Genomic portrait of resectable hepatocellular carcinomas

Genomic portrait of resectable hepatocellular carcinomas: Implications of RB1 and FGF19 aberrations for patient stratification

Hepatocellular carcinoma (HCC, 간암)에서의 유전적 양상을 보기 위한 논문. 주로 RB1, FGF19 유전자에 대해 관찰함.

First published: 22 September 2014

Abstract

간암 초기 간 절제술은 일반적인 치료이기는 하나 재발율이 높음.

- 수술 후 5년 내 재발이 50% 이상

- 해당 단계의 유전적 원인이 어떤 것이 있는지 연구함.

- Whole Exome Sequencing을 진행하고 CNV 분석을 수행함

- 대상: 231 hepatocellular carcinomas (72% with hepatitis B viral infection)

분석

1. Unsupervised genomic analyses: genetic aberrations과 수술 후 clinical outcomes 간의 연관성을 분석

- Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected.

2. 해당 유전자를 대상으로 Pathway 분석 수행 

- p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways

RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). 

RB1 Gene: The protein encoded by this gene is a negative regulator of the cell cycle and was the first tumor suppressor gene found. The encoded protein also stabilizes constitutive heterochromatin to maintain the overall chromatin structure. The active, hypophosphorylated form of the protein binds transcription factor E2F1. Defects in this gene are a cause of childhood cancer retinoblastoma (RB), bladder cancer, and osteogenic sarcoma. [provided by RefSeq, Jul 2008]

FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). 

Conclusion: RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti-FGF19 treatment in these patients. (Hepatology 2014;60:1971–1981)

CNV: copy number variation

HBV: hepatitis B virus

HCC: hepatocellular carcinoma

HCV: hepatitis C virus

NBNC: neither HBV nor HCV

Materials and Methods

Study Design and Clinical Samples

- 샘플: Fresh-frozen tissues of 231 HCCs + matched nonneoplastic liver tissues

- Clincal 특징은 Table 1에 명시

Table1

- Etiology of the liver disease: 간질환의 원인

- Microvascular invasion: 미세혈관성 침입

- AFP: 간암표지자 검사(https://labtestsonline.org/understanding/analytes/afp-tumor/tab/test/)

- Hepatic fibrosis stage: 간 장애의 결과 간에 섬유가 증가한 상태를 말한다.

- Edmondson-Steiner grade: 간세포의 분화도

- Sequencing 후 Tumor purity 는 PurBayes 프로그램으로 측정(tumor-normal tissue samples 대상으로 분석)

- Fibrosis staging은 METAVIR scoring system으로 측정

- Table 1의 tumor size, microvascular invasion, early recurrence, the Edmondson-Steiner histological grade, the fibrosis stage of the nonneoplastic liver tissue, and the serum alpha-fetoprotein level의 추가 정보는 아래 링크에서 확인

http://onlinelibrary.wiley.com/store/10.1002/hep.27198/asset/supinfo/hep27198-sup-0001-suppinfo01.pdf?v=1&s=e503ac40ca5341ac4da74d00e80deac5e67a9ec4 

Whole Exome Sequencing and Copy Number Variation (CNV) Analysis

- Agilent SureSelect 50 Mb system

- Illumina HiSeq 2000

- Reference Genome: UCSC hg19

- Mutation: MuTect, VarScan2, GATK somatic indel detector

- Validation: Sanger sequencing

- Annotation: 

    - Gene: MutSig CV

    - Functional enrichment: Metacore (GeneGo)

    - CNV: Affymetrix Cytoscan HD platform(Nexus Copy Number software)

Tissue Microarray and Immunohistochemistry

Quantitative Real-Time Polymerase Chain Reaction (PCR)

Statistical Analysis

- Association testing was performed using Fisher's exact tests between mutation and clinicopathological factors. 

- Recurrence-free survival was determined using the Kaplan-Meier method and survival curves were compared using the log-rank test. 

- Logistic regression analysis was performed to evaluate patients with early recurrence according to clinicopathological risk factors and RB1 gene aberration status. 

- The Cox proportional hazards model was used to evaluate the relationships between clinicopathologic factors and survival. 

- The hazard ratio (HR) or odds ratio (OR), along with 95% confidence interval (CI), were assessed for each factor. 

All tests were two-sided and P < 0.05 was considered statistically significant. Statistical analyses were performed using the R program (v. 2.14.2, www.r-project.org) and Stata/IC statistical software (v. 12, StataCorp, College Station, TX).

Sequencing Data Deposition

Results

Clinical Features

The median age of the 231 HCC patients was 55 years (range, 26-80), and 75% of the patients were male. All patients had well-preserved liver function (Child-Pugh class A). Among them, 167 cases were associated with HBV infection, 22 with HCV infection, and 42 with neither HBV nor HCV (NBNC). The median tumor size was 3.8 cm, and 66 (30%) of the 231 tumors were less than 3 cm in size. Thirteen patients had multiple tumors. Sixty-eight (30%) of the 231 patients had microvascular invasion and 185 (80%) patients had advanced hepatic fibrosis (stage 3 or 4). Approximately two-thirds of the patients had Edmondson-Steiner grade I or II tumors (Table 1; Supporting Table 1). The mean tumor purity, estimated based on sequencing data of paired tumor-normal tissue samples, was 58%. The estimated tumor purity of each tumor sample is summarized in Supporting Table 3. There was no significant relationship between tumor purity and the number of somatic mutations.