Development And Application Of Reference Materials In Companion Diagnostics Of Targeted Drugs For Breast Cancer And Lung Cancer Based On High-throughput Sequencing

Background:Studies showed that targeted drug could kill cancer cells with specil gene mutations.However,individual difference in genetics influence sensitivity of cancer cells to the targeted drugs.Therefore,companion diagnosis before targeted therapy could promote the individual diagnosis and treatments of cancers.Next-generation sequencing(NGS)technology can realize parallel detection of genetic mutation that linked with individualized diagnosis and treatment of cancer.This is of great help for auxiliary diagnosis of clinic therapies.However,the detection error existed on NGS could derectly cause the failure of targeted patients selecting for targeted therapies.Therefore,laboratories with NGS technology should take verification test by using reference materials(RMs)for the quality control of testing procedures and performance evaluation.At present,China,s IVD companies were hastening exploitation of NGS ditection kits of targeted drug genes for breast cancer and lung cancer.Thus the relavent RMs were in urgent need.However,the RMs applied for quality control management of clinical molecular diagnostic laboraries is limited and expensive at the moment.Consequently,this study was performed to establish a steady and repeatable procedure of preparing RMs for tumor targeted therapy and companion diagnosis.Methods:(l)The guidelines for variant interpretation from the American College of Medical Genetics and Genomics(ACMG)and the characteristics of BRCA mutations in China were used to reach a consensus to establish interpretation rules for BRCA variant detection.BRCA1/2 mutations identified in cell lines and clinical cases were selected to establish RMs.Variant call format files and standard variant data sets obtained by NGS of clinical cases were collected and annotated to build the RD.The RMs and RD were validated for accuracy of BRCA1/2 mutation detection and variant interpretation.(2)NGS and digital PCR were used to determine and validate the variants and the mutation frequency of samples,which were then used to establish RMs in targeted drugs of breast cancer and lung cancer.In addition,participant laboratories were invited to validate the accuracy and specificity of the RMs.NGS were used to validate the stability and uniformity.(3)Tumor variants detection reagents base on Ion Torrent platform were adopted to screen oncogenetic mutations from 35 cell lines.Formalin-fixed paraffin-embedded(FFPE),cell lines and plasmid samples were used to prepare compounded samples with gradient mutation frequency.The detected mutation frequency of compounded samples from FFPE,cell lines and plasmid were compered to estimate the accuracy and retianlity of these samples for establishing RMs.Screened cell lines and simulated circulating tumor DNA(ctDNA)preparation process were used to try to prepare ctDNA RMs.Results:(l)Interpretation rules for BRCA variants in the Chinese population were generated as a standard for BRCA variant interpretation in these RMs and RD.The RMs consisted of 25 DNA samples harboring 31 BRCA variants.A total of 750 distinct BRCA variants formed the BRCA variant database in China were included in the RD.An online system based on the RD and the interpretation rules for BRCA variants in China was built to evaluate the performance of BRCA variant interpretation according to the BRCA variant database.(2)Thirty common genetic alterations with KRAS,NRAS variants,BRAF,PIK3CA,EGFR,ALK and MET mutations were designed based on these RMs.RMs consisted of a positive reference,a limit of detection(LOD)reference,and a negative reference.(3)Twenty-four cell lines were screened to be suitable for oncogene RMs preparation and relevant variants and mutation frequency were detected.ctDNA preparation process were established preliminarily,and ctDNA RMs of EGFR,KRAS and BRAF mutation were prepared.Conclusion:The BRCA variant reference system based on NGS and the RMs for lung cancer targeted therapy and companion diagnosis were used for the quality control and performance evaluation of relavent gene mutation detection.Screened cell lines and explored ctDNA preparation process could be used for the preparation of oncogene mutation RMs and as the foundation of RMs research.These will provide research foundation for breast cancer and lung cancer targeted therapies and companion diagnosis.In conclusion,this study provides reference for the development of tumor targeted therapies and companion diagnosis,and promots the development of NGS in clinical molecular diagnosis.