Vol. 50, No. 4
Printed in U.S.A.
T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Copyright © 1968 by The Williams & Wilkins Co.
IMMUNOFLUORESCENT STUDIES IN GONADOTROPIN-SECRETING
BRONCHOGENIC CARCINOMA
JOHN C. COTTRELL, M.D., KENNETH L. BECKER, M.D., PH.D., AND CHARLES F. MOORE
Veterans Administration Hospital, Washington, D. C. 20J$2
Since the original observations of Coons
and co-workers in 1942,3 immunofluorescent
staining technics have been found useful in
the identification and localization of hormones in tissues. Immunofluorescent studies
have demonstrated thyrocalcitonin within
the thyroid gland,2' 5 thyroid stimulating
hormone, corticotropin, and gonadotropins
within the pituitary, 4, 6i 9_u and insulin
in the islets of Langerhans. 7 In the present
investigation, we have used the immunofluorescent technic to identify the presence of
gonadotropin and to study its distribution
in the tumor tissue of two patients with
bronchogenic carcinoma who had elevated
gonadotropin levels in the urine. The clinical
histories, hormone assays, autopsy findings,
and postmortem tissue assays will be reported elsewhere and are only summarized
here. The purpose of the present report is to
describe in detail the study of gonadotropinsecreting bronchogenic carcinomas for the
first time by an immunofluorescent method.
SUMMARY O F CLINICAL H I S T O R I E S AND
AUTOPSY F I N D I N G S
Case 1. E. H., a 74-year-old Negro male
complaining of cough and weight loss, was
noted to have bilateral gynecomastia and a
left upper lobe lung mass in November
1963. The sputum was positive for malignant cells, and an exploratory thoracotomy
revealed an epidermoid carcinoma of the
lung. The patient persistently excreted between 1500 and 7000 international units
Received November 29, 1967.
Presented at the Annual Meeting of the American Society of Clinical Pathologists in Chicago,
111., September 22 to 30, 1967.
This study was supported in part by Research
Grant AM-10196, National Institute of Arthritis
and Metabolic Diseases, U. S. Public Health Service.
Requests for reprints should be addressed to
Dr. Becker.
of human chorionic gonadotropin (HCG) per
24 hr. of urine, and the serum contained
105 I.U. of HCG per ml. He died in May
1965. Postmortem examination revealed a
tumor of the lung which originated from
the left upper lobe bronchus and infiltrated
the mediastinal structures. Solitary metastases were noted in the left kidney and lung.
Microscopically (Fig. 1) there were sheets,
nests, and cords of large cells with eosinophilic cytoplasm, hyperchromatic nuclei,
and atypical mitotic figures. There were
areas resembling epithelial pearls and many
intercellular bridges. No areas were seen
resembling choriocarcinoma. The metastases
revealed a similar microscopic appearance.
Extensive examination of serial sections of
both testes revealed no evidence of a testicular tumor.
Case 2. I. H., a 70-year-old Caucasian
male with chronic cough, was noted to
have a persistent pulmonary infiltrate of
the left lung in January 1966. Bronchoscopic washings and sputum analysis revealed a Class 3 sputum cytology. Cerebral
arteriogram, performed because of his confusion and right hemiparesis, revealed a left
frontal lobe mass which, on surgical exploration, was found to be metastatic epidermoid
carcinoma. Prior to his death in April 1967,
the patient was noted to have bilateral
gynecomastia. A postmortem sample of
urine from the bladder contained 46 I.U.
of HCG per ml. Autopsy demonstrated a
tumor of the left lower lobe of the lung with
metastases to the mediastinal lymph nodes,
liver, kidney, brain, and left adrenal gland.
Microscopically (Fig. 2) the primary tumor
in the lung consisted of nests, sheets, and
cords of small cells with large hyperchromatic nuclei, abundant
abnormal
mitotic figures, and scant cytoplasm. Intercellular bridges and keratin were absent.
Serial sections of the testes revealed no evidence of choriocarcinoma.
422
FIG. 1 (upper left). Section of a representative area of the tumor of Case 1. Note the "epithelial
pearl" in the upper right corner. Hematoxylin and eosin. X 100.
FIG. 2 (upper right). Section of a representative area of the tumor of Case 2. Hematoxylin and eosin.
X 100.
FIG. 3 (lower left). PAS stain of an immature placenta, showing positive staining trophoblast.
FIG. 4 (lower right). PAS stain of the primary tumor of patient of Case 1, showing positive staining
cytoplasm.
423
424
COTTRELL ET
MATERIALS AND METHODS
Antibody. The specific antihormone used
in these studies was a commercially available
rabbit antihuman chorionic gonadotropin
(Ortho Pharmaceutical Corporation, Raritan, N. J.) in which in vivo hormone inhibition studies in rats had been performed.13
Conjugate. Commercially available fluorescein-conjugated goat antirabbit globulin
(Ortho) which had been adsorbed with
human nuclear sediment was mixed with
commercially available albumin-conjugated
rhodamine (Ortho) in a ratio of 1 part of
rhodamine to 20 parts of fluorescein conjugate. The mixture was stored frozen in
0.1-ml. aliquots in glass test tubes. Immediately prior to use, it was thawed and
diluted to the desired volume.
Tissue. Representative areas of the primary tumors and metastases of both patients, two human placentas obtained at
spontaneous deliveries near the end of the
first trimester, one human placenta obtained
at spontaneous delivery at term, grossly
normal human liver, lung, brain, and
pituitary obtained at autopsy, and a nongonadotropin-secreting bronchogenic carcinoma were fresh frozen and stored for
further study.
m
"
J
Fluorescent staining method. Immediately
before staining, 3-M sections of all tissues
were cut on a cryostat at —20 C. and
mounted on acid-alcohol-cleaned glass slides.
The sections were dried in an incubator at
37 C. for 10 min. Antibody was then overlaid on the sections designated for study.
Duplicate sections to be used as controls
were overlaid with nonimmune homologous
serum instead of the specific antiserum,
heterologous human serum, or buffer, as
appropriate. The slides were placed in a
humidity chamber and incubated at 37 C.
for 30 min. At the end of the incubation
period, the tissues were washed for 60 min.
in tap water and then were immersed in
phosphate-buffered saline for 10 min., pH
7.2. During this time the conjugate was
thawed and diluted. All of the pertinent
tissues were overlaid with the conjugate
and incubated in a humidity chamber for
30 min. The tissues were then washed in tap
water for 60 min. and mounted with 50%
glycerine, and coverslips were applied. A
Vol. 50
AL.
Leitz Labolux microscope with Osram
HBO 200 mercury arc lamp and BG12 exciting filter was utilized with an OGl
barrier filter. A micro-IBSO shutter attachment equipped with a Leica M2 body was
used to photograph the observations on
Kodachrome II film.
Histologic study methods. Hematoxylin and
eosin and periodic acid-Schiff (PAS) stains
following diastase predigestion were performed on adjacent sections of each tissue
studied by the immunofluorescent technic.
The hematoxylin and eosin-stained tissues
were reviewed in order to assure the absence of pathologic changes in the placentas
and other "normal" control tissues. The
cytoarchitecture of the tumor was studied
and nonnecrotic, nondegenerated areas were
selected for immunohistochemical study.
The diastase-digested, PAS-stained sections
were examined for the presence of reacting
polysaccharides.
Gonadotropin assays. Bioassay of gonadotropin was performed by the ovarian hyperemia technic of Albert and Berkson.1
The immunoassay of gonadotropin used
was a modification12,15 of the agglutination
inhibition method of Wide and Gemzell.16
Unconcentrated urine and plasma specimens were assayed by bioassay and immunoassay. The primary and metastatic tumors
were homogenized in cold saline and, after
centrifugation, the sediment was discarded.
To 10 ml. of extract, 1 ml. of 10% sodium
tungstate was added, followed by 1 ml. of
0.67 N sulfuric acid. The solution was mixed,
allowed to stand for 10 min., and centrifuged, and the supernatant was discarded.
The sediment was dissolved in 1 ml. of
Tris-HCl buffer, 0.1 M, pH 8.4, containing
0.154 M NaCl and 0.11 M CaCl 2 , centrifuged, and the precipitate was discarded.
All tumor extracts were then tested for
gonadotropin content by bioassay and
agglutination inhibition assay. All data were
expressed in terms of the Second International HCG standard, kindly supplied by
Dr. D. R. Bangham, London, England.
RESULTS
Gonadotropin assays. The results of the
assay of gonadotropin of the tumor and a
metastasis of each patient are shown in
Table 1.
Oct. 1968
IMMUNOFLUORESCENT STUDIES OF CARCINOMA
Histochemical studies. Diastase-fast PASpositive staining was evident in syncytiotrophoblast and cytotrophoblast of the
control placentas (Fig. 3). The cells of both
of the primary tumors and all of the metastases contained similar PAS-positive material (Fig. 4). Intense PAS positivity was
observed in a pulmonary metastasis of
Patient E. H. and a cerebral metastasis of
Patient I. H.
TABLE 1
R E S U L T S OF ASSAY OF GONADOTROPINS OF
PRIMARY LUNG TUMOR AND K I D N K Y
METASTASIS*
Agglutination Inhibition Assay
E. 1-1.
I. H.
Bioassay
Lung
tumor
Metastases
Lung
tumor
Metastases
50
53
So
100
427
40
271
40
" Given in international units of ITCG
Gram of wet tissue.
per
425
Immunofluorescent studies. During the
preliminary study of the placental tissues, 8,14
it was found that diluting the conjugate to a
final dilution of 40:1 and the addition of
rhodamine-albumin resulted in nearly complete suppression of nonspecific fluorescence
(Fig. 5).
Tables 2 and 3 summarize the results of
the immunofluorescent studies. The fluorescent staining controls were consistently
negative (Table 2). The tumor cells of both
of the primary lesions and those of all but
two metastases showed varying degrees of
regional fluorescence (Fig. 6 and Table 3).
The two aforementioned metastases which
departed from this regional pattern (a
solitary pulmonary nodule of E. H. and a
cerebral metastasis of I. H.) exhibited
brilliant apple-green fluorescence of the
cytoplasm of nearly every cell (Fig. 7 and
Table 3). No specific fluorescence was seen
in the grossly normal liver, brain, and lung
tissues or in the non-gonadotropin-secreting
bronchogenic carcinoma, although placentas
F I G . 5 (left). Immunofluorescent stain of placenta control, showing specific fluorescence of trophoblast
F I G . G (right). Immunofluorescent stain of tumor of Case 1, showing regional specific fluorescence
426
COTTRELL ET AL.
Vol. 50
TABLE 2
R E S U L T S O F C O N T R O L STUDY
Specific Fluorescence
Controls
Tissue only
Tissue and rabbit serum
Tissue and human serum
Tissue and antibody
Tissue and rabbit serum and conjugate
Tissue and human serum and conj ugate
Tissue and antibody and con.ugate
0
0
0
0
0
0
++++
TABLE 3
RESULTS
OF
FLUORESCENT
STAINING
O P THE
T I S S U E S STUDIED
Tissue Studied
Patient E. II.
Primary
Renal metastasis
Lung metastasis
Patient I. H.
Primary
Liver metastasis
Kidney metastasis
Cerebral metastasis
Normal pituitary
Term placentas
Immature placentas
Nongonadotropin-secreting lung
tumor
Normal liver
Normal brain
Normal lung
Fluorescence
Regional
Regional
Diffuse
Regional
Regional
Regional
Diffuse
Regional
Regional
Regional
Negative
Negative
Negative
Negative
processed along with these tissues as positive controls fluoresced as expected. Distinctly regional fluorescence was also observed in the pars anterior of the normal
pituitary.
DISCUSSION
Immunofluorescent studies were undertaken to determine the site of production of
gonadotropic hormone by these tumors.
The postmortem study of the tumor from
E. H. was undertaken after extensive premortem hormone assays demonstrated hypergonadotropinemia and hypergonadotropinuria. Shortly afterward, the second
patient (I. H.) was admitted to the hospital
with gynecomastia and bronchogenic car-
F I G . 7. Immunofluorescent stain of metastasis
of Case 1, showing diffuse brilliant specific fluorescence.
cinoma. This patient died of his tumor
before extensive endocrine studies could be
undertaken. Bladder urine, obtained at
postmortem examination, revealed an elevated titer of gonadotropin. At autopsy,
representative samples of tumor tissue were
removed for later bioassay and immunoassay, and immunofluorescent studies were
immediately performed to confirm the
presence of a gonadotropin-secreting bronchogenic carcinoma. These studies were
positive before quantitative gonadotropin
assays were performed upon the tumor.
The presence of PAS-positive material
in the tissues served to pinpoint the areas in
which to expect fluorescence. PAS positivity
in itself is not prima facie evidence of the
presence of gonadotropin. For example,
glycogen, luteinizing hormone, and thyroidstimulating hormone all contain glycol
groups which react positively by the PAS
reaction. It was the immunofluorescent
technic which identified and precisely localized the gonadotropin.
Oct. 1968
I M M U N O F L U O R E S C E N T S T U D I E S OF CARCINOMA
The distinctly regional positive fluorescence observed in the tumors and most of
the metastases is of interest. One can only
speculate as to the explanation for this
phenomenon. Perhaps the intensity of
fluorescence in each cell is proportional to
its secretory activity. Alternately, one
could postulate that the brilliantly fluorescing cells of the tumor are those which
store gonadotropin and that the less brilliantly fluorescing tumor cells are actively
secreting hormone. Whatever the reason, it
is certain that the population of cells comprising the tumor are not uniform in their
gonadotropin activity or content.
The observation of the solitary, brilliantly
fluorescing metastasis in each patient might
be explained by assuming their origin from
a single cell or clone of cells within the
primary tumor.
SUMMARY
Immunofluorescent studies have been
performed on the primary and metastatic
tumors of two patients with gonadotropinproducing bronchogenic carcinoma. The
technic has been found valuable in identifying the tumors as the specific source of
gonadotropin and in studying the precise
intracellular localization and distribution
of this hormone.
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