The Research Of Concentration And Integrity Of Cell-free DNA In Non-small Cell Lung Cancer Patients

Part 1 The establishment and performance evaluation of DNA-extraction-free technique for determination of concentration and integrity of cf DNA BackgroundNon-small cell lung cancer(NSCLC) is the most common type of lung cancer, including adenocarcinoma and squamous carcinomas, bronchioloalveolar carcinoma and cell large carcinoma. About 70% of the NSCLC patients are at a late stage or distant metastasis when they are diagnosed so that they cannot get proper treatment. Therefore, early finding and early treatment are essential for NSCLC. The cell-free DNA(cf DNA) in blood of cancer patients include circulating tumor DNA(ct DNA), which is mainly derived from apoptosis and necrosis. Cancer cells and distant metastasis of cancer cells in the circulation also can release cf DNA; we can estimate the tumor load and evaluate the effect of the treatment by detecting the amount of cf DNA. The integrity of circulating tumor DNA is another significant feature of cancer patients. The first step of quantitative PCR method for cf DNA is to extract it, and error is easy to occur in this step. If we do not take the factor of the operator into consideration,different extraction methods and reagents used will also make a huge difference.ObjectiveTo explore a method without DNA extraction and quantify the circulating DNA directly,and then we can avoid the differences of DNA extraction kit and the DNA extraction error of operators by using this new method.MethodsWe quantify the cf DNA by both extracting DNA and DNA-extraction-free technique and then compare the amplification efficiency, precision, and accuracy of this two approaches.ResultsWe quantify the cf DNA directly by quantitative PCR reaction without affecting the PCR reaction through the particular technology processing, and the amplification efficiency and purification of cf DNA are consistent with traditional methods. The new method canaccurately detect the level of cf DNA even it is low to nanogram. The detection range is1-10000 ng/ml and this approach has higher accuracy and precision compared with the kit extracted cf DNA firstly. What's more, the stability and repeatability of quantitative results had improved significantly than traditional ones.Part 2 The research of concentration and integrity of cell-free DNA in non-small cell lung cancer patients BackgroundIn recent years, cell-free DNA(cf DNA) has attracted increasing attention as a potential tumor marker for its minimally invasive, convenient, and readily accepted properties. The amount of cf DNA in plasma or serum was significantly higher in NSCLC patients than that in healthy controls or patients with benign diseases. The integrity of circulating tumor DNA is another important feature of cancer patients. These findings demonstrated that the cf DNA could serve as a viable tool to diagnose NSCLC.ObjectiveBy comparing the cf DNA concentration and integrity of NSCLC patients and healthy people, we evaluate the clinical value of circulating DNA content and integrity in the diagnosis of NSCLC.MethodsWe use the DNA-extraction-free PCR technique created by our laboratory to detect the cf DNA concentration and integrity of non-small cell lung cancer patients and healthy people,and then compare the difference between this two groups.Results1. The median(quartile range) of concentrations of circulating DNA in NSCLC patients and healthy people was 212(622.25) ng/ml and 65.4(290.65) ng/ml, respectively. The difference was significant(P<0.05). While the median(quartile range) of DNA integrity index(DII)was 0.0764(0.276) and 0.066(0.186), there was no significant difference between this two groups( P>0.05).2. cf DNA concentrations and DNA integrity index( DII) varied significantly withpathological stage(P<0.05) and no differences were found in gender, age or pathological type3. The AUC of the cf DNA concentration for distinguishing primary NSCLC patients from healthy controls was 0.659, and the sensitivity and specificity were 72.3% and 59.5%,respectively. The cut-off value was 87.5ng/ml.Part 3 The detection of gene mutations related to EGFR signaling pathways in the tumor tissues of non-small cell lung cancer patients BackgroundNowadays, several signaling pathways associated with NSCLC targeted therapy have been studied for many years, and the most common of them is the epidermal growth factor receptor(EGFR) signal pathway. The medicine working on EGFR signaling pathway is tyrosine kinase inhibitor(TKI), and the standard drugs have researched and developed are erlotinib, gefitinib, cetuximab and so on, they have all obtained an excellent effect in clinical application. Whether there are gene deletions, chromosomal rearrangements or point mutations in EGFR, KRAS, PIK3 gene of EGFR signaling pathway can significantly affect the efficacy of above-targeted drugs. Therefore, we usually detect the mutations of relevant target genes before applying targeted drugs.ObjectiveTo detect some gene mutations related to EGFR signaling pathway which often occur in NSCLC patients and then analyze the relationship between variation conditions and clinical factors in patients.MethodsWe collected the fresh tumor tissues during the operations in NSCLC patients firstly and then extracted the genomic DNA from tissues. Finally, we did the PCR amplification by using specific primers and detected the gene mutation with pyrophosphate sequencing.Results1. In the tumor tissue, there were nine patients presented with the EGFR Exon 19 deletion, eight patients with the positive Exon 21 L858 R mutation and one patient with the positive Exon 18 G719S/C. What's more, the presence of KRAS mutation was tested in 2patients and PIK3 CA mutation was detected from one patient. The mutation rate of the EGFR?KRAS?PIK3CA was 32.14%,3.57% and 1.79%.2. The mutation rate of the EGFR varied significantly with pathological stage and pathological type, the P value were all less than 0.05.Conclusions1. The DNA-extraction-free quantitative PCR technique has a high accuracy and precision, the detection range of it can be from 1 to 10000 ng/ml. Comparing the new methods to the extraction one, the stability and repeatability of the quantitative results by using the DNA-extraction-free quantitative PCR technique are improved significantly.2. The DNA-extraction-free quantitative PCR technique based on the LINE1 gene is good at the determination of concentration and integrity of cf DNA in NSCLC patients.3. The cf DNA concentration of non-small cell lung cancer patients was significantly higher than that of healthy controls, the difference was statistically significant between two groups. The cf DNA level and DNA integrity index(DII) of NSCLC patients were both related to the pathological stage, and when the stage was earlier, the cf DNA concentration and DII were both lower. This result showed cf DNA could be a marker to evaluate disease states.When the cf DNA concentration was used for the diagnosis of NSCLC, the cut-off value was87.5ng/ml, the area under the ROC curve is 0.659, the sensitivity is 72.3% and the specificity is 59.5%.4. EGFR mutations were the most common gene mutations detected in NSCLC, and the KRAS, PIK3 CA mutation rate were lower. The EGFR mutation rate varied significantly in pathological stages and pathological types. When the stage is later; the mutation rate was higher; the mutation rate in adenocarcinoma was the highest.