Previous studies have confirmed that tropomyosin-related kinase B (TrkB) plays a critical role in the occurrence, development, and metastasis of many kinds of malignant tumour. More recently, TrkB was found to be overexpressed in head and neck squamous cell carcinoma (SCC) and to be involved in multistep tumour progression. In this study, the expression of TrkB was investigated in 27 cases of sinonasal SCC using an immunohistochemical method. The clinical significance and possible role of TrkB as a prognostic marker in these tumours was also explored. The results showed that TrkB was overexpressed in all cases of sinonasal SCC. A high level of expression of TrkB was significantly related with poor-to-moderate differentiation of SCC ( P = 0.026), high clinical stage ( P = 0.023), and the presence of local recurrence ( P = 0.004). Analysis by Kaplan–Meier method indicated that patients with high levels of TrkB expression had shorter overall survival ( P = 0.006) and disease-free survival ( P = 0.018). Multivariate analysis revealed that the level of TrkB expression was an independent prognostic factor for both overall and disease-free survival ( P = 0.019 and P = 0.048, respectively). These data suggest that the overexpression of TrkB may play a significant role in sinonasal SCC and that TrkB may be used as a potential prognostic marker for the clinical outcome.
Malignant tumours arising from nasal cavity and paranasal sinuses are not common, accounting for 3% of malignancies in the head and neck region and less than 1% of all malignant tumours. Sinonasal malignancies are aggressive tumours; they are characterized by their local aggressiveness and high rates of recurrence and mortality. Squamous cell carcinoma (SCC) is the most common histological type of sinonasal malignant tumour. It is difficult to detect these tumours at an early stage because of their insidious growth pattern and non-specific symptoms during the initial phase of growth. At the time of diagnosis, the majority of patients already have an advanced-stage tumour with involvement of key structures (orbit, skull base, brain, cranial nerve, etc.), which makes it difficult to remove these neoplasms completely. Currently, treatments for sinonasal SCC include surgery, radiation, and chemotherapy. Studies have shown that even with the application of multimodality therapies, the prognosis is still relatively poor: the 5-year survival rates of sinonasal SCC range from 38% to 60%. The molecular mechanisms of tumourigenesis and progression of sinonasal SCC remain poorly understood. Therefore, it is essential to clarify the molecular mechanisms of sinonasal SCC pathogenesis and to investigate new therapies for this malignancy.
Tropomyosin-related kinase B (TrkB), a tyrosine kinase receptor that is a member of the Trk family, is important in the development of the nervous system. TrkB was initially found to be widely expressed in the central nervous system. Accumulating evidence has also revealed that TrkB is involved in various human malignancies, such as Wilms’ tumour, neuroblastoma, lung adenocarcinoma, pancreatic cancer, gastric carcinoma, colon cancer, head and neck SCC (HNSCC), and hepatocellular carcinoma. Some studies have suggested that TrkB might play an essential role in the course of tumourigenesis and the progression and metastasis of malignancies. Recent studies have reported that the overexpression of TrkB is sufficient to transform normal epithelial cells into highly tumourigenic cells and to give them the ability to resist anoikis.
The role of TrkB as a prognostic marker has also been demonstrated, and increasing numbers of studies are showing that high levels of TrkB expression are correlated with more aggressive behaviour and a poor prognosis. Some studies have suggested that brain-derived neurotrophic factor (BDNF)/TrkB signalling could be an anti-tumour target. Other recent studies have reported that TrkB is significantly overexpressed in HNSCC specimens and cell lines. Kupferman et al. found that the overexpression of TrkB resulted in altered expression of molecular mediators of epithelial-to-mesenchymal transition (EMT), including down-regulation of E-cadherin and up-regulation of Twist. Furthermore, in a mouse model of HNSCC, they also found that down-regulation of TrkB could suppress tumour growth. Zhu et al. demonstrated that TrkB plays a significant role in HNSCC, presumably by rendering HNSCC cells resistant to anoikis.
Taken together, these findings indicate that TrkB plays an important role in HNSCC and may be a crucial component in the multistep tumour progression of HNSCC. However, the specimens tested in these previous studies mainly included oral cavity, pharynx, larynx, and neck SCC. Therefore, the suggestion that TrkB may be involved in sinonasal carcinogenesis has been proposed. In spite of these studies, neither the expression of TrkB in sinonasal SCC patients nor the potential clinical significance of TrkB in sinonasal SCC appears to have been reported. The objective of the present study was to determine the expression of TrkB in sinonasal SCC using immunohistochemistry, and to investigate the relationship between the expression of TrkB and various clinical features of these patients. Furthermore, it was sought to explore the clinical significance, possible role as a prognostic marker, and potential therapeutic benefits of TrkB in sinonasal SCC.
Materials and methods
Patients and tissue samples
This study included 27 patients with histologically confirmed SCC in the nasal cavity and sinuses, who underwent surgery at a university hospital in Beijing, China from 2001 to 2007. No patient in this study received preoperative radiation therapy or chemotherapy. The medical records and follow-up data of the patients were collected by retrospective chart review. Follow-up information was obtained up until the time of death, or until the time that contact was lost.
Tumours were assessed based on the degree of differentiation according to the World Health Organization classification. The TNM stage was defined according to the American Joint Committee on Cancer (AJCC 2009) staging system postoperatively. The brain tissue of a male Wistar rat and the tumour-free sinonasal mucosa (surgical margin) were also examined as the positive and negative control, respectively.
The immunohistochemistry of TrkB was studied using the procedures reported previously. All of the samples examined were fixed in 10% neutral formalin, embedded in paraffin, and sliced at a thickness of 4 μm. Sections were deparaffinized and rehydrated in a routine manner. The sections were then immersed in 3% hydrogen peroxide for 10 min to quench endogenous peroxidase. Subsequently, an antigen retrieval process was accomplished with microwave treatment (98–100 °C) in citrate buffer (pH 6.0) for 20 min. After rinsing with phosphate-buffered saline (PBS) three times and 5% goat serum treatment at ambient temperature for 30 min, these slices were incubated with primary rabbit polyclonal antibody anti-TrkB (1:100; Santa Cruz, CA., USA) at 4 °C overnight. On the following day, the slices were rinsed with PBS three times and treated with secondary antibody (1:100; Boster, Wuhan, China) in an incubator for 30 min at 37 °C. After that, they were rinsed and treated with streptavidin–biotin complex (SABC-kit; Boster) for 30 min at 37 °C. Following rinsing, the slices were visualized with 3,3′-diaminobenzidine (DAB; Dako, Glostrup, Denmark) for 1 min. Finally, the slices were washed with distilled water and counterstained with haematoxylin.
Analysis of immunohistochemical expression
These slices were evaluated under an optical microscope; cells with brown staining in the cytoplasm or cell membrane were regarded as positive. The sinonasal mucosa was used as the TrkB-negative control and the rat brain was used as the TrkB-positive control. To evaluate the immunoreactivity of TrkB, five consecutive microscopic fields were observed at 400× magnification. The percentage of positive cells (<10% = 0, 10–25% = 1, 26–50% = 2, 51–100% = 3) and the intensity of TrkB immunostaining (0 = negative, 1 = weak, 2 = intense) were assessed. The score for each sample was calculated by multiplying the intensity score and percentage score to give a final score of 0, 1, 2, 3, 4, or 6. TrkB expression was finally determined as negative (score 0), low (score ≤3), or high (score >3).
IBM SPSS Statistics version 21.0 software (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. The results were recorded as the mean ± standard deviation. The correlation between clinical parameters and TrkB expression detected by immunohistochemistry was analyzed using Fisher’s exact probability test. Disease-free survival and overall survival were established by Kaplan–Meier method, and prognostic factors were calculated by log-rank test. Multivariate analysis was performed by Cox regression model. If the P -value was less than 0.05, the result was considered statistically significant.