Free Human Chorionic Gonadotropin β Subunit in Gonadal and

Nongonadal Neoplasms
Isabelle Marcillac, Frédéric Troalen, Jean-Michel Bidart, et al.

Cancer Res 1992;52:3901-3907.

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[CANCER RESEARCH 52, 3901-3907, July 15, 1992]

Free Human Chorionic Gonadotropin ßSubunit in Gonadal and Nongonadal

Neoplasms1
Isabelle Mamilar, FrédéricTroalen, Jean-Michel Bidart, Pascale Giuliani, Vincent Ribrag, Bernard Escudier,
Benoit Malassagne, Jean-Pierre Droz, Catherine I homme, Philippe Rougier, Pierre Duvillard, Michel Prade,
Pierre-Marie Lugagne, FrançoisRichard, Thierry Poynard, Claude Bohuon, Jack Wands, and Dominique Bellet2
Departments of Molecular Immunology [I. M., F. T., J-M. B., P. G., B. M., C. B., D. B.J. Medicine ¡J-P.D., C. L., P. R.]. and Pathology B ¡P.D., M. P.], and the
Unit of Intensive Care [f. R., B. E.], Institut G. Koussy, 38 Rue Camille Desmoulins, 94805 Villejuif, France; Hôpital de la Pitié,Boulevard de l'hôpital, 75013
Paris, France /P-M. L., F. R.]; Hôpital Antoine Béclère,
157 Rue de la Porte de Trivaux, 92140 Claman, France [T. P.J; Department of Immunology, CNRS, URA
1484, Facultéde Pharmacie, 6 Avenue de l'Observatoire, 75006 Paris, France [J-M. B., T. P., D. B./; and the Molecular Hepatology Laboratory, Massachusetts
Cenerai Hospital Cancer Center, Charlestown, Massachusetts 02129 ¡J.H'.]

ABSTRACT by trophoblastic cells in addition to the biologically active hCG

during normal pregnancy (2-4).
The diagnostic value of elevated human chorionic gonadotropi There is an extensive documentation of the presence of either
(hCG) and its free a (hCGa) and .; (hCG/3) subunit serum levels as hCG, hCG-like material, or free hCG subunits in the serum of
specific tumor markers for nongonadal malignancies is controversial. In
the present report, different monoclonal based immunoradiometric as
normal adults as well as in patients with either trophoblastic or
nontrophoblastic tumors (5-13). For example, several studies
says specific for hCG and its free hCGa and hCGtf subunits have been
used to reevaluate the presence of these molecules in the serum of have shown that such molecules may be localized to a variety of
patients with a wide variety of tumors. Serum samples from patients nontrophoblastic tissues and further was extractable from vir
with newly diagnosed, persistent, or recurrent malignancies of either tually all normal and malignant human tissues (14-23). How
known (n = 717) or unknown in = 32) primary site, healthy blood ever, most of these studies were based on the recognition of
donors (n = 309), and nonmalignant disease controls (n = 86) were hCG, hCG-like material, or free hCG subunits by either poly-
studied using four highly specific and sensitive monoclonal based im clonal or monoclonal antibodies that were not totally specific
munoradiometric assays to hCG and its free subunits. Low level hCG for these glycoproteins (24, 25). Consequently, the production
elevations (< 11)0(1pg/ml) were found to be common in cancer patients, of hCG-related molecules by nontrophoblastic cells remained
normal subjects, and disease controls. However, serum levels > 1(100
pg/ml were highly diagnostic of gonadal tumors and specifically iden
controversial and only indirect evidence has been provided that
a hCG-like material was secreted by these cells. Recently, the
tified nonseminomatous testicular tumors. Significant serum elevations
of free hCGor subunit (as high as 3000 pg/ml) were found in approxi utilization of monoclonal antibodies highly specific for hCG
mately 96% of cancer patients, normal individuals, and disease controls. provided direct evidence that this hormone was secreted by the
In contrast, free hCG/3 subunit levels ( •¿
100 pg/ml) were detected in 70 normal human pituitary and was present in the serum of normal
and 50% of patients with nonseminomatous and seminomatous testicu adults (26-29). Moreover, monoclonal antibodies specific for
lar cancers, respectively, and in 47% of bladder, 32% of pancreatic, and either the free hCGa or hCG/3 subunit have proved to be useful
30% of cervical carcinomas. All normal subjects and disease controls in assessing the production of free subunits by normal and
had free hCG0 levels <100 pg/ml. Thus, the detection of the free hCG/8 malignant trophoblastic cells (4, 30). In the present investiga
subunit in serum of nonpregnant subjects was highly diagnostic of ma tion, different m-IRMAs specific for hCG, and its free hCGa
lignancy in general and specifically defines a subgroup of aggressive
nongonadal malignancies.
and hCG/3 subunits, respectively, have been utilized to reeval
uate the presence of these molecules in the serum of patients
with a wide variety of nontrophoblastic tumors. In the present
INTRODUCTION report, we will present direct evidence demonstrating that the
Human chorionic gonadotropin is a glycoprotein hormone detection of the free hCG/3 subunit in serum of nonpregnant
normally secreted by the placenta and composed of two nonco- subjects is highly diagnostic of malignancy in general and
valently linked molecules, the a and the •¿'> subunits. It is one specifically defines a subgroup of aggressive nongonadal
malignancies.
member of a family of glycoproteins including the three pitu
itary hormones, namely human luteinizing, human follicle-
stimulating, and human thyroid-stimulating hormones. The MATERIALS AND METHODS
amino acid sequences of the a subunits of the hormones are
identical whereas the individual /3-subunit sequences diverge Subjects. Serum samples from 309 apparently healthy blood donors
were obtained through the blood transfusion services of Creteil, France.
(1), and it is the ßsubunit which confers the biological and Healthy donors included men (rti= 147) and women (n = 162) with a
immunological specificity to each hormone. These subunits are median age of 41 years. Among these women, 44 subjects were older
encoded by independent genes and consequently the free than 45 years of age. From our serum banks at HôpitalAntoine Béclère
hCGa1 and hCG0, which are biologically inactive, are produced and at Institut Gustave Roussy, we selected a panel of 86 serum samples
from patients with nonmalignant diseases including gynecological
(n = 19), pulmonary (n = 3), and gastrointestinal (n = 64) disorders.
Received 2/26/92; accepted 5/6/92. Serum samples from patients with newly diagnosed, persistent, or
The costs of publication of this article were defrayed in part by the payment of recurrent malignancies of either known (n = 717) or unknown (n = 32)
page charges. This article must therefore be hereby marked advertisement in accor
dance with 18 U.S.C. Section 1734 solely to indicate this fact. primary site were retrospectively obtained from the serum banks of the
1 Supported in part by grants from Association pour la Recherche sur le Cancer. Institut Gustave Roussy and the Hôpital Antoine Béclère as well as
Villejuif, and CA-35711 and HD-20469 from the N1H. prospectively from patients treated at the Institut Gustave Roussy using
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: hCG. human chorionic gonadotropin: hCG«,n protocols previously approved by the human studies committee. The
subunit of hCG; hCG/i, ß subunit of hCG; m-IRMA, monoclonal immunoradio diagnoses were confirmed by review of operative notes and pathology
metric assay: hill, human luteinizing hormone; hill... .; subunit of hill reports. Table 1A depicts the primary site of disease and the histology
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 SERUM FREE hCGff MEASUREMENT AND CANCER

Table 1 Primary site and histology of (A) cancer patients included in this study and labeled with 12SIserves as a radiolabeled indicator probe (Fig. 1).
and (B) patients with detectable serum level (> 100 pg/ml) of free HCGfi subunit Assay standards consisted of hCG (CR123, NIH) diluted in fetal calf
as measured with the m-IRMA 11-9
serum and the preparation had been found previously to lack hCG/3
imiminon-acti\ ily (31). The assay designated m-IRMA 9-12-13 had a
with lower limit of sensitivity of 50 pg/ml for hCG using a 3-h incubation
siteTestisOvaryBladderUterusCervixCorpusBreastLungHead no.)1219113726514338324207411202327284712326445743314311322129B(No.
Primary free hCG/3)6111299181742381511511324611
protocol and demonstrated no cross-reaction with the free a and ß
subunits of either hCG or hLH, nor with intact hLH, human follicle-
stimulating hormone, or human thyroid-stimulating hormone.
carcinomaChoriocarcinomaTeratocarcinomaMixed
Monoclonal Immunoradiometric Assay for the Free hCGa Subunit.
To measure the free a subunit of hCG, we used a specific assay (in
tumorGerm IRMA 20-13) as described previously (4). Briefly, this assay is based on
cellDysgerminomaTeratocarcinomaMixed a monoclonal antibody AHT20 which captures the free hCGa subunit
on a solid phase support and uses the 125I-Iabeled HT 13 described
above as a radiolabeled detecting antibody (Fig. 1). Assay standards
tumorEpithelialTransitional consisted of hCGa (CR-123, NIH) diluted in fetal calf serum. This
assay has a lower limit of sensitivity of 50 pg/ml of hCGa and showed
carcinomaAdenocarcinomaEpidermoid
cell no cross-reactivity with the free hCG/3 subunit or with other intact
glycoprotein hormones.
Monoclonal Immunoradiometric Assays for the Free hCG0 Subunit.
Two different specific assays were used for the measurement of the free
carcinomaAdenocarcinomaLeiomyosarcomaAdenocarcinomaNon-Hodgkin's
li( (..; subunit. Both assays were based on monoclonal antibody FBT11
as described previously (4, 32). This antibody has been demonstrated to
be highly specific for the free hCG/3 subunit with no cross-reactivity
lymphomaAdenocarcinomaLarge-cell with hCGa or with hCG or hLH and displayed a cross-reactivity of less
than 0.2% with the free beta chain of hLH. The first assay used was a
commercial kit (Cis-Bio, Marcoules, France) which utilized FBT11 as
carcinomaSmall-cell
carcinomaEpidermoid the capture antibody and a monoclonal antibody directed against the
carcinomaPoorly central portion of hCG/3 (HN9) as radiolabeled indicator antibody (Fig.
carcinomaEpidermoid
differentiated 1). This assay (m-IRMA 11-9) was performed according to manufac
turer's instructions and had a limit of sensitivity of 100 pg/ml of highly
carcinomaAdenocarcinomaPoorly
andneckProstateColorectalLiverPancreasStomachEsophagusUnknownHistologySeminomaNon-seminomaEmbryonal
purified hCG|8 (l.IRP 75/551 for hCG/3). Its pattern of cross-reactivity
carcinomaAdenocarcinomaAdenocarcinomaHepatocellular
differentiated was identical to that displayed by monoclonal antibody FBT11. In order
to avoid a potential cross-reactivity with the free ß subunit of hLH and
to ascertain the presence of the carboxyl terminal portion on the free 0
subunit of hCG, serum samples demonstrating a high level of free
hCG/3 in the m-IRMA 11-9 were retested with a second assay for free
carcinomaHepatoblastomaAdenocarcinomaIslet hCG/3. In this assay, the radiolabeled indicator antibody was a mono
clonal antipeptide antibody designated FBI9 as described previously
(33). This antibody is directed to the 110-116-amino acid carboxyl-
carcinomaLeiomyosarcomaAdenocarcinomaNon-Hodgkin's
cell terminal portion of hCG/3 and does not bind to the corresponding

m-IRMA 9-12-13-
lymphomaRhabdomyosarcomaEpidermoid forhCQ

carcinomaAdenocarcinomaAdenocarcinomaEpidermoid

carcinomaPoorly
differentiated carcinomaA(total

Total 749 139

m lorflMhCQß
-IRMA 11-W

of the tumor in the 749 patients. There were 380 females and 369 males
in this study. The primary tumor site was either the testis (n = 70), the
ovary (n = 156), the bladder (n = 38), the uterine cervix (n = 27), the
uterine corpus (n = 27), the breast (n = 42), the lung (n = 102), the head
or the neck (n = 10), the prostate (n = 32), the colon or the rectum
(n = 64), the liver (n = 52), the pancreas (n = 47), the stomach (n = 45),
the esophagus (n = 5), or unknown origin (n = 32). Serum samples were
stored at -20'C before analysis.
m-IRMA 20-13* m-IRUAII-9-
Monoclonal Immunoradiometric Assay for hCG. To measure hCG, for lar
iTMhCOa frwhCOß
we used a highly sensitive and specific monoclonal immunoradiometric
assay as described previously (31). The assay is based on two mono Fig. 1. Localization of the antigenic regions recognized by monoclonal anti
clonal antipeptide antibodies designated FBI2 and FB09 that are di bodies within the hCG«subunit (a) and the li( <..; subunit (ß)and schematic
rected against the carboxyl-terminal portion of the hCG/3 subunit. FBI 2 representation of m-IRMAs utilized to measure either the free hCGa, the free
h< <,,;. or the intact o/(j dimeric hormone. D, antigenic sites located on the
and FB09 serve as capture antibodies bound to a solid phase support carboxyl terminal portion (CTP) of the hCGff subunit: *, antibody used as radi
whereas monoclonal antibody HT 13 directed against the hCGa subunit olabeled indicator in a given assay.
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 SERUM FREE hCGtÃ-MEASUREMENT AND CANCER

region of hLHß(data not shown). Consequently, the m-IRMA 11-19 to

based on FBT11 as capture antibody and FBI9 as tracer was entirely
specific for hCGßwith no cross-reactivity with hLHfi. Moreover, this
assay will measure any free hCG/3 subunit containing the carboxyl-
terminal portion of hCGi) (Fig. 1). The coefficient of variation within
the assays (m-IRMA 11-19) averaged 8.4%; the coefficient of variation
between assays averaged 6.4%. The sensitivity was 100 pg/ml of highly
purified hCGß(1.IRP 75/551 for hCG/3).

RESULTS
Serum Levels of hCG, Free hCGa, and hCG/8 in Normals, in
Disease Controls, and in Patients with Various Tumors. Figs.
2, 3, and 4 present the serum levels of hCG and its free hCGa
and hCG/îsubunits as measured by m-IRMA 9-12-13,
m-IRMA 20-13, and m-IRMA 11-9, respectively. It is notewor
thy that hCG was present at low levels (< 1000 pg/ml) in serum
of numerous cancer patients as well as in healthy subjects,
including females older than 45 years, and patients with benign V
diseases (Fig. 2). Moreover, the free hCGa subunit was detect
able at a level up to 3000 pg/ml in serum of most normal
individuals (87.3%); the highest values were observed in healthy 47 45 5 32

women older than 45 years. Significant serum levels of free

hCGa as high as 3000 pg/ml were also found in 93% of patients
with a benign disease and in 96% of patients with a malignant
tumors (Fig. 3). In contrast, it was striking that none of the
serum samples from normal individuals or patients with benign
diseases contained a detectable amounts (>100 pg/ml) of the
free hCG/3 subunit whereas this subunit was present in the Fig. 3. Serum levels of free hCGa in healthy males and females and in patients
serum of 139 patients (18.5%) with neoplasms (Table IB). with benign diseases or tumors from various origins. D, limit of detection of the
assay (m-IRMA 20-13). - limit of values found in healthy subjects or patients
with benign diseases. Symbols are as described in the legend to Fig. 2. It., uterine.

10
Thus, neither the presence of hCG nor free hCGa in serum was
K specific of malignant diseases. The presence of detectable serum
levels of the free hCG/3 subunit was highly diagnostic of malig
nancies in this patient population.
Diagnosis of Gonadal and Nongonadal Neoplasms. Fig. 5
10
presents the percentages of cancer patients with serum levels of
hCG, free hCGa, and hCGßhigher than those observed in both
normal individuals and patients with benign diseases. It is ap
parent that 58.6% of patients with a nonseminomatous testic-
ular tumor, 16% with a seminomatous testicular tumor, and
1.3% with an epithelial tumor of the ovary displayed a hCG
a It serum level >1000 pg/ml. All other cancer patients (95%) had
hCG serum levels <1000 pg/ml (Fig. 5A). Thus, the presence
of hCG serum level >1000 pg/ml was highly diagnostic of
T gonadal tumors and specifically identified nonseminomatous
testicular cancer patients. We found that 24 of 749 cancer pa
5x10 tients (3.2%) had a free hCGa serum level of >3000 pg/ml.
Moreover, the highest percentage (13.8%) of patients with free
. hCGa > 3000 pg/ml was found in those with a testicular non
seminomatous tumor (Fig. SB). Consequently, the measure
ment of free hCGa subunit in serum did not appear useful for
the diagnosis of either gonadal or nongonadal neoplasms. In
contrast, 70 and 50% of patients with nonseminomatous and
seminomatous testicular cancers, respectively, and as high as
47% of those with a nongonadal neoplasm had a serum level of
free hCG/3 > 100 pg/ml (Fig. 5C). In such patients, the presence
Fig. 2. Serum levels of hCG in healthy males and females and in patients with of free hCG/3 was most frequently detected in the serum of
benign diseases or tumors from various origins. G, limit of detection of the assay
(m-IRMA 9-12-13); O, upper limit of values found in either healthy subjects or individuals with bladder (47%), pancreatic (32%), and cervical
patients with benign diseases. D, 100 cases; O, 10 cases; •¿.
1 case. Ut., uterine. carcinomas (30%).
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 SERUM FREE hCGji MEASUREMENT AND CANCER

aggressive nontrophoblastic neoplasms such as tumors of the

pancreas, lung, or bladder. It is noteworthy that these nongo
nadal tumors are generally resistant to low risk chemotherapy
and often require high risk cytotoxic protocols (34). It will be
important to determine if such treatment is warranted for pa
tients with free hCGßsubunit-producing neoplasms including
tumors of unknown primary site (35, 36). In our study, we
found that 25% of such patients had an isolated production of
free hCG/8. Furthermore, we believe that the utilization of
highly specific and sensitive assays capable of detecting pg
amounts of free hCG/3 subunit may facilitate the follow-up of
patients undergoing surgery, radiation, and chemotherapy.
These results provide new information on the production of
hCG and its subunits by malignant tumors. Following the ini
tial report (20), numerous studies have provided indirect evi
dence for the production of hCG, hCG-like substances, or free
subunits by nontrophoblastic cancers. However, these results

u? tie 44 ee 12 se 13 143 3e 2? 2? 42 102 10 32 64 52 47 45 s 32

hCG

60

40
Fig. 4. Serum levels of free h<'<;., in healthy males and females and in patients
with benign diseases or tumors from various origins. D, limit of detection of the 20
assay (m-IRMA 11-9). Symbols are as described in the legend to Fig. 2. Ut..
uterine.
0 lili

Presence of Free hCG/î with the Carboxyl-Terminal Portion 80

hCGa
of hCG/8 in Cancer Patients. In order to provide additional
evidence of the presence of free hCG/3 in the serum of cancer 60
patients, 24 samples which displayed a free hCGßserum level >
a>
O)
1000 pg/ml using the m-IRMA 11-9 were restudied using the
m-IRMA 11-19 which measures free hCG/S with its carboxyl- 40

terminal portion. This assay, therefore, is entirely specific for O)

the free hCG/3 subunit. We found that 67% of serum samples O- 20
previously found positive with the m-IRMA 11-9 were also
positive with the m-IRMA 11-19. In contrast, 3 of 8 patients
O
with a cancer of the bladder, 1 of 2 with an adenocarcinoma of
the uterine corpus, 2 of 3 with a large-cell carcinoma of the 80
hCGß
lung, and 3 of 4 with an adenocarcinoma of the pancreas had
unmeasurable serum levels using m-IRMA 11-19. Thus, it ap 60
pears that a majority of sera from cancer patients do contain the
free hCG/3 with the carboxyl-terminal portion of hCG/3 exten
sion while 33% of sera contain an altered form of free hCG£i 40
devoid of the carboxyl-terminal portion of hCG/3.
20
DISCUSSION
O
The present observations have clinical importance with re
spect to the diagnosis, treatment, and follow-up of cancer
patients. First, it is striking that elevated free hCGßin serum of
nonpregnant subjects was highly diagnostic of patients with
several common malignancies of nongonadal and gonadal
origin. Indeed, detection of the free hCG/3 identified 70, 50, and
6% of patients with germ cell nonseminomatous testicular tu
mors, testicular seminomas, and epithelial tumors of the ovary,
Fig. 5. Percentage of cancer patients with serum values of hCG (A), free hCGn
respectively. If we exclude tumors of gonadal origin, however, (B), or free hCGtf (Q above the values found in the serum of healthy subjects and
the secretion of free hCG/3 subunit was highly correlated with patients with benign diseases. Ut., uterine.
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 SERUM FREE hCG/i MEASUREMENT AND CANCER

were controversial since most investigations used polyclonal free hCG/3 or a predominant production of this free subunit in
and monoclonal antibodies lacking the required specificity. One excess of hCG. This pattern of secretion contrasts with that
of the major problems was that most antibodies cross-reacted observed in pregnant women who display a predominant secre
with hLH. For example, antibody SB6 had a cross-reactivity of tion of hCG in large excess of free hCG/3 (4). Since the initial
7 to 11% with hLH and therefore was not a highly specific case report in 1973 (8), we have found only three other well-
reagent for hCG and hCG/3 (9, 24, 25). Recently developed documented published examples of either an isolated or pre
assays based on monoclonal antibodies still displayed a low but dominant production of free hCG/ìin cancer patients (24, 50,
significant cross-reactivity with hLH (37-39). More impor 51). In these four prior cases, such a production was associated
tantly, most assays did not distinguish between the intact hCG with a pancreatic adenosquamous carcinoma (8), an adenocar-
molecule and the free hCG/3 subunit (14, 37-41). Finally, the cinoma of the stomach (24), a carcinoma of the uterine cervix
nomenclature for "hCG0" assays has been ambiguous since the
(50), and an epidermoid carcinoma of unknown primary site
same designation has been given to at least two types of assays (51). Using the highly specific and sensitive mIRMAs, it ap
with widely different specificities including those measuring pears that the presence of elevated serum levels free hCG/3 is
only the free hCGßsubunit and those measuring both intact not rare and may be relatively common particularly in patients
hCG and the free hCG/3 subunit (34). Similarly, the nomencla with bladder tumors. This finding was not totally unexpected
ture for "hCG" assays may be misinterpreted since these assays
since the in vivo production of either hCG or free hCG/3 by such
may also measure the free hCG/3 subunit in addition to hCG neoplasms had been reported previously (52-56). Moreover, in
(42). vitro studies performed on culture media derived from urothe-
Recently, mIRMAs entirely specific for intact hCG and the lial cell lines strongly suggest that the material excreted by these
free hCGa or hCG/3 subunit have enabled investigators to re-
cells consisted principally of free hCG0 subunit or fragments
evaluate the production of hCG and free subunits by normal thereof (57, 58).
individuals, during pregnancy, and in patients with gestational There is accumulating evidence indicating that the isolated
trophoblastic diseases (4, 26, 29-31). The present study has
production of free hCG/3 may be associated with aggressive
taken advantage of the specificity of such assays and assessed nongonadal and nontrophoblastic malignancies. For example,
the production of hCG and free subunits by gonadal as well as we have found that the isolated production of free hCG/3 sub-
nongonadal and nontrophoblastic malignancies. In this con unit was associated with tumors of poor prognosis such as
text, we have used four different assays that are entirely specific cancers of the lung, pancreas, and liver. This observation is in
for the molecules of interest, namely hCG, free hCGa, and agreement with prior documented cases in which the presence
hCG/3 and the free hCG/3 with the CTP extension. The latter of free hCG/3 was associated with aggressive malignancies as
assay is the first described for free hCG/3 that displays no cross-
measured by a duration of survival of 2 weeks to 3 months (8,
reaction with the free hLH/3 subunit. The results from this
investigation demonstrate using these four highly specific mlR- 51).
The histológica! origin of free hCG/3-producing cells in these
MAs that neither the production of free hCGa nor low level
tumors (Table 1B) deserves comment. Several hypotheses have
hCG was specific of malignant tumors. Indeed, the levels of been proposed to explain the origin of hCG/3-producing cells
both free hCGa and hCG were marginally raised in most pa
and include the following: (a) displaced totipotential or gonadal
tients with tumors but were within the range of values generally
cells (extragonadal choriocarcinomas); (b) metastasis from an
found in healthy individuals and those with benign diseases (29,
intrauterine or gonadal lesion; and (c) an evolution from a
30). In this regard, a common cause of marginally raised hCGa
levels has been shown to be due to production of free a subunits somatic cell that underwent a morphological and functional
by the pituitary gland which will cross-react in the free hCGa transformation (metaplasia) into a cell functionally similar to
assay (38). In addition, recent studies have provided direct ev the trophoblast. Since the pioneering work of Pick (59) in 1926,
idence for production of the intact hCG molecule by the pitu most authors favor the concept of a trophoblastic metaplasia
within the carcinomatous tissues (54, 60-63). Indeed, the path
itary (27, 28, 43). It is highly likely that the low level hCG and
the free hCG« subunit found in the blood of patients with way of cytotrophoblast differentiation ensures that hCGa
various tumors originates from the pituitary gland and not from mRNA accumulates before hCG/3 mRNA, resulting in increas
the tumor (44-48). This observation may explain in part the ing hCG production as hCG/3 synthesis is initiated (39). Our
finding of elevated free hCG«and hCG levels in females older results are in agreement with this pathway; most gonadal and
than 45 years with gynecological or breast cancers since serum extragonadal choriocarcinomas as well as metastatic choriocar
levels relating to a pituitary origin have been found to increase cinomas produce both hCG and the free hCG/3 subunit and thus
with age (29, 30). Finally, in contrast to the insignificance of a differ from nongonadal cells producing predominantly the free
low level hCG in patients with malignancy with respect to its hCG/3 subunit (34, 39, 40). In addition, it has been shown that
value to a specific tumor marker, we found that a serum level of in the early stages of implantation as well as in trophoblastic
hCG >1 ng/ml was highly suggestive of a testicular tumor (see disease, the origin of free hCG/3 production may be due to
Figs. 2 and 5A). poorly differentiated trophoblastic tissue (40, 64). These obser
The finding of the free hCGßsubunit in patients with non vations suggest that the origin of nongonadal malignant cells
gonadal and nontrophoblastic malignancies is at odds with pre involves a metaplasia of the carcinomatous tissues into a tissue
vious results suggesting that the free hCG0 is rarely, if ever, similar to poorly differentiated trophoblasts. Finally, recogni
produced by such tumors and that the free hCG/3 secretion tion of a secretion of free hCG/3 by nongonadal tumors such as
occurs only in association with the synthesis of high levels of bladder may become increasingly important inasmuch as this
intact hCG (37, 49, 50). In the present investigation, 139 pa free subunit may represent a unique biological marker of ma
tients with various tumors had elevated free hCG0 levels. lignancies that do not produce and secrete an identifiable tumor
Among these patients, 92 had either an isolated secretion of cell product.
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 SERUM FREE hCGf) MEASUREMENT
ACKNOWLEDGMENTS
We are indebted to the National Pituitary Program of the National
Institute of Arthritis and Metabolic Diseases and the National Institute
of Child Health and Human Development for supplying hCG and its
subunits used in these studies and to Christine Bombled and Josyane Le
Calvez for their excellent technical assistance.
REFERENCES
1. Pierce, J. G.. and Parsons. T. F. Glycoprotein hormones: structure and
function. Annu. Rev. Biochem.. 50:465-495. 1981.
2. Fiddes. J. C.. and Goodman, H. M. The gene encoding the common a subunit
of the four human glycoprotein hormones. J. Mol. Appi. Genet., /: 3-18,
1981.
3. Boorstein, W. R., Vamvakopoulos. N. C., and Fiddes, J. C. Human chorionic
gonadotropi •¿
-.nimmt is encoded by at least eight genes arranged in tandem
and inverted pairs. Nature (Lond.). 300: 419-423, 1982.
4. Ozturk. M., Bellet. D., Manil, L., Hennen. G., Frydman, R., and Wands, J.
R. Physiological studies of human chorionic gonadotropi (hCG), nhCG, and
ÕJhCGas measured by specific monoclonal immunoradiometric assays. En
docrinology. 120: 549-558. 1987.
5. Borkowski. A., and Muquardt. C. Human chorionic gonadotropi in the
plasma of normal nonpregnant subjects. N. Engl. J. Med.. 301: 298-302.
1979.
6. Kahn. C. R.. Rosen, S. W„Wcintraub. B. D.. Fajans. S. S., and Corden. P.
Ectopie production of chorionic gonadotropin and its subunits by islet-cell
tumors. N. Engl. J. Med.. 297: 565-569, 1977.
7. Rosen. S. W., and Weintraub. B. D. Ectopie production of the isolated a
subunit of the glycoprotein hormones. N. Engl. J. Med., 290: 1441-1447,
1974.
8. Weintraub. B. D.. and Rosen. W. S. Ectopie production of the isolated lì
subunil of human chorionic gonadotropin. J. Clin. Invest.. 52: 3135-3142,
1973.
9. Tormey, D. C, Waalkes. T. P., and Simon, R. M. Biological markers in
breast carcinoma. Cancer (Phila.), 39: 2391-2396, 1977.
10. Gailani. S., Chu, T. M., Nussbaum, A.. Ostrander. M., and Christoff. N.
Human chorionic gonadotrophins (hCG) in nontrophoblastic neoplasms.
Cancer (Phila.), 38: 1684-1686. 1976.
11. Blackman, M. R.. Weintraub. B. D., Rosen, S. W., and Kourides. I. A.,
Steinwascher. K., and Gail, M. H. Human placenta! and pituitary glycopro
tein hormones and their subunits as tumor markers: a quantitative
assessment. J. Nati. Cancer Inst.. 65: 81-93. 1980.
12. Hussa, R. O. Clinical applications of hCG tests: tumors. In: R. O. Hussa
(ed.). The Clinical Marker hCG, pp. 119-136. New York: Praeger. 1987.
13. Hussa, R. O. Subunits of hCG. In: R. O. Hussa (ed.). The Clinical Marker
hCG, pp. 161-178. New York: Praeger, 1987.
14. Hussa. R. O. Tissue localization of hCG. In: R. O. Hussa (ed.). The Clinical
Marker hCG. pp. 151-160. New York: Praeger, 1987.
15. Burg-Kurland. C. L., Purnell. D. M., Combs, J. W.. Hillman, E. A., Harris.
C. C., and Trump. B. F. Immunocytochemical evaluation of human esoph-
ageal neoplasms and preneoplastic lesions for ^-chorionic gonadotropin. pla-
cental lactogen. a-fetoprotein, carcinoembryonic antigen, and nonspecific
cross-reacting antigen. Cancer Res., 46: 2936-2943, 1986.
16. Campo, E.. Palacin, A., Benasco. C., Quesada, E., and Cardesa, A. Human
chorionic gonadotropin in colorectal carcinoma. Cancer (Phila.). 59: 1611-
1616. 1987.
17. Yakeishi. Y., Mori. M.. and Enjoji. M. Distribution of (¡-humanchorionic
gonadotropin-positive cells in noncancerous gastric mucosa and in malignant
gastric tumors. Cancer (Phila.). 66: 695-701. 1990.
18. Braunstein. G. D.. Rasor, J., and Wade, M. E. Presence in normal human
testes of a chorionic-gonadotropin-like substance distinct from human lutein-
izing hormone. N. Engl. J. Med., 29.J: 1339-1343, 1975.
19. McGregor. W. G., Kühn.R. W.. and Jaffe, R. B. Biologically active chorionic
gonadotropin: synthesis by the human fetus. Science (Washington DC), 220:
306-308. 1983.
20. Reeves. R. L., Tesluk. H., and Harrison, C. E. Precocious puberty associated
with hepatoma. J. Clin. Endocrinol. Metab., 19: 1651-1660, 1959.
21. Odell, W. D.. Wolfsen, A., Yoshimoto, Y.. Weitzman, R., Fisher. D., and
Hirose, F. Ectopie peptide synthesis: a universal concomitant of neoplasia.
Tr. Assoc. Am. Physicians. 90: 204-227. 1977.
22. Yoshimoto. Y., Wolfsen, A. R.. and Odell. W. D. Human chorionic gona-
dotropin-like substance in nonendocrine tissues of normal subjects. Science
(Washington DC), 797: 575-577. 1977.
23. Braunstein, G. D., Kamdar, V.. and Rasor, J.. Swaminathan, N.. and Wade,
M. E. W ¡despreaddistribution of a chorionic gonadotropin-like substance in
normal human tissues. J. Clin. Endocrinol. Metab., 49: 917-925. 1979.
24. Papapetrou. P. D., Sakarelou. N. P., Braouzi. H., and Fessas, P. H. Ectopie
production of human chorionic gonadotropin (hCG) by neoplasms: the value
of measurements of immunoreactive hCG in the urine as a screening
procedure. Cancer (Phila.), 45: 2583-2592. 1980.
25. Monteiro, J. C. M. P.. Ferguson. K. M., McKinna, J. A., Greening, W. P.,
and Neville. A. M. Ectopie production of human chorionic gonadotrophin-
3906
Downloaded from cancerres.aacrjournals.org on December 7, 2013. © 1992 American Association for Cancer
Research.
AND CANCER
like material by breast cancer. Cancer (Phila.), 53: 957-962, 1984.
26. Griffin, J., and Odell, W. D. Ultrasensitive immunoradiometric assay for
chorionic gonadotropin which does not cross-react with luteinizing hormone
nor free ß chain of hCG and which detects hCG in blood of non-pregnant
humans. J. Immunol. Methods. 103: 275-283. 1987.
27. Odell. W. D.. Griffin, J.. Bashey, H. M., Snyder, P. J. Secretion of chorionic
gonadotropin by cultured human pituitary cells. J. Clin. Endocrinol. Metab.,
71: 1318-1321, 1990.
Hammond. E.. Griffin, J.. and Odell, W. D. A chorionic gonadotropin-
28.
secreting human pituitary cell. J. Clin. Endocrinol. Metab.. 72: 747-754,
1991.
29. Odell. W. D., and Griffin. J. Pulsatile secretion of human chorionic gona
dotropin in normal adults. N. Engl. J. Med.. 317: 1688-1691, 1987.
30. Ozturk. M., Berkowitz, R., Goldstein. D., Bellet. D., and Wands, J. R.
Differential production of human chorionic gonadotropin and free subunits
in gestational trophoblastic disease. Am. J. Obstet. Gynecol., 158: 193-198,
1988.
31. Bellet, D. H.. Ozturk, M.. Bidart, J. M., Bohuon. C. J., and Wands, J. R.
Sensitive and specific assay for human chorionic gonadotropin (hCG) based
on ami-peptide and anti-hCG antibodies: construction and clinical
implications. J. Clin. Endocrinol. Metab., 63: 1319-1327. 1986.
32. Bidart. J. M.. Troalen. F., Salesse, R.. Bousfield. G. R., Bohuon, C, and
Bellet, D. Topographic antigenic determinants recognized by monoclonal
antibodies on human choriogonadotropin (J-subunit. J. Biol. Chem., 262:
8551-8556, 1987.
33. Bidart. J. M.. Bellet. D. H.. Alberici, G. F.. Van Besien. F., and Bohuon. C.
The immune response to a synthetic peptide analogous to the 109-145 (ihCG
carboxyl-terminus is directed against two major and two minor regions. Mol.
Immunol.. 24: 339-345, 1987.
Case records of the Massachusetts General Hospital (case 34-1983). N. Engl.
J. Med.. 309: 477-487. 1983.
Richardson. R. L., Schoumacher, R. A., and Fer, M. F. The unrecognized
extragonadal germ cell cancer syndrome. Ann. Intern. Med., 94: 181-186,
1981.
36. Greco, A., Vaughn, W. K., and Hainsworth. J. D. Advanced poorly differ
entiated carcinoma of unknown primary site: recognition of a treatable
syndrome. Ann. Intern. Med.. 104: 547-553. 1986.
37. Sailer. B.. Clara. R.. Spottl, G., Siddle, K., and Mann. K. Testicular cancer
secretes intact human choriogonadotropin (hCG) and its free ß subunit: ev
idence that hCG (+hCG-rf) assays are the most reliable in diagnosis and
follow-up. Clin. Chem., 36: 234-239. 1990.
38. Hay, D. L., and Murphy. J. R. Evaluating human choriogonadotropin het
erogeneity in nongestational malignancy with monoclonal immunoassays.
Oncology (Basel). 44: 174-179, 1987.
39. Hay, D. L. Histological origins of discordant chorionic gonadotropin secre
tion in malignancy. J. Clin. Endocrinol. Metab.. 66: 557-564. 1988.
40. Fan, C., Goto, S., Furuhashi, Y., and Tomoda. Y. Radioimmunoassay of the
serum free /i-subunit of human chorionic gonadotropin in trophoblastic
disease. J. Clin. Endocrinol. Metab., 64: 313-318, 1987.
41. Mann. K.. and Siddle. K. Evidence for free /J-subunit secretion in so-called
human chorionic gonadotropin-posilive seminoma. Cancer (Phila.), 62:
2378-2382. 1988.
42. Cole. L. A., and Kardana. A. Discordant results in human chorionic gona
dotropin assays. Clin. Chem., 38: 263-270. 1992.
43. Hoermann, R.. Spoettl. G., Moncayo. R., and Mann. K. Evidence for the
presence of human chorionic gonadotropin (hCG) and free iJ-subunit of hCG
in the human pituitary. J. Clin. Endocrinol. Metab., 71: 179-186, 1990.
44. Fusco. F. D., and Rosen. S. W. Gonadotropin-producing anaplastic large-cell
carcinomas of the lung. N. Engl. J. Med.. 275: 507-515, 1966.
45. Broder. L. E., Weintraub, B. D., Rosen, S. W., Cohen, M. H., and Tejada. F.
Placental proteins and their subunits as tumor markers in prostatic
carcinoma. Cancer (Phila.), 40: 211-216, 1977.
46. Yamaguchi, A.. Ishida. T.. and Nishimura. G. Human chorionic gonadotro
pin in colorectal cancer and its relationship to prognosis. Br. J. Cancer, 60:
382-384. 1989.
47. Morinaga. S.. Yamaguchi. M.. Watanabe, I., Kasai, M., Ojima, M., and
Sasano, N. An immunohistochemica! study of hepatoblastoma producing
human chorionic gonadotropin. Cancer (Phila.). 51: 1647-1652, 1983.
48. Hattori. M., Yoshimoto, Y.. Matsukura, S.. and Fujita. T. Qualitative and
quantitative analyses of human chorionic gonadotropin and its subunits pro
duced by malignant tumors. Cancer (Phila.). 46: 355-361, 1980.
49. Heitz. P. U., von Herbay, G., and Klöppel,G. The expression of subunits of
human chorionic gonadotropin (hCG) by nontrophoblastic, nonendocrine.
and endocrine tumors. Am. J. Clin. Pathol.. 88: 467-472. 1987.
50. Papapetrou, P. D., and Nicopoulou, S. Ch. The origin of a human chorionic
gonadotropin tf-subunit-core fragment excreted in the urine of patients with
cancer. Acta Endocrinol.. 112: 415-422. 1986.
51. Nagelberg, S. B., Marmorstein, B., Khazaeli, M. B., and Rosen, S. W. Iso
lated ectopie production of the free beta subunit of chorionic gonadotropin by
an epidermoid carcinoma of unknown primary site. Cancer (Phila.), 55:
1924-1930, 1985.
52. Dexeus. F.. Logothetis. C.. Hossan, E.. and Samuels, M. L. Carcinoembry
onic antigen and .<huni.ui chorionic gonadotropin as serum markers for
advanced urothelial malignancies. J. Urol.. 136: 403-407, 1986.
53. lies. R. K., Jenkins. B. J., Oliver. R. T. D.. Blandy, J. P., and Chard, T. ß
 SERUM FREE hCGfJ MEASUREMENT AND CANCER

human chorionic gonadotrophin in serum and urine. A marker for metastatic
urothelial cancer. Br. J. Urol., 64: 241-244, 1989.
54. Vahlensieck. W., Riede. U., Wimmer, B., and Ihling. C. rf-Human chorionic
gonadotropin-positive extragonadal germ cell neoplasia of the renal pelvis.
Cancer (Phila.), 67: 3146-3149, 1991.
55. lies. R. K., and Chard. T. Human chorionic gonadotropin expression by
bladder cancers: biology and clinical potential. J. Urol., 145:453-458, 1991.
56. Fetissof, F., Bellet, D.. Guilloteau, D„and Maillot, O. Hormone gonado-
tropine chorionique et carcinome Ãcellules traditionnelles de la vessie. Ann.
Pathol.. 8: 276-280, 1988.
57. Iles. R. K., Oliver. R. T. D.. Kitau, M., Walker. C., and Chard. T. In vitro
secretion of human chorionic gonadotropin by bladder tumour cells. Br. J.
Cancer, 55: 623-626, 1987.
58. lies, R. K., and Chard, T. Immunochemical analysis of the human chorionic
gonadotrophin-like material secreted by "normal" and neoplastic urothelial
cells. J. Mol. Endocrino!., 2: 107-112, 1989.
59. Pick, L. Über die chorionepitheliomätnlich metastasierende Form des
Magencarcinoms. Klin. Wochenschr., 5: 1728-1729, 1926.
60. Park, C. H., and Reid, J. D. Adenocarcinoma of the colon with choriocarci-
noma in its métastases.Cancer (Phila.), 46: 570-575, 1980.
61. Obe, J. A., Rosen, N., and Koss, L. G. Primary choriocarcinoma of the
urinary bladder. Cancer (Phila.). 52: 1405-1409. 1983.
62. Kubosawa, H.. Nagao, K., Kondo. Y., Ishigc, H., and Inaba, N. Coexistence
of adenocarcinoma and choriocarcinoma in the sigmoid colon. Cancer
(Phila.). 54:866-868. 1984.
63. Campo, E., Algaba, F., Palacin, A., Germa, R., Sole-Balcells, F. J., and
Cardesa, A. Placental proteins in high-grade urothelial neoplasms. An im-
munohistochemical study of human chorionic gonadotropin, human placen-
tal lactogen, and pregnancy-specific /J-1-glycoprotein. Cancer (Phila.), 63:
2497-2504. 1989.
64. Hay, D. L. Discordant and variable production of human chorionic gonado
tropin and its free «-and /J-subunits in early pregnancy. J. Clin. Endocrinol.
Metab., 61: 1195-1200, 1985.

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