Prognostic Value Of FDG In Laryngeal Carcinoma And The Relationship With The Expression Of GLUT-1,HIF-1α And The PI3K/Akt Pathway

Background18F-2-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (FDG PET/CT) imaging has been used widely for the diagnosis, preoperative staging, restaging, prognosis prediction, and detection of unknown primary tumors. Increased uptake of FDG, a glucose analog, directly reflects the higher glucose metabolic rate in malignant tumor cells as compared to their nonmalignant counterparts.Many mechanisms have been proposed for the accelerated glucose use in growing tumors and in transformed and malignant cells:passive diffusion, Na+-dependent glucose transport, oncogenes, and facilitative glucose transporter (GLUT). Many studies, including ours, have revealed that GLUT-1plays a significant role in malignant glucose metabolism and that it might contribute to the increased FDG uptake. Some authors consider GLUT-1a possible intrinsic marker of hypoxia in malignant tumors. Hypoxia of solid tumors has been associated with therapy resistance and a poor clinical prognosis. Biological markers that predict tumor hypoxia might be useful for selecting treatment and predicting the prognosis of patients. Some studies have demonstrated that FDG indirectly reflects the hypoxic status of malignant tumors, since FDG is associated with hypoxia markers such as GLUT and hypoxia inducible factor (HIF). High FDG uptake by human carcinomas is also associated with a poor prognosis. Previously, we showed that the FDG uptake detected by single photon emission computed tomography in head and neck cancers was related to increased GLUT-1expression. Our recent preliminary study also revealed that the SUVmax of FDG PET/CT in cervical lymph nodes predicted cervical metastasis of carcinoma from an unknown primary tumor. There is some controversy, however, as some studies did not find any association between FDG uptake and the hypoxic status of some carcinomas. The expression of HIF-la and correlated target genes such as GLUT-1is regulated by the phosphatidylinositol3-kinese/protein kinase B (PI3K/Akt) pathway. It is still unclear whether the PI3K/Akt signal pathway is involved in regulation of FDG uptake. Consequently, the relationship between FDG and hypoxia markers must be studied further. Therefore, we investigated the correlation between FDG uptake and the hypoxia markers GLUT-1, HIF-1α, PI3K, and Akt using immunohistochemistry to clarify whether FDG PET/CT can be used to predict tumor hypoxia and the prognosis of patients with laryngeal carcinoma.Materials and MethodsPatients:Using FDG PET/CT,26consecutive patients (23male,3female; mean age61.5(range37-80) years with laryngeal carcinoma were examined preoperatively. No patient received tumor-directed therapy preoperatively. Our study was approved by the Institutional Review Board of The First Affiliated Hospital, School of Medicine, Zhejiang University. Written informed consent was obtained from each patient before inclusion.Immunohistochemical Analysis and Evaluation:For immunohistochemical evaluation, paraffin blocks of formalin-fixed specimens were obtained via biopsies of the predominant lesions in each subject. Formalin-fixed, paraffin-embedded tissue blocks from the primary lesions were cut into4-μm sections, and representative sections were analyzed immunohistochemically (EliVisionTM Plus IHC kit; Fuzhou Maixin Biotechnology Development, China), using a rabbit polyclonal antibody against GLUT-1(1:50, Santa Cruz Biotechnology), a mouse monoclonal antibody against HIF-1α (1:100, Santa Cruz Biotechnology), a rabbit monoclonal antibody against PI3K (1:100, Santa Cruz Biotechnology), and a rabbit polyclonal antibody against p-Akt (1:50, Santa Cruz Biotechnology). Briefly, antigen retrieval was performed after the sections were deparaffinized with xylene and dehydrated through an ethanol series. Endogenous peroxidase activity was blocked by incubating the slides in3%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 for30min. The reaction products were visualized using 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 instead of the primary antibody for negative controls. Erythrocytes, which were present in all sections, served as internal controls for GLUT-1to confirm constant immunostaining intensity.GLUT-1, HIF-1α, PI3K, and p-Akt were evaluated by the same investigator, 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 fields and100tumor cells were counted in each high-power field for each case and for all antibodies used. 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.Follow-up:The patients were asked to come back for follow-up at1-,3-, and6-month intervals in the first, second, and third to fifth years after the initial surgery, respectively, and then annually thereafter. Follow-up consisted of a routine physical examination, strobolarynoscopy, and CT or magnetic resonance imaging (MRI) of the primary site. Patient follow-up was reported up to the date last seen in the clinic.Statistical Analyses:The statistical analyses were performed using SPSS for Windows, version15.0. Associations among SUVmax, GLUT-1, HIF-1α, PI3K, and p-Akt protein expression and the other pretreatment parameters were analyzed using the chi-square test and Fisher’s exact test. Cox’s proportional hazards regression model was used for the multivariate analysis. The correlation analysis was performed using Pearson correlation.ResultsPatients’ Characteristics:The clinicopathological findings (including sex, age, site, pathological type, TNM stage, recurrence, metastasis and follow-up) are listed in Table1. Of the twenty-six patients, twenty-four (92.3%) had squamous cell carcinomas and two (7.7%) had neuroendocrine carcinomas. twenty (76.9%), five (19.2%), and one (3.9%) patients had tumors located in the glottis, supraglottis and subglottis, respectively. All patients underwent surgery and eleven received postoperative radiotherapy. The average follow-up period was39.0(range6-81) months. Two patients were lost to follow-up. Three patients (11.5%) developed local recurrence and two (7.7%) developed distant metastases. The median overall survival was46.8months. The3-year survival rate was59.2%, the5-year survival rate was36.3.SUVmax and Survival Analysis:No significant difference in SUVmax was found betweensex, age, tumor sites, pathological types, differentiations status, TNM stages and treatment in our patients. The mean SUVmax was11.1(range6.2-23.9). When the mean SUVmax was defined as the cutoff, there was a significant difference in mean survival between the higher (≥11.1, n=10) and lower (<11.1, n=16) SUVmax subgroups (28.8vs.58.8months, p=0.004)(Fig.9).Expression of GLUT-1, HIF-1α, PI3K and p-Akt:In our series,57.7%(15/26),50.0%(13/26),30.8%(8/26) and53.8%(14/26) of the laryngeal carcinomas were positive for GLUT-1, HIF-la, PI3K and p-Akt protein, respectively (Table1).Pearson analysis showed significant correlations between GLUT-1and HIF-1α (r=0.701, p<0.001), GLUT-1and PI3K (r=0.402, p=0.042), GLUT-1and p-Akt (r=0.613, p=0.001), HIF-la and PI3K (r=0.667, p<0.001), HIF-1α and p-Akt (r=0.772, p<0.001), and PI3K and p-Akt (r=0.617, p=0.001).Correlation between SUVmax and Hypoxic Markers:Pearson analysis showed significant correlations between SUVmax and GLUT-1(r=0.517, p=0.007), HIF-1α (r=0.474, p=0.014) expression.ConclusionsAlthough we cannot draw any definite conclusion because of the small sample size, our results suggest that a high SUVmax predicts a poor prognosis in laryngeal carcinoma.A high SUVmax might be associated with increased GLUT-1,HIF-1αexpression. We suggest that FDG PET/CT can be used as a marker of tumor hypoxia and the prognosis of patients with laryngeal carcinoma. Further study is needed to confirm these findings.