osteosarcoma , the most common malignant bone tumor , has the potential of forming lung metastasis at an early stage , and is characterized by poor prognosis . surgical resection and multi - agent chemotherapy is essential for a standard strategy , but it is currently limited by multi - drug resistance to chemotherapy . clinical trials are ongoing to verify whether patients with advanced diseases will benefit from more aggressive therapeutic regimens ( such as trial euramos-1 ) . many attempts , such as trying to add ifosfamide and etoposide or ifn--2b to the ordinary regimen , have not revealed promising results yet . however , things may change if there are ways to predict prognosis at an early stage . thus , biomarkers which can be easily examined will be of great value for predicting outcomes . single - nucleotide polymorphisms ( snps ) are believed to be related with malignancy risk and progression . emerging studies on different kinds of cancers have provided abundant evidence for snps serving as predicting roles . further studies on snps in osteosarcoma will also be valuable in identifying potential prognostic biomarkers . the ras - association domain family 1 isoform a ( rassf1a ) gene , whose locus is located at 3p21.3 chromosome , is a verified tumor suppressor gene . rassf1a is involved in multiple cellular processes , including microtubule stabilization , apoptosis , and cellular motility . the rassf1a protein showed ability in inhibiting the accumulation of cyclin d1 , and consequently induced cell cycle arrest . although mutations on rassf1a gene are not common , many polymorphisms have been discovered within this region . several studies reported that different snps in rassf1a are related to risk or outcome of malignancies , such as lung cancer , breast cancer , and renal cell carcinoma . however , to the best of our knowledge , there is still no report on the relationship between rassf1a polymorphisms and risk or outcome of osteosarcoma . in consideration of the favored role rassf1a plays in osteosarcoma , it is rational to build a hypothesis that rassf1a polymorphisms are associated with osteosarcoma . we analyzed 5 rassf1a tagging snps and tried to find evidence that rassf1a polymorphisms were related with osteosarcoma . a total of 279 primary osteosarcoma individuals younger than 20 years old and 286 tumor - free healthy controls treated in different institutions were involved in this study . all patients were diagnosed by pathologic examination during the period from february 2007 to november 2012 , and blood samples were collected before performing chemotherapy and were consequently preserved . all cases underwent surgical operations by qualified orthopedists and were followed up for at least 36 months . . signed informed consent to participate in this research was acquired from all participants or their guardians . genomic dna was extracted using a dna blood mini kit from qiagen , berlin , germany . five snps ( rs2236947a / c , rs2073497a / c , rs1989839c / t , rs72932987c / t , and rs4688728g / t ) were tested in this study . abi steponeplus system and software ( thermo fisher scientific , waltham , ma , usa ) were used to perform pcr analysis and to collect data . the amplification condition was set as : initial denaturing step at 95c for 10 min , followed by 40 cycles at 95c for 15 s , 60c for 1 min , and 72c for 1 min . the 5 candidate snps were analyzed on shesis to find the most frequent haplotypes ( proportions over 3% ) . shesis ( http://analysis.bio-x.cn/myanalysis.php ) was reported to be a powerful online platform for analyses of genetic correlation at polymorphisms . the standard  test was utilized to assess the potential differences in the distributions of subject characteristics , variables , and genotypes of rassf1a alterations between the osteosarcoma cases and tumor - free controls . odds ratios ( ors ) and 95% confidential intervals ( 95% cis ) were calculated to evaluate the relationship between the 5 selected snps and the prognosis and risk of osteosarcoma . to estimated crude ors , logistic regression analysis was performed and was subsequently adjusted for age and sex . the hardy - weinberg equilibrium was assessed using pearson s  test . all of the statistical analyses were 2-sided , and p<0.05 was regarded as statistically significant . data analyses were performed by using spss ( v21.0 ; ibm , ny , usa ) . a total of 279 primary osteosarcoma individuals younger than 20 years old and 286 tumor - free healthy controls treated in different institutions were involved in this study . all patients were diagnosed by pathologic examination during the period from february 2007 to november 2012 , and blood samples were collected before performing chemotherapy and were consequently preserved . all cases underwent surgical operations by qualified orthopedists and were followed up for at least 36 months . . signed informed consent to participate in this research was acquired from all participants or their guardians . genomic dna was extracted using a dna blood mini kit from qiagen , berlin , germany . five snps ( rs2236947a / c , rs2073497a / c , rs1989839c / t , rs72932987c / t , and rs4688728g / t ) were tested in this study . abi steponeplus system and software ( thermo fisher scientific , waltham , ma , usa ) were used to perform pcr analysis and to collect data . the amplification condition was set as : initial denaturing step at 95c for 10 min , followed by 40 cycles at 95c for 15 s , 60c for 1 min , and 72c for 1 min . the 5 candidate snps were analyzed on shesis to find the most frequent haplotypes ( proportions over 3% ) . shesis ( http://analysis.bio-x.cn/myanalysis.php ) was reported to be a powerful online platform for analyses of genetic correlation at polymorphisms . the standard  test was utilized to assess the potential differences in the distributions of subject characteristics , variables , and genotypes of rassf1a alterations between the osteosarcoma cases and tumor - free controls . odds ratios ( ors ) and 95% confidential intervals ( 95% cis ) were calculated to evaluate the relationship between the 5 selected snps and the prognosis and risk of osteosarcoma . to estimated crude ors , logistic regression analysis was performed and was subsequently adjusted for age and sex . the hardy - weinberg equilibrium was assessed using pearson s  test . all of the statistical analyses were 2-sided , and p<0.05 was regarded as statistically significant . data analyses were performed by using spss ( v21.0 ; ibm , ny , usa ) . the median ages of cases and controls were 15.92 years and 16.24 years , respectively . no statistical significance was found for age or sex between osteosarcoma cases and tumor - free controls ( p=0.241 and 0.305 , respectively ) . pooled data of distributions of 5 tested rassf1a snps ( rs2236947a / c , rs2073497a / c , rs1989839c / t , rs72932987c / t , and rs4688728g / t ) in osteosarcoma cases and tumor - free controls are shown in table 2 . genotype distributions of the 5 selected snps were all in hardy - weinberg equilibrium in the control group ( p=0.079 , 0.730 , 0.102 , 0.132 , and 0.260 , respectively ) . in rs1989839c / t , the ct genotype showed elevated risk of osteosarcoma ( ct vs. cc : crude or=1.69 , 95% ci=1.182.41 , p=0.004 ; adjusted or=1.66 , 95% ci=1.162.38 , p=0.006 ) when the cc homozygote genotype was set as the reference group . in addition , the tt genotype was also associated with increased risk of osteosarcoma ( tt vs. cc : crude or=1.77 , 95% ci=1.052.99 , p=0.034 ; adjusted or=1.74 , 95% ci=1.032.95 , p=0.039 ) when compared with cc genotype . furthermore , a stronger statistical significance was revealed in the dominant model ( ct / tt vs. cc : crude or=1.70 , 95% ci=1.222.38 , p=0.002 ; adjusted or=1.68 , 95% ci=1.202.35 , p=0.003 ) . the ac genotype displayed a tendency toward elevated risk of osteosarcoma ( p=0.080 ) , as did cc genotype ( p=0.054 ) and dominant model ( p=0.055 ) . the other 3 snps ( rs2073497a / c , rs72932987c / t , and rs4688728g / t ) did not show any association for osteosarcoma risk in young chinese individuals . data on clinical features of osteosarcoma cases were collected to investigate the associations between the examined snps and prognosis of osteosarcoma . tumor location , enneking stage , operation method , and metastasis was compared , and rs1989839c / t was found to be correlated with some of these prognostic factors ( table 3 ) . in rs1989839c / t , the frequencies of the ct and tt genotypes at enneking stage ii or iii ( 50.00% and 15.68% , respectively ) were greater when compared with ia or ib stage cases ( 34.88% and 9.30% , respectively ) , and a significant difference was found ( p=0.027 ) . the genotype tt displayed a much higher frequency ( 28.30% ) in metastatic cases compared with metastasis - free cases ( 11.50% ) , and a significant significance was found in frequency distribution ( p=0.007 ) . the confounding variables , which were not statistically significant , are shown in tables 4 and 5 . analyses of the 5 candidate snps picked out 11 frequent ( frequency over 3% ) haplotypes : caccg , cacct , cactt , cattt , ccccg , cccct , ccctg , ccctt , cctcg , cctct , and ccttt ( table 6 ) . among them , ccccg , ccctg , cctct , and ccttt showed statistically significant differences between osteosarcoma cases and tumor - free controls ( p<0.001 , 95% ci=0.180.80 , p<0.001 , 95%=1.494.85 , p=0.03 , 95% ci=1.0483.648 and p=0.02 , 95% ci=1.0762.726 , respectively ) . the median ages of cases and controls were 15.92 years and 16.24 years , respectively . no statistical significance was found for age or sex between osteosarcoma cases and tumor - free controls ( p=0.241 and 0.305 , respectively ) . pooled data of distributions of 5 tested rassf1a snps ( rs2236947a / c , rs2073497a / c , rs1989839c / t , rs72932987c / t , and rs4688728g / t ) in osteosarcoma cases and tumor - free controls are shown in table 2 . genotype distributions of the 5 selected snps were all in hardy - weinberg equilibrium in the control group ( p=0.079 , 0.730 , 0.102 , 0.132 , and 0.260 , respectively ) . in rs1989839c / t , the ct genotype showed elevated risk of osteosarcoma ( ct vs. cc : crude or=1.69 , 95% ci=1.182.41 , p=0.004 ; adjusted or=1.66 , 95% ci=1.162.38 , p=0.006 ) when the cc homozygote genotype was set as the reference group . in addition , the tt genotype was also associated with increased risk of osteosarcoma ( tt vs. cc : crude or=1.77 , 95% ci=1.052.99 , p=0.034 ; adjusted or=1.74 , 95% ci=1.032.95 , p=0.039 ) when compared with cc genotype . furthermore , a stronger statistical significance was revealed in the dominant model ( ct / tt vs. cc : crude or=1.70 , 95% ci=1.222.38 , p=0.002 ; adjusted or=1.68 , 95% ci=1.202.35 , p=0.003 ) . the ac genotype displayed a tendency toward elevated risk of osteosarcoma ( p=0.080 ) , as did cc genotype ( p=0.054 ) and dominant model ( p=0.055 ) . the other 3 snps ( rs2073497a / c , rs72932987c / t , and rs4688728g / t ) did not show any association for osteosarcoma risk in young chinese individuals . data on clinical features of osteosarcoma cases were collected to investigate the associations between the examined snps and prognosis of osteosarcoma . tumor location , enneking stage , operation method , and metastasis was compared , and rs1989839c / t was found to be correlated with some of these prognostic factors ( table 3 ) . in rs1989839c / t , the frequencies of the ct and tt genotypes at enneking stage ii or iii ( 50.00% and 15.68% , respectively ) were greater when compared with ia or ib stage cases ( 34.88% and 9.30% , respectively ) , and a significant difference was found ( p=0.027 ) . the genotype tt displayed a much higher frequency ( 28.30% ) in metastatic cases compared with metastasis - free cases ( 11.50% ) , and a significant significance was found in frequency distribution ( p=0.007 ) . the confounding variables , which were not statistically significant , are shown in tables 4 and 5 . analyses of the 5 candidate snps picked out 11 frequent ( frequency over 3% ) haplotypes : caccg , cacct , cactt , cattt , ccccg , cccct , ccctg , ccctt , cctcg , cctct , and ccttt ( table 6 ) . among them , ccccg , ccctg , cctct , and ccttt showed statistically significant differences between osteosarcoma cases and tumor - free controls ( p<0.001 , 95% ci=0.180.80 , p<0.001 , 95%=1.494.85 , p=0.03 , 95% ci=1.0483.648 and p=0.02 , 95% ci=1.0762.726 , respectively ) . osteosarcoma is a relatively rare malignancy , with the morbidity rate of about 35 per million people every year . however , most osteosarcoma patients are children or adolescents , and this malignancy profoundly harms the whole family , especially in china , where most parents have only 1 child . nevertheless , none of these new strategies , including immunotherapy , have become a standard method in clinical treatment so far . thus , exploring evidence relevant to traditional chemotherapy is still of great value . emerging reports about genomic factors correlated with osteosarcoma tumorigenesis and/or progress have provided some chances to predict osteosarcoma risk and outcome , especially studies on gene polymorphisms , such as her2 , , ercc , and grm4 . however , we believe that in predicting cancer risk and outcome , referring to only a few predicting roles is far from satisfactory . given that osteosarcoma is not common , we launched a series of projects involving over 10 high - rank hospitals in the eastern coastal area of china , which has a population of over 100 million , to profile possible genomic predictors . in this series of projects , we discovered that wwox and nat2 polymorphisms may be possible predictors for risk of osteosarcoma . herein , we provide evidence that rassf1a polymorphisms are related to the risk and metastatic potential of osteosarcoma . we analyzed 5 candidate snps with recorded clinical data , and discovered that rs1989839c / t was associated with elevated osteosarcoma risk . furthermore , this snp was related to high - grade osteosarcoma and elevated risk of forming metastasis . another snp , rs2236947a / c , also showed value in predicting risk and outcome of osteosarcoma . we believe that including more cases will help to further analyze the role of this snp . rassf1a has been widely studied in different kinds of tumors . as a tumor suppressor gene , rassf1a gene promoter methylation was found to be related with carcinogenesis of non - small - cell lung cancer   and breast cancer . furthermore , its genotypes have been revealed to be associated with cancers in japanese and korean populations . in these studies , similarly , rs1989839c / t was also found to be correlated with worse survival and/or higher risk of cancer . although we were not able to perform some molecular biological experiments to validate the mechanism underlying this phenomenon , it is reasonable to reach this conjecture according to the role rassf1a plays in controlling cancer . in addition , haplotype analyses showed that the haplotype cccgg was much more frequent in tumor - free individuals , while ccctg , cctct , and ccttt were more common in osteosarcoma patients . we were not able to avoid inherent bias in our study , as all the blood samples were collected in hospitals . the sample size was limited due to the low morbidity of osteosarcoma , and results for homozygotic cases might not be as reliable . we are trying to recruit more osteosarcoma cases from additional institutions and plan to publish updated data . this is the first study to disclose the correlation between rassf1a polymorphism and risk and/or outcome of osteosarcoma . this multi - center study recruited a relatively large number of blood samples from young osteosarcoma patients . our results show that rassf1a rs1989839c / t snp indicates an elevated risk of osteosarcoma ( p=0.004 for ct genotype , and p=0.034 for tt genotype ) and lung metastasis ( tt genotype , p=0.007 ) in young chinese individuals .
backgroundthe ras - association domain family 1 isoform a ( rassf1a ) gene serves as a bona fide tumor suppressor gene . 
 the polymorphisms in rassf1a were previously reported to be associated with the risk of solid malignant tumors . 
 we hypothesized herein that rassf1a gene polymorphisms are involved in the risk and prognosis of osteosarcoma ( os).material / methodswe recruited 279 young os cases and 286 tumor - free controls from the east chinese population . 
 five tagsnps of rassf1a gene ( rs2236947a / c , rs2073497a / c , rs1989839c / t , rs72932987c / t , and rs4688728g / t ) were genotyped . 
 dna was isolated from blood samples and then underwent pcr analysis for genotyping.resultsrs1989839c/t is an important predictor of osteosarcoma risk and outcome . 
 the ct genotype of rs1989839 is highly related to elevated risk of osteosarcoma . 
 furthermore , rs1989839c / t is also associated with the enneking stage of osteosarcoma and risk of lung metastasis . 
 one of the other 4 snps , rs2236947a / c , shows a borderline significance in predicting osteosarcoma risk.conclusionsour study is the first to prove that rassf1a gene polymorphisms may potentially be predictive for osteosarcoma risk and prognosis .