| BackgroundLung cancer is currently the most common cause of cancer deaths worldwide.More than 1 million people die each year from the disease,and its incidence and mortality are still on the rise.NSCLC accounts for 80-85%of the total number of lung cancers.It is mainly based on comprehensive treatments such as surgery and/or radiotherapy,which improves the survival and prognosis of patients to some extent.However,in the actual clinical diagnosis and treatment,75%of lung cancer patients are diagnosed in the middle and late stages,and the treatment effect is not satisfactory.Overall,the patient's 5-year survival rate is only about 13%,which puts a heavy burden on the individual family and society.Many studies have shown that the occurrence and development of lung cancer is closely related to gene mutations.The state of gene mutation can be used as an important predictor of targeted therapeutic effects.Therefore,the comprehensive detection of genetic mutations in lung cancer patients is a key step in targeted therapy.With the deepening of research,the value of molecular detection,especially gene detection,has become more and more prominent in NSCLC,and it has become a research hotspot for scholars.Traditional gene detection technologies include Amplification Refractory Mutation System PCR(ARMS-PCR),high performance liquid chromatography,Sanger sequencing,etc.,but it is impossible to study the changes in the number and type of genomes,and the requirements of extraction and preservation for cancer tissues and blood samples are high.Next Generation Sequencing(NGS)technology has high-throughput properties,which can simultaneously detect most gene changes in a single tissue or blood sample,and continuously monitor the dynamics and prognosis of lung cancer in combination with clinical pathological features,and find certain potential or rare mutation sites have certain clinical value.Tissue-based genetic testing remains the gold standard in clinical diagnostics.However,in actual clinical work,the high risk of tumor tissue samples,the difficulty of repeated biopsy and the heterogeneity of tumors have brought challenges to the acquisition of tissue samples.Compared with tissue biopsy,body fluid testing has the advantages of non-invasiveness,low risk,and convenient material selection.It mainly includes circulating tumor DNA(ctDNA),circulating cell-free DNA(cfDNA),Circulating Tumor Cell(CTC)in body fluids such as blood,sputum,urine and exosomes.Additionally,ctDNA can be a suitable alternative when tissue samples are difficult to obtain,providing a more comprehensive genetic summary for the diagnosis,treatment and monitoring of lung cancer.Objective1.Based on NGS technology to detect the related gene mutations and site distribution of lung cancer tissue samples,analyze the relationship between mutant genes and clinicopathological features.2.Based on NGS technology to detect the related gene mutations and site distribution of lung cancer blood samples,analyze the relationship between mutant genes and clinicopathological features.3.Based on NGS technology to evaluate the consistency of lung cancer tissue and blood tumor related gene mutations.Methods1.Prospectively included patients with lung cancer who were admitted to the Department of Respiratory Medicine of Tangdu Hospital from December 2017 to July 2018.A total of 53 tissue samples,194 blood samples,and 33 samples of tissue and blood paired samples were obtained.2.Place the EDTA anticoagulant tube containing the blood sample in a centrifuge at4°C and 6000 r/min for 10 minutes,and extract the supernatant in the console for 3centrifuge tubes(an average of 2 mL);The 2 mL centrifuge tube was centrifuged at 4°C,16000 r/min centrifuge for 10 minutes,and the supernatant was extracted from the console in two 2 mL cryotubes(Corning 430659 2.0 mL externally frozen cryotube).The total volume of the liquid is not less than 3 ml.The frozen storage tube is sealed with a self-sealing membrane and stored in a-80°C low temperature refrigerator.All processes are completed in 2 hours.All samples were transported by dry ice to the laboratory of zhengu Co.,Ltd.,and the ctDNA was extracted by blood DNA extraction and separation kit(Aide,China),and the test was completed within 7 days.The collected fresh tissue specimens will be placed in a 2 mL cryotube(Corning 430659 2.0 mL externally-circulated cryotube)with a total volume of not less than 25 mg and a tumor cell ratio greater than 30.00%(the tissue is divided equally into two halves,half used for pathological biopsy,and half is used for NGS testing.If the tumor cell ratio is>30%,the sample is included in the study,if<30%,the study is excluded)and stored in a-80°C low temperature refrigerator.All tissue samples were transported by dry ice to the laboratory of zhengu Co.,Ltd.3.The ctDNA extraction was performed according to the QIAGEN QIAamp Circulating Nucleic Acid Kit manual.The extracted free DNA content was determined by Qubit?3.0 fluorescence meter and Qubit dsDNA High Sensitivity assay kit.The quantification method was based on the Qubit?3.0 operating instructions.The constructed DNA library was sequenced using a Miseq sequencer according to the Preparing Libraries for Sequencing on the MiSeq and MiSeq System User Guide,and the sequencing read length was PE150.The DNA extracted from the tissue samples was constructed using the SLIMampTM Lung&Colon Cancer Hotspot Panel kit(zhengu Co.,Ltd.),including 2rounds of PCR,targeting amplification and library amplification.The DNA library was quantified using a Qubit?3.0 fluorescence meter and a Qubit dsDNA High Sensitivity assay kit according to the Qubit?3.0 protocol.Finally,the constructed DNA library was sequenced by a Miseq sequencer according to Preparing Libraries for Sequencing on the MiSeq and MiSeq System User Guide,and the sequencing read length was PE150.NGS technology screened for gene mutations and annotated(gene mutation abundance threshold of 1%and positive reads?5),and finally obtained the corresponding results.4.SPSS19.0 software was used for data analysis.The chi-square test was used to compare the difference between groups and the correlation between genes.The statistically significant index of the single factor analysis was Logistic two(multiple)element regression analysis.Analyze the line Kappa calculation.P<0.05 was statistically different.Results1.Mutation analysis of tumor-associated genes in lung cancer tissue samples.In the tissue samples of 53 patients with lung cancer,there were no tumor-associated gene mutations in 9 cases(9/53,16.98%),and tumor-related gene mutations occurred in 44 cases(44/53,83.02%),of which 20 cases occurred with single gene mutations(20/53,37.74%),22 patients(22/53,41.51%)with double gene mutation,and 2 patients(2/53,3.77%)with simultaneous mutation of three or more genes.A total of 19 tumor-related gene mutations were detected,and the number of gene mutations was 71.Among them,the number of TP53gene mutations was 33(33/71,46.48%),and both the number of EGFR and CDKN2A gene mutations were 6(6/71,8.45%).There were 74 tumor-related gene mutation sites,among which TP53,EGFR and CDKN2A mutation sites were more.In terms of clinicopathological significance,the difference in EGFR gene mutations among patients with different pathological types was statistically significant(?2=7.447,P=0.024),and the difference between adenocarcinoma and squamous cell carcinoma was statistically significant(?2=7.422,P=0.006),the difference between small cell lung cancer and adenocarcinoma and squamous cell carcinoma was not statistically significant(P>0.05);the difference of EGFR gene mutation in patients with different clinical stages was statistically significant(?2=9.272,P=0.037).There were no significant differences in TP53 and CDKN2A gene mutations between different ages,genders,smoking history,pathological type,differentiation,metastasis and clinical stage(P>0.05).Logistic regression analysis showed that in the pathological type,small cell lung cancer was used as the reference standard,and the risk of EGFR gene mutation in adenocarcinoma was 2.484×10~7 times that of small cell lung cancer.The difference between the two groups was statistically significant(P=0.000).The risk of EGFR gene mutation in squamous cell carcinoma was 2.484×10~7 times that of small cell lung cancer,and the difference between the two groups was statistically significant(P=0.000).In the clinical stage,the risk of EGFR gene mutation in patients with stage IV lung cancer was 9.091 times that of stage III lung cancer patients,and the difference between the two groups was statistically significant(P=0.014).2.Mutation analysis of tumor-associated genes in blood samples from lung cancer.In the blood samples of 194 patients with lung cancer,there were 33 tumor-related gene mutations(33/194,17.01%),and 161 tumor-related gene mutations(161/194,82.99%),including 39 single gene mutations(39/194,20.10%),63 patients(63/194,32.47%)with double gene mutation,59 patients with three or more genes(59/194,30.41%).A total of 174tumor-associated genes were detected,including 154 mutations in TP53(154/407,37.84%),75 EGFR mutations(75/407,18.43%),and the number of KRAS gene mutations was 23(23/407,5.65%).There are 502 mutation sites of blood tumor related genes,among which TP53 and EGFR have more mutation sites.TP53 gene mutations were significantly different in different pathological types(P=0.001).Among them,small cell lung cancer had higher TP53 mutation than adenocarcinoma and squamous cell carcinoma;EGFR gene mutation was statistically significant in gender and smoking history,especially in females.The frequency of gene mutations was higher in lung cancer patients(P=0.007)and non-smokers(P=0.034);the difference between KRAS gene mutations and non-metastatic patients in metastatic patients was statistically significant(P=0.029).There were no significant differences in other tumor-related gene mutations between different ages,genders,smoking history,pathological type,degree of differentiation,metastasis,and clinical stage(P>0.05).Multivariate logistic regression analysis showed that the risk of adenocarcinoma mutation was 0.244 times(P=0.000)for small cell lung cancer compared with that of unmutated patients with TP53 gene mutation,and the risk of squamous cell carcinoma was 0.333 times that of small cell lung cancer(P=0.010),and the differences were statistically significant.Binary logistic regression analysis between EGFR and KRAS gene mutations and gender,smoking history and metastasis showed that the risk of EGFR gene mutation in female patients was 2.406 times higher than that in males(P=0.007),and the risk of EGFR gene mutation in non-smokers was 1.938 for smokers.Times(P=0.035),both were statistically significant;the risk of mutation in the KRAS gene in metastasis was 2.654×10~8 times that of non-metastatic patients,which was statistically significant(P=0.000).In the correlation between TP53,EGFR,and KRAS gene mutations,there was no correlation between EGFR gene mutation and KRAS and TP53 gene mutations(P=0.165,P=0.132),between KRAS gene mutation and TP53 gene mutation.Significantly correlated(P=0.009).3.Consistency analysis of tumor-associated gene mutations in lung cancer tissues and blood specimens.Combining the results of the part?and part?,the differences in the frequency of TP53,EGFR and KRAS gene mutations between tissues and blood were further analyzed.The frequency of TP53 gene mutation was statistically significant in tissue and blood(?2=13.683,P=0.000),and there was no consistency;the mutation frequency of EGFR and KRAS gene was not statistically significant but with consistency in tissue and blood(?2=2.011,0.053;P=0.156,0.818).Among the mutation types,the difference between the missense mutation,the frameshift mutation,the splice donor mutation and the splice acceptor mutation was statistically significant between the tissue and the blood specimen(?2=14.907,P=0.000;?2=9.677,P=0.002;?2=7.083,P=0.008;?2=35.663,P=0.000),and the difference between nonsense mutation,deletion mutation,and insertion mutation in tissue and blood samples was not statistically significant(?2=0.560,P=0.454;?2=0.890,P=0.346;?2=0.142,P=0.707).The same gene mutation site consistency analysis was performed on tissues and blood,and the results showed that the EGFR gene exon 19Glu746Ala750del deletion mutation was statistically significant between tissues and blood(?2=11.546,P=0.001).There was no significant difference in TP53 between tissue and blood in the missense mutation of exon 8 Arg273Leu(?2=2.838,P=0.092).Comparison of the frequency of EGFR gene mutations between tissues and blood samples with different clinicopathological features showed that EGFR gene mutations in male,smoking and squamous cell carcinoma patients were statistically significant in tissues and blood samples(?2=5.341,P=0.021;?2=5.768,P=0.016;?2=6.913,P=0.009);there was no statistically significant difference in EGFR gene mutation between women and non-smoking,adenocarcinoma and small cell lung cancer patients(?2=2.966,P=0.085;?2=3.358,P=0.067;?2=1.068,P=0.301;?2=0.830,P=0.360).In 33 cases of tissue and blood paired samples,there were 16 cases of the same gene mutation(16/33,48.48%),there were no mutations in the same gene in 17 cases(17/33,51.52%).EGFR gene mutations were consistent in tissue and blood samples(Kappa=0.338;P=0.043)and KRAS gene mutations were consistent in tissue and blood samples(Kappa=0.353;P=0.038),while TP53 gene mutations were not consistent in tissues and blood samples(Kappa=-0.124;P=0.466);1patient with both TP53 and EGFR in tissue and blood samples Two gene mutations.Conclusions1.NGS technology can effectively detect tumor-associated gene mutations in lung cancer tissues.The frequency of TP53,EGFR and CDKN2A mutations is higher in tissues,accounting for more than 60%of the total mutation,the lord of which more than 80%are single-gene and double-gene mutations.Mutations in the EGFR gene of tissue samples may be more common in patients with advanced adenocarcinoma.There are no significant differences in TP53 and CDKN2A gene mutations in tissue samples at different ages,genders,smoking history,pathological type,differentiation,metastasis,and clinical stage.2.NGS technology can detect more than 80%of gene mutations in lung cancer blood samples,and the ratio of double gene and multi-gene mutation is large,suggesting that the detection of tumor-related gene mutations in blood samples based on NGS technology can better reveal the full spectrum of genes during tumor development and its'change.The TP53gene mutation in blood samples has significant differences in different pathological types,among which TP53 mutations in small cell lung cancer are the most common;EGFR gene mutation in blood samples is higher in female and non-smoking lung cancer patients;KRAS gene mutation in blood samples in patients with metastasis were more common;other tumor-associated gene mutations in blood samples were not significantly different among different clinical features;The TP53 gene mutation in blood sample has a significant correlation with the KRAS gene mutation.3.The consistency analysis of tissue and blood tumor related gene mutations in lung cancer showed that the mutation frequency of EGFR and KRAS genes was consistent in tissue and blood,while the frequency of TP53 gene mutation was not consistent.Among the mutation types,nonsense mutation,deletion mutation,insertion mutation was consistent in tissue and blood samples,while missense mutations,frameshift mutations,splice donor mutations,and splice acceptor mutations were inconsistent in tissue and blood.With identical gene mutation sites in tissue and blood samples,the TP53 gene exon 8 Arg273Leu missense mutation is consistent,while the EGFR gene exon 19 Glu746Ala750del deletion mutation is inconsistent.Among the frequency of EGFR gene mutations in different clinicopathological features,EGFR gene mutations in women,non-smokers,adenocarcinoma and small cell lung cancer patients are consistent in tissue and blood samples,while there is no consistency with the blood sample of EGFR gene mutations in male,smoking and squamous cell carcinoma patients are organized.In the paired samples,the tumor-associated gene mutations in the tissue and blood samples were in good agreement,like EGFR and KRAS genes.The detection of tumor-related gene mutations in tissue and blood samples based on NGS technology is consistent,but different tumor-related genes have different mutation frequencies in tissue and blood samples.Blood samples are more likely to detect tumor-related genes than tissue samples,which can better reflect the clinicopathological features of the patient.Therefore,simultaneous detection of tumor-related gene mutations in tissue and blood samples can complement each other,and more comprehensively discover the genetic mutation status of patients,and better provide a basis for clinical decision-making. |
PDF Full Text Request