Whole genome sequencing discriminates hepatocellular carcinoma with intrahepatic metastasis from multi-centric tumors.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) have a high-risk of multi-centric (MC) tumor occurrence due to a strong carcinogenic background in the liver. In addition, they have a high risk of intrahepatic metastasis (IM). Liver tumors withIM or MC are profoundly different in their development and clinical outcome. However, clinically or pathologically discriminating between IM and MC can be challenging. This study investigated whether IM or MC could be diagnosed at the molecular level. METHODS: We performed whole genome and RNA sequencing analyses of 49 tumors including two extra-hepatic metastases, and one nodule-in-nodule tumor from 23 HCC patients. RESULTS: Sequencing-based molecular diagnosis using somatic single nucleotide variation information showed higher sensitivity compared to previous techniques due to the inclusion of a larger number of mutation events. This proved useful in cases, which showed inconsistent clinical diagnoses. In addition, whole genome sequencing offered advantages in profiling of other genetic alterations, such as structural variations, copy number alterations, and variant allele frequencies, and helped to confirm the IM/MCdiagnosis. Divergent alterations between IM tumors with sorafenib treatment, long time-intervals, or tumor-in-tumor nodules indicated high intra-tumor heterogeneity, evolution, and clonal switching of liver cancers. CONCLUSIONS: It is important to analyze the differences between IM tumors, in addition to IM/MC diagnosis, before selecting a therapeutic strategy for multiple tumors in the liver. LAY SUMMARY: Whole genome sequencing of multiple liver tumors enabled the accuratediagnosis ofmulti-centric occurrence and intrahepatic metastasis using somatic single nucleotide variation information. In addition, genetic discrepancies between tumors help us to understand the physical changes during recurrence and cancer spread.

[Two Cases of HER2-Positive Gastric Cancer with Multiple Liver Metastases Leading to Conversion Therapy with Chemotherapy].

Case 1: A 69-year-old man underwent chemotherapy with capecitabine plus cisplatin plus trastuzumab to treat advanced gastric cancer that was diagnosed as cStage IV adenocarcinoma(T3N2M1[P0, CYX, H1]). After 12 courses, liver metastases were absent on computed tomography images. The patient underwent total gastrectomy with D2 lymphadenectomy. It has been 22 months since the patient had gastrectomy without recurrence of the cancer. Case 2: A 70-year-old man underwent chemotherapy with capecitabine plus cisplatin plus trastuzumab for treatment of advanced gastric cancer that was diagnosed as cStage IV adenocarcinoma(T4aN1M1[P0, CY0, H1]). After 12 courses, regrowth of multiple liver metastases led to a treatment with weekly paclitaxel plus trastuzumab as a second-line chemotherapy. After 9 courses of second-line chemotherapy, multiple liver metastases were absent in computed tomography images. The patient underwent total gastrectomy with D2 lymphadenectomy. A small white nodule on the surface of S2 and S3 of the liver led to the patient receiving a partial liver resection. The pathological finding of the resected liver specimen was a metastasis of an adenocarcinoma. During continuous chemotherapy with weekly paclitaxel plus trastuzumab after gastrectomy, multiple liver metastases were revealed. The patient died 19 months after gastrectomy.

[Efficacy of low-dose CDDP and CPT-11 for patients with intestinal type of gastric adenocarcinoma].

BACKGROUND: Preclinical studies have shown that irinotecan (CPT-11) and cisplatin (CDDP) can act synergistically. Several chemotherapy regimens combining CPT-11 and CDDP for advanced gastric cancer have been reported to demonstrate high response rates and high incidence of severe toxicity. PURPOSE: We conducted a combination chemotherapy regimen of low-dose CDDP and CPT-11 to prolong the time to progression with less toxicity. PATIENTS AND METHODS: Seven patients with histologically-confirmed intestinal type of gastric adenocarcinoma were enrolled in this study. All patients were male, and their age at diagnosis ranged from 52 to 76 with a mean age of 64.8. Six patients received combination chemotherapy with CPT-11 and CDDP after the gastrectomy (stage I b: 1, II : 3, III b: 1, IV: 1). Only chemotherapy was administered in one patient because of a far advanced primary lesion and metastatic tumors. Low-dose CDDP (20 mg/body) and CPT-11 (65 mg/m(2)) were administered intravenously once every two weeks. RESULTS: The overall response rate was 43% including 1 complete response and 2 partial responses. One patient had grade 3 myelosuppression. Other adverse reactions were mild. CONCLUSION: The combination of low-dose CDDP and CPT-11 has mild therapeutic toxicities and may achieve a prolonged median survival time in patients with intestinal- type gastric adenocarcinoma.

Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer.

Liver cancer, which is most often associated with virus infection, is prevalent worldwide, and its underlying etiology and genomic structure are heterogeneous. Here we provide a whole-genome landscape of somatic alterations in 300 liver cancers from Japanese individuals. Our comprehensive analysis identified point mutations, structural variations (STVs), and virus integrations, in noncoding and coding regions. We discovered mutational signatures related to liver carcinogenesis and recurrently mutated coding and noncoding regions, such as long intergenic noncoding RNA genes (NEAT1 and MALAT1), promoters, CTCF-binding sites, and regulatory regions. STV analysis found a significant association with replication timing and identified known (CDKN2A, CCND1, APC, and TERT) and new (ASH1L, NCOR1, and MACROD2) cancer-related genes that were recurrently affected by STVs, leading to altered expression. These results emphasize the value of whole-genome sequencing analysis in discovering cancer driver mutations and understanding comprehensive molecular profiles of liver cancer, especially with regard to STVs and noncoding mutations.

A combination of targeted enrichment methodologies for whole-exome sequencing reveals novel pathogenic mutations.

Whole-exome sequencing (WES) is a useful method to identify disease-causing mutations, however, often no candidate mutations are identified using commonly available targeted probe sets. In a recent analysis, we also could not find candidate mutations for 20.9% (9/43) of our pedigrees with congenital neurological disorder using pre-designed capture probes (SureSelect V4 or V5). One possible cause for this lack of candidates is that standard WES cannot sequence all protein-coding sequences (CDS) due to capture probe design and regions of low coverage, which account for approximately 10% of all CDS regions. In this study, we combined a selective circularization-based target enrichment method (HaloPlex) with a hybrid capture method (SureSelect V5; WES), and achieved a more complete coverage of CDS regions (~97% of all CDS). We applied this approach to 7 (SureSelect V5) out of 9 pedigrees with no candidates through standard WES analysis and identified novel pathogenic mutations in one pedigree. The application of this effective combination of targeted enrichment methodologies can be expected to aid in the identification of novel pathogenic mutations previously missed by standard WES analysis.

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.

Integrated analysis of whole genome and transcriptome sequencing reveals diverse transcriptomic aberrations driven by somatic genomic changes in liver cancers.

Recent studies applying high-throughput sequencing technologies have identified several recurrently mutated genes and pathways in multiple cancer genomes. However, transcriptional consequences from these genomic alterations in cancer genome remain unclear. In this study, we performed integrated and comparative analyses of whole genomes and transcriptomes of 22 hepatitis B virus (HBV)-related hepatocellular carcinomas (HCCs) and their matched controls. Comparison of whole genome sequence (WGS) and RNA-Seq revealed much evidence that various types of genomic mutations triggered diverse transcriptional changes. Not only splice-site mutations, but also silent mutations in coding regions, deep intronic mutations and structural changes caused splicing aberrations. HBV integrations generated diverse patterns of virus-human fusion transcripts depending on affected gene, such as TERT, CDK15, FN1 and MLL4. Structural variations could drive over-expression of genes such as WNT ligands, with/without creating gene fusions. Furthermore, by taking account of genomic mutations causing transcriptional aberrations, we could improve the sensitivity of deleterious mutation detection in known cancer driver genes (TP53, AXIN1, ARID2, RPS6KA3), and identified recurrent disruptions in putative cancer driver genes such as HNF4A, CPS1, TSC1 and THRAP3 in HCCs. These findings indicate genomic alterations in cancer genome have diverse transcriptomic effects, and integrated analysis of WGS and RNA-Seq can facilitate the interpretation of a large number of genomic alterations detected in cancer genome.

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.

Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in ∼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs.

Whole-genome sequencing and comprehensive variant analysis of a Japanese individual using massively parallel sequencing.

We report the analysis of a Japanese male using high-throughput sequencing to × 40 coverage. More than 99% of the sequence reads were mapped to the reference human genome. Using a Bayesian decision method, we identified 3,132,608 single nucleotide variations (SNVs). Comparison with six previously reported genomes revealed an excess of singleton nonsense and nonsynonymous SNVs, as well as singleton SNVs in conserved non-coding regions. We also identified 5,319 deletions smaller than 10 kb with high accuracy, in addition to copy number variations and rearrangements. De novo assembly of the unmapped sequence reads generated around 3 Mb of novel sequence, which showed high similarity to non-reference human genomes and the human herpesvirus 4 genome. Our analysis suggests that considerable variation remains undiscovered in the human genome and that whole-genome sequencing is an invaluable tool for obtaining a complete understanding of human genetic variation.