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Technical Briefs |
-Fetoprotein Concentration in Hepatocellular Carcinoma Patients
1
Departments of Anatomical and Cellular Pathology,
2
Chemical Pathology,
3
Surgery, and
4
Clinical Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region
a author for correspondence: fax 852-2712-2719, e-mail wkc-mok{at}wkc.hkcampus.net
Detection of tumor-derived genetic and epigenetic alterations in
plasma and serum may have a profound impact on the noninvasive
diagnosis of cancers among high-risk populations (1)(2)(3).
Chronic hepatitis infection and cirrhosis are well-documented risk
factors for hepatocellular carcinoma (HCC) (4). However,
patients with chronic hepatitis/cirrhosis may develop HCC only after
many years. One long-sought goal in this field, therefore, has been the
development of sensitive, specific, and noninvasive blood tests for the
early detection and monitoring of HCC (5). A
well-established conventional marker for HCC, serum 
-fetoprotein
(AFP), has limitations for cancer screening because high-risk
individuals with AFP concentrations within the reference interval may
have already developed HCC at an advanced stage
(6)(7). Hypermethylation of p16, a
cyclin-dependent kinase inhibitor gene that regulates cell cycling, has
been found frequently in human cancers, including HCC
(3)(8). Using methylation-specific PCR (MSP), we
analyzed p16 promoter methylation status in 100 plasma and
serum samples from HCC patients, controls with chronic
hepatitis/cirrhosis, and healthy subjects. Of particular diagnostic
interest, we investigated the relationship of p16
methylation status and the serum AFP concentration.
With informed consent and ethics approval, peripheral blood samples
were obtained from 45 HCC patients before surgical resection, 35
non-HCC patients with chronic hepatitis/cirrhosis, and 20 healthy
volunteers. The diagnosis of HCC was confirmed histopathologically in
liver biopsy and was based on serum AFP concentration 
500 µg/L,
ultrasound examination with computed tomography, or hepatic angiography
as appropriate. Tumor specimens were collected from the HCC patients
after surgery, and DNA was extracted using a QIAamp Tissue Kit
(Qiagen). Peripheral blood samples were centrifuged at
3000g, and plasma and serum samples were collected from
EDTA-containing and plain tubes, respectively. DNA was extracted from
400 µL of plasma or serum by a QIAamp Blood Kit (Qiagen).
Bisulfite modification of DNA was conducted using the CpGenome DNA Modification Kit (Intergen). Bisulfite-treated DNA was amplified using primers for the methylated p16 sequence (3)(9). The sense and antisense primers for the methylated p16 sequence were 5'-TTA TTA GAG GGT GGG GCG GAT CGC-3' and 5'-GAC CCC GAA CCG CGA CCG TAA-3', respectively. All bisulfite-converted DNA samples were also amplified using primers for the unmethylated p16 sequence. The sense and antisense primers for the unmethylated p16 sequence were 5'-TTA TTA GAG GGT GGG GTG GAT TGT-3' and 5'-CAA CCC CAA ACC ACA ACC ATA A-3', respectively. PCR was conducted using the GeneAmp DNA Amplification Reagent Kit and AmpliTaq Gold polymerase (Perkin-Elmer). Using 35 cycles for tumor DNA and 55 cycles for circulating DNA, 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 (3)(10). PCR products were loaded onto 2% agarose gel and stained with ethidium bromide. Each sample was analyzed in duplicate, in parallel with a methylated cell line (see below), unmethylated controls, and multiple negative water blanks. The identity of the PCR product for the methylated p16 sequence was confirmed by nonradioactive Southern blot analysis (11). The probe sequence designed to hybridize to the methylated p16 sequence was 5'-GAG TAG TAT GGA GTT TTC GGT TGA TTG GTT G-3'.
A plasmacytoma-derived cell line, HS-Sultan (CRL-1484; American Type Culture Collection), previously shown to have p16 methylation by Southern blot analysis (12), 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. The lower detection limit for the methylated p16 allele was 50 pg of HS-Sultan DNA in 1000 ng of buffy coat DNA from a healthy volunteer.
Preoperative serum AFP concentrations were measured by a two-site sandwich immunoassay (Automated Chemiluminescence System 180; Bayer Diagnostics) and were correlated with the p16 methylation status in plasma or serum from the 45 HCC patients and the 35 non-HCC patients with chronic hepatitis/cirrhosis. The relationship between the p16 methylation status and the serum AFP concentration was assessed using ROC curves and the Fisher exact test.
We demonstrated the presence of methylated p16 sequences in
plasma or serum from 80% (24 of 30) of HCC patients with tumoral
p16 methylation (Table 1
). Among the 30 HCC patients with tumoral p16
methylation, we found a significant association between the
p16 methylation status in circulating DNA and the
preoperative serum AFP concentration. A serum AFP concentration of 45
µg/L was found to give the best discrimination between HCC patients
with or without p16 methylation in circulating DNA (MedCalc
5.0). Thus, all 16 cases (100%) with serum AFP concentrations >45
µg/L showed p16 methylation in circulating DNA (Fig. 1
), whereas only 57% (8 of 14) of cases with AFP concentrations
45 µg/L (range, 4–45 µg/L) had p16 methylation
(specificity = 100%; sensitivity = 67%; MedCalc 5.0). The
difference in the proportion of HCC patients with circulating
methylated p16 sequences between the two groups with higher
or lower AFP concentrations was statistically significant (Fisher exact
test, P = 0.005). None of the 20 healthy volunteers
studied had p16 methylation in plasma or serum. None of the
35 non-HCC patients with chronic hepatitis/cirrhosis had p16
methylation in circulating DNA. Four patients of the latter control
group had serum AFP concentrations >45 µg/L, whereas the remaining
31 had AFP concentrations 45 µg/L.
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Clinically, serum AFP concentration alone cannot reliably differentiate
HCC from benign liver diseases, especially when the concentration is
<500 µg/L. In this study, only 29% (13 of 45) of HCC patients had
serum AFP concentrations 500 µg/L (Table 1
). For the remaining
71%, diagnostic confirmation based on ultrasonography and angiography
was required. Of diagnostic relevance, the application of the
p16 methylation marker enabled us to detect circulating
tumor DNA in 80% of HCC patients with tumoral p16
methylation. Aberrant p16 methylation may thus serve as a
valuable molecular marker for the noninvasive and early diagnosis of
HCC among high-risk populations. Indeed, the peripheral blood MSP assay
for p16 could identify 53% (24 of 45) of HCC patients,
including those who were not detectable by AFP screening alone. The
combination of the MSP assay with the AFP test further improved the
rate of HCC detection to 62% (28 of 45). Therefore, addition of the
p16 methylation marker, detectable in plasma or serum from
patients with well-differentiated or early-stage tumors (3),
would possibly permit early detection of HCC and hence earlier
treatment among high-risk populations. Our data suggest the diagnostic
implications of p16 methylation for the noninvasive
diagnosis of cancer with potentially higher sensitivity and
specificity.
For disease monitoring, an increased serum AFP concentration (>10 µg/L) is generally applied as one of the useful criteria. However, clinical metastasis and recurrence might also be present in HCC patients with serum AFP concentrations within the reference interval. We currently are studying the usefulness of the peripheral blood MSP analysis for monitoring minimal residual tumor or micrometastasis, especially among HCC patients with serum AFP concentrations within the reference range during clinical follow-up.
Of biological interest, we report for the first time a statistically
significant correlation between the p16 methylation status
in circulating DNA and the preoperative serum AFP concentration. Among
the HCC patients with tumoral p16 methylation, 100% of the
cases with serum AFP concentrations >45 µg/L had circulating
methylated p16 sequences, whereas only 57% of patients with
AFP concentrations 45 µg/L had p16 methylation. None of
the 35 non-HCC patients with chronic hepatitis/cirrhosis, regardless of
their AFP concentrations, had p16 methylation in circulating
DNA. Although the biological basis of this relationship remains unclear
at present, our findings may stimulate further investigations into the
correlations of conventional tumor markers with the new generation of
molecular markers based on DNA analysis in plasma and serum.
Acknowledgments
This work was partially supported by grants from the Hong Kong Research Grants Council, the Direct Grants Scheme from the Chinese University of Hong Kong, the Industrial Support Fund, and the Kadoorie Charitable Foundation for the Hong Kong Cancer Genetics Research Group.
References
-fetoprotein in the early stage of human hepatocellular carcinoma. Gastroenterology 1984;86:1404-1409.[Web of Science][Medline]
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The following articles in journals at HighWire Press have cited this article:
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  C. Zhang, Z. Li, Y. Cheng, F. Jia, R. Li, M. Wu, K. Li, and L. Wei CpG Island Methylator Phenotype Association with Elevated Serum {alpha}-Fetoprotein Level in Hepatocellular Carcinoma Clin. Cancer Res., February 1, 2007; 13(3): 944 - 952. [Abstract] [Full Text] [PDF]
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 S.-Y. Kuang, P. E. Jackson, J.-B. Wang, P.-X. Lu, A. Munoz, G.-S. Qian, T. W. Kensler, and J. D. Groopman Specific mutations of hepatitis B virus in plasma predict liver cancer development PNAS, March 9, 2004; 101(10): 3575 - 3580. [Abstract] [Full Text] [PDF]
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 P. E. Jackson, S.-Y. Kuang, J.-B. Wang, P. T. Strickland, A. Munoz, T. W. Kensler, G.-S. Qian, and J. D. Groopman Prospective detection of codon 249 mutations in plasma of hepatocellular carcinoma patients Carcinogenesis, October 1, 2003; 24(10): 1657 - 1663. [Abstract] [Full Text] [PDF]
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I. H. N. Wong, J. Zhang, P. B. S. Lai, W. Y. Lau, and Y. M. Dennis Lo Quantitative Analysis of Tumor-derived Methylated p16INK4a Sequences in Plasma, Serum, and Blood Cells of Hepatocellular Carcinoma Patients Clin. Cancer Res., March 1, 2003; 9(3): 1047 - 1052. [Abstract] [Full Text] [PDF]
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, methylation-positive and
AFP 
, methylation-negative and AFP