Gene Variations In Multiple Primary Breast Cancer Based On Targeted Sequencing

Objective: In recent years,the incidence of breast cancer has increased year by year,and it has ranked first among morbidity and mortality among female cancers.Among them,multiple primary breast cancer also showed a gradual increase,which seriously endangered women's physical and mental health.In clinical treatment,there is no specific treatment for multiple primary breast cancer,and most of them are completely resected according to the primary cancer treatment,which not only increases the burden on the patient's body but may also cause over-treatment.Next-generation sequencing is widely used in the study of tumor genomes,but artifacts may be introduced during library construction and sequencing,resulting in inaccurate sequencing results and excessive noise,and resulting in a certain false positive rate of the obtained mutations.The project hopes to use a high-precision next-generation sequencing technology to study the genetic variation of multiple primary breast cancer.Methods: In this study,high-precision molecular barcode next-generation sequencing technology was used to perform targeted sequencing on totally 9 samples including paraffin-embedded tissue and white blood cells of three primary breast cancer patients.The target region covers 72 tumor hotspot mutation gene,including genes with high incidence of breast cancer in situ cancer.The experiment marks the original DNA molecules by ligated with the molecular barcodes;the data is grouped by molecular barcodes during the analysis,and the bases at each position are calculated to construct a new single-stranded consensus sequence,and finally the mutations are detected,and the high-confidence mutations are selected.To analyze the type and frequency of mutations in different cancer foci between the multiple tumors in multiple primary breast cancers,and to explore the homogeneity and heterogeneity,and to find out the clinically significant variation from the genomics level.Results:(1)Successfully construct a single-srand molecular barcode library;(2)The quality of the sequencing data is good.After extracting the molecular barcode sequence,the peak value of the family size is between 10-20,which can satisfy the following the constraction of single srand concensus sequence;(3)After detecting the mutation,the VMF of molecular barcodes in the leukocytes of the control sample was 0-5%,45-55%,and 95-100% of the variation was used as a control matching tissue section sample.Most of the variations are missense and synonymous.Comparing the VMF of mutations in different lesions of the same patient,it was found that the mutations shared between the cancerous lesions in the same patient were not significant,and the unique mutations were significant.(4)The number of mutations with high impact on cancer was counted.It was found that MET mutated in all four samples,and the types of mutations were base deletions or duplications rather than substitutions.Conclusion: This study found that different cancerous lesions in the same patient are homogenous and heterogeneous.There is no difference in the VMF of shared variations between different cancerous lesions in the same patient,and the unique variations are significant(P<0.01);copy number variation may be repulsive with the occurrence of gene mutations.Some genes such as MET and APC have opposite number of copy number variations and mutations;GSEA performs pathway enrichment analysis,and most of the genes are enriched in cancer,cell apoptosis,regulation of phosphate metabolism,and adhesive junctions.