Clinicopathological Findings And Expression Of Glut-1 And PI3K/Akt Signal Pathway In Adenoid Cystic Carcinomas

Background:Adenoid cystic carcinomas(ACC) in ear, nasal cavity, paranasal sinus, larynx and trachea are uncommon. These malignant tumours often are asymptomatic or mimic inflammatory diseases, leading to a delay in diagnosis. ACC was noted for its slow growth and indolent course, like ACCs in general, patients with ACC of these sites often experienced recurrence and distant metastasis many years after diagnosis and definitive treatment. Patterns of disease included perineural and vascular invasion. The most important modalities for primary treatment of local and locoregional disease are surgery and/or irradiation. However, the outcome of these treatment is unfavorable. Several studies have identified clinicopathological factors in ACC with an unfavorable effect on survival, including old age, tumor location, advanced stage, solid histological subtype, high grade, TNM staging, major nerve involvement, the presence of perineural invasion, a positive surgical margin, and lymph node metastasis. However, real prognostic factors are still unknown. Thus, more studies are needed for pathogenesis, prognostic factors, new diagnostic and therapeutic methods, especially in the molecular level.Like other malignant tumors, cells of ACC exhibit increased glucose uptake and utilization in comparison to their nonmalignant counterparts. The phenomenon has been demonstrated by positron emission tomography (PET),using 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG).Many mechanisms of FDG uptake have been proposed for accelerated glucose use in growing tumors and in transformed and malignant cells: passive diffusion, Na+-dependent glucose transport, and via facilitative glucose transporters (GLUT). The latter is considered to be the most important mechanism for enhancing glucose influx into cells. Gluts are membrane proteins that facilitate the transport of glucose across cellular membranes. Thirteen (14) members of the facilitative sugar transporter family are now recognized (Glut-1-Glut-12 and HMIT; genename SLC2A).The human genes encoding these proteins are named Glut 1-5 and Glut 7-13; Glut-6 and Glut-14 are now known to be pseudogenes. Of the 14 isoforms, Glut-1 appears to be the most ubiquitously distributed. Increased Glutl levels and glucose uptake correlate with increased cellular growth and proliferation. This isoform is overexpressed in many human cancer cells, and its appearance is correlated with aggressive biological behavior. Therefore, control of Glutl trafficking and activity are also key elements regulating glucose uptake. Similar to the insulin-responsive glucose transporter, Glut-4, Glut-1 cell surface localization is controlled by extrinsic signals. Among signaling pathways initiated in cell activation, the PI3K/Akt pathway has been shown to promote both Glut-1 cell surface trafficking and activity.PI3K is a heterodimeric enzyme important for growth and proliferation and Akt is a downstream serine-threonine kinase that transmits survival signals from growth factors. The PI3-K/AKT pathway is frequently overactivated in a wide range of tumors and triggers a cascade of responses, from cell growth and proliferation to cell survival and motility, which drive tumor progression. It has been shown that the PI3K/Akt pathway is involved in translocating the GLUT-1 glucose transporter from the cytosol to the plasma membrane in some endocrine organs such as the thyroid gland, pancrea.Our previous study revealed that FDG uptake was higher in malignant lymphoma than in other head and neck malignant tumors by SPECT/CT, and a significant correlation was found between FDG levels and glucose transporter-1 (Glut-1) mRNA or Glut-1 protein levels. We also showed that the increased expression of Glut-1 in head and neck carcinomas was correlated with lymph node metastasis, poor survival and clinical stage.Then,we revealed that Glut-1 overexpression in vitro is associated with cell proliferation and increased glucose uptake by laryngeal carcinoma Hep-2 cells. Conversely, the suppression of Glut-1 expression by antisense oligodeoxynucleotides (AS-ODNs) may decrease glucose uptake and inhibit the proliferation of Hep-2 cells. Therefore, we propose the suppression of Glut-1 expression as a new therapeutic target for laryngeal carcinoma.To date, only a few studies have investigated the expression of Glut-1 in ACC,especilly in the effect of regulation of expression of Glut-1 by PI3K/Akt signal pathway.In present study, we retrospectively investigated the clincopathigical features of external auditory canal, sinonasal, laryngeal and tracheal ACC and we also assessed expression of Glut-1, PI3K and p-Akt protein in these ACCs by immunohistochemistry. Additionally, we examined possible correlations between Glut-1, PI3K and p-Akt protein and clinicopathological parameters in this cohort of patients. Materials and methods:14 patients with ACC from Otolaryngology Department, the First Affiliated Hospital, College of Medicine, Zhejiang University were collected between January 1993 and February 2010. The institutional review board approved this study and written informed consent was obtained from the patients before inclusion. The clinicopathological findings (including age, sex, site, TNM stage, pathological type, recurrence, metastasis, follow-up) were analysed. The expression of Glut-1, PI3K and p-Akt proteins were examined in ACC tissue samples by an EliVisionTM plus IHC Kit (Maixin Biological, Fuzhou, China) immunohistochemical analysis.5 paraffin-embedded archival tissue blocks from patients with nasal polyps,10 paraffin-embedded archival tissue blocks from patients with vocal cord polyps,5 tissue blocks from patients with nasal benign schwannomas,1 tissue block from patient with laryngeal benign chondroma and 7 tissue blocks from patients with nasal inverted papilloma were also obtained. Formalin fixed, paraffin-embedded archival tissues were obtained from institutional and consultation files.Statistical analyses:Analyses were performed using SPSS 15.0 software package. Associations between Glut-1, PI3K and p-Akt protein expression and the other pretreatment parameters were analyzed using the chi-squared test. A P-value of<0.05 was deemed to indicate statistical significance.The correlation analysis using Kappa test. A value of 1 indicates perfect agreement. We calculated weighted k-values to describe concordance in reporting as slight ( 0.4), moderate(0.4-0.75), or almost perfect (>0.75). Results:Patient Characteristics:Of the 14 ACC tumor tissues,4 cases (28.6%) were located in the external auditory canal,3 (21.40%) in the nasal cavity,3 (21.4%) in the paranasal sinus,3 (21.4%) in larynx and 1(7.2%) in the trachea. The median age of the 14 patients was 54 (range, 32-80).6 patients were males and 8 patients were females. All patients had no lymph node and distant metastasis. Six of the patients (42.9%) had cancer classified as T1 stage, 5 (35.7%) with T2 stage and 3(21.4%) with T3 stage. Surgery was performed in all patients. Ten patients were received radiotherapy postoperative. The patients'average follow-up period was 74 months (range,6-288 months). Three patients were lost to follow-up.Of 10 patients received radiotherapy, two patients suffered disease recurrence(one patient had local recurrence 2 years after initial surgery and another had local recurrence 5 years after initial surgery) and they received several surgeries. The two patients were alive over 24 years,9 years, respectively, one was living without evidence disease and one was living with recurrence. Other eight patients were living without evidence disease at a mean follow-up time of 8.6 years after diagnosis (range,1.4-24 years). Three patients were not received radiotherapy after surgery. Of these patients, one was lost to follow-up 24 months after surgery, one was lost to follow-up six months after surgery, and one was living without evidence disease over twenty months.One was performed operation at another hospital 9 months ago and lost to follow-up after surgery in our hospital.Glut-1, PI3K and Aktprotein expression in ACCThe positive rate of Glut-1,PI3K and Akt protein in ACC was 35.7%(5/14), 28.6%,71.4%, respectively. The expression of Glut-1 or PI3K or Akt protein in ACC was higher than that in inflammatory lesions or benign tumor (p<0.05).The correlation between Glut-1, PI3K and Akt protein expressionKappa test showed that correlation between Glut-1 and PI3K expression, between Glut-1 and p-Akt expression, between p-Akt and PI3K expression was slight correlation(K=0.186,0.364,0.276, respectively).Association of Glut-1, PI3K and Akt with Clinicopathological ParametersThere was no statistically significant association between Glut-1,PI3K and Akt protein in ACC and all clinicopathological variables examined (P>0.05).Conclusions:Clinicopathological findings1,The presnt study showed that there were high recurrent rate in the sinonasal and external auditory canal ACC. had no lymph node and distant metastasis.2,This study also revealed that it was very important for the initial treatment and radiotherapy may be effective on control local recurrenc.3,Additionally, this study suggested that adenoid cystic carcinoma shoule be as differentiatial diagnosis in diseases of the external auditory canal, nasal cavity, paranasal sinus,larynx and trachea. The implication of Glut-1 expression and PI3K/AKT signal pathway in ACC1. Our results showed that over-expression of Glut-1 may be associated with the occurrence of ACC.2,Our data also suggested that abnormal activation of PI3K/Akt signaling pathways may play a role in the process of occurrence of ACC.