Radioimmunoassay of free ~-subunit of human chorionic gonadotropin as a prognostic test for persistent trophoblastic disease in molar pregnancy

M. B. Khazaeli, Ph.D., M. M. Hedayat, M.D., K. D. Hatch, M.D., A. C. W. To, Ph.D., S.-j. Soong, Ph.D., H. M. Shingleton, M.D., L. R. Boots, Ph.D., and A. F. LoBuglio, M.D.

Birmingham, Alabama

A new radioimmunoassay system was established with a monoclonal antibody (1 E5) that distinguishes the free l3-subunit of human chorionic gonadotropin in the presence of intact human chorionic gonadotropin, showing only 0.23% cross-reactivity with the intact human chorionic gonadotropin molecule and virtually no cross-reactivity with other glycoprotein hormones or their l3-subunits. Serum samples, taken at initial diagnosis from nine patients with hydatidiform mole and spontaneous remission and 12 patients with subsequent progression to persistent trophoblastic disease, were assayed for free and total levels of the 13-subunit of human chorionic gonadotropin. The assay results were expressed as a ratio of nanograms of free l3-subunit per 1000 mlU of total l3-subunit. Eight of nine patients with mole and spontaneous remission had a ratio value <4 whereas 10 of 12 patients with subsequent persistent disease had a ratio value >4. Statistical analysis with x2 showed a highly significant correlation of high ratios with eventual progressive

disease (p = 0.0009). This study suggests that excessive production of the free l3-subunit of human chorionic gonadotropin may identify patients with a high likelihood of developing persistent trophoblastic

disease. (AM J DesTET GVNECOL 1986;155:320-4.)

Key words: Monoclonal antibodies; free l3-hCG, gestational trophoblastic disease, persistent

Human chorionic gonadotrophin (hCG) is a glyco­protein hormone composed of two dissimilar non­covalently linked subunits of a- and l3-chains. The a-chain is nearly identical to and immunologically in­distinguishable from the a-subunit of some other pi­tuitary glycoprotein hormones, including luteinizing hormone, follicle-stimulating hormone, and thyroid­stimulating hormone. The l3-subunits of these hor­mones differ in their amino acid sequences and in this way confer unique immunologic and biologic activity to each hormone. I The l3-subunit of hCG (l3-hCG) con­tains an extended COOH terminal of 30 amino acid residues not present in luteinizing hormone or other hormones. Conventional l3-hCG assay systems use an­tibodies that are raised against the structural differ­ences conferred on l3-hCG by these unique amino acid sequences.2 Today, these conventional l3-hCG assays, while specific for l3-hCG, do not discriminate the free l3-subunit of hCG from intact hCG molecules. We have

From the Division of HematologylOncology and the Biostatistic Core Unit, Comprehensive Cancer Center, and the Divisions of Gyne­cologic Oncology and Reproductive Endocrinology and Biology, Department of Obstetrics and Gynecology, University of Alabama at Birmingham.

Received for publication January 28, 1986; revised April 1, 1986; accepted April 16, 1986.

Reprint requests: M. B. Khazaeli, Ph.D., Division of Hematology/ Oncology, Comprehensive Cancer Center, 223H Tumor Institute, University of Alabama at Birmingham, Birmingham, AL 35294.

320

developed a monoclonal antibody (l E5) that distin­guishes the free l3-hCG in the presence of hCG and shows only 0.23% cross-reactivity with the intact hCG molecule and virtually no cross-reactivity with other glycoprotein hormones or their l3-subunits. A radio­immunoassay system has been established with this an­tibody.'

At the present time, serial determination of serum l3-hCG concentrations, including measurement of both intact and free l3-hCG, is the standard follow-up pro­cedure after the initial diagnosis and suction curettage of patients with hydatidiform mole.1-7 Approximately 85% of these patients undergo spontaneous remission as evidenced by a gradual fall in the serum l3-hCG level while 15% of patients develop persistent trophoblastic disease with a progressive rise in the serial levels. In either case, the level of l3-hCG is a function of the half life of serum hCG and the rate of production of hCG by the trophoblastic tissue remaining in the uterus.

The hCG polypeptide and variable quantities of its subunits have been found in tumors containing tro­phoblastic tissue. Some tumors secrete only one form of the hCG subunits and others secrete a combination of the hormone and its subunits. Secretion of only the a-subunit of hCG in the absence of either l3-subunit or the intact hCG molecule has been reported in a tumor of the stomach.8 Metastatic testicular choriocarcinoma also demonstrates differential production of hCG sub-

Volume 155 Number 2

Free l3-hC~ in persistent trophoblastic disease 321

Table I. Patients with hydatidiform mole who underwent spontaneous remission

Free {3-hCG Total {3-hCG ng Free Patient No. (nglml) (mlUlml) {3-hCGI1000 mlU keG

I 10.5 5,100 2.1 2 84.2 52,290 1.6 3 143.3 462,100 0.3 4 139.9 165,560 0.9 5 14.1 8,716 1.6 6 8.9 3,927 2.3 7 11.2 1,902 5.9 8 27.5 23,000 1.2 9 7.4 9,600 0.7

Mean ± SD 49.6 ± 57.3 81,355 ± 152,009 1.8 ± 1.7

Table II. Patients with hydatidiform mole who developed persistent trophoblastic disease

Free {3-hCG Patient No. (nglml)

I 4550 2 41.8 3 47.7 4 86.8 5 501 6 27.2 7 44.6 8 41.3 9 19.7

10 107.5 II 24.2 12 46.1

Mean ± SD 461 ± 1294

units with a high ratio of free J3-hCG to total hCG molecule compared with that of normal pregnant sub­jects." The intact hCG secreted by these neoplastic tis­sues has not been shown to be different from that found secreted by the normal placenta.

The known clinicopathologic features of hydatidi­form mole coupled with the knowledge that unbal­anced synthesis of hCG subunits can occur in tumors with trophoblastic elements suggested that the quan­titative measurement of hCG subunits might be useful in the laboratory evaluation of patients with molar pregnancy. This retrospective study was designed to provide preliminary data on whether measurement of the free J3-hCG fraction in sera taken at the time of molar evacuation is useful in predicting which patients will develop persistent trophoblastic disease.

Material and methods Serum samples obtained at initial diagnosis of molar

pregnancy from 21 patients were selected from the serum bank of the Southern Regional Trophoblastic Disease Center. The selection was based on the patient having adequate serial follow-up to clearly delineate the eventual outcome in regard to spontaneous regression (nine patients) or persistent trophoblastic disease (12 patients). The serum specimens had been stored at

Total {3-hCG ng Free (mlUlml) {3-hCGI1000 mlU hCG

401,900 11.3 5,927 7.1

14,670 3.3 12,674 6.8

480,000 1.0 3,236 8.4 8,295 5.4 6,102 6.8 4,308 4.6

20,640 5.2 3,049 7.9 3,865 13.7

80,388 ± 169,322 6.8 ± 3.4

- 20° C for periods ranging from 2 to 5 years. Samples were assayed for free J3-hCG and total J3-hCG individ­ually without knowledge of clinical outcome of the pa­tients or assay results of either component; that is, free J3-hCG was assayed without knowledge of total J3-hCG determination.

The free J3-hCG assay was carried out by means of the double-antibody technique of Midgley. IO All re­agents were diluted in phosphate-buffered saline con­taining 0.1 % gelatin. The monoclonal antibody to free J3-hCG produced in mice ascites was used at a dilution previously found to bind 30% of the added iodinated free J3-hCG. The total assay volume was kept constant at 1.0 ml and included the following: 0.1 ml of unla­beled free J3-hCG (standard) or samples; 0.1 ml of io­dine 125-labeled free J3-hCG (50,000 cpm); 0.2 ml of lE5 monoclonal antibody diluted as mentioned above; 0.1 ml of normal mouse serum diluted at 1 : 100 in gel­phosphate-buffered saline; 0.1 ml of normal human serum. Gel-phosphate-buffered saline was used to bring the total volume to 1.0 ml. For standards and nonspecific binding controls, normal human serum was diluted to match the dilution of samples used in the assay. This reaction mixture was incubated for a min­imum of 4 hours at room temperature. Then 1 ml of the second antibody, sheep anti mouse )'-globulin, was

322 Khazaeli et al.

100

c 0 80 :; .J:l

",E 60 '" 0

Q) > . .;::: 40 E :J E :J

20 U

~ 0

0 0 2 3 4 5

hCG (mIU/ml) x 10 5

Fig. 1. Cumulative distribution of total hCG concentration in serum of patients with spontaneous remission (0) and those with subsequent persistent trophoblastic disease (D).

added at a dilution of 1: 100 in 2.5% polyethylene glycol and the incubation was continued for a minimum of 30 minutes at room temperature. Assay tubes were cen­trifuged at 7000 X gat 40 C for 20 minutes, decanted, and blotted, then counted in a Micromedics gamma counter (Micromedics Systems, Inc., Horsham, Penn­sylvania). Data plots and calculations were performed on an MACC Micromedics computer interfaced with the gamma counter, with the use of a logit plot as pre­viously described.'

Total/3-hCG was assayed with a commercially avail­able immunoradiometric system for hCG (Serono Di­agnostics, Randolph, Massachusetts). Patient samples, standards, and controls, respectively, were reacted with a mixture of three monoclonal antibodies to hCG. The antibodies reacted with both free /3-subunit and the intact hCG molecule. Two of the antibodies were la­beled with 1251 and the third linked to fluorescein. In­cubation was 15 minutes at 37° C. After incubation, antifluorescein coupled to a magnetic solid phase was added in large excess to bind to the hCG-monocional antibodies complex, which was precipitated in a mag­netic field. The sensitivity of the assay is 5 mlU/ml of hCG. Calibration was against the first international ref­erence preparation 75/537. Cross-reaction with lutein­izing hormone and follicle-stimulating hormone was 0.55% and 0.68%, respectively. Precision of the assay, as expressed by interassay and intra-assay coefficients of variation, was < 10% each.

The results on each sample were analyzed as the amount of free /3-hCG (nanograms per milliliter), the amount of total /3-hCG (milli-international units per milliliter), and a ratio of nanograms of free /3-hCG per 1000 mIU ofhCG. The sensitivity (or true positive rate) of the test was defined as the proportion of patients who had progressive disease and who were correctly identified by the test. The specificity (or true negative

c S? :J

.J:l . .:

100

80

August 1986 Am J Obstet Gynecol

1

Vi 60 0 Q) >

40 0

:J E :J

20 U

~ 0

O~~~~~~~~~~~~~-L~~~~

o 20 40 60 80 100 120 140 160 180 200

Free (3 hCG (ng/mg)

Fig. 2. Cumulative distribution of free l3-hCG concentration in serum of patients with spontaneous remission (0) and those with subsequent persistent trophoblastic disease (D).

rate) of the test was defined as the proportion of pa­tients who had spontaneous regression and were cor­rectly identified by the test. The significance of the sensitivity and specificity of the test was evaluated with the use of X2 and Wilcoxon two-sample test statistical analysis. 11

Results

The serum /3-hCG values from patients who even­tually developed spontaneous remission or persistent trophoblastic disease are listed in Tables I and II, re­spectively. Samples of serum had been stored for vary­ing periods ranging from 2 to 5 years without signifi­cant differences in storage time between the two groups of patients. A wide range of free and total/3-hCG values were noted in the two groups of patients. Both groups had similar mean total /3-hCG values. The group of patients with progression had a higher mean value for free /3-hCG, but it was not statistically significant (p = 0.10). The higher mean ratios of free /3-hCG per 1000 mlU of hCG in the group with progression were highly significant when analyzed by the Wilcoxon two­sample test (p = 0.002).

When the cumulative distributions of patients with spontaneous remission (0) were compared with those of patients with persistent disease (D) with the use of the values for total /3-hCG (Fig. I), free /3-hCG (Fig. 2), and the ratio of nanograms of free /3-subunit 1000 mlU hCG (Fig. 3), only the ratio values produced sig­nificant discrimination by the Wilcoxon two-sample test (p = 0.002).

Table III examines the sensitivity and specificity of identification of patients who will develop persistent trophoblastic disease with the use of cutoff ratio values of either 3.0 or 4.0 ng of free /3-hCG per 1000 mIU of total /3-hCG as selected from the cumulative distribu­tion curves in Fig. 3. With a cutoff of 3.0, the true

Volume 155 Number 2

Table III. Prognostic specificity of the assay of free l3-hCG11000 mlU of hCG with the use of cutoff values of 4.0 or 3.0

Spontaneous remission Persistent trophoblastic

disease Total

p = 0.0009 (0.0002).

Free (3-hCG/J 000 mlU hCG

<4.0 «3.0) I >4.0 (>3.0)

8 (8) I (I) 2(1) 10(11)

10 (9) 11 (12)

Total

9 12

21

positive rate was 11112 (92%) and the true negative rate was 8/9 (89%) with a p value of 0.0002 by the X" test. With a cutoff of 4.0, the true positive rate was 10112 (83.3%) and true negative rate was 8/9 (89%) with a p value of 0.0009.

Comment

Hydatidiform mole occurs in approximately 1 in 1200 pregnancies in the United States.' Approximately 80% to 85% of women with hydatidiform mole will undergo spontaneous remission and 15% to 20% of women will develop persistent trophoblastic disease. The diagnosis and treatment of patients with persistent trophoblastic disease are based on the serial serum 13-hCG titers. Patients with nonmetastatic and low-risk metastatic persistent trophoblastic disease are ulti­mately cured with single-agent or multiagent chemo­therapy. 12. 13 High-risk metastatic trophoblastic disease develops in approximately 4% of patients with hyda­tidiform mole. Survival for high-risk patients is about 60%. High-risk patients are treated with multiagent chemotherapy with resultant significant morbidity.13

Goldstein et al. 5 treated 247 patients with prophy­lactic chemotherapy at the time of evacuation of the hydatidiform mole and had a 4% persistent disease rate while 858 patients who did not receive prophylactic chemotherapy had an 18.6% persistent disease rate. Those patients who developed persistent disease after receiving prophylactic chemotherapy all achieved com­plete remission with only one subsequent course of che­motherapy and none developed metastatic disease. It is apparent therefore that prophylactic chemotherapy can: (1) reduce the incidence of persistent trophoblastic disease, (2) decrease or eliminate metastatic tropho­blastic disease, and (3) decrease the number of che­motherapy cycles needed if persistent disease does oc­cur. Despite these demonstrated advantages, prophy­lactic chemotherapy has received little enthusiasm since 80% to 85% of patients undergo spontaneous remission and therefore would be treated unnecessarily.

For this reason, several investigators have attempted to identify a subset of patients with molar pregnancy

Free [3-hCG in persistent trophoblastic disease 323

100

c 80 S? "'3 .Q

Vi 0 Q)

>

a :J

E :J

U

~ 0

3 6 9 12 15

Free f3 hCGI 1000 mlU hCG

Fig. 3. Cumulative distribution ratio of nanograms of free [3-hCG per 1000 mlU of hCG in serum of patients with spon­taneous remission (0) and those with subsequent persistent trophoblastic disease (D).

who are at high risk of developing persistent tropho­blastic disease in order to justify prophylactic chemo­therapy at the time of evacuation. ' · 5. 11 These classifi­cation schemes are based on clinical parameters. The size of the uterus at the time of diagnosis, relationship of the uterine size to the date from the last menstrual period, presence or absence of variant theca-lutein cysts, and initial level of l3-hCG have been used most often. In these authors' retrospective analyses the pre­dictive accuracy rates are 39.8%, 57%, and 61.7%. These levels of accuracy are not judged sufficient to justify the use of prophylactic chemotherapy by most investigators and only one author uses the scoring sys­tem as a rationale for prophylactic chemotherapy.5

The results of this study are clearly preliminary be­cause of its retrospective nature and modest patient numbers. We could find no statistical correlation be­tween the ratio of free f3-hCG to total f3-hCG and the and the interval between uterine evacuation and date of serum sample (a few days to a few weeks) in these 21 patients, but a prospective study should provide data on the disappearance curve of free and total hCG af­ter uterine evacuation in both patients with eventual regression and those with progressive disease. This would allow delineation of the optimal timing of plasma sampling for discrimination of high- and low-risk patients.

Despite the retrospective nature of this study, the preliminary data are very encouraging with a high per­centage of patients with eventual progression being identified and a true positive rate of about 90%. A prospective study to gather large numbers of patients, standardize serum sampling times, and characterize free and totall3-hCG disappearance curves is underway in the Gynecologic Oncology Group. If these prelimi­nary results can be confirmed, prophylactc therapy can

Khazaeli et al.

be reevaluated with this assay to select high-risk patients and eventually change the clinical care of patients with molar pregnancy.

REFERENCES

I. Saxena BB, Rathman P. Activity relationships of protein and polypeptide hormones. In: Margulies M, Greenwood FC, eds. Amsterdam: Excerpta Medical Foundation, 1971:122.

2. Vaitukaitis JL, Braunstein GD, Ross CT. A radioimmu­noassay which specifically measures human chorionic go­nadotropin in the presence of human luteinizing hor­mone. AMJ OBSTET GYNECOL 1972;113:751-8.

3. Khazaeli MB, England BG, Dieterle RC, Nordblom GD, Kabza GA, Beierwaltes WHo Development and charac­terization of a monoclonal antibody which distinguishes the ~-subunit of human chorionic gonadotropin (~ hCG) in the presence of the hCG. Endocrinology 1981; 109: 1290-2.

4. Curry SL, Hammond CB, Tyrey L, Creasman WT, Parker RT. Hydatidiform mole: diagnosis, management and long-term follow-up of 347 patients. Obstet Gynecol 1975;45: 1-8.

5. Goldstein DP, Berkowitz RS, Bernstein MR. Management of molar pregnancy. J Reprod Med 1981 ;26:208-12.

6. Goldstein DP. Prevention of gestational trophoblastic dis­ease by use of actinomycin D in molar pregnancies. Obstet GynecoI1974;43:475-9.

August 1986 Am J Obstet Gynecol

7. Hatch KD, Shingleton HM, Austin JM, Boots LR, Youn­ger JB, Soong SJ. Southern regional trophoblastic disease center. South Med J 1978;71: 1334-40.

8. Rosen SW, Weintraub BD. Ectopic production of the iso­lated a-subunit of the glycoprotein hormone. N Engl J Med 1974;290:1441-7.

9. Khazaeli MB, Haghighi-Rad F, LaFeria JJ, Beierwaltes WHo Comparison of serum levels of human chorionic gonadotropin (hCG) and its free ~-subunit in normal and gestational trophoblastic disease using monoclonal antibodies [Abstract). J Reprod Immunol 1983;(suppl 112)5:102-3.

10. Midgley ARJr. Radioimmunoassay: a method for human chorionic gonadotropin and human luteinizing hormone. Endocrinology 1966;79: 105-18.

II. Ott L. In: An introduction to statistical methods and data analysis. North Scituate, Massachusetts: Duxbury Press, 1977:317.

12. Hammond CB, Parker RT. Diagnosis and treatment of trophoblastic disease. A report from the Southeastern Re­gional Center. Obstet Gynecol 1970;35: 132-41.

13. Hammond CB, Weed JC, Currie JL. The role of operation in the current therapy of gestational trophoblastic disease. AM J OBSTET GYNECOL 1980; 136:844-55.

14. Morrow CP, Kletzky OA, Disaia PJ, Townsend DE, Mishell DR, Nakamura RM. Clinical and laboratory correlates of molar pregnancy and trophoblastic disease. AMJ OBSTET GYNECOL 1977; 128 :424-9.

Methotrexate inhibition of normal trophoblasts in vitro

Peter K. Sand, M.D., Patricia A. Stubblefield, Ph.D., and Steven J. Ory, M.D.

Chicago, Illinois

Methotrexate is a folic acid inhibitor useful in the treatment of gestational trophoblastic disease. Its effect on normal trophoblast in vitro was studied to help establish a minimum effective dose in vivo for the

conservative treatment of ectopic pregnancy. In vitro inhibition of normal trophoblastic cell growth was measured after 48 hours of exposure to variable concentrations of methotrexate (10- 2 to 10-9moI/L) and rescue with leucovorin. A modification of Cotte's method of trophoblastic cell enrichment was used. Long­term (10 days) linear growth inhibition was observed at concentrations >10- 5mol/L. Leucovorin control cultures showed no inhibition of cell growth. The study demonstrates that methotrexate is effective in inhibiting normal trophoblastic cell culture growth in vitro at concentrations 1000 times those reported necessary to inhibit deoxyribonucleic acid synthesis in choriocarcinoma cell cultures. (AM J OaSTET

GVNECOL 1986;155:324-9.)

Key words: Methotrexate, trophoblast, tissue culture, and heG

The drug methotrexate (4-amino-4-deoxy-N 'O-methyl folic acid) is a tetrahydrofolate reductase inhib­itor that blocks the formation of reduced folates nec-

From the Division of Endocrinology and Infertility, Department of Obstetrics and Gynecology, Northwestern University Medical School.

Receivedforpublicatianjanuary23, 1986; revised March 7,1986; accepted April 22, 1986.

Reprint requests: Peter K. Sand, M.D., 2880 Atlantic Ave., Suite 130, Long Beach, CA 90806.

324

essary for the transfer of one-carbon molecules. They are important in the biosynthesis of thymidylic acid and inosinic acid. Methotrexate, while S-phase specific in its inhibition of deoxyribonucleic acid synthesis, also acts throughout the cell cycle to inhibit amino acid inter­conversions. Its effects in vivo and in vitro on malignant trophoblasts have been studied extensively; however, no data are available regarding the effect of metho­trexate on normal trophoblasts in vitro.

In vitro effects of methotrexate on choriocarcinoma