Association Study Between The MDM4 Gene Polymorphism And Squamous Cell Carcinoma Of The Oropharynx

BackgroundIncidence rate in Squamous cell carcinoma of the oropharynx(SCCOP)has recently been increasing significantly in the world.Human papillomavirus(HPV)infection has been confirmed to be the major cause for the increased incidence of SCCOP.HPV E6 protein can binds p53 for degradation,leading to the risk of SCCOP.HPV associated SCCOP has been shown to have unique epidemiologic,molecular,biologic characteristics,and better prognosis compared with non-HPV related SCCOP.Recurrences are the major cause of poor prognosis and mortality of SCCOP.However,prognostic models based on current TNM staging system may not effectively predict the outcome of SCCOP.A single-nucleotide polymorphism,often abbreviated to SNP,can be used as a reliable prognostic molecular marker to identify patients at high risk of recurrence.However,characteristic SNPs associated with the recurrence of SCCOP remain unclear.Mouse double minute 4(MDM4 or MDMX)is a key gene for negatively regulating p53.MDM4 polymorphisms have been reported to be associated with the risk of developing gastric cancer,prostate cancer.Given the crucial role of both HPV and MDM4 in p53 pathway,thus,in the present study,we investigated the associations of 3 tagging functional MDM4 variants with HPV status and the likelihood of recurrence among SCCOP patients.Materials and Methods1.A total of 552 incident patients with SCCOP were diagnosed and treated at The University of Texas(U.T.)M.D.Anderson Cancer Center were recruited.The SCCOP patients' demographic characteristics as well as smoking and alcohol history were collected.Paraffin-embedded tumor tissue samples were analyzed for HPV status using specific PCR assay.2.We used the public HapMap SNP database to identify the MDM4 tagging SNPs.Two MDM4 polymorphisms are located in the 3' untranslated region(3f UTR)[rs11801299 G>A and rs10900598 G>T]and one in intron 1[rsl380576 C>G]among three tagging variants of MDM4.All 552 enrolled SCCOP patients with known HPV status donated blood samples,of which 1ml was used for genomic DNA extraction with a DNA blood Mini Kit.Three polymorphisms in MDM4 were genotyped from blood genomic DNA samples using Applied Biosystems TaqMan genotyping platform.We further performed stratified analyses.3.Demographic,epidemiological,and clinical data of 1,008 SCCOP patients at The University of Texas(U.T.)M.D.Anderson Cancer Center were obtained.The primary endpoint of this study was recurrence.Genomic DNA was extracted from blood sample of 1008 SCCOP patients.Three polymorphisms in MDM4 were genotyped using Applied Biosystems TaqMan genotyping platform.Three separated/combined variants of MDM4 and recurrence risk of SCCOP in 1,008 incident patients were investigated.4.We further evaluated the separated and combined effect of the 3 polymorphisms on recurrence risk among HPV-positive SCCOP patients.5.After plasmid constructs,transfection,we perform luciferase assays to determine the A/G and T/G allele difference for MDM4rs11801299 and MDM4rs10900598 in both head and neck cell lines:UMSCC4(HPV-negative)and UMSCC47(HPV-positive).Result:1.Among 552 patients with SCCOP,439(79.5%)were positive for tumor HPV16 DNA.HPV16-positive patients have higher possibility of man and never-smokers than HPV16-negative patients.2.Three MDM4 variant genotypes were significantly associated with HPV16 tumor status among SCCOP patients compared with the common homozygous genotypes.Such a significant difference was also observed when genotypes were combined.In multivariable analyses,a significant difference was also noticed for all three polymorphisms.Additionally,modifying effect of MDM4 risk genotypes was more pronounced among patients under 54 years old,non-Hispanic white,never-smokers,and never-drinkers.3.A total of 1008 patients were followed up.The overall incidence of SCCOP recurrence was approximately 20%.The univariate Kaplan-Meier analysis showed that patients,who were older than 57 years old,other ethnicity,ever smokers,and alcohol drinkers,had moderate to severe comorbidity,and received combined treatment of surgery with chemoradiotherapy,respectively,were significantly associated with DFS.'Univariable and multivariable analysis showed that significantly different recurrence risks were found among patients with MDM4-rs10900598 GT/TT and rs11801299AG/AA variant genotypes compared with their corresponding common homozygous genotypes.Furthermore,after combining the risk genotypes of the three SNPs,patients among low-risk group had a significantly lower risk of SCCOP recurrence than those in high-risk group.4.A total of 45 SCCOP patients(13.9%)with HPV16-positive tumors experienced recurrence among 324 cases whose tumor specimens were available for tumor HPV status determination.The risk for both individual SNPs and combined risk genotypes was more significant among HPV-positive SCCOP patients.5.We replaced the 3'-UTR of a luciferase reporter gene with the 633 or 675-bp MDM4 3'-UTR containing either rs10900598 T or rs10900598 G and rs11801299A or rsl 1801299G,respectively.Luciferase assays showed significantly lower levels of luciferase expression were observed when UMSCC4 and UMSCC47 cells were cotransfected with MDM4 3'UTR luciferase reporter plasmids carrying the rs11801299A allele than with those plasmids carrying the G allele in both cell lines,While borderline significantly higher levels of luciferase expression were observed when UMSCC4 and UMSCC47 cells were cotransfected with MDM4 3'UTR luciferase reporter plasmids carrying the rs10900598 T allele than with those plasmids carrying the G allele in both cell lines.Conclusion:1.The majority of SCCOP were found to be HPV positive tumor.2.Potential functional polymorphisms in MDM4 may serve as biomarkers for predicting tumor HPV16 status among SCCOP patients,particularly in non-Hispanic white,never-smokers and never-drinkers.3.Our findings suggest that the MDM4 polymorphisms may,individually or in combination,confer an independent risk of SCCOP recurrence,particularly in HPV-positive SCCOP patients.4.The association between MDM4 polymorphisms and SCCOP recurrence risk may be more pronounced in HPV-positive SCCOP patients.5.These three SNPs in MDM4 may alter expression of MDM4,eventually affect recurrence risk of SCCOP.