Clinical Features And Hypoxic Marker Expression Of Neuroendocrine Carcinoma Of Head And Neck

Neuroendocrine carcinoma (NEC) of the head and neck is rare. Approximately180cases of the larynx and75cases of nasal and paranasal cavities have been reported in the English-language literature:mostly males, around age50, and heavy smokers. The prognosis of NEC in the nasal cavity and larynx is poor. Extra-pulmonary small cell carcinoma is usually a fatal disease, with a13%5-year survival rate. In the larynx, the2-year survival rate was16%and the5-year rate was only5%. In the nasal cavity and sinus, the median survival time was2-3years. The survival rates are similar to those for small-cell lung cancer. Unfavorable prognostic factors of NEC in the head and neck are correlation with invasion of the lamina cribosa, ectopic hormone syndrome, recurrence, and distant metastasis. However, tumor size and number of mitoses show no correlation with recurrence, metastasis, or survival. Conclusive prognostic factors remain unclear because of the limited number of SCNEC cases in the head and neck. Thus, prognostic factors need further study using greater numbers of cases of SCNEC in the head and neck.Some studies have investigated the prognostic factors for NEC, looking at molecular markers, such as Hypoxia-inducible factor-la (HIF-la). HIF-la has been studied in neuroendocrine carcinoma (NEC). Hypoxia in solid tumors has been associated with therapy resistance and poor clinical prognosis. HIF-1α is upregulated in a wide range of solid tumors in humans, and over-expression of HIF-1α is associated with tumor aggressiveness and poor prognosis. In small-cell lung cancer, a few studies have revealed that high HIF-1α is correlation with poor survival. GLUT-1, as a major protein of cellular glucose uptake, has been studied in NEC. Hypoxia promotes the chemo-radioresistance of carcinomas.GLUT-1is overexpression in hypoxic status. HIF-1α, a transcription factor associated with the cellular response to hypoxia, upregulates the expression of several hypoxia response genes, including GLUT-1. We have first reported that a significant correlation between GLUT-1and HIF-la expression in laryngeal carcinoma.The phosphatidylinositol3-kinase (PI3K)/protein kinase B (Akt) pathway may regulate HIF-la and GLUT-1. To our knowledge, there is no previous report of these hypoxic markers in NEC.In the present study, we investigated the clinicopathological features of nasal and laryngeal SCNEC retrospectively. We also used immunohistochemistry to determine the expression of HIF-1α, GLUT-1, PI3K, and p-Akt protein in these SCNEC.Materials and Methods1. Patients:The subjects were13consecutive patients at The First Affiliated Hospital with histologically demonstrated sinonasal and laryngeal SCNEC between2003and2014, and15sinonasal and laryngeal precancerous lesions were also obtained as a control group. Data were obtained from the hospital surgical pathology files.Our study was approved by the Institutional Review Board of The First Affiliated Hospital, College of Medicine, Zhejiang University. Written informed consent was obtained from each patient before inclusion.2. ImmunohistochemistryFormalin-fixed and paraffin wax-embedded tissue blocks from primary lesions were cut into4-μm sections, and representative sections were analyzed immunohistochemically(EliVision Plus IHC kit; Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou, China). Briefly, the sections were deparaffinized with xylene and dehydrated through an ethanol series. Then, antigen retrieval was performed with a microwave oven over two10-min cycles. Endogenous peroxidase activity was blocked by incubating the slidesin1.5%hydrogen peroxide in absolute methanol at room temperature for10min. Primary antibodies were applied for1h at room temperature, followed by50μL of polymer enhancer for20min and50μL of polymerized horseradish peroxidase-anti-mouse immunoglobulin G (IgG)(DAB Kit; Maixin Biological) for30min. The reaction products were visualized using3,3’ diaminobenzidine (DAB Kit; Maixin Biological), and the sections were counterstained with hematoxylin and eosin, dehydrated, and examined under a light microscope. Tris-buffered saline was used in place of the primary antibody for negative controls.GLUT-1, HIF-1α, PI3K, and p-Akt levels were evaluated by the same investigator (QH You), who was blinded to the clinical and follow-up data. GLUT-1expression was considered positive only if distinct membrane staining was present. HIF-la, PI3K, and p-AKT proteins were observed in the nucleus and cytoplasm. Protein analysis was performed in10random high-power fields within each of which,100tumor cells were counted for each case and for all antibodies. The percentage of positive cells was calculated by dividing the number of positive tumor cells by the total number of tumor cells counted. A sample was considered negative if<25%of the cells were stained. 3. Follow-upThe patients were scheduled forfollow-up visits every3months after the initial surgery. Follow-up consisted of a routine physical examination and a computed tomography (CT) or magnetic resonance imaging (MRI) scan of the primary site. Patient follow-up was reported up to the date last seen in the clinic.4. Statistical analysisThe SPSS software (ver.19for Windows; SPSS Inc., Chicago, IL, USA) was used to conduct all statistical tests. Associations among GLUT-1, HIF-1α, PI3K, and p-Akt protein expression and pretreatment clinical parameters were analyzed using the chi-squared and Fisher’s exact tests. Survival rateOverall survival (OS), defined as the time from surgery until death from any cause, was plotted as a Kaplan-Meier curve. Univariate survival analysis was performed using a log-rank test, and multivariate analysis was performed using Cox proportional-hazards regression analysis. Ap-value <0.05was deemed to indicate statistical significance. The correlation analysis was performed using Spearman’s rank correlation.Results1. Clinicopathological findings:The subjects included nine males and four females with a mean age of61(range,50-71) years. Tumors were located in the maxillary sinus (n=3), right tonsil (n=1), the larynx (n=8) and cervical trachea (n=1). In the larynx, six cases were located in the supraglottic area, one in the glottis area and one in the subglottic area. No patient presented with paraneoplastic endocrine syndrome.2. Radiological images:The NEC radiological images available varied. CT images were available for seven patients and showed homogenous (two cases) and heterogeneous (five cases) soft-tissue masses. Contrast-enhanced CT images were available for seven patients:mild enhancement was found in the two cases of laryngeal NEC and strong enhancement in five cases. MRI data were available for six patients (two in the nasal cavity, one in cervical trachea and three in the larynx). The T1-weighted signals were hyperintense in three patients, isointense in one patient, hypointense in two patients.The T2-weighted signals were hypointense in all six patients.Contrast-enhanced T1-weighted MRI images showed strong enhancement in all six patients. Diffusion-weighted MRI (DWI) was available for two patients. DWI in four patients was high-signal and ADC was0.793±0.33×10-3mm2/s,1.78±0.16x10-3mm2/s,1.14±0.22×10-3mm2/s,3.48±0.1×10-3mm/s, respectively.3. Treatment and follow-up:In nasal NEC, two patients received concurrent chemo-radiotherapy (CCRT) and one patient received partial maxillectomy and postoperative radiotherapy. In the larynx, two patients underwent horizontal supraglottic laryngectomy and two patient received vertical hemilaryngectom. Four patients underwent total laryngectomies. Six patients died of disease and five patients were alive without disease. In this small series, the3-year survival rate was35.1%.4. Immunohistochemical findings:Expression of GLUT-1, HIF-1α, PI3K, and p-Akt was seen in sinonasal and laryngeal SCNEC in81.8%(9/11),54.5%(6/11),45.5%(5/11) and36.4%(4/11) of cases, respectively. Expression of GLUT-1, HIF-1α, PI3K, and p-Akt in control precancerous lesions was seen in6.7%(1/15),0%(0/15),0%(0/15), and0%(0/15) cases, respectively. The expression of GLUT-1, HIF-1α, PI3K, and p-Akt was higher in NEC of head and neck than in precancerous lesions (p=0.001,0.005,0.017, and0.017, respectively)..ConclusionsIn this small series, univariate analysis showed that poor survival was significantly associated with distant metastasis GLUT-1,HIF-1α, PI3K, and p-Akt expression were not correlation with survival. In a multivariate analysis, these markers were not predictors of OS.