Research On Cell-free DNA In Colorectal Cancer Patients

Part 1 The feasibility of using circulating free DNA for the detection of KRAS mutation status in colorectal cancer: a meta-analysis BackgroundColorectal cancer(CRC)is one of the common malignant cancers in the world.KRAS mutation has been reported as a reliable biomarker for epidermal growth factor receptor(EGFR)targeted therapy in colorectal cancer(CRC)patients.However,limitations of detecting KRAS mutations in tissues are obvious.KRAS mutations in the peripheral blood can be detected as an alternative to tissue analysis.The objective of this meta-analysis was to evaluate the diagnostic value of cfDNA,compared with tissues.MethodsSearches were performed in PubMed,Embase and Cochrane Library for published studies.We extracted true-positive(TP),false-positive(FP),false-negative(FN),and true-negative(TN)values and calculated accuracy measures(pooled sensitivity and specificity,positive/negative likelihood ratios [PLRs/NLRs],diagnostic odds ratios [DORs],and corresponding 95% confidence intervals [95%CIs]).We also generated a summary receiver operating characteristic(SROC)curve to evaluate the overall diagnostic value.ResultsTotally,22 relevant studies were recruited and used for the meta-analysis on the efficacy of cfDNA testing in detecting KRAS mutations.The pooled sensitivity,specificity,PLR,NLR,and DOR were 0.782(95%CI: 0.753–0.805),0.821(95%CI:0.795–0.846),7.728(95%CI: 4.436–13.464),0.293(95%CI: 0.220–0.388),and31.107(95%CI: 17.498–55.299),respectively.The area under the SROC curve was0.8972.Totally,22 relevant studies were recruited and used for the meta-analysis on the efficacy of cfDNA testing in detecting KRAS mutations.The pooled sensitivity,specificity,PLR,NLR,and DOR were 0.782(95%CI: 0.753–0.805),0.821(95%CI:0.795–0.846),7.728(95%CI: 4.436–13.464),0.293(95%CI: 0.220–0.388),and31.107(95%CI: 17.498–55.299),respectively.The area under the SROC curve was0.8972.Compared with prior probability(20%),the positive posttest probability(75%)is much higher and the negative posttest probability is quite lower(<0.1),indicating a high diagnostic potential of cfDNA in detecting KRAS status in CRC patients.cfDNA is located in the right upper quadrant(PLR>10,NLR>0.1)in the likelihood ratio scatter matrix,demonstrating that cfDNA could act as a test to confirm KRAS mutation.ConclusionscfDNA has a high diagnostic accuracy in detecting KRAS mutation status in colorectal cancer patients and could be used as an alternative to tumor tissues.Part 2 Detection of KRAS mutations in plasma DNA of colorectal cancer patients by an ultra-sensitive PNA-mediated PCR combined with pyrophosphate sequencing technology BackgroundColorectal cancer(CRC)is one of the common gastrointestinal cancer in our country and the incidence rate has rised in the past few years.KRAS mutation status has been reported to associate with the occurrence,development and prognosis of CRC patients.However,the application of detecting KRAS mutation status was limited because of complicated procedures and low sensitivity of the present approaches.We established a hypersensitive method which combined PNA(peptide nucleic acid)-mediated PCR(polymerase chain reaction)with pyrosequencing technique to detect KRAS mutation in codon 13 in cell free DNA(cfDNA).Then detecting KRAS mutation status among 20 colorectal cancer patients before and afteroperation with this new method in plasma.ResultsOur results showed that the sensitivity of the assay which combined PNA-PCR with pyrosequencing technique reached 0.4%.Our results revealed that 8 out of 20 turned out to have KRAS mutation in codon 13.7 out of these 8 CRC patients turn to hold wild type KRAS after operation.1 out of these 8 CRC patients still remain to have KRAS mutations after surgical treatment.There are three patients whose KRAS status were wild type but turn out to be mutation type after operation.Conclusions1?PNA-mediated PCR combined with pyrophosphate sequencing technology has high sensitivity in detecting KRAS mutation status in CRC patients.While the sensitivity reaches 0.4%,the specificity turns out to be lower.2?There is a change in KRAS mutation status after surgical operation among KRAS-mutated CRC patients.Therefore,it is necessary to monitor KRAS mutation status continuously in the management of CRC patients to ensure reasonable application of the medication.Part 3 Changes of concentration of cell-free DNA in colorectal cancer patients receiving surgical treatment BackgroundColorectal cancer is a common gastrointestinal cancer,accounting for the second place.Although the treatment measures,including surgery,radiotherapy and chemotherapy have improved recently,the prognosis for patients with CRC tends to be poor,mainly because most diagnosed CRC cases are at advanced stages,losing opportunities of receiving standard treatment.Early diagnosis and monitoring is crucial in the management of CRC patients.The level of cf DNA is higher in CRC patients than that in healthy individuals,therefore,cf DNA level could be used as a diagnostic tool.Exploring changes of cf DNA level among CRC individuals whoreceived surgical treatment could provide information on outcomes of surgical operation and be used as a sensitive biological indicator for prognostic evaluation.Therefore,more valuable informaton could be obtained in clinical practice.MethodsCompare the changes of cf DNA levels and the content of tumor biomarker before and after operation after finishing the detection of the concention of cf DNA and CEA?CA19-9 in 38 CRC patients who received surgical operation.ResultsThe level of cfDNA in CRC individuals before and after operation were 408.545(1171.81)ng/ml and 313.75(845.31)ng/ml,respectively.And we find no statistically difference between them(P>0.05).However,the content of CEA and CA19-9 decreased dramatically after operation compared with preoperative values(P<0.05).ConclusionsCompared with changes of cfDNA levels,changes of the content of CEA ?CA19-9 may provide more valuable guidance in assessing surgical treatment outcomes as well as in the judgement of prognosis.However,this conclusions should be warranted in further studies.