Genomic characterization of biliary tract cancers identifies driver genes and predisposing mutations.

BACKGROUND & AIMS: Biliary tract cancers (BTCs) are clinically and pathologically heterogeneous and respond poorly to treatment. Genomic profiling can offer a clearer understanding of their carcinogenesis, classification and treatment strategy. We performed large-scale genome sequencing analyses on BTCs to investigate their somatic and germline driver events and characterize their genomic landscape. METHODS: We analyzed 412 BTC samples from Japanese and Italian populations, 107 by whole-exome sequencing (WES), 39 by whole-genome sequencing (WGS), and a further 266 samples by targeted sequencing. The subtypes were 136 intrahepatic cholangiocarcinomas (ICCs), 101 distal cholangiocarcinomas (DCCs), 109 peri-hilar type cholangiocarcinomas (PHCs), and 66 gallbladder or cystic duct cancers (GBCs/CDCs). We identified somatic alterations and searched for driver genes in BTCs, finding pathogenic germline variants of cancer-predisposing genes. We predicted cell-of-origin for BTCs by combining somatic mutation patterns and epigenetic features. RESULTS: We identified 32 significantly and commonly mutated genes including TP53, KRAS, SMAD4, NF1, ARID1A, PBRM1, and ATR, some of which negatively affected patient prognosis. A novel deletion of MUC17 at 7q22.1 affected patient prognosis. Cell-of-origin predictions using WGS and epigenetic features suggest hepatocyte-origin of hepatitis-related ICCs. Deleterious germline mutations of cancer-predisposing genes such as BRCA1, BRCA2, RAD51D, MLH1, or MSH2 were detected in 11% (16/146) of BTC patients. CONCLUSIONS: BTCs have distinct genetic features including somatic events and germline predisposition. These findings could be useful to establish treatment and diagnostic strategies for BTCs based on genetic information. LAY SUMMARY: We here analyzed genomic features of 412 BTC samples from Japanese and Italian populations. A total of 32 significantly and commonly mutated genes were identified, some of which negatively affected patient prognosis, including a novel deletion of MUC17 at 7q22.1. Cell-of-origin predictions using WGS and epigenetic features suggest hepatocyte-origin of hepatitis-related ICCs. Deleterious germline mutations of cancer-predisposing genes were detected in 11% of patients with BTC. BTCs have distinct genetic features including somatic events and germline predisposition.

Circulating Tumor DNA Analysis for Liver Cancers and Its Usefulness as a Liquid Biopsy.

BACKGROUND & AIMS: Circulating tumor DNA (ctDNA) carrying tumor-specific sequence alterations has been found in the cell-free fraction of blood. Liver cancer tumor specimens are difficult to obtain, and noninvasive methods are required to assess cancer progression and characterize underlying genomic features. METHODS: We analyzed 46 patients with hepatocellular carcinoma who underwent hepatectomy or liver transplantation and for whom whole-genome sequencing data was available. We designed personalized assays targeting somatic rearrangements of each tumor to quantify serum ctDNA. Exome sequencing was performed using cell-free DNA paired primary tumor tissue DNA from a patient with recurrent liver cancer after transcatheter arterial chemoembolization (TACE). RESULTS: We successfully detected ctDNA from 100 µL of serum samples in 7 of the 46 patients before surgery, increasing with disease progression. The cumulative incidence of recurrence and extrahepatic metastasis in the ctDNA-positive group were statistically significantly worse than in the ctDNA-negative group (P = .0102 and .0386, respectively). Multivariate analysis identified ctDNA (OR 6.10; 95% CI, 1.11-33.33, P = .038) as an independent predictor of microscopic vascular invasion of the portal vein (VP). We identified 45 nonsynonymous somatic mutations in cell-free DNA after TACE and 71 nonsynonymous somatic mutations in primary tumor tissue by exome sequencing. We identified 25 common mutations in both samples, and 83% of mutations identified in the primary tumor could be detected in the cell-free DNA. CONCLUSIONS: The presence of ctDNA reflects tumor progression, and detection of ctDNA can predict VP and recurrence, especially extrahepatic metastasis within 2 years. Our study demonstrated the usefulness of ctDNA detection and sequencing analysis of cell-free DNA for personalized treatment of liver cancer.

Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity.

Intrahepatic cholangiocarcinoma and combined hepatocellular cholangiocarcinoma show varying degrees of biliary epithelial differentiation, which can be defined as liver cancer displaying biliary phenotype (LCB). LCB is second in the incidence for liver cancers with and without chronic hepatitis background and more aggressive than hepatocellular carcinoma (HCC). To gain insight into its molecular alterations, we performed whole-genome sequencing analysis on 30 LCBs. Here we show, the genome-wide substitution patterns of LCBs developed in chronic hepatitis livers overlapped with those of 60 HCCs, whereas those of hepatitis-negative LCBs diverged. The subsequent validation study on 68 LCBs identified recurrent mutations in TERT promoter, chromatin regulators (BAP1, PBRM1 and ARID2), a synapse organization gene (PCLO), IDH genes and KRAS. The frequencies of KRAS and IDHs mutations, which are associated with poor disease-free survival, were significantly higher in hepatitis-negative LCBs. This study reveals the strong impact of chronic hepatitis on the mutational landscape in liver cancer and the genetic diversity among LCBs.

GROWTH OF LENS AND OCULAR ENVIRONMENT: ROLE OF NEURAL RETINA IN THE GROWTH OF MOUSE LENS AS REVEALED BY AN IMPLANTATION EXPERIMENT.

The lens of 6-day-old normal mouse was implanted into the lentectomized eye of adult mouse to examine the effect of retina upon the growth of the implanted lens in vivo. The implanted lens grew normally and its transparency was kept for more than 5 months after implantation. The connection between the implanted lens and the ciliary part of the recipient iris was well established with the regeneration of zonular fibers from the recipient. In young lenses implanted reversely into adult eyes, the epithelial cells facing the retina elongated and a new epithelium was formed on the corneal side of the lens within 5 days. Young lenses implanted either in normal or reverse orientation into eyes from which the retina was previously removed did not grow. The cells of the original lens epithelium of these lenses were randomly accumulated beneath the posterior lens capsule, while the anterior portion of the implanted lenses became an epithelial structure without cell elongation. These results suggest that the growth of the implanted lens may be dependent on the retina of the adult eye.

A practical method to detect SNVs and indels from whole genome and exome sequencing data.

The recent development of massively parallel sequencing technology has allowed the creation of comprehensive catalogs of genetic variation. However, due to the relatively high sequencing error rate for short read sequence data, sophisticated analysis methods are required to obtain high-quality variant calls. Here, we developed a probabilistic multinomial method for the detection of single nucleotide variants (SNVs) as well as short insertions and deletions (indels) in whole genome sequencing (WGS) and whole exome sequencing (WES) data for single sample calling. Evaluation with DNA genotyping arrays revealed a concordance rate of 99.98% for WGS calls and 99.99% for WES calls. Sanger sequencing of the discordant calls determined the false positive and false negative rates for the WGS (0.0068% and 0.17%) and WES (0.0036% and 0.0084%) datasets. Furthermore, short indels were identified with high accuracy (WGS: 94.7%, WES: 97.3%). We believe our method can contribute to the greater understanding of human diseases.