplified by MSP.

Sensitivity of MSP.
For methylated p15 alleles, the lower detection limit of MSP using 35 cycles was 1 ng of HS-Sultan DNA in 1000 ng of DNA from normal PBNCs (Fig. 1A) . The sensitivity for detecting methylated p15 alleles has reached 2.5 x 10^-4 when using higher-cycle MSP (14) . The sensitivity for detecting methylated p16 alleles was 5-fold higher, reaching 5 x 10^-5 with a higher-cycle profile (11) .

View larger version (35K): [in this window] [in a new window]   Fig. 1. Detection of aberrant p15 methylation (p15M) in tumor and plasma/serum from HCC patients by MSP and Southern blot analysis. A: Lanes 1–19 (corresponding to the patient number in Table 1 ), tumors; Lane N, normal blood cell DNA; right, Lanes 0.1, 1, 10, 100, and 200, 0.1, 1, 10, 100, and 200 ng of HS-Sultan DNA (HS). B: Lanes 1–25 (corresponding to the patient number in Table 1 ), patient plasma/serum samples.

	RESULTS

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Frequent p15 Methylation in HCC and the Association between Concurrent Methylation and Clinical Metastasis/Recurrence.
Aberrant p15 promoter methylation was demonstrated in 64% (16 of 25) of HCCs using 35 MSP cycles (Fig. 1A ; Table 1 ). Concurrent p15 and p16 methylation was found in 48% (12 of 25) of tumors (Table 1) . Methylation of p15 alone was demonstrated in 16% (4 of 25) of HCCs, whereas methylation of p16 alone was found in 28% (7 of 25) of HCCs. Overall, 92% (23 of 25) of HCC patients had p15 and/or p16 methylation in tumors. None of the 35 healthy subjects and non-HCC patients with chronic hepatitis/cirrhosis showed p15 or p16 methylation in PBNCs. In all samples analyzed, unmethylated p15 and p16 alleles were detected by MSP. During a median follow-up time of 14 months postsurgery, 75% (9 of 12) of HCC patients with concurrent p15 and p16 methylation in tumors developed liver recurrence or lung metastasis (Table 1) . In contrast, only 4 of the remaining 13 HCC patients developed recurrence or metastasis (² test; P = 0.027).

Eleven of 12 patients (92%) with concurrent p15 and p16 methylation in HCCs showed methylation of at least one of the two genes in plasma/serum (Table 1 ; Fig. 2 ). Among cases with tumor methylation in only one of the genes, 25% (1 of 4) of patients with tumor methylation of p15 alone exhibited methylated p15 sequences in plasma. Seventy-one percent (5 of 7) of patients with tumor methylation of p16 alone demonstrated methylated p16 sequences in plasma/serum. In other words, circulating tumor DNA was detected in 74% (17 of 23) of plasma/serum samples from patients with tumor methylation.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Detection of aberrant p16 methylation (p16M) in plasma/serum of HCC patients by MSP and Southern blot analysis. Lanes 1–13 (corresponding to the patient number in Table 1 ), plasma/serum samples from HCC patients; Lane HS, HS-Sultan DNA. Patients 1, 2, 5, 6, 7, 8, and 11 showed concurrent p16 and p15 methylation in tumors, and all of these patients had methylation of at least one of the two genes in the plasma/serum.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Aberrant p15 methylation (p15M) in PBNCs from HCC patients. Lane 1kb, molecular weight standard; Lanes 1–3 and 12–17 (corresponding to the patient number in Table 1 ), buffy coat samples from HCC patients; Lane HS, HS-Sultan DNA; Lane N, normal blood cell DNA. PCR products were stained with ethidium bromide after agarose gel electrophoresis.

	DISCUSSION

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During cell cycling, differential function of p15 or p16 protein in a selective manner has been indicated (13) . Expression of p15 appears to be independent of pRb protein but is induced by extracellular growth inhibitors IFN- and TGF-ß (13 , 16) . There is evidence suggesting that IFN- treatment may decrease the rate of heaptocarcinogenesis in cirrhosis patients (17) . On the other hand, disruption of TGF-ß-mediated apoptosis and growth arrest is crucial in hepatocarcinogenesis (18) . Thus, p15 inactivation via hypermethylation as reported here could possibly abrogate cell cycle control and confer resistance to the growth-inhibitory effect of TGF-ß that is usually overexpressed in HCC cells (19) .

The selective pressure that TGF-ß imposes on p15 inactivation might potentially account for the high frequency and tumor type specificity of aberrant p15 methylation in HCC. Of particular interest, this incidence rate is comparable to that seen in acute leukemias, which arise in the bone marrow with highly expressed TGF-ß (the potent growth inhibitor of hematopoietic stem cells or early progenitors; Ref. 14 ). The identification of hepatic oval cells of bone marrow origin and hematopoietic stem cells of liver origin may lend credence to the possibility that HCC and hematological malignancies might have distinctive genetic and epigenetic lesions in common (20 , 21) . One of the possible abnormalities appears to be aberrant p15 methylation, as reported here.

Expression of p16 is repressed in part by pRb protein, whereas Rb expression, in turn, is inhibited by p53 protein (22 , 23) . There has been evidence indicating that p16 inactivation is closely associated with aberrant p53 expression (24) , suggesting a collaborating role for p16 in apoptosis (25) . In contrast to Rb and p53 abnormalities that were inconsistently detected in HCC, aberrant p16 methylation has been found frequently (4 , 5 , 11 , 12) . In this study, we demonstrated methylation of p16 alone without involving p15 in 28% of the HCCs examined, suggesting an important role for p16 inactivation in hepatocarcinogenesis.

On the other hand, progressive p16 methylation has been associated with metastasis and invasive phenotypes in cancers (26 , 28) . Dual p15 and p16 methylation has been found almost exclusively in hematological malignancies such as Burkitt’s lymphoma and T-cell acute leukemia (7 , 29) . The latter two diseases generally have very high proliferative indices. Of note, we detected concurrent p15 and p16 methylation in 48% of HCCs, an unusual phenomenon among solid tumors. It has been shown previously that overall survival was significantly shortened for patients with high proliferative indices and low degrees of apoptosis and necrosis in HCCs (30) . To augment the selective growth advantage, p16 methylation may act in concert with p15 methylation during hepatocarcinogenesis. Additional p16 methylation might also contribute to immortalization and inhibition of apoptosis (31) . In this first attempt to investigate the clinical relevance of p15 and p16 methylation in HCC, we found a significant association between concurrent tumor methylation and the development of recurrence or metastasis. The functional significance of p15 and p16 methylation may thus be implicated in tumor progression, in that methylation could be an initiating event leading to progressive inactivation of the cell cycle-regulatory genes (10 , 26 , 32) . Impaired p15 and p16 expression, which confers a selective growth advantage to tumor cells capable of clonal expansion, might promote stepwise transformation and neoplastic progression.

Tumors that have metastasized may not shed many cells into the peripheral blood but might release tumor DNA into the circulation. Detection of genetic alterations and methylation abnormalities in the plasma/serum of cancer patients may create a profound impact on noninvasive diagnosis of cancers among high-risk populations (11 , 12 , 33 , 34) . In plasma/serum samples, we detected methylated p15 sequences in 25% of HCC patients with p15 methylation in tumor. Of note, nearly all patients (92%) showing concurrent p15 and p16 methylation in HCCs had detectable methylation abnormalities in plasma/serum. The lower detection rate of p15 methylation may be related to the lower sensitivity of p15 methylation detection (2.5 x 10^-4) compared with p16 methylation detection (5 x 10^-5). The detectability for p15 methylation can be enhanced if a larger amount of plasma/serum is used.

The application of dual p15 and p16 methylation markers allowed us to detect circulating tumor DNA in 74% (17 of 23) of plasma/serum samples from 92% (23 of 25) of HCC patients with tumor p15/p16 methylation. In addition, p15 methylation was shown in PBNCs from three additional cases. Regardless of the tumor size (range, 1.4–11 cm in diameter), we found methylation abnormalities in the peripheral circulation of 87% (20 of 23) of HCC patients with tumor methylation. Our findings may form the basis for noninvasive diagnosis of small HCC among high-risk populations at an early stage and for disease monitoring. The mechanism of DNA release from the tumor into plasma/serum remains unknown but may be related to cellular turnover, necrosis, or apoptosis. The methylation analysis of peripheral blood may prove valuable for studying the pathophysiological basis for cell-free tumor DNA liberation and tumor cell dissemination into the patient’s circulation.

With regard to the molecular detection of circulating HCC cells, albumin and -fetoprotein mRNAs have been analyzed previously using reverse transcription PCR (1 , 15) . However, "illegitimate transcription" is a potential problem that needs to be addressed. In PBNCs, we showed methylated p15 sequences in all HCC patients with tumor p15 methylation, in no HCC patients without tumor p15 methylation, and in no healthy subjects and non-HCC patients with chronic hepatitis/cirrhosis. These results indicate that MSP for p15 enables specific detection of circulating HCC cells in addition to cell-free tumor DNA.

Dual p15 and p16 methylation abnormalities as diagnostic and prognostic markers for HCC can be used generally for a wide variety of other cancers with aberrant p15/p16 methylation. Furthermore, this approach may be applied to many other tumor suppressor genes or metastasis suppressor genes, which are methylated in different tumor types. The peripheral blood MSP analysis is sensitive and specific and can be conducted easily for widespread cancer screening and monitoring of patient response to therapies. Patients with chronic hepatitis or cirrhosis, from whom liver tissues would not be easily accessible, may be screened for early detection of HCC.

	ACKNOWLEDGMENTS

 
We thank Dr. S. Ho for support during the course of this project and E. Wong for helpful advice on statistical analysis of data.

	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 grants from the Hong Kong Research Grants Council, the Direct Grants Scheme of the Chinese University of Hong Kong, and the Industrial Support Fund.

² To whom requests for reprints should be addressed, at 25F, 4B, The Tolo Place, Ma On Shan, Shatin, New Territories, Hong Kong SAR. Fax: 852-2712-2719; E-mail: wkc-mok{at}wkc.hkcampus.net

³ The abbreviations used are: HCC, hepatocellular carcinoma; MSP, methylation-specific PCR; PBNC, peripheral blood nucleated cells; IFN-, interferon-; TGF-ß, transforming growth factor ß.

Received 4/10/00; revised 6/16/00; accepted 6/23/00.

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