Diagnostic Value Of Lung Cancer Based On Non-invasive Determination Of Cell-free DNA Concentration And Integrity In Plasma

Lung cancer is one of the most common malignancies that threaten human health. The mortality of the patients with lung cancer was 40.1 per hundred thousand in male and 17.2 per hundred thousand in female.There are over 1 million new cases each year globally.In China,among all patients with cancer,1/4 of them died from lung cancer. Further more,both incidence and mortality show increasing trend.Therefore, prevention of lung cancer is very important.Up to now,the diagnosis of lung cancer relies mainly on imageology,sputum cytology,bronchial endoscopy and biopsy.The smallest size of node in lung tissue that could be detected by chest X-ray examination is 1cm,however,which may be large enough to invade tracheal epithelium or blood vessel endothelium.Small nodes can be detected more easily by low dose than by X-ray,the former has a higher detection ration of peripheral lung cancer than the latter,however,is less sensitive in finding small central nodes.Sputum cytology,especially multiple sputum cytological examination is helpful to diagnose central tumor developed from large bronchia,for example squamous cell carcinoma,small cell carcinoma.However,it will be of less value for detecting tumors developed from small bronchia,for example pancreatic carcinoma,especially when the tumor size is less than 2cm.Furthermore,it is not sensitive to detect early lung cancer by sputum cytological examination.Precancerous lesions and carcinoma in situ could be more easily found by bronchial endoscopy examination than other ways, however,the detection range is confined to carcinoma of bronchia epithelium nearby lung segments and peripheral lung cancer could not be detected.Biopsy is very traumatic,in spite of high sensitivity and specificity,therefore it could not be applied to screen lung cancer in early stage.In fact,most patients with cancers have developed to late stage while they are diagnosed.The clinical study demonstrated that the early diagnosis can make 80%of patients with lung cancer survive longer.Therefore,early diagnosis and treatment is very important to decrease lung cancer mortality as well as to increase survival.Given the limits of traditional ways for lung cancer diagnosing,we need to explore and establish an effective,less traumatic method for early lung cancer diagnosis,thus to achieve the goal of early diagnosing and treatment.In 1947,Mendel was the first to find circulating free DNA(CFDNA) in serum/plasma,after which many researchers demonstrated the phenomenon that the level of CFDNA was higher in serum/plasma of malignant carcinoma patients than that in normal people.The higher content of CFDNA may be associated with tumor cell necrosis and apoptosis.During the process of necrosis,large amount of DNA is released to peripheral blood,which leads to high DNA content.In addition,DNA fragment released during necrosis is usually of good intensity;however,in serum of normal people,the CFDNA was released because of apoptosis and the intensity of the CFDNA was not good.Our purpose of the study was to evaluate the diagnostic value of DNA content and intensity,based on the distinct level of CFDNA in normal people and lung cancer patients.It was reported that the CFDNA increased in serum of SLE,RF and diabetes patients.Thus,we also recruited these patients in our study.We applied Real-time PCR to quantify CFDNA.Firstly,genomic DNA was extracted,and quantified by UV-spectrometer.Then the quantified DNA was diluted in a serial of concentration(2000ng/ml,500ng/ml,100ng/ml,20ng/ml,and 5ng/ml),which will be served as standard DNA.The genomic standard DNA was used as template to amplify actin100 and actin400 by Real-time PCR.The coefficient of determination of genomic standard curve was 0.9990 and 0.9997.The detection range was 5-2000 ng/ml. The lowest detection level of CFDNA was 1ng/ml,when the volume of DNA template was 5μl in each reaction system.In 5 repeated tests using 100ng/ml template DNA,the largest difference between measured value and true value was 1.51%,and 0.7%on average.The largest coefficient of variation was 5.61%,and 3.68%on average,which suggested that the accuracy and repetitiveness of genomic DNA standard curve was qualified in quantification of CFDNA.Due to its short fragment,the CFDNA was easy to be lost or degrade,thus to establish an effective and appropriate method of serum/plasma DNA extraction was a key point in study of CFDNA.We made comparison among four normally used DNA extraction kit reagents.The Genomic DNA Purification Kit turned out to be the best. Then,we evaluated the efficiency of the extraction method.The extraction efficiency was 56.6%,95%CI was(53.8%-9.3%),the mean relative loss ratio was 43.4%,95%CI was(41.7%-47.1%),the mean relative inhibition ratio was-1.67%,95%CI was(-7.8 %-4.4%),the CV was 5.8%.By primer actin100,we estimated the plasma CFDNA concentration in different groups.The CFDNA concentration was 31.36(17.86-80.22) ng/ml in plasma of lung cancer patients,32.79(13.35-90.90)ng/ml in that of groups of other diseases and 11.20 (5.51-20.66)ng/ml in that of normal people.The CFDNA integrity in plasma of lung cancer patients was 0.67(0.33-0.89),in that of group of other diseases was 0.58 (0.28-0.87),and in that of normal people was 0.25(0.20-0.30).The CFDNA integrity was better in lung cancer patients than that in patients of other diseases.However,in normal people,the CFDNA integrity was worse than that in patients of other diseases.When we used CFDNA concentration as a diagnosis index of lung cancer,the largest Youden's index was 0.49,the area under ROC curve was 0.805,and the 95%CI was(0.764-0.845).When we took CFDNA integrity as a diagnosis index of lung cancer,the largest Youden's index was 0.608,the area under ROC curve was 0.812,and the 95%CI was(0.767-0.857).Therefore,CFDNA integrity was better than CFDNA concentration when applied as a lung cancer diagnosis index.Then we combined CFDNA concentration and integrity.The largest Youden's index was 0.651,the area under ROC curve was 0.873,and the 95%CI was(0.836-0.909).Compared with CFDNA concentration or CFDNA integrity as a single diagnosis index,the largest Youden's index of the combined index was increased by 32.9%and 7.1%respectively. We combined normal and other disease groups as control group and made lung cancer the test group.Then we got its area under ROC curve(0.677),and 95%CI (0.593-0.762).Therefore,as our data showed,CFDNA has certain value in lung cancer diagnosis,which may be applied in lung cancer screening.However,it has less utility in differentiation between lung cancer and other diseases.