Tell me about the Truth of hCG in Nonpregnant Patients Chul Hoi Jeong, MD., PhD. Assistant professor in OB & GYN Inje University

hCG  a glycoprotein composed of 2 dissimilar subunits :α- and β-subunit, coded by separate genes on separate chromosomes, held together by charge interactions.  the 8 oligosaccharide side chains : comprise approximately 30% of the M.W. of hCG

Structure of Intact hCG Chromosomal 19Chromosomal 6

Structure of hCG-Related Molecules Detected, to Different Extents, by Commercial hCG Immunoassays in Serum and Urine Samples in Pregnancy and Trophoblastic Diseases

Representation of the structures of hCG and related molecules in the placenta, blood, and urine Cole LA Clin Chem 1997

Concentrations of hCG and its metabolites in pregnancy serum The concentration of nonnicked hCG ( ), nicked hCG ( ), free α ( ), and free β ( ) Cole LA Clin Chem 2001; 47:308-15

The 10 common hCG variants in pregnancy, GTD, or other malignancy

Occurence of the 10 Common Variants of hCGβ in Serum and Urine Sample Cole LA, et al. J Reprod Med 2006

Terminology to described assays for hCG related biochemical markers

Ability of Common Brands of Professional Laboratory Serum hCG Assays to Measure hCG Metabolic Products Commonly Found in Individuals with Gestational Trophoblastic Diseases

Commonly identified epitopes on hCG

Commonly identified antibody binding sites (epitopes) on hCG, its free subunits, and degradation products

False positive hCG results in 9 common commercial immunoassays 106 women have been referred to the USA hCG Reference Service for investigating potential false positive hCG results 44 women were shown to have had false positive results

False positive hCG results in 9 common commercial immunoassays

Persistent low level hCG outside of pregnancy

Causes of persistent low level hCG outside of pregnancy (n=170) Khanlian SA, Cole LA. J Reprod Med 2006;51:812–818 (10%) ( 41%) (42%) ( 7.6%)

Causes of false-positive HCG results

Schematic representation of a sandwich assay detecting human chorionic gonadotropin (hCG)

Schematic representation of a false-positive assay caused by the presence of heterophilic antibodies

The criteria of false-positive hCG results 1. The finding of more than 5-fold differences in serum hCG results with alternative immunoassays (essential criterion) 2. The presence of hCG in serum and absence of detectable hCG or hCG related molecule in urine (essential criterion) 3. The observation of false positive results in other tests for molecules not normally present in serum, such as urine β-core fragment (essential criterion) 4. The finding that a heterophilic antibody blocking agent (Scantibodies Inc. HBR) prevented or limited false detection (supporting criterion) 5. The finding that dilution of samples 2- and 10-fold does not diluted results close to 2- and 10- fold (supporting criterion)

 GTN - active GTN, choriocarcinoma - quiescent GTN  placental site trophoblastic tumors (PSTTs) Trophoblastic causes of low level hCG

Active GTN, Choriocarcinoma  the major proportion of total hCG forms produced at trophoblast invasion : Hyperglycosylated hCG (hCG-H) use to absolutely discriminate active disease requiring therapy (L.A. Cole et al. Gynecol Oncol 2006; 102, L.A. Cole Placenta 2007; 28)

Sites of attachment of oligosaccharides on hCG αβ dimer L.A. Cole Placenta 28 (2007)

Principal structures of O- and N-oligosaccharides attached to regular hCG and hCG-H L.A. Cole Placenta 28 (2007)

 functions poorly in this hormonal capacity  is produced in invasive conditions; i.e. - by the cytotrophoblast cells that invade the decidua at implantation of pregnancy - involved in choriocarcinoma invasion  promote cytotrophoblast and chorioca. cell growth & invasion  a virtual absolute marker of ongoing invasion or malignancy in these cancers and in early pregnancy Hyperglycosylated hCG (hCG-H) L.A. Cole et al. Gynecol Oncol 102 (2006) 145–150

The proportion of total hCG immunoreactivity in urine samples due to hCG-H in early pregnancy L.A. Cole Placenta 28 (2007)

Use of total hCG and hCG-H(%) (hCG-H as a proportion of total hCG) to discriminate gestational trophoblastic diseases L.A. Cole et al. Gynecol Oncol 102 (2006) 151–159 a Measuring hCG, no significant difference is observed between quiescent gestational trophoblastic disease or self-resolving hydatidiform mole cases (control categories) and the “early” choriocarcinoma/GTN cases (P > 0.05). Measuring hCG-H(%), a significant difference is observed (P < and P < ).

Use of hCG and hCG-H(%) tests for differentiating Quiescent GTD from active malignancy and for differentiating self-resolving H-mole (Mole-SR) from active malignancy L.A. Cole et al. Gynecol Oncol 102 (2006) 151–159

Quiescent GTN  constant, low level of hCG, without evidence of a primary or metastatic malignancy  premalignant state (the differentiated syncytiotrophoblast cells) : hold the potential to transform into active GTN  no respond well to chemotherapy or Surgery  continuous low serum hCG results, persisting for periods ranging from 3 months to 16 years in the absence of hCG-H  need to followed with frequent hCG levels - if the hCG level is rising, confirm by measuring hCG-H

Quiescent GTN case with persistent low levels of serum hCG

1. Clinicians should refrain from treating quiescent GTD cases with chemotherapy, hysterectomy, or other surgery. Therapy should probably be limited to cases with at least two consecutive, rising hCG titers 2. Quiescent GTD cases may develop into a malignancy. this is indicated by an abrupt rise in hCG titers months or years after the detection of persistent low-level hCG titers. 3. Quiescent GTD appears to be a premalignant condition for GTN, choriocarcinoma, or PSTT - rigorous (monthly) monitoring with serum or urine hCG measurements is recommended. - measurement of ITA supplements hCG determinations in the management of quiescent GTD Guidelines for the management and monitoring of Quiescent GTD Khanlian SA, et al Am J Obstet Gynecol 2003

Placenta site trophoblastic tumor ( PSTT)  amenorrhea or irregular vaginal bleeding - often remotely following a normal pregnancy, spontaneous abortion, or hydatidiform mole  The interval between the occurrence of PSTT and the antecedent gestational event: long compared with choriocarcinoma &other GTN The mean interval: 3.4 years (2 ~ 5 years)  significantly lower hCG levels than choriocarcinoma - < 200 mIU/ml that fail to rise sharply over time

 the accuracy of the initial pathologic: limited by the small amounts of tissue obtained by endometrial curettage  the definitive diagnosis: difficult to achieve short of performing a hysterectomy  lacks this tendency for early and widespread vascular invasion  The hCG subunits produce in insufficient concentrations to produce αβ dimers leading to hCG-free β-subunit production high proportions of β-core fragment in urine  predominant form of hCG; free β subunit- useful marker (L.A. Cole et al. Gynecol Oncol 2006; 102) Placenta site trophoblastic tumor (PSTT)

Pituitary hCG  in 1987, hCG was demonstrated to be secreted in the serum of normal men and women in a pulsatile fashion that paralleled the secretion of LH (Odell WD, et al. N Engl J Med 1987)  in these nonpregnant individuals: suppressed by estrogen and progestin therapy (Stenman UH, et al. J Clin Endocrinol Metab 1987) suppressed by OCPs: establishing the diagnosis of pituitary hCG  identification of cells in the anterior pituitary which secrete hCG ( Hammond E, et al. J Clin Endocrinol Metab 1991)  identification of β–hCG mRNA in the pituitary gland (van Strien A, et al. Nucleic Acids Res 1989)

 higher levels of hCG in postmenopausal women than nonpregnant premenopausal women because of increased GnRH secretion and pituitary response - appropriate cutoffs for a “negative hCG” premenopausal women: 5 IU/L postmenopausal women:14 IU/L (Snyder JA, et al. Clin Chem 2005)  If peri- or post-menopause or BSO, with low level hCG, should take HRT or oral contraceptives after 2–3 weeks, suppress hCG production if pituitary origin Pituitary hCG

Nongynecologic tumors with positive serum β-hCG levels

rule out pregnancy and ectopic pregnancy determine if the hCG is biologically real  False-positive hCG - 1. the presence of hCG immunoreactivity in serumbut not urine - 2. varying hCG results (more than 5-fold) or negative results in 3 or more hCG tests - 3. the suppression of result by a heterophilic antibody blocking agent determine if active GTN, PSTT, or non-trophoblastic malignancy is present  hCG-H is detectable, >1 ng/ml: active GTN/choriocarcinoma  hCGfree β-subunit is more than one third of hCG: PSTT or non-trophoblastic malignancy  hCG-H(-) or no significant hCG free β-subunit: quiescent GTD  peri- or post-menopause or BSO: pituitary hCG L.A. Cole et al Gynecol Oncol 102 (2006) Guidelines for the management of patients with persistent low level hCG

Thank you for your attention!