α-Fetoprotein in Human Hepatoma: Improved Detection in Serum, and Quantitative Studies using a New Sensitive Technique

GASTROENTEROLOGY(J) Official Publication of the American Gastroenterologica/ Association ('()pytti(;IIT 19il

VOLUME

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August 1971

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a-FETOPROTEIN IN HUMAN HEPATOMA: IMPROVED DETECTION IN SERUM, AND QUANTITATIVE STUDIES USING A NEW SENSITIVE TECHNIQUE ELLIOT ALPERT, M.D., RoBERT HERSHBERG, M .D. , PETER H. ScHuR, M.D., AND KURT J. lsSELBACHER, M.D.

Departments of Medicine and Pathology, Harvard Medical School, and Massachusetts General Hospital (Gastrointestinal Unit), and the Robert Breck Brigham Hospital, Boston, Massachusetts

a-Fetoprotein (AFP) was measured in the sera of 62 patients from the United States and 58 foreign patients with hepatoma. AFP was detected in 50.5% of these patients by double immunodiffusion in agar gel. Cminterimmunoelectrophoresis detected 1/40 the amount of AFP detectable by double immunodiffusion and indicated the presence of AFP in 72.4% of patients. A significant ethnic variation was noted with 51 % of 41 Caucasian patients being positive and 75 to 95% of 64 nonCaucasian patients from different areas being AFP-positive, depending on their ethnic origin. Age and sex factors had a slight effect on the frequency of AFP. Although not apparent in any individual patient, AFP presence tended to correlate with the presence of larger more undifferentiated tumors. The quantitative level of AFP varied widely and was of no clinical or prognostic significance. The level, however, tended to generally stabilize or increase with time and may prove to be of value in assessing therapy. 1963 3 that one of the hepatoma-specific antigens was immunochemically identical to AFP suggested the reversion by the malignant liver cell to the fetal biosynthetic state and resynthesis of this fetal protein.

a-Fetoprotein (AFP), a unique a-1 globulin, is synthesized by embryonic liver cells and is normally present in serum only during fetal life. 1• 2 The demonstration in Received February 3, 1971. Accepted March 18, 1971. Address requests for reprints to: Dr. Elliot Alpert, Massachusetts General Hospital, Boston, Massachusetts 02114. This work was supported by grants from the United States Public Health Service (T1-AM-05146 and AM11414) and The John A. Hartford Foundation, Inc. The authors gratefully acknowledge the reference antisera supplied by Dr. J. Uriel. They are also grateful for the advice and encouragement of Dr. Kurt Bloch which enabled this study to be initiated. The expert technical assistance of Ruth L. Coston was in-

valuable and appreciated. They also wish to thank the many doctors, including Drs. M. Warnock (Chicago); M. Kaplan, L. Stolbach, E. Thomas, D. Trey, and W. McDermott (Boston); S. Winawer (New York); R. Propp (Albany); and R. Seeler (Chicago) for providing clinical information and sera from their patients. Several of the serum specimens came from the Leary Laboratory through the help of Dr. Norman Zamcheck, Stella M. Goldsmith, and Tom Boenisch, and the Formosan specimens were kindly supplied by Dr. E. Rosenberg (United States Public Health Service). 137

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ALPERT ET AL.

Many experimental studies have confirmed the production of a homologous speciesspecific fetal protein by all types of hepatoma in a variety of mammalian species, although it was not detected in every case. 4 A similar a-1 fetal specific protein was demonstrated in the serum of patients with hepatoma in 1965. 5 Subsequently, a number of investigators used double immunodiffusion in agar gel to detect the presence of human AFP and reported it to be present in the sera of 60 to 80% of histologically confirmed hepatoma patients. •- 12 These patients, however, have been almost exclusively African or Chinese, groups known to have a high incidence of hepatic cancer, usually presenting with a large tumor mass. In 1968, we first reported that AFP was detectable in only about 30% of sera from hepatoma patients in the United States where the tumors tend to be small and occult.' 3 This report describes a more sensitive method for the detection of a-1 fetoprotein (AFP) in the sera of 124 patients with primary carcinoma of the liver, including 74 patients in the United States, by means of the more sensitive technique of counterimmunoelectrophoresis. In addition, a quantitative assay has been used in order to determine the relationship of the serum concentration of AFP to various clinical, prognostic, and pathologic features of this tumor. Methods Production of antisera. Rabbits were immunized with pooled serum from several human fetuses of 17 to 21 weeks of gestation. One milliliter of this pooled human fetal serum was mixed with equal volumes of complete Freund's adjuvant and injected into the footpads of several rabbits. Booster injections were given at 3 and 5 weeks with 1 ml of the same mixture in multiple subcutaneous injections. Sera were collected two to three times per week for several months after antibodies appeared and were pooled. The entire pool was absorbed with lyophilized normal human serum. Specificity of the antisera was documented by immunoelectrophoresis and double immunodiffusion in agar gel against fetal sera hepatoma sera. A single precipitin line between fetal sera and hepatoma sera was noted with complete identity and no reaction was detectable against a pool of normal human sera.

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Methods of assay. Sera were tested by Ouchterlony double immunodiffusion in agar gel" and by quantitative radial immunodiffusion, 15 modified to increase sensitivity. 16 Counterimmunoelectrophoresis (CIEP) was adapted from a previously published method."· 18 Ten milliliters of 0.9% agarose in barbital buffer (0.05 M, pH 8.6) were layered on an 8- by 10-cm glass plate on a level surface. After cooling, double rows of 3-mm holes were cut, 1.0 em apart. Sera from patients and control subjects were placed in one row and anti-AFP was placed in the other row. The plates were inserted into an electrophoresis chamber, and Whatman no. 1 paper, saturated with buffer, was used as wicks. Counterelectrophoresis was performed in 0.05 M barbital buffer, pH 8.6, for 60 min at 30 rna, with the antibody-filled wells placed closer to the anode than the wells filled with sera. Although most precipitin lines were readily visible without further treatment, the plates were dried and stained with Amido-Black before final reading. This method permitted the detection of 'l.lo the concentration of AFP needed to produce a visible line by double immunodiffusion in agar. The concentration of AFP was determined by the method of Gitlin et al. 1 Serum from a 17-week fetus was electrophoresed on cellulose acetate. A unique a-1 fetoprotein region in the serum was quantitated by densitometry and used as the standard for radial immunodiffusion. Although this method can only approximate the true AFP concentration, results using this as a standard are expressed as mg per 100 ml of serum, according to criteria previously described. 1 The antiserum pool could detect as little as 1 mg per 100 ml of AFP by Ouchterlony double immunodiffusion. The CIEP method increased the sensitivity 40fold to a lower limit of approximately 0.025 mg per 100 ml (25 ng per ml) . Patients. Sera were obtained from 124 patients with histologically confirmed primary hepatic neoplasms. Control sera were obtained from 337 other patients with various liver diseases. Clinical records of 63 of the 124 hepatoma patients were examined and chemical and biochemical data analyzed. Duration of survival was available from 67 patients who were followed by their physician. Twenty-eight of the patients in this series originally were studied in Uganda and were the subject of previous reports. 8 • 1 9 Necropsy data was available on 19 patients.

Results AFP was detected in serum by double immunodiffusion in 59 of the 117 patients

August 1971

(50.5%) with primary hepatocellular carcinoma. Three children with hepatoblastoma were all AFP-positive. Negative results were obtained in 4 patients with cholangiocarcinoma who were excluded from any further study. Using the CIEP method, 71.2% of the 120 patients with hepatocellular malignancy were AFP-positive. No positive reactions were noted in any of the 337 patients with other forms of liver diseases, including metastatic carcinoma. The ethnic origin of 105 patients is shown in table 1. As previously reported, 13 29.2% of the 41 Caucasian patients with hepatoma from the United States were AFP-positive, whereas 67.2% of the 64 non-Caucasians from the United States, Uganda, and Taiwan were positive. Using the CIEP technique, the frequency of AFP-positive paTABLE

1. a-Fetoproteinin-histologically confirmed hepatoma patients Ouchterlony CIEP positive posi· tive

Caucasian Non-Caucasian . Ugandan (Bantu) . Formosan (Chinese)

-.

~

Total (nonCaucasian)

Total

Total assayed

positive

12 13

21 16

41 21

,. ,, 51.2a 76.2

17

24

28

85.7

11

14

15

93.3

43

54

64

84 . 3a

• The difference between Caucasians from the United States (51.2%) and non-Caucasians (84.3%) is significant with a P < 0.01 .

tients increased to 51.2% of Caucasian patients from the United States and 84.3% of non-Caucasian patients. The frequency of AFP-positive Caucasian patients with hepatoma remains significantly lower (P < 0.01) than in non-Caucasian patients, despite the ability to detect AFP in 20% more patients with hepatoma. Age and sex. The age was available from 89 patients (see table 2A). Although younger patients appeared to produce AFP more frequently than older patients, the differences were not statistically significant. No difference was apparent in the AFP distribution by age in the different ethnic groups studied. The sex of 90 of the patients is shown in table 2B. Although males were more frequently AFP-positive than females in all the populations studied, this difference was not statistically significant. Clinical features. Adequate clinical information was available on 75 patients. There appeared to be nq correlation between the presence of AFP in the serum and cirrhosis (present in approximately 60% of both Caucasian and non-Caucasian patients) or to any other biochemical tests, such as serum bilirubin, serum glutamic oxaloacetic transaminase, alkaline phosphatase, albumin, and globulin levels. Survival. The duration of survival from onset of symptoms to death in the Caucasian series was virtually identical to that found in the Bantu series. 19 In addition, survival from date -of diagnosis to death was similar, indicating that the differences in AFP production in the two racial groups

2. a-Fetoprotein by age and sex

TABLE

B. Sex distri bution

A. Age distribution Pooitive

No. tested

'

0-20 21-40

5 20

7 27

71 .4 74.1

41-60

20

31

64.5

15

24

62.5

60

89

67 .4

Total

Positive

Pos itive

,.

yr

> 60

139

a-FETOPROTEIN IN HEPATOMA

Caucasian Male Female Non-Caucasian Male Female Total Male Female

No. tested

Posit ive

,. '

17 4

30 11

56.7 36.4

31 9

35 14

88.6 64.3

48 13

65 25

73.8 52. 0

ALPERT ET AL.

140

25

• Potltlve (OueMerlony PoLl o Trott Pos • Neqotlve

SURVIVAL TIME (Ooys)

FIG. 1. Serum a -fetoprotein and the survival of 58 patients with hepatoma related to serum a-fetoprotein results.

are not related to the clinical stage of the disease at diagnosis. The duration of survival was estimated in 58 patients in both racial groups from the date of the blood assay-usually corresponding closely with the date of diagnosis-to the date of death (see fig. 1). Nine additional patients were followed for a short period and were still alive at the conclusion of this study. AFPnegative patients appeared to have a longer survival, whereas the strongly AFP-positive patients all died within 4 months (see fig. 1). However, there was no statistically significant difference between the groups when subjected to computer analysis and when the number of survivors was compared at 30-day intervals. Pathologic features. Histologic slides of the tumor were evaluated on a blind-coded basis (by R. H.). Material was available from 67 patients, of which 57 specimens were deemed adequate for interpretation. This included material from 23 Ugandan and 24 patients from the United States. The parameters evaluated (on a semiquantitative 1 to 3+ scale) included degree of eosinophilia, cell size, number of mitoses, degree of anaplasia, necrosis, steatosis, and bile stasis. In addition, a separate coded evaluation estimated the general degree of dedifferentiation on a 1 to 3 scale. There was no correlation between bile stasis, steatosis, extent of necrosis, and the degree of differentiation or AFP production. However, as shown in figure 2, the well differentiated tumors tend to be AFP-negative, whereas the poorly differentiated tumors

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tend to be AFP-positive. In general, the less differentiated tumors were associated with less eosinophilia, greater cell size, more mitoses, and a greater degree of pleomorphic anaplasia. Size of tumor. The weights of the livers with hepatoma at necropsy are indicated in table 3. The mean weight of the tumors from patients who were strongly AFP-positive was more than 1 kg heavier than the mean weight of the tumors from AFP-negative patients. Although the numbers are small, the difference is significant (P < 0.05) . The mean weight of the tumors in patients with only a trace of AFP (0.025 to 0.1 mg per 100 ml) was intermediate. Serum concentrations. The serum concentration of AFP ranged widely, from the lower limit of sensitivity, 0.025 mg per 100 ml to 600 mg per 100 ml. As indicated in figure 3, most of the AFP levels were low. There was no correlation between the absolute serum concentration in any individual patient and any of the parameters studied. The serum concentration tended gen-

NUMBER HEPATOMA PATIENTS

POS NEG

POS NEG

a, -FETO·PROTEIN

POS

NEG

ASSAY

.Trace Pot

FIG. 2. Serum a-fetoprotein and the degree of dedifferentiation of 57 hepatomas. T ABLE

3. Relatiom;hip of fetoprotein to tumor size Size of liver at autopsy

Fetoprotein Mean

Positive (11'g/ ml) Trace (0.02-1 l'g/ml) N egative a

, 25· 2.47 2 . 14"

r I

±SE

0.41 0.43 0.15

Statistically significant difference, P < 0.05.

No.

9 7 3

141

a-FETOPROTEIN IN HEPATOMA

August 1971

o Caucasian (U.S.) • Nan Caucasian (U.S.)

NUMBER HEPATOMA PATIENTS

• Bantu

0.1

10

100

600

a,-FETO-PROTEIN CONCENTRATION (mg/IOOml)

FIG. 3. Range of serum · a-fetoprotein concentrations in 120 patients with hepatoma.

erally to increase with time but at variable rates. In the 1 patient who received intraarterial chemotherapy, · the serum concentration of AFP fell progressively as the patient went into deepening hepatic coma and finally died (see fig. 4). Discussion The detection · of a -fetoprotein in serum has been found helpful in the diagnosis of primary carcinoma of the liver in man. 6 - 12 AFP is a normal serum a-1 globulin synthesized by the embryonic hepatocyte in early fetal life. 2 . It becomes undetect
25

OAYS FIG. 4. Serial a-fetoprotein measurements in 7 patients with hepatoma.

collaborative study involving 231 hepatoma patients and over 500 control subjects has confirmed the specificity of AFP for histologically confirmed hepatoma patients. 1 2 Previous reports of large series of hepatoma patients, which showed that 60 to 80 ~[ were AFP-positive, have consisted mainly of non-Caucasian patients from areas of the world where the incidence of hepatoma is very high. 6 - 1 2 In a study from the United States, we reported that only 30 ~;. of Caucasian patients with hepatoma were AFPpositive. 13 This ethnic rather than geographic variation has since been noted by others although the number of patients in these studies was small. 22 · 2 3 This investigation, using the same antisera but with a more sensitive technique than used previously, resulted in 20r;. more AFPpositive sera, confirming the significance of this ethnic variation. The reason for this ethnic variation in AFP production is un-

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ALPERT ET AL.

known. Whether the difference is a qualitative or quantitative phenomenon will require the use of even more sensitive techniques, such as radioimmunoassay. Several reports of small groups of padents have suggested that age 24 • 25 and sex 22 influence AFP production and may account for the variation in AFP production. In our experience, younger males with hepatoma tended to have AFP more frequently. However, this trend was not statistically significant and cannot account for the differences seen in our series. The incidence of AFP production by experimentally induced hepatomas has varied with the type of carcinogenic agent used 26 • 27 and with the growth rate or histologic degree of malignancy of the tumors. 28 Environmental carcinogens such as mycotoxins have been suggested as a cause for the marked geographic variation in hepatoma incidence. 29 Indeed, Aflatoxin, the most carcinogenic of the mycotoxins, was found in significant amounts in Africa 30 and was correlated to hepatoma incidence. 31 However, Aflatoxin does not appear to be a factor in the United States where hepatoma is uncommon. Thus, the variation in type and amount of these ingested carcinogens could be responsible for the variation in hepatoma incidence, but this variation may also be related to the frequency of AFP production by the tumors. To investigate the possibility that AFP might be related to the degree of malignancy, as suggested experimentally, 28 we examined hepatoma size, histology, and survival time in coded AFP-positive and AFP-negative patients. The AFP-positive tumors, as a group, were indeed significantly larger and less differentiated. Several AFP-negative patients had unusually long survival times, but the number of survivors was too small at any point to be statistically significant. These differences were not apparent in any individual case and were not appreciated previously, possibly because the number of AFP-negative patients was too small to observe the trend. 9 • 23 It is possible that AFP production is related to the stage of biochemical dedifferentiation, rather than to the gross

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histologic appearance. However, quantitative studies in tissue culture of cell lines of variable malignancy will be needed to confirm this hypothesis. The quantitative level of AFP varied widely and, in any given patient, had no relationship to any clinical, biochemical, or prognostic features studied. Nevertheless, the level generally stabilized or increased with time in untreated patients. The latter findings have proven useful in following the efficacy of potential therapeutic measures such as liver transplantation. 32 As previously suggested, 3 3 the use of techniques more sensitive than double immunodiffusion should serve to increase the infrequency of detection of AFP in patients with hepatomas. In this study, using counterimmunoelectrophoresis we were able to increase the sensitivity 40-fold. This resulted in a statistically significant increase (20%) in AFP-positive sera from patients with hepatoma. No false positives were found with this new method. AFP can now be detected in 50% of Caucasian and 75 to 95% of non-Caucasian patients with hepatoma. REFERENCES 1. Gitlin D, Boesman ML: Serum a-feto-protein, albumin and globulin in the human conceptus. J

Clin Invest 45:1826-1838, 1966 2. Gitlin D, Boesman M: Sites of serum a-feto-protein synthesis in the human and in the rat. J Clin Invest 46:1010-1016, 1967 3. Abelev Gl, Perova SD, Khramkova NI, et a!: Production of embryonal a"globulin by transplantable mouse hepatomas. Transplantation 1: 174-180, 1963 4. Abelev GI: Production of embryonal serum aglobulin by hepatomas: review of experimental and clinical data. Cancer Res 28:1344-1350, 1968 5. Tatarinov YS: Content of embryo-specific a-globulin in fetal and neonatal sera and sera from adult humans with primary carcinoma of the liver. Vop Med Khim 11:20, 1965. (Fed Proc Trans! (Suppl) 25:T344-T345, 1966) 6. Uriel J, deNechaud B, Birencwajg MS, et a!: Antigenes embryonnaires et cancer du foie chez l'homme: Association de Ia alpha-1-fetoproteine serique avec l'hepatome primarie. CR Acad Sci, Paris, 265:75-77, 1967 7. Abelev GI, Assecritova IV, Kraevsky NA, et a!: Embryonal serum globulin in cancer patients: diagnostic value. Int J Cancer 2:551-558, 1967

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alpha-foeto-protein in adults during hepatitis treated by steroids. Presse Med 78:1107-1109, 1970 22. Hull E, Moertel C, Carbone P: Serum a-fetoprotein (A.F.P.) in cancer patients. Clin Res 17:403, 1969 23. FoliA, Sherlock S, Adinolfi M: Serum a-fetoprotein in patients with liver disease. Lancet 2:12671269, 1969 24. Mawas C, Buffe D, Burtin P: Influence of age on a-fetoprotein incidence. Lancet 1:1292, 1970 25. Bagshawe A, Parker AM: Age distribution of a-fetoprotein in hepatocellular carcinoma. Lancet 2:268, 1970 26. Stanislawski-Birencwajg M, Uriel J, Grabar P: Association of embryonic antigens with experimentally induced hepatic lesions in the rat. Cancer Res 27:1990-1996, 1967 27. Abelev GI: Production of embryonal serum a globulin by hepatomas: review of experimental and clinical data. Cancer Res 28:1344-1350, 1968 28. Abelev G: Antigenic structure of chemically-induced hepatomas. Progr Exp Tumor Res 7:104157, 1965 29. Alpert E, Davidson CS: Mycotoxins: a possible cause of primary carcinoma of the liver. Amer J Med 46:325-329, 1969 30. Alpert E, Wogan G, Davidson CS: Aflatoxin and hepatoma in Uganda. Gastroenterology 54:149, 1968 31. Alpert E, Hutt MSR, Wogan GN, et a!: The correlation of aflatoxin content of food with hepatoma frequency in Uganda. Cancer (in press) 32. Alpert E , Starzl T, Schur PH, eta!: Serum a-fetoprotein in hepatoma patients after liver transplantation. Gastroenterology 61:144-148, 1971 33. Purves LR, MacNab M, Bersohn I: Serum a-fetoprotein. Lancet 2:634-635, 1968