Based On Next Generation Sequencing Technology Screening Radiotherapy Sensitivity Related Genes In Cervical Squamous Carcinoma

Objective: Using the next-generation sequencing technology to discover whole exon sequencing of hot spot gene in cervical squamous carcinoma,exploring the genetic level differences of different individuals,finding genes associated with radiation sensitivity.Methods: Collect 45 cervical squamous carcinoma patients who are prescribed radiotherapy in the radiotherapy department of the second Affiliated Hospital of Dalian Medical University,20 patients of stage ?B and 25 of stage ?B.Before treatment,all patients are extracted serum cf-DNA for Qu-bit2.0 fluorescence quantitative and record value,and the determination of serum squamous cell antigen?SCCA?value,pelvic cavity enhanced CT or enhanced MRI images to measure cervical lesions.All patients are prescribed 6MV-X photons,Volumetric Modulated Arc Therapy?VMAT?,giving the plan target dose 4500cGy/25 f,using 3-D192 Ir source after-loading therapy,making "A" dose of 3000cGy/5f,the whole course of "A" point total dose about 75 Gy,with cisplatin based concurrent chemotherapy?DDP 30 mg/m2,once a week?.According to the clinical stages,patients with cervical squamous carcinomas are grouped.Using SSPS spearman analysis the relationship of serum cf-DNA and SCCA with clinical stage and the effect of radiotherapy.A month after radiotherapy,all patients are re-taken pelvic enhanced CT or enhanced MRI images,evaluating the effect of radiotherapy.According to the World Health Organization?WHO?solid tumor curativeeffect evaluation standard,divide the patients into radiation sensitive group and radiation resistance group.In the two groups,selecting the same clinical stage and differentiation degree of cervical squamous carcinoma patients,build library of patients with ultra-low concentration serum cf-DNA by PCR,white blood cells?WBC?in blood as a control.Using NGS?Next-generation sequencing?and sequencing platform Hiseq X-TEN Illumina,sequencing strategy PE150,sequencing whole exon of 48 tumor hot spot genes.Using Linux systems analyze data,searching for genes associated with radiation sensitivity.Results: 45 patients with cervical squamous cell carcinomas,including 35 patients?77.8%?achieved PR defined as the radiation sensitive group.15 patients of stage ?B in radiation sensitive group,2 patients of well-differentiated squamous carcinoma,6patients of middle-differentiated squamous carcinoma,7 patients of poorlydifferentiated squamous carcinoma.20 patients of stage ?B in radiation sensitive group,5 patients of well-differentiated squamous carcinoma,11 patients of middle-differentiated squamous carcinoma,4 patients of poorly-differentiated squamous carcinoma.10 of 45 patients with cervical squamous cell carcinomas?22.2%?don't achieve PR defined as radiation resistance groups.5 patients of stage ?B in radiation resistance groups,3 patients of well-differentiated squamous carcinoma,1 patient of middle-differentiated squamous carcinoma,1 patient of poorly-differentiated squamous carcinoma.5 patients of stage ?B in radiation resistance groups,2 patients of well-differentiated squamous carcinoma,1 case of middle-differentiated squamous carcinoma,2 patients of poorly-differentiated squamous carcinoma.Cervical squamous carcinoma patients of stage ?B before radiotherapy,value of SCCA is higher than that of ?B patients,SCCA has relationship with clinical stage?r=0.785,P=0.011?.Concentration of serum cf-DNA in stage ?B patients is higher than that of stage ?B patients.Serum cf-DNA has relationship with clinical stage?r=0.987,P=0.002?.Cervical squamous carcinoma patients of radiotherapy resistance group,the value of SCCA is higher than that of radiotherapy sensitivity group,SCCA has relationship with radiotherapy effect?r=0.732,P=0.014?.Concentration of serumcf-DNA in radiotherapy resistance group is higher than that of radiotherapy sensitivity group.Serum cf-DNA has relationship with radiotherapy effect?r=0.943,P=0.007?Select 6 cervical poorly-differentiated squamous carcinoma patients of stage ?B,4 patients in radiation sensitive group and 2 patients in radiation resistance group.Next-generation sequencing results show that the sequencing depth of cf-DNA achieves10000 X and WBC achieves 2000 X.However,4 patients in radiation sensitive group do not have the same genetic mutations,the NGS results show the mutation of ABL1,JAK,NOTCH1,CSF1 R,FLT3 genes.The NGS results of 2 patients in radiation resistance group show ch13₄9051481 RB1 gene loci from T to A with mutation rate is18.81%.The APC gene,ch5₁12162854 loci from C to T with mutation rate is17.92%.The ALK gene,ch2₂9940529 loci from A to T with mutation rate is 11.09%,The NOTCH1 gene,ch9₁39400388 loci from G to C with mutation rate is 16.06%,The NPM1 gene,Ch5₁70819887 loci from G to A with mutation rate is 18.67%,The KDR gene,Ch4₅5968016 loci from T to A with mutation rate is 13.76%,The TP53 gene,Ch17₇577427 loci from G to A with mutation rate is 13.76%,The HNF1 A gene,Ch12₁21437221 loci from T to C,ch12₁21431300 loci from C to T with mutation rate is 21.71% and 24.79% respectively.A patients in radiation sensitive group,NGS results show ALK,NOTCH1,TP53,HNF1 A,APC genes mutations,which has some cross genes with patients in radiotherapy resistance group.After Nine months,this patient's lung CT images present lung metastasis.This case indicates ALK,NOTCH1,TP53,HNF1 A,APC gene mutations of cervical squamous carcinoma patients with poor prognosis.Conclusion: The later clinical stage,the higher value of SCCA and higher concentration of cf-DNA in squamous cell carcinoma patients.The value of SCCA and the concentration of serum cf-DNA both correlated with clinical stages.Serum cf-DNA concentration may be a potential biomarker of cervical squamous carcinoma.The higher value of serum SCCA and cf-DNA concentration,the less sensitivity of cervical squamous carcinoma patients to radiotherapy.The value of SCCA and the concentration of serum cf-DNA both correlated with the effect to radiotherapy.Serumcf-DNA concentration may predict the effect to radiotherapy of cervical squamous carcinoma patients.Cervical squamous carcinoma patients have point mutations in RB1 gene,the APC gene,ALK gene,NOTCH1 gene,NPM1 gene,KDR gene,TP53 gene and HNF1 A gene,these genes may be associated with cervical squamous carcinomas radiotherapy resistance and the poor prognosis.NGS technology detecting radiotherapy sensitivity related genes has the advantage of high throughput,high sensitivity,and low price.It's expected to be applied in precision therapy in cervical squamous carcinoma.