Immunohistochemical Localization of Keratin-Type Proteins in
Epithelial Neoplasms
Correlation with Electron Microscopic Findings
CARMEN G. ESPINOZA, M.D. AND HENRY A. AZAR, M.D.
A rabbit antiserum prepared against human keratins isolated
from calluses was applied to sections of 108 neoplasms using
indirect immunofluorescence and immunoperoxidase technics.
The vast majority of epithelial neoplasms were strongly pos-
itive for keratin-type proteins, even in the absence of obvious
keratinization or squamous differentiation as revealed by light
microscopy. This keratin-positivity was invariably correlated
with the identification of intermediate-sized filaments arranged
in loose or dense bundles in the cytoplasm of neoplastic epi-
thelial cells. Keratin-negative neoplasms included nevi, malig-
nant melanomas, carcinoid tumors, malignant lymphomas, and
a variety of connective-tissue tumors. Immunologic identifi-
cation of keratin-type proteins was particularly helpful in es-
tablishing the epithelial nature of "undifferentiated" malignant
tumors, including oat cell carcinomas. (Key words: Immuno-
chemistry; Immunoperoxidase; Electron microscopy; Keratins;
Intermediate filaments; Epithelial neoplasms) Am J Clin Pa-
thol 1982; 78: 500-507
KERATIN-TYPE PROTEINS or prekeratins, chemi-
cally and immunologically related to intermediate-sized
(average 10 nm in diameter) epidermal tonofilaments,
have been identified in a variety of human and animal
epithelial cells with the use of immunofluorescence and
immunoperoxidase techics.51013"15 In recent years, an-
tibodies to keratin-type proteins have been found useful
in the identification of a number of epithelial tumors,
and in their differentiation from tumors of lymphoid
and mesenchymal origin.'•2'6'9,11 Generally, neoplasms
appear to maintain the type of intermediate-sized fila-
ments found in cells they derive from. Whereas keratin-
type proteins are found in a variety of human epithelial
neoplasms, mesenchymal tumors contain vimentin and/
Received November 5, 1981; received revised manuscript and ac-
cepted for publication December 1, 1981.
Supported by the Veterans Administration and an educational fund
of the Department of Pathology, University of South Florida College
of Medicine.
Presented in part at the 70th annual meeting of the International
Academy of Pathology (U. S.-Canadian Division), Chicago, March
1981.
Address reprint requests to Dr Espinoza: Laboratory Service (113),
James A. Haley Veterans Hospital, Tampa, Florida 33612.
0002-9173/82/1000/0500 $01.25 © American Society of Clinical Pathologists
500
or desmin.6 Specific intermediate-sized neurofilaments
have been found in neurons, and a glial fibrillary acid
protein has been identified in astrocytes.8,17 Antibodies
to those specific intermediate-type filaments will be used
more frequently by surgical pathologists as an aid in the
differentiation between epithelial, mesenchymal, and
neuroectodermal neoplasms.
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This paper deals with an immunohistochemical study
of 108 neoplasms from humans, using a rabbit antise-
rum prepared against human keratin-type proteins ex-
tracted from calluses. Our results indicated that the
anti-keratin reaction is particularly useful in the iden-
tification of poorly differentiated or "undifferentiated"
carcinomas of most sites. Keratin-positivity was invari-
ably correlated with the finding of cytoplasmic inter-
mediate-sized filaments associated with desmosome
(macula adherens) type junctions in neoplastic epithelial
cells. Our preliminary results are reported briefly else-
where.4
Materials and Methods
Antigen Preparation
The isolation, purification, and identification of ker-
atin proteins was performed according to the method
of Sun and Green14 with some minor modifications.
Briefly, 900 mg of human calluses were finely minced,
and extracted at room temperature as follows: (1) the
sample was vigorously homogenized at 4°C in a Pyrex®
homogenizer, then in a Polytron, using 20 mM Tris-HCl
(pH 7.4). The suspension was centrifuged at 12,000 rpm
for 20 minutes in a Beckman® J21 B centrifuge. The
pellet was resuspended in fresh buffer, and after two
additional extractions with this buffer, the pellet was
extracted three more times with 20 mM Tris buffer con-
taining 8 M urea, leaving a pellet without water-soluble
proteins. Then, the pellet was resuspended in 20 mM
Tris buffer (pH 7.4) containing 8 M urea and 10 mM
dithiothreitol (the latter to solubilize the disulfide cross-
linked protein), sonicated for 20 seconds at a setting of
Vol. 78 • No. 4 KERATINS IN EPITHELIAL NEOPLASMS
50 in an Artek® sonic 300, and ceritrifuged at 12,000
rpm for 20 minutes. The supernatant fluid was used as
antigen.
To prove the presence of keratin in the supernatant,
the formation of filaments in vitro was observed by elec-
tron microscopy after a portion of the supernatant sam-
ple was dialyzed against distilled water and mounted in
carbon-coated grids. In addition, a sample was run in
one-dimensional polyacrylamide gel electrophoresis with
11 % SDS, and found to contain three keratin bands of
molecular weights ranging from 43K (ovalbumin) to
68K (bovine serum albumin).
Antibody Preparation
Keratin proteins purified from human calluses as de-
scribed above were emulsified with Freund's complete
adjuvant and injected subcutaneously into three New
Zealand white rabbits. One milligram of protein was
used for the primary injection. This was followed with
a second injection given a month later, using 1 mg of
protein mixed with incomplete Freund's adjuvant.
Serum was collected at weekly intervals following the
second injection. Two additional injections were needed
to achieve a high titer. Antisera was run against the ker-
atin proteins using an Ouchterlony double diffusion test.
A precipitin line was obtained. The plates that were used
contained 1% agar, 0.1% sodium dodecyl sulfate, and
0.5% triton X-300 in PBS (pH 7.2) with thiomerosol.
The antibody was absorbed with normal human serum.
In addition, a portion was absorbed with keratin proteins
for control studies.
Immunoperoxidase Staining
Immunoperoxidase staining was performed according
to previously published procedures'2,16 with some mod-
ifications. Fresh tissue was fixed in 10% buffered for-
malin and then routinely embedded in paraffin. These
paraffin-embedded tissues from tumors accessed at the
James A. Haley Veterans Hospital and the Medical Clin-
ics of the University of South Florida College of Med-
icine, were cut into 4-/im thick sections, mounted in
glass slides, and dried at 60°C for 30 minutes. After the
sections were deparaffinized in xylol and placed in al-
cohol, endogenous peroxidase activity was blocked with
methanol containing 0.3% hydrogen peroxide for 30
minutes. Subsequently, the specimens were hydrated
and digested with 0.05% trypsin in 0.1% calcium chlo-
ride adjusted to pH 7.8 with 0.1 N sodium hydroxide for
35 minutes. The specimens were rinsed in distilled water
and then placed in Tris saline. Nonspecific staining was
reduced with application of normal swine serum at 1/
501
20 dilution for 10 minutes. Rabbit antihuman keratin
antiserum was used in dilutions of 1/40 to 1/100 for 30
minutes. Swine antirabbit serum protein 1/20, and rab-
bit peroxidase antiperoxidase complex 1/20 also were
applied for 30 minutes. All dilutions of antisera were
made in Tris buffer 0.1 M/?H 7.6. After each incubation,
the sections were washed three times in Tris saline. Tris
saline is a 1:10 dilution of Tris buffer 0.1 M/?H 7.6 in
saline. Antibody localization was determined by detec-
tion of peroxidase activity, effected by a 10-minute in-
cubation of the sections with 3'3' diaminobenzidine te-
trahydrochloride (Sigma Chemical Company, St. Louis,
MO), 6 mg in 10 mL of Tris buffer containing 0.01%
of freshly added hydrogen peroxide. The sections were
washed in water, counterstained with hematoxylin, de-
hydrated, and mounted in Permount.® This method
yielded a brown reaction product. All reagents for per-
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oxidase antiperoxidase staining were obtained from
Dako, Copenhagen, Denmark, and supplied by Accu-
rate Chemical and Scientific corporation, Hicksville,
New York.
Immunofluorescence Staining
The indirect immunofluorescence technic was used.
Human tissue used for immunofluorescence was either
frozen, sectioned andfixedin acetone, or formalin-fixed.
Paraffin-embedded tissue was first dewaxed and then
digested with trypsin according to the technic described
by Huang and co-workers.7 After thorough washing with
phosphate-buffered saline (PBS), the tissue was incu-
bated with rabbit antikeratin antiserum at 1/40 dilution,
and then with fluoresceinated goat antirabbit antisera
at 1/10 dilution (Behring Diagnostics, Somerville, N. J.).
Between incubations, the tissue was washed with PBS
pH 7.2. The sections were examined with a fluorescence
microscope, and a hematoxylin-eosin-stained section
was used for orientation. Control sections were incu-
bated with the primary antibody absorbed with keratin,
and in some instances, a nonimmunized rabbit serum
was used.
Electron Microscopic Studies
In selected cases, 2-mm fresh tissue fragments were
fixed in 2.5% buffered gluteraldehyde, post-fixed in 1%
osmium tetroxide, and embedded in Epon. One-mi-
crometer thick sections stained with methylene blue
were examined for orientation, and representative areas
were selected for ultrathin sectioning. The ultrathin sec-
tions were mounted in copper grids, stained with uranyl
acetate and lead citrate, and examined with a Phillips®
301 electron microscope.
502 ESPINOZA AND AZAR A.J.C.P. • October 1982

Table 1. Keratin Positivity Using Immunofluorescence and Immunoperoxidase Technics

and Correlation with Electron Microscopic Findings
Number of Cases
(with No. Keratin- EM Findings* Desmosome-
positive) Antibody Reactivity Tonofilament Complex
Keratin positive tumors
Skin and conjunctival tumors
Basal'cell carcinoma 14(14) 4+ 4+0)
Squamous cell carcinoma 5(5) 4+ 4+(5)
Sebaceous carcinoma 4(4) 4+ 4+(2)
Lung and upper respiratory tumors
Squamous cell carcinoma 12(12) 4+ 4+(12)
Adenocarcinoma and adenosquamous carcinoma 7(7) 1+ to 4+ 1+ to 3+ (7)
Large cell (undifferentiated) carcinoma 6(3) - to 4+ - to 3+ (6)
Small ceil carcinoma, oat cell type 5(4) - to 3+ (focal) 3+(5)
In few cells
Salivary mucoepidermbid carcinoma 2(2) 4+ 2+(2)
Bladder transitional cell carcinoma 3(3) 4+ 2+(3) .
Breast adenocarcinoma .2(2) 2+ • Not done
Colonic adenocarcinoma 10(2) - to ± focal ±(10)
Prostatic adenocarcinoma

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2(2) - to ± focal Not done
Extramammary Paget's disease KD 1+ Not done
Keratin negative tumors
Epithelial neoplasms
Gastric poorly differentiated adenocarcinoma 2 -(2)
Renal cell carcinoma 5 - -(5)
Seminoma 1 — -d)
Neuroectodermal (including melanocyte tumors)
Nevus 2 — Not done
Malignant melanoma 6 - -(6)
Carcinoid 3 — -(3)
Malignant lymphomas (Hodgkin's and non-
Hodgkin's) 6 ' - -(6)
Atypical fibrous histiocytoma 9 - Not done
Dermatofibroma 1 - Not done
s studied are in parentheses.

Results idase technics gave comparable results; The immuno-

fluorescence technic was less time-consuming. However,
Immunohistochemical Localization of Keratin Proteins immunoperoxidase stainings offered the advantages of
The findings are summarized in Table 1. In general, permanent sections, better localization of cellular land-
both indirect immunofluorescence and immunoperox- marks, and easier photography.

FIG. \A (upper, left). Well-differentiated squamous cell carcinoma. All tumor cells are strongly positive for keratin (immunoperoxidase X400).

Fie \B (upper, right). Moderately differentiated squamous cell carcinoma. Most tumor cells stain strongly
for keratin (immunofluorescence, X250).
FIG. \C (upper middle, left). Metastatic poorly differentiated squamous cell carcinoma in cervical lymph node originating in nasopharynx
(Schmincke's tumor). In spite of their poor differentiation, all tumor cells stain strongly for keratin in contrast to the negative staining for
lymphoid cells (immunoperoxidase, X400).

FIG. \D (upper middle, right). Moderatly differentiated transitional cell carcinoma of urinary bladder. Most tumor cells stain for keratin with
varying degrees of intensity, with the superficial cells showing more intense staining (immunoperoxidase, X400).
FIG. \E (lower middle, left). Metastatic mucoepidermoid carcinoma in cervical lymph node. Several malignant cells show strong
staining for keratin (immunoperoxidase, X400).
FIG. 1F (lower middle, right). Poorly differentiated adenocarcinoma of the breast. A variable degree of staining for keratin is shown, with
most tumor cells demonstrating a weak, diffuse staining (immunoperoxidase, X400).

FIG. \G (lower, left). Papillary adenocarcinoma of the lung. Tumor cells show weak and variable staining for keraun (immunoperoxidase, X400).

FIG. \H (lower, right). Malignant lymphoma of the skin, infiltrating adjacent epidermis. Note negative staining of lymphoma cells
in contrast to the strong staining of epidermal cells (immunoperoxidase, X400).
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T * £f*,r
KERATINS IN EPITHELIAL NEOPLASMS
Vol. 78 • No. 4
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A.J.C.P. • October 1982
ESPINOZA AND AZAR
504
Vol. 78 • No. 4 KERATINS IN EPITHELIAL NEOPLASMS 505
FIG. 2 (upper). Electron micrograph of transitional cell carcinoma cell (same as Fig. ID). Note the presence of numerous delicate and slightly
interlacing intracytoplasmic filaments without formation of dense tonofilament bundles. Uranyl acetate and lead citrate (X 16,530).

FIG. 3 (lower). Electron micrograph of a mucoepidermoid carcinoma cell. Discrete intracytoplasmic filaments, similar to those seen in the
transitional cell carcinoma, are found without condensation into dense tonofilaments. Uranyl acetate and lead citrate (X43,500).

The tumors that were studied were placed in two
broad categories according to their reaction with the
anti-keratin serum: keratin-positive and keratin-nega-
tive. There was, however, a considerable overlapping
between the degree of positive immunostaining which
was graded on a scale of 0 (negative) to 4 +. In the
keratin-positive group, a strong reaction was observed
in all squamous cell carcinomas regardless of tissue of
origin (i.e., ectodermal of skin and conjunctiva, endo-
dermal of esophagus and lung). In well-differentiated
and moderately differentiated squamous cell carcino-
In the weakly or focally positive group, the pattern
of staining for keratin was either limited to a few tumor
cells, or it was diffuse but weakly positive. This applied
to two examples of adenocarcinoma of the breast and
to adenocarcinomas of the lung (Figs. IF, \G). Occa-
sional tumor cells of adenocarcinomas of the colon and
prostate, and of an extramammary Paget's disease gave
weakly positive reactions for keratin.
Keratin-negative tumors included two examples of
poorly differentiated gastric adenocarcinomas, five renal
cell carcinomas, and a seminoma. All nevi, melanomas,

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mas, every single tumor cell stained for keratin. The
reaction was strongly positive even in poorly differen-
tiated squamous cell carcinomas. Basal cell carcinomas
and skin adnexal tumors, urothelial transitional cell car-
cinomas, and salivary mucoepidermoid tumors were
also strongly positive for keratin (Figs. I A, IB, \C, ID,
and IE).
carcinoid tumors, malignant lymphomas, and fibrohis-
tiocytic tumors were negative for keratin.
Correlation with Electron Microscopic Findings
With electron microscopy, heavy bundles of tonofila-
ments as well as developed macula adherens type of

FIG. 4. Electron micrograph of renal cell carcinoma. The cytoplasm of two neighboring tumor cells contain empty spaces formerly occupied
by glycogen and a variety of organelles but no fibrils. Note also absence of intercellular junctions. Uranyl acetate and lead citrate (X43.500).
 ESPINOZA AND AZAR
506
intercellular junctions were readily seen in squamous
cell carcinomas of all sites, and regardless of degree of
differentiation, in basal cell carcinomas and in sebaceous
carcinomas. Thesefindingscorrelate well with the strong
immunohistochemical staining for keratin of the tu-
mors.
In contrast to skin and adnexal tumors, urothelial
transitional cell carcinomas (Fig. 2), and salivary mu-
coepidermoid carcinomas (Fig. 3) showed only delicate
aggregates of 10-nm filaments, as well as discrete fila-
ments, during electron microscopic examination. It ap-
pears, therefore, that the antikeratin serum can detect
the keratin cytoskeleton of epithelial cells prior to the
aggregation of keratin proteins into heavy bundles of
tonofilaments. Keratin-negative epithelial tumors such
as renal cell carcinomas failed to show, after careful
search, any cytoplasmic intermediate-sized filaments or
any macula adherens type junctions (Fig. 4). Likewise,
in the few instances in which 10-nm filaments or finer
fibrils were observed in non-epithelial tumors, they were
not clustered in bundles or linked to desmosomes. It
was assumed that these fibrils and filaments did not be-
long to the class of keratin proteins.
Discussion
From the initial studies of localization of keratin in
normal human epithelial tissues, it was anticipated that
tumors of epithelial origin would be positive, while non-
epithelial tumors should give a negative staining for ker-
atin.10 Recent studies of the localization of keratin in
human neoplasms of diverse origin have confirmed this
hypothesis to a large extent.1'3,6'9" Schlegel and col-
leagues,9 in their survey of a large number of neoplasms
from humans, found that the use of keratin antisera was
helpful in establishing the epithelial nature of primary
or metastatic poorly differentiated neoplasms of the
lung. Battifora and associates3 found the use of antikera-
tin antibody helpful for distinguishing thymomas from
lymphomas and seminomas. They observed positive
staining for keratin only in the epithelial cell of the thy-
momas and thymic hyperplasias. Their findings showed
a good correlation with electron microscopic studies.
Asa and co-workers1 also reported the usefulness of the
localization and differential diagnosis of craniopharyn-
giomas from pituitary adenomas. The results obtained
in the present study agreed with those of Schlegel and
colleagues,9 and the more recent observations of Sieinski
and associates," and Gabbiani and co-workers.6 Positive
staining for keratin was observed in the vast majority
of epithelial tumors studied. All basal cells, squamous
cell, sebaceous, transitional cell, and mucoepidermoid
A.J.C.P. • October 1982
carcinomas were positive for keratin. Focal staining for
keratin was distinctly observed in adenocarcinoma of
lungs, carcinomas of the breast, as well as in carcinomas
of the colon and prostate. However, renal cell carcino-
mas stained negatively for keratin. Without exception,
tumors of nonepithelial origin (i.e., malignant lympho-
mas, nevi, malignant melanomas, carcinoid, and sem-
inoma) gave a negative staining for keratin.
The use of immunohistochemical localization of ker-
atin proteins with immunofluorescence and immuno-
peroxidase in human tumors provides the pathologist
with a reliable and reproducible procedure for the di-
agnosis of epithelial tumors. Combined with electron
microscopy, the use of immunohistochemical technics
has helped in reaching a more precise histopathologic
diagnosis in the majority of neoplasms that were con-
sidered to be "undifferentiated" by routine light micro-
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scopic examination.2 With the increasing use of anti-
bodies to specific intermediate-sized filaments and other
stable cellular antigens, the surgical pathologist will be
better prepared to clarify the histogenesis of tumors still
called "large," "small," "pleomorphic," "spindle-
shaped," "myxoid," or "organoid." Antibodies raised
against intermediate-sized filaments appear to be di-
rected against groups of cells with shared embryologic
origin or similar differentiation (i.e., epithelium, con-
nective tissue or mesenchyme, glial, and neural cells),
and are not species specific. There is, however, some
merit in raising antibodies against individual polypep-
tide bands of purified human intermediate-size filaments
and determining their presence or absence in individual
epithelial and non-epithelial neoplasms.
Acknowledgments. The authors thank Dr. John U. Balis and Dr.
Paul Byvoet for their advice and guidance in this study. The technical
assistance of Miss June E. Paciga, Mr. Walter McAllister, Mr. George
Harrison, and Mr. George Kasnic, Jr., is gratefully acknowledged. The
secretarial assistance of Mrs. Bernadette Farnsworth and Mrs. Lynn
Hubbard is greatly appreciated.
References
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