Inverted papilloma (IP) is a frequent benign sinonasal tumor that is characterized histologically by squamous metaplasia, epithelial acanthosis, and hyperplasia of the nasal epithelium. Because of its high recurrence rate and malignant transformation potential, careful long-term follow up is necessary.
The purpose of the current report was to study the expression of squamous cell carcinoma (SCC) antigen in sinonasal IPs and to evaluate the usefulness of SCC antigen as a biologic marker for the follow-up of patients with sinonasal IP. The expression of SCCA1 in three sinonasal IP cases, three sinonasal SCC cases, and cases of normal nasal epithelium were examined by Western blot analysis, and the SCCA1 expression pattern in 31 IP specimens and 4 carcinoma in IP specimens were evaluated immunohistochemically. The serum levels of SCC antigen in 11 patients with sinonasal IP also were analyzed.
SCCA1 was overexpressed in all three sinonasal IP tissues compared with sinonasal SCC tissues or normal nasal epithelium. SCCA1 cytoplasmic immunoreactivity was detected in the suprabasal epidermal keratinocytes of all 31 sinonasal IP cases. In the four carcinoma in IP specimens, SCCA1 expression in the papillomatous lesion was more intense than in the cancerous lesion. The serum SCC antigen level was high in 10 of 11 patients with IP (91%) and significantly decreased after surgical resection of the tumors.
Inverted papilloma (IP) is a proliferative lesion of the squamous epithelium lining the sinonasal tract. This tumor histologically is comprised of markedly thickened, well differentiated columnar or ciliated respiratory epithelium with variable squamous differentiation. Although IP is the most frequent benign mucosal neoplasm of the sinonasal tract, it features a high recurrence rate that may exceed 70% after surgical treatment,1–3 and occasionally is associated with squamous cell carcinoma (SCC). Evolution to SCC has been reported in 3–24% of IP cases.4–6 Therefore, the postoperative long-term follow-up of these patients is recommended, and the establishment of a biologic marker reflecting the extent of disease can be of great help to the clinician. In the search for new biologic markers for IP, we focused on the SCC antigen.
The SCC antigen first was isolated biochemically from SCC tissue of the uterine cervix.7 Serum levels of this antigen in those patients with gynecologic, head and neck (HN), lung, and esophageal SCCs are elevated, and the SCC antigen has been well used as a tumor marker against SCC patients.8–14 However, serum levels of this antigen also have been reported to be elevated in patients with nonmalignant skin or lung disease.15–25
Molecular studies have demonstrated that SCC antigen is transcribed by two nearly identical genes (SCCA1 and SCCA2),26 and is produced mainly by SCCA1.27 SCCA1 and SCCA2 are protease inhibitors that map to the serine protease inhibitor (serpin) cluster at 18q21.3.26, 28 These gene products are expressed in SCC tissues as well as normal squamous epithelium.29, 30
Recently, we found that SCCA1 was expressed not only in tumor cells but also in T-lymphocytes peripheral to tumor cells. In addition, T-lymphocytes peripheral to tumor cells might be responsible for serum SCC antigen production in HNSCC patients.31 Conversely, high levels of SCC antigen present in the serum of patients with benign diseases such as psoriasis appear to be due mainly to its direct release from squamous epithelium into the circulation, because SCC antigen has been demonstrated in only suprabasal keratinocytes of the squamous epithelium.17 There may be multiple mechanisms for the elevation of serum SCC antigen in benign and malignant diseases.
In the current study, we evaluated the usefulness of serum SCC antigen as a marker in patients with IP of the sinonasal tract. In addition, we also compared the SCCA1 expression pattern in IP with carcinoma in IP and normal nasal mucosa.
MATERIALS AND METHODS
Patients and Tissue Samples
Surgically removed specimens of 31 cases of sinonasal IP were obtained from patients who were treated between 1991 and 1995 at the Department of Otorhinolaryngology of Kyushu University or the Division of Head and Neck of the National Kyushu Cancer Center. The patients (25 men and 6 women) had a mean age of 61.1 years (range, 35–77 years). None of the patients had any other disease known to cause an elevated serum SCC antigen level. Four carcinoma in IP and four normal nasal mucosa specimens were used as controls. All surgical samples had been fixed in 10% neutral formalin solution and embedded in paraffin; 4-μm consecutive sections were cut and mounted on glass slides. For Western blot analysis, fresh tissue samples from 3 sinonasal IP cases, 3 sinonasal SCC cases, and normal nasal epithelium cases were frozen in liquid nitrogen and stored at −80 °C until use.
Western Blot Analysis
Western blot analysis was performed to detect SCCA1 expression in frozen tissue samples. Briefly, proteins were extracted by M-PER lysis buffer (Pierce, Rockford, IL). The protein concentration was determined using the Pierce protein assay kit (Pierce). Equal amounts (100 μg) of cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Atto, Tokyo, Japan). The membrane was blocked for 1 hour with 5% nonfat dry milk in Tris-buffered saline-0.05% Tween (Wako, Osaka, Japan) and then incubated with the monoclonal antibody against SCCA1 (clone 8H11, 1:1000) or β-actin antibody (AC-15, 1:2000) (Sigma Chemical Company, St. Louis, MO) for 1 hour at room temperature. The membranes then were incubated with the horseradish peroxidase-conjugated antimouse immunoglobulin (Ig) G secondary antibody (1:75000) for 50 minutes at room temperature.
Immunohistochemistry was performed on paraffin sections using the streptavidin-biotin-peroxidase (SAB) complex (Histofine SAB-PO kit; Nichirei, Tokyo, Japan) and techniques using the anti-SCCA1 (clone 8H11, 1:1000) monoclonal antibody. Briefly, tissue sections were deparaffinized and dehydrated in a graded series of alcohol. They then were digested in 0.05% trypsin for 10 minutes, and blocked in 0.3% hydrogen peroxide in methanol for 30 minutes and in normal rabbit serum for 10 minutes. The slides were incubated for 1.5 hours at room temperature with the anti-SCCA1 monoclonal antibody. After extensive washing in phosphate-buffered saline (PBS), the slides were incubated for another 30 minutes at room temperature with the SAB complex. The peroxidase-catalyzed product was visualized with 3,3′-diaminobenzidine chromogen stock solution. The sections were counterstained with hematoxylin. A section of normal oral epithelium previously identified as demonstrating strong staining was used as a positive control with each batch. As a negative control, PBS-treated sections were used instead of the SCCA1 antibody. The experiment was performed in a serial section to compare the specificity of the primary antibody. Two investigators evaluated staining independently. The evaluation was performed with the investigators blind to clinical information.
Evaluation of Serum SCC Antigen Level
Blood samples for the analysis of serum SCC antigen were taken from 11 of 31 sinonasal IP patients before and after surgical resection. Serum levels of the marker were assayed with a solid phase immunoradiometric assay using monoclonal antibody (Dainabot Ltd., Tokyo, Japan). A level of 1.5 ng/mL was used as the upper limit of normal, representing the 95th percentile in a control group.
Statistical analysis of the correlation between the serum SCC antigen level before and after surgery was performed by the Student t test for paired data. Differences with a P value < 0.05 were considered to be significant.
Western Blot Analysis of Frozen Tissues
To evaluate the expression of SCCA1 in sinonasal IPs, Western blot analysis was performed. As shown in Figure 1, all three sinonasal IP samples revealed elevated expression of SCCA1 in IP tissue compared with sinonasal SCC tissues or normal nasal mucosa tissue.
SCCA1 cytoplasmic immunoreactivity was detected in the suprabasal epidermal keratinocytes of all 31 sinonasal IP cases. The cells of the basal layer did not demonstrate positive staining (Fig. 2A and 2B). The normal nasal mucosal epithelium was stained weakly with anti-SCCA1 antibody. In the four carcinoma in IP specimens, SCCA1 expression in the papillomatous lesion was more intense than in the cancerous lesion (Fig. 3).
Serum SCC Antigen Level
Ten of 11 cases (91%) showed evaluated serum SCC antigen levels above the upper limit of normal (1.5 ng/mL) (Table 1). The median serum SCC antigen level (and 90% ranges [5th to 95th percentile]) was 3.6 ng/mL (range, 0.8–8.9 ng/mL). A comparison of serum SCC antigen levels before and after surgical resection is shown in Figure 4. The serum SCC antigen level was found to have decreased in all cases. There was a significant decrease in the serum SCC antigen level after surgical resection (P = 0.0018).
Table 1. Characteristics of Sinonasal IP Patients with Measured Serum SCC Antigen
Sinonasal IP is the most frequent mucosal neoplasm of the sinonasal tract,1–3 and typically presents as a large polypoid nasal mass unilaterally involving contiguous regions. Sinonasal IPs are characterized by squamous metaplasia, epithelial acanthosis, and hyperplasia, forming undulating ribbons and inverting fingers of growth. Although histologically benign, these are clinically ominous lesions because of their high recurrence rates after surgical resection (range, 33–74%), risk of malignant transformation (range, 10–15%), tendency toward multicentricity, and local aggressiveness.2, 32 Therefore, careful postoperative follow-up of these patients is recommended. However, it occasionally is difficult to detect the presence of tumor in asymptomatic patients.
Accurate tumor markers are useful for detecting the development or recurrence of tumor. To our knowledge to date, many tumor markers have been reported to be clinically helpful in various malignant neoplasms. In the search for new biologic markers for IP, we focused on the SCC antigen.
In 1977, Kato and Torigoe presented what to our knowledge is the first report of the detection of circulating tumor-associated SCC antigen in patients with SCC of the uterine cervix.7 Serum levels of this antigen have been well known to be a tumor-associated marker against cervical SCC, esophageal SCC, lung SCC, and HNSCC.8–14 However, some investigators have found that the SCC antigen is a protein expressed during the proliferating process of the normal squamous epithelium.29, 30 The serum level of this antigen also is elevated in patients with nonmalignant diseases, and is used as a marker for assessing the severity and clinical course of these diseases.17 Pulmonary diseases (tuberculosis, adult respiratory distress syndrome, pulmonary infiltration with eosinophilia, sarcoidosis, and bronchogenic cyst) and skin diseases (eczema, pemphigus, erthroderma epidermis, and psoriasis) have been reported to be associated with elevated serum levels of this antigen.15–25 In the current study, the majority of the patients (91%) with sinonasal IPs were found to have elevated serum SCC antigen levels. In addition, the serum SCC antigen level was decreased significantly after surgical resection. To the best of our knowledge, the current study is the first to report that the serum SCC antigen is elevated in patients with sinonasal IP.
Using Western blot analysis, we found that SCCA1 frequently was overexpressed in sinonasal IP tissues compared with the normal human nasal epithelium. We also analyzed the expression pattern of SCCA1 in 31 IP specimens. As a result, SCCA1 cytoplasmic immunoreactivity was detected in the suprabasal epidermal keratinocytes in IP tissues. We previously reported that SCCA1 was expressed not only in tumor cells but also in T-lymphocytes peripheral to tumor cells in patients with SCC of the tongue, and T-lymphocytes peripheral to tumor cells might be responsible for serum SCC antigen production in HNSCC patients.31 In sinonasal IPs, the squamous epithelium itself is overexpressing SCCA1. High serum SCC antigen levels in sinonasal IP patients may be due to its direct release from the squamous epithelium into the circulation. The serum SCC antigen may have the potential to be a useful biologic marker in patients with sinonasal IP. We propose that postoperative IP patients with elevated serum SCC antigen levels should be scanned by computed tomography (CT) more frequently.
The results of the current study also demonstrated that SCCA1 frequently is decreased in sinonasal SCC tissues compared with sinonasal IP tissues. In addition, SCCA1 expression in the papillomatous lesions was more intense than in the cancerous lesions in the four carcinoma in IP specimens. Expression of other serpins located at 18q21.3, such as maspin and headpin, is down-regulated in SCC tissues compared with normal squamous epithelium.33, 34 Transcriptional regulation has been implicated as the mechanism of this event. However, to our knowledge, the true mechanisms of the down-regulation of SCCA1 in SCC still is unknown. Cataltepe et al.,29 reported that SCCA1 is related to epithelial cell differentiation and is a marker for squamous differentiation in SCC. SCCA1 is an inhibitor of papain-like cysteine proteases such as cathepsins L, S, and K.35 Imbalance between proteases and their inhibitors can affect tumor cell motility, invasiveness, proliferation, and death.36 SCCA1 expression may affect the potential for malignant transformation of sinonasal IP. Further studies would be needed to define the biologic role of SCCA1 overexpression in patients with sinonasal IP.
The results of the current study indicate that SCCA1 is highly expressed in sinonasal IP tissues compared with sinonasal SCC tissues or normal nasal mucosa tissue. Serum SCC antigen may be a reliable biologic marker for sinonasal IP, and may have the potential to serve as a useful tool for clinical decision-making.