Arch Iranian Med 2003; 6 (4): 278 – 281
IMMUNODETECTION OF SIALOSYL-TN (MUCIN-
ASSOCIATED ANTIGEN) ON MESOTHELIAL AND
CARCINOMATOUS CELLS IN SEROSAL CAVITY
Tofigh Javadzadeh MD , Shahram Sane MD
Department of Pathology, Shaheed Beheshti University of Medical Scienses,Tehran, Iran
Background – Discrimination of malignant cells from reactive mesothelium in body cavity
effusions is a diagnostic problem in some cases. In an attempt to resolve this problem, several
accessory techniques have been tried without general acceptance.
Sialosyl-Tn (STn) is a mucin carbohydrate-associated antigen, which represents an aberrant
glycosylation product of cellular mucin carcinomas. This antigen may sparsely be detected in
normal epithelial cells such as goblet cells of small and large intestinal mucosa, gallbladder,
urinary bladder, salivary glands, and basal cells in normal laryngeal stratified epithelium;
besides, it is frequently expressed in dysplastic epithelium and epithelial carcinomas. We
investigated the STn monoclonal antibody for its clinical utility as an isolated stain to
distinguish metastatic carcinomatous cells from benign mesothelium.
Methods – In this study, cell block materials from the serosal cavity fluids of 76 cases (48
with malignant and 28 with nonmalignant effusions) were immunostained for detecting STn.
Specimens were retrieved from the archives of the pathology departments of the teaching
hospitals of Shaheed Beheshti University of Medical Sciences, Tehran, Iran from March 2000 to
Results – Strong immunoreactivity was noted in 42 (95%) and negative staining was
observed in 2 (4.5 %) of the 44 carcinoma cases. Benign effusions showed no strong staining
but weak response was seen in 5 (18%) of the 28 benign cases. No immunoreactivity was noted
in the 4 noncarcinomatous malignant effusions. The diagnostic value indexes of STn
immunostaining test were as follows: specificity: 100%, sensitivity: 95%, positive predictive
value: 100%, negative predictive value: 93%, and accuracy rate: 92%.
Conclusion – This method is of great value in discriminating metastatic carcinomatous cells
in body cavity effusions. STn immunostaining can also be used to differentiate carcinomatous
from noncarcinomatous malignant effusions.
Archives of Iranian Medicine, Volume 6, Number 4, 2003: 278 – 281.
Keywords • carcinoma • effusion • mesothelium • Sialosyl-Tn
Introduction tatic carcinomatous cells making their discrimina-
tion difficult. 1, 2
Cytoplasmic vacuoles, high nuclear/ cyto-
I n most cases, malignant cells are easily
identified in serous fluids because they
make up a second population of cells
histologically distinct from the mesothelial cells. In
some benign effusions, the mesothelial cells may
plasmic ratios, hyperchromasia, and rare mitotic
figures in reactive mesothelial cells can mimic
malignancy. Glandular or acinar arrangement of
mesothelium and the formation of papillary
display marked morphologic similarity to metas- structures may occur in some benign mesothelial
proliferations, which make the pathologist regard
•Correspondence: T. Javadzadeh MD, Department of Pathology,
Shaheed Beheshti University of Medical Sciences, Tehran, Iran. Fax:
them with a high degree of suspicion. However,
+98-21-8088330, E-mail: firstname.lastname@example.org. the single cells of some malignant tumors such as
278 Archives of Iranian Medicine, Vol 6, No 4, October 2003
T. Javadzadeh, S. Sane
most lymphomas, some adenocarcinomas of breast Seventy-six cases of benign and malignant
and stomach, and melanoma exfoliate may be so effusions were collected from the archives of
monomorphous that a careful examination of pathology departments of three teaching hospitals
cellular details would be crucial for an accurate affiliated to Shaheed Beheshti University of
diagnosis.2, 3 Medical Sciences, Tehran, Iran (Shohada,
A host of diagnostic adjuncts have been Modarres, and Labbafinejad Hospitals) from
investigated as a means of discriminating benign March 2000 to February 2001. All specimens were
mesothelial cells from malignant ones. fixed in formalin prior to embedding in paraffin.
Specifically, flowcytometry, Argyrophilic All cellular cell blocks containing diagnostic cells
Nucleolar Organizer Region (AgNOR) staining, in on the last hematoxylin- and eosin-stained slides
situ hybridization, and detection of K-ras mutation were cut from the block. The diagnosis was based
in fluids supernate by polymerase chain reaction on cellular morphology and confirmed by available
(PCR) have been examined but have not gained clinical history. Immunohistochemistry was
widespread acceptance.4 – 6 performed using avidin-biotin complex through
Multiple monoclonal antibodies against indirect method. In brief, 4-µ sections on coated
antigens such as carcinoembryonic antigen (CEA), slides were deparaffinized in xylene after a 48-
Leu Ml, Ber EP4, AuA-l, HMFG2, epithelial hour incubation period at 37°C and then rehydrated
membrane antigen (EMA), and Vimentin have in 100%, 96%, and 70% alcohol. The slides were
been evaluated as diagnostic aids, however, no
consensus has been reached regarding an optimal
antibody or antibody panel.1, 3
Sialosyl-Tn is the product of aberrant
glycosylation of the Tn glycoprotein, a mucin
washed in phosphate-buffered saline (PBS) for 5
minutes. Endogenous peroxidase activity was
blocked by dipping the slides in 3% hydrogen
peroxide for 5 minutes.
After successive washes in PBS and deionized
carbohydrate-associated antigen, which has gained water, the slides were placed in a citrate solution
increasing attention in recent years as a diagnostic (pH = 6) and heated in an autoclave at 120°C for
and therapeutic target in epithelial carcinomas.7 10 minutes. STn antibody was applied at a 1:50
Studies have demonstrated STn expression in a dilution and incubated for 1 hour at room
large percentage of carcinomas while limited temperature. After washing in PBS again, the
expression was detected in nonmalignant glandular sections were incubated with biotinylated
tissues (e.g. mucinous salivary gland cells, goblet secondary antimouse antibody for 10 minutes.
cells of the small intestine, and bronchus).8, 9 After the slides were washed again in PBS, they
Studies of colonic polypoid dysplasia and colonic were overlied with diaminobenzidine substrate as a
adenocarcinoma have shown increased levels of
chromogen for 15 minutes. After washing in
STn expression with progressively severe distilled water, the sections were counterstained
dysplasia. The STn antigen is also present in a with hematoxylin and mounted.
wide variety of other human carcinomas like the
carcinomas of pancreas, prostate, ovary, uterus,
Quantitation of STn staining
stomach, esophagus, breast, and gallbladder.9, 10
After the percentage of cells with strong
STn antigen can also be used as a promising
staining was estimated, the slides were graded
marker of cancer diagnosis in patients with
according to the degree of staining intensity into 3
ulcerative colitis.11 In addition, increased STn is
categories of strong, moderate, or weak staining.
associated with tumor metastases and with
Strong staining was a staining so clearly distinctive
decreased survival. It is often secreted into the
that could not be mistaken for background staining
blood stream and its circulating levels correlate
or endogenous peroxidase activity in macrophages.
with tumor burden and prognosis. If STn is not
Moderate staining was a staining greater than
expressed in normal mesothelium, this antibody
background but not as decisively positive as strong
may be of value in identifying malignant cells in
staining. Weak staining was indistinguishable from
body cavity effusions. We examined the diagnostic
background or macrophage staining. Statistical
utility of the STn antibody in detecting malignant
analysis (Chi-square and Fisher's exact tests) was
cells in this cytologic setting.
only performed on the specimens showing strong
staining. The specimens with moderate or weak
Materials and Methods staining were excluded.
Archives of Iranian Medicine, Vol 6, No 4, October 2003 279
Immunodetection of Sialosyl-Tn on Mesothelial and Carcinomatous Cells
Table 1. Distribution of effusion specimens, by origin Table 2. Clinical histories of the benign and malignant
and cytological diagnosis. effusion cases.
Effusion (total no.) Benign Malignant History Number
Pericardial (n = 3) 1 2 Heart disease 3
Peritoneal (n = 27) 15 12 Liver disease 7
Benign (n = 28)
Infectious disease 14
Pleural (n = 46) 12 34
Breast carcinoma 18
Lung carcinoma 11
Malignant (n = 48) GI tract adenocarcinoma 10
Immunohistochemistry was successfully Ovarian carcinoma 5
performed on all of the samples. The Mesothelioma 2
characteristics of the 76 specimens, their clinical Hematologic malignancy 2
histories, and cytologic diagnoses are summarized been employed to discriminate benign
in Tables 1 and 2. The number of strongly-positive mesothelium from malignant epithelial cells.
cases by the type of the malignancy is summarized Commonly used antigens whose monoclonal
in Table 3. antibodies are used for staining include CEA, Leu
Strongly-positive tumor cells were identified in
Ml, EMA, and desmin. However, these tests do not
42 of 44 (95%) cases of metastatic carcinoma. Of always give reliable results. The mesothelium has
these, 14 (33%) had fewer than 10% of tumor cells been shown to immunoreact with EMA and
strongly immunoreactive for STn. Ten to 50
nonspecifically with CEA.12 Other immunostains
percent of tumor cells were strongly such as Ber-EP4 and B72.3 have also been
immunoreactive in 21 (50%) while in 7 (17%) evaluated. Ber EP4 is 87% to 100% sensitive for
more than 50% of tumor cells were positive for detecting lung carcinoma, but it has a lower
STn staining. The staining was primarily sensitivity in cell block specimens and will stain up
cytoplasmic with associated occasional
to 13% of benign mesothelial cases and up to 15%
membranous staining seen in some cases. of mesotheliomas.13 – 15 Given the limitations of the
Staining varied among cells in the same block currently available individual stains, a better
and among cases of the same type of carcinoma. immunostain for discriminating epithelium from
The highest rate of positivity for STn was observed mesothelium would be valuable.
in gastrointestinal and lung carcinomas. One Mucins are high-molecular-weight
(5.5%) of the 18 cases of breast carcinoma and one glycoproteins with a high content of O-
(20%) of the 5 cases of ovarian carcinoma were glycosidically-linked carbohydrate. Synthesis and
negative for STn staining. The samples from other secretion of mucins are common features of
types of malignancy in this study, namely
glandular epithelial tissues. Sialosyl-Tn (NeuAc α;
mesothelioma and hematologic malignancies,
2 – 6 GalNAcαl-O-Ser or Thr) is a mucin
showed no reaction to STn antibody. Staining was
carbohydrate-associated antigen that carries
seen only in the samples of metastatic carcinoma.
aberrant or truncated carbohydrate side chains that
Of the 28 benign effusions, 5 (18%) showed weak
can be recognized by the immune system as novel
immunoreactivity. The mesothelial cells of the
epitopes.16, 10 It is expressed in a large number of
benign cases showed no immunoreactivity.
adenocarcinomas in many kinds of organs. These
Overall, the diagnostic value indexes for strong
findings indicate that STn antigen is a useful tumor
staining for STn were as follows: sensitivity: 95%,
marker especially in adenocarcinoma of the mucin-
specificity: 100%, positive predictive value: 100%,
producing organs, and suggest that the regulation
and negative predictive value: 93%.
of Sialosyl-Tn antigen synthesis in carcinomas is
Discussion different from that in normal tissues.7, 9 Expression
of STn was seen in the majority (95%) of
In recent years, monoclonal antibodies have carcinoma cases in our study. 1 – 3 Staining varied
Table 3. Number of malignant cases with strongly-positive tumor cells.
Diagnosis Negative < 10% positive 10 – 50% positive > 50% positive
Carcinoma (n = 44) 2 14 21 7
Breast (n = 18) 1 5 9 3
Lung (n = 11) 0 3 6 2
GI (n = 10) 0 4 5 1
Ovary (n = 5 ) 1 2 1 1
Mesothelioma (n = 2 ) 2 0 0 0
Hematologic malignancy (n = 2 ) 2 0 0 0
280 Archives of Iranian Medicine, Vol 6, No 4, October 2003
T. Javadzadeh, S. Sane
among cells in the same block and among cases of flowcytometric DNA analysis in detecting the
the same type of carcinoma. These findings are in presence of malignant cells in ovarian carcinoma
peritoneal fluids. Br J Obstet Gynaecol. 1995 ; 102:
line with the reports for other malignancies.12 656 – 9.
Similar to other studies,9 – 12 our results 5 Chen LM , Lazcano O, Katzmann JA, et al. The role
demonstrate that STn is not generally expressed or of conventional cytology, immunocytochemistry, and
only weakly expressed in normal mesothelial cells. flowcytometric DNA ploidy in the evaluation of body
cavity fluids: a prospective study of 52 patients. Am J
In fact, 5 (18%) of 28 benign effusions exhibited Clin Pathol. 1998; 109: 712 – 2l.
weak STn expression that could be due to 6 Zojer N, Fiegl M, Angerler J, et al. Interphase
nonspecific binding of the antibody to the protein fluorescence in situ hybridization improves the
deposited on the mesothelial cell surface. A second detection of malignant cells in effusions from breast
explanation could be the expression of STn in cancer patients. Br J Cancer. 1997; 75: 403 – 7.
7 Cho SH, Sahin A, Hortobagyi GN, et al. Sialosyl-Tn
benign mesothelium because of a reaction to the antigen expression occurs early during human
inflammatory background, a phenomenon similar mammary carcinogenesis and is associated with high
to that seen in the colonic mucosa of the patients nuclear grade and aneuploidy. Cancer Res. 1994; 54:
with ulcerative colitis. However, the sensitivity of 6302 – 5.
8 Therkildsen MH, Mandel U, Thorn J, et al. Simple
STn immunostaining in detection of carcinomatous mucin-type carbohydrate antigens in major salivary
cells is higher than that of B72.3 and Ber-Ep4 glands. J Histochem Cytochem. 1994; 42: 1251 – 9.
reported in previous investigations.13 – 15 9 Yonezawa S, Tachikawa T, Shin S, et al. Sialosyl-Tn
We conclude that STn can be used as a part of antigen. Its distribution in normal human tissues and
an antibody panel for differentiation of metastatic expression in adenocarcinomas. Am J Clin Pathol.
1992; 98: 167 – 74.
carcinomatous cells from reactive mesothelial and 10 Itzkowitz SH, Yuan M, Montgomery CK ,et al.
noncarcinomatous malignant cells in serosal cavity Expression of Tn, Sialosyl-Tn, and T-antigens in
effusions. Nevertheless, a precise assessment of the human colon cancer. Cancer Res. 1989; 49:
prevalence of malignant effusions along with 197 – 204.
11 Itzkowitz SH, Marshall A, Kornbluth A, et al.
additional studies on excess cell blocks should be Sialosyl-Tn antigen: initial report of a new marker of
performed to utilize STn immunostaining as an malignant progression in long-standing ulcerative
isolated stain. colitis. Gastroenterology. 1995; 109: 490 – 7.
12 Zimmerman RL, Fogt F, Bibbo M. Diagnostic utility
Acknowledgment of Sialosyl-Tn in discriminating carcinomatous cells
from benign mesothelium in body cavity effusions.
Acta Cytol. 1999; 43: 1079 – 84.
The authors wish to thank Dr. Forouzan 13 Bailey ME, Brown RW, Mody DR, et al. Ber-EP4 for
Mohammadi and Dr. Shirin Karimi from the differentiating adenocarcinoma from reactive and
Deptartment of Pathology, Masih Daneshvari neoplastic mesothelial cells in serous effusions.
Comparison with carcinoembryonic antigen, B72.3
Hospital. This investigation was supervised by and Leu-Ml. Acta Cytol. 1996; 40: 1212 – 6.
Prof. Moslem Bahadori. This study was sponsored 14 Garcia-Prats MD, Ballestin C, Sotelo T, et al. A
by the Vice Chancellor of Research, Shaheed comparative evaluation of immunohistochemical
Beheshti University of Medical Sciences, to whom marker for the differential diagnosis of malignant
pleural tumors. Histopathology. 1998; 32: 462 – 72.
the authors wish to express their gratitude. 15 Ordonez NG. Value of the Ber-EP4 antibody in
differentiating epithelial pleural mesothelioma from
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Archives of Iranian Medicine, Vol 6, No 4, October 2003 281