BACKGROUND & AIMS Dietary exposure to aflatoxin is an important risk factor for hepatocellular carcinoma (HCC). However, little is known about the genomic features and mutations of aflatoxin-associated HCCs compared with HCCs not associated with aflatoxin exposure. We investigated the genetic features of aflatoxin-associated HCC that can be used to differentiate them from HCCs not associated with this carcinogen. METHODS We obtained HCC tumor tissues and matched non-tumor liver tissues from 49 patients, collected from 1990 through 2016, at the Qidong Liver Cancer Hospital Institute in China-a high-risk region for aflatoxin exposure (38.2% of food samples test positive for aflatoxin contamination). Somatic variants were identified using GATK Best Practices Pipeline. We validated part of the mutations from whole-genome sequencing and whole-exome sequencing by Sanger sequencing. We also analyzed genomes of 1072 HCCs, obtained from 5 datasets from China, the United States, France, and Japan. Mutations in 49 aflatoxin-associated HCCs and 1072 HCCs from other regions were analyzed using the Wellcome Trust Sanger Institute mutational signatures framework with non-negative matrix factorization. The mutation landscape and mutational signatures from the aflatoxin-associated HCC and HCC samples from general population were compared. We identified genetic features of aflatoxin-associated HCC, and used these to identify aflatoxin-associated HCCs in datasets from other regions. Tumor samples were analyzed by immunohistochemistry to determine microvessel density and levels of CD34 and CD274 (PD-L1). RESULTS Aflatoxin-associated HCCs frequently contained C>A transversions, the sequence motif GCN, and strand bias. In addition to previously reported mutations in TP53, we found frequent mutations in the adhesion G protein-coupled receptor B1 gene (ADGRB1), which were associated with increased capillary density of tumor tissue. Aflatoxin-associated HCC tissues contained high-level potential mutation-associated neoantigens, and many infiltrating lymphocytes and tumors cells that expressed PD-L1, compared to HCCs not associated with aflatoxin. Of the HCCs from China, 9.8% contained the aflatoxin-associated genetic features, whereas 0.4%-3.5% of HCCs from other regions contained these genetic features. CONCLUSIONS We identified specific genetic and mutation features of HCCs associated with aflatoxin exposure, including mutations in ADGRB1, compared to HCCs from general populations. We associated these mutations with increased vascularization and expression of PD-L1 in HCC tissues. These findings might be used to identify patients with HCC due to aflatoxin exposure, and select therapies.

The aim of our study was to determine the impact of genetic polymorphisms in the caspase (CASP) genes on prognosis of hepatocellular carcinoma (HCC). We genotyped 7 potentially functional polymorphisms in CASP3, CASP7, CASP8, CASP9, CASP10 genes in 362 HCC patients of receiving surgical resection of HCC tumor. The associations of genotype and haplotype with overall survival (OS) and disease free survival (DFS) were analyzed by using the Cox proportional hazards model. We found that the CASP9 rs4645981 C allele was significantly associated with positive effect on DFS (P = 0.011 and 0.016 for CT+CC vs. TT in univariate and multivariate analysis, respectively), CT genotype was associated with a better OS of HCC than the TT genotype both in univariate and multivariate analysis (P = 0.048 and 0.041, respectively). Moreover, the CASP3 rs2705897 GT genotype showed marginally significant association with decreased OS and DFS, compared with the GG genotype. One haplotype TT/TG in CASP3 (constructed by rs12108497 T>C and rs2705897 T>G) was significantly associated with decreased OS and DFS, compared to the common haplotype TT/TT both in univariate analysis (P = 0.021 and 0.026, respectively) and multivariate analysis (P = 0.025 and 0.030, respectively). The haplotype GT/GT in CASP9 (constructed by rs4645978 A>G and rs4645981 C>T) was significantly associated with decreased DFS both in univariate and multivariate analysis (P = 0.012 and 0.010, respectively). In conclusion, the CASP9 rs4645981 polymorphism, CASP3 and CASP9 haplotypes may be useful prognosis markers for HCC patients with surgical resection of tumor.

Single nucleotide polymorphisms (SNPs) within microRNAs (miRNAs) are considered potential markers for risk and prognosis of various cancers. In the current study, we aimed to determine whether miR-608 rs4919510 affected hepatocellular carcinoma (HCC) prognosis. We genotyped rs4919510 using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. Associations between rs4919510 and overall survival (OS) and demographic characteristics and clinical features were estimated using the Cox proportional hazards model. Results showed that HCC patients who carried the rs4919510 CC genotype had a significantly longer OS compared to those who carried the GG genotype ( P = 0.013, hazard ratio [HR] = 0.600, 95 % confidence interval [CI] 0.402–0.897) and the CG + GG genotype ( P = 0.033, HR = 0.681, 95 % CI 0.479–0.970) in univariate analysis. Similar results were obtained in multivariate analysis. Further stratification analysis indicated that rs4919510 was significantly associated with OS in patients who were satisfied with one of the following criteria: male gender, HbsAg-positive, α-fetoprotein (AFP)-positive, tumor size >5 cm, cirrhosis, solitary tumor, I + II pTNM stage, or no tumor capsule. Finally, a significantly higher frequency of rs4919510 CC genotype was observed in patients with cirrhosis (22.9 %, 55/240) than those without cirrhosis (14.0 %, 17/121) ( P = 0.047). In conclusion, our results illustrated the potential role of miR-608 rs4919510 as a prognostic marker for HCC patients undergoing surgical resection of the tumor.

Single-nucleotide polymorphisms (SNPs) of microRNAs (miRNAs) are considered potential markers of cancer risk and prognosis in various cancers. In the current study, the primary aim is to determine whether the miR-492G>C polymorphism (rs2289030) altered hepatocellular carcinoma (HCC) prognosis. The SNP rs2289030 of miR-492 was genotyped using DNA from blood samples of 362 HCC patients that had undergone surgical resection of a HCC tumor. The associations between overall survival and demographic characteristics, clinical features, and the SNP rs2289030 were estimated using the Cox proportional hazards model. Results showed that patients who carried the CG genotype (P  = 0.015, hazard ratio [HR] = 0.704, 95 % confidence interval [CI] 0.530–0.934) and CG+GG genotype (P = 0.011, HR = 0.703, 95 % CI 0.536–0.924) had significantly decreased risk of death compared to those with the CC genotype. Similar results were found in the multivariate analysis adjusted by tumor size and venous invasion. Further stratification analysis indicated that the effect of rs2289030 had more prominence in patients ≤50 years old and that reported ever using alcohol, male gender, a family history of HCC, being HbsAg or alpha fetoprotein (AFP) positive, differentiation I + II, presence of venous invasion or cirrhosis, multiple tumors, and pTNM stage I + II. Results from this study illustrate the potential use of miR-492 rs2289030 as a prognostic marker for HCC patients that have undergone a surgical resection of the tumor.

The apoptotic pathway is important in the control of vital processes of hepatocellular carcinoma (HCC). In the current study, we aimed to determine whether apoptotic gene-related polymorphisms modified HCC prognosis. We genotyped 16 single nucleotide polymorphisms (SNPs) in 10 core genes (TP53, TP53INP1, TP53BP1, CDKN2A, CDKN1A, CDKN1B, MDM2, BAX, CCDN1 and BCL2) in the apoptotic pathway by using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. The associations between genotypes/haplotypes of the 10 genes and overall survival (OS) of HCC patients were assessed using the Cox proportional hazards model. We found one CDKN1B haplotype CCT/ACT (constructed by rs36228499 C>A, rs34330 C>T and rs2066827 T>G) significantly associated with decreased OS of HCC patients, compared to the common haplotype ACT/CTT both in univariate analysis (P=0.013, HR=1.198, 95% CI: 1.039-1.381) and multivariate analysis (P=0.006, HR=1.224, 95% CI: 1.059-1.413). We also find two SNPs (rs560191 G>C and rs2602141 T>G) in TP53BP1 shown to be marginally significantly associated with decreased OS of HCC patients. However, none of the other SNPs or haplotypes were significantly associated with HCC OS. Our results illustrated the potential use of CDKN1B haplotype as a prognostic marker for HCC patients with surgical resection of tumor.

Qidong City, China, has had high liver cancer incidence from endemic hepatitis B virus (HBV) infection and dietary exposure to aflatoxin. Based on etiologic studies, we began interventions in 1980 to reduce dietary aflatoxin and initiate neonatal HBV vaccination. We studied trends in liver cancer incidence rates in the 1.1 million inhabitants of Qidong and examined trends in aflatoxin exposure, staple food consumption, HBV infection markers and annual income. Aflatoxin exposure declined greatly in association with economic reform, increased earnings and educational programs to shift staple food consumption in the total population from moldy corn to fresh rice. A controlled neonatal HBV vaccination trial began in 1983 and ended in November, 1990, when vaccination was expanded to all newborns. Liver cancer incidence fell dramatically in young adults. Compared with 1980-83, the age-specific liver cancer incidence rates in 2005-08 significantly decreased 14-fold at ages 20-24, 9-fold at ages 25-29, 4-fold at ages 30-34, 1.5-fold at ages 35-39, 1.2-fold at ages 40-44 and 1.4-fold at ages 45-49, but increased at older ages. The 14-fold reduction at ages 20-24 might reflect the combined effects of reduced aflatoxin exposure and partial neonatal HBV vaccination. Decrease incidence in age groups >25 years could mainly be attributable to rapid aflatoxin reduction. Compared with 1980-83, liver cancer incidence in 1990-93 significantly decreased 3.4-fold at ages 20-24, and 1.9-fold at ages 25-29 when the first vaccinees were <11 years old.

Background: Most of the reports on tumor relapse are recurrence of the same type of tumor after months to few years of a successful initial cancer treatment. It is generally unusual and unexpected that a different type of the second tumor occurs after several decades of the curative treatment of the original tumor. Case Presentation: We report a case of 74-year-old man with intrahepatic cholangiocarcinoma (ICC) diagnosed 38 years after curative resection of hepatocellular carcinoma (HCC). In addition to the uniqueness of longer survival and developed a new type of tumor, both the original HCC and the later occurred ICC were detected through a cancer surveillance program by screening alpha-fetoprotein (AFP) and ultrasonography of the liver for the general population and/or high risk group of people who were asymptomatic. Conclusion: This report provides evidence demonstrating occurrence of new type of tumor following initial curative therapy of the original tumor. In addition, this case report also highlights the importance of cancer surveillance program for earlier detection of the tumors to achieve a remarkably improved prognosis of the cancer patients for a prolonged cancer free survival time.

Objective To explore the relationship between clinical pathological characteristics and long survival in hepatocarcinoma(HCC) patients who survived more than 30 years after resection.Methods 17 HCC patients who survived more than 30 years after resection(group A) were compared with those who survived less than 3 years(group B).Results In group A,10 cases were male and 7 were female,aged from 17 to 46 years,mean age of 29.One died at the 34thyear after resection,16 were alive.88.24%(15 /17) were diagnosed in routine physical checkup,72.22%(13 /17) were at clinical stage of T 2 N 0 M 0,70.5%(12/17) tumour were ≤5cm,94.12%(16/17) AFP converted negative after resection,94.12%(16 /17) had fibre membrane around tumour,94.12%(16 /17) had no embolism and remains of tumour,82.35%(14 /17) had no or light cirrhosis,76.47%(13 /17) were resected more than 1 cm far from the edge.All the above characteristics of group A were better than that of group B(P 0.01),while the clinical symptom,hepatitis history,family history,tumour number and histological stage of two groups had no significant difference(P 0.05).Conclusion Diagnosed in routine physical checkup,at lower stage than T 2 N 0 M 0,tumour ≤5cm,with fibrous membrane,without tumour embolism and remains,without cirrhosis,AFP conversion and resected 1cm far from the edge,are closely related with long surival.Enhancement of the early diagnosis and early treatment of liver cancer recurrence,paying more attention to the treatment,and refusing tobacco and alcohol,are important for long surviral after liver cancer resection.

Objective To explore the factors affecting the survival after liver cancer surgery in order to provide refer ence for the clinical development of therapeutic measures.Methods Seventy-eight patients receiving liver cancer surgery were divided into the more than 5 years’ survival group(group A) and the death within 3 years group(group B).The clinical pathological factors were compared.Results The two groups had no statistical significant differences in the preoperative AFP(+),HBs Ag(+),clinical symptoms,tumor single nodules,distance of tumor to cutting edge > 1 cm and histological grade of cancer(level I and II).Group A showed a postoperative AFP negative rate of 82.05%(32/39),clinical TNM(I-II) staging of 87.18%(34/39),general survey detection of 69.23%(27/39),peritumoral capsule of 76.92 %(30/39),long tumor diameter ≤ 5 cm of 53.85%(21/39),non-cancer embolus rate of 87.18%(34/ 39) and peritumoral moderate and severe sclerosis of 15.38%(6/39),which were significantly or highly significantly superior to the postoperative AFP negative rate of 25.64%(10/39),clinical TNM(I-II) staging of 30.77%(12/39),general survey detection of 35.90%(14/39),peritumoral capsule of 20.51%(8/39),long tumor diameter ≤ 5 cm of 20.51%(8/39),non-cancer embolus rate of 30.77% 12/39) and peritumoral moderate and severe sclerosis of 35.90%(14/39)(P < 0.05 or P < 0.01).Conclusion Apart from some clinical pathological factors irrelevant to the length of postoper ative survival time,most clinical pathological factors are closely related to the postoperative survival and consistent with the report.

To identify genetic susceptibility loci for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in the Chinese population, we carried out a genome-wide association study (GWAS) in 2,514 chronic HBV carriers (1,161 HCC cases and 1,353 controls) followed by a 2-stage validation among 6 independent populations of chronic HBV carriers (4,319 cases and 4,966 controls). The joint analyses showed that HCC risk was significantly associated with two independent loci: rs7574865 at STAT4, P(meta) = 2.48 × 10(-10), odds ratio (OR) = 1.21; and rs9275319 at HLA-DQ, P(meta) = 2.72 × 10(-17), OR = 1.49. The risk allele G at rs7574865 was significantly associated with lower mRNA levels of STAT4 in both the HCC tissues and nontumor tissues of 155 individuals with HBV-related HCC (P(trend) = 0.0008 and 0.0002, respectively). We also found significantly lower mRNA expression of STAT4 in HCC tumor tissues compared with paired adjacent nontumor tissues (P = 2.33 × 10(-14)).

Objective To investigate the long-term survival factors in patients with hepatocellular carcinoma(HCC) after the operation to provide evidence for clinical treatment and prognostic judgement.Methods Clinical pathologic characteristics and the expression of p53 and c-myc were analyzed in 37 operated cases with HCC survival for more than 20 years(group A) and those survival for less than 3 years at the same term(group B).Results 67.6% of the patients in group A were from general survey.The surgical margin was more than 1 cm(67.6%),and negative conversion rate of AFP was 96.8% in the first month after the surgery.There were significant differences compared with group B.However there were no significant differences between the two groups in the history of hepatitis and the family history.In the tumor-adjacent tissue of the two groups,expression of p53 and c-myc was 24.3% vs 71.4% and 37.8% vs 80.8%,and there were significant differences between the two groups.Conclusions In the patients from general survey,the important long-term survival factors are as follows: single tumor diameter less than 5 cm,complete envelop,no tumor thrombosis,Ⅰ~Ⅱ of histopathologic grades,and no or mild cirrhosis around the cancer.The expression of p53 and c-myc has a negative correlation with prognosis.

目的:探讨肝癌术后长期生存患者临床病理及P53、c-myc癌基因表达的特点和意义.方法:以启东肝癌高发区34例肝癌术后生存10年以上的病例(A组)作为研究对象,42例肝癌术后5年内死亡的病例(B组)为对照,进行临床病理特点观察,并用免疫组化方法检测肝癌及癌旁P53和c-myc表达. 结果:肝癌组织中P53、c-myc表达阳性率分别为56.58%(43/76)和60.53%(46/76),A组中分别为47.06%(16/34)和50.00%(17/34),B组中分别为64.29%(27/42)和69.05%(29/42),两组相比无显著差异.癌旁组织中P53、c-myc表达阳性率分别为42.11%(32/76)和57.89%(44/76),A组中分别为26.47%(9/34)和41.18%(14/34),与B组中54.76%(23/42)、71.43%(30/42)相比差异有统计学意义.A组85.29%(29/34)的病人有完整包膜,显著多于B组的21.43%(9/42);5.88%(2/34)有门脉栓塞,38.24%(13/34)伴有肝硬化,显著低于B组(69.05%,78.57%).结论:癌周组织中P53、c-myc表达与术后生存呈负相关;肝癌包膜完整、无门脉栓塞、无肝硬化的单结节病人,预后好,术后生存期明显延长.

目的:探讨C-myc基因蛋白在肝癌及癌周肝内的表达与肝癌术后复发的关系。方法:应用免疫组化方法,在58例肝癌存档石蜡切片上标记和观察C-myc基因蛋白的表达。结果:C-myc基因蛋白在肝癌组织及其癌周肝组织中的阳性率分别为68.97%(40/58)及65.52%(38/58),表抗阳性的肝癌及癌周阳性表达分别为70.27%(26/37)、67.57%(25/37),伴有肝硬化的肝癌及癌周肝组织中分别为86.49%(32/37)、81.08%(30/37),伴有肝细胞不典型增生的肝癌及癌周肝组织中阳性率分别为90.91%(20/22)、90.91(20/22,),有包膜的癌内和癌周阳性率分别为60.47%(26/43)、55.81%(24/43),术后复发组的癌内及癌周阳性率分别为79.49%(31/39)、79.49%(31/39)。结论:C-myc基因蛋白表达的增加与HBsAg阳性、肝硬化、肝细胞不典型增生、癌周肝内转移及肿瘤有无包膜密切相关,与肿瘤复发有密切关系。

Objective To detect the location and geographical features of gene mutation of the cancer based on 30 709 clinical tumor biopsies.Methods Thirty thousand seven hundred and nine cases of tumor biopsy materials were collected from the Department of Pathology of Nantong Cancer Hospital between June 1974 and December 1987. The address of the village and county of patients was collected and statistical analysis was performed on data from lab examination. DNA sequencing of 31 cases of hepatocellular carcinoma (HCC) was performed at the Department of Pathology of the Medical College of Tokyo University, Japan.Results The data suggested that different carcinomas occurred predominantly in some districts and the frequency and type of mutation of the P53 gene in the HCC were different in different districts.Conclusion It seems that based on the clinical biopsy materials, carcinomas of the uterus cervix (CC) and nasopharynx tend to occur in some districts of Nantong City and the frequency and type of mutation of the gene P53 of the HCC were different in different districts. The reason for this is not understood and further study will be done.

目的分析和探讨p53异常对人肝细胞癌(HCC)术后复发与生存的影响,以及其在HCC发生发展中的作用机制.方法手术切除HCC标本202例.分别来自启东HCC高发区和上海等一般地区,其中行二次或多次手术患者54例,组织学检测HBsAg阳性患者138例(68.3%),HBV DNA原位杂交检测阳性患者86例(42.6%),采用PCR-SSCP和RFCP技术结合免疫组化示综显示,分析p53基因5,6,7,8外显子突变及其编码蛋白的过度表达.结果 p53基因突变率为44.6%(33/74),其中89.8%(27/73)表现为点突变,18.2%(6/33)为片段性碱基缺失,长度10bp～16bp.等位基因杂合性缺失(LOH)主要集中于外显子7和8,各与39.4%(13/33)和27.3%(9/33).外显子5和6的突变发生率各为18.2%(6/33)和15.1%(5/33).外显子7RFLP分析证实第249位密码子有颠换突变(53.8%,7/13)外,248位密码子也有点突变发生(30.8%,4/13).免疫组化检测显示P53蛋白过度表达的总检出率为70.3%(142/202),pAb1801与CM-1 mAb检测P53蛋白过度表达率分别为69.5%(89/128)和67.2%(86/128),而采用DO-7和pAb240单抗检测的阳性率仅为39.8%(51/128)和19.5%(25/128).蛋白检测的地区差异明显.启东高发区肝癌的P53蛋白过度表达率为81.2%(92/114),而上海等一般地区肿瘤组织的阳性表达率为56.8%(50/88).p53基因突变检测与上述结果雷同,分别为57.1%(16/28)和37.1%(17/46),二者差异显著(P＜0.05).并显示与HBV感染,肿瘤去分化和侵袭行为,以及术后复发及生存时间有关联.结论国人HCC p53基因突变多为非定点错义突变,外显子7上249位密码子仅为HCC常见位点之一,反映除AFB1诱变作用外,还可能有其他环境诱变因素的参与,尤其是HBV感染.由于基因-蛋白质表达上既存在一致性,也可出现不一致性,表明P53蛋白的构型变异不仅来自其编码基因的转录失常,蛋白质自身的变化更是其正常的功能失活的重要机制.地理差异反映病因与致癌机制的复杂性.采用CM-1和pAb1801株mAb检测P53蛋白的过度表达比用PCR-SSCP分析p53基因变化更能反映人HCC的术后复发与预后.

Immunohistochemical deteation of C-myc protein expression in 58 cases of primary hepato-cellular carcinoma(HCC)has been conducted in paraffin sections.It suggested that the positive rates of C-myc protein expression in HCC and its peripheral tissue were 68.97 %(40/58)and 65.52%(38/58),respec-tively.The increase of C-myc protein expression was closely related with HBsAg positive cirrhosis,atypicalproliferation of hepatocytes,metastasis in tumor surrounding tissue,and liver neoplasm without fibrous en-capsulation,C-myc protein expression increased significantly in HCC patients after hepatectomy,and thetumor recurrence rate was high.The possible action of C-myc protein expression in carcinogenesis of pri-mary HCC were.

应用免疫组化ＡＢＣ法，对肝癌高发区６２例肝细胞癌组织及癌周肝组织进行了Ｐ５３和Ｃ－ｍｙｃ基因产物的标记。结果显示，肝细胞肝癌组织中Ｐ５３与Ｃ－ｍｙｃ基因蛋白均过量表达，阳性率分别为７４．１９％与６７．７４％，均高于有关文献报道的检测率。癌周肝组织中Ｐ５３与Ｃ－ｍｙｃ阳性检出率均低于癌组织。ＨＢｓＡｇ阳性病例、癌周伴肝硬变病例Ｐ５３与Ｃ－ｍｙｃ基因异常表达率均显著高于相应的阴性病例。本文随访病例中，术后肿瘤复发组Ｐ５３与Ｃ－ｍｙｃ基因表达的阳性率极明显地高于未复发尚生存组。文中还讨论了Ｐ５３和Ｃ－ｍｙｃ基因产物的表达在肝细胞癌发生中的可能作用