Research On Improving The Accuracy Of FFPE Molecular Diagnosis

Formalin-fixed paraffin-embedded(FFPE)tissue contains a large amount of diseaserelated information and is the most common material for retrospective studies.Due to the FFPE manufacturing method,the quality of DNA extracted from FFPE tissue is poor,and C:G>T:A false mutations often occur during detection.In detection methods based on suppression effects such as LNA or PNA,false mutations occur more frequently.In this thesis,the purpose of eliminating false mutations in the detection of FFPE specimens by the suppression system was studied.Cod UNG enzyme repair combined with high-fidelity DNA polymerase pre-enrichment technology was used to establish a pretreatment method for FFPE specimens.The first chapter is the introduction,which introduces the process of making FFPE specimens,the type of damage,the impact on mutation detection,and methods to improve the quality of FFPE specimens,and puts forward the research content of this paper.The second chapter analyzes fragmentation of colorectal cancer(CRC)FFPE specimens stored for 4 years,3 years,2 years,and 1 year.Capillary electrophoresis(CE)was performed on the specimens.It was found that the longer the storage time,the smaller the main peak of DNA.Then qPCR test was performed,and it was found that as the amplicon length increased,the Ct value of the specimens gradually decreased.These two phenomena indicate that the longer the FFPE is stored,the worse the quality is and it is not suitable for long fragment amplification.The third chapter uses the PIK3CA LPO-MMCA system established in this laboratory as the detection technology,establishes a FFPE pretreatment scheme and performs application evaluation.Investigating the ability of UNG enzyme to eliminate false mutations,it was found that the mutation rate of colorectal cancer FFPE specimens after treatment with UNG enzyme decreased by 19.5%,indicating that UNG enzyme can effectively eliminate non-specific mutations.Screen different UNG enzymes and select Cod UNG enzymes with better effect.The pretreatment method of Cod UNG enzyme repair combined with high-fidelity DNA polymerase pre-enrichment was investigated.The cancer tissues and adjacent tissues of FFPE specimens from 19 breast cancer(BC)patients were used as research objects,and 19 cancers were found.The non-specific disappearance of the adjacent tissues after pretreatment.PIK3CA mutations were detected in 11 of the 19 matched cancer tissues,and all except A18 were verified by digital PCR.It shows that this method can eliminate false mutations without affecting the detection of real mutations.According to the PCR efficiency of the specimen,a detection scheme for FFPE specimens was proposed,and clinical specimens were tested.Fourteen positive specimens were detected in 77 valid colorectal cancer FFPE specimens,and the consistency,sensitivity and specificity with digital PCR were all 100%.Ten positive specimens were detected from 17 valid breast cancer FFPE specimens,and the coincidence rate with digital PCR was 94.1%,the sensitivity was 100%,and the specificity was 87.5%.The LPO-MMCA system detected one more positive result than the digital PCR,presumably because the copy number was still low after pre-enrichment,and the sampling error caused the missed mutation detection.In summary,this paper studies the degree of fragmentation of FFPE specimens and establishes a pretreatment method of Cod UNG enzyme repair combined with highfidelity DNA polymerase pre-enrichment technology,which improves the accuracy of FFPE specimens in the detection of the compression system and solves The problem of false mutations caused by the quality of specimens was solved.This technology strategy is also expected to be applied to other molecular diagnostic techniques with higher sensitivity,and has broad application prospects in the field of tumor tissue biopsy.