【精品论文】The role of Fascin-1 and E-cadherin expression in laryngeal squamous cell carcinoma.doc

精品论文the role of fascin-1 and e-cadherin expression in laryngeal squamous cell carcinomazou jian, yang hui, chen fei, li wen, wang lihong, liu shixi5(department of otolaryngology, west china hospital of sichuan university, chengdu 610041) abstract: fascin-1 and e-cadherin which are related with cell motility and cell adhesiveness are important factors in the progression and metastasis of cancers. the objective of this study was to explore the association of fascin-1 and e-cadherin expression level with clinical characteristics and prognosis of patients with laryngeal squamous cell carcinoma. in our study, tumor tissue samples from10150 patients with laryngeal squamous cell carcinoma were determined for fascin-1 and e-cadherin expression by immunohistochemistry. the statistical analysis was performed to evaluate the associationof fascin-1 and e-cadherin expression with clinical characteristics and patients prognosis. as a result, fascin-1 expression was an independent predictive factors for recurrence in patients with laryngeal squamous cell carcinoma (p=0.021) andindependently related to disease-free survival(p=0.010).15although e-cadherin expression status was not the independent predictive factors for recurrence (p=0.055) or disease-free survival (p=0.063), the subgroup with fascin-1 high expression/e-cadherin low expression has the poorest prognosis (p=0.000) using subgroup analysis. in conclusion, fascin-1 expression could be a potential prognostic predictor for patients with laryngeal squamous cellcarcinoma. simultaneous analysis of fascin-1 and e-cadherin expression could be more effective to20evaluate the prognosis of patients with laryngeal squamous cell carcinoma.keywords: fascin-1; e-cadherin; laryngeal squamous cell carcinoma; immunohistochemistry; recurrence0introduction25laryngeal cancer is the most common head and neck malignancy in the worldwide, with a tendency towards an increasingly occurrence of new cases and deaths annually1-3. more than 95% of all laryngeal cancers are squamous cell carcinomas (scc). despite advances in detection and treatment options in laryngeal squamous cell carcinoma (lscc) the survival has not improved much over the last thirty years1-2. most carcinoma cells remain in the same area for a long time,30while invasion and metastasis usually develops only in a late stage of the disease. however, there is a subgroup of aggressive tumors that disseminates fast during the process of tumorigenesis, which make this subgroup with aggressiveness difficult to treat. potential characteristics of invasion and metastasis in carcinoma cells are the very important prognostic factors for tumorrecurrence4-6. although many clinicopathological factors related to invasiveness of carcinoma35cells have been proposed, satisfactory predictive factors have not yet been determined. so there is a strong need to evaluate the tumor invasiveness and determine biomarkers that can help clinicians decide how to appropriately manage individual patients.fascins, are globular actin cross-linking proteins that have a major function in forming parallel actin bundles in cell protrusions, was originally found in the extracts of unfertilised sea40urchin eggs as an actin-binding protein7. in humans, the fascins family consists of three isoforms.fascin-1, is widely expressed by mesenchymal tissues and in the nervous system; fascin-2 is expressed by retinal photoreceptor cells; and fascin-3 is testis specific8-9. the expression of fascin-1 is low or absent in the majority of normal adult epithelia, yet up-regulation of the protein has been reported in several human epithelial neoplasms such as colonic, gastric, breast, skin and45nonsmall cell lung carcinomas, and markedly associated with metastasis or poor prognosis10-15.meanwhile, adhesion processes are involved in all levels of the metastatic cascade. e-cadherin is afoundations: specialized research fund for the doctoral program of higher education.(no.20090181120101) brief author introduction:zou jian, (1979-), male, associate professor, the disease in throat. e-mail: - 12 -transmembrane glycoprotein located at adherens junctions that mediates the selective adhesion of epithelial cells, which is required for the interaction and maintenance of normal integrity16. it has also been shown that linkage between e-cadherin and the cytoskeleton is necessary for functional50cell-cell adhesion17. loss or reduction of e-cadherin expression has been associated with advancedstages of tumor growth, increased metastatic potential and a shortened disease-free and overall survival in a variety of epithelial neoplasms, with a concomitant poor prognosis18-21.to our knowledge, correlations between fascin-1 and e-cadherin expression in lscc tissues and prognosis of lscc have not been reported up to date. in the present study, we examined the55immunoreactivity of fascin-1 and e-cadherin in patients with lscc and evaluated the importance of the two proteins in terms of their relation to both the recurrence and survival.1methods1.1 patients and tissue samplesthe institutional review board and human ethics committee of the regional cancer centre60approved the study. one hundred and fifty-nine consecutive patients (155 male and 4 female) with lscc who received surgery in the department of otolaryngology at the west china hospital of sichuan university from 2003 to 2004 were investigated. patients who received preoperative treatment were excluded in this study. the routine formalin-fixed and paraffin-embedded material blocks were retrieved from the files of the department of pathology at the west china hospital of65sichuan university. the tumor type and the grade of cell differentiation were designated based on the criteria of world health organization (who), whereas the pathological stage of each tumor was determined by the international union against cancer (uicc) tnm classification22. details of clinical and pathological characteristics of the patients were summarized in table 1.follow-up focused specifically on recurrence, disease-free survival, overall survival and70cancer-related death. patients were scheduled for physical examination, fibrolaryngoscope, abdominal ultrasound examination and chest x-ray or ct scan every 3 months during the first year following surgery, then every 6 months for the second years, and annually thereafter. follow-up time for all the patients ranged from 4 to 66 months, with a median time of 49 months. there were 7 patients were lost to follow-up, 2 cases of death occurred because of cerebral75hemorrhage and heart failure respectively. therefore the remaining 150 patients were the subjects of our retrospective study.1.2 immunohistochemistryimmunohistochemical studies were performed to detect the expression of fascin-1 ande-cadherin using the standard streptavidin-biotin complex method. one hundred and fifty80formalin-fixed, paraffin-embedded lscc tissue samples were cut into sections of 4 m, dried overnight at 56c. then these sections were dewaxed in xylol, hydrated in ethanol with descending concentration (absolute, absolute, 96%, 70%, 40%, 5 minutes each) ending with pure water. endogenous peroxidase activity was blocked by incubation in a 3% hydrogen peroxide for10 min at room temperature. antigen retrieval was carried out by microwave treatment of the85slides in sodium citrate buffer (ph 6.0) for 20 minutes. nonspecific background staining was reduced by incubation in goat serum for 10 minutes at 37c. the sections were incubated overnight at 4.0c with anti-fascin-1(fcn01, 1:200, neomarkers, fremont, ca, usa) or anti-e-cadherin (4a2c7, 1:100, zymed laboratory, san francisco, ca, usa) antibody. they were then washed three times in pbs for 5 min each and incubated with biotinylated second90antibody (boster biologicals co., wuhan, china) for 30 min at room temperature in a humidified95100105110115120125130chamber, then rinsed again in pbs and incubated with streptoavidin-horseradish peroxidase for 10 min. immunostaining was visualized with 3, 3-diaminobenzidine (dab) for about 2 min, and the reaction was stopped by washing with pbs. counterstaining was performed with hematoxylin, then dehydrated and mounted.1.3 evaluation of immunohistochemistrythe sections without the primary antibody were used as a negative control for fascin-1 and e-cadherin in each case. fascin-1 immunoreactivity in each tumor was verified by the labelling of endothelial cells of microvessels in each specimen9, while the expression of e-cadherin in the normal pharyngeal mucous membranes was used as positive control for e-cadherin. allimmunostainings were observed under high magnification (400). cytoplasmic expression of fascin-1 and membranous expression of e-cadherin in tumor cells were determined by two independent observers who assessed semi-quantitatively the percentage of stained tumor cells as well as staining intensity. the followed scoring criteria used for assessment were as previously reported. the percentage of positive cells was rated as follows: 1 point, less than 10%; 2 points,11-50% positive tumor cells; 3 points, 51-80% positive cells; and 4 points, 81% positive cells. staining intensity was rated as follows: 1 point, weak intensity; 2 points, moderate intensity; and 3 points, strong intensity. points for expression and percentage of positive cells were added, andspecimens were attributed to four groups according to their overall scores : (1) negative, 10%of cells stained positive, regardless of intensity; (2) weak expression, 3 points; (3) moderate expression, 45 points; and (4) strong expression, 6-7 points. group (1) and (2) were classified as low expression of fascin-1or e-cadherin, whereas group (3) and (4) were classified as highexpression of fascin-1or e-cadherin.1.4 statistical analysisspss 12.0 software package was used to do the statistical analysis. comparisons of sample means were evaluated using analysis of variance. categorical variables were evaluated using the chi-square test. associations between clinicopathologic predictors and immunohistochemical fascin-1 or e-cadherin expression were analyzed using the logistic regression model with the presence of recurrence as the dependent variable. multivariate survival analysis was performed with the cox proportional hazards model. survival curves were constructed according to the kaplan-meier method. a log rank test was performed to evaluate the statistical significance of the differences in survival.a p value less than 0.05 was considered statistically significant.2results2.1 clinicopathologic features and follow-up outcometable 1 presents a summary of primary location, tumor stage (t-stage), lymph node metabasis as well as histological grade. forty-four patients underwent total laryngectomy, and 106 patients underwent partial laryngectomy. radical neck dissection was performed in 14 patients, modified radical neck dissection in 18 patients, and selective dissection in 35 patients. the median time to recurrence was 13 months, with a range of 4-52 months. fifty-two patients with recurrence occurred. recurrences were confirmed by histopathology. thirty-one cancer-related deaths were reported. the median time to death was 16 months, with a range of 5-45 months. the reasons for deaths were local recurrence in 24 patients, pulmonary metastases in 3 patients and stomal recurrence or mediastinum metastases in 4 patients respectively.135table 1. association between clinicopathologic characteristics of patients with lscc and immunohistochemical features in tumor tissues. age(years)n fascin-1 highlow e-cadherin phigh low pgender62 77 45 32 38 3962 73 34 39 0.595 41 32 0.403male 146 76 70 77 69female 4 3 1 2 2course(months)6 63 30 33 28 356 87 49 38 0.523 51 36 0.399primary locationsupraglottical 16 8 8 9 7glottical 122 63 59subglottical 12 8 4early stage (i-ii)7926535326advanced stage 0.000(iii-iv) 7153182645t-stage0.59464 586 60.9420.000lymph node metabasispositive 67511623 440.000 56 27 0.000negative 832855histological gradewell31724229morderately6836320.00035330.048poorly51361522291401451501552.2 immunohistochemical expression of fascin-1 or e-cadherinpositive staining for fascin-1 was observed in endothelial cells of microvessels in tumor tissue, while strongly positive staining for e-cadherin was found in normal epithelial cells of pharyngeal mucous membrane. the positive expression site of fascin-1 was mainly localized in cytoplasm of tumor cells (fig. 1a), and e-cadherin were mainly immunostained in tumor cell membrane or cytoplasm near cell membrane (fig. 1c). the fascin-1 immunoreactivity was uniform in the center of the tumor, whereas increased immunoreactivity was seen at the edge of the tumor. in the central tumor areas, the majority of the e-cadherin immunoreactivity was reduced. especially at the invasion front of tumor, the membranous staining of e-cadherin was usually lost. the distribution of the scores of the fascin-1 or e-cadherin immuno-stain could be seen in table 2. the high expression rate of fascin-1 protein in 150 patients with lscc was52.7% (79/150). concurrently, 71 (47.3%) of the primary tumor tissues were low immunohistochemical expression for e-cadherin. when analyzing relationship between clinicopathological features and fascin-1 or e-cadherin expression in lscc tissues, a significant association between fascin-1 immunostaining and t-stage (p=0.000), cervical lymph nodes metastasis (p=0.000) or histological grade (p=0.000) was observed (table 1). meanwhile, there was a significant association between e-cadherin immunostaining and t-stage (p=0.000), cervical lymph nodes metastasis (p=0.000) or histological grade (p=0.048) (table 1). furthermore, the combination of fascin-1 high expression / e-cadherin low expression in lscc tissues was mostly seen in patients with advanced tumor stage (p=0.001, data not shown).160165fig. 1 immunohistochemical staining for fascin-1 and e-cadherin expression in lscc. brown grains stood for positive signal (dab staining). the fascin-1 staining was mainly localized in cytoplasm. e-cadherin were mainly immunostained in cell membrane or cytoplasm near cell membrane. (a) fascin-1 high expression in tumor cells; (b) fascin-1 low expression in tumor cells (arrow); (c) e-cadherin high expression in tumor cells; (d) e-cadherin low expression in tumor cells. (original magnification 400). table 2. the distribution of the scores of the fascin-1 or e-cadherin immuno-stain. fascin-1e-cadherin score(n)(n)percentage+intensityexpression classification1+x911negative2+12225low2+23531low2+31518high3+154low3+23129high3+31915high4+100high4+2911high4+356hightotal (n)1501501701752.3 association with recurrenceunivariate analysis showed a significant association of tumor recurrence with t-stage (p=0.000), cervical lymph node metastasis (p=0.000) or histological grade (p=0.000). no association was found between tumor recurrence and patients age (p=0.374) and disease course (p=0.151). patients with lscc tissues demonstrated fascin-1 high expression had a significantly increased tumor recurrence (58.2% in fascin-1 high expression versus 8.5% in fascin-1 low expression, p=0.000). a significant correlation between tumor recurrence with fascin-1 (p=0.000) status in lscc tissues was found (table 3). moreover there was a significant correlation between tumor recurrence with e-cadherin (p=0.000) status in tumor tissues. e-cadherin low expression showed a trend for development of recurrence (54.9% in e-cadherin low expression versus 16.5%180in e-cadherin high expression, p=0.000) (table2). analyzed by subgroup assay, the highest recurrence was documented in the fascin-1 high expression / e-cadherin low expression subgroup (recurrence rate=64.3%, p=0.000), whereas the lowest incidence of recurrence was related to the fascin-1 low expression / e-cadherin high expression subgroup (recurrence rate 9.3%, p=0.000) (table 4).table 3. clinicopathological variables and immunohistochemical features in relation to tumour recurrenceage(years) case without case with nrecurrence(n=98) recurrence(n=52) pgender62 77 51 2662 73 47 26 0.374male 146 95 51female 4 3 1course(months)6 63 46 176 87 52 35 0.151primary locationsupraglottical 16 11 5glottical 136 82 40subglottical 12 5 7t-stageearly stage (1-2) 79 71 80.198advanced stage (3-4) 71 27 44 0.000lyphm node metabasispositive 67 25 42negative 83 73 10 0.000histopathologic differentiationwell 31 29 2morderately 68 44 24poorly 51 25 26fascin-1 statushigh 79 33