[CANCER RESEARCH
46,866-876,
February 1986]
Immunohistochemical Localization of Placental Alkaline Phosphatase,
Carcinoembryonic Antigen, and Cancer Antigen 125 in Normal and
Neoplastic Human Lung1
Etienne J. Nouwen, Dirk E. Rollet, Marlene W. Eerdekens, Patricia G. Hendrix, Tony W. Briers, and
Marc E. De Broe2
Department of Nephrology and Hypertension, University Hospital Antwerp, Wilrijkstraat 10, B-2520 Edegem, Belgium
ABSTRACT
interest in view of the increased serum levels of HPLAP in
cigarette smokers (9-11), for whom the increased risk for lung
Human placental alkaline phosphatase (HPLAP), carcinoembryonic antigen (CEA), and cancer antigen 125 (CA 125) were
localized immunohistochemically in paraffin sections of normal
lung tissue from 16 patients, using monoclonal antibodies and
an indirect avidin-biotin-peroxidase staining procedure. HPLAP
and CEA were present in epithelial cells of respiratory bronchioli
and alveolar type I pneumocytes. CEA was also observed in the
trachea!, bronchial, and bronchiolar epithelium. CA 125 was
present in the trachea!, bronchial, bronchiolar, and terminal bron
chiolar epithelium; in the trachea! and bronchial glands; and in
the pleura! mesothelium. Normal and hyperplastic type II pneu
mocytes were negative for HPLAP, CEA, and CA 125 but were
histochemically positive for nonspecific alkaline phosphatase.
Fetal lung tissue between 11 and 15 weeks of gestation was
negative for HPLAP, CEA, and CA 125. The fetal trachéaland
bronchial epithelium, trachea! glands, and pleural mesothelium
were positive for CA 125. For ten malignant pulmonary tumors
investigated, HPLAP staining was observed in five, CEA in nine,
and CA 125 in seven. The localization of HPLAP, CEA, and CA
125 in apparently normal constituents of all pulmonary speci
mens is in disagreement with the concept that the expression of
these substances in the lung is indicative of abnormal cellular
activity.
cancer is well documented (12). Furthermore the expression of
HPLAP in the lung and other normal tissues may restrict the
future application of monoclonal antibodies to HPLAP for tumor
immunoscintigraphy and immunotherapy.
The link between CEA and cancer, including pulmonary cancer,
has been extensively investigated. However, serum CEA levels
are also increased in a number of nonneoplastic diseases and,
as for HPLAP, circulating levels of CEA are slightly elevated in
healthy cigarette smokers (13,14).
Cancer antigen 125 (CA 125) is a glycoprotein derived from
an ovarian serous cystadenocarcinoma. It is present in large
amounts in most nonmucinous ovarian neoplasms and is rec
ognized by the mouse monoclonal antibody OC 125 (15). Serum
levels of CA 125 are strongly increased in ovarian cancer pa
tients, in a number of nonovarian cancer patients including lung
cancer, and in some benign chronic pathologies (16). Interference
with cigarette smoking has not been reported.
Little or no information is available on the histological distri
bution of HPLAP, CEA, and CA 125 in the normal lung, although
the presence of two of these substances has been demonstrated
biochemically. In the present study, we used monoclonal anti
bodies and sensitive immunohistochemical techniques to deter
mine the localization of these antigens on adjacent sections of
normal, pathological, and neoplastic lung tissue. The distribution
pattern for HPLAP was compared with that of nonspecific AP
activity. The presence of HPLAP, CEA, and CA 125 in the fetal
lung was also investigated.
INTRODUCTION
Since the original report of Fishman ef al. (1) on the ectopie
production of HPLAP3 in a patient with bronchiogenic squamous
cell carcinoma, the clinical significance of HPLAP as a tumor
marker for a variety of malignant tumors has been extensively
investigated by the use of different biochemical techniques. The
recent introduction of monoclonal antibodies to HPLAP has made
it possible to obtain a hitherto unreachable degree of specificity
for HPLAP with respect to the other isoenzymes of alkaline
phosphatase (AP). This has opened new perspectives for the
clinical application of HPLAP in cancer, but it has also led to the
discovery of trace expression of HPLAP in a number of clinically
normal organs, i.e., the ovary (2, 3), testis (4, 5), cervix (2, 6),
tuba (7), and lung (2, 8). The latter observation is of particular
Received 4/10/85; revised 9/9/85; accepted 9/11/85.
1This work was supported by grants from the Fonds voor Kankeronderzoek
van de Algemene Spaar-en Ujfrentekas, Nationale Loterij-FGWO (Grant 9.0005.84),
the National Program for Reinforcement of the Scientific Research (PREST/UIA
04), and a research grant from the University of Antwerp.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: AP, alkaline phosphatase; CA 125, cancer antigen
125; CEA, carcinoembryonic antigen; HPLAP, human placental alkaline phospha
tase.
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MATERIALS AND METHODS
Patients and Tissues. The whole protocol of this study was examined
and found to be ethically acceptable by the Ad Hoc Committee at the
University Hospital Antwerp.
All tissues were surgical biopsies, except the samples from Patient
16, which were obtained 12 h postmortem. Normal lung tissue was
obtained from organ donors (Patients 1 to 8) and at the occasion of a
pulmonary Echinococcus cyst resection (Patient 9). Separate samples
from the upper, intermediate, and lower pulmonary lobes were taken
from Patients 3, 4, and 5. The other tissues were taken during surgical
resection of a pathological lung with benign disease or a malignant lung
tumor. In the latter, separate samples were taken from (a) the center of
the tumor, (b) the tumor periphery including invaded lung parenchyma,
and (c) macroscopically normal lung tissue distally from the tumor. Fetal
lung tissue was obtained from intact fetuses within 12 h after prostaglandin induced expulsion.
The tissues were cut into 1.5-mm-thick slices, which were sequentially
allotted for histology and biochemical analysis. The samples were proc
essed for histology or frozen in liquid nitrogen within 1 h after receipt.
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Determination of HPLAP in Tissue Butanol Extracts. The amount of
HPLAP in tissue samples was determined by an enzyme-antigen immunoassay, based on a mouse monoclonal antibody against HPLAP (E6)
(17), after homogenization and extraction with butanol. Extraction and
assay procedures have been described in detail elsewhere (8). The
antibody does not cross-react with liver, bone, kidney, or intestinal AP.
The assay detection limit was 0.02 unit/liter. The within-assay and
between-assay coefficients of variation were 6.1 and 7.0%, respectively.
Calculation of sample estimates was performed using a spline function
program.
Determination of Nonspecific AP Activity in Tissue Extracts. The
nonspecific AP activity in tissue butanol extracts was determined by the
method of Van Belle ef al. (18). One unit of enzyme activity is defined as
the activity liberating 1 Mmol of 4-nitrophenol (£405
= 14,600 liter mor1
cm"1) from 5 mmol of 4-nitrophenyl phosphate per min in 0.1 M Nethylaminoethanol buffer, pH 10.2, at 30°C.
Immunohistochemical Localization of HPLAP, CEA, and CA 125.
The tissue distribution of HPLAP, CEA, and CA 125 was determined in
adjacent sections. The immunohistochemical
staining procedures for
CEA and CA 125 were the same as described previously for HPLAP (3).
Briefly the tissues were fixed during 1.5 h in buffered 4% formol (0.1 M
sodium cacodylate buffer, pH 7.4, containing 1% CaCfe) and were
embedded in Paraplast or in low melting point paraffin. Five-fim sections
were treated with trypsin and binding of the monoclonal antibodies was
revealed by an indirect avidin-biotin-peroxidase
method. Staining for
peroxidase was performed with 3-amino-9-ethylcarbazole. Two specific
monoclonal antibodies were used for the localization of HPLAP, i.e., E6
(17) and H317 (19). E6 and H317 culture supernatant was diluted 1/50
and 1/100, respectively. The Hybritech monoclonal antibody to CEA (Lot
360791), was used at a dilution of 1/100,000. Mouse ascitic fluid
containing the monoclonal antibody OC 125, recognizing the antigen CA
125 (20), was diluted 1/3000. Treatment of sections with trypsin yielded
significantly stronger and more consistent staining with each of the four
mouse monoclonal antibodies. The sections were counterstained with
methyl green and were mounted in glycerin-gelatin. Negative control
staining was performed on adjacent sections using a monoclonal anti
body of irrelevant specificity or normal mouse serum. Full-term placenta,
a colon carcinoma, and an ovarian carcinoma, processed in an identical
way, were used as positive controls for HPLAP, CEA, and CA 125
staining, respectively.
Histochemical Localization of Nonspecific AP. Paraffin sections
adjacent to those for the immunohistochemical demonstration of HPLAP
were used to localize nonspecific AP by the method of Gossrau (21) with
5-bromo-4-chloro-3-indoxylphosphate-p-toluidine
salt as the substrate
and tetranitroblue tetrazolium as the capturing agent. Histochemical
inhibition studies were performed by adding 1 mw D-p-bromotetramisole,
1 ITIM L-p-bromotetramisole (18), 50 mw o-phenylalanine, or 50 rriM Lphenylalanine to the incubation mixture. The sections were counterstained with methyl green and mounted in glycerin-gelatin.
Routine Histology. Adjacent sections were stained either with Carazzi's hematoxylin and eosin, with periodic acid-Schiff reagent and
hematoxylin, or with Masson's trichrome staining.
RESULTS
The pertinent clinical and histopathological data of the patients
are presented in Table 1.
Table 2 summarizes the nonspecific AP and HPLAP content
of butanol extracts from normal and nonneoplastic lung tissues,
the histochemical grading for nonspecific AP, and immunohisto
chemical grading for HPLAP, CEA, and CA 125 in adjacent
histológica! sections from the same tissue samples. There was
a broad correlation between the extent of HPLAP staining in
histological sections and the amount of HPLAP measured in
extracts. Normal lung tissue contained 58.8 ±54.2 (SD) milliunits
HPLAP/g tissue (n = 16), corresponding to 4.6 ±5.6% of the
nonspecific AP content. No difference could be found between
the biopsies taken from the upper, intermediate, and lower
pulmonary lobes (Patients 3, 4, and 5). HPLAP and CEA staining
was present in all nontumoral lung tissues, ranging from scarce
to abundant.
HPLAP staining with monoclonal antibody E6 was always
more intense; hence more cells were positive than when mono
clonal antibody H317 was used. Staining with H317 was located
in regions which were also positively stained with E6. Regions
which were immunohistochemically positive for HPLAP were also
histochemically positive for nonspecific AP.
Fig. 1 gives a schematic representation of the histological
distribution patterns for nonspecific AP, HPLAP, CEA, and CA
125 in the normal lung. Immunostaining was absent in all tissues
when the primary monoclonal antibodies were replaced by a
mouse monoclonal antibody of irrelevant specificity or by normal
mouse serum (negative controls). The apical trophoblastic
plasma membrane of full-term placenta was strongly stained for
HPLAP and was negative for CEA and CA 125; the colon
carcinoma contained strong cytoplasmic staining for CEA and
was negative for HPLAP and CA 125; in the ovarian carcinoma,
the plasma membrane of all carcinoma cells was strongly positive
for CA 125 but was negative for HPLAP and CEA (positive
controls).
The normal trachéal,bronchial, and bronchiolar epithelium; the
trachéaland bronchial glands; and the endothelium of small blood
vessels were positively stained for nonspecific AP but were
negative for HPLAP. All other constituents of trachea, bronchi,
and bronchioli were negative for nonspecific AP as well as for
HPLAP. Intense CEA and CA 125 staining (Fig. 7A) was present
in trachea! and bronchial epithelial cells. CEA staining was local
ized on the apical surface and in the cytoplasm of some of these
cells. CA 125 staining was present in the mucus of goblet cells
and also in ¡ntracellulargranules and on the apical surface of
some other epithelial cells. The bronchiolar epithelium stained
weakly for CEA and strongly for CA 125. In addition, strong
cytoplasmic CA 125 staining was observed in the trachéaland
bronchial glands and their ducts (Fig. IB).
In the terminal bronchioli of the normal lung (Fig. 2), nonspecific
AP staining was present on the surface and in the apical cyto
plasm of some of the columnar epithelial cells lining the mucosa!
folds. Neither HPLAP nor CEA was present in these cells. Scarce
positive HPLAP and CEA staining was observed ¡nrounded or
flattened cells which were occasionally present between the
columnar ciliated cells and goblet cells. This staining was local
ized predominantly on the apical plasma membranes. Strong CA
125 staining was present in the apical cytoplasm and glycocalyx
of some terminal bronchiolar epithelial cells (Fig. 2, B to D). Most
of these CA 125 positive cells are goblet cells. CA 125 staining
was also observed at the level of the cellular and amorphous
debris in the terminal bronchiolar lumen (Fig. 2ß).
In the respiratory bronchioli (Fig. 3), the columnar ciliated or
unciliated epithelial cells were sometimes positive for nonspecific
AP. Ciliated cells were negative for HPLAP. In contrast, the low
columnar unciliated cells with bulging apical cytoplasm (Clara
cells) were occasionally moderately positive for HPLAP (Fig. 3D).
Cuboidal and rounded cells lining the respiratory bronchioli were
heavily stained for HPLAP and for AP. Some of them were also
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Table 1
Patients' clinical data and histopathology
PatientNormal123456789Age(yr)272234202017141730SexFMMMMMMFFS/NS"SNSNSNSNSNSNSNSNSPathologyNormal
Grading0
lungNormal
lungNormal
lungNormal
lungNormal
lungNormal
lungNormal
lungNormal
lungPulmonary
Echinococcus cystStaging"
Benign pulmonary pathology
10
46
Bronchiectasis
NS
Malignant
tumors1112131415161718192070557254506363564761MMMMFFMMMMSSSsssssssEpidermoid
lung
carcinomaEpidermoid
carcinomaEpidermoid
carcinomaCombined
epidermoid/adenocarcinomaBronchiogenic
adenocarcinomaBronchiogenic
adenocarcinomaBronchiogenic
adenocarcinomaBronchioloalveolar
carcinomaLarge
carcinomaSmall
cell
cell carcinoma132223133122-333133333
" S, cigarette smoker; NS, nonsmoker.
6 According to the TNM (tumor-nodes-metastasis) classification (47).
c 1, well-differentiated; 2, moderately well-differentiated; 3, poorly differentiated.
positive for CEA. CEA staining in respiratory bronchiolar cells
was in general less intense than HPLAP staining. HPLAP and
CEA staining was localized predominantly at the luminal plasma
membranes. No staining for CA 125 could be observed in the
respiratory bronchioli.
Fig. 4 illustrates the alveolar nonspecific AP staining patterns
in the normal lung (Patient 3) without inhibitors (Fig. 4A; Fig. 5A)
and in the presence of L-p-bromotetramisole (Fig. 46) or Lphenylalanine (Fig. 4C). Staining for nonspecific AP was present
in isolated protruding cells, suggestive for type II pneumocytes,
and less frequently in epithelial cells bearing attenuated cytoplasmic extensions, suggestive for type I pneumocytes. L-pBromotetramisole addition suppressed nonspecific AP staining
in type II pneumocytes, without affecting staining of type I
pneumocytes. In contrast L-phenylalanine suppressed nonspe
cific AP staining in type I pneumocytes without affecting staining
of type II pneumocytes. D-p-Bromotetramisole and o-phenylalanine had no effect on the histochemical pattern for nonspecific
AP. The pattern of alveolar HPLAP ¡mmunohistochemical staining
(Fig. 5, B and C) grossly resembles that of AP staining with L-pbromotetramisole inhibition. Indeed positive HPLAP staining was
observed only in attenuated cytoplasmic extensions along small
or extended areas of the alveolar surfaces and their perinuclear
apical plasma membrane. Positive cells were present on one or
both surfaces of the alveolar septa. Small to extended focal
alveolar surface areas were stained for CEA (Fig. 5D). The picture
is identical to that of observed alveolar localization of HPLAP.
However, the ratio of HPLAP and CEA staining intensity varied
greatly between different biopsies, although all stainings were
performed simultaneously. All components of normal alveoli were
negative for CA 125 staining.
Macrophages were in general negative for nonspecific AP,
HPLAP, CEA, and CA 125. However, in the respective positive
regions, diffuse intracellular staining was observed. Granulocytes
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in alveolar capillaries and alveolar lumina were positively stained
for CEA. The pulmonary interstitium and arteries and the alveolar
capillary endothelium were devoid of HPLAP, CEA, and CA 125
staining, whereas a diffuse and weak nonspecific AP staining
was sometimes present.
The visceral pleura was negative for HPLAP and CEA, but the
visceral pleural mesothelium was positive for CA 125 (Fig. 9).
Lung tissue distantly from the tumors was in all aspects
identical to normal lung tissue from healthy organ donors. In
contrast, lung tissue at the tumoral invasion front was atelectatic
and affected by retrotumoral obstructive pneumonia. It was
characterized by proliferation of the alveolar interstitium and by
the presence of numerous nonspecific AP positive cells lining the
alveolar lumina (Fig. 6). HPLAP, CEA, and CA 125 staining was
absent or very scarce in these strongly AP positive regions.
Table 3 summarizes the nonspecific AP and HPLAP content
of lung tumor extracts; the histochemical grading for nonspecific
AP; and the ¡mmunohistochemical grading for HPLAP, CEA, and
CA 125 in adjacent histológica! sections from the same tissue
specimens as used for biochemical analysis. Fig. 11 illustrates
the staining pattern for CEA and CA 125 in a poorly differentiated
bronchioloalveolar carcinoma (Patient 18). Figs. 12 and 13 dem
onstrate the histological distribution patterns for nonspecific AP,
HPLAP, CEA, and CA 125 in a moderately well differentiated
epidermoid carcinoma (Patient 11) and in a poorly differentiated
bronchiogenic adenocarcinoma (Patient 16), respectively. Of the
10 malignant lung tumors 5 were immunohistochemically positive
for HPLAP. Some HPLAP negative tumors contained appreciable
nonspecific AP staining. Nine tumors contained CEA staining;
the histological distribution ranged from scarce to virtually uni
form. CEA staining in the carcinoma cells was localized on the
apical plasma membranes of acini or was combined membranous-cytoplasmic. Seven tumors were immunohistochemically
positive for CA 125. Staining was localized on the plasma mem-
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TUMOR ANTIGENS
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Table 2
Normal lung tissue and benign lung pathologies
contentsPatientNormal
distribution*HPLAP
Tissue
AP22.1
CA125++++
CEA
++++
+++++
++
++
++++
++
++
++
+—
+
+
+++
++
+
+
+
+
+
tissue123456789Normal
lung
+++7.3
+++11.2
+++1.1
+++1.9
++4.4
+++0.8
+++0.6
++++1.1
+++1.2
bronchus3516Normal
+++0.9
+++1.1
ND0.4
+++—
+++ND
ND—
+++3.4
+++++
+++
++8.2
+++0.2
+++2.7
+++3.5
++1.6
++2.8
+++0.2
+-(•++
"+
++
+
++
++++
++
+++
++
+++
++
+++
+
+
+++0.8
++++ND
++++0.5
++++1.2
++2.4
++1.3
++
-"+++
+++
"_6++
++
-1+++
+
trachea3Normal
++
++
ND
distantfrom
lung tissue
lungtumor11121314151718Lung
malignant
—
tissue at tumoral in
front111213141718Lung
vasion
+++
—
+
benignlung
tissue from
pathology10*
++++not
+
+
-, absent; +, scarce; ++, weak;AP(milliunits/g)82195215041956151917132287350229432121123814418501466838164712822920147113661770ND17501917548690+++,
moderately strong;HPLAP(milliunits/g)18169.816820.624.274.817.221.73.23.71.92.70.662.71201.445.244.645.241.62.514.8ND9.422.513.28.7+
strong; ND,% determinedHistological
branes and in intracellular inclusions.
As shown in Table 4, human fetal lung tissue at a pregestational age of 11 to 15 weeks contained considerably less non
specific AP than did adult normal lung tissue, and only small
amounts of HPLAP could be detected. The fetal lung was neg
ative for HPLAP and CEA staining. In the 11-week-old fetus, a
few pulmonary tubules were moderately positive for CA 125,
whereas the other fetuses were negative. The fetal visceral
pleural mesothelium was positive for CA 125 (Fig. 10) but was
negative for HPLAP and CEA. The apical plasma membrane of
the fetal trachea! and bronchial epithelium and the trachéalglands
were also positive for CA 125 (Fig. 8). Only in Fetus 5 was the
trachéalepithelium moderately positive for CEA.
DISCUSSION
The link between HPLAP and the human lung dates from
1968, when Fishman ef al. (1) described its presence in serum
of a lung carcinoma patient. In addition to the association of
HPLAP with pulmonary cancer, this enzyme has also been
detected biochemically in normal lung tissue (22). Using rabbit
polyclonal antisera and several inhibitors, these authors demon
strated that most of the AP activity in lung tissue extracts is of
the liver-bone-kidney type. The heat-stable fraction of AP
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showed polymorphic phenotype differences corresponding to
those of HPLAP. The presence of HPLAP in extracts from normal
lung tissue has been confirmed subsequently by the use of
specific monoclonal antibodies (8,10).
Nonspecific AP activity was demonstrable histochemically in
the epithelia lining the entire respiratory system, i.e., from the
trachea up to the alveoli. This is in agreement with available data
(23). In contrast, HPLAP immunohistochemical staining was ob
served only in the peripheral respiratory lung parenchyma (i.e.,
respiratory bronchioli, alveolar ducts, alveolar sacs, and alveoli).
The absence of HPLAP staining in the fetal lung at a pregestational age between 11 and 15 weeks, i.e., in the pseudoglandular
stage, is in agreement with our data on the histological distribu
tion of HPLAP in the adult lung, since in this stage only the
conducting airways up to the terminal bronchioli are formed (24,
25). Respiratory bronchioles are observed only after the 16th
week and saccules are not present before the 24th week. Further
study will be necessary to determine if HPLAP is present in the
canalicular and terminal sac stages. Whether these observations
are due to a differential embryonic origin, i.e., a presumed
mesodermal origin for the epithelium lining respiratory lung par
enchyma and the entodermal origin of the epithelium in the
conducting airways (25), remains an open question.
We have demonstratedpreviouslythat in clinicallynormal
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HPLAP
Fig. 1. Schematic representation of the histochemical distribution of nonspecific AP and
immunohistochemical distribution of HPLAP,
CEA, and CA 125 in the adult normal lung.
Thick lines or dots indicate presence of positive
staining. A, alveolar sac with alveoli; AD, alveo
lar duct; B, bronchus; Br, bronchiolus; M, pleural
mesothelium; RB, respiratory bronchiolus; HG,
respiratory glands; T, trachea; 7B, terminal
bronchiolus.
CEA
Table 3
Malignant lung tumors
Histológica! distribution8
Tissue contents
Patient
AP
(milliunits/g)
HPLAP
(milliunits/g)
AP
HPLAP
CEA
CA125
Table 4
Fetal lung parenchyma
contentFetus1
Tissue
CA125
(milliunits/g)8.7
11121314151617181920232189925191907296514255237223154203.92.12.88.027.33221.0338.78.60.20.20.10.40.922.60.24.62.
2
34
13
13
1414
57
121
148
5
82
6Age(wk)1115AP(milliunits/g)170
184HPLAP
1.6
3.1
0.5%5.1
1.0
<0.5
1.13.8
0.3
isoenzymes of AP in different celltypes of the alveolar epithelium.
L-p-Bromotetramisole sensitive, L-phenylalanine resistant non
" The histological grading concerns only the tumoral elements in the sections.
First number indicates histological distribution (% positive cells): 1, scarce (<5%);
2. focal (5-<50%); 3, predominant (50-<90%); 4, uniform (90-100%). Second
number indicates staining intensity: 1, faint; 2, weak; 3, moderately strong; 4,
strong. Letter code indicates cellular localization: c, diffuse cytoplasmic; g, intracellular granules; a, glycocalyx; m, plasma membranes.
ovarian tissue HPLAP was confined to germinal inclusion cysts
(3), considered as the origin of benign and malignant serous
ovarian tumors. In contrast, the presence of HPLAP in clinically
normal lung tissue was always associated with apparently normal
constituents of this organ. Normal lung tissue contains more
HPLAP than do most malignant lung tumors investigated. More
over HPLAP staining was present in all normal lung tissues from
smokers as well as nonsmokers. This indicates that the expres
sion of HPLAP in the lung is not involved in mechanisms of
carcinogenesis.
The difference between histochemical nonspecific AP and
immunohistochemical HPLAP staining in normal alveoli, along
with the observed patterns for nonspecific AP obtained by the
use of two inhibitors for AP, demonstrates the presence of two
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0.6<0.6
specific AP, not recognized by the monoclonal antibodies E6 and
H317 against HPLAP, is found in isolated protruding cells, com
patible with type II pneumocytes. Nonspecific AP staining in type
II pneumocytes has also been reported by other authors for the
animal (26-28) and human lung (23). In contrast, L-phenylalanine
sensitive, L-p-bromotetramisole resistant AP, recognized by the
monoclonal antibodies E6 and H317 against HPLAP, is found in
epithelial cells presenting cytoplasmic extensions, compatible
with type I pneumocytes. CEA staining was also present exclu
sively in the latter type of cells. These results show that type II
pneumocytes in normal lung tissue are negative for HPLAP,
CEA, and also CA 125. They also demonstrate that histochemical
staining for nonspecific AP in the presence of L-phenylalanine
can be used to distinguish type II pneumocytes at the light
microscopic level.
Moderate interstitial fibrosis in atelectatic lung parenchyma is
accompanied by alveolar type II pneumocyte proliferation and
hypertrophy (29,30). The peritumoral lung tissue presented this
type of histological alteration. Histochemical nonspecific AP
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ANTIGENS IN THE HUMAN LUNG
staining of individual cells was more intense than in nonprolifer-
based. Elevated plasma CEA levels have also been described in
smokers (13, 14, 38). They can be explained by the same
mechanism as proposed for HPLAP, in view of their similar
histological distribution.
CA 125 staining was observed in the central airways (trachea,
bronchi, bronchioli, and terminal bronchioli) from smokers and
nonsmokers, in the trachea! and bronchial glands, and in the
pleural mesothelium. Since this antigen was, unlike HPLAP and
CEA, never present in close contact with the pulmonary capillary
network, this may explain why, in contrast to HPLAP and CEA,
an elevation of the serum CA 125 levels in cigarette smokers
has not been reported. The expression of CA 125 in the adult
trachea, bronchi, and respiratory glands is consistent with its
presence in the corresponding fetal structures. Positive CA 125
staining in the adult pleural mesothelium demonstrates that the
expression of this antigen by the fetal coelomic epithelium (39)
continues in the adult. These observations support the hypoth
esis that the elevated serum levels of CA 125 in patients with
benign chronic liver disease (16) are probably of extrahepatic
origin and are caused by a reduced hepatic clearance of CA 125.
Our data indicate that HPLAP is a poor marker for lung
carcinomas. This is in agreement with available serological data
(19, 40, 41), using polyclonal and monoclonal antibodies and
heat stability criteria. The observed CEA staining patterns for
the different histological types of pulmonary carcinomas are in
agreement with available data (34, 42, 43). CA 125 staining,
present in seven of the ten pulmonary neoplasms, was in general
scarce. These findings confirm the limited value of serum CA
125 levels for the detection of lung cancer (20).
The observed HPLAP immunohistochemical staining patterns
are almost surely due to the presence of HPLAP because of the
well-documented specificity of the two monoclonal antibodies
used with respect to the other isoenzymes of AP and since
nonspecific AP activity could always be demonstrated histochemically in the HPLAP positive sites. For CEA and CA 125,
however, it cannot be excluded that cross-reacting substances
ating type II pneumocytes in histologically normal alveoli. How
ever, again these cells were negative for HPLAP, CEA, and CA
125 staining.
The finding that type I pneumocytes contain the tumor asso
ciated antigens HPLAP and CEA whereas their progenitor cells,
the type II pneumocytes (31, 32), are negative is intriguing. One
explanation could be that HPLAP and CEA, which are observed
on the luminal surface of type I pneumocytes, are of bronchiolar
origin, reaching the alveoli via the bronchioloalveolar surface
fluid. However, the presence of strong HPLAP and CEA staining
on well defined portions of the alveolar septa, adjacent to nega
tive zones, is a strong argument against this hypothesis. A
second hypothesis could be that some (if not all) type I pneu
mocytes are descendants from HPLAP and CEA producing
bronchiolar epithelial progenitor cells instead of type II pneumo
cytes. The continuity of HPLAP positive staining in the respiratory
bronchiolar epithelium, from the unciliated low columnar epithelial
cells with bulging apical cytoplasm (Clara cells) over the cubic
and rounded cells to the neighboring flattened bronchioloalveolar
cells adds further support to the latter hypothesis. This hypoth
esis is, however, in contradiction with the currently accepted
type II pneumocyte origin of type I pneumocytes (26, 31-33)
although a common undifferentiated precursor cell has also been
suggested (26).
Bronchiolar and alveolar staining of CEA was found in the
same type of pulmonary epithelial cells as those which contained
HPLAP. However, the histological distribution of CEA and
HPLAP staining on adjacent sections was not overlapping. Con
sequently the expression of HPLAP and CEA by single cells
seems to be independent. In addition to this difference in the
expression of CEA and HPLAP, CEA was also observed in the
conducting airways, in contrast to HPLAP. The described local
ization of CEA grossly resembles that reported by Sun ef al.
(34), using a polyclonal antiserum.
It has been reported that serum levels of HPLAP are elevated
in cigarette smokers (9-11). One may speculate on the origin of
with closely related or shared epitopes may be responsible for
the observed staining (44-46).
these elevated levels. Our results, indicating that HPLAP is
localized exclusively on the luminal plasma membrane of respi
ratory bronchiolar and probably also of alveolar epithelial cells
and the abundance of HPLAP in normal lung tissue compared to
other normal tissues (8, 10), suggest a pulmonary origin for
HPLAP in smokers. The occurrence, in the strongly HPLAP
positive zones, of diffuse HPLAP staining in the bronchiolar and
alveolar lumina and in the content of these lumina, shows that
part of the HPLAP produced by the lung parenchyma is secreted
into the bronchioloalveolar fluid. Several authors have reported
that the respiratory epithelial permeability is significantly in
creased in cigarette smokers (35-37). Consequently the in
In conclusion, the present study demonstrates that three
tumor associated antigens, HPLAP, CEA, and CA 125, are
present in clinically normal lung tissue. The immunohistochemical
staining patterns for these three antigens were not correlated.
HPLAP was localized exclusively in the peripheral airways, CEA
both in the peripheral and the central airways, and CA 125 only
in the central airways, the respiratory glands, and the pleural
mesothelium. Fetal lung tissue at the age of 11 to 15 weeks was
negative for HPLAP, CEA, and CA 125, except the pleural
mesothelium which was positive for CA 125. The presence of
these tumor associated antigens in all normal lung tissues from
smokers and nonsmokers and its association with apparently
normal pulmonary constituents do not support the hypothesis
that the expression of these antigens is indicative for precancer-
creased serum levels of HPLAP in smokers may be due to the
increased leakage of HPLAP containing bronchioloalveolar liquid
into the pulmonary capillaries. However, we were unable to
detect any significant difference in the serum levels of HPLAP
between smokers and nonsmokers,4 using the same monoclonal
ous alterations.
antibody E6 on which the present immunohistochemical data are
ACKNOWLEDGMENTS
4 D. E. Rollet, E. J. Nouwen, M. W. Eerdekens, P. G. Hendrix, and M. E. De
Eroe. Characterization of alkaline phosphatase isoenzymes from normal and neopiastic human lung tissue, submitted for publication.
Dr. Andre Van de Voorde (Department of Molecular Biology, State University of
Ghent, Belgium) is gratefully acknowledged for providing us the monoclonal anti
body E6 to HPLAP. We would like to thank Dr. P. Jeremy McLaughlin (Department
CANCER RESEARCH VOL
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TUMOR
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IN THE
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of Immunology, University of Liverpool, United Kingdom) for the batch of monoclonal
antibody H317 and Dr. Robert C. Bast (Department of Medicine, Duke University
Medical Center, Durham, NC) for the batch of OC 125 ascitic fluid. Dr. J. Lauweryns
(Laboratory of Histopathology, Catholic University of Leuven, Belgium) is gratefully
acknowledged for fruitful discussions. Drs. P. J. den Houwdyker, R. Roelens, E.
Schoofs, J. Tombeur, W. Vaneerdeweg, and R. Van Hee are gratefully acknowl
edged for providing some of the biopsies. Finally we would like to thank Simonne
Dauwe, Erik Martens, Rita Marynissen, and Denise Schellemans for their skillful
technical assistance.
CANCER
HUMAN
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1986
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Fig.2. Normal terminal bronchiolus.A, AP staining, x 160. ß,C, and D, CA 125 staining. S, x 160; C, x 200; D, x 500.
Fig. 3. Normal respiratory bronchiolus. A, C, and D, HPLAP staining. D, continuity of positive HPLAP staining from the low columnar unciliated cells over the cubic
and rounded cells to the flattened bronchioloalveolarcells; arrow, HPLAP positive Clara cell. A, x 160; C, x 500; D, x 500. B, CEA staining, x 160.
CANCER RESEARCH
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1986
873
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•.l.»A
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Fig. 4. Normal alveoli. Comparison of nonspecific AP staining on adjacent sections. /Arrows,nonspecific AP positive type II pneumocytes; arrowheads, AP positive
type I pneumocyte.A, without inhibitors, x 250. B, with 1 mw L-p-bromotetramisole.x 250. C, with 50 mw t-phenylalanine. x 250.
Fig. 5. Normal alveoli. A and B, comparison on adjacent sections of nonspecific AP staining and immunohistochemicalHPLAP staining, respectively.Arrows, type II
pneumocytes; arrowheads, type I pneumocytes.A and B, x 250. C, HPLAP staining, x 250. D, CEA staining, x 250.
Fig. 6. Atelectatic alveoli. AP staining, x 250.
CANCER RESEARCH
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1986
874
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Fig. 7. Normal bronchus with bronchial glands. CA 125 staining. A, x 96. B, detail of bronchial glands, x 230.
Fig. 8. Fetal trachea with respiratory glands. CA 125 staining, x 160.
Fig. 9. Normal visceral pleura. CA 125 staining. Arrows, nuclei of mesothelial cells, x 250.
Fig. 10. Fetal visceral pleura. CA 125 staining, x 285.
Fig. 11. Poorly differentiated bronchk>toalveolar carcinoma (Patient 18). Arrows, ¡ntracellularCA 125 positive staining. A, CEA staining, x 260. B and C, CA 125
staining. B, x 260; C, x 340.
CANCER RESEARCH
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TUMOR ANTIGENS
IN THE HUMAN LUNG
13D
Fig. 12. Moderately well-differentiated epidermoid carcinoma (Patient 11). Arrows, regions of positive staining. A, AP staining, x 150. B, HPLAP staining, x 150. C,
CEA staining, x 150. D, CA 125 staining, x 150.
Fig. 13. Poorly differentiated bronchiogenic adenocarcinoma (Patient 16). A, AP staining, x 100. B, HPLAP staining, x 100. C, CEA staining, x 100. D, CA 125
staining. Arrow, intracellularpositive staining. D, x 100.
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1986
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Immunohistochemical Localization of Placental Alkaline
Phosphatase, Carcinoembryonic Antigen, and Cancer Antigen
125 in Normal and Neoplastic Human Lung
Etienne J. Nouwen, Dirk E. Pollet, Marlène W. Eerdekens, et al.
Cancer Res 1986;46:866-876.
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