CtDNA In The Diagnosis Of Colorectal Cancer

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research.However,while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment,the error rate of ?1% results in hundreds of millions of sequencing mistakes.These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures,such as tumors or mixed microbial populations.Circulating tumor DNA(ctDNA)holds promise as a non-invasivemeans for tumor monitoring in solid malignancies.Assays with high sensitivity andmultiplexed analysis of mutations are needed to enable broad application.To overcome limitations in sequencing accuracy,we have used a method termed Duplex Sequencing(we called Error suppressed random-index technology,ESRiT).This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex.As the two strands are complementary,true mutations are found at the same position in both strands.In contrast,PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error.Across multiple characterization experiments,the assay demonstrateda limit of detection of 0.01%(1 molecule in 10,000)for the majority of the 60 mutations in the panel.In CRC patient samples(n=50),detected mutations were concordant in tissue and plasma for 90% of metastatic patients.To verify whether the concentrations and integrity index of circulating DNA(ctDNA)in serum may be clinically useful for the diagnosis and progression monitoring of colorectal cancer(CRC)patients.Serum samples were collected from 104 with primary CRC,63 patients with intestinal polyps and 110 normal controls.Long(247 bp)and short(115 bp)DNA fragments in serum were detected by real-time quantitative PCR by amplifying the ALU repeats(ALU-qPCR).The median absolute serum ALU115 and ALU247/115 in primary CRC group was significantly higher than those in intestinal polyp and normal control groups,in recurrent/metastatic CRC was significantly higher compared with primary CRC.Combined detection of ALU115,ALU247/115 and CEA could improve the diagnostic efficiency for CRC.Serum DNA concentrations and integrity index may be valuable in early complementary diagnosis and monitoring of progression and prognosis of CRC.