Laurence A. Cole

False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations

BACKGROUND 12 women were diagnosed of having postgestational choriocarcinoma on the basis of persistently positive human chorionic gonadotropin (hCG) test results in the absence of pregnancy. Most of the women had extirpative surgery or chemotherapy, or both, without significant diminution in hCG titre. Our aim was to assess whether the hCG concentrations were false-positive test results. METHODS Samples were tested for hCG, hCG free beta subunit, and hCG beta-core fragment. Assay kinetics were also assessed, and samples were tested independently by competitive RIA. False-positive hCG concentrations were identified by two criteria: detection of hCG in serum and lack of detection of hCG and its degradation products in urine; and wide variations in results for different hCG assays. We corroborated false-positive hCG values by the lack of parallel changes in hCG results when serum was diluted, by false detection of other antigens, and by failure to detect hCG with in-house assays. FINDINGS All 12 women met both criteria for false-positive hCG, and all had corroborating findings. In all 12 cases, a false diagnosis had been made, and most of the women had been subjected to needless surgery or chemotherapy. Assay kinetics indicated that heterophilic antibodies were responsible for the false-positive results. As a result of our findings all further therapy was stopped. INTERPRETATION Current protocols for the diagnosis and treatment of choriocarcinoma should be modified to include a compulsory test for hCG in urine.

Carbohydrate and peptide structure of the α- and β-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the “nicking” of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG α- and β-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease.Peptide-bond nicking was restricted to a single linkage (β47–48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma β-subunit. In the carbohydrate moiety, α-subunit from normal pregnancy hCG contained non-fucosylated, mono-and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma α-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P<0.0001). The β-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in β-subunit from normal pregnancy, disaccharidecore structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in β-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidi-form mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy.Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or “hyperbranched” glycoforms on both α- and β-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, α1,6fucosyltransferase andN-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.

Biological functions of hCG and hCG-related molecules

BackgroundhCG is a term referring to 4 independent molecules, each produced by separate cells and each having completely separate functions. These are hCG produced by villous syncytiotrophoblast cells, hyperglycosylated hCG produced by cytotrophoblast cells, free beta-subunit made by multiple primary non-trophoblastic malignancies, and pituitary hCG made by the gonadotrope cells of the anterior pituitary.Results and discussionhCG has numerous functions. hCG promotes progesterone production by corpus luteal cells; promotes angiogenesis in uterine vasculature; promoted the fusion of cytotrophoblast cell and differentiation to make syncytiotrophoblast cells; causes the blockage of any immune or macrophage action by mother on foreign invading placental cells; causes uterine growth parallel to fetal growth; suppresses any myometrial contractions during the course of pregnancy; causes growth and differentiation of the umbilical cord; signals the endometrium about forthcoming implantation; acts on receptor in mothers brain causing hyperemesis gravidarum, and seemingly promotes growth of fetal organs during pregnancy.Hyperglycosylated hCG functions to promote growth of cytotrophoblast cells and invasion by these cells, as occurs in implantation of pregnancy, and growth and invasion by choriocarcinoma cells. hCG free beta-subunit is produced by numerous non-trophoblastic malignancies of different primaries. The detection of free beta-subunit in these malignancies is generally considered a sign of poor prognosis. The free beta-subunit blocks apoptosis in cancer cells and promotes the growth and malignancy of the cancer. Pituitary hCG is a sulfated variant of hCG produced at low levels during the menstrual cycle. Pituitary hCG seems to mimic luteinizing hormone actions during the menstrual cycle.

New discoveries on the biology and detection of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG.For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis.While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy.New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic disease, diagnosing placental site trophoblastic tumor, managing testicular germ cell malignancies, and monitoring other human malignancies. There are very few molecules with such wide and varying functions as regular hCG and its variants, and very few tests with such a wide spectrum of clinical applications as total hCG.

hCG, the wonder of today's science

BackgroundhCG is a wonder. Firstly, because hCG is such an extreme molecule. hCG is the most acidic glycoprotein containing the highest proportion of sugars. Secondly, hCG exists in 5 common forms. Finally, it has so many functions ranging from control of human pregnancy to human cancer. This review examines these molecules in detail.ContentThese 5 molecules, hCG, sulfated hCG, hyperglycosylated hCG, hCG free beta and hyperglycosylated free beta are produced by placental syncytiotrophoblast cells and pituitary gonadotrope cells (group 1), and by placental cytotrophoblast cells and human malignancies (group 2). Group 1 molecules are both hormones that act on the hCG/LH receptor. These molecules are central to human menstrual cycle and human pregnancy. Group 2 molecules are autocrines, that act by antagonizing a TGF beta receptor. These molecules are critical to all advanced malignancies.ConclusionsThe hCG groups are molecules critical to both the molecules of pregnancy or human life, and to the advancement of cancer, or human death.

The normal variabilities of the menstrual cycle

OBJECTIVE To address conflicts in the normal variabilities of the menstrual cycle using the newest generation test methods and to establish normal ranges for use in clinical practice. DESIGN Daily urine samples were collected from 167 women eager to achieve pregnancy. Samples were tested prospectively for LH and total hCG. A total of 458 nongestational and 111 gestational menstrual cycles were evaluated. SETTING Division of Womens Health Research, University of New Mexico. PATIENT(S) One hundred sixty-seven women desiring pregnancy. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Levels of hCG and LH. RESULT(S) Menstrual cycles were 27.7 +/- 2.4 days in length. The LH peak indicated the onset of the presumed ovulatory window, which occurs at 14.7 +/- 2.4 days. Implantation (first day of sensitive detection of hCG) occurred in gestational menstrual cycles at 24.6 +/- 3.1 days, or 4.3 +/- 2.2 days before missing the expected onset of menses. CONCLUSION(S) Our data confirm epidemiological studies on menstrual cycle length and variability and hormonal studies on timing of the ovulatory window and its variability. They dispute, however, the published data on the timing and variance of implantation. As shown, implantation is limited to a 10-day interval culminating in the day of the expected onset of menses. Reference range data provide guidelines for differentiating normal and problem menstrual cycles.

Hyperglycosylated hCG (invasive trophoblast antigen, ITA) a key antigen for early pregnancy detection

OBJECTIVES Hyperglycosylated human chorionic gonadotrophin (hCG) is an hCG variant with extra-large O-linked oligosaccharides, produced by phenotypically invasive cytotrophoblast cells in choriocarcinoma and pregnancy. It is the principal form of hCG produced in the first weeks of gestation. We investigated the importance of hyperglycosylated hCG in pregnancy testing and its detection by current hCG tests. DESIGN AND METHODS We measured the concentration of hyperglycosylated hCG and total hCG in 512 pregnancies throughout gestation. We assessed and compared the abilities of 14 commonly used commercial laboratory hCG tests and 18 home pregnancy tests to detect regular and hyperglycosylated hCG. RESULTS Hyperglycosylated hCG is the principal source of hCG-related immunoreactivity in early pregnancy. In the week following missing menses, hyperglycosylated hCG measurements may be more sensitive than regular hCG measurements in detecting pregnancy. Of 14 commercial laboratory hCG tests, 3 appropriately detected hyperglycosylated hCG standard. Of 18 different home pregnancy products 11 poorly or very poorly detected this key antigen. CONCLUSIONS Hyperglycosylated hCG may be the key molecule in the detection of early pregnancy. However, the majority of tests poorly detected or failed to detect this key antigen. New pregnancy tests are needed that either solely detect hyperglycosylated hCG or equally detect regular hCG and hyperglycosylated hCG.

Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient: generation of antibodies that differentiate between pregnancy hCG and choriocarcinoma hCG.

Human chorionic gonadotropin (hCG) exists in blood and urine as a variety of isoforms one of which contains peptide bond cleavages within its β-subunit loop 2 and is referred to as nicked hCG (hCGn). This hCG isoform appears to be more prevalent in the urine of patients with certain malignancies and possibly in some disorders of pregnancy. Until now, only indirect immunoassays could be used to quantify hCGn. We report the development of two monoclonal antibodies (MAbs) to a form of hCGn isolated from a choriocarcinoma patient. This hCG isoform was not only 100% nicked, but also contained 100% tetrasaccharide-core O-linked carbohydrate moieties in its β COOH-terminal region. Two-site immunometric assays have been developedusing these new antibodies, B151 and B152. The former exhibits good specificity for hCGn independent of the source of the hCGn, the form excreted by choriocarcinoma patients or the form of hCGn from normal pregnancies. The latter antibody, B152, is sensitive to the carbohydrate moieties and possibly other differences in hCG isoforms, but is not for nicking of the β-subunit. These two immunometric assays provide potential novel diagnostic tools for direct measurement of hCG isoforms which could not be accurately quantified earlier before development of the assays using these newly generated antibodies.

Hyperglycosylated hCG, a review

Hyperglycosylated hCG (hCG-H) is a glycoprotein with the same polypeptide structure as hCG, and much larger N- and O-linked oligosaccharides. The oligosaccharides increase the molecular weight of hCG from 36,000 - 37,000 u to 40,000 - 41,000 u, depending on the extent of hyperglycosylation. hCG-H has triantennary N-linked oligosaccharides and double molecular size O-linked oligosaccharides (hexasaccharide compared with predominantly trisaccharide structures). hCG is produced by syncytiotrophoblast cells while hCG-H is made by extravillous cytotrophoblast cells. hCG-H promotes trophoblast invasion during choriocarcinoma, growth of cytotrophoblast cells and placental implantation in pregnancy. hCG-H is an independent molecule to hCG with totally separate biological functions. hCG has numerous functions during pregnancy, it promotes progesterone production, promotes angiogenesis in uterine vasculature, immuno-suppresses the invading placental tissue, promotes the growth of the uterus in line with the growth of the fetus during pregnancy, promotes the differentiation of growing cytotrophoblast cells, promotes the quiescence of contractions in the uterine myometrium during the course of pregnancy, and also has function in growth and development of fetal organs. Monoclonal antibody B152 uniquely binds hCG-H. Using this monoclonal antibody in immunometric assays permits detection of pregnancy. It also permits management of gestational trophoblastic diseases and detection of quiescent gestational trophoblastic disease. This same test can be used to differentiate of aggressive and minimally-aggressive gestational trophoblastic disease, and discrimination of patients that respond to chemotherapy and who are chemorefractory. The hCG-H test can be used to screen for Down syndrome pregnancies and predict patients likely to generate hypertensive disorder in pregnancy. It also can be used to differentiate pregnancies that will miscarry and pregnancies that will go to term.

Hyperglycosylated hCG, a potential alternative to hCG in Down syndrome screening

Hyperglycosylated hCG (H‐hCG) is a minor variant of hCG with abnormal oligosaccharide side chains. It is the principal gonadotropin detected in the serum and urine of patients with gestational choriocarcinoma. A monoclonal antibody was produced against this antigen and an immunoassay developed. Levels of hCG and H‐hCG were determined in 142 urine samples from normal pregnancies from 10 to 21 weeks of gestation. Levels were normalized to urine creatinine concentration, and were each plotted against gestational age. Bi‐weekly median values were calculated, the best‐fitting regression lines were determined, and multiples of the normal median (MoM) were computed.