Application Of MeltArray Analysis In The Detection Of 30 Mutationsin EGFR Gene

Lung carcinoma is the leading cause of cancer-related death worldwide,and the mortality rate remains at a high level.Over the past few decades,the emergence of molecular targeted therapy has fundamentally changed the treatment of lung cancer.The lung cancer patient with the EGFR mutations has great treatment effect by using the corresponding targeted medicines.And genetic testing is usually required to identify the specific gene mutation types for improved therapeutic effects.The positive EGFR mutations are prerequisites for the use of the corresponding targeted medicines.In this thesis,the MeltArray technology was used to develop a simple,rapid,and cost-effective EGFR mutations screening system for clinical use.In the first chapter,we briefly reviewed non-small cell lung caner and its molecular targeted therapy,the relationship between EGFR mutation and tumor cell proliferation,and targeted medicines currently being used.The significance of EGFR mutations detection was described and those common EGFR mutations detection methods were compared regarding their advantages and disadvantages.Finally,we put forword the purpose,content and significance of this paper.The second chapter is Materials and Methods.It includes the source of the template,the design and the selection of the primers and the probes,the establishment of the uniplex PCR assay,the establishment of MeltArray system.The formed 2-reaction assay could detect 30 common EGFR mutations in 4 exons,accounting for 95%of the total EGFR mutations.In the Results and Analysis of Chapter 3,the assay performance was evaluated in terms of its sensitivity and selectivity.Using linear plasmid as templates,the limits of detection were determined to be 3-30 copies/reaction.By spiking different percentages of linear plasmids(mutant types)into genomic DNA prepared from 293T cell(wild-type),the detectable mutant allele frequency(MAF)for all the mutations were 1?5%.To verify the clinical reliability and efficacy of the system,we tested 190 formalin-fixed paraffin-embedded(FFPE)lung cancer tissue samples,and the results were comparaed with Sanger sequencing.A total of 56 samples were found positive(29.5%)with 100%concordance with Sanger sequencing.In summary,the new EGFR mutation assay proposed in this assay involves 2 reaction tubes to cover 30 EGFR mutations,which proved to be more efficient than those with much more reactions for the same number of EGFR mutations.It proves to be a convenient and cost-effective method with noticeable clinical significance.In the meantime,the design principles applied in this thesis also have relevant implications and insights for the testing of other mutations,and holds high potential to be used in conjunction with other cancer-associated genes to realize combined gene mutation tests in different types of cancer.