Abstract
Objective
Cyclooxygenases (COXs) are enzymes that catalyze the conversion of arachidonic acid to prostaglandins. Many studies have suggested that COX-2, the inducible form of COX, is important in carcinogenesis. However, little is known about the pattern of expression of COX-2 in a multistep process of malignant transformation of sinonasal inverted papilloma (IP). In this study, we investigated COX-2 expression in IPs, IPs with dysplasia, IPs with squamous cell carcinoma (SCC), and primary SCCs of sinonasal tract.
Study design
A retrospective study was conducted.
Setting
The setting was a tertiary care referral center.
Subjects and methods
The expression of COX-2 was evaluated by immunohistochemistry in 56, 7, 18, and 17 cases of IPs, IPs with dysplasia, IPs with SCC, and primary SCCs, respectively. Furthermore, we investigated the possible correlation between the expression of COX-2 and clinicopathologic variables in patients with IPs with SCC and primary SCC patients.
Results
Positive immunoreactivity for COX-2 was observed in 3 (5.4%) of 56 IPs, 7 (38.9%) of 18 IPs with SCC, and 7 (41.2%) of 17 primary SCCs, whereas it was not observed in IPs with dysplasia. The percentage of tumors with COX-2–positive immunostaining was significantly higher in IPs with SCC and primary SCCs compared with benign IPs. There was no significant correlation between the expression of COX-2 and clinicopathologic variables, such as tumor stage, histologic differentiation, and the proportion of malignant areas in patients with IPs with SCC.
Conclusion
Cyclooxygenase-2 may play an important role in the process of malignant transformation from IP to SCC.
1
Introduction
A sinonasal inverted papilloma (IP) is a benign epithelial tumor arising from the sinonasal mucosa. Although IPs are rare tumors, clinicians have focused on IPs because of the tendency of IPs to recur, multicentricity, and the potential for malignant transformation . It has been suggested that IPs can progress to dysplasia, and then to squamous cell carcinoma (SCC) through a stepwise series of histologic changes . Recently, many studies have been conducted to elucidate the mechanism underlying the malignant transformation of IPs. A variety of molecular abnormalities including defects in cell cycle regulation, apoptosis, and loss of intercellular adhesion molecules have been suggested to contribute to the malignant transformation from IP to SCC . Some investigators have suggested that a high-risk type of human papilloma virus infection may be an important etiologic factor in the carcinogenetic process of IPs . However, the mechanisms underlying the malignant transformation of IPs have not been fully understood.
Cyclooxygenases (COXs) are enzymes that catalyze the synthesis of prostaglandins from arachidonic acid. There are 2 isoforms of the COX enzymes, COX-1 and COX-2, and most tissues have constitutive expression of COX-1. In contrast, COX-2 is inducible and is often found to be overexpressed in inflammation and in many types of cancer. The COX-2 gene is considered to be an immediate early response gene with multiple inducers such as growth factors, oncogenes, carcinogens, and tumor-promoting phorbol esters . The constitutive isoform, COX-1, is essentially unaffected by these factors.
Recently, a large body of evidence from a variety of experimental studies suggests that COX-2 is important in carcinogenesis. Oshima et al provided first direct genetic evidence that formation of intestinal polyps in a murine model of familial adenomatous polyposis was dramatically suppressed by knocking out the COX-2 gene. Enhanced synthesis of prostaglandins, a consequence of up-regulation of COX-2, has been implicated in carcinogenesis by stimulating cell proliferation , promoting new vessel formation , inhibiting apoptosis , and increasing malignant cell invasion . Elevated COX-2 expression has been demonstrated in numerous neoplasms, including colorectal , breast , lung , esophageal , thyroid , head and neck , and sinonasal cavity cancer .
To date, however, there have been no studies to determine the expression of COX-2 in a multistep process of malignant transformation of IPs. In the current study, we determined the immunohistochemical expression of COX-2 in benign IPs, IPs with dysplasia, and IPs with SCC. We also determined COX-2 expression in primary SCCs of sinonasal tract to compare with those of IPs with SCC. Furthermore, we investigated for possible correlations between COX-2 expression and clinicopathologic variables in patients with IPs with SCC and primary SCC patients.
2
Materials and methods
2.1
Patients
A total of 98 patients who underwent surgery in the Department of Otorhinolaryngology at Chungnam National University Hospital for IPs (n = 56), IPs with dysplasia (n = 7), IPs with SCC (n = 18), and primary SCCs (n = 17) of sinonasal tract between 1992 and 2010 were selected for the study. This study was approved by the Institutional Review Board of Chungnam National University Hospital. The age and sex distributions of the patients are summarized in Table 1 .
| No. of cases | Age (y) | Sex | |||
|---|---|---|---|---|---|
| Mean | Range | Male | Female | ||
| IP | 56 | 51.3 | 16–80 | 47 | 9 |
| IP with dysplasia | 7 | 58.6 | 32–81 | 6 | 1 |
| IP with SCC | 18 | 59.3 | 42–86 | 14 | 4 |
| Primary SCC | 17 | 62.8 | 39–82 | 13 | 4 |
Formalin-fixed, paraffin-embedded tissue specimens were obtained from pathology archives. Hematoxylin and eosin–stained sections were reexamined to confirm the original diagnosis and histologic grading. The following cytologic features observed within IPs were used to establish the diagnosis of dysplasia: nuclear atypia, hyperchromasia, increased mitotic rate and mitoses above the basal layer, and abnormal mitotic figures.
In IPs with SCC and primary SCC patients, histologic grading of malignant cells was performed according to the World Health Organization classification as well, moderately or poorly differentiated. Tumors were staged according to the tumor staging system adopted by the American Joint Committee on Cancer in 2010 . In IPs with SCC, the percentage of malignant areas in the entire tumor tissue was estimated in representative tissue section slides and then classified as low grade (≤50%) and high grade (>50%).
2.2
Immunohistochemistry
All immunohistochemical staining was carried out on formalin-fixed, paraffin-embedded tissue. Sequential tissue sections were cut by using a microtome, set at 3 μ m thick. The tissue sections on the microslides were deparaffinized in xylene, hydrated in serial dilutions of alcohol. Antigen retrieval was done in a PTLink machine (Dako, Glostrup, Denmark) with prewarmed 10 mM sodium citrate buffer with a pH 6.0 at 97°C for 20 minutes. Endogenous peroxidase was blocked by incubation in 3% H 2 O 2 for 10 minutes. To minimize nonspecific staining, sections were incubated with Serum-Free Protein block solution (Dako, Carpinteria, CA) for 20 minutes. The sections were then incubated with primary antibodies overnight at 4°C. A mouse monoclonal antibody against human COX2 (Dako, Glostrup, Denmark) was used at a dilution of 1:100. After slides were washed with TBS, they were further incubated with EnVision anti-mouse (Dako, Glostrup, Denmark) polymer for 30 minutes. After rinsing, antigen-antibody reactions were detected with 3, 3′-diaminobenzidine tetrachloride solution for 5 minutes. The slides were lightly counterstained with Meyer hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted with Canada balsam.
2.3
Analysis of immunohistochemical staining
The percentage of immunoreactive cells was estimated in 10 random 400 microscopic fields. The positivity for COX-2 was defined as positive staining in 10% or more of the tumor cells. To achieve a more precise assessment of positive expression, 3 pathologists, blinded to the stage and patient profiles, reviewed the immunohistochemically stained sections.
2.3.1
Statistical analysis
SPSS (version 14.0; SPSS, Inc, Chicago, IL) was used for statistical analysis. The significance of immunoreactivity of COX-2 in different groups or with different clinicopathologic variables was investigated using the χ 2 test. P < .05 was considered statistically significant.
2
Materials and methods
2.1
Patients
A total of 98 patients who underwent surgery in the Department of Otorhinolaryngology at Chungnam National University Hospital for IPs (n = 56), IPs with dysplasia (n = 7), IPs with SCC (n = 18), and primary SCCs (n = 17) of sinonasal tract between 1992 and 2010 were selected for the study. This study was approved by the Institutional Review Board of Chungnam National University Hospital. The age and sex distributions of the patients are summarized in Table 1 .
| No. of cases | Age (y) | Sex | |||
|---|---|---|---|---|---|
| Mean | Range | Male | Female | ||
| IP | 56 | 51.3 | 16–80 | 47 | 9 |
| IP with dysplasia | 7 | 58.6 | 32–81 | 6 | 1 |
| IP with SCC | 18 | 59.3 | 42–86 | 14 | 4 |
| Primary SCC | 17 | 62.8 | 39–82 | 13 | 4 |
Formalin-fixed, paraffin-embedded tissue specimens were obtained from pathology archives. Hematoxylin and eosin–stained sections were reexamined to confirm the original diagnosis and histologic grading. The following cytologic features observed within IPs were used to establish the diagnosis of dysplasia: nuclear atypia, hyperchromasia, increased mitotic rate and mitoses above the basal layer, and abnormal mitotic figures.
In IPs with SCC and primary SCC patients, histologic grading of malignant cells was performed according to the World Health Organization classification as well, moderately or poorly differentiated. Tumors were staged according to the tumor staging system adopted by the American Joint Committee on Cancer in 2010 . In IPs with SCC, the percentage of malignant areas in the entire tumor tissue was estimated in representative tissue section slides and then classified as low grade (≤50%) and high grade (>50%).
2.2
Immunohistochemistry
All immunohistochemical staining was carried out on formalin-fixed, paraffin-embedded tissue. Sequential tissue sections were cut by using a microtome, set at 3 μ m thick. The tissue sections on the microslides were deparaffinized in xylene, hydrated in serial dilutions of alcohol. Antigen retrieval was done in a PTLink machine (Dako, Glostrup, Denmark) with prewarmed 10 mM sodium citrate buffer with a pH 6.0 at 97°C for 20 minutes. Endogenous peroxidase was blocked by incubation in 3% H 2 O 2 for 10 minutes. To minimize nonspecific staining, sections were incubated with Serum-Free Protein block solution (Dako, Carpinteria, CA) for 20 minutes. The sections were then incubated with primary antibodies overnight at 4°C. A mouse monoclonal antibody against human COX2 (Dako, Glostrup, Denmark) was used at a dilution of 1:100. After slides were washed with TBS, they were further incubated with EnVision anti-mouse (Dako, Glostrup, Denmark) polymer for 30 minutes. After rinsing, antigen-antibody reactions were detected with 3, 3′-diaminobenzidine tetrachloride solution for 5 minutes. The slides were lightly counterstained with Meyer hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted with Canada balsam.
2.3
Analysis of immunohistochemical staining
The percentage of immunoreactive cells was estimated in 10 random 400 microscopic fields. The positivity for COX-2 was defined as positive staining in 10% or more of the tumor cells. To achieve a more precise assessment of positive expression, 3 pathologists, blinded to the stage and patient profiles, reviewed the immunohistochemically stained sections.
2.3.1
Statistical analysis
SPSS (version 14.0; SPSS, Inc, Chicago, IL) was used for statistical analysis. The significance of immunoreactivity of COX-2 in different groups or with different clinicopathologic variables was investigated using the χ 2 test. P < .05 was considered statistically significant.
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