cutaneous squamous cell carcinoma ( cscc ) is the second most common type of non - melanoma skin cancer ( nmsc ) globally , with a constantly increasing incidence .
the known risk factors for cscc include aging , fair complexion , homosexual intercourse , chronic skin ulcers , burn scars , immune suppression , and exposure to ultraviolet light and chemical carcinogens .
the vast majority of csccs are curable with simple surgical resection , and prognosis is usually favorable .
however , approximately 4% of the patients develop metastatic disease and 1.5% eventually die from the disease .
there are few treatments for patients with advanced csccs , primarily due to the lack of knowledge regarding genomic alternations that induce metastatic cscc .
therefore , it is important to identify specific molecules involved in metastatic cscc that can potentially become targets for diagnosis and enable the development of new therapy strategies .
angiogenesis , the formation of new blood vessels from pre - existing vasculature , plays a key role in tumor development , invasion , and metastases formation .
angiogenesis is a complicated process stimulated by a variety of factors , among which vascular endothelial growth factor ( vegf ) is crucial .
vegf is one of the most potent endothelial cell growth factors ; it promotes formation of new blood vessels and stimulates vascular permeability of endothelial cells .
vegf initiates intracellular signal transduction pathways by specifically binding 2 transmembrane vegf receptors on endothelial cells . while initiating intracellular signal transduction pathways , vegf supplies the newly formed blood vessels with nutrients and oxygen .
this induces aggressive local growth and metastases formation , resulting in poor prognosis and low survival rates of cancer patients .
the vegf gene is located at chromosome 6p21.3 and consists of 8 exons and 7 introns .
it is very polymorphic , with at least 30 functional single - nucleotide polymorphisms ( snps ) in the 5-untranslated region , 3-untranslated region ( utr ) and promoter .
single - nucleotide polymorphisms ( snps ) of vegf that can be found in the promoter or other regulatory regions may regulate vegf expression or activity .
the involvement of vegf polymorphisms in cancer progression has been demonstrated in several types of tumors [ 1518 ] .
vegf polymorphisms have previously been described in solid carcinomas , containing lung cancer , non - hodgkin s lymphoma , cervical cancer , colorectal cancer , esophageal squamous cell carcinoma ( escc ) [ 10 , 23 ] , and oral squamous cell carcinoma ( oscc ) [ 9,2426 ] . the impact of the vegf snps on clinical outcomes of patients with cancer has been previously described .
however , the impact of vegf polymorphism during follow - up has been much less studied .
the -1154460c > t snp ( rs833061 ) and -1154g > a snp ( rs1570360 ) are located at the promoter region and may regulate promoter activity .
however , the effect of vegf gene -460c > t snp ( rs833061 ) and -1154g > a snp ( rs1570360 ) on cscc risk and prognosis remains unclear . in the current study ,
the -460c > t snp ( rs833061 ) and -1154g > a snp ( rs1570360 ) within the vegf gene were detected in cscc patients ; a regular follow - up was conducted for all the patients .
this study aimed to further clarify the potential association of these 2 snps with the cscc risk and to evaluate the prognostic impact of vegf - related snps on cscc patients .
they were treated in the department of surgery at shandong provincial hospital affiliated to shandong university between 2010 and 2014 ; the group included 55 men and 45 women .
the ages of patients ranged from 34 to 76 years old , with the average age of 56.0410.41 years old .
the control group contained 124 healthy subjects who came from our hospital s physical checkup center during the same period of time .
written permission was obtained from all the participants and the study was approved by the research ethics committee of shandong provincial hospital affiliated to shandong university . peripheral venous blood samples ( 5 ml )
were acquired from all the participants and stored at 20c until the extraction of genomic dna .
genomic dna was isolated with the qiaamp dna mini kit ( qiagen , hamburg germany ) according to the manufacturer s protocol .
the primers of -1154460c > t were 5-cgagagtgaggacgtgtgtg-3(forward ) and 5-attggaatcctggagtgacc-3(reverse ) ; the primers for -11541154g > a were 5-tcctgctccctcctcgccaatg-3(forward ) and 5-ggcggggacaggcgagcatc-3(reverse ) , purchased from sangon biotech ( shanghai , china ) .
the pcr reaction was performed in a 20-l reaction mixture containing 40160 ng dna template , 0.25 units taq dna polymerase ( sangon biotech ) , 250 m each deoxyribonucleotide triphosphate ( dntp ) ( sangon biotech ) , 0.5 m forward primer , 0.5 m reverse primer , and 1pcr buffer with 1.2 mm mgcl2 .
the pcr reaction was carried out in a thermocycler ( mj research , usa ) and reaction conditions consisted of 94 c pre - denaturizing for 5 min , 94c denaturizing for 45 s , 61c annealing for 45 s , 72c extension for 45 s , and 72c continuous extension for 7 min after 35 cycles .
the amplified products were digested with bshl236i and mnli restriction endonuclease ( takara , japan ) incubated at 37c for 16 h. the pcr and digestion products were analyzed with 3% agarose gels .
follow - up files were created for all the cscc patients upon admission , including complete pathological , clinical , and follow - up data .
a regular follow - up of 2~5 years for 100 patients was performed by hospital visit , treatment , and telephone interview .
follow - up start time was defined as the discharge time of patients after system treatment and the follow - up deadline was january 2015 .
follow - up time was counted monthly until death or the end of the follow - up time .
hardy - weinberg equilibrium ( hwe ) was calculated with hardy - weinberg equilibrium theory ( p = allele frequency , q=1p , p+q=1 ) .
the different distributions of allele frequencies and genotypes in the case and control group were analyzed using the chi - square test .
logistic regression analysis was used to calculate the odds ratios ( ors ) and 95% confidence intervals ( cis ) .
kaplan - meier method was used to plot survival curves and the statistical differences were analyzed with the log - rank test .
they were treated in the department of surgery at shandong provincial hospital affiliated to shandong university between 2010 and 2014 ; the group included 55 men and 45 women .
the ages of patients ranged from 34 to 76 years old , with the average age of 56.0410.41 years old .
the control group contained 124 healthy subjects who came from our hospital s physical checkup center during the same period of time .
written permission was obtained from all the participants and the study was approved by the research ethics committee of shandong provincial hospital affiliated to shandong university .
peripheral venous blood samples ( 5 ml ) were acquired from all the participants and stored at 20c until the extraction of genomic dna .
genomic dna was isolated with the qiaamp dna mini kit ( qiagen , hamburg germany ) according to the manufacturer s protocol .
genotyping of the vegf gene -460c > t and -1154g > a polymorphisms were detected using pcr 
the primers of -1154460c > t were 5-cgagagtgaggacgtgtgtg-3(forward ) and 5-attggaatcctggagtgacc-3(reverse ) ; the primers for -11541154g > a were 5-tcctgctccctcctcgccaatg-3(forward ) and 5-ggcggggacaggcgagcatc-3(reverse ) , purchased from sangon biotech ( shanghai , china ) .
the pcr reaction was performed in a 20-l reaction mixture containing 40160 ng dna template , 0.25 units taq dna polymerase ( sangon biotech ) , 250 m each deoxyribonucleotide triphosphate ( dntp ) ( sangon biotech ) , 0.5 m forward primer , 0.5 m reverse primer , and 1pcr buffer with 1.2 mm mgcl2 .
the pcr reaction was carried out in a thermocycler ( mj research , usa ) and reaction conditions consisted of 94 c pre - denaturizing for 5 min , 94c denaturizing for 45 s , 61c annealing for 45 s , 72c extension for 45 s , and 72c continuous extension for 7 min after 35 cycles .
the amplified products were digested with bshl236i and mnli restriction endonuclease ( takara , japan ) incubated at 37c for 16 h. the pcr and digestion products were analyzed with 3% agarose gels .
follow - up files were created for all the cscc patients upon admission , including complete pathological , clinical , and follow - up data .
a regular follow - up of 2~5 years for 100 patients was performed by hospital visit , treatment , and telephone interview .
follow - up start time was defined as the discharge time of patients after system treatment and the follow - up deadline was january 2015 .
follow - up time was counted monthly until death or the end of the follow - up time .
hardy - weinberg equilibrium ( hwe ) was calculated with hardy - weinberg equilibrium theory ( p = allele frequency , q=1p , p+q=1 ) .
the different distributions of allele frequencies and genotypes in the case and control group were analyzed using the chi - square test .
logistic regression analysis was used to calculate the odds ratios ( ors ) and 95% confidence intervals ( cis ) .
kaplan - meier method was used to plot survival curves and the statistical differences were analyzed with the log - rank test .
there were 55 men and 45 women in the patient group ; 46 patients were under 60 years old and 54 patients were 60 years old or older ( average age : 56.0410.41 years old ; range : 3476 years old ) . there were 40 poorly - differentiated cases ( the percentage of differentiated cells < 50% ) , 34 moderately - differentiated cases ( the differentiated cells between 5075% ) , and 26 well - differentiated cases ( the differentiated cells > 75% ) , according to broders classification .
thirty - five cases with lymph node involvement and 65 cases without lymph node involvement were identified .
the control group contained 124 healthy subjects attending the hospital physical checkup center during the same period .
there were 66 males and 58 females in the control group ; 59 subjects were under 60 years old and 65 patients were 60 years old or older ( average age : 53.7911.89 years ; range : 3078 years ) .
age and sex in the two groups showed no significant differences ( p>0.05 ) ( table 1 ) .
genotypes of the vegf-460c > t and -1154g > a were successfully detected in all the dna samples using the pcr - rflp method .
the homozygous t / t genotype remained uncut at 175bp ; the homozygous c / c genotype was completely digested into 155 bp and 20 bp fragments ; 175 bp , 155 bp , and 20 bp fragments corresponded to the heterozygous c > t genotype ( figure 1 ) . the 206-bp pcr product of vegf-1154g > a was digested with mnli restriction endonuclease and the g allele
the homozygous a / a genotype was digested into 184bp and 22bp fragments ; the homozygous g / g genotype was completely digested into 150 bp , 34 bp and 22 bp fragments ; all 4 fragments ( 184 bp , 150 bp , 34bp , and 22 bp ) corresponded to the heterozygous g > a genotype ( figure 2 ) .
the distribution of the vegf-460c > t snp genotype in patients is presented in table 2 .
the genotype frequencies of cc , ct , and tt in the well - differentiated group was 46.15% , 38.46% , and 15.38% , respectively ; in the moderately - differentiated group , the genotype frequencies of cc and ct were 44.11% and 55.89% , respectively ; the genotype frequencies of cc and ct were 95.00% and 5.00% , respectively , in the poorly - differentiated group .
there were hardly any carriers of homozygote tt in either moderately- or poorly - differentiated grades .
differences between the 3 groups were significant ( p<0.001 ) . the genotype distribution of vegf-1154 g > a was also associated with histological differentiation of cscc ( p<0.001 ) .
the genotype frequencies of gg in the well - differentiated group , the moderately - differentiated group , and the poorly - differentiated group were 96.15% , 47.06% , and 25.00% , respectively ; the genotype frequencies of ga in the well - differentiated group , the moderately - differentiated group , and the poorly - differentiated group were 4.00% , 47.06% , and 60.00% , respectively .
the genotype distribution of vegf-460c > t and vegf-1154 g > a showed no significant correlation with age , sex , or lymph node metastasis ( all p>0.05 ) ( table 2 ) .
the vegf-460c > t and -1154g > a genotype and allele distribution of the cscc patients and of the control group are presented in table 3 .
the genotype distribution of the vegf-460c > t and -1154g > a polymorphism did not deviate from hardy - weinberg equilibrium ( p>0.05 ) .
the ct and cc genotype frequencies of vegf-460c > t in the control group were 50.81% and 41.13% , which were significantly different from those in the patient group ( 31.00% and 65.00% , respectively ) ( ct vs. cc , or=0.39 , 95%ci : 0.220.68 , p=0.001 ) . compared to the cc genotype
, the tt+ct genotype significantly decreased the risk of cscc ( or=0.36 , 95% ci : 0.210.63 , p<0.001 ) .
the c allele frequencies in the control group and patient group were 66.53% and 80.50% , respectively ; while the t allele frequencies in the control group and patient group were 33.47% and 19.50% , respectively ; the differences in the 2 groups were statistically significant ( t vs. c , or=0.48 , 95%ci : 0.310.75 , p=001 ) . comparing the genotype distributions of the vegf gene -1154
g > a polymorphism in cscc patients to that in the controls revealed no significant differences ( p>0.05 ) and the differences in allele frequencies were also not statistically significant .
the follow - up deadline was january 2015 ; the longest follow - up time was 60 months and the shortest follow - up time was 24 months .
eleven patients were lost during the follow - up period ; the follow - up rate was 89% .
for vegf-460c > t snp , the mean survival time of cscc patients with cc , ct , and tt genotype were 28.92 , 53.54 , and 58.75 months , respectively .
the cc genotype was an adverse factor that negatively affected the survival time of cscc patients when compared with heterozygote ct and homozygote tt ( p<0.001 , figure 3 ) . for vegf-1154 g > a snp , the mean survival time of cscc patients with gg , ga , and aa genotypes were 41.19 , 36.41 , and 23.88 months , respectively ; compared to the gg and ga genotypes , the aa genotype had a significant correlation with decreased overall survival time ( p=0.009 , figure 4 ) .
there were 55 men and 45 women in the patient group ; 46 patients were under 60 years old and 54 patients were 60 years old or older ( average age : 56.0410.41 years old ; range : 3476 years old ) . there were 40 poorly - differentiated cases ( the percentage of differentiated cells < 50% ) , 34 moderately - differentiated cases ( the differentiated cells between 5075% ) , and 26 well - differentiated cases ( the differentiated cells > 75% ) , according to broders classification .
thirty - five cases with lymph node involvement and 65 cases without lymph node involvement were identified .
the control group contained 124 healthy subjects attending the hospital physical checkup center during the same period .
there were 66 males and 58 females in the control group ; 59 subjects were under 60 years old and 65 patients were 60 years old or older ( average age : 53.7911.89 years ; range : 3078 years ) .
age and sex in the two groups showed no significant differences ( p>0.05 ) ( table 1 ) .
genotypes of the vegf-460c > t and -1154g > a were successfully detected in all the dna samples using the pcr - rflp method .
the homozygous t / t genotype remained uncut at 175bp ; the homozygous c / c genotype was completely digested into 155 bp and 20 bp fragments ; 175 bp , 155 bp , and 20 bp fragments corresponded to the heterozygous c > t genotype ( figure 1 ) .
the 206-bp pcr product of vegf-1154g > a was digested with mnli restriction endonuclease and the g allele was replaced by the a allele with a loss of digestion site .
the homozygous a / a genotype was digested into 184bp and 22bp fragments ; the homozygous g / g genotype was completely digested into 150 bp , 34 bp and 22 bp fragments ; all 4 fragments ( 184 bp , 150 bp , 34bp , and 22 bp ) corresponded to the heterozygous g > a genotype ( figure 2 ) .
the distribution of the vegf-460c > t snp genotype in patients is presented in table 2 .
the genotype frequencies of cc , ct , and tt in the well - differentiated group was 46.15% , 38.46% , and 15.38% , respectively ; in the moderately - differentiated group , the genotype frequencies of cc and ct were 44.11% and 55.89% , respectively ; the genotype frequencies of cc and ct were 95.00% and 5.00% , respectively , in the poorly - differentiated group .
there were hardly any carriers of homozygote tt in either moderately- or poorly - differentiated grades .
differences between the 3 groups were significant ( p<0.001 ) . the genotype distribution of vegf-1154 g > a was also associated with histological differentiation of cscc ( p<0.001 ) .
the genotype frequencies of gg in the well - differentiated group , the moderately - differentiated group , and the poorly - differentiated group were 96.15% , 47.06% , and 25.00% , respectively ; the genotype frequencies of ga in the well - differentiated group , the moderately - differentiated group , and the poorly - differentiated group were 4.00% , 47.06% , and 60.00% , respectively .
the genotype distribution of vegf-460c > t and vegf-1154 g > a showed no significant correlation with age , sex , or lymph node metastasis ( all p>0.05 ) ( table 2 ) .
the vegf-460c > t and -1154g > a genotype and allele distribution of the cscc patients and of the control group are presented in table 3 .
the genotype distribution of the vegf-460c > t and -1154g > a polymorphism did not deviate from hardy - weinberg equilibrium ( p>0.05 ) .
the ct and cc genotype frequencies of vegf-460c > t in the control group were 50.81% and 41.13% , which were significantly different from those in the patient group ( 31.00% and 65.00% , respectively ) ( ct vs. cc , or=0.39 , 95%ci : 0.220.68 , p=0.001 ) . compared to the cc genotype ,
the tt+ct genotype significantly decreased the risk of cscc ( or=0.36 , 95% ci : 0.210.63 , p<0.001 ) .
the c allele frequencies in the control group and patient group were 66.53% and 80.50% , respectively ; while the t allele frequencies in the control group and patient group were 33.47% and 19.50% , respectively ; the differences in the 2 groups were statistically significant ( t vs. c , or=0.48 , 95%ci : 0.310.75 , p=001 ) . comparing the genotype distributions of the vegf gene -1154
g > a polymorphism in cscc patients to that in the controls revealed no significant differences ( p>0.05 ) and the differences in allele frequencies were also not statistically significant .
the follow - up deadline was january 2015 ; the longest follow - up time was 60 months and the shortest follow - up time was 24 months .
eleven patients were lost during the follow - up period ; the follow - up rate was 89% .
for vegf-460c > t snp , the mean survival time of cscc patients with cc , ct , and tt genotype were 28.92 , 53.54 , and 58.75 months , respectively .
the cc genotype was an adverse factor that negatively affected the survival time of cscc patients when compared with heterozygote ct and homozygote tt ( p<0.001 , figure 3 ) . for vegf-1154 g > a snp , the mean survival time of cscc patients with gg , ga , and aa genotypes were 41.19 , 36.41 , and 23.88 months , respectively ; compared to the gg and ga genotypes , the aa genotype had a significant correlation with decreased overall survival time ( p=0.009 , figure 4 ) .
angiogenesis is an important process for tumor development , invasion , and metastasis ; it is stimulated by a variety of factors .
recently , several vegf snps have been identified and their important effects have attracted considerable attention .
correlation between vegf snps and susceptibility or prognosis in various forms of cancers has been reported in many studies . in the present study , the -460
c > t and -1154 g > a polymorphisms of vegf in 100 cscc patients and 124 healthy subjects were observed . in order to analyze the relation of vegf gene polymorphisms with cscc risk
, the patients were stratified by age , sex , histopathological grade , and lymph node involvement .
the results demonstrate that the genotype distribution of vegf-460 c > t shows no correlation with age , sex , or lymph node metastasis ( all p>0.05 ) , but there was association with histological differentiation of cscc ( p<0.001 ) .
a significant difference was found between the genotype distribution and allele frequency of the vegf gene -460 c > t polymorphism of the control group and that of the patients with cscc .
furthermore , a higher percentage of the t allele and the tt homozygote were distributed in cscc patients compared to the controls .
therefore , we can reason that there was a much lower cscc risk for individuals with the ct heterozygote genotype in their vegf-460 c > t region , indicating that the vegf-460 c > t polymorphism could act as a candidate genetic biomarker in cscc patients .
the association of the vegf-460 c > t polymorphism with clinical outcomes in cscc patients has not been evaluated .
however , there were several studies concerning vegf-460 c > t polymorphism in other cancers .
indicated that the t allele and tt genotype of vegf-460 c > t are significantly associated with oscc .
borase et al . demonstrated that the t allele and tt genotype of vegf-460 c > t can greatly increase the risk for oscc patients . in the analysis of a correlation between clinical characteristics and the vegf gene -1154
g > a polymorphism , the results demonstrate that the genotype distribution of vegf-1154 g > a shows no relation with age , sex , or lymph node metastasis ( all p>0.05 ) , but there was an association with histological differentiation of cscc ( p<0.001 ) .
the decreased frequency of the vegf-1154 a allele ( aa and ga genotypes ) in cscc patients did not differ significantly from that of the control . in a previous study ,
kmmerer et al . demonstrated that the genotype distribution of vegf-1154 g > a in oscc patients and controls had no significant difference .
unal et al . examined 89 healthy subjects and 57 patients with laryngeal squamous cell carcinoma ; they found that the -1154 gg genotype of vegf appears to be a higher risk for laryngeal scc patients .
prognostic factors play an essential role when come to dividing patients into different risk groups in order to predict outcomes and provide adequate therapy strategies . in the current study ,
the relationship between vegf gene -460 c > t polymorphism , -1154 g > a polymorphism , and the survival of patients with cscc was examined for the first time .
the results demonstrate that the tt genotype of -460 c > t is a protective factor in comparison to genotypes of ct and cc , positively affecting the survival time of cscc patients ( p<0.001 ) .
the aa genotype of -1154 g > a has a significant correlation with the decreased overall survival time in cscc patients when compared with homozygote gg and heterozygote ga ( p=0.009 ) .
observed that the vegf-460 c > t snp had no significant impact on survival in advanced osccs , while the -1154 aa genotype was associated with significantly worse survival in the univariate analysis .
in contrast , supic et al . found that the -1154 gg genotype may be a prognostic marker associated with significantly worse survival in advanced - stage oscc patients with a similar sample size .
other studies revealed the -1154 gg genotype to be an adverse survival factor in colorectal cancer , but it was associated with a higher overall survival of the 1154 a allele in advanced breast cancer . to sum up , results of clinical studies are inconsistent , possibly due to differences in ethnic groups studied , tumor locations , and sample sizes . in this study , only 2 snps in the promoter were analyzed , which might be a limiting factor
. the results of 1 specific snp may be influenced by the other snps in the angiogenesis pathway .
in addition , the sample sizes of the present study are somewhat small , even though similar studies were previously performed .
hence , more studies , including larger sample sizes and more snps , distributed not only in the promoter but also in the 5 and 3 un - translated region , should be conducted .
our data suggest that the vegf gene -460 c > t polymorphism is related to the risk of cscc , and both vegf gene -460 c > t polymorphism and -1154 g > a polymorphism are correlated with the prognosis of cscc patients .
therefore , the vegf gene -460 c > t polymorphism and -1154 g > a polymorphism may serve as a potential genetic marker for the risk and prognosis of cscc .
 backgroundcutaneous squamous cell carcinoma ( cscc ) is the second most common type of non - melanoma skin cancer ( nmsc ) globally . 
 the aims of this study were to further systematically clarify the potential association of rs833061 ( -460 c > t ) and rs1570360 ( -1154 g > a ) , two snps of vegf , with the risk of cscc and the prognostic impacts on cscc patients.material/methodsthis hospital - based case - control study analyzed peripheral venous blood collected from 100 cscc patients and 124 healthy controls , and gathered personal information on patients . 
 genotypes of the vegf gene -460c > t and -1154g > a polymorphism were detected using polymerase chain reaction ( pcr)-restriction fragment length polymorphism ( rflp ) method . 
 different distributions of allele frequencies and genotype in the case and control group were measured , comparing different genotype differences in the survival of patients with cscc.resultsdistributions of allele frequencies and genotype of -460 c > t in the case and control group were statistically different ; the tt + ct genotype was significantly correlated with a decrease risk of cscc ( or=0.36 , 95% ci=0.210.63 , p<0.001 ) . 
 there was no difference in the distribution of allele frequencies and genotype of -1154 g > a between control and case groups . 
 for -1154460c > t , the cc genotype was an adverse factor , associated with a significant decrease in the survival status of cscc patients ( p<0.001 ) . for vegf-1154 g > a 
 , the aa genotype was significantly correlated with the reduced overall survival in cscc patients , with the mean survival time of 23.88 months ( p=0.009).conclusionsthe vegf gene -460 c > t polymorphism and -1154 g > a polymorphism may serve as potential genetic markers for the risk and prognosis of cscc . 