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					                                                                                   Arch Iranian Med 2003; 6 (4): 278 – 281




                                                                           ORIGINAL ARTICLE
      IMMUNODETECTION OF SIALOSYL-TN (MUCIN-
      ASSOCIATED ANTIGEN) ON MESOTHELIAL AND
      CARCINOMATOUS CELLS IN SEROSAL CAVITY
                    EFFUSIONS
                                                                      •
                                         Tofigh Javadzadeh MD , Shahram Sane MD




                                                                                  D
       Department of Pathology, Shaheed Beheshti University of Medical Scienses,Tehran, Iran




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           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,
                                                           of
       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
                                        ive

       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
       February 2001.
           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
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       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
             Ar




       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: tofighjave@yahoo.com.                             the single cells of some malignant tumors such as


278 Archives of Iranian Medicine, Vol 6, No 4, October 2003
                                                                                                                www.SID.ir
                                          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




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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
                                                           SI
                                                            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
                                              of
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
                                ive

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
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                                                            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,
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                                                            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
                                                                                                      www.SID.ir
                   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
                                                                                         Unknown                          4
                                                                                         Breast carcinoma                 18
                            Results
                                                                                         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




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                                                                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


                                                               SI
                                                                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
                                                  of
                                                                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.
                                  ive

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;
                           ch



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
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                                                                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
                                                                                                                   www.SID.ir
                                              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.




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                                                                 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




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    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:
                                                   of
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
                                   ive

                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.
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                                                                      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
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                                                                      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
                                                                                                               www.SID.ir

				
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