adenoid cystic carcinoma ( acc ) is uncommon malignancies , which commonly arise in the salivary glands , although nearly half of acc occurs in other glandular areas , particularly in the nasal cavity and paranasal areas . they also arise in the tongue and the minor salivary glands , and , very rarely , originate at other sites , such as the external auditory canal , trachea , lung , breast , and bartholin s glands . typically the natural course of acc is slow ; however , local recurrence and hematogenous spread to the lungs often occur during the course of the disease . despite the indolent growth pattern , once metastatic disease is present , disease progression becomes more rapid ; one - third of patients die within 2 years of developing multiple metastases . treatment remains limited to surgery and radiation , and no systemic chemotherapeutic agent has been proven to be effective . due to its rarity clinical stage and solid histologic type were poor prognostic indicators of survival in several studies . in immunohistochemistry , expression of myb proto - oncogene protein ( myb ) , c - kit , vascular endothelial growth factor ( vegf ) , p53 , and ki-67 are known poor prognostic factors of acc ; however , the relationship between these markers and survival of patients is not well - known [ 9,11 - 15 ] . in addition , there are many potential biomarkers whose significance has not yet been determined . a previous study reported that fibroblast growth factor ( fgf ) , fibroblast growth factor receptor ( fgfr ) , and platelet - derived growth factor receptor beta ( pdgfr - beta ) represented dna copy number gain in acc by microarray - based comparative genomic hybridization . the aim of this study was to evaluate the prognostic value of potential biomarkers related to acc . the medical records of 188 patients diagnosed with acc at seoul national university hospital between 1990 and 2012 were reviewed . among these patients , 68 patients who underwent curative surgery and had available tissue this study was approved by the institutional review board of seoul national university hospital ( irb approval no . demographics , clinical and pathologic data , and treatment - related factors with regard to recurrence and patient survival were collected from medical records . sixteen patients ( 24% ) were followed up until death , and the median follow - up period was 68 months ( range , 4 to 263 months ) . core tissues ( 2 mm in diameter ) were taken from representative formalin - fixed paraffin - embedded tissue blocks and tissue microarrays were constructed for further immunohistochemical analysis . the antibodies used were basic fgf ( bfgf ; 1:100 , calbiochem , san diego , ca ) , fgfr2 ( h2263-m01 , 1:3,000 , abnova , taipei , taiwan ) , fgfr3 ( sc-13121 , 1:50 , santa cruz biotechnology , santa cruz , ca ) , c - kit ( a2502 , 1:200 , dako , carpinteria , ca ) , myb ( # 1792 - 1 , 1:80 , epitomics , burlingame , ca ) , pdgfr - beta ( sc-713 , 1:100 , santa cruz biotechnology ) , vegf ( sc-7269 , 1:1,000 , santa cruz biotechnology ) , and ki-67 ( m7240 , 1:100 , dako ) . immunostaining was evaluated semi - quantitatively for intensity ( 0 , negative ; + 1 , weak positive ; + 2 , moderate positive ; and + 3 , strong positive ) and extent ( 0 , 0% ; + 1 , 1%-25% ; + 2 , 26%-50% ; + 3 , 51%-75% ; and + 4 , 76%-100% ) by two experienced pathologists ( s.j.n . , y.k.j . ) ; normal salivary gland samples were used as controls . positivity of expression was defined as a score of 4 or more , and high expression was defined as a score of 8 or more . conversely , c - kit was graded only by extent ( 0 to + 4 ) because its expression was strong enough in all positive specimens , and positivity was defined as a grade of 2 or more . the immunostained slides for ki-67 were submitted to virtual microscope scanning under high - power magnification ( 200 ) using scanscope cs2 eslide ( aperio technologies , vista , ca ) . estimation of ki-67 expression was based on the proportion of positive cells in all tumor cells using the nuclear v9 algorithm of imagescope software ( aperio technologies ) . statistical analyses of categorical variables were performed using pearson s  test or fisher exact probability test where appropriate . the median duration of survival was calculated using the kaplan - meier method , and comparisons between groups were made using the log - rank tests . to determine the important prognostic factors , cox proportional hazards the medical records of 188 patients diagnosed with acc at seoul national university hospital between 1990 and 2012 were reviewed . among these patients , 68 patients who underwent curative surgery and had available tissue this study was approved by the institutional review board of seoul national university hospital ( irb approval no . demographics , clinical and pathologic data , and treatment - related factors with regard to recurrence and patient survival were collected from medical records . sixteen patients ( 24% ) were followed up until death , and the median follow - up period was 68 months ( range , 4 to 263 months ) . core tissues ( 2 mm in diameter ) were taken from representative formalin - fixed paraffin - embedded tissue blocks and tissue microarrays were constructed for further immunohistochemical analysis . the antibodies used were basic fgf ( bfgf ; 1:100 , calbiochem , san diego , ca ) , fgfr2 ( h2263-m01 , 1:3,000 , abnova , taipei , taiwan ) , fgfr3 ( sc-13121 , 1:50 , santa cruz biotechnology , santa cruz , ca ) , c - kit ( a2502 , 1:200 , dako , carpinteria , ca ) , myb ( # 1792 - 1 , 1:80 , epitomics , burlingame , ca ) , pdgfr - beta ( sc-713 , 1:100 , santa cruz biotechnology ) , vegf ( sc-7269 , 1:1,000 , santa cruz biotechnology ) , and ki-67 ( m7240 , 1:100 , dako ) . immunostaining was evaluated semi - quantitatively for intensity ( 0 , negative ; + 1 , weak positive ; + 2 , moderate positive ; and + 3 , strong positive ) and extent ( 0 , 0% ; + 1 , 1%-25% ; + 2 , 26%-50% ; + 3 , 51%-75% ; and + 4 , 76%-100% ) by two experienced pathologists ( s.j.n . , y.k.j . ) ; normal salivary gland samples were used as controls . positivity of expression was defined as a score of 4 or more , and high expression was defined as a score of 8 or more . conversely , c - kit was graded only by extent ( 0 to + 4 ) because its expression was strong enough in all positive specimens , and positivity was defined as a grade of 2 or more . the immunostained slides for ki-67 were submitted to virtual microscope scanning under high - power magnification ( 200 ) using scanscope cs2 eslide ( aperio technologies , vista , ca ) . estimation of ki-67 expression was based on the proportion of positive cells in all tumor cells using the nuclear v9 algorithm of imagescope software ( aperio technologies ) . statistical analyses of categorical variables were performed using pearson s  test or fisher exact probability test where appropriate . the median duration of survival was calculated using the kaplan - meier method , and comparisons between groups were made using the log - rank tests . to determine the important prognostic factors , cox proportional hazards regression models were used in univariate and multivariate analyses . a two - sided value of p < 0.05 was considered statistically significant . all analyses were performed using spss for windows ver . 20.0 ( ibm co. , armonk , ny ) . of the 68 patients , 26 patients were male and 42 were female , with median age of 55 years ( range , 26 to 84 years ) . primary sites were major salivary glands in 36 cases ( 53% ) , nasal cavity and paranasal sinuses in 16 ( 24% ) , oral cavity and tongue in seven ( 10% ) , lung and trachea in four ( 6% ) , and other sites in five ( 7% ) . other sites included the lacrimal gland , bartholin s gland , and external auditory canal . twenty - three patients ( 34% ) had perineural invasion and 39 ( 57% ) had involvement of resection margins . local recurrence was detected in 21 patients ( 31% ) , regional recurrence in three ( 4% ) , distant metastases in 35 ( 51% ) , and any kind of recurrence in 39 ( 57% ) . of patients with distant metastases , 32 patients ( 47% ) had lung metastasis , nine ( 13% ) had liver metastasis , six ( 9% ) had bone metastasis , and two ( 3% ) had central nervous system metastasis . analysis of patients was based on clinicopathological factors , including sex , age , primary site , local treatment modality including postoperative radiotherapy , systemic chemotherapy , perineural invasion , and positive resection margins . survival analysis was performed in each subgroup using the cox proportional hazards regression model . among these clinicopathological variables , primary site of nasal cavity and paranasal sinus was a poor prognostic factor for disease - free survival ( dfs ; hazard ratio [ hr ] , 2.34 ; 95% confidence interval [ ci ] , 1.13 to 4.84 ; p=0.022 ) . perineural invasion was the only significant prognostic factor of poor overall survival ( os ; hr , 3.05 ; 95% ci , 1.10 to 8.44 ; p=0.032 ) ( table 1 ) . in immunohistochemistry , overexpression of bfgf was observed in 48 patients ( 71% ) , c - kit in 52 ( 77% ) , fgfr2 in six ( 9% ) , fgfr3 in four ( 6% ) , myb in 24 ( 35% ) , pdgfr - beta in 43 ( 63% ) , and vegf in 62 ( 91% ) . the median ki-67 value was 4% ( range , 0% to 33% ) ( table 2 , fig . positive expression with a score of 4 or more for bfgf , fgfr2 , myb , pdgfr - beta , and vegf and a score of 2 or more for c - kit did not show correlation with dfs and os ( table 2 ) . on the other hand , high expression with a score of 8 or more for vegf was a poor prognostic factor for os ( hr , 3.45 ; 95% ci , 1.11 to 10.71 ; p=0.033 ) , and ki-67 expression of more than 7% was significant as a poor prognostic factor for both dfs ( hr , 3.25 ; 95% ci , 1.58 to 6.68 ; p=0.001 ) and os ( hr , 4.47 ; 95% ci , 1.52 to 13.18 ; p=0.007 ) ( table 3 , figs . although a score of 4 or more for fgfr3 and a score of 8 or more for fgfr2 expression also showed a significant p - value , due to the few patients with expression , further validation of the prognostic values is needed in a larger sample size . in multivariate analysis , primary site of nasal cavity and paranasal sinus ( hr , 2.21 ; 95% ci , 1.09 to 4.50 ; p=0.028 ) and ki-67 expression ( hr , 3.05 ; 95% ci , 1.43 to 6.54 ; p=0.004 ) were independent risk factors for poor dfs . high expression of vegf ( hr , 5.44 ; 95% ci , 1.48 to 19.98 ; p=0.011 ) and ki-67 ( hr , 4.83 ; 95% ci , 1.44 to 16.21 ; p=0.011 ) were independently significant factors of poor os . however , perineural invasion was not statistically significant for os ( hr , 2.90 ; 95% ci , 1.00 to 8.41 ; p=0.051 ) ( table 4 ) . conduct of further studies to determine relationship between perineural invasion and os will be needed . patients who had both high expression of vegf and ki-67 expression of more than 7% showed poorer os than patients who had high expression of only one of the two proteins ( hr , 8.58 ; 95% ci , 1.47 to 50.01 ; p=0.017 ) ( fig . of the 68 patients , 26 patients were male and 42 were female , with median age of 55 years ( range , 26 to 84 years ) . primary sites were major salivary glands in 36 cases ( 53% ) , nasal cavity and paranasal sinuses in 16 ( 24% ) , oral cavity and tongue in seven ( 10% ) , lung and trachea in four ( 6% ) , and other sites in five ( 7% ) . other sites included the lacrimal gland , bartholin s gland , and external auditory canal . twenty - three patients ( 34% ) had perineural invasion and 39 ( 57% ) had involvement of resection margins . local recurrence was detected in 21 patients ( 31% ) , regional recurrence in three ( 4% ) , distant metastases in 35 ( 51% ) , and any kind of recurrence in 39 ( 57% ) . of patients with distant metastases , 32 patients ( 47% ) had lung metastasis , nine ( 13% ) had liver metastasis , six ( 9% ) had bone metastasis , and two ( 3% ) had central nervous system metastasis . analysis of patients was based on clinicopathological factors , including sex , age , primary site , local treatment modality including postoperative radiotherapy , systemic chemotherapy , perineural invasion , and positive resection margins . survival analysis was performed in each subgroup using the cox proportional hazards regression model . among these clinicopathological variables , primary site of nasal cavity and paranasal sinus was a poor prognostic factor for disease - free survival ( dfs ; hazard ratio [ hr ] , 2.34 ; 95% confidence interval [ ci ] , 1.13 to 4.84 ; p=0.022 ) . perineural invasion was the only significant prognostic factor of poor overall survival ( os ; hr , 3.05 ; 95% ci , 1.10 to 8.44 ; p=0.032 ) ( table 1 ) . in immunohistochemistry , overexpression of bfgf was observed in 48 patients ( 71% ) , c - kit in 52 ( 77% ) , fgfr2 in six ( 9% ) , fgfr3 in four ( 6% ) , myb in 24 ( 35% ) , pdgfr - beta in 43 ( 63% ) , and vegf in 62 ( 91% ) . the median ki-67 value was 4% ( range , 0% to 33% ) ( table 2 , fig . positive expression with a score of 4 or more for bfgf , fgfr2 , myb , pdgfr - beta , and vegf and a score of 2 or more for c - kit did not show correlation with dfs and os ( table 2 ) . on the other hand , high expression with a score of 8 or more for vegf was a poor prognostic factor for os ( hr , 3.45 ; 95% ci , 1.11 to 10.71 ; p=0.033 ) , and ki-67 expression of more than 7% was significant as a poor prognostic factor for both dfs ( hr , 3.25 ; 95% ci , 1.58 to 6.68 ; p=0.001 ) and os ( hr , 4.47 ; 95% ci , 1.52 to 13.18 ; p=0.007 ) ( table 3 , figs . although a score of 4 or more for fgfr3 and a score of 8 or more for fgfr2 expression also showed a significant p - value , due to the few patients with expression , further validation of the prognostic values is needed in a larger sample size . in multivariate analysis , primary site of nasal cavity and paranasal sinus ( hr , 2.21 ; 95% ci , 1.09 to 4.50 ; p=0.028 ) and ki-67 expression ( hr , 3.05 ; 95% ci , 1.43 to 6.54 ; p=0.004 ) were independent risk factors for poor dfs . high expression of vegf ( hr , 5.44 ; 95% ci , 1.48 to 19.98 ; p=0.011 ) and ki-67 ( hr , 4.83 ; 95% ci , 1.44 to 16.21 ; p=0.011 ) were independently significant factors of poor os . however , perineural invasion was not statistically significant for os ( hr , 2.90 ; 95% ci , 1.00 to 8.41 ; p=0.051 ) ( table 4 ) . conduct of further studies to determine relationship between perineural invasion and os will be needed . patients who had both high expression of vegf and ki-67 expression of more than 7% showed poorer os than patients who had high expression of only one of the two proteins ( hr , 8.58 ; 95% ci , 1.47 to 50.01 ; p=0.017 ) ( fig . the goal of the current study was to identify prognostic markers of acc by evaluating the relationship between clinicopathological and immunohistochemical data and dfs and os . the markers bfgf , c - kit , fgfr2 , fgfr3 , myb , pdgfr - beta , vegf , and ki-67 were evaluated by immunohistochemistry . in univariate analysis , nasal cavity and paranasal sinus as primary site and ki-67 expression of more than 7% were risk factors for poor dfs , and presence of perineural invasion and high expression of vegf and ki-67 were significant prognostic factors for poor os . among them , high expression of vegf and ki-67 were independently significant prognostic factors for os in multivariate analyses . acc has diverse primary sites and there are varying levels of difficulty of operation according to primary sites . ciccolallo et al . , who analyzed survival of acc in a large population using the eurocare database , showed that acc originating from nasal cavity , pharynx , and larynx have poorer 5-year survival rate than oral cavity and major salivary glands . in the current study , although no significant difference of os was observed among primary sites , nasal cavity and paranasal sinus were the sites showing poor dfs . in the current study , perineural invasion was observed in 23 patients ( 34% ) , and it was the only significant prognostic factor for os among the clinicopathological values evaluated . its importance as a poor prognostic factor is well - established in the literature . in the study reported by garden et al . , in addition to the presence of perineural invasion , positive resection margin was an adverse prognostic factor in terms of local control , which was not the case in this study . vegf , a critical factor in tumor angiogenesis , has been widely assessed in various types of cancer . association of vegf expression with poor prognosis of acc has been reported in a few studies . in an analysis with 29 patients of salivary gland carcinomas , lim et al .   reported a significant association of high expression of vegf with poor os . reported correlation of high vegf expression with tumor stage in 55 patients of salivary accs , but not with local recurrence and survival rate . high expression of vegf was an independently important factor for poor os in the current study . although vegf expression showed no statistically significant difference for dfs , it showed a poor trend in the kaplan - meier curve . conduct of further studies with a large population will be needed in order to verify correlation of vegf expression with poor dfs . several studies identified high expression of ki-67 as a negative prognostic factor in salivary gland carcinomas . nordgard et al . , who analyzed 44 patients diagnosed with accs , reported a significant difference of poor dfs with ki-67 expression of more than 4% . lim et al . , who analyzed survival of 29 patients , 15 of salivary accs and 14 of mucopeidermoid carcinomas , reported correlation between high expression of ki-67 and poor os . in addition , ettl et al .   demonstrated relationship between ki-67 expression and poor os in 101 patients of salivary gland carcinomas . however , that population only included 25 acc patients . in the current study , ki-67 was the strongest prognostic marker for both poor dfs and os in acc . this is the largest study to date composed only of acc patients , and proves significance of ki-67 expression . in our analysis one of the most studied biomarkers of acc is the translocation between myb oncogene and nfib translocation factor . approximately 50% of patients have a myb - nfib translocation , and these patients tend to have a higher risk for local relapse . myb over - expression has been associated with myb - nfib translocation , although its relationship with acc prognosis was uncertain . in our study , myb expression was not a significant factor in predicting poor dfs or os . another biomarker , c - kit , has been the subject of several research studies in acc . in a recent study , c - kit expression showed correlation with clinical stage , perineural invasion , locoregional recurrence , and distant metastases ; however , there was little data regarding the relationship between c - kit expression and os . c - kit expression was not a significant prognostic factor of dfs and os in the current study . pdgfr - beta expression is known to be a poor prognostic factor in breast cancer . similarly , dna copy numbers of pdgfr - beta on chromosome 5 are increased in acc and it is a possible factor contributing to the progression of acc . however , the relationship between pdgfr - beta expression and the prognosis of acc has not been previously reported . the current study showed that high expression of pdgfr - beta was not significant as a prognostic factor in acc . dna copy number gain of fgf and fgfr in acc was also reported in an earlier study ; however , there was almost no data on the immunohistochemical expression of fgf and fgfr which was investigated in this study . bfgf expression of more than 70% was observed , however expression of fgfr2 and fgfr3 was rare . among them , bfgf had no significant value as prognostic markers , and conduct of further studies on fgfr2 and fgfr3 will be needed in order to provide patients with practical recommendations . most studies conducted in the past analyzed the utility of immunohistochemical markers with respect to acc invasiveness and recurrence . however , the current study was conducted in a large population and investigated the correlation between several immunohistochemical markers and survival , which is more critical for acc prognosis . we acknowledge that there are some limitations to this study , including the lack of validating the immunohistochemical cut - off values used , and the fact that this is a retrospective study . in conclusion , high expression of vegf and high expression of ki-67 are independent prognostic factors of poor os in acc . therefore , more aggressive and differentiated treatment should be provided to patients who show high expression of these markers . conduct of further prospective studies in larger populations will be necessary in order to confirm these prognostic factors and to elucidate appropriate treatment modalities .
purposethe purpose of this study was to evaluate potential prognostic factors in patients with adenoid cystic carcinoma ( acc).materials and methodsa total of 68 patients who underwent curative surgery and had available tissue were enrolled in this study . 
 their medical records and pathologic slides were reviewed and immunohistochemistry for basic fibroblast growth factor , fibroblast growth factor receptor ( fgfr ) 2 , fgfr3 , c - kit , myb proto - oncogene protein , platelet - derived growth factor receptor beta , vascular endothelial growth factor ( vegf ) , and ki-67 was performed . 
 univariate and multivariate analysis was performed for determination of disease - free survival ( dfs ) and overall survival ( os).resultsin univariate analyses , primary site of nasal cavity and paranasal sinus ( p=0.022 ) and ki-67 expression of more than 7% ( p=0.001 ) were statistically significant factors for poor dfs . 
 regarding os , perineural invasion ( p=0.032 ) , high expression of vegf ( p=0.033 ) , and high expression of ki-67 ( p=0.007 ) were poor prognostic factors . 
 in multivariate analyses , primary site of nasal cavity and paranasal sinus ( p=0.028 ) and high expression of ki-67 ( p=0.004 ) were independent risk factors for poor dfs , and high expression of vegf ( p=0.011 ) and ki-67 ( p=0.011 ) showed independent association with poor os.conclusionhigh expression of vegf and ki-67 were independent poor prognostic factors for os in acc .