laryngeal cancer is common worldwide ; it is the third most common head and neck malignancy , after nasopharyngeal and sinonasal tumors . every year , approximately 12,760 new cases of laryngeal cancer are diagnosed in the united states , and estimated 3560 deaths are caused by the disease . the most common type of laryngeal cancer is squamous cell carcinoma ( scc ) , which can range from carcinoma in situ to poorly differentiated carcinoma . laryngeal cancer is three times more likely to arise in the glottis than the supraglottis ; cancer in the subglottis is extremely rare and accounts for just 2% of all laryngeal cancers . a number of factors are believed to contribute to survival after laryngeal cancer diagnosis ; the tumor stage , tumor site , treatment strategy , and patient 's age and comorbidities are all thought to play a role . the causes of hypoxia are multifactorial and include abnormal or chaotic tumor vasculature , impaired blood perfusion , reduced oxygen consumption , and anemia . severe tumor hypoxia ultimately leads to tissue necrosis , but nonlethal levels of hypoxia can impact tumor cell biology . hypoxia - inducible factor-1 ( hif-1 ) is a transcription factor that mediates adaptive responses to hypoxia . hif-1 activity is increased as a result of genetic alteration or intratumoral hypoxia in many human cancers . hif-1 activates gene transcription to increase oxygen availability ; hif-1 can stimulate angiogenesis or reprogram cellular metabolism to adapt to reduced oxygen availability . the regulation of hif-1 subunits forms part of the oxygen response pathway regulation . in the presence of oxygen , however , in hypoxic conditions , they are not hydroxylated ; hif-1 is stabilized and can stimulate gene expression . hif-1 regulates several important biological pathways , including those involved in cellular proliferation , angiogenesis , cell metabolism , apoptosis , and migration . however , the role of hif-1 activity in laryngeal cancer is poorly understood , and very few studies regarding hif-1 in indonesian laryngeal cancer patients have been published . the aim of this study was to determine hif-1 expression in laryngeal scc ( lscc ) . the ethics committee of faculty of medicine of universitas gadjah mada , yogyakarta , approved this cross - sectional study . the study included paraffin - embedded tissue from 47 histologically diagnosed lscc patients that were seen from january 2010 to april 2014 . the study was conducted by the departments of otorhinolaryngology head and neck surgery and anatomical pathology from the faculty of medicine at universitas gadjah mada , yogyakarta , indonesia . the inclusion criteria were a patient age > 40 years and no previous chemotherapy , radiotherapy , or surgery . sections of 4 - 5  m were cut from the paraffin - embedded tissue blocks . endogenous peroxidase activity was blocked by incubating the slides in 1.5% hydrogen peroxide in absolute methanol at room temperature for 10 minutes . a mouse monoclonal immunoglobulin g ( igg ) anti - hif-1 antibody ( r&d systems , usa ) was used to detect hif-1 protein expression in the nucleus and cytoplasm . primary antibodies were applied for 1 hour at room temperature and sections were washed three times with 50  mm tris - buffered saline , ph 7.2 ( tbs ) , prior to incubation with 50  l of a polymerized , horseradish peroxidase conjugated , anti - mouse igg antibody for 30 minutes at room temperature . sections were washed three times with 50  mm tbs and protein expression was visualized using diaminobenzidine ( dab kit , thermo scientific , usa ) . sections were subsequently counterstained with hematoxylin and eosin , dehydrated , and evaluated using a light microscope . samples were incubated with 10  mm tbs instead of a primary antibody as a negative control . hif-1 expression in the nucleus and cytoplasm was only scored as positive ( 1 + ) or negative ( 0 ) . positive staining was defined as being hif-1 expression in > 10% of the tumor area . the associations between hif-1 expression and clinical stage and differentiation of lscc were analyzed using the chi square test . the clinical stage of the patients was determined by computed tomography scans , chest x - rays , and abdominal ultrasound imaging . the patient characteristics , including their gender , age , clinical stage , and histopathological differentiation ( well , moderate , or poor ) are shown in table 1 . there were 24 ( 51.1% ) patients that were < 60 years old and 23 ( 48.9% ) patients that were 60 years old . positive hif-1 staining and negative hif-1 staining were observed in 29/47 ( 61.7% ) and 18/48 ( 38.3% ) of patients , respectively . of the 29 hif-1-positive samples , 28/29 ( 96.5% ) of the patients were male and 1 ( 3.5% ) patient was female . the patients ' clinical stage was defined as early ( stages i and ii ) or advanced ( stages iii and iv ) . there were 4 ( 8.5% ) early stage patients and 43 ( 91.5% ) advanced stage patients . the primary tumor location , defined as being either the glottis or supraglottis , was predominantly the glottis ; there were 42 ( 89.4% ) patients with cancer in the glottis . histopathological analysis indicated that 15/47 ( 31.9% ) tumors were well differentiated , 24/47 ( 51.1% ) tumors were moderately differentiated , and 8/47 ( 17.0% ) tumors were poorly differentiated . no statistically significant difference in hif-1 expression was observed in tumors of different differentiations ( table 2 ) . we proceeded to examine the association between hif-1 expression and lscc clinical stage . of the 4 ( 8.5% ) early stage patients , 2 patients were positive for hif-1 protein expression and 2 were negative for hif-1 expression . in the 43 ( 91.5% ) advanced stage patients , there were 27 ( 62.8% ) patients that were positive for hif-1 protein expression and 16 ( 37.2% ) patients that were negative for hif-1 expression . however , the statistical analysis did not indicate any significant associations between hif-1 expression and lscc clinical stage ( p = 0.631 ; table 2 ) . previous studies have reported inconsistent results regarding the association between hif-1 expression and lscc clinical stage . wu et al .   found a significant relationship between hif-1 expression and a t3-t4 stage lscc ( p = 0.027 ) in china . however , another study by schrijvers et al .   demonstrated no significant relationship between hif-1 expression and an early clinical stage in scc of the glottis . however , the difference in clinical stage between their study , which focused on early stage patients , and our study , which included predominantly advanced stage patients , makes it difficult to compare our results . in other malignancies , cao et al .   have demonstrated a significant correlation between hif-1 expression and an advanced clinical stage in colorectal carcinoma . lin et al .   have also reported a statistically significant correlation between hif-1 expression and tumor size , regional metastasis , and an advanced clinical stage in oral cavity scc . another study by no et al .   examined the relationship between hif-1 and vascular endothelial growth factor ( vegf ) expression in cervical carcinomas . they found no statistically significant relationship between hif-1 expression and tumor size or regional metastasis , but they concluded that cervical carcinoma carcinogenesis is influenced by hif-1 , although the exact mechanism remained unclear . in this study , we found no associations between hif-1 expression and lscc stage or differentiation , indicating that other biomarkers may be important in defining lscc carcinogenesis . hif-1 is induced by hypoxia in solid tumors and enables tumor cell adaptation to low oxygen conditions . however , hif-1 expression is regulated by tightly controlled expression and degradation cycles . toffoli and michiels   have shown that there are hypoxic and nonhypoxic regions contained within individual tumors . furthermore , hypoxic areas can undergo a process of reoxygenation to restore normoxic conditions ; hif-1 that is stable in hypoxic conditions would be degraded during the process of reoxygenation . as such , tumor areas that have undergone reoxygenation to restore oxygen conditions would stain negative for hif-1 expression . complex mechanisms of carcinogenesis underlie the progression of lscc from an early clinical stage to an advanced clinical stage . during this transition , complex changes occur in the tumors , and many studies are being conducted to improve our understanding of these processes . when solid tumors encounter hypoxic conditions and hif-1 is expressed , a number of processes occur to achieve tumor progression and an improved clinical stage . the expression of hif-1 in hypoxic conditions triggers genomic instability , angiogenesis , apoptosis , immune evasion , tumor invasion and metastasis , and the activation of glycolysis . all of these processes produce changes in the cellular and molecular environment and are underpinned by further complex mechanisms [ 13 , 14 ] . according to poon et al . , the activation of hif-1 requires , in addition to hypoxic conditions , changes in tumor microenvironment stimuli from a number of factors , including growth factors , the von - hippel - lindau tumor suppressor protein , succinate dehydrogenase , p53 , and other proteins . protein expression in tumor cells triggers malignant progression through various processes , including angiogenesis , which is achieved through vegf ; alterations in glucose metabolism , which is achieved through glucose transporter 1 ; tumor cell survival , which is achieved though insulin - like growth factor-1 ; and metastasis , which is achieved through lysyl oxidase and plasminogen activator inhibitor-1 . this study only measured the expression of hif-1 protein , and many studies have failed to show a relationship between hif-1 expression and clinical stage . however , given the complexity of the mechanisms that underlie the progression and development of lscc , we anticipate that other factors will be shown to be correlated with lscc clinical stage . a study by no et al . has shown no statistically significant relationship between hif-1 protein expression and lscc tumor size or metastasis . it is assumed that the progression of a malignancy , which is usually characterized by an increased clinical stage , occurs through a number of complex mechanisms , not the action of one or two factors alone . have proposed that oxygen diffusion is sufficient for oxygen to travel to 100180  m between the distal end of a capillary and a cell . an increased tumor volume , indicated by a higher t stage , will decrease the efficacy of oxygen diffusion in maintaining normoxia ; hypoxic conditions and hif-1 stabilization will occur in tumor cells that are located far from the distal end of the capillary . our study did not show any significant association between hif-1 protein expression and lscc metastasis . this adds strength to the argument that hif-1 is not the only protein that plays a role in lscc carcinogenesis ; hif-1 may play an active role in the progression of malignancy , but other proteins may be required to accelerate tumor development and aggressiveness . the only statistically significant relationship demonstrated in this study was the relationship between hif-1 expression and gender ( p = 0.047 ) . in contrast , cao et al .   found no relationship between these variables , but their study cohort had a male  :  female patient ratio of approximately 1.5  :  1 . in the present study , the male  :  female patient ratio was approximately 7  :  1 . since the gender distribution was not homogenous in our study , further studies with a more homogeneous distribution of patients are required to validate our finding . overall , our results emphasize the role of hif-1 in carcinogenesis . despite finding no statistically significant associations in the present study , hif-1 and other biomarkers
objectives . this research aimed to determine the association between hypoxia - inducible factor-1 ( hif-1 ) expression and 
 laryngeal squamous cell carcinoma clinical stage . methods . 
 we retrospectively analyzed paraffin - embedded tissue from 47 laryngeal squamous cell carcinoma ( lscc ) patients from 2011 to 2014 . 
 hif-1 expression was analyzed by immunohistochemistry using an anti - hif-1 mouse monoclonal antibody . 
 the association between hif-1 expression and clinical stage was analyzed using the chi square test . 
 results . 
 the glottis was the predominant site of laryngeal squamous cell carcinoma occurrence , and 43/47 ( 91.5% ) patients presented at an advanced stage . 
 of the advanced stage patients , 27/43 stained positive for hif-1 expression and 16/43 stained negative . 
 of the early stage patients , 2/4 stained positive for hif-1 expression and 2/4 stained negative . 
 statistical analysis did not demonstrate significant association of hif-1 expression . 
 conclusion . 
 there was no statistically significant association between hif-1 expression and the clinical stage or histological differentiation of lscc .