| Objective Lung cancer is the leading cause of cancer-related death worldwide terribly harmful to human health.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer,accounts for about 80% of the total number of lung cancer patients.Currently,targeted therapy based on genotype has become one of the standard therapies for NSCLC.Before the administration of target drugs,genotype of target gene need to be detected to select the patient benefited from targeted therapy.There are some limitations in traditional tissue biopsy for mutation detection.Recently,circulating tumor DNA(ctDNA)as a "liquid biopsy" material has attracted much attention.Lots of literatures compared the consistency of mutation detection by using tissue and ctDNA,but the results of each laboratory are quite different.The main reason attributed to the difference of plasma ctDNA concentration.The present study intends to analyze the relationship between ctDNA concentration and the accuracy of mutation detection,and determined the level of ctDNA concentration that could get reliable mutation detect results.In addition,in order to improve the accuracy of gene mutation detection,we research on the feasibility of using chemotherapy to elevate ctDNA concentration.Methods Thirty-six nude mice were xenografted with human NSCLC cell line H1975 which harbored the EGFR T790 M mutant.After tumor spontaneous growing in diverse volume,the mice were sacrificed and blood were collected to extract cfDNA.We used four pair of PCR primers which amplified the 81 bp or 197 bp fragments of human LINE-1gene and120 bp or 338 bp fragment of mouse ACTB gene.Q-PCR method was used to determine the concentration of ctDNA which released from human H1975 cancer cells(expressed as hctDNA)and cfDNA which released from nude mice itself(expressed as mcfDNA).cfDNA integrity index(DII)were calculated as the ratio of the length hctDNA(or mctDNA)to the short hctDNA(or mcfDNA).T790 M mutation were detected by using Droplet Digital PCR.The relationship between mutation and ctDNA concentration or tumor weight was analyzed.In addition,cisplatin was added in cultured NSCLC cells or intraperitoneal injected into tumor-bearing mice,respectively.The concentration of ctDNA in culture supernatant and mouse plasma was determined by above methods.Analyze the regularity of enhancing ctDNArelease in vitro and in vivo by chemotherapeutic drugs.Statistical analysis was done with SPSS17.0 software.Results 1.The tumor weight of 36 xenografts varied from 0.06 g to 2.26 g.hct DNA in plasma were skewed distribution,the concentration(ng/ml)were expressed as the median±interquartile range(range): short hctDNA concentration were 6.43±25.93(0.37~186.76),long hctDNA concentration were 0.34±0.95(0.01~25.46),DII were0.040±0.044(0.009~0.268).mcfDNA in plasma were normal distribution,the concentration(ng/ml)were expressed as the mean±SD(range): short mcfDNA concentration were 76.71±34.64(6.41~147.44),long mcfDNA concentration were 28.13 ±12.01(4.72~46.66),DII were 0.397 ± 0.121(0.187~0.736).Short fragment cfDNA concentration were as the real cfDNA concentration.The ratio of hct DNA to mcfDNA were expressed as median±interquartile range(range):0.086±0.406(0.004~3.918).2.The levels of plasma mcfDNA concentration and DII were independent on tumor weight.On the contrary,plasma hctDNA concentration were positively correlated with tumor weight,rs=0.85,P<0.001.DII were negatively correlated with tumor weight,rs=-0.34,P=0.042.hctDNA/mcfDNA ratio increased with the increasing of tumor weight,were positively correlated with tumor weight,rs=0.76,P<0.001.3.In the 36 cases of samples,EGFR T790 M mutations were detected in 22 cases and not detected in 14 cases.In the detectable samles,the mutation copies per milliliter of plasma were 105.00±231.86(21~4140),the tumor weight ranged from 0.22 g to 2.26 g,hctDNA concentration median±interquartile range(range)were18.24±39.23(1.40~186.76),hctDNA/mcf DNA ratio were 0.236±0.465(0.039 ~3.918).In the mutation undetectable samples,the tumor weight ranged from 0.06 g to 0.55 g,hctDNA concentration were 2.92±2.91(0.37~16.82),hctDNA/mcfDNA ratio were 0.053±0.058(0.004~0.462).4.In the mutation detected samples,the copies numbers were positively correlated with tumor weight(rs=0.42,P=0.049)and hctDNA concentration(rs=0.432,P=0.045).The hctDNA concentration,tumor weight and hctDNA/mcfDNA ratio were significantly higher than that of the mutation undetectable samples(P <0.05).While hctDNA concentration over 6ng/ml,the mutation detection rate was 89.47%,and while hctDNA concentration lower than 6ng/ml,the mutation detection rate was 29.41%.If the tumor weight was bounded by 0.45g(or hctDNA/mcfDNA ratio was bounded by 0.08),mutation detectionrate were 84.21% and 35.29%.5.Cisplatin can promote A549 and H520 cells release of ctDNA,the highest level was in the 36 hours to 48 hours after cisplatin treatment,which was181~260ng/ml.6.After cisplatin injected to A549 xenografts mice,the plasma ctDNA level changed over time,and gradually increased,the peak appeared at 48 hours after chemotherapy,which was 416.81±62.25ng/ml.Then ctDNA level decreased and tended to normal.Conclusions 1.There was no correlation between mcfDNA concentration and tumor weight,so was the DII.On the contrary,hctDNA was positively correlated with tumor weight,and hctDNA DII was negatively correlated with tumor weight.With the increase of tumor weight,the hctDNA concentration increased and hctDNA integrity decreased.The results showed that in xenografts,the plasma cfDNA diverse from nude mice itself were relatively stable and the ctDNA diverse from tumor were positive proportion to tumor burden.2.When using cfDNA as the mutation detecting material,mutation detecting rate was positively correlated with tumor weight and hctDNA concentration.Only when hctDNA concentration reached a definite level,the mutation detecting results were reliable.In consideration that part of cfDNA diverse from normal cells were relatively stable,in clinical practice,hctDNA concentration can be replaced by total cfDNA in mutation detecing.3.Cytotoxic drugs enhanced the release of ctDNA.Collecting blood at the time of ctDNA releasing peak can increase ct DNA concentration,and then improve the reliability of mutation detection. |
PDF Full Text Request