| With the rapid development of experimental medicine,the disease from cognition to treatment has been improved significantly.The detection methods of clinical diseases have developed from cell morphology diagnosis,biochemical diagnosis and immunodiagnosis to molecular diagnosis.Since the 1970 s,molecular diagnosis has become one of the most important aspects in the field of in vitro diagnosis.Molecular diagnosis technology mainly includes molecular diagnosis technology based on molecular hybridization and molecular conformation,nucleic acid sequencing technology,quantitative polymerase chain reaction(PCR)technology and so on.Molecular diagnosis technology is widely used in infectious diseases,genetic diseases,blood screening and tumor molecular diagnosis.Molecular diagnosis technology has become a hot spot in the field of tumor research,and the core technology is PCR technology,sequencing technology and related liquid biopsy technology.The exploration of circulating tumor cells,circulating tumor DNA,exosomes and other tumor information carriers in liquid biopsy is changing with each passing day,which provides a research direction for the realization of noninvasive liquid molecular biopsy.Our research is represented by the latest three molecular detection methods,including the next generation gene sequencing technology(represented by Miseq Illumina),digital PCR technology(represented by Droplet Digital PCR),and detection of circulating tumor cells(represented by folate receptor PCR detection),to explore the application of these technologies in hepatobiliary tumors.The main histological types of primary liver cancer(PLC)include hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma(ICC)and mixed liver cancer.Biliary tract tumor is a group of heteromorphic tumors formed by differentiation of biliary tract or gallbladder epithelial bile duct cells.Both of them are malignant and lethal in digestive system tumors.Part 1: Next generation sequencing analysis of HBV multidrug resistance sites and HBs Ag + / HBs Ab + related mutationsThe next generation gene sequencing technology plays an important role in the accurate diagnosis and treatment of tumor,which has the advantages of short detection cycle,high flux and high sensitivity.The massive data results obtained after sequencing are conducive to deeper data analysis and mining,and ultimately achieve accurate etiological diagnosis of patients.Hepatitis B virus(HBV)infection is the most common cause of liver cancer.At present,antiviral treatment is the only choice to control and prevent the further development of patients with chronic HBV infection.It is generally believed that anti hepatitis B surface antibody(HBs Ab)can completely neutralize hepatitis B surface antigen(HBs Ag).However,the coexistence of HBs Ab and HBs Ag may occur in chronic HBV infection.In this study,1066 patients with chronic hepatitis B(CHB)and HBV related HCC were enrolled in our hospital and multi center.The drug resistance sites of HBV RT region and the sequence characteristics of HBs Ag + / HBs Ab + double positive patients were analyzed by NGS.The drug resistance analysis of five antiviral drugs in RT region showed that the highest drug resistance rate(low frequency resistance + resistance)of LAM and Ld T was 61.66% and 61.34%;the drug resistance rate of ETV and adv was 21.25% and 15.34%,respectively;the drug resistance rate of TDF genotype was the lowest,which was 13.10%.LAM,Ld T,ETV produced resistance(mutation rate > 20%);low frequency mutations(mutation rate of 5-20%)in rt S78 T,rt A181 T / V and rt N236 T sites accounted for 59.09%,18.75% and 38.46% of the positive mutations(mutation rate ≥ 5%);the low-frequency resistance of TDF accounted for 79.27% of the total drug resistance,and the low-frequency mutations of rt S78 T and rt A181 T / V sites(mutation rate ≥ 5%)related to adv resistance were 59.09%,18.75% and 38.46%,respectively The mutation rate of 5-20% was 59.09% and 18.75% respectively.There were significant differences in rt236 and rt184 between the two groups(P < 0.05).The distribution of amino acid mutation in S region indicated that the average mutation rate of HBs Ag + HBs Ab + double positive(DP)patients and HBs Ag + HBs Ab-single positive(SP)patients in the main hydrophilic region(MHR)was higher than that in other regions,and the "a" determinant region within MHR in DP group was significantly higher than that in SP group(P < 0.05).Grouping analysis found that in HCC group,P46 L,L104F,T118 P,R/K122 T,T125P,I126 T,Q129N,T131 N,M133S,M133 T,V168A,I195 M,P203R,Y206 C,F220C,V224 A there was significant difference in mutation rate between DP and SP group(P < 0.05);in CHB group,T47 K,P62L,L77 R,Q101H,M103 L,L104F,I126 S,M133T,G145 R,M198I,L216* there was significant difference in mutation rate between DP and SP groups(P < 0.05);Among the 16 loci in HCC group and 11 loci in CHB group,the common difference sites were M133 T and L104 F,while the others were different.The results showed that the 16 heterotopic points of HCC patients were negatively correlated with HBs Ag results;T125P,L104 F,I195M,T118 P,R / K122 T,Y 206 C,Q129N,V168 A were negatively correlated with HBs Ab results,while I126T、V224A、P46L、F220C、M133T、T131N、M133S、P203R were positively correlated with HBs Ab.However,M103 L,L104F,Q101 H,G145R,L77 R,T47K,P62 L,M133T and L216* in CHB patients were negatively correlated with HBs Ag results,while I126 S and M198 I were positively correlated with HBs Ag;M103L,L104 F,Q101H,L77 R,G145R,L216*,T47 K,M133T,P62 L were negatively correlated with HBs Ab,while I126 S and M198 I were positively correlated with HBs Ab.The correlation analysis between the mutation frequency of 16 loci(P46L,L104 F,T118P,R / K122 T,T125P,I126 T,Q129N,T131 N,M133S,M133 T,V168A,I195 M,P203R,Y206 C,F220C,V224A)in HCC and serum HBs Ag neutralization rate were analyzed,showed that there was a negative correlation between mutation frequency and neutralization rate of Q129 N heterotopic point(r =-0.41,P < 0.05).In conclusion,NGS technology was used to analyze the resistance sites in RT region and HBs Ag / HBs Ab double positive mutations in S region.Quantitative detection of drug resistance is the advantage of this study,and the discovery of low-frequency(5-20%)drug resistance has an important predictive value for the occurrence of drug resistance in the future.Quantitative and hypersensitive drug resistance detection and the existence of differential drug resistance sites of HCC and CHB provide guidance for clinical medication to implement more accurate anti-virus treatment;mutation analysis of HBV S region in HBs Ag + HBs Ab + double positive patients showed that HCC and CHB could produce different mutations in the course of disease progression.The difference sites of HCC patients mainly concentrated in the "a" determinant region,which affected the neutralization reaction of HBs Ag and HBs Ab,leading to new HBV superinfection.The discovery of these mutations may be an important reason for HBV immune escape,HBV transmission and even infection in vaccinated population.Part2: Preliminary clinical study on the diagnosis and prognosis of biliary tract cancers by quantitative detection of TP53 gene p.R273 H mutation by digital PCRThe advantages of digital PCR technology in the detection of micro mutation difference in micro nucleic acid samples are generally recognized.We use d PCR technology to detect the mutation of tumor suppressor gene TP53 in patients with biliary tract tumor,and explore its clinical application value.We analyzed the data of TP53 gene in human cancer mutation information database(COSMOC),and found that the mutation rate of several tumor driving gene hot spot mutation sites(p.R175 H,p.R248 Q,p.R248 W,p.R273H)was generally high.In this study,a total of 45 pairs of bile duct tumor and its adjacent tissues were collected from the laboratory of Shanghai Oriental hepatobiliary surgery hospital.After DNA extraction,quantitative detection of p.R175 H,p.R248 Q,p.R248 W,p.R273 H was performed by using dd PCR technology.76 patients with biliary tract tumor and 40 patients with cholecystitis were selected as experimental group.In the control group,40 healthy people were served as the healthy control group.Cell free DNA(cf DNA)was extracted from plasma samples,and four mutation sites were detected by dd PCR.The quantitative analysis of TP53 gene mutation sites in cancer and adjacent tissues of 45 pairs of patients with BTC showed that the mutation rate(%)of p.R175 H in biliary tumor tissues was significantly different from that in adjacent tissues(P < 0.05),but there was no significant difference in the pairing of p.R273 H,p.R248 W and p.R248 Q in other sites(P > 0.05).According to whether there was p.R175 H mutation or not,the clinical indexes of the two groups were compared.It was found that there were significant differences in albumin and total protein between the two groups(P < 0.05).These indexes of patients with p.R175 H mutation were lower than those without mutation.Peripheral blood cf DNA TP53 study showed that the mutation frequency(%)of p.R273 H site in 76 patients with biliary tract tumor and 40 patients with cholecystitis(gallbladder cancer,intrahepatic cholangiocarcinoma,biliary tract tumor group)was higher than that of disease control group,disease control group and healthy control group(P < 0.05).Further combined with pathological features including tumor size,number,metastasis and TNM stage,p.R273 H mutation was associated with tumor number(P < 0.05),while p.R248 W,p.R248 Q and p.R175 H mutation were not associated with tumor number(P > 0.05).When the cut-off value was 0.547,the sensitivity,specificity and ROC of binary logistic stepwise regression model combined with p.R273 H,CA19-9 and AFP were 73.7%,90.0% and 0.845 [95% CI(0.775-0.914)].When the cut-off value was 0.177,the sensitivity,specificity and ROC of p.R273 H and CA19-9 binary logistic stepwise regression model were 79.2%,95.0% and 0.908 [95% CI(0.831-0.985)].When the cut-off value was 0.547,the sensitivity,specificity and ROC of p.R273 H,p.R248 W,CA19-9 and AFP binary logistic stepwise regression model were 75.0%,90.0% and 0.844 [95% CI(0.764-0.924)].Survival curve analysis showed that the survival rate of patients with positive p.R273 H mutation was significantly lower than that of patients with negative p.R273 H mutation(P < 0.05).In conclusion,the TP53 gene mutation analysis based on d PCR technology has certain clinical significance for the diagnosis and prognosis analysis of biliary system tumors.The quantitative detection of p.R273 H mutation can effectively improve the diagnostic efficiency of biliary tract tumors,and can be used as an effective indicator for postoperative prognosis.Part3: Preliminary application of circulating tumor cell detection technology in clinical diagnosis of hepatobiliary tumorCirculating tumor cell(CTC)detection technology is the first found marker of effective real-time monitoring method in liquid biopsy.The isolated cells are considered to be the real "complete" markers of liquid biopsy,which is the frontier technology in the field of tumor diagnosis.Studies have found that folate receptors(FRs)are highly expressed in tumor tissues.Previous studies have taken FRs as a target for CTC detection in tumors,especially in patients with lung cancer.Whether FRs can be used as a target for CTC detection in hepatobiliary and other tumors needs further research or confirmation.This study focused on the expression of FRs in peripheral blood and tissues of patients with hepatobiliary tumor.We collected 28 whole blood samples from hepatobiliary tumor patients,14 disease control patients and 14 healthy control subjects from Shanghai Oriental hepatobiliary surgery hospital in 2020.We also selected 45 pairs of tumor tissues from HCC and BTC patients from 2012-2014 and their matched paracancerous samples to study the expression of folate receptor protein.The results showed that the number of folate receptors per 3 ml of blood in hepatobiliary tumors was significantly higher than that in the control group(P < 0.05).In HCC group,the expression of folate receptor α m RNA was correlated with carbohydrate antigen 19-9(r = 0.636,P < 0.05),g-glutamyltransferase(r = 0.477,P < 0.05)and aspartate transferase(r = 0.5,P < 0.05).Western blotting(30 pairs of tissues)and immunohistochemistry(45 pairs of tissues)showed significant difference in the expression of folate receptor α protein between carcinoma and paracancerous tissues(P < 0.05).In conclusion,the results of this study suggest that folate receptor as a target of CTC may also be suitable for blood detection in patients with hepatobiliary tumors,but the value of folate receptor for prognosis evaluation needs further study.The expression of folate receptor α was found in hepatobiliary tumors compared with the adjacent control tissues,and its pathological significance is worthy of further study. |
