Detection Of Gene Mutation In Serum Exosome DNA And RNA From Patients With Colorectal Cancer

Colorectal cancer(CRC)is the third most common malignancy in the world,with a fatality rate of fourth.Its occurrence and development are closely related to many factors such as environment and heredity with a multi-genes,multi-steps accumulation process.In recent years,the incidence and mortality of CRC in China have increased year by year,and the age of onset has become younger.Due to the absence of early pathological symptoms,most CRC patients were metastatic colorectal cancer(mCRC)at the first diagnosis,even if some patients underwent surgical treatment,there would be recurrence and metastasis after surgery.Therefore,chemotherapy in CRC,especially mCRC is essential.FOLFOX and FOLFIRI,as the current first-line treatment of chemotherapy,can effectively alleviate the patients' syndromes,but the median survive is still less than 2 years.Targeted EGFR monoclonal antibodies,such as cetumimab and panitumumab,are a major breakthrough in the treatment of CRC.The targeted EGFR monoclonal antibodies,competitively binds to the extracellular domain of EGFR with EGFR ligand epidermal growth factor(EGF),transforming growth factor alpha(TGF-?),etc.by targeting EGFR to inhibit cell growth,invasion and metastasis of tumor cells.However,not all patients can benefit from targeted therapy,the current study suggested that treatment of targeting EGFR monoclonal antibody can be affected by genes in the EGFR signaling pathway,patients with drug resistance can be screened by the gene mutation test of KRAS,NRAS,BRAF,PIK3 CA,etc.Therefore,gene mutation test before and after targeted treatment is essential.Gene mutation detection is currently mainly relying on histological examination,based on specimens from surgery,colonoscopy or biopsy,but these invasive tests are not acceptable for all patients.Moreover,in patients with CRC who have undergone radical resection of the primary tumor,it is difficult for obtaining the tumor tissues of metastases in clinical due to the restricted size and location of metastases,which couldnot only cause some damage for patients but also exists a greater risk.Therefore,a noninvasive examination instead of histological examination will be an ideal choice for detecting tumors.Exosomes are bilayer vesicles with 30-150 nm,widely distributed in a variety of body fluids.As a kind of important intercellular communication molecules,exosomes are rich in protein,lipids and a variety of nucleic acids,the inclusions of which are consistent with the source cells.In the tumor,the secretion of exosomes increased significantly as the increased demand for signal exchange between tumor cells and surrounding cells.Therefore,analysis and detection of inclusions of serum exosomes from patients with tumor can assist the early diagnosis of cancer,efficacy evaluation and prognosis analysis.In this study,we hoped that comparison the detection results of DNA,mRNA in serum exosomes from CRC patients to the detection of tumor tissues,to achieve the aim that mutations detected from DNA,mRNA in exosomes can assist the early diagnosis,guidance for clinical medication and evaluation.In this study,whole blood samples from 90 cases of clinically diagnosed mCRC were collected to extract blood cell DNA,exosome DNA and exosome mRNA.PCR was carried out on the corresponding fragments of KRAS gene G12,G13,Q61,BRAF V600,NRAS Q61,and PIK3 CA genes E542,E545,H1047,respectively.The mutations of DNA,serum exosome DNA and mRNA in 90 cases of CRC were detected by next-generation sequencing of PCR products,and compared with the results of clinical histological examination.Through next-generation sequencing,we found that 8 kinds of gene mutations were detected in exosome DNA and mRNA in 90 cases of CRC,they were KRAS gene G12 D,G12V,G12 A,G12S,G13 D,PIK3CA gene E542 K,E545K,H1047 R.In exosome DNA,a total of 54 patients with mutations were detected(mutation rate was 60%),among them,51 cases were KRAS mutation(mutation rate was 56.67%),42 cases were PIK3 CA mutation(mutation rate was 46.67%),39 cases were KRAS combined with PIK3 CA mutations(mutation rate was 43.33%).In KRAS gene,42 cases were G12 D mutation(mutation rate was 46.67%),1 case was G12 V mutation(mutation rate was 1.11%),1 case was G12 A mutation(mutation rate was 1.11%),1case was G12 S mutation(mutation rate was 1.11%),25 cases were G13 D mutation(mutation rate was 27.78%);in PIK3 CA gene,20 cases were E542 K mutation(mutation rate was 22.22%),10 cases were E545 K mutation(mutation rate was 11.11%),27 cases were H1047 R mutation(mutation rate was 30%).In exosome mRNA,a total of 49 patients with mutations were detected(mutation rate was 54.44%),among them,40 cases were KRAS mutation(mutation rate was44.44%),39 cases were PIK3 CA mutation(mutation rate was 43.33%),30 cases were KRAS combined with PIK3 CA mutations(mutation rate was 33.33%).In KRAS gene,32 cases were G12 D mutation(mutation rate was 35.56%),1 case was G12 V mutation(mutation rate was 1.11%),1 case was G12 A mutation(mutation rate was 1.11%),1case was G12 S mutation(mutation rate was 1.11%),17 cases were G13 D mutation(mutation rate was 18.89%);in PIK3 CA gene,16 cases were E542 K mutation(mutation rate was 17.78%),9 cases were E545 K mutation(mutation rate was 10%),24 cases were H1047 R mutation(mutation rate was 26.67%).We further compared the frequency of mutations of corresponding genes in exosome DNA and blood cell DNA in 90 patients with colorectal cancer.We found that the frequency of mutation in blood cell DNA was all < 1%.These results suggested that the mutant DNA in the exosomes is derived from tumor cells and can be used to detect mutations in the tumor tissue by detecting mutations in exosomes.In order to further determine whether the mutation detection of DNA and mRNA in exosomes was reliable,we compared the results of mutations in KRAS gene of exosome DNA and m RNA respectively with histological test results.In exosome DNA,the results showed that the detection sensitivity of 12 codons mutations of KRAS gene was90.63%,the specificity was 75.86%,and the coincidence degree with the histological detection was 81.11%;the detection sensitivity of 13 codons mutations of KRAS gene was 100%,the specificity was 82.28%,and the coincidence degree with the histological detection was 84.44%.In exosome mRNA,the detection sensitivity of 12 codons mutations of KRAS gene was 68.75%,the specificity was 81.03%,and the coincidence degree with the histological detection was 76.67%;the detection sensitivity of 13 codons mutations of KRAS gene was 63.33%,the specificity was 87.34%,and the coincidence degree with the histological detection was 84.44%.We believed that both detection methods have shown a potential value for clinical,especially the detection ofexosome DNA mutation,which has shown a better sensitivity and consistency.In the comparison of exsome DNA and mRNA,we found that patients with mutations in the mRNA all can be detected in DNA mutation,and the type of mutation is consistent,however,the detection rate of mRNA mutation was 70% ~ 85% of DNA's detection.Additionally,in each gene,frequency of DNA mutation was all higher than that of mRNA,the number of patients was over 85%.The above two aspects all suggested that there was mutant gene with a low expression or no expression in exosome mRNA,the effect of mutant genes in the transcriptome by detecting mutations in exosome mRNA.We futher compared the frequency of mutations of KRAS G12,KRAS G13,PIK3 CA E542,PIK3 CA E545 and PIK3 CA H1047 in exosome DNA and mRNA detection,results suggested that there was no statistacally difference in each mutant gene between detection of exosome mRNA and DNA.Additionally,20 serum samples from patients with colorectal cancer treated with cetuximab were collected.During the treatment of cetuximab,through continuous monitoring of DNA mutations in the exosome DNA,we found that the mutation rate of exosome DNA was closely related to the efficacy of cetuximab,the increased mutation frequency could predict the drug resistance of cetuximab in advance,and the predictive ability of which was superior to the current clinical tumor markers.The detection of exosome DNA mutations can be a real-time minimally invasive monitor method of tumors in clinic and may serve as a new method to guide the clinical treatment of EGFR monoclonal antibody.In summary,the detection of exosome DNA and mRNA can reflect the mutant state of the tumor tissue,and as the content of exosomes is rich and which are easy to extract,the detection of tumor mutations,monitoring can be achieved,it has a huge potential value in clinical applications.