Yonago Acta medica 2000;43:47–57

α-Fetoprotein Messenger RNA in the Blood Predicts Poor Prognosis of
the Patients with Hepatocellular Carcinoma
Manri Kawakami, Masahiko Koda*, Yoshikazu Murawaki*, Hironaka
Kawasaki* and Shiro Ikawa
Department of Clinical Laboratory Medicine and *Second Department of Internal Medicine,
Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
α-Fetoprotein (AFP) messenger RNA (mRNA) in the peripheral blood of patients with
hepatocellular carcinoma (HCC) may indicate hematogenous spread of HCC. This
study examined the presence of AFP mRNA in the blood of 148 patients, in terms ofclinical parameters, tumor metastasis and survival rate. For the prospective study, 109
patients with HCC were followed in the period between March 1996 and March 1999.
AFP mRNA in the blood was examined by means of nested reverse transcription polymerase chain reaction. AFP mRNA was detected in the blood in 23 (15.5%) of 148
patients with HCC. AFP mRNA in the blood was significantly correlated with protein
induced by vitamin K absence or antagonist II level, higher AFP level (200 IU/mL ormore) and extrahepatic metastases, but not with tumor size, number of tumor nodules
or tumor-nodule-metastasis stage. This prospective study confirmed that intra- andextra-hepatic metastases developed more frequently in the 22 AFP mRNA-positive
patients than in the 87 AFP mRNA-negative patients (P < 0.01). The cumulative survival
rate was significantly lower in the former than in the latter (P < 0.01). In conclusion,
AFP mRNA in the blood is closely related to hematogenous spread and might be a goodpredictor of metastasis and poorer survival rate in HCC patients.
Key words: α-fetoprotein messenger RNA; hepatocellular carcinoma; metastasis; tumor marker

micrometastases in order to choose effective
therapeutic approaches.
α-Fetoprotein (AFP), a serum protein produced in large amounts during fetal life, rapidly
reduces from late fetal life and is essentiallyscarce in normal adults. The synthesis of AFP
is often associated with the development of
HCC and yolk sac tumors (Gitlin and Boesman,
1966; Adinolfi et al., 1975). The detection of serum AFP provides a useful marker for diagnosis
and prognosis of these tumors (Adinolfi et al.,1975). However, the serum AFP level does notalways correspond to the clinical stage of HCC(Okuda et al., 1975; Nomura et al., 1989; Inoueet al., 1994; Suehiro et al., 1994; Nakagawa etal., 1999). Recent molecular biological techniques have provided a method for detecting

Hepatocellular carcinoma (HCC) is a common
malignancy throughout the world, especially in
Asian countries. Despite advances in diagnostic tools and therapeutic options, intra- or extrahepatic tumor metastases are frequently foundafter surgical and medical treatments such as
hepatic resection, transcatheter arterial embolization , percutaneous ethanol injection and livertransplantation (Yokoyama et al., 1990; Behghiti
et al., 1991; Okuda, 1992; Nagasue et al., 1993;
Adachi et al., 1995; Zhou, et al, 1996). HCC metastases include intrahepatic metastases and/orde novo tumors (Chen et al., 1989; Heu et al.,
1991; Sheu et al., 1993; Nakano et al., 1994;
Takenaka et al., 1994; Tarao et al., 1994;
Toyosaka et al., 1996; Kumada et al., 1997; Kubo
et al., 1998). Therefore, it is important to detect

Abbreviations: AFP, α-fetoprotein; bp, base pair; CT, computed tomography; HCC, hepatocellular carcinoma; mRNA, messenger RNA; PCR, polymerase chain reaction; PIVKA-II, protein induced by vitamin K
absence or antagonist II; RT-PCR, reverse transcription-PCR; TNM, tumor-nodule-metastasis

47

M. Kawakami et al.

malignant cells in the peripheral blood byamplification of messenger RNA (mRNA) ofvarious genes specific to a particular cell typefrom peripheral blood mononuclear cells(Smith et al., 1991; Mattano et al., 1992;
Moreno et al., 1992; Tada et al., 1993; Brandt etal., 1996; Krüger et al., 1996; Nomoto et al.,
1996; Ishikawa et al., 1998). AFP mRNA has
been demonstrated to be one of the candidate
molecules for detecting HCC cells in the blood(Matsumura et al., 1994, 1995, 1999; Komeda etal., 1995; Jiang et al., 1997; Lemoine et al., 1997;
Louha et al., 1997; Nambu et al., 1997; OmichiFunaki et al., 1997, 1998; Wong et al., 1997; ; Luoet al., 1999; Okuda et al., 1999). With thisbackground, this study examined AFP mRNAin patients with HCC using nested reverse
transcription polymerase chain reaction (RTPCR), in terms of clinical parameters, tumor
metastasis and survival rate.

Table 1. Profiles of HCC patients
Number of patients148
Age (year)
66 ± 9Gender (male/female)
94/54
Etiology of liver disease
HBV-related33
HCV-related
96
HBV- and HCV-related
4
Alcoholic
5
Cryptogenic
10
Underlying liver disease
Chronic hepatitis
19
Cirrhosis
Child-Pugh grade A
67
Child-Pugh grade B
36
Child-Pugh grade C
26
TNM stage*
I
10
II
30
III
27
IVA
54
IVB
27
Tumor differentiation†Well
40 (34.5%)
Moderate
65 (56.0%)
Poor
11 ( 9.5%)
Serum AFP (ng/mL)
Median159
Range
1 – 332,500
Serum PIVKA-II (mAU/mL)‡
Median80
Range
4 – 1,270,000
AFP, α-fetoprotein; HBV, hepatitis B virus; HCC,
hepatocellular carcinoma; HCV, hepatitis C virus;
PIVKA-II, protein induced by vitamin K absenceor antagonist II; TNM, tumor-nodule-metastasis.
* Clinical staging of the tumor was assessed according to the TNM system of the General Rules for
the Clinical and Pathological Study of PrimaryLiver Cancer in Japan on the basis of imaging
studies.
† Tumor biopsies were performed in 116 lesions andclassified according to the General Rules for theClinical and Pathological Study of Primary Liver
Cancer in Japan.
‡ Serum PIVKA-II levels were measured in 126 patients.

Subjects and Methods

Patients and cell line
All patients were admitted to Tottori University
Hospital between March 1996 and March 1999.
Samples were obtained from the 148 patientswith HCC. Informed consent was obtained
from each patient or from family members if thepatient could not make a decision. Table 1 summarized the patients’ profiles. In 116 of 148 patients, HCC was histologically classified according to the General Rules for the Clinical andPathological Study of Primary Liver Cancer inJapan (Liver Cancer Study Group of Japan, 1989)
by tumor biopsies under the guidance of ultrasonography. The remaining patients were diagnosed by several imaging modalities such as
ultrasonography, computed tomography (CT),magnetic resonance imaging or angiography.
On the basis of these findings, the tumors wereclassified according to the tumor-nodulemetastasis (TNM) classification of the General
Rules for the Clinical and Pathological Study ofPrimary Liver Cancer in Japan (Liver Cancer
Study Group of Japan, 1989) when the sample
was obtained. Extrahepatic metastases were48

AFP mRNA in blood of HCC patients

Germany), which was based on the acidguanidium phenol-chloroform method and stored at –80°C until use.

detected by means of interviews, physical examinations and imaging techniques such as
chest X-ray, scintigraphy or CT. The mean diameters of the main tumors were 1.7 ± 0.4 cm instage I, 2.5 ± 1.3 cm in stage II, 3.7 ± 1.6 cm instage III, 3.3 ± 1.8 cm in stage IVA and 4.6 ± 4.1
cm in stage IVB. No HCC patients underwent
treatment in the 2 months before the blood
samples were obtained.
Samples were also obtained from healthy
volunteers, who agreed to donate blood. A
human hepatic carcinoma cell line, HepG2 cells
known to produce AFP, was kindly supplied by
Prof. Kenzo Sato of the Dept. of Molecular Biochemistry, School of Life Sciences, Faculty of
Medicine, Tottori University. For the prospective study, 109 patients (72 males and 37 females) with a median age of 66 ± 9 years and arange of 34–80 years were enrolled. Four
(3.7%) of 109 patients underwent hepatic resection, 71 (65.1%) were treated medically bytranscatheter arterial embolization and/or
percutaneous ethanol injection and 33 (30.3%)
remained untreated because of advanced liver
dysfunction, advanced liver cancer or the decision of each patient. These patients were examined every 1 or 2 months at our outpatient
clinic for the detection of metastasis by themeasurement of tumor markers, AFP and protein induced by vitamin K absence or antagonist
II (PIVKA-II), every 1 or 2 months, ultrasonography every 3 months and CT every 6 months.
The patients who developed a diffuse, infiltrative HCC were not treated. The patients were
followed up for a mean period of 16.2 ± 11.2
months.

Synthesis of complementary DNA(cDNA)
One microgram of RNA was heated at 65°C for
10 min and cooled rapidly on ice, diluted to 13mL buffer with 45-mmol/L Tris (pH 8.3), 68mmol/L KCl, 9-mmol/L MgCl2, 15-mmol/L 1,4dithiorethreitol, 0.2-mg random hexadeoxynucleotides, RNA guard (porcine), 0.8-µg/mL
RNase/DNase-free bovine serum albumin, 1.8mmol/L each dNTP, and Moloney Murine Leukemia Virus reverse transcriptase (First-Strand
cDNA synthesis kit, Amersham-PharmaciaBiotech). cDNA was synthesized by means of
incubation at 37°C for 60 min. It was thenheated at 94 °C for 5 min for inactivation of reverse transcriptase, cooled rapidly and stored at–20°C until use for the 1st PCR.

Sequence of primers used in nested
PCR
The sequences of primers used in the experiment were as follows. The primers for the AFP
gene were i) 5'-ACA GCA GCC ACT TGTTGC CAA-3' (nucleotides 1413 to 1433) andii) 5'-CTC TTC AGC AAA GCA GAC TTC-3'(nucleotides 1809 to 1829) for the outer primers; and iii) 5'-GCT GAC ATT ATT ATC GGA
CAC-3' (nucleotides 1473 to 1493) and iv) 5'AGC CTC AAG TTG TTC CTC TGT-3' (nucleotides 1734 to 1754) for the inner primers.
The PCR amplification of the β-globin gene
was done to confirm the successful extractionof mRNA from blood nuclear cells and HepG2
cells. The primer sequences for the β-globin
gene were v) 5'-TGG TCT CCT TAA ACC TGTCTT G-3' and vi) 5'-ACA CAA CTG TGT TCA
CTA GC-3' for the outer primers; and vii) 5'GTC TCC TTA AAC CTG TCT TG-3' and viii)
5'- ACA ACT GTG TTC ACT AGC-3' for the
inner primers. The sense and antisense primers
described above were selected from different
exons to distinguish amplification of RNA fromcontamination of DNA (Saiki et al., 1988).

Extraction of RNA
Five milliliters of heparinized blood were collected from each patient. RNA was extracted
from blood nuclear cells and HepG2 cells. Peripheral mononuclear cells that may have contained tumor cells were separated by discontinuous gradient centrifugation using a FicollPaque (Amersham-Pharmacia-Biotech,
Buckinghamshire, United Kingdom). Total
RNA from mononuclear cells was extractedwith an RN easy Mini Kit (QIAGEN, Hilden,
49

M. Kawakami et al.

NA
mR
P
AF

Nested RT-PCR

er
h
nte
wit
u
s
l
nt
o
ell
e
v
i
t
2c
hy
pa
t
l
G
C
a
p
HC
He
He

One microliter of cDNA solution was mixed
with 9 µL of the PCR reaction mixture containing 20-mmol/L Tris-HCl (pH 8.0), 100-mmol/L
KCl, 0.1-mmol/L EDTA, 0.5% Tween 20, 1mmol/L 1,4-dithiothreitol, 50% glycerol, 15mmol/L Mg2+, 1 mmol of each primer (Nos. i, ii, v
and vi) and 0.25-unit Taq polymerase (Gene Taq;
Wako Pure Chemical Ind., Osaka, Japan). The
reaction mixture was overlaid with mineral oil(Perkin Elmer, Foster City, CA) and heated at
95°C for 5 min. It was subjected to a total of 35
cycles of heat at 94°C for 30 s, 54°C for 30 s and
72°C for 1 min with a thermal cycler (Gene Amp
PCR System 9600-R, Perkin-Elmer). The reaction terminated by heating at 72°C for 7 min
and cooling at 4°C. One microliter of the amplified product was then used in the 2nd PCR.
One microliter of the amplified sample was
mixed with the same buffer as in the 1st PCR
for the 2nd PCR, except that the primers wereinner primers (Nos. iii, iv, vii and viii). The amplification program was also the same as for the
1st PCR. Three microliters of the above secondary amplified products were electrophoresed on a 1.8% agarose gel containing ethidium
bromide and photographed on a UV transilluminator (ATTO densitograph, ATTO Corporation, Tokyo, Japan). The amplified productsof AFP and β-globin were 240 and 167 basepair (bp), respectively.

AFP
240 bpβ-Globin
167 bpFig. 1. Nested reverse transcription-polymerase
chain reaction of α-fetoprotein (AFP) messenger
RNA (mRNA). bp, base pair; HCC, hepatocellular
carcinoma.

Meier method. Survival curves were comparedwith a generalized Wilcoxon test. A P valueless than 0.05 was considered significant.

Results
A specific band for the amplified AFP gene(240 bp) was noted in the samples obtained
from HepG2 cells and an HCC patient with
AFP mRNA, but not in the sample from ahealthy volunteer (Fig. 1).
AFP mRNA in the peripheral blood was
detected in 23 (15.5%) of 148 patients with
HCC. The frequency of positive patients ineach TNM stage was as follows: 1 of 10 patients(10.0%) in stage I, 4 of 30 patients (13.3%) instage II, 4 of 27 patients (14.8%) in stage III, 7
of 54 patients (13.0%) in stage IVA and 7 of 27
patients (25.9%) in stage IVB (Table 2). Although the frequency tended to be higher instage IVB, there was no statistical significance.
HCC patients with AFP mRNA in the
peripheral blood were analyzed in relation to tumor size, number of tumor nodules, the presence of portal vein thrombosis, portocaval
shunt, intrahepatic metastasis, extrahepatic

Assay for AFP and PIVKA-II
Serum AFP and PIVKA-II concentrations were
measured with a chemiluminescence assay(Wako Pure Chemical) and an electrochemiluminescence assay (Eizai Pharmaceutical Co.,Tokyo), respectively.

Statistics
Statistical analysis was performed with theMann-Whitney U test. The χ2 test was used forstatistical analysis between group frequencies.
The cumulative overall and metastasis-freesurvival rates were calculated by the Kaplan50

AFP mRNA in blood of HCC patients

Table 2. AFP mRNA in the blood of 148 HCC patients

Tumor diameter
Number of tumors
AFP
PIVKA-II*TNM stage†

Portal thrombosis
Portocaval shunt
Extrahepatic metastasis≥ 5 cm< 5 cm≥3<3≥ 200 ng/mL< 200 ng/mL≥ 40 mAU/mL< 40 mAU/mL
I
II
III
IVA
IVB
Present
AbsentPresent
AbsentPresent
AbsentNumberofpatients

Positive
[23]

26
122
90
58
64
84
72
54
10
30
27
54
27
27
121
37
111
27
121

5
18
17
6
15
8
10
8
1
4
4
7
7
7
16
3
20
8
15

AFP mRNA in blood
P value
Negative Positive rate
[125]
(%)
21
104
73
52
49
76
62
46
9
26
23
47
20
20
105
34
91
19
106

19.2
14.8
18.9
10.3
23.4
9.5
13.9
14.8
10.0
13.3
14.8
13.0
25.9
25.9
13.2
8.1
18.0
29.6
12.4

NS
NS
< 0.05NS
NS
NSNS< 0.05[ ], number of patients.
AFP, α-fetoprotein; HCC, hepatocellular carcinoma; mRNA, messenger RNA; NS, not significant;
PIVKA-II, protein induced by vitamin K absence or antagonist II; TNM, tumor-nodule-metastasis.
* Serum PIVKA-II levels were measured in 126 patients.
† Clinical staging of the tumor was assessed according to TNM system of the General Rules for Clinical andPathological Study of Primary Liver Cancer in Japan on the basis of imaging studies.
(ng/mL)metastasis and serum tumor markers (AFP and
PIVKA-II) (Tables 2 and 3). The largest tumor
was described in cases with two or more tumors
in the liver. Tumor diameter was not significantly different between the HCC patients with
AFP mRNA in the blood and those without
AFP mRNA. The incidence of AFP mRNA in
the blood was not correlated with tumor size.
The number of tumor nodules in the liver
was not significantly different between the
HCC patients with AFP mRNA in the blood andthose without AFP mRNA. The incidence of
AFP mRNA in the blood was not correlatedwith the number of tumor nodules (Table 2).
Serum AFP concentration was higher in the
HCC patients with AFP mRNA (median: 1209
ng/mL, range: 9–319,900 ng/mL) than in thepatients without AFP mRNA (median: 125 ng/
mL, range: 1–332,500 ng/mL), the value being
not statistically significant (Fig. 2, Table 3).
When the patients were divided into 2 groups
according to serum AFP concentration, the inci-

Median106

AFP concentation

105

104

103

102

10

1

Positive

Negative

[23]

[125]

AFP mRNA
Fig. 2. Serum α-fetoprotein (AFP) concentrations in 23
patients with AFP messenger RNA (mRNA) in the
blood (median: 1209 ng/mL, range; 9–319,900 ng/mL)
and 125 patients without AFP mRNA (median: 125 ng/
mL, range; 1–332,500 ng/mL). [ ], number of patients.

51

M. Kawakami et al.

Table 3. Profiles of 148 patients with or without AFP mRNA
AFP mRNA in blood
Positive
Negative
Number of patients
Age(year)
Gender (male/female)
Etiology of liver disease

Underlying liver disease

HBV-related
HCV-related
HBV- and HCV-related
Alcoholic
Cryptogenic
Chronic hepatitis
Cirrhosis
Child-Pugh grade A
Child-Pugh grade B
Child-Pugh grade C

23
65.7 ± 8.8
14/9
7
15
0
0
1
4

125
66.1 ± 8.6
80/45
26
81
4
5
9
15

P value

NS
NS
NS

6
61
< 0.05432
917
Tumor diameter(cm)
3.3 ± 2.9
3.3 ± 2.3
NS
Number of tumors
8.9 ± 8.4
7.2 ± 7.8
NS
TNM stage*
I, II
5
35
NS
III , IV
18
90
Tumor differentiation†Well
8 (40.0%)32 (33.3%)
NS
Moderate or poor
12 (60.0%)64 (66.7%)
Serum AFP
(ng/mL) Median
1209
125
NS
Range
9 – 319,900
1 – 332,500
Serum PIVKA-II (mAU/mL)‡ Median151
73.5< 0.05Range
10 – 1,270,000
4 – 409,000
AFP, α-fetoprotein; HBV, hepatitis B virus; HCV, hepatitis C virus; mRNA, messenger RNA; NS, not significant; PIVKA-II, protein induced by vitamin K absence or antagonist II; TNM, tumor-nodule-metastasis.
* Clinical staging of the tumor was assessed according to the TNM system of the General Rules for theClinical and Pathological Study of Primary Liver Cancer in Japan on the basis of imaging studies.
† Tumor biopsies were performed in 116 lesions (20 cases in the AFP mRNA-positive group and 96 cases in
the AFP mRNA-negative group) and classified according to the General Rules for the Clinical andPathological Study of Primary Liver Cancer in Japan.
‡ Serum PIVKA-II was measured in 126 patients (18 in the AFP mRNA-positive group and 108 in the AFP
mRNA-negative group).

dence of AFP mRNA in the blood was significantly higher in the patients with 200 ng/mL or
more (23.4%) than those with less than 200 ng/
mL (9.5%; P < 0.05; Table 2). PIVKA-II concentration in the serum was significantly higher in
HCC patients with AFP mRNA in the blood(76,443 ± 298,178 mAU/mL) than in the patients
without AFP mRNA (12,209 ± 50,415 mAU/mL;
P < 0.05). However, when the patients were divided into 2 groups according to PIVKA-II concentration, the incidence of AFP mRNA was
not different between the patients with PIVKAII concentrations of 40 mAU/mL or more andthose with less than 40 mAU/mL (Table 2).
The presence of portal thrombosis wasfound in 7 (30.4%) of the 23 patients with AFP
mRNA in the blood and in 20 (16.0%) of the

125 patients without AFP mRNA (Table 2).
The presence of a portocaval shunt was found in
3 (13.0%) of the 23 patients with AFP mRNA in
the blood and in 34 (27.2%) of the 125 patients
without AFP mRNA (Table 2). Thus, AFP
mRNA in the blood did not correlate with portal
thrombosis or a portocaval shunt.
Extrahepatic metastases were noted in 8
(34.8%) of the 23 patients with AFP mRNA in
the blood and 19 (15.2%) of the 125 patients
without AFP mRNA, the value being significantly higher in the former than in the latter (P <0.05; χ2 test). When the patients were classified
according to the presence or absence of extrahepatic metastases, AFP mRNA in the blood
was detected in 8 (29.6%) of the 27 patientswith extrahepatic metastases and in 15 (12.4%)
52

AFP mRNA in blood of HCC patients

bleeding and 4 from other diseases. AFP mRNA
was noted in 8, 4, 0 and 0 patients, respectively.
Extrahepatic metastasis was observed in 13(11.9 %) patients (7 in the AFP mRNA-positiveand 6 in the AFP mRNA-negative group).
Tumor metastases were found in the lung in 6
patients, the bone in 3, the adrenal glands in 4,
the spleen in 1 and the pharynx in 1.
Next, the following parameters were analyzed: the cumulative rate of the extrahepatic
metastasis, the cumulative rate of intra- andextra-hepatic metastases and the cumulative
survival rate for each AFP mRNA status. The
cumulative rate of extrahepatic metastasis was
similar between the 2 groups (data not shown).
However, the cumulative rate of intra- andof the 121 patients without extrahepatic metastases, the value being statistically significant (P< 0.05; Table 2).
Aspartate aminotransferase and alanine
aminotransferase concentrations in the serum
were significantly higher in the 23 HCC patientswith AFP mRNA in the blood (aspartate aminotransferase; 203 ± 297 IU/L, alanine aminotransferase; 113 ± 134 IU/L) than in the 125 patients without AFP mRNA (104 ± 116, P <0.01,72 ± 61 IU/L, P < 0.05).
Table 4 summarizes the profiles of 109 patients undergoing follow-up studies which
disclosed the cause of death in 51 patients(46.8%); 35 died from HCC progression, 9from hepatic failure, 3 from gastrointestinal

Table 4. Profiles of the 109 HCC patients with or without AFP mRNA
AFP mRNA in blood
Positive
Negative
Number of patients
Age(year)
Gender (male/female)
Etiology of liver disease

Underlying liver disease

HBV-related
HCV-related
HBV- and HCV-related
Alcoholic
Cryptogenic
Chronic hepatitis
Cirrhosis
Child-Pugh grade A
Child-Pugh grade B
Child-Pugh grade C

22
65.4 ± 8.9
13/9
7
14
0
0
1
3

87
65.6 ± 8.9
59/28
18
54
3
4
8
12

P value

NS
NS
NS

6
48
< 0.05421
9
6Tumor diameter(cm)
3.3 ± 3.0
3.5 ± 2.5
NS
Number of tumors
9.0 ± 8.6
6.1 ± 7.3
NS
TNM stage*
I, II
5
28
NS
III , IV
17
59
Tumor differentiation†
Well
7 (36.8%)
23 (36.5%)
NS
Moderate or poor
12 (63.2%)
40 (63.5%)
Serum AFP
(ng/mL) Median
1951
63
NS
Range
9 – 319,900
1 – 332,500
Serum PIVKA-II (mAU/mL)‡ Median
101
30
NS
Range
10 – 1,270,000
4 – 409,000
AFP, α-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; mRNA,
messenger RNA; NS, not significant; PIVKA-II, protein induced by vitamin K absence or antagonist II;
TNM, tumor-nodule-metastasis.
* Clinical staging of the tumor was assessed according to the TNM system of the General Rules for theClinical and Pathological Study of Primary Liver Cancer in Japan on the basis of imaging studies.
† Tumor biopsies were performed in 82 lesions (19 cases in the AFP mRNA-positive group and 63 cases in
the AFP mRNA-negative group) and classified according to the General Rules for the Clinical andPathological Study of Primary Liver Cancer in Japan.
‡ Serum PIVKA-II was measured in 94 patients (17 in the AFP mRNA-positive group and 77 in the AFP
mRNA-negative group).

53

Intra- and extra-hepatic metastases rate

M. Kawakami et al.

(%)
100

Discussion

80

In this study, AFP mRNA in the blood was
detected in the 23 (15.5%) of the total 148 HCC
patients. Previous studies showed the positive
rate of AFP mRNA in the peripheral blood to befrom 25 to 95% of HCC patients (Matsumura etal., 1994, 1999; Komeda et al., 1995; Jiang et al.,
1997; Lemoine et al., 1997; Louha et al., 1997;
Nambu et al., 1997; Luo et al., 1999; Okuda et al.,1999). The lower frequency in the present study
might be partly due to the selection of patients,
in whom extrahepatic metastases were noted
only in 27 patients. Furthermore, there weremore patients with AFP non-producing HCC in
the present study than in the previous reports(Matsumura et al., 1994; Jiang et al., 1997).
The next possible reason is the difference in thetiming of the examination of blood samples.
Louha et al. (1997) reported that AFP mRNA
producing cells in the blood were detected after
locoregional therapy such as transcatheter
arterial embolization or percutaneous ethanol
injection. Matsumura et al. (1994) and Okuda
et al. (1999) also showed that tumor cells do notalways circulate in the blood, but may appear
intermittently. We analyzed the blood sample
before therapy or at least 2 months after therapy.
Next, we investigated the relationship between AFP mRNA in the blood and the clinicalparameters of HCC. The AFP mRNA in the
blood did not correlate with tumor size, numberof tumor nodules, presence of portal thrombosis
and portocaval shunt, or TNM stage. Clinicalsignificance of AFP mRNA in the blood is controversial even now (Matsumura et al., 1994,
1999; Komeda et al., 1995; Lemoine et al., 1997;
Louha et al., 1997; Nambu et al., 1997; Luo et al.,1999). What is interesting is that this study
confirmed a significant correlation between serum aspartate aminotransferase or alanine aminotransferase concentration and AFP mRNA in
the blood. The present study also demonstrated
that the HCC patients with high levels of serum
AFP showed higher detectable rates of AFP
mRNA in the blood than those with low levelsof serum AFP. The frequency of AFP mRNA

60

40

AFP mRNA-positive [22]20

AFP mRNA-negative [87]P < 0.01

0

0

1

2

3

(year)

Fig. 3. Rates of intra- and extra-hepatic metastases
calculated by the Kaplan-Meier method for 22 patients with positive α-fetoprotein (AFP) messenger
RNA (mRNA) and 87 patients with negative AFP
mRNA. The difference is statistically significant (P< 0.01). [ ], number of patients.

extra-hepatic metastases was significantly
higher in the 22 AFP mRNA-positive patients
than that in the 87 AFP mRNA-negative patients (P < 0.01; Fig. 3). The cumulative survival rate was significantly lower in the 22 AFP
mRNA-positive group than in the 87 AFP
mRNA-negative group (P < 0.01; Fig. 4).

(%)

Culumative survival rate

100

80

60

40

AFP mRNA-positive [22]20

AFP mRNA-negative [87]P < 0.01

0

0

1

2

3

(year)

Fig. 4. Cumulative survival curve calculated by the
Kaplan-Meier method for 22 patients with positive
α-fetoprotein (AFP) messenger RNA (mRNA) and87 patients with negative AFP mRNA. The
difference is statistically significant (P < 0.01). [ ],number of patients.

54

AFP mRNA in blood of HCC patientsin the blood, however, was not so much higher
in the HCC patients with a high AFP level(23.4%). This result suggests that the serum
AFP level does not necessarily reflect the presence of circulating cells with AFP mRNA. In
other words, the serum AFP level is not a useful
test to indicate the presence of circulating HCC
cells. Nambu et al. (1997) and Luo et al. (1999)
showed that the PIVKA-II value may be a good
indicator of AFP mRNA in the blood. The positive rate of AFP mRNA in the blood was notdifferent between PIVKA-II-positive patients(40 mAU/mL and more) and -negative patients(less than 40 mAU/mL) in this study, although
HCC patients with AFP mRNA-positive in the
blood had higher PIVKA-II values than thosewith lower PIVKA-II. It is conceivable that thepositivity of AFP mRNA did not correlate withthe serum PIVKA-II value, because PIVKA-IIpositive and AFP-negative HCCs are commonly
experienced and the nested RT-PCR in the present study does not detect AFP non-producing
cells.
In agreement with some previous research(Matsumura et al., 1994, 1999; Komeda et al.,
1995; Louha et al., 1997), we found that thepositivity of AFP mRNA in the blood was associated with extrahepatic metastasis. The detection of AFP mRNA was clearly more frequentin the patients with extrahepatic metastasis thanthose without. This suggests that circulating
AFP producing cells might be responsible forextrahepatic metastasis.
The present study compared the survival rateand intra- and extra-hepatic metastases, in which
there were no differences in serum AFP levels,
PIVKA-II levels or TNM stages. Matsumura etal. (1999) reported that the incidence of extrahepatic metastasis was significantly higher inthe AFP mRNA-positive group than that in the
AFP mRNA-negative group. However, theextrahepatic metastasis was not significantly
different between the 2 groups in the present
study. The cumulative rate of intra- and extrahepatic metastases was significantly higher inthe patients positive with AFP mRNA in the
blood than in the patients without. This result
may indicate that tumor cells in the systemic
circulation are responsible not only for extra-

hepatic metastasis but also intrahepatic metastasis, which have been considered to be caused
mainly by the spreading of cancer cells via theportal vein (Toyosaka et al., 1996). Funaki etal. (1997) reported that cancer cells released
from the primary tumor to the systemic circulation might participate in intrahepatic metastasis. Since the liver provides an essentially favorable environment for HCC cells, the incidence of intrahepatic metastasis through thesystemic circulation might be more frequent
than previously considered. Therefore, it isreasonable to assume that HCC patients who
undergo liver transplantation do not always
have good prognoses and often experience metastases in the transplanted livers (Zhou et al.,1996).
In the present follow-up study, we foundthat the cumulative survival rate was significantly poorer in the AFP mRNA-positive groupthan that in the AFP mRNA-negative group.
Matsumura et al. (1999) found that overall survival was similar between patients in AFP
mRNA-positive and -negative groups, but thesurvival rates in continuous AFP mRNA-positive patients was poorer than that in continuous
AFP mRNA-negative patients. This resultshows that the presence of AFP mRNA cells inthe blood might be a predictive factor in HCC
patients. Since the present AFP mRNA-positive group had more severe underlying liver disease than the AFP mRNA-negative group, thismight affect the survival rate. However, it isindisputable that the high frequency of intraand extra-hepatic metastases in the AFP
mRNA-positive group was responsible for theirpoor prognoses.
In conclusion, AFP mRNA in the blood of
HCC patients related to PIVKA-II levels andwas significantly detectable in patients with an
AFP level of 200 IU/mL or more, or with extrahepatic metastases. Thus, the detection of AFP
mRNA in the blood might be a predictor for
tumor metastasis and survival of HCC patients.

55

M. Kawakami et al.

1996;122:679–686.
13 Kubo S, Nishiguchi S, Hirohashi K, Shuto T,
Kuroki T, Minamitani S, et al. Clinicopathological criteria for multicentricity of hepatocellular carcinoma and risk factors for suchcarcinogenesis. Jpn J Cancer Res 1998;89:419–
426.
14 Kumada T, Nakano S, Takeda I, Sugiyama K,
Osada T, Kiriyama S, et al. Pattens of recurrence
after initial treatment in patients with small
hepatocellular carcinoma. Hepatology 1997;25:
87–92.
15 Lemoine A, Le Bricon T, Salvucci M, Azoulay
D, Pham P, Raccuia J, et al. Prospective evaluation of circulating hepatocytes buy alpha-fetoprotein mRNA in humans during liver surgery. Ann
Surg 1997;226:43–50.
16 Louha M, Poussin K, Ganne N, Zylberberg H,
Nalpas B, Nicolet J, et al. Spontaneous and iatrogenic spreading of liver-derived cells into peripheral blood of patients with primary liver cancer.
Hepatology 1997;26:998–1005.
17 Luo W, Yatsuhashi H, Hamada R, Matsumoto T,
Inoue O, Koga M, et al. Analysis of α-fetoproteinmRNA in peripheral blood: detection and semiquantitation by reverse transcription polymerase
chain reaction. Hepatol Res 1999;14:1–12.
18 Matsumura M, Niwa Y, Hikida Y, Okano K,
Kato N, Shiina S, et al. Sensitive assay for detection of hepatocellular carcinoma associatedgene transcription (alpha-fetoprotein mRNA) inblood. Biochem Biophys Res Commun 1995;
207:813–818.
19 Matsumura M, Niwa Y, Kato N, Komatsu Y,
Shiina S, Kawabe T, et al. Detection of αfetoprotein mRNA, an indicator of hematogenous
spreading hepatocellular carcinoma, in thecirculation: a possible predictor of metastatic
hepatocellular carcinoma. Hepatology 1994;20:
1418–1425.
20 Matsumura M, Shiratori Y, Niwa Y, Tanaka T,
Ogura K, Okudaira T, et al. Presence of α-fetoprotein mRNA in blood correlates with outcome
in patients with hepatocellular carcinoma. J
Hepatol 1999;31:332–339.
21 Mattano LA Jr, Moss TJ, Emerson SG. Sensitive
detection of rare circulating neuroblastoma cellsby the transcriptase-polymerase chain reaction.
Cancer Res 1992;52:4701–4705.
22 Moreno JG, Croce CM, Fischer R, Monne M,
Vihko P, Mulholland SG, et al. Detection ofhematogenous micrometastasis in patients withprostate cancer. Cancer Res 1992;52:6110–6112.
23 Nagasue N, Uchida M, Makino Y, Takemoto Y,
Yamanoi A, Hayashi T, et al. Incidence and factors associated with intrahepatic recurrence following resection of hepatocellular carcinoma.
Gastroenterology 1993;105:488–494.
24 Nakagawa T, Seki T, Shiro T, Wakabayashi M,

References
1 Adachi E, Maeda T, Matsumata T, Shirabe K,
Kinukawa N, Sugimachi K, et al. Risk factors for
intrahepatic recurrence in human small hepatocellular carcinoma. Gastroenterology 1995;108:768–775.
2 Adinolfi A, Adinolfi M, Lessof MH. Alpha-fetoprotein during development and in disease. J
Med Genet 1975;12:135–151.
3 Behghiti J, Panis Y, Farges O, Benhamou JP,
Fekete F. Intrahepatic recurrence after resectionof hepatocellular carcinoma complicating cirrhosis. Ann Surg 1991;214:114–117.
4 Brandt B, Junker R, Griwatz C, Heidl S,
Brinkmann O, Semjonow A, et al. Isolation of
prostate-derived single cells and cell clusters
from human peripheral blood. Cancer Res 1996;
56:4556–4561.
5 Chen P-J, Chen D-S, Lai M-Y, Chang M-H,
Huang G-T, Yang P-M, et al. Clonal origin ofrecurrent hepatocellular carcinomas. Gastroenterology 1989;96:527–529.
6 Gitlin D, Boesman M. Serum α-fetoprotein,
albumin, and γG-Globulin in the human conceptus. J Clin Invest 1966;45:1826–1838.
7 Heu H-C, Chiou T-J, Chen J-Y, Lee C-S, Lee PH, Peng S-Y. Clonality and clonal evaluation ofhepatocellular carcinoma with multiple nodules.
Hepatology 1991;13:923–928.
8 Inoue S, Nakao A, Harada A, Nonami T, Takagi
H. Clinical significance of abnormal prothrombin (DCP) in relation to postoperative survival
and prognosis in patients with hepatocellular
carcinoma. Am J Gastroenterol 1994;89:2222–
2226.
9 Ishikawa T, Kashiwagi H, Iwakami Y, Hirai M,
Kawamura T, Aiyoshi Y, et al. Expression of αfetoprotein and prostate-specific antigen genes inseveral tissues and detection of mRNA in normal
circulating blood by reverse transcriptase-polymerase chain reaction. Jpn J Clin Oncol 1998;28:
723–728.
10 Jiang SY, Shyu R-Y, Huang M-F, Tang H-S, Young
H-S, Roffler SR, et al. Detection of alphafetoprotein-expressing cells in the blood of patientswith hepatoma and hepatitis. Br J Cancer 1997;
75:928–933.
11 Komeda T, Fukuda Y, Sando T, Kita R, Furukawa
M, Nishida N, et al. Sensitive detection of
circulating hepatocellular carcinoma cells inperipheral venous blood. Cancer 1995;75:2214–
2219.
12 Krüger W, Krzizanowski C, Holweg M,
Stockschläder M, Kröger N, Jung R, et al. Reverse
transcriptase/polymerase chain reaction detection ofcytokeratin-19 mRNA in bone marrow and blood ofbreast cancer patients. J Cancer Res Clin Oncol

56

AFP mRNA in blood of HCC patients

Imamura M, Itoh T, et al. Clinicopathologic significance of protein induced vitamin K absence or
antagonist II and a-fetoprotein in hepatocellular
carcinoma. Intern J Oncol 1999;14:281–286.
25 Nakano S, Haratake J, Okamoto K, Takeda S. Investigation of resected multinodular hepatocellular carcinoma: assessment of unicentric or
multicentric genesis from histological and prognostic viewpoint. Am J Gastroenterol 1994;89:
189–193.
26 Nambu S, Nishimori H, Maekawa M, Higuchi K,
Watanabe A. Plasma PIVKAII values as anindicator of AFP mRNA in the circulation in patients with advanced hepatocellular carcinoma.
Hepatol Res 1997;8:28–36.
27 Nomoto S, Nakao A, Kasai Y, Harada A, Nonami
T, Takagi H. Detection of ras gene mutations inperioperative peripheral blood with pancreatic
adenocarcinoma. Jpn J Cancer Res 1996;87:793–
799.
28 Nomura F, Ohnishi K, Tanabe Y. Clinical features and prognosis of hepatocellular carcinoma
with reference to serum alpha-fetoprotein levels.
Cancer 1989;64:1700–1707.
29 Okuda K. Hepatocellular carcinoma: recent
progress. Hepatology 1992;15:948–963.
30 Okuda K, Kotoda K, Obata H, Hayashi N,
Hisamitsu T, Tamiya M, et al. Clinical observations during a relatively early stage of hepatocellular carcinoma, with special reference toserum α-fetoprotein levels. Gastroenterology
1975;69:226–234.
31 Okuda N, Nakao A, Takeda S, Oshima K,
Kanazumi N, Nonami T, et al. Clinical significance of α-fetoprotein mRNA during perioperative period in HCC. Hepatogastroenterology
1999;46:381–386.
32 Omichi-Funaki N, Tanaka J, Imamura M. Quantitative analysis of alpha fetoprotein mRNA incirculating peripheral blood of patients with
hepatocellular carcinoma and alpha fetoprotein
producing gastric carcinoma. Life Sci 1998;62:
1973–1984.
33 Omichi-Funaki N, Tanaka J, Seto S, Kasamatsu
T, Kaido T, Imamura M. Hematogenous spreading of hepatocellular carcinoma cells: possible
participation in recurrence in the liver. Hepatology 1997;25:564–568.
34 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ,
Higuchi R, Horn GT, et al. Primer-directed enzymatic amplification of DNA with a thermostable

DNA polymerase. Science 1988;239:487–491.
35 Sheu J-C, Huang G-T, Chou H-C, Lee P-H,
Wang J-T, Lee H-S, et al. Multiple hepatocellular carcinomas at the early stage have different
clonality. Gastroenterology 1993;105:1471–
1476.
36 Smith B, Selby P, Southgate J, Pittman K,
Bradley C, Blair GE. Detection of melanoma
cells in peripheral blood by means of reverse
transcriptase and polymerase chain reaction.
Lancet 1991;338:1227–1229.
37 Suehiro T, Sugimachi K, Matsumata T, Itasaka
H, Taketomi A, Maeda T. Protein induced byVitamin K absence or antagonist II as prognostic
marker in hepatocellular carcinoma. Cancer
1994;73:2464–2471.
38 Tada M, Omata M, Kawai S, Saisho H, Ohto M,
Saiki RK, et al. Detection of ras gene mutation inpancreatic juice and peripheral blood of patientswith pancreatic adenocarcinoma. Cancer Res
1993;53:2572–2574.
39 Takenaka K, Adachi E, Nishizaki T, Hiroshige K,
Ikeda T, Tsuneyoshi M, et al. Possible multicentric occurrence of hepatocellular carcinoma:a clinicopathological study. Hepatology 1994;
19:889–894.
40 Tarao K, Hoshino H, Shimizu A, Ohkawa S,
Nakamura Y, Harada M, et al. Role of increased
DNA synthesis activity of hepatocytes in multicentric hepatocarcinogenesis in residual liver ofhepatectomized cirrhotic patients with hepatocellular carcinoma. Jpn J Cancer Res 1994;85:
1040–1044.
41 Toyosaka A, Okamoto E, Mitsunobu M, Oriyama
T, Nakao N, Miura K. Intrahepatic metastasis in
hepatocellular carcinoma: evidence for spread
via the portal vein as an efferent vessel. Am J
Gastroenterol 1996;91:1610–1615.
42 Wong IH-N, Leung T, Ho S, Law WY, Chan M,
Johnson PJ. Semiquantification of circulating
hepatocellular carcinoma cells by reverse transcriptase polymerase chain reaction. Br J Cancer
1997;76:628–633.
43 Yokoyama I, Todo S, Iwatsuki S, Starzl TE.
Liver transplantation in the treatment of primaryliver cancer. Hepatogastroenterology 1990;37:
188–193.
44 Zhou X-D, Tang Z-Y, Yang B-H, Yu Y-Q, Lin ZY, Lu J-Z, et al. Long-term results of surgery forsmall primary liver cancer in 514 adults. J Cancer
Res Clin Oncol 1996;122:59–62.

(Received December 16, 1999, Accepted January 5, 2000)

57