Persistent low-level "real" human chorionic gonadotropin: a clinical challenge and a therapeutic dilemma.

OBJECTIVE: The finding of persistent low-level human chorionic gonadotropin (hCG) with or without a preceding pregnancy event presents a rare but clinically important challenge and a therapeutic dilemma. These are patients with "real" hCG shown by the positive test in both serum and urine or by specialized testing. The problems associated with "phantom" hCG have been recognized and should now be clinically resolvable. Four cases of low-level "real" hCG are described to illustrate the problems encountered, the management, and the resolution achieved. METHODS: Two patients presented with persistent low-level hCG after hydatidiform mole pregnancy, one after an early pregnancy loss and one as amenorrhea and irregular bleeding. A detailed clinical description is provided to illustrate the difficulties encountered. RESULTS: All patients have real hCG. The hCG level of Patient 1 was responsive to hormonal contraception and disappeared with such medication. Over a period of 3 years hCG reappeared whenever estrogen was stopped. Patient 2 achieved two pregnancies and the hCG subsequently disappeared. The hCG in Patient 3 persisted over a period of 6 years although she is now menopausal. Patient 4 developed metastatic placental site trophoblastic tumor after 2 1/2 years of observation of low-level hCG. CONCLUSIONS: The finding of unexplainable low-level hCG in a patient without evidence of a uterine lesion or of trophoblastic metastases provides a therapeutic challenge. The administration of single-agent chemotherapy had no effect on the level of hCG in the three patients to whom it was administered. The administration of multiple-agent chemotherapy appears unjustified in the absence of a demonstrable trophoblastic tumor. A small number of trophoblastic cells must be providing this hCG and these cells may be quiescent for years. Nevertheless these cells may proliferate and manifest themselves as trophoblastic tumor. Continuing long-term surveillance of these patients is necessary.

Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented.

Early pregnancy human chorionic gonadotropin (hCG) isoforms measured by an immunometric assay for choriocarcinoma-like hCG.

Human chorionic gonadotropin (hCG) exhibits molecular heterogeneity in both its protein and carbohydrate moieties. This communication describes changes in hCG isoforms detected directly in clinical samples. These isoforms, quantified in blood or urine specimens, show a progression of change throughout normal pregnancy. Early pregnancy produces a type of hCG that resembles, in terms of immunoreactivity, a major form of hCG excreted in choriocarcinoma. The isoforms predominate for the first 5-6 weeks of gestation and then diminish, being replaced with the hCG isoforms which predominate throughout the remainder of pregnancy. The alteration in hCG isoform content occurs in both blood and urine. The progression of isoforms is best delineated by calculating the change in the ratio of the two forms, as many hCG assays either do not detect or fail to discriminate among these isoforms. An analogous pattern of hCG isoforms was observed in patients with in vitro fertilization pregnancies. hCG isolated from the pituitary displayed binding characteristics similar to those of the hCG derived from normal pregnancy urine. The early pregnancy hCG isoforms appear to have a differential expression in normal pregnancy as opposed to pregnancies which will not carry to term, suggesting that a determination of the relative balance of hCG isoforms may have diagnostic application in predicting pregnancy outcome.

Molecular heterogeneity of hCGbeta--related glycoproteins and the clinical relevance in trophoblastic and non-trophoblastic tumors.

We analyzed immunoreactive hCG/hCGbeta (IR-beta) in the sera and urine of patients with trophoblastic diseases and non-trophoblastic tumors by using enzyme immunoassays (EIAs) specific for intact hCG, free hCG beta, and beta-core fragment of hCG (beta-CF). In trophoblastic diseases, while intact hCG and free hCGbeta were contained in both serum and urine, the beta-CF could be detected only in the urine of the patients. The relative contribution of the beta-CF to the total urinary IR-beta accounted for about 30-50% in normal early pregnancy and hydatidiform mole, and more than 60% in choriocarcinoma. We conclude that intact hCG should be measured in the serum rather than in the urine as a tumor marker for trophoblastic diseases, and suggested that the ratios of intact hCG, free hCGbeta, and beta-CF to each other may be useful indices in the differential diagnosis of trophoblastic diseases. Ectopic IR-beta was also investigated in the sera and urine of the patients with cervical, endometrial, ovarian, lung, and bladder carcinomas. We found that even when IR-beta could not be detected in the serum, the urine of the same patients with cancer often contained the significant amounts of IR-beta. The chromatographic study indicated that these urinary IR-beta were essentially attributed to beta-CF, leading to the evaluation of urinary beta-CF as a tumor marker. The positive rated of urinary beta-CF were 48% for cervical, 38% for endometrial, and 84% for ovarian, 40% for lung, and 42% for bladder carcinomas. We conclude that ectopic production of hCG beta by non-trophoblastic tumors is not a rare phenomenon and it can be recognized as a tumor marker when beta -CF is measured in urine of the patients.

hCG, its free subunits and its metabolites. Roles in pregnancy and trophoblastic disease.

OBJECTIVE: To examine the structure and metabolism of human chorionic gonadotropin (hCG) and the effect of molecular heterogeneity on the immunodiagnosis and monitoring of gestational trophoblastic disease. STUDY DESIGN: A review of the current medical literature concerning measurement of hCG and hCG-related molecules. RESULTS: hCG molecules in gestational trophoblastic disease are more heterogeneous or degraded in serum and urine samples than in normal pregnancy. CONCLUSION: Appropriate measurement and monitoring of hCG levels in gestational trophoblastic disease require an understanding of hCG structure and metabolism.

[Urinary gonadotropin fragment measurement in the monitoring of trophoblastic disease].

Urinary gonadotropin fragment (UGF) is a small peptide which is present in the urine of pregnant women and of women with trophoblastic diseases as well as with certain nontrophoblastic malignancies. 275 samples each of urine and blood from 46 patients with trophoblastic diseases were taken for UGF and hCG measurements and compared. 24 samples from 12 healthy, nonpregnant women were taken as control. Cut-off values of UGF and hCG used for measuring the sensitivity of trophoblastic diseases were respectively > 0.2 microgram/L and above 20 micrograms/L. It was found that 64.0% of the urine samples gave UGF values > 0.2 microgram/L and 66.5% of the blood samples showed hCG levels above 20 micrograms/L (P > 0.1). No false-positive rate was observed in the control group. However, among patients who were found to have low or negative hCG values, 57.6% showed positive UGF levels. These findings suggest that in patients with positive levels of both UGF and hCG, the UGF measurement may not be necessary. But for patients with low or negative blood hCG values, certain percentage of urine UGF could still be detected.

Detecting and monitoring trophoblastic disease. New perspectives on measuring human chorionic gonadotropin levels.

The human chorionic gonadotropin (hCG) molecules in trophoblastic disease serum and urine samples are more heterogeneous, or degraded, than those in pregnancy samples. HCG immunoassays, particularly some of the new multiantibody sandwich tests, are designed primarily for pregnancy application and do not necessarily detect the degraded molecules found in trophoblastic disease samples. This leads to erroneous results and possibly false diagnoses. Care is needed in choosing the hCG test for monitoring trophoblastic disease.

Separation of nicked human chorionic gonadotropin (hCG), intact hCG, and hCG beta fragment from standard reference preparations and raw urine samples.

hCG is found in pregnancy urine and in urine from some cancer patients in a variety of forms whose concentrations have clinical importance. Recently, concerns about accurate measurement of these forms have been raised because of the finding that hCG with peptide bond cleavages within the beta-subunit is not recognized by commonly used antibodies. Such nicked forms of hCG are biologically inactive or of very low activity. They are present in normal pregnancy urine and to varying extents in the urine of patients with trophoblastic disease. International reference preparations of hCG contain nicked forms of hCG. Previously, it was not possible to separate nicked hormone from the intact form of hCG. This was a serious impediment to producing improved reference standards from natural pregnancy hormone. We now report that a simple hydrophobic purification scheme separates intact hCG from nicked hCG as well as from hCG beta core fragment. This scheme is a modification of the method of Hiyama et al. The order of elution from low to high hydrophobicity is hCG beta core fragment, nicked hCG, and lastly, intact hCG. Nicking of the putative amphipathic helix loop, hCG beta 38-57, apparently renders the hormone significantly less hydrophobic despite the equal molar content of sialic acid. The hCG CR 127 nicked preparation was only 10% as potent as the reference preparation in a heterodimer-directed assay. The nicked-depleted hCG CR 127 was 30% more potent in this assay. Improved hCG reference standards should display similar increases in immunopotency (20-30%) with most antiheterodimeric antibodies and similar increases in bio-potency assays. It should now be possible to make reference preparations of these forms of hCG directly from the raw urine of normal pregnant patients and those with trophoblastic disease.

Immunosuppressive 30-kDa protein in urine of pregnant women and patients with trophoblastic diseases.

Urine samples obtained from normal pregnant women and patients with trophoblastic diseases contain 30-kDa protein that suppresses phytohemagglutinin-induced T cell proliferation. The immunosuppressive protein was measured by a newly developed radioimmunoassay. The 30-kDa protein was demonstrated in almost all urine samples examined, fluid from hydatid vesicles and chorionic extracts, but not in any serum samples except at low levels in some sera from patients with choriocarcinoma. During pregnancy, the level of urinary 30-kDa protein was higher in the first (1625.5 +/- 1212.0 ng/ml, mean +/- S.D.) and second (1457.4 +/- 1332.4 ng/ml) trimesters than in the third trimester (460.6 +/- 419.0 ng/ml). The urinary 30-kDa protein/hCG ratios in patients with choriocarcinoma (8.3 +/- 10.9) were significantly higher than those in patients with hydatidiform mole (0.67 +/- 1.00, P < 0.01) and in all trimesters than those of normal pregnant women (0.54 +/- 0.44 in first trimester, P < 0.05; 0.63 +/- 0.46 in the second trimester, P < 0.05; 0.24 +/- 0.17 in the third trimester, P < 0.01). There is no significant difference between the ratios in hydatidiform mole and normal pregnancy. These findings and the fast disappearance of the 30-kDa protein from the circulation suggest that the 30-kDa protein plays a part in proliferation of trophoblastic cells in, or their invasion into the host by locally suppressing the immune reaction of the host and that the increase in the urinary 30-kDa protein level, in cases of choriocarcinoma, may be due to the malignant transformation of trophoblastic cells resulting in their rapid invasion.

The biological and clinical significance of nicks in human chorionic gonadotropin and its free beta-subunit.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits, alpha and beta. Nicks or missing peptide linkages have been found in the beta 44-52 region of the beta-subunit of hCG, whether from pregnancy or trophoblast disease. This article reviews recent reports about the location of nicks in hCG, their origin and occurrence, their effects on the steroidogenic and receptor-binding activities of hCG, and on the immunological activities of hCG and its free beta-subunit. Taken together, the reports show: (1) nicks occur primarily between beta 47 and beta 48, and to a lesser extent between beta 44 and beta 45; (2) the extent of nicking in hCG samples varies widely, from undetectable to 100 percent of molecules; (3) nicks greatly reduce the steroidogenic activity of hCG in vitro (nicked molecules have less than 20 percent of the activity of the intact hormone); (4) nicks may occur at the trophoblast-myometrial interface or in the circulation by the action of human leucocyte elastase or similar leucocytic protease; (5) hCG testing kits using dimer-specific antibodies may not detect nicked molecules and may give different results from those using other antibodies; (6) hCG international reference preparations and the CR series of hCG standards are variably nicked (10 percent to 20 percent), complicating the problem of discordant hCG results in nick-sensitive assays; (7) results from commonly used immunoassays for measurement of the hCG free beta-subunit vary by as much as tenfold because some of the antibodies employed do not detect nick free beta-subunit.

Experience of human chorionic gonadotropin assays in gestational trophoblastic tumors.

From May 1979 through December 1988, 146 patients with gestational trophoblastic tumors (71 hydatidiform mole, 3 partial mole, 15 choriocarcinoma and 57 persistent trophoblastic tumors) were studied. A total of 1178 daily urine samples were collected before and/or after treatment, and in the course of follow-up. H93 RIA (an HCG specific assay), H80 RIA (an assay detecting hCG and hLH) and a hCG alpha assay measured levels in the urine specimens. Three hCG declining patterns (pattern D, P and R) based on the H93 RIA assay were noted. Patients showing pattern D had the most favorable outcome (no mortality at all). However, pattern P and R had a 10% and 14.3% mortality rate, respectively. The ratios of H80/H93, hCG alpha/H93, hCG alpha/H80 in the urine specimens were similar in both pattern D and R (excluding samples from a patient who expired later). However, the ratios of H80/H93, hCG alpha/H93, hCG alpha/H80 of samples from the patient (CK) who expired later were significantly different from those of the pattern D and R. This was suggestive of a marked unbalanced secretion of hCG and its subunit in the urine specimens of patient CK. The molecular forms in pattern D were similar to the standard hCG. However, the molecular form in pattern R of 3 fatal choriocarcinomas showed a great variation, from smaller to larger than the standard hCG. The isoelectric points of hCG in pattern D and R were all acidic. In clinical practice, we can measure the ratios of H80/H93, hCG alpha/H93 and hCG alpha/H80, molecular forms, and isoelectric points of hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

[The serum concentration of specific beta 1-glycoprotein in trophoblastic disease]. / Prouchvaniia na serumnata kontsentratsiia na spetsifichniia beta 1-glikoprotein pri trofoblastna bolest.

Twelve women with hydatidiform mole were studied between 12 and 20 weeks-gestation. The level of SP1 was determined by radioimmunologic method. The author found statistically significant increase of SP1 over 90th percentile of gestational age (p less than 0.001). Examining the level of SP1 after evacuation of hydatidiform mole it was established that SP1 was eliminated more quickly than human chorionic gonadotropin (HCG) in urine during early pregnancy. Quite the contrary was discovered in women with advanced pregnancy, in whom the concentration of SP1 disappeared more slowly, while HCG was eliminated for a shorter time. The observed dissociation in elimination of SP1 and HCG showed that the combined examination of HCG and SP1 provided better information in the follow-up of women with trophoblastic disease. The author think that treatment of trophoblastic disease should continue till complete elimination both of HCG and SP1.

Structural changes induced in the sugar chains of glycoproteins by malignant transformation of producing cells and their clinical application.

Altered glycosylation is widely observed in glycoproteins produced by tumors. One of the most consistently observed alterations is the increase of larger asparagine-linked sugar chains in the plasma membrane glycoproteins. This phenomenon is brought about by the increase of N-acetylglucosaminyltransferase V, which is responsible for the formation of the GlcNAc beta 1----6Man alpha-1----6 group. The enrichment of the complex-type sugar chains containing the -GlcNAc beta 1----6(-GlcNAc beta 1----2)Man alpha 1----6 group is correlated with tumorigenicity and metastasic potential of tumor cells. Comparative study of the sugar chains of human chorionic gonadotropin isolated from the urine of pregnant women and of patients with trophoblastic diseases including choriocarcinoma revealed that many new oligosaccharides are included in the tumor hCG. The altered glycosylation of hCG is brought about by the ectopic expression of N-acetylglucosaminyltransferase IV. With use of this altered glycosylation, a novel method useful for the diagnosis of choriocarcinoma was established.

[Molecular weight and fragmentation of human chorionic gonadotropin in urine in patients with trophoblastic diseases].

Molecular weights (MW) of urinary human chorionic gonadotropin (hCG) from trophoblastic diseases were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting using specific antibodies. Although the MW of hCG beta from hydatidiform mole and invasive mole were identical to that of standard hCG beta, those from choriocarcinoma were individually different. These results indicate that the structural changes of carbohydrates, which may be induced by malignant transformation of trophoblast, can be reflected as differences in MW. In SDS-PAGE under nonreducing conditions. Western blotting using anti-hCG beta-carboxy-terminal peptides (CTP) revealed a single band corresponding to hCG beta. Under reducing conditions with dithiothreitol (DTT), however, a second low MW material (CTP') could be observed with anti-hCG beta-CTP. All hCG samples from invasive mole and choriocarcinoma were much more susceptible than standard hCG to release of CTP' by DTT. Estimation of MW and susceptibility to DTT reducing of urinary hCG by Western blotting are useful in the diagnosis of invasive mole and choriocarcinoma.

[Biochemical study on the thyrotropic activity in urinary immunoreactive-human chorionic gonadotropin of patients with trophoblastic disease].

We have studied the biochemical properties of urinary immunoreactive-human Chorionic Gonadotropin (IR-hCG) using the human thyroid gland in vitro slice method to investigate the thyrotropic activity in purified IR-hCG of patients with thyroid hyperfunction associated with trophoblastic disease. IR-hCG was extracted using a kaolin-acetone-alcohol concentration and purified by DEAE Cellulose, Sephadex G-100 and DEAE Sephacel column chromatographies according to Nishimura et al. with slight modifications. The substance with thyrotropic activity (hCT) was found in 3 cases of trophoblastic disease with hyperthyroidism among the 14 cases investigated. HCT consists of two dissimilar subunits, namely, hCT alpha-subunit and hCT beta-subunit like hCG, whose molecular weights are estimated at about 15,000 and 20,000 respectively and have carbohydrate moiety bound to concanavalin A. The thyrotropic activity was shown by the recombinant hybrid molecules, hCT beta-subunit and hCG alpha-subunit, whereas no potency was found in hCT beta-subunit only. The carboxyterminal residues of hCT (Ile-Leu-Pro-Glu) were not digested by carboxypeptidases Band Y. These results suggest that hCT is an intrinsic activity of hCG and cross-reacts immunologically, while the amino acid residues of the carboxyterminus of the hCT beta-subunit are different from those of the hCG beta-subunit.

Detection of desialylated forms of human chorionic gonadotropin.

Urinary forms of human chorionic gonadotropin (hCG) with oligosaccharides deficient in sialic acid content (ashCG) have been reported to be excreted by patients with choriocarcinoma in greater amounts than by healthy, pregnant women. Although ashCG potentially could be a useful marker for the diagnosis and management of gestational trophoblastic neoplasia, the methods previously used for its detection were not suitable for routine clinical application. Therefore, we have devised a simpler method which can provide specific and sensitive measurements of ashCG in urine. This method, which is designated as a lectin-immunoradiometric assay (LIRMA), employs an agarose-coupled lectin to selectively extract the ashCG, which is then quantified directly with a purified and radiolabelled rabbit antibody. The LIRMA has been applied to demonstrate that there is an increased excretion of ashCG by choriocarcinoma patients. It is also applicable, in principle, for the study of any glycoprotein which has a reduced content of sialic acid in its carbohydrate side chains.

[Lectin immunoradiometric assay versus improved radioimmunoassay: a comparison of methods for determination of desialylated forms of human chorionic gonadotropin].

Two different assay systems have been developed for the measurement of asialo hCG(ashCG). One of them is an immunoradiometric assay which utilizes peanut lectin (PNA) to selectively extract ashCG and then directly quantify it with purified radiolabeled antiserum which is generated against carboxy terminal peptide of hCG beta (beta-CTP). PNA is highly specific for the terminal carbohydrate linkage Gal beta-(1----3)-Gal NAc which is only exposed to hCG when the O-serine linked oligosaccharide of its unique beta-CTP region is deficient in sialic acid. The other is an improved RIA using a specific antiserum to as beta-CTP, in which extraction of hCG with monoclonal antibody-Sepharose 4B conjugate is used to obtain increased sensitivity with retention of specificity. The ashCG concentration can be measured as low as 0.01 and 0.05 pmoles/ml in the presence of an excess amount of hCG in each of these assays. These assays were applied to detect ashCG in the urine of patients with trophoblastic tumors and normal pregnant women. The proportion of ashCG to total hCG in choriocarcinoma was significantly higher than that in normal pregnancy in both assays. However, the proportion was dependent upon the amount of total hCG and it decreased as total hCG increased.

[Detection of hCG production in trophoblastic diseases by HCG beta carboxyl-terminal peptide].

A radioimmunoassay (CTP-RIA) for urinary human chorionic gonadotropin (hCG) with the use of an antiserum to be carboxyl-terminal peptide of hCG beta subunit was employed to detect hCG production in patients with gestational trophoblastic disease. In urine samples obtained from normal subjects, the upper limit of hCG-immunoactivity detected by this assay system was 1.1 IU/24h. More than 90% of the subjects tested had values lower than 0.5 IU/24h. Based on these data, we selected urinary hCG levels below 1.1 IU/24 h as the normal range for clinical applications. The utility of this new assay system was assessed in 50 cases of gestational trophoblastic disease. In patients with hydatidiform mole, invasive mole and undetermined cases, the urinary hCG level declined to be normal range following the therapy and stayed there afterwards without any sign of recurrence. However, in a woman with a long history of metastatic choriocarcinoma, we noted the reappearance of hCG even after the hCG level once declined to the normal range. It therefore seems that cell viability will persist even in the normal range determined by CTP-RIA. Therefore, therapeutic decisions should take into account these points. This specific and sensitive CTP-RIA method for the detection of hCG production was found to improve the ability to diagnose persistent or recurrent trophoblastic disease.