Alpha-Fetoprotein inthe Differential Diagnosis of. Benign and Malignant Liver Disease. Paul K. Buamah,1. Ronald Harris,1. Oliver F.W. James,2 and Andrew W.
CLIN. CHEM. 32/11, 2083-2084
(1986)
Lentil-Lectin-Reactive Alpha-Fetoprotein Benign and Malignant Liver Disease Paul K. Buamah,1
Ronald
Oliver F.W. James,2 and Andrew
Harris,1
Affinity chromatography of serum on lentil lectin bound to Sepharose 4B has been used to identify different forms of alpha-fetoprotein in serum of patients with liver disease. We studied eight patients with non-malignant liver disease, finding that 7-15% of the serum alpha-fetoprotein bound to the lectin. In contrast, for 15 patients with malignant liver disease, 25-83% of the serum aipha-fetoprotein bound to the
lectin. Evidently, this simple technique can be used effectively to differentiate these two conditions. AddItional chronic
Keyphrases: active
affinity chromatography primary hepatocellular
hepatitis
metastatic liver disease disease
Liver
such
isoprotein as cirrhosis
cirrhosis carcinoma
cancer or chronic
active
hepatitis,
where there is no evidence of malignancy but where hepatic regeneration occurs, can lead to an increased concentration of alpha-fetoprotein (AFP) in serum. Malignant liver disease, either primary hepatocellular carcinoma or metastatic liver disease, is usually associated with a much greater increase in the AFP concentration in serum. There is no clear-cut serum AFP concentration that is characteristic of benign or malignant liver disease, and thus this assay is of little
value
(1). The serum
in
the differential
in the Differential Diagnosis of
diagnosis
of hepatic
disease
concanavalin A binding characteristics of AFP in afford a means of differentiating between the AFP
from primary hepatocellular carcinoma and metsstatic liver disease (2), but this technique is ineffective in distinguishing between the AFP produced in benign liver disease from that found in malignant liver disease. Affinity chromatography on lentil lectin, which has an affinity for glycopeptides with both a fucose residue and N-acetylglucosamine (3), has been used as a means of achieving the latter separation, and we present our preliminary findings.
of serum was loaded onto a (0.5 x 10 cm) column of lectin-Sepharose, which had been previously equilibrated with Ti-is HC1 (50 mmoIJL, pH 7.2) containing 150 mmol of NaC1, 1 mmol of MgCl2, and 1 mmol of CaCl2 per liter. The column was first eluted with 10 mL of the same buffer and then with 20 mL of Ti-is-buffered isotonic saline containing 0.5 mol of methyl-i-mannopyranoside per liter. The column was then regenerated with 50 mL of the Ti-isbuffered saline. We collected 0.5-mL fractions directly into plastic tubes and estimated the AFP concentrations in them by using an enzyme immunoassay kit from Abbott Diagnostics Limited (Wokingham, Berkshire). The non-reactive (unbound) fraction was designated I and the reactive (retarded) fraction II. Analytical recovery of AFP from the columns ranged from 95 to 106% (mean 98%). The AFP that was bound to the column was termed the “lentil-lectinaliquot lentil
reactive” total
and Method
Serum was obtained
from eight patients with benign liver chronic active hepatitis and six with cirrhosis) 15 patients with malignant liver disease (six metastatic liver disease and nine with primary hepatocellular carcinoma). The serum samples were collected disease
(two
with and from
before stored
the histology was established, and samples were at -20 #{176}C until analysis. The nature of the disease confirmed by histological examination of biopsy speci-
was
mens. We did the affinity using
lentil
Co. Limited,
‘Department
lectin
Poole,
chromatography bound to Sepharose Dorset
of Clinical
BH17
Biochemistry High Heaton,
at room temperature, 4B (Sigma Chemical 7NH, U.K.). An 0.1-mL
and2 Department of MediNewcastle upon Tyne, NE7
cine, Freeman Hospital, 7DN, U.K. 3Department of Clinical Biochemistry, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, U.K. Received June 17, 1986; accepted August 6, 1986.
form
and
was
expressed
of the
as a percentage
AFP.
Results Table 1 summarizes the percentage patients
with
the AFP
in the
concentration
lentil-lectin-reactive
malignant
liver
disease
disease. The AFP concentrations grams per liter, with use of IARC on Cancer)
Research
Table
arising
Patients
W. Skillen3
AFP
in serum
are expressed (International
reference
in
1. Lentli-Lectln-Reactlve AFP or Malignant
micro-
Agency
preparation
Patients with Benign
and
form for the and benign liver for
72/225
as
In Serum of 23 Liver Disease
AFP Total
Sex
LentIl
lectin
jcig/L
%
Disease
1265
65
PLC
2980 2167 1040 6308
83 39
PLC
67
1950 3600 5600 2520 14500
74
1280
780
61
PLC PLC PLC PLC
F
63
PLC
59 63 56
1466 6233 258
60
M M M
2460 7900 435
79
428
106
59 25
PLC PLC MLD
F F F M
34 40 36 65
369 130 17800 430
95 97 6372 110
26 75 36 26
MLD MLD MLD MLD
M
920 2150 161
376
41
MLD
140
7
C
F
67 83 32
23
14
CAH
M M
62 68
435 389
62 58
14 15
C
M
40
F
83
139 1710
18 253
13 15
C C
F
56
1495
F
35
145 37
10 10
CAH
Age,
M
75
M F F
71 59 49
M M
F
Concn,
reactive
y
364
PLC. primary liver cancer; and C, cirrhosis.
M LD, metastic
41 43
C
C
liver dise ase; CAN, ch ronic active
hepatitis;
CLINICAL CHEMISTRY,
Vol. 32, No. 11, 1986
2083
C
PRIMARY
HEPATOCELLULAR
CARCINOMA
APP
pill.
S
CIRRHOSIS
A,,
.10.
A
SIRONK
ACTIVO
MOTAITATIC
LIVIN
DISEAAO
‘PP
HRPATITIS
.18.
II
ISO
‘SO
S0 i0 40
a
S0
3
5
7
5
Il
13
0MM.,
IA
It
II
SI
2
24
2t
l,MM..,
#{163}1.11.,I,tMn
Fig. 1. Representative elution patterns of serum AFP from patients and (L metastatic liver disease I, non-reactive (unbound) fraction; II, reactive (retarded) fraction
with (A) chronic active hepatitis, (
cirrhosis,
(C
primary
hepatocellular
carcinoma,
the standard, the upper limit of the normal reference interval being 10 jg/L. The percentage of AFP in serum that reacted with the lentil lectin ranged from 25 to 83% (mean 51%, median 42%) in malignant liver disease and from 7 to 15% (mean 12%, median 14%) in benign liver disease. Statistical analysis (Mann-Whitney) differences in reactivity of AFP towards
lentil
showed lectin
these
liver
to be
the sugar chain as the molecular basis for this heterogeneity of AFP (6), and our own studies have shown that the test is useful in monitoring the efficacy of chemotherapy in patients with primary liver cancer (7). We believe that the estimation of this fraction may be useful in detecting primary liver cancer at an early stage, particularly in patients with chronic liver diseases associat-
highly
significant (p 0.02). Figure 1 compares representative elution patterns for serum
AFP
sorts of liver-disease
from various
(39-83%, mean 59%), this difference was not statistically significant. This preliminary study has shown that estimation of the lentil-lectin-reactive isoprotein of AFP is a useful test for differentiating between patients with benign and malignant
patients.
disease.
ed with
Previous
a malignant
work
has identified
flicosylation
of
transformation.
Discussion Several investigations have shown that there are isoproof AFP with different lectin-binding affinities. Some
thins
have reported that estimation of concanavalin A non-reactive isoprotein of AFP in amniotic fluid provides a useful means for the and Buamah nique affords with primary
prenatal detection of neural tube defects et al. (2) have demonstrated that this a means
of distinguishing
between
(4,5),
tech-
patients
hepatocellular carcinoma and hepatic secondaries. The two groups of patients could not be differentiated on the basis of their serum AFP concentrations, for there is considerable overlap of values (Table 1). The lentil-lectinreactive AFP isoprotein, however, clearly distinguishes between the two groups of patients. With patients with benign liver disease the lentil-lectin-reactive isoprotein comprised less than 16% of the total AFP, whereas with patients with malignant liver disease the lentil-lectin-reactive fraction exceeded 24% (Table 1). Although the lentil-lectin-reactive
form was a smaller proportion of the total AFP in the case of patients with metastatic liver disease (26-75%, mean 38%) than was true for those with primary hepatocellular cancer
2084
CLINICAL
CHEMISTRY,
Vol. 32, No. 11, 1986
References Y, Isemura M, et al. Differential reactivity of lectins and evaluation of its usefulness in the diagnosis of hepatocellular carcinoma. Gann 1984;75:809-15. 2. Buaniah PK, Gibb I, Bates G, Milford Ward A. Serum alpha fetoprotem heterogeneity as a means of differentiating between primary hepatocellular carcinoma and hepatic secondaries. Clin Chim Acts 1984;139:313-6. 3. Kornfeld K, Reitman ML, Kornfeld R. The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J Biol Chem 1981;256:6633-40. 1. Aoyagi
Y, Suzuki
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4. Smith
with
(11, Kelleher PC, Belanger L, Dallaire fluid alpha-fetoprotein with concanavalin of neural tube defects. Br Med J 1979;i:920-1. amniotic
L. Reactivity of A in diagnosis
5. Buamah PK, Taylor P, Milford Ward A. Concanavalin A binding of a-fetoprotein in amniotic fluid as an aid in the diagnosis of neural
tube defects. Clin Chem 1981;27:1658-60. 6. Aoyagi Y, Isemura M, Yosizawa Z, et al. Fucosylation
of serum a-fetoprotein in patients with primary hepatocellular cancer. Biochim Biophys Acts 1985;830:217-23. 7. Buamah PK, Cornell C, Cassells-Srnith AJ, Harris AL. Fucosylation of a-fetoprotein in hepatocellular carcinomas. Lancet
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