a-FETOPROTEIN LEVELS IN NORMAL MALES FROM SEVEN ETHNIC GROUPS WITH DIFFERENT HEPATOCELLULAR CARCINOMA RISKS P. Sizaret," A. Tuyns,* N. Martel,* A. Jouvenceaux,? A. Levin, $ Y. W. Ong. 8 and J. Rive 6

INTRODUCTION The initial observations of elevated a-fetoprotein (AFP) values in various pathological conditions such as hepatocellular carcinoma (HCC) in mouse,' man,' or rat,". human teratoblastomas," and serum hepatitis i, have been confirmed and extended by various authors." As long as crude techniques were used, AFP was thought to be present only in pathological processes, but with sensitive techniques such as immunoautoradiography or radioimmunoassay it was also found in pregnant women l o as well as in normal persons.l1 Purves et al.'? published the results of a study including more than 1,000 sera from normal individuals and found that people originating from areas where HCC occurs in high incidence appear to have higher AFP levels compared with people living in low HCC-risk areas, MATERIAL AND METHODS Material Examined

Venous blood samples were collected into plain tubes from 1,335 males . Samples were obtained from fasting blood donors in the first three locations, but not all subjects of the two representative samples of the IC rural populations (forest in the South and savanna in the North) were fasting. Sera were separated, frozen at -20°C without the addition of preservatives, and shipped with dry ice. Before testing, all sera were decomplemented by heating 15 minutes at 56" C. The number and age distribution of samples for each of the seven groups are given in TABLE1. All ages were well represented in the two samples from IC, whereas the older age-groups were poorly or not all represented among the blood donors of the other groups. The Chinese, Indians, and Malays from Singapore had very similar age structures. ( 2 15 years) in Singapore, Nairobi, Lyon, and the Ivory Coast (IC)

* International

Agency f o r Research on Cancer, 69008 Lyon, France

i Centre de Transfirsion Sangrrine, H6pital E. Herriot, 69003 Lyon, France $ International Agency for Research on Cancer, Nairobi Research Centre, PO BOX 46831, Nairobi, Kenya § Singapore Blood Transfirsion Service, General Hospital, Singapore rl Direction Ge'nkralr d e la PrPwntion et Service des Rrandes Endkmies, Ministhre t i e le Santr', Abidjan, Ivorv Coast.

136

137

Sizaret et al.: AFP in Different Ethnic Groups TABLEI AGE STRUCTURES OF MALESFROM SEVENPOPULATIONS ~~-

Groups

No. of Individuals

Singapore Malays Chinese Indians

199 197 149

Age Un(years) (years) (years) (years) (years) (years) known

< 25 40 36 36

25-34

35-44

45-54

55-64

66 65 50

63 59 43

26 29 19

4 7 1

265

-

-

1

L

Lyon

174

24

50

35

52

11

-

Nairobi

128

46

59

19

2

1

1

Ivory coast Forest Savanna Total

226 262

31 35

28 53

47 55

67 61

30 35

23 23

-

1335

248

371

321

256

89

48

2

2 -

A F P Dosages

Dilutions were made with Eppendorff pipettes and plastic tubes 13 X 75 mm. Specimens were assayed 1 3 by the double antibody radioimmunoassay (RIA). The purified AFP was prepared in this laboratory from a pool of cord sera by the method of Nishi," using a pH 2.8 buffer for dissociation of the AFP immune complex. Purity was checked by electrophoresis in 9% acrylamide 1 ) with gel l B and antigenicity by comparing the dose-response curves (FIGURE

95 90

110

rrn 4 I0 I 50

i"

-

pm 10 10

5 2 1

DO'.

FIGURE 1. Dose-responses of various AFP-containing preparations.

138

Annals New York Academy of Sciences

those of 721 225 and of two other AFP preparations of HCC origin (one purified in this laboratory, the second of HCC ascitic fluid origin kindly provided by Dr. Nishi). A total of 4 micronuits ( M U ) (see ADDENDUM) of purified AFP were labeled by the same person on five separate occasions, following Hunter's technique''; and using 2.5 mCi of Amersham 125 iodine. The yield was 17% and the intensity of labeling was 60,000 countslminlnanounit ( N U ) ; 97% of the labeled antigen was precipitated by 10% trichloracetic acid and 90% by the specific rabbit antiserum. Precipitation of the radioactive immune complex was achieved by using sheep antirabbit immunoglobulin (Institut Pasteur) and normal rabbit serum. The 72/225 preparation was used for the reference inhibition curves; it was assayed at 9 (initial) dilutions from 32 to 0.125 NU/ml with two tubes/dilution. 0.05 M pH 8.6 borate buffer with 10% normal chicken serum" was used for diluting all sera except rabbit anti-human AFP, which was diluted with plain borate buffer. Standard dilutions were prepared by the same person on four different occasions, split into aliquots, and kept frozen until used. Assaying all specimens required 24 RIA: coded sera were randomized and assayed ( 1 tube/specimen) by three persons (one person/assay) at 92' , and where necessary, at higher dilutions. Two coded controls of known AFP content were included in each dosage. All the reagents (100 p1 vol) were added in the following order: First day: specimen and antiserum (50% precipitin dose: 1/45,000); Second day: radioactive AFP: 10,000 cpm (circa 160 picounits); Third day: sheep anti-rabbit immunoglobulin ( 1 / 8 ) and normal rabbit serum ( 1 /90). The tubes were maintained at room-temperature for three days; on the fourth day, 1 ml of saline was introduced in each tube; sedimentation of immune complexes was achieved by centrifugation for one hour at 1,850 g in a refrigerated MSE Mistral Centrifuge holding 144 tubes. Supernatants were poured off and tubes were counted for two minutes in a Packard gamma spectrometer counter. Each run included a series of 72/225 dilutions, and the corresponding inhibition curve was drawn on log-logit paper, using the following equation : % inhibition =

maximum precipitation - observed precipitation maximum precipitation - nonspecific precipitation

x

100.

Only the figures corresponding to the dots located on the linear part of the curve were kept for AFP determination. They usually ranged from 0.125 to 4 NUIml. After transformation of standard percentage inhibition in logits and standard AFP values in log values and calculation of the regression line, crude titers were obtained by feeding a Hewlett Packard 9810 A programmed machine with raw counts. Hepatitis-Associated Antigen Determinations

The IC sera were previously tested by RIAIS and the Singapore sera by counter immunoelectroph~resis.~~

Sizaret et al.: AFP in Different Ethnic Groups

139

Statistical Analysis In each subgroup examined, the distribution of A F P values was normalized by log transformation and calculations were made on log values. Mean and variance were computed in each subgroup of at least 20 individuals, with the exception of two subgroups of 19 subjects (Indians in Singapore, age 45-54 and Africans in Nairobi, age 35-44). The observed differences between groups were tested for significance by variance analysis, and differences between pairs were further tested by t-test.

989-

95

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-

: Ec s40302 o0 10

52. 1-

asn201

I I

I I I

: ; I:

I I I

I I

I I

I I I I

I I I I

I I I I

II I I

I I I

1I I I

I

I

I I

I I

I I I

b

+

4

i

4

0.25

0.6

1

2

4

A F P Nono

FIGURE 2. Comparison of dose-responses of 12 preparations: *=pure AFP from cord origin; x =72/225 reference preparation; and 0 = 1 0 sera from normal blood donors (1 Caucasian from Lyon, 1 Indian and 1 Chinese from Singapore, 3 Black from Nairobi, 4 Black from Ivory Coast). Depending upon specimens, dilutions varied from !h to 1/32.

RESULTS A F P Dose Responses In FIGURES 1 and 2, dose responses of several AFP-containing preparations 3 illustrates the mean percentage inhibition 2 standard are shown. FIGURE deviation of serial dilutions of the standard which were run on 24 different occasions.

Annals New York Academy of Sciences

140

511-

0s02-

ai

'

TABLE 2 MEANAFP LEVELS(IN NANOUNITS/ML) k SD IN VARIOUSAGE GROUPS OF SEVENPOPULATIONS OF MALES Ethnic Groups

16-24

25-34

35-44

45-54

55-64

2 65

(years)

(years)

(years)

(years)

(years)

(years)

Singapore Malays Chinese Indians

1.722.10 2.68k.14 2.062.14 2.282.11 2.062.1 1 2.672.20

3.24k.19 2.992.18 2.902.23

3.482.31 3.442.35 3.382.41

Total

1.932.07

2.522.09

3.062.11

3.442.20

Lyon

1.872.14

2.712.22

3.112.25

3.532.28

Nairobi

2.812.26

3.532.32

3.582.63

Ivory coast Forest Savanna

2.202.27 2.582.23

3.142.33 2.84k.19

3.242.25 4.222.24 3.23k.19 2.99k.14

4.23e.47 3.102.31

4.652.74 3.70k.63

2.39k.11

2.942.17

3.232.16

3.582.27

4.052.47

+

Forest Savanna

3.582.17

*Each age group included at least 20 individuals, except Chinese, 45-54, and Nairobi, 35-44, which included 19 individuals each.

Sizaret et al.: AFP in Different Ethnic Groups

141

Mean A F P Values The mean A F P values for age groups in each population are indicated in TABLE 2. It is apparent that A F P levels increased with age and varied between populations. Distribution of A F P Values by A g e When age subgroup histograms based on crude A F P values were drawn, a skewing phenomenon appeared that precluded statistical analysis of the results (FIGURE4,A ) . This difficulty was overcome by using log A F P values; distribu-

TABLE3 COMPARISON OF F VALUES(VARIANCE ANALYSIS) BETWEEN DIFFERENTMALE AGE GROUPSO F SEVENPOPULATIONS? Ethnic Groups

3 Singapore Groups 2 Ivory Coast Groups

Singapore/Lyon Ivory Coast/Nairobi Singapore/Ivory Coast Singapore/Nairobi Lyon/Ivory Coast Lyon/Nairobi Singapore +Lyon/Africa (Ivory Coast Nairobi)

+

*p