Comparison Of Circulating Tumor DNA In Peripheral And Portal Vein Blood Of Patients With Colorectal Cancer And Its Value In Tracking Minimal Residual Disease

With the development of medicine,in addition to the original imaging,tumor markers,and other methods for diagnosis of tumors,there are new gene detection techniques.The circulating tumor cells(CTC)and circulating tumor DNA(ctDNA)based on blood specimens are the research hotspots,and as the "star" ctDNA of blood tests,more and more researchers are paying attention.ctDNA refers to DNA that is released into the circulatory system after tumor cells have fallen off or become apoptosis,and is a characteristic tumor biomarker.With ctDNA detection,tumor traces in the blood can be detected.Compared with other traditional tumor markers,ctDNA has the characteristics of short half-life,low false-positive rate,and real-time reflection of the tumor itself.However,the content of ctDNA itself is small,and extraction and detection are difficult.With the development of quantitative detection technology,the detection sensitivity of DNA has gradually increased,and people have continued to study in a variety of tumor types,demonstrating the value of ctDNA in the diagnosis and treatment of cancer.In spite of this,the current distribution of ctDNA in peripheral blood and portal vein blood in patients with colorectal cancer and its value in tracking minimal residual disease remains unclear.Considering that there is a common portal vein regurgitant system in gastrointestinal tumors,this study collected the peripheral blood and portal vein blood from patients with colorectal cancer and gastric cancer,and then performed an advanced second-generation sequencing of 61 gene pannels by Accu-Act to compare and analyze ctDNA.Compare the differences in content,gene mutation patterns,fragment length,and abundance.In addition,a fragment carrying the mutation site K-ras gene was injected into the animal body to examine the metabolic dynamics of ctDNA in the animal body.Finally,we established the follow-up time points of patients with colorectal cancer,combined with tumor recurrence,metastasis,and death data,to investigate the value of ctDNA in tracking blood residual microsurgical lesions in patients with colorectal cancer.The study found that,compared with peripheral venous blood,the ctDNA(cfDNA containing mutation sites)in the portal vein blood had no significant difference in the content,tumor mutation map,and fragment length(P>0.05).However,the minimum allele frequency(MAF)of somatic mutation was higher in portal vein blood(P < 0.05).Further,we calculated that the half-life of ctDNA in peripheral venous blood varied from 30 seconds to 45 minutes.In addition,the average length of ctDNA was shorter than that of full-length cfDNA(P<0.05).To further explore the metabolism of K-ras gene fragments carrying mutation sites in animals,we found that the average half-life of ctDNA in animals is approximately 2.22 minutes,which is different from the metabolic time estimated by ctDNA in venous blood of clinical colorectal cancer patients.Detection of ctDNA in patients with colorectal cancer can effectively predict tumor recurrence within 2 years.ctDNA plays a crucial role in the assessment of minimal residual disease in patients with resectable colorectal cancer.It is a good NCCN guideline.supplement.Part ? Comparative Study of ctDNA in Peripheral and Portal Vein Blood of Patients with Colorectal CancerObjective:Taking into account the common portal vein reguration system of gastrointestinal cancer,and at the same time,stomach cancer and colorectal cancer can be compared,we collected peripheral blood and portal vein blood in patients with colorectal cancer and gastric cancer,compared ctDNA content,gene mutation patterns,fragment length and abundance,and Further investigate the metabolic dynamics of the fragment ctDNA in animals.Method:Since gastrointestinal cancer has the same system of portal venous return,we collected peripheral venous blood and portal venous blood,performed 64-gene pannel NGS second-generation sequencing by Accu-Act technique,and compared and analyzed ctDNA content,the mutation rate,length,and MAF,and the half-life of their metabolism was estimated.The K-ras gene fragment carrying the mutation site was injected into the animal.The CL-187 digested product with a concentration of 51.27 ng/uL was diluted in physiological saline and injected into the left femoral vein of the lower extremity of the dog.Blood samples were taken 3-5 ml from the right femoral vein at 1 minute,2.5 minutes,5 minutes,10 minutes,and 30 minutes.Plasma was separated,Trans magnetic beads were extracted for ctDNA,and ddPCR.The frequency of ctDNA mutations at each time point was calculated to calculate the half-life.Result:There was no difference in the content of ctDNA(cfDNA containing mutation sites)from peripheral venous and portal venous blood(P=0.566).We detected no difference in the ctDNA mutation patterns of peripheral blood and portal vein blood.The mutation rate of ctDNA(the number of mutations in blood/the number of mutations in tumor tissue)was 42% and 47% in peripheral venous and portal venous blood,respec-tively,and there was no significant difference between the two(P=0.35).We detected 6 patients(C4,C8,C9,C10,C11,and S5)tumor-matched venous blood ctDNA fragment lengths that were significantly shorter than those of cfDNA without mutations in the same chromosomal region and the difference was statistically significant(P <0.001).We compared tumor-matched peripheral venous blood and portal vein ctDNA MAF.Using a binary linear regression model,MAF in portal vein blood was significantly higher than in peripheral venous blood(P=0.000679).Assessing the degradation rate of cfDNA is dependent on the estimation of tumor MAF mutations,and because of tumor heterogeneity,changes in copy number,and aneuploidy,MAF is difficult to obtain integrallty.However,the maximum and minimum degradation rates can be estimated from multiple genomic loci and the exponential decay can be calculated.The rate of degradation was estimated by estimating 9 genomic locations in 5 patients with colorectal cancer and 1 patient with gastric cancer.There were 6 or more UC readers per genome location.Our results showed that the cfDNA half-life of the patient's blood was 30 seconds to 45 minutes.The mutation frequencies of animal experiment dog 1 at each time point were 3/986,1.4/1274,1.4/1254,0/610,0/1166,respectively;the mutation frequencies of dog 2 at each time point were 11.2/430,4.8/466,2.6/ The mutation frequencies of 352,1.4/502,0/848,and Dog 3 at each time point were 24/800,9.8/538,1.6/588,0/658,and 0/928,respectively.The half-life of ctDNA in each dog was calculated by R software programming,which was 2.479011 minutes,2.794444 minutes,1.38862 minutes,with an average of 2.220692 minutes.Conclusion:There is no significant difference in the content of ctDNA in peripheral blood and portal vein blood,and in the tumor mutation spectrum,and fragment size,but the frequency of ctDNA gene mutation in portal vein blood is higher than peripheral blood.After calculation,the half-life of ctDNA in human blood is an average of 1.98 minutes.The average half-life in animals is 2.2 minutes.Peripheral blood ctDNA has a good circulatory system representative,and short half-life,can monitor tumors in real time.Part ? The value of ctDNA in tracking minimal residual disease in patients with colorectal cancerObjective:To establish the follow-up time points of patients with colorectal cancer,combined with tumor recurrence,metastasis,and death data,to investigate the value of ctDNA in tracking small residual blood lesions in colorectal cancer.Method:We collected 517 preoperative/post-operative blood/clinical tumor specimens from patients with colorectal cancer in the previous period.Exclude unmatched specimens,exclude follow-up visits for ?12 months,exclude stage IV tumors,and finally include 85 with complete preoperative blood,Postoperative blood and clinical tumor specimens,moreover 22 patients with stage IV metastatic colorectal cancer with intact matching clinical specimens were studied as controls.All 85 patients underwent radical R0 radical surgery and were regularly followed up.Result:We performed the NGS sequencing by Accu-Act technology.The results showed that the top five genes detected in colorectal cancer tumor tissue,preoperative peripheral venous blood and postoperative venous blood were:TP53,KRAS,APC,BRAF and PIK3 CA,which are similar to the highly mutated genes detected in WES tumor gDNA(The Cancer Genome Atlas Network,Nature 2012)in various tumors.Further,we selected TP53,KRAS,APC,and BRAF.We found that the mutated patterns of gDNA and plasma in colorectal cancer tissues were similar to those of full exon sequencing(TCGA).Therefore,the captured Panel is also reasonable and can cover 90% of patients with colorectal cancer(1-2 mutations).There was no significant difference between the patient population and the reported population.The ctDNA test was accurate and consistent with the tissue DNA test results.This shows the feasibility and accuracy of the NGS second-generation sequencing with Accu-Act.We included a total of 85 colorectal cancer patients,including 52 males(61.2%)and 33 females(38.8%)with an average age of 62.87 years.Left colon cancer accounted for 28.23%,right colon represented 29.41%,and rectal cancer accounted for 42.36%.Of the 85 patients,only 8(9.4%)had postoperative ctDNA,4 patients had recurrence or metastasis;77 patients had no ctDNA detected after surgery,and only 6 patients had recurrence or metastasis.To compare the distribution of ctDNA,the distribution of MAF in different stages of colorectal cancer patients,and the prediction of postoperative ctDNA by preoperative ctDNA,we included information analysis of patients with stage IV colorectal cancer,whether preoperative ctDNA or Postoperative ctDNA was not associated with the stage of colorectal cancer(P>0.05).The level of preoperative cfDNA did not affect the detection of postoperative ctDNA(P>0.05).Therefore,ctDNA content is not related to the total amount of cfDNA but is related to micrometastasis.The preoperative ctDNA MAF was positively correlated with the stage(P<0.05),and the preoperative ctDNA MAF could not simply predict postoperative ctDNA detection(P>0.05).Neither preoperative cfDNA nor postoperative cfDNA content can predict tumor recurrence within 2 years.The MAF in Preoperative ctDNA and in tumor tissue cannot predict tumor recurrence within 2 years,but postoperative MAF in ctDNA predicts 2 Tumor recurrence during the year(HR:1.33;P=0.0256).We found that preoperative CEA,postoperative CEA,tumor size,lymph node detection,and tumor thrombosis did not effectively predict tumor recurrence within 2 years.Only postoperative ctDNA detection can effectively predict tumor recurrence within 2 years.We further conducted a subgroup analysis and found that for patients with stage II colorectal cancer who did not use adjuvant chemotherapy after surgery,there was a clear survival advantage for patients who did not have ctDNA detected after surgery.It is seen that ctDNA is a good biomarker for the prognosis assessment of MRD.In patients with negative ctDNA after stage III colorectal cancer,adjuvant chemotherapy has no significant effect on progression-free survival,which may be related to the fact that the total number of stage III patients with postoperative ctDNA negative and no chemotherapy is too small,and the follow-up time is short,so the reliability of the conclusion needs to be further confirmed.Finally,we used the small panels of the TP53,KRAS,APC,and BRAF4 genes to verify the possibility of detecting MRD,confirming that the small panel's post-operative ctDNA detection is also a good predictor of recurrence risk.Conclusion:Detection of ctDNA in patients with colorectal cancer can effectively predict tumor recurrence within 2 years.ctDNA plays a crucial role in the assessment of minimal residual disease after resection of colorectal cancer patients.It is a good supplement to the NCCN guide.