Genomic Alterations Of Hepatocellular Carcinoma By Panel-based Sequencing

Primary hepatocellular carcinoma(hepatocellular,carcinoma,HCC)is one of the most common cancer worldwide each year,involving more than 700 thousand lives,to become the second largest cause of death after lung cancer tumor associated[1,2].The pathogenic factors of HCC are complex,the common risk factors of HCC are chronic hepatitis virus infection,including HBV and HCV infection,long-term large amounts of alcohol intake and exposure to carcinogens,such as aflatoxin B [3].In recent years,the incidence of obesity and diabetes caused by nonalcoholic fatty liver disease associated withhas increased by HCC[4].HCC occult onset,clinical symptoms are not typical,the low rate of early diagnosis,early treatment for the main HCC is surgical resection,radiofrequency ablation,chemoembolization combined with chemotherapy,but the recurrence rate is high,50%-70% of patients in postoperative recurrence or metastasis within 5 years [5,6].In patients with advanced HCC treatment options are limited;liver transplantation is an effective way to treat HCC,but because of the shortage of donors,beneficiaries with limited scope;therefore studies show that surgical treatment combined with immune therapy,molecular targeted therapy HCC with comprehensive treatment is the future development direction of HCC precise treatment of[7,8].Because the etiology of HCC complex,forming a highly heterogeneous tumor microenvironment,manifested in the differences among different patients and different tumor patients with the same tumor nodule lesions and internalheterogeneity of cells[9],therefore need to develop individualized treatment for patients with HCC individual tumor molecular pathological type,can avoid the blind use of drugs and unnecessary side effects.Individualized treatment of cancer needs to develop a targeted treatment plan before treatment,but also need to be in the treatment of cancer patients in real-time,dynamic monitoring.At present the common clinical HCC monitoring methods including the determination of serum AFP and ultrasonography,but the early diagnosis of HCC sensitivity of only 40%-50%,CT,MRI in the clinic is optional,but due to high cost,check itself the role of strong radiation and patient dependence is poor and not easy to be repeated several times for[6,10].In addition,the traditional HCC monitoring method can not reflect the heterogeneity between the tumor and the tumor,so it is urgent to develop biomarkers for early diagnosis and real-time dynamic monitoring of liver cancer.Objective:To evaluate whether the detection of liver cancer related gene mutation and second generation sequencing techniques can be used to analyze liver cancer molecular classification based on the observed variation of tumor related genes in hepatocellular carcinoma cell line,whether it can be used with the molecular diagnosis of liver cancer.Materials and methods: This study is divided into two parts,the first part,the use of gene sequencing technology based on target to target the second generation sequencing technology,325 of 7 common tumor cell line HCC DNA gene sequencing capture.Analysis of tumor associated mutations in DNA gene and mutation load rate,compared to the difference of genes,as well as the difference between the gene mutation trend.In the second part,the samples of 29 HCC patients were collected,including plasma samples,peripheral blood leukocytes(PBMC)and tumor tissues and adjacent tissues in the 1 days before operation.The cover the first part of the 50 common cancer oncogene and tumor suppressor gene amplification sub kit,Illumina HiSeq X Ten sequencing platform based on plasma cfDNA and DNA in tumor tissuesand adjacent tissues of DNA target amplicon sequencing into the mutant allele frequency(mutant allele frequency,MAF)is higher than that of gene site 1%.To compare the variation of the high frequency mutations of cfDNA,tumor tissue DNA and adjacent tissue DNA in patients with HCC before surgery,and the genomic PBMC from the DNA source as a control for the exclusion of germ cell variation.At the same time,the correlation between the gene mutation in plasma cfDNA and the tumor load was analyzed.Results:In the first part of this study,this study is based on the detection of tumor gene trapping of high-throughput sequencing of 7 hepatoma cell lines(HuH-7,Hep3 B,SK-HEP-1,MHCC97 H,MHCC97L,HepG2,HepG2.2.15)somatic mutations.Using a custom sequence capture system containing 325 tumor related genes panel,NimbleGen,the target region of the cell line was captured and sequenced.The results showed that the average coverage rate of the target area was 99.7%,and the average sequencing depth was about 1000 x,and 100 genes were detected,including 344 non synonymous mutations.There are 38 closely related mutations in cancer genes enriched in 5 oncogenic signaling pathways: chromatin remodeling,Notch,MAPK,p53,and Wnt/ beta-catenin pathways.Compared with the previous reports of somatic mutations in HCC patients,the chromatin remodeling and gene mutations in the Notch signaling pathway were higher in HCC cell lines.The four HCC cell lines(HuH-7,Hep3 B,SK-HEP-1,HepG2,)from different HCC patients had different mutation modes,but the same clone derived HCC cell lines showed a large similarity.In the second part of this study,we prospectively observed the tumor related gene.28 cases of patients with tumors in 27 cases(96%)samples detected at least 2 mutations(median mutation number 6,ranging from 2-18),1 cases of tumor tissue were detected any gene mutation,35 mutation in tumor tissue was detected by TP53,ATM,and ALK the mutation frequency is the highest,respectively,50%(14/28),39%(11/28)and 39%(11/28);all 29 cases(100%)tumor related gene mutation was detected in the plasma of patients with cfDNA(median number of 27 mutations,ranging from 6-47),including a large number of low frequency mutation.Compared with the same tumor tissue of patients with DNA and preoperative plasma cfDNA gene mutation found in 28 patients with paired samples,21 cases(21/28,75%)mutations detected at least one genetically identical serum cfDNA and tumor tissue DNA,among which TP53,PDGFRA,KIT,and NPM1 mutation frequency the highest,respectively is 33%(7/21),23.8%(5/21),19%(4/21)and 19%(4/21);7cases(7/28,25%)serum cfDNA and tumor tissue DNA was not detected in the same gene mutation,including 1 cases of tumor tissue of patients with DNA mutation was not detected in any gene mutation.Detection of gene CDKN2 A,including low frequency EGFR,plasma cfDNA.In addition,28 cases of HCC patients with paired samples,17 cases(17/28,61%)to detect identical gene mutation and tumor tissue of DNA patients to DNA in paracarcinoma tissues,including 14 cases(14/17,82%)DNA in tumor tissue and cancer adjacent tissues DNA gene mutations can be detected in plasma in cfDNA,the high frequency of mutations in PDGFRA,KIT and NPM1 genes.Comparison of AFP level and plasma cfDNA mutation detection results showed that 29 patients had 14 cases(48.3%)patients with positive AFP(>20ng/ml),24 cases(82.7%)in the plasma of patients with cfDNA detected tumor related gene mutations and 15 cases of AFP negative patients in 13 cases(86.7%)tumor related gene mutation was detected in plasma in patients with cfDNA;and plasma cfDNA and tumor tissue DNA have uniform mutation serum level of AFP was significantly higher than that of non uniform mutation group,AFP level in patients with two median value were 144ng/ml and 2.5ng/ml(P=0.016).In this study,no direct correlation between plasma cfDNA concentrations in patients with clinical and pathological features,and the diameter of the tumor patients in the >5cm group of TP53,CTNNB1,PIK3 CA and CDKN2 A gene MAF level is higher than the diameter of the tumor patients in the <5cm group,and the tumor or tumor patients with multiple metastases in TP53,RET,FGFR3 and APC gene MAF levels solitary tumor patients.Conclusion:1 this study was conducted to capture sequencing of tumor related genes in hepatocellular carcinoma cell line,the commonly used laboratory hepatocellular carcinoma cell model also revealed somatic mutations,mutation patterns of hepatocellular carcinoma cell lines and different sources of the same clone.2 patients with HCC cfDNA might replace tumor biopsies as molecular markers for screening HCC gene mutation;in this study,MAF levels were significantly associated with HCC gene mutation in plasma in cfDNA with tumor burden,the plasma cfDNA may become a molecular marker for HCC diagnosis.