This document discusses immunohistochemistry and in situ hybridization detection of COX2 in salivary gland tumors. It provides background on immunohistochemistry and in situ hybridization techniques. It then describes a study that examined COX2 expression in pleomorphic adenoma and mucoepidermoid carcinoma salivary gland tumor samples using immunohistochemistry. The study found higher COX2 expression in mucoepidermoid carcinoma samples compared to pleomorphic adenoma samples.

1. immunohistochemical and
in situ hybridization
detection of cox2 in
salivary gland tumour

2. Content

3. Immunohistochemistry
is the most common application of immunostaining. which is
widely used in the diagnosis of abnormal cells such as those
found in cancerous tumors.
. immunohistochemistry can provide a powerful adjunct tool for
pathologists to identify the cellular differentiation and assign
correct classifications in difficult tumor cases

4. In situ hybridization
is a technique that allows for precise localization of a specific segment of nucleic acid within a
histologic section.
Labelling techniques
• Radioactive isotopes
• 32P
• 35S
• 3H
• Non-radioactive labels
• biotin
• digoxigenin
• fluorescent dye (FISH)

5. cyclooxygenase (COX) enzymes,
COX-1 and COX-2, perform the rate-limiting step in the synthesis of eicosanoids from
arachidonic acid.
COX-2 overexpression is associated with the progression, proliferation, and metastasis as
well as inhibition of apoptosis in various tumors.
Moreover, COX-2 is known to induce the expression of several growth factors through the
mediation of prostaglandins (9-12).
Therefore, COX-2 has a variety of activities in various tumors.

6. Introduction
• Most salivary gland tumors originate from glandular parenchyma and occur
in the parotid gland; a few arise in the minor salivary glands.1
• The salivary glands include 3 major pairs of salivary glands (parotid,
submandibular, and sublingual) and many minor salivary glands, which can
be located throughout the upper respiratory tract
• They account for 2–6.5% of all neoplasms of the head and neck.2
• Their peak incidence is in the sixth and seventh decades of life.3
• Pleomorphic adenoma is the most common benign tumor, while
mucoepidermoid carcinoma is the most common malignancy.
• Adenoid cystic carcinoma and carcinoma in pleomorphic adenoma also
occur frequently.
• 21% to 46% of salivary gland tumors are histologically malignant.2
• Although irradiation,4 heavy smoking5 and alcohol consumption6 are
associated with tumor development and incidence.

7. Pathogenesi
s

8. Materials
Twenty-eight cases of pleomorphic adenoma and 18 cases of mucoepidermoid
carcinoma were collected from the 1992–2004 pathological files of the
Chonbuk National University Hospital and H&E-stained sections were examined
by light microscopy
. All available clinical data were obtained from a review of the patients’ medical
records. The histopathological diagnosis was verified by an independent
evaluation of all H&E slides by two pathologists. The mucoepidermoid
carcinomas were graded histopathologically based on the criteria reported by
Goode et al. (18) as follows: cystic component ≤20%, four or more mitotic
figures per 10 high-power fields, neural involvement, necrosis, and anaplasia.
Of the 18 mucoepidermoid carcinomas, 11 cases were low grade and the
remaining seven were high grade. The tumor size and nodal status were
classified according to the American Joint Committee on Cancer (AJCC) staging
system. Twelve cases were T1 and the remaining six cases were T2. There was
no case of lymph node or distant metastasis. Follow-up data were obtained
from 10 mucoepidermoid carcinoma cases. The mean follow-up period was 5.2
years and there was no recurrence or cancer-related death during this period.

9. Methods
Immunohistochemistry
• Immunohistochemical staining was carried out using an EnVisionTM Detection Kit System
(Dako, Glostrup, Denmark), which is based on the peroxidase-catalyzed deposition of
biotinylated tyramide.
• Briefly, 4-μm sections were mounted onto positively charged slides, dewaxed in xylene, and rehydrated
with a graded series of ethanol
• The sections were immersed in a 0.1 M citrate buffer solution at pH 6.0. “antigen retrieval “
• After boiling twice in a microwave oven for 5 min, the slides were cooled to room temperature and
rinsed in phosphate-buffered saline (pH 7.4) for 10 min.
• The endogenous peroxidase activity was quenched by treating the specimens with 3% hydrogen
peroxide for 5 min at room temperature.
• The sections were incubated with a COX-2 specific monoclonal “1ry antibody “(Cayman Chemical, Ann
Arbor, MI, USA; diluted 1:100), or with a mouse HuR monoclonal antibody (Zymed, South San Francisco,
CA, USA; diluted 1:100) for 1 h at 37°C.
• The sections were then incubated with the secondary antibody for 30 min at room temperature,
visualized using diaminobenzidine and counterstained with Mayer's hematoxylin. The negative control
was performed by omitting the antibody.

10. Scoring
Two investigators scored the intensity of COX-2 staining in a blinded manner. All specimens with
discordant scores were re-evaluated and the consensus score was used for statistical analysis.
COX-2 expression was scored using the following scale: 0 = no staining, 1 = weak diffuse
cytoplasmic staining (may contain a stronger intensity in less than 10% of the cells), 2 =
moderate granular cytoplasmic staining in more than 10% of the cells, and 3 = strong
granular cytoplasmic staining in more than 50% of cells.
Statistical analysis
The COX-2 scores 0 and 1 were combined to represent low COX-2 expression, and scores 2
and 3 were combined to represent high COX expression.

11. High COX-2 (score 2–3) expression was observed in 13 (72.2%) of the 18 mucoepidermoid carcinoma
cases (Fig. 1a–c).
A-Strong COX-2 expression in the high grade mucoepidermoid carcinoma.
B-Moderate
C-weak COX-2 expression in the low-grade mucoepidermoid carcinomas
Results

12. High COX-2 expression was observed in eight (28.6%) of the 28 pleomorphic adenoma casas
(Fig. 1d and e).

13. COX-2 was expressed in the cytoplasm of the ductal epithelium of the normal salivary
glands, particularly in the striated ducts (Fig. 1f)

14. Increased expression of
cyclooxygenase-2 in human salivary
gland tumors

15. MATERIALS
Tissue specimens
We examined 30 specimens of SGA and 40 of SGC surgically removed from 70 patients.
The patients ranged in age from 19 to 90 years (mean: 62.7 years), and men outnumbered
women by a ratio of 1.6:1.
Forty-eight specimens were obtained from major salivary glands (including an accessory
parotid gland), 21 from minor salivary glands, and one from a mandibular osteocentric
aberrant or heterotopic salivary gland.
Histologically
The SGA included 14 pleomorphic adenomas, six Warthin tumors, three myoepitheliomas,
three basal cell adenomas and four other types of adenomas.
The SGC included eight mucoepidermoid carcinomas, eight adenoid cystic carcinomas, six
carcinoma in pleomorphic adenomas, three epithelial–myoepithelial carcinomas, three acinic
cell carcinomas and 12 other types of carcinomas

16. Antibodies and immunohistochemical studies:
Tissue specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4 μm-thick sections according to conventional
procedures.
For immunohistochemical studies, a modification of the streptavidin–biotin–peroxidase-complex (SABC) method35 was used.
• The tissue sections were deparaffinized and rehydrated in a graded series of alcohols.
• The sections were microwaved three times for 5 min each in citrate phosphate buffer (pH 6.0) for antigen retrieval.
• Endogenous peroxidase activity was blocked with 0.3% H2O2 for 15 min.
• The sections were incubated with 10% normal bovine serum albumin in phosphate-buffered saline (PBS; 1:20) for 10 min to block
non-specific background staining.
• Rabbit polyclonal antibody specific for human COX-2 (9E-825; IBL, Gunma, Japan) was then applied as a primary antibody at a
dilution of 1:10 (10 μg/mL) at 4°C overnight.
• Non-immunized rabbit serum was used as a negative control.
• Then, the streptoavidin–biotin complex (LSAB-2; Dakopatts, Copenhagen, Denmark) immunoperoxidase reaction was carried out
for 1 h at 4°C. After washing with PBS, biotinylated goat antirabbit IgG (Vector, Burlingame, CA, USA) was applied to the sections,
which were then incubated for 30 min at room temperature. Peroxidase reactivity was detected with the use of 3,3′-
diaminobenzidine tetrahydrochloride containing 0.03% H2O2. Sections were lightly counterstained with hematoxylin and were
dehydrated and mounted with glycerol gelatin.

17. RESULTS
 COX-2 expression in NSG
In all NSG specimens, grade 1 expression of COX-2 protein was observed selectively and homogeneously
in ductal epithelial cells.
COX-2 protein appeared to be localized in the cytoplasmic region consistent with the endoplasmic matrix,
but was seen in neither serous nor mucous acinar cells.

18. COX-2
expression in
SGA and SGC

19. Cyclooxygenase (COX)-2 immunostaining in various
malignant salivary gland tumors.
 (A) Adenoid cystic carcinoma and (B) acinic cell
type; right, papillary–microcystic subtype) shows
grade 3 and 4 intensity in most tumor cells. Similarly,
expression is observed in (C) squamous cell carcinoma
carcinoma.
 (E) Carcinoma in pleomorphic adenoma shows
2 and 3 intensity in the foci of undifferentiated
 (F) In contrast, positive staining of grade 1 and 2 is
number of tumor cells located at the center of the
epithelial–myoepithelial carcinoma.

20. Intensity of cyclooxygenase (COX)-2
expression in normal salivary gland (NSG),
salivary gland adenoma (SGA) and salivary
gland carcinoma (SGC).
COX-2 expression intensity differed
significantly between NSG and SGA (P <
0.05), between NSG and SGC (P < 0.001),
and between SGA and SGC (P < 0.05).
When the staining intensity of COX-2 in
SGA was compared with that in SGC, SGC
was stronger.
Only Warthin tumors showed strong
reactivity, but other SGA showed weak or
moderate reactivity. In contrast, most SGC,
except for mucoepidermoid carcinomas,
demonstrated strong reactivity.

21. Conclusion
13

22. References
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23. Thank You