Abstract
Expression of Id-1 (inhibitors of DNA binding/differentiation protein 1) and TSP-1 (thrombospondin-1) in mucoepidermoid carcinoma and their relationship to pathological features and prognosis was studied. Moderately and poorly differentiated groups had significantly higher Id-1 positive expression rate ( p < 0.05) than well differentiated carcinoma. Stages III-IV showed significant increase of Id-1 positive expression rate ( p < 0.05) compared with stages I and II. Id-1 positive expression was significantly higher in patients with cervical lymph node metastasis or relapse at 5 years ( p < 0.05). After that, patients with negative Id-1 expression had significantly higher tumor-free survival than patients with positive expression ( p < 0.05). Correlation between the expression of Id-1 and TSP-1 in mucoepidermoid carcinoma was negative ( p < 0.05). Poorly differentiated groups show significantly lower TSP-1 positive expression rate than well differentiated groups ( p < 0.05). No significant differences of TSP-1 positive expression were detected with clinical stage. TSP-1 positive expression was significantly lower in patients with cervical lymph node metastasis or relapse at 5 years ( p < 0.05). After 5 years, patients with positive TSP-1 expression had significantly higher tumor-free survival than patients with negative TSP-1. Positive Id-1 expression is associated with high malignancy/poor prognosis; positive TSP-1 expression is associated with low malignancy/good prognosis. Protein expression status may help assess tumor malignancy and patient prognosis.
Mucoepidermoid carcinoma is the most common primary oral and maxillofacial malignant salivary gland tumor in children and adults in Western populations . According to a pathological analysis of 6539 cases of oral and maxillofacial malignant tumors, the incidence of mucoepidermoid carcinoma was rated top among all malignant salivary gland tumors in the Chinese population . Mucoepidermoid carcinoma is usually composed of mucous, epidermal-like and intermediate cells. In the latest classification of cancers by WHO (Section of Pathology and Genetics of Head and Neck Tumor, 2005), mucoepidermoid carcinoma was divided into three groups based on the degree of differentiation: well (low malignancy), moderately (middle malignancy) and poorly differentiated (high malignancy). Owing to different invasive capacities and biological characteristics, mucoepidermoid carcinomas with different degrees of differentiation are linked to different prognoses and require different treatments .
Inhibitors of DNA binding/differentiation (Id) proteins are a group of small proteins with helix-loop-helix (HLH) DNA binding domains. Id proteins bind basic/helix-loop-helix (bHLH) bHLH transcription factors and form heterodimers, preventing them from forming homodimers and activating downstream transcriptional activities . Id-1 is highly expressed in epithelial carcinoma cell lines and plays an important role in the development of several tumors . Id-1 expression is associated with tumor metastasis and poor prognosis .
Thrombospondin-1 (TSP-1), also known as thrombin response protein (TRP), is the strongest suppressive factor of tumor angiogenesis . TSP-1 exists in abundance in platelet alpha particles and also presents in many normal cells as well as the extracellular matrix. TSP-1 plays a key role in suppressing the adhesion and migration of cancer cells and inhibiting tumor angiogenesis . To date, the expression and roles of Id-1 and TSP-1 in mucoepidermoid carcinoma have not been studied.
The authors examined the expression of Id-1 and TSP-1 in well, moderately and poorly differentiated mucoepidermoid carcinoma using immunohistochemistry (IHC) staining techniques. They analyzed the relationship between Id-1/TSP-1 expression and clinical stage, metastasis, relapse and patient survival. The data indicate that increased Id-1 and decreased TSP-1 expression is associated with high malignancy and poor prognosis, suggesting that examination of the expression status of these two proteins in malignant mucoepidermoid carcinoma may help to assess the prognosis and guide treatment:
Patients and methods
Patients
Between January 1997 and January 2006, 352 cases of mucoepidermoid carcinoma were diagnosed in West China Hospital of Stomotology (Sichuan University, China). The authors reviewed the information from these patients retrospectively. The following patients were excluded: those who received anticancer therapy before or after surgery; those whose surgically removed salivary gland had been histologically confirmed to have positive margin (presence of tumor tissue in the resection edge); those whose paraffin-embedded tissues were not properly stored. 70 patients were recruited into the study: 36 males and 34 females, with an average age of 46 years (range 25–78 years). Two experienced pathologists performed histological analysis of hematoxylin-eosin stained sections. In reference to the latest version of the WHO classification of cancers (2005) , the patients and their tissues were assigned to three differentiation groups: well (n = 22), moderately (n = 20) and poorly (n = 28); and three stage groups: stage I (n = 38), stage II (n = 20) and stage III–IV (n = 12). Metastasis in cervical lymph nodes was found in 10 patients. Normal salivary gland tissues (parotid and submandibular gland) from 20 patients, who had been confirmed histologically as having normal tissues with no tumor lesions, were included as a control group. In the 5-year follow-up, relapse occurred in 18 patients. When relapse occurred or the patient died, follow-up ceased in the current study.
Written informed consent was obtained from all the patients/family to include their data in this study, which was approved by the ethics committee of West China College of Stomatology, Sichuan University.
Tissue processing and IHC staining
Archival formalin-fixed, paraffin-embedded tumor or normal salivary gland tissues were sectioned at a thickness of 3–4 μm. The high pressure antigen retrieval method was used to recover Id-1 and TSP-1 antigen, as previously reported . A commercial IHC staining kit purchased from Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd (Beijing, China) was used to detect Id-1 and TSP-1 expression, following the manufacturer’s protocol. The primary antibodies were rabbit anti-human Id-1 polyclonal Ab ( 1:80, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-human TSP-1 mAb (Fuzhou Maxin Bio technology Co., Ltd, Fujian China). Phosphate buffered saline (PBS) solution replaced primary antibody in the blank control. The secondary antibodies were horseradish peroxidase conjugated goat anti-rabbit and rat anti-mouse polyclonal antibodies ( 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The remaining reagents were included in the IHC staining kit. The positive sections in the staining kit (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd (Beijing, China) were used as the positive control. The resultant sections were further counterstained with hematoxylin, and subjected to microscopic examination with a microscope equipped with a digital camera (Nikon, Janpan).
Microscopicic examination of IHC staining results and classifications
The resultant sections were first examined at low magnifications (×40 and ×100) to identify the areas without folding and edge effect. Within this area, five random ×400 visual fields were examined. Cells with stained brown cytoplasm with buffy granules in the cytoplasm (occasionally in the nucleus) were considered as positive cells. According to C haiyarit et al’s method , the immunostaining intensity distribution index, the proportion of positively stained cells and staining intensity was assessed. Tissues without positive cells were marked 0, and those with 1–25%, 26–50% and more than 50% positive cells were marked 1, 2 and 3, respectively. The immunoreactivity of staining was defined as follows: no staining –, mild staining +, moderate staining ++, and intensive staining +++. During the correlation analysis for Id-1 and TSP-1 expression, the data from the immunoreactivity groups were used. In the remaining analyses, considering the implications from the clinical perspectives, groups – and + were combined and named the ‘negative expression’ group, and groups ++ and +++ were combined and named the ‘positive expression’group. The specific tests in each analysis are listed below.
Statistical analysis
Statistic analysis was accomplished with SPSS 13.0 software. Quantitative data were compared with the rank sum test. The correlation between two variances was analyzed with Spearman’s rank correlation analysis. The survival rate was compared with survival analysis (log-rank test). The differences were considered significant if they reached p < 0.05.
Results
Expression of Id-1 and TSP-1 in normal salivary gland tissues
To study the abnormality of Id-1 and TSP-1 expression in tumors, it is critical to determine their baseline expression in normal salivary glands. Conduct epithelial cells stained positively for Id-1 and TSP-1, but salivary gland cells were barely stained ( Fig. 1 ). Only one case of positive staining for Id-1 (5%) and one case of positive staining for TSP-1 (5%) were detected in 20 normal salivary gland samples, significantly lower than their positive rate in malignant mucoepidermoid carcinoma ( p < 0.05 for both Id-1 and TSP-1, Table 1 ).
| Salivary gland tissues | Id-1 positive expression a Case (%) | TSP-1 positive expression a Case (%) | |
|---|---|---|---|
| Normal n = 20 | 1 (5.00%)* | 1 (5.00%)* | |
| Carcinoma n = 70 | Well n = 22 | 5 (22.73%) | 12 (54.55%) |
| Moderately n = 20 | 15 (75.00%)** | 8 (40.00%) | |
| Poorly n = 28 | 23 (82.14%)** | 5 (17.86%)** | |
| Total | 43 (61.43%) | 25 (35.71%) | |
a Positive group consists of samples with immunoreactivity of ++ and +++.
Expression of Id-1 in mucoepidermoid carcinoma with different degrees of differentiation
The positive expression rates of Id-1 in well, moderately and poorly differentiated malignant mucoepidermoid carcinoma ( Fig. 2 ) were 23%, 75% and 82%, respectively ( Table 1 , left column). Compared with well differentiated groups, Id-1 positive expression rates were significantly increased in the moderately and poorly differentiated groups ( p = 0.001 and 0, respectively). Statistical difference was not detected between moderately and poorly differentiated groups ( p =0.548). These results indicated that Id-1 expression is associated with the degree of differentiation, showing an increasing tendency with a decrease in differentiation.
Expression of the TSP-1 in mucoepidermoid carcinoma with different degrees of differentiation
TSP-1 expression was studied in well, moderately and poorly differentiated malignant mucoepidermoid carcinoma ( Fig. 3 ); its positive expression rates were 55%, 40% and 18%, respectively ( Table 1 , right column). Compared with well differentiated groups, TSP-1 positive expression rates were lower in the moderately differentiated group without significance ( p = 0.346) and significantly lower in the poorly differentiated group ( p = 0.007). No significant difference of TSP-1 positive expression rate was detected between the moderately and poorly differentiated groups ( p = 0.089). These results indicated that TSP-1 expression is associated with the degree of differentiation, but shows a decreasing tendency with a decrease in differentiation, which is opposite to that of Id-1.
Correlation analysis of Id-1 and TSP-1 expression
Both of the positive expression rates of Id-1 and TSP-1 in mucoepidermoid carcinoma were associated with the degree of differentiation, so the authors analyzed the relationship between the expression of Id-1 and TSP-1 with Spearman’s rank correlation analysis. There was a negative correlation between Id-1 and TSP-1 expression in mucoepidermoid carcinoma ( r = –0.394, p = 0) ( Table 2 ).
| TSP1 | Id-1 | Total | |||
|---|---|---|---|---|---|
| +++ | ++ | + | − | ||
| +++ | 0 | 0 | 0 | 5 | 5 |
| ++ | 0 | 8 | 4 | 4 | 16 |
| + | 8 | 7 | 5 | 3 | 23 |
| − | 10 | 10 | 2 | 4 | 26 |
| Total | 18 | 25 | 11 | 16 | 70 |
Clinical significance of Id-1/TSP-1 expression in mucoepidermoid carcinoma
Once the expression of Id-1 and TSP-1 in normal and mucoepidermoid carcinoma at different grades had been determined, the authors assessed their clinical implications in predicting prognosis by analyzing the relationships between their expression with several clinical parameters including clinical stage, metastasis, relapse and patient survival. The positive expression rate of Id-1 in stage I, II, and III–IV was 42%, 80% and 92%, respectively ( Table 3 ). Compared with stage I, both stages II and III–IV showed a significant increase in Id-1 positive expression rate ( p = 0.006 and 0.003, respectively), which is in agreement with its pattern in different degrees of differentiation. Id-1 positive expression rate was significantly higher in patients who showed cervical lymph node metastasis than those who did not (100% vs. 55%, p = 0.005). Id-1 positive expression rate was significantly higher in patients who showed relapse at the 5-year follow-up than in those who did not (83% vs. 54%, p = 0.023). At the end of the 5-year follow-up, 89% (24/27) of patients with negative Id-1 expression survived without relapse, in contrast, only 65% (28/43) of patients with positive Id-1 expression survived without relapse ( Fig. 4 A , p = 0.022).
