Publications by authors named "Yasushi Totoki"

65 Publications

Impaired tumor immune response in metastatic tumors is a selective pressure for neutral evolution in CRC cases.

PLoS Genet 2021 01 21;17(1):e1009113. Epub 2021 Jan 21.

Department of Surgery, Kyushu University Beppu Hospital, Beppu, Japan.

A Darwinian evolutionary shift occurs early in the neutral evolution of advanced colorectal carcinoma (CRC), and copy number aberrations (CNA) are essential in the transition from adenoma to carcinoma. In light of this primary evolution, we investigated the evolutionary principles of the genome that foster postoperative recurrence of CRC. CNA and neoantigens (NAG) were compared between early primary tumors with recurrence (CRCR) and early primary tumors without recurrence (precancerous and early; PCRC). We compared CNA, single nucleotide variance (SNV), RNA sequences, and T-cell receptor (TCR) repertoire between 9 primary and 10 metastatic sites from 10 CRCR cases. We found that NAG in primary sites were fewer in CRCR than in PCRC, while the arm level CNA were significantly higher in primary sites in CRCR than in PCRC. Further, a comparison of genomic aberrations of primary and metastatic conditions revealed no significant differences in CNA. The driver mutations in recurrence were the trunk of the evolutionary phylogenic tree from primary sites to recurrence sites. Notably, PD-1 and TIM3, T cell exhaustion-related molecules of the tumor immune response, were abundantly expressed in metastatic sites compared to primary sites along with the increased number of CD8 expressing cells. The postoperative recurrence-free survival period was only significantly associated with the NAG levels and TCR repertoire diversity in metastatic sites. Therefore, CNA with diminished NAG and diverse TCR repertoire in pre-metastatic sites may determine postoperative recurrence of CRC.
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http://dx.doi.org/10.1371/journal.pgen.1009113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864431PMC
January 2021

Ependymoma with C11orf95-MAML2 fusion: presenting with granular cell and ganglion cell features.

Brain Tumor Pathol 2021 Jan 22;38(1):64-70. Epub 2020 Nov 22.

Department of Neurosurgery, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8641, Japan.

C11orf95-RELA fusion or, less frequently, YAP1 fusion is recurrently detected in most cases of supratentorial ependymoma. Other fusions have rarely been reported in some cases of supratentorial ependymoma, and little is known about their pathological or clinical features. Here, we present a case of supratentorial ependymoma with unusual pathological findings and C11orf95-MAML2 fusion. A 23-year-old man was admitted to our hospital because of headache and vomiting. Magnetic resonance imaging revealed a cystic lesion in the right frontal lobe, and gross total resection of the tumor was performed. Pathologically, the tumor was mainly composed of typical ependymal lesions with perivascular pseudorosettes and contained some atypical lesions, with granular and ganglion cell features. The tumor was diagnosed as anaplastic ependymoma, which was classified as grade III on the World Health Organization scale, and found to be RELA fusion-positive in the DNA methylation analysis. However, the tumor was negative for C11orf95-RELA fusion, and RNA sequencing detected C11orf95-MAML2 fusion. The patient has not received adjuvant therapy and has remained alive without any evidence of disease for 30 months, suggesting that the prognosis might be better than that of typical C11orf95-RELA fusion-positive ependymoma.
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http://dx.doi.org/10.1007/s10014-020-00388-6DOI Listing
January 2021

Whole-exome Sequencing Reveals New Potential Susceptibility Genes for Japanese Familial Pancreatic Cancer.

Ann Surg 2020 Aug 18. Epub 2020 Aug 18.

Department of Cancer Genome Informatics, Graduate School of Medicine, Osaka University, Osaka, Japan.

Objective: The primary objective of this study was to identify novel genes that predispose people in the Japanese population to FPC.

Summary Of Background Data: Familial history of pancreatic cancer is an important risk factor but, to date, few genes predisposing individuals to increased risk of developing FPC have been identified.

Methods: We performed whole-exome sequencing of germline DNA from 81 Japanese FPC patients. We also investigated somatic gene alterations in 21 matched tumor tissues through whole-exome sequencing and copy number analysis.

Results: Our germline variants identified previously known FPC susceptibility genes such as ATM and BRCA2, and several novel tumor suppressor genes with potentially deleterious variants for FPC. Interestingly, somatic whole-exome analysis demonstrated that most tumor samples with suspicious loss of heterozygosity of candidate genes were KRAS wild-types, implying that these cases may not have required KRAS activation as a driver event for carcinogenesis.

Conclusions: Our findings indicate that FPC patients harbor potentially deleterious causative germline variants in tumor suppressor genes, which are known to acquire somatic mutations in pancreatic cancer, and that somatic loss of heterozygosity of some FPC susceptibility genes may contribute to the development of FPC in the absence of somatic KRAS-activating mutation. Genetic testing for a wider variety of FPC-predisposition genes could provide better screening approach for high-risk groups of pancreatic cancer.
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http://dx.doi.org/10.1097/SLA.0000000000004213DOI Listing
August 2020

Defined lifestyle and germline factors predispose Asian populations to gastric cancer.

Sci Adv 2020 May 6;6(19):eaav9778. Epub 2020 May 6.

Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan.

Germline and environmental effects on the development of gastric cancers (GC) and their ethnic differences have been poorly understood. Here, we performed genomic-scale trans-ethnic analysis of 531 GCs (319 Asian and 212 non-Asians). There was one distinct GC subclass with clear alcohol-associated mutation signature and strong Asian specificity, almost all of which were attributable to alcohol intake behavior, smoking habit, and Asian-specific defective allele. Alcohol-related GCs have low mutation burden and characteristic immunological profiles. In addition, we found frequent (7.4%) germline variants among Japanese GCs, most of which were attributed to a few recurrent single-nucleotide variants shared by Japanese and Koreans, suggesting the existence of common ancestral events among East Asians. Specifically, approximately one-fifth of diffuse-type GCs were attributable to the combination of alcohol intake and defective allele or to variants. These results revealed uncharacterized impacts of germline variants and lifestyles in the high incidence areas.
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http://dx.doi.org/10.1126/sciadv.aav9778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202881PMC
May 2020

Post-mortem Plasma Cell-Free DNA Sequencing: Proof-of-Concept Study for the "Liquid Autopsy".

Sci Rep 2020 02 7;10(1):2120. Epub 2020 Feb 7.

Department of Cellular and Organ Pathology, Graduate School of Medicine, Akita University, Akita, Japan.

Recent genomic studies on cancer tissues obtained during rapid autopsy have provided insights into the clonal evolution and heterogeneity of cancer. However, post-mortem blood has not been subjected to genetic analyses in relation to cancer. We first confirmed that substantial quantities of cell-free DNA were present in the post-mortem plasma of 12 autopsy cases. Then, we focused on a pilot case of prostate cancer with multiple metastases for genetic analyses. Whole-exome sequencing of post-mortem plasma-derived cell-free DNA and eight frozen metastatic cancer tissues collected during rapid autopsy was performed, and compared their mutational statuses. The post-mortem plasma cell-free DNA was successfully sequenced and 344 mutations were identified. Of these, 160 were detected in at least one of the metastases. Further, 99% of the mutations shared by all metastases were present in the plasma. Sanger sequencing of 30 additional formalin-fixed metastases enabled us to map the clones harboring mutations initially detected only in the plasma. In conclusion, post-mortem blood, which is usually disposed of during conventional autopsies, can provide valuable data if sequenced in detail, especially regarding cancer heterogeneity. Furthermore, post-mortem plasma cell-free DNA sequencing (liquid autopsy) can be a novel platform for cancer research and a tool for genomic pathology.
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http://dx.doi.org/10.1038/s41598-020-59193-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005783PMC
February 2020

DNA Adductome Analysis Identifies -Nitrosopiperidine Involved in the Etiology of Esophageal Cancer in Cixian, China.

Chem Res Toxicol 2019 08 9;32(8):1515-1527. Epub 2019 Jul 9.

Cancer Institute , The Fourth Hospital of Hebei Medical University/The Tumor Hospital of Hebei Province , Shijiazhuang 050011 , China.

Esophageal cancer is prevalent in Cixian, China, but the etiology of this disease remains largely unknown. Therefore, we explored this by conducting a DNA adductome analysis. Both tumorous and nontumorous tissues were collected from patients who underwent surgical procedures at Cixian Cancer Hospital and the Fourth Hospital of Hebei Medical University, which is in a low-incidence area. -(3,4,5,6-Tetrahydro-2-pyran-2-yl)deoxyguanosine (THP-dG) was the major adduct detected in samples from esophageal cancer patients in Cixian. The precursor of THP-dG, -nitrosopiperidine (NPIP), exhibited a strong mutagenic activity under metabolic activation in the Ames test and a significant dose-dependent increase in mutation frequency during an mutagenicity test with guanine phosphoribosyltransferase () delta rats. The NPIP-induced mutation was dominated by A:T to C:G transversions, followed by G:C to A:T and A:T to G:C transitions, in the liver and esophagus of animal samples. A similar mutational pattern was observed in the mutational signature of esophageal cancer patients that demonstrated weak correlation with THP-dG levels. These findings suggested that NPIP exposure is partly involved in the development of esophageal cancer in Cixian residents.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00017DOI Listing
August 2019

Transcriptome analysis of soybean (Glycine max) root genes differentially expressed in rhizobial, arbuscular mycorrhizal, and dual symbiosis.

J Plant Res 2019 Jul 5;132(4):541-568. Epub 2019 Jun 5.

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Soybean (Glycine max) roots establish associations with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. Both rhizobia and AM fungi have been shown to affect the activity of and colonization by the other, and their interactions can be detected within host plants. Here, we report the transcription profiles of genes differentially expressed in soybean roots in the presence of rhizobial, AM, or rhizobial-AM dual symbiosis, compared with those in control (uninoculated) roots. Following inoculation, soybean plants were grown in a glasshouse for 6 weeks; thereafter their root transcriptomes were analyzed using an oligo DNA microarray. Among the four treatments, the root nodule number and host plant growth were highest in plants with dual symbiosis. We observed that the expression of 187, 441, and 548 host genes was up-regulated and 119, 1,439, and 1,298 host genes were down-regulated during rhizobial, AM, and dual symbiosis, respectively. The expression of 34 host genes was up-regulated in each of the three symbioses. These 34 genes encoded several membrane transporters, type 1 metallothionein, and transcription factors in the MYB and bHLH families. We identified 56 host genes that were specifically up-regulated during dual symbiosis. These genes encoded several nodulin proteins, phenylpropanoid metabolism-related proteins, and carbonic anhydrase. The nodulin genes up-regulated by the AM fungal colonization probably led to the observed increases in root nodule number and host plant growth. Some other nodulin genes were down-regulated specifically during AM symbiosis. Based on the results above, we suggest that the contribution of AM fungal colonization is crucial to biological N-fixation and host growth in soybean with rhizobial-AM dual symbiosis.
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http://dx.doi.org/10.1007/s10265-019-01117-7DOI Listing
July 2019

Initial and crucial genetic events in intestinal-type gastric intramucosal neoplasia.

J Pathol 2019 04 16;247(4):494-504. Epub 2019 Jan 16.

Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan.

Gastric cancer (GC) is one of the most common and life-threatening malignancies. The course of disease and tumor aggressiveness vary among GCs, although how early fate is determined and by what factors remains elusive. To solve this question, we collected 43 gastric intramucosal neoplasias (GINs), comprising dysplasia/intraepithelial neoplasia (D/IEN; a premalignant lesion) and minute GC (miGC; ≤10 mm) of intestinal histotype and performed targeted deep DNA sequencing of 67 GC-related genes derived from large-scale data. Gastric D/IEN was classified into low or high grade (LG-D/IEN or HG-D/IEN). The most frequent mutations in D/IENs included APC (19/25; 76%), ARID2 (6/25; 24%) and MUC6 (5/25; 20%). All LG-D/IENs had APC mutation (12/12) and APC hotspot mutations affecting R1450 and E1554 were noted in both LG-D/IEN and HG-D/IEN. ARID2 mutation always co-occurred with APC mutation, whose tumor variant allele frequency (TVAF) was higher than that of ARID2 in D/IEN. APC and TP53 mutations were mutually exclusive in D/IEN (p = 0.031 [main cohort], p = 0.025 [expanding cohort]) and TP53-mutated D/IEN was exclusively HG-D/IEN (4/4). TP53 mutations were highly recurrent (11/14; 79%) in MLH1-positive miGCs and were detected even in two microscopic lesions measuring 1 and 3 mm, respectively. Furthermore, TVAF analyses suggested that TP53 mutation is the initial event in the TP53-mutated miGCs. In contrast, TP53 mutation was absent (0/4) in MLH1-negative small intramucosal carcinoma (8-24 mm). Advanced GC data suggested that early mutations (APC and TP53) may affect the potential of cancerous progression from D/IEN. This study revealed somatic mutational landscape and initial mutations of GINs, and we report for the first time that TP53 mutations precede other mutations in intestinal-type GC. Our results also indicate that molecular subtyping based on APC/TP53 mutations would be a high-priority approach for determining and predicting the malignant potential of GIN, including D/IEN. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5208DOI Listing
April 2019

Integrated genetic and epigenetic analysis of myxofibrosarcoma.

Nat Commun 2018 07 17;9(1):2765. Epub 2018 Jul 17.

Division of Cancer Genomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.

Myxofibrosarcoma (MFS) is a common adult soft tissue sarcoma characterized by an infiltrative growth pattern and a high local recurrence rate. Here we report the genetic and epigenetic landscape of MFS based on the results of whole-exome sequencing (N = 41), RNA sequencing (N = 29), and methylation analysis (N = 41), using 41 MFSs as a discovery set, and subsequent targeted sequencing of 140 genes in the entire cohort of 99 MFSs and 17 MFSs' data from TCGA. Fourteen driver genes are identified, including potentially actionable therapeutic targets seen in 37% of cases. There are frequent alterations in p53 signaling (51%) and cell cycle checkpoint genes (43%). Other conceivably actionable driver genes including ATRX, JAK1, NF1, NTRK1, and novel oncogenic BRAF fusion gene are identified. Methylation patterns cluster into three subtypes associated with unique combinations of driver mutations, clinical outcomes, and immune cell compositions. Our results provide a valuable genomic resource to enable the design of precision medicine for MFS.
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http://dx.doi.org/10.1038/s41467-018-03891-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050269PMC
July 2018

Epigenome mapping of human normal purified hepatocytes: personal epigenome variation and genome-epigenome correlation.

Epigenomics 2018 07 4;10(7):955-979. Epub 2018 Jul 4.

Department of Pathology, Keio University School of Medicine, Tokyo 160-8582, Japan.

Aim: The aim of this study was to reveal the epigenome landscape of human normal hepatocytes.

Materials & Methods: Cells purified from partial hepatectomy specimens of Japanese patients were subjected to whole-genome bisulfite sequencing using postbisulfite adaptor tagging, chromatin immunoprecipitation sequencing, RNA sequencing and whole-genome sequencing.

Results: CHG and CHH methylations were inversely associated with gene expression. Histone modification profiles of personal differentially methylated regions (pDMRs) differed considerably among samples. pDMRs were observed around the transcription start sites of genes whose expression is reportedly regulated by CpG methylation. pDMRs were frequently observed in the vicinity of single-nucleotide variations and insertions/deletions.

Conclusion: Genetic variations may induce epigenetic variations, generating individual differences in the phenotypes of normal hepatocytes through variations in expression.
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http://dx.doi.org/10.2217/epi-2017-0111DOI Listing
July 2018

A computational tool to detect DNA alterations tailored to formalin-fixed paraffin-embedded samples in cancer clinical sequencing.

Genome Med 2018 06 7;10(1):44. Epub 2018 Jun 7.

Division of Cancer Genomics, National Cancer Center Research Institute, Chuo-ku, Tokyo, 104-0045, Japan.

Advanced cancer genomics technologies are now being employed in clinical sequencing, where next-generation sequencers are used to simultaneously identify multiple types of DNA alterations for prescription of molecularly targeted drugs. However, no computational tool is available to accurately detect DNA alterations in formalin-fixed paraffin-embedded (FFPE) samples commonly used in hospitals. Here, we developed a computational tool tailored to the detection of single nucleotide variations, indels, fusions, and copy number alterations in FFPE samples. Elaborated multilayer noise filters reduced the inherent noise while maintaining high sensitivity, as evaluated in tumor-unmatched normal samples using orthogonal technologies. This tool, cisCall, should facilitate clinical sequencing in everyday diagnostics. It is available at https://www.ciscall.org .
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http://dx.doi.org/10.1186/s13073-018-0547-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992758PMC
June 2018

Epigenetic landscape influences the liver cancer genome architecture.

Nat Commun 2018 04 24;9(1):1643. Epub 2018 Apr 24.

Division of Cancer Genomics, National Cancer Center Research Institute, Chuo-ku, Tokyo, 104-0045, Japan.

The accumulations of different types of genetic alterations such as nucleotide substitutions, structural rearrangements and viral genome integrations and epigenetic alterations contribute to carcinogenesis. Here, we report correlation between the occurrence of epigenetic features and genetic aberrations by whole-genome bisulfite, whole-genome shotgun, long-read, and virus capture sequencing of 373 liver cancers. Somatic substitutions and rearrangement breakpoints are enriched in tumor-specific hypo-methylated regions with inactive chromatin marks and actively transcribed highly methylated regions in the cancer genome. Individual mutation signatures depend on chromatin status, especially, signatures with a higher transcriptional strand bias occur within active chromatic areas. Hepatitis B virus (HBV) integration sites are frequently detected within inactive chromatin regions in cancer cells, as a consequence of negative selection for integrations in active chromatin regions. Ultra-high structural instability and preserved unmethylation of integrated HBV genomes are observed. We conclude that both precancerous and somatic epigenetic features contribute to the cancer genome architecture.
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http://dx.doi.org/10.1038/s41467-018-03999-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915380PMC
April 2018

Molecular genomic landscapes of hepatobiliary cancer.

Cancer Sci 2018 May 28;109(5):1282-1291. Epub 2018 Apr 28.

Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan.

Hepatocellular carcinoma (HCC) and biliary tract cancer are more frequent in East Asia including Japan than in Europe or North America. A compilation of 1340 multi-ethnic HCC genomes, the largest cohort ever reported, identified a comprehensive landscape of HCC driver genes, comprised of three core drivers (TP53, TERT, and WNT signaling) and combinations of infrequent alterations in various cancer pathways. In contrast, five core driver genes (TP53, ARID1A, KRAS, SMAD4, and BAP1) with characteristic molecular alterations including fusion transcripts involving fibroblast growth factor receptor 2 and the protein kinase A pathway, and IDH1/2 mutation constituted the biliary tract cancer genomes. Consistent with their heterogeneous epidemiological backgrounds, mutational signatures and combinations of non-core driver genes within these cancer genomes were found to be complex. Integrative analyses of multi-omics data identified molecular classifications of these tumors that are associated with clinical outcome and enrichments of potential therapeutic targets, including immune checkpoint molecules. Translating comprehensive molecular-genomic analysis together with further basic research and international collaborations are highly anticipated for developing precise and better treatments, diagnosis, and prevention of these tumor types.
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http://dx.doi.org/10.1111/cas.13582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980207PMC
May 2018

Whole exome sequencing to identify genetic markers for trastuzumab-induced cardiotoxicity.

Cancer Sci 2018 Feb 24;109(2):446-452. Epub 2018 Jan 24.

Division of Genetics, National Cancer Center Research Institute, Tokyo, Japan.

Although trastuzumab-induced cardiotoxicity is an important determinant to limit the use of this drug, the molecular mechanism of risk for this toxicity is not well understood. To identify genetic variants determining the risk of trastuzumab-induced cardiotoxicity, we carried out whole exome sequencing of germline DNA samples from 9 patients with trastuzumab-induced cardiotoxicity, and conducted a case-control association study of 2258 genetic variants between 9 cases (with trastuzumab-induced cardiotoxicity) and general Japanese population controls registered in the Human Genetic Variation Database (HGVD). The top variant which showed the lowest P-value in the screening study was rs139503277 in PHD Finger Protein 3 (P = .00012, odds ratio [OR] = 51.23). To further validate the result of screening study, we carried out a replication study of 10 variants showing P < .001 in the screening study using 234 independent patients treated with trastuzumab, including 10 cases and 224 controls (without trastuzumab-induced cardiotoxicity). In the replication study, we observed that three variants had an effect in the same direction as in the screening study (rs78272919 in exon 2 of Keratin 15, rs5762940 in exon 2 of zinc and ring finger 3, and rs139944387 in exon 44 of Eyes shut homologs [EYS]). A combined result of the screening and the replication studies suggested an association of a locus on chromosome 6q12 with trastuzumab-induced cardiotoxicity (rs139944387 in EYS, combined P = .00056, OR = 13.73). This finding provides new insights into personalized trastuzumab therapy for patients with human epidermal growth factor receptor 2 (HER2)-positive cancer.
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http://dx.doi.org/10.1111/cas.13471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797809PMC
February 2018

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma.

Cancer Discov 2017 10 30;7(10):1116-1135. Epub 2017 Jun 30.

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in amplifications and mutations; conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and / expression, or epigenetic mutations () and /-related gene rearrangements. Whole-genome analysis highlighted 3' untranslated region deletion as a mechanism of upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores-mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics, and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer. Integrated whole-genome and epigenomic analysis of CCA on an international scale identifies new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628134PMC
October 2017

Integrative analysis of genomic alterations in triple-negative breast cancer in association with homologous recombination deficiency.

PLoS Genet 2017 Jun 21;13(6):e1006853. Epub 2017 Jun 21.

Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Triple-negative breast cancer (TNBC) cells do not express estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2. Currently, apart from poly ADP-ribose polymerase inhibitors, there are few effective therapeutic options for this type of cancer. Here, we present comprehensive characterization of the genetic alterations in TNBC performed by high coverage whole genome sequencing together with transcriptome and whole exome sequencing. Silencing of the BRCA1 gene impaired the homologous recombination pathway in a subset of TNBCs, which exhibited similar phenotypes to tumors with BRCA1 mutations; they harbored many structural variations (SVs) with relative enrichment for tandem duplication. Clonal analysis suggested that TP53 mutations and methylation of CpG dinucleotides in the BRCA1 promoter were early events of carcinogenesis. SVs were associated with driver oncogenic events such as amplification of MYC, NOTCH2, or NOTCH3 and affected tumor suppressor genes including RB1, PTEN, and KMT2C. Furthermore, we identified putative TGFA enhancer regions. Recurrent SVs that affected the TGFA enhancer region led to enhanced expression of the TGFA oncogene that encodes one of the high affinity ligands for epidermal growth factor receptor. We also identified a variety of oncogenes that could transform 3T3 mouse fibroblasts, suggesting that individual TNBC tumors may undergo a unique driver event that can be targetable. Thus, we revealed several features of TNBC with clinically important implications.
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http://dx.doi.org/10.1371/journal.pgen.1006853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500377PMC
June 2017

Highly recurrent H3F3A mutations with additional epigenetic regulator alterations in giant cell tumor of bone.

Genes Chromosomes Cancer 2017 10 27;56(10):711-718. Epub 2017 Jul 27.

Division of Cancer Genomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.

Recurrent H3F3A and IDH2 mutations have been reported in giant cell tumor of bone (GCTB). However, the reported incidences have varied, and other molecular genetic alterations have not been identified due to the small number of cases analyzed with comprehensive methods. Moreover, the relative sensitivities of Sanger sequencing and next-generation sequencing (NGS) for the detection of H3F3A mutations in DNA extracted from archival formalin-fixed paraffin-embedded (FFPE) samples for clinical diagnosis have not been assessed. To address these issues, we conducted whole-exome sequencing of 7 GCTBs and integrated the previously published genomic sequencing data of 6 GCTBs. We subsequently performed targeted sequencing of an additional 39 GCTBs, including 2 atypical cases and an extremely rare case of primary malignant transformation of GCTB. We also evaluated the sensitivity of Sanger sequencing for detecting H3F3A mutations in FFPE samples that are usually used for clinical diagnosis. H3F3A glycine hotspot mutations were the most frequently detected mutations (96%) in the 52 GCTBs by NGS. Of the 50 hotspot mutations, p.G34W was observed in 48 cases and p.G34L/G34R was detected in one. One of two atypical GCTB cases with wild-type H3F3A had a H3F3B mutation (p.G34V). Other mutated genes were not recurrent. Sanger sequencing did not detect H3F3A mutations in 10 of 15 H3F3A NGS mutation-positive FFPE samples. In conclusion, we confirmed that H3F3A is the most frequently mutated GCTB driver gene, and that H3F3A mutations are not present in atypical GCTBs. Sanger sequencing was much less sensitive than targeted NGS for detecting H3F3A mutations in FFPE samples.
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http://dx.doi.org/10.1002/gcc.22469DOI Listing
October 2017

Genome-wide methylation profiles in primary intracranial germ cell tumors indicate a primordial germ cell origin for germinomas.

Acta Neuropathol 2017 03 11;133(3):445-462. Epub 2017 Jan 11.

Division of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka, 540-0006, Japan.

Intracranial germ cell tumors (iGCTs) are the second most common brain tumors among children under 14 in Japan. The World Health Organization classification recognizes several subtypes of iGCTs, which are conventionally subclassified into pure germinoma or non-germinomatous GCTs. Recent exhaustive genomic studies showed that mutations of the genes involved in the MAPK and/or PI3K pathways are common in iGCTs; however, the mechanisms of how different subtypes develop, often as a mixed-GCT, are unknown. To elucidate the pathogenesis of iGCTs, we investigated 61 GCTs of various subtypes by genome-wide DNA methylation profiling. We showed that pure germinomas are characterized by global low DNA methylation, a unique epigenetic feature making them distinct from all other iGCTs subtypes. The patterns of methylation strongly resemble that of primordial germ cells (PGC) at the migration phase, possibly indicating the cell of origin for these tumors. Unlike PGC, however, hypomethylation extends to long interspersed nuclear element retrotransposons. Histologically and epigenetically distinct microdissected components of mixed-GCTs shared identical somatic mutations in the MAPK or PI3K pathways, indicating that they developed from a common ancestral cell.
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http://dx.doi.org/10.1007/s00401-017-1673-2DOI Listing
March 2017

Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment.

Nat Commun 2016 12 6;7:13616. Epub 2016 Dec 6.

Department of Epigenomics, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.

Targeting self-renewal is an important goal in cancer therapy and recent studies have focused on Notch signalling in the maintenance of stemness of glioma stem cells (GSCs). Understanding cancer-specific Notch regulation would improve specificity of targeting this pathway. In this study, we find that Notch1 activation in GSCs specifically induces expression of the lncRNA, TUG1. TUG1 coordinately promotes self-renewal by sponging miR-145 in the cytoplasm and recruiting polycomb to repress differentiation genes by locus-specific methylation of histone H3K27 via YY1-binding activity in the nucleus. Furthermore, intravenous treatment with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system induces GSC differentiation and efficiently represses GSC growth in vivo. Our results highlight the importance of the Notch-lncRNA axis in regulating self-renewal of glioma cells and provide a strong rationale for targeting TUG1 as a specific and potent therapeutic approach to eliminate the GSC population.
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http://dx.doi.org/10.1038/ncomms13616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150648PMC
December 2016

Mutational signatures associated with tobacco smoking in human cancer.

Science 2016 11;354(6312):618-622

Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, Cambridgeshire, UK.

Tobacco smoking increases the risk of at least 17 classes of human cancer. We analyzed somatic mutations and DNA methylation in 5243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clocklike mutational process. Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.
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http://dx.doi.org/10.1126/science.aag0299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141049PMC
November 2016

Clinical Utility of Circulating Tumor DNA for Molecular Assessment and Precision Medicine in Pancreatic Cancer.

Adv Exp Med Biol 2016 ;924:13-17

Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect molecular characteristics of tumors, supporting the concept of "liquid biopsy".We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR in 259 patients with PDAC, retrospectively. Furthermore, we constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA in 48 patients who had ≥1 % mutant allele frequencies of KRAS in plasma cfDNA.Droplet digital PCR detected KRAS mutations in plasma cfDNA in 63 of 107 (58.9 %) patients with inoperable tumors. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2 %) examined by cfDNA sequencing.Our two-step approach with plasma cfDNA, combining droplet digital PCR and targeted deep sequencing, is a feasible clinical approach. Assessment of mutations in plasma cfDNA may provide a new diagnostic tool, assisting decisions for optimal therapeutic strategies for PDAC patients.
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http://dx.doi.org/10.1007/978-3-319-42044-8_3DOI Listing
September 2017

Comprehensive mutation profiling of mucinous gastric carcinoma.

J Pathol 2016 10 19;240(2):137-48. Epub 2016 Sep 19.

Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan.

Mucinous gastric carcinoma (MGC) is a unique subtype of gastric cancer with a poor survival outcome. Comprehensive molecular profiles and putative therapeutic targets of MGC remain undetermined. We subjected 16 tumour-normal tissue pairs to whole-exome sequencing (WES) and an expanded set of 52 tumour-normal tissue pairs to subsequent targeted sequencing. The latter focused on 114 genes identified by WES. Twenty-two histologically differentiated MGCs (D-MGCs) and 46 undifferentiated MGCs (U-MGCs) were analysed. Chromatin modifier genes, including ARID1A (21%), MLL2 (19%), MLL3 (15%), and KDM6A (7%), were frequently mutated (47%) in MGC. We also identified mutations in potential therapeutic target genes, including MTOR (9%), BRCA2 (9%), BRCA1 (7%), and ERBB3 (6%). RHOA mutation was detected only in 4% of U-MGCs and in no D-MGCs. MYH9 was recurrently (13%) mutated in MGC, with all these being of the U-MGC subtype (p = 0.023). Three U-MGCs harboured MYH9 nonsense mutations. MYH9 knockdown enhanced cell migration and induced intracytoplasmic mucin and cellular elongation. BCOR mutation was associated with improved survival. In U-MGCs, the MLH1 expression status and combined mutation status (TP53/BCL11B or TP53/MLL2) were prognostic factors. A comparative analysis of driver genes revealed that the mutation profile of D-MGC was similar to that of intestinal-type gastric cancer, whereas U-MGC was a distinct entity, harbouring a different mutational profile to intestinal- and diffuse-type gastric cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.4761DOI Listing
October 2016

Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers.

Nature 2016 06 23;534(7607):402-6. Epub 2016 May 23.

Department of Immunology, Institute for Frontier Medical Science, Kyoto University, Kyoto 606-8507, Japan.

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3' region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3'-untranslated region (UTR). Disruption of the Pd-l1 3'-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3'-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.
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http://dx.doi.org/10.1038/nature18294DOI Listing
June 2016

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

Nat Genet 2016 05 11;48(5):500-9. Epub 2016 Apr 11.

Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

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.
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http://dx.doi.org/10.1038/ng.3547DOI Listing
May 2016

Recurrent neomorphic mutations of MTOR in central nervous system and testicular germ cell tumors may be targeted for therapy.

Acta Neuropathol 2016 06 8;131(6):889-901. Epub 2016 Mar 8.

Department of Neurosurgery, Tokyo Medical and Dental University Graduate School of Medical and Dental Sciences, Tokyo, Japan.

Germ cell tumors constitute a heterogeneous group that displays a broad spectrum of morphology. They often arise in testes; however, extragonadal occurrence, in particular brain, is not uncommon, and whether they share a common pathogenesis is unknown. We performed whole exome sequencing in 41 pairs of central nervous system germ cell tumors (CNS GCTs) of various histology and their matched normal tissues. We then performed targeted sequencing of 41 selected genes in a total of 124 CNS GCTs, 65 testicular germ cell tumors (tGCTs) and 8 metastatic GCTs to the CNS. The results showed that mutually exclusive mutations of genes involved in the MAPK pathway were most common (48.4 %), typically in KIT (27.4 %), followed by those in the PI3K pathway (12.9 %), particularly in MTOR (6.5 %), among the 124 CNS GCTs. Pure germinomas and non-germinomatous germ cell tumors (NGGCTs), as well as CNS and testicular GCTs, showed similar mutational profiles, suggesting that GCTs share a common molecular pathogenesis. Mutated MTOR identified in CNS GCTs upregulated phosphorylation of the AKT pathway proteins including AKT and 4EBP1 in nutrient-deprived conditions and enhanced soft-agar colony formation; both events were suppressed in a dose-dependent manner by addition of the MTOR inhibitor pp242. Our findings indicate that the dominant genetic drivers of GCTs regardless of the site of origin are activation of the MAPK and/or PI3K pathways by somatic point mutations. Mutated MTOR represents a potential target for novel targeted therapies for refractory GCTs.
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http://dx.doi.org/10.1007/s00401-016-1557-xDOI Listing
June 2016

Genomic Sequencing Identifies ELF3 as a Driver of Ampullary Carcinoma.

Cancer Cell 2016 Feb 21;29(2):229-40. Epub 2016 Jan 21.

Department of Surgery, Institute of Gastroenterology, Tokyo Women's Medical University, Tokyo 1628666, Japan.

Ampullary carcinomas are highly malignant neoplasms that can have either intestinal or pancreatobiliary differentiation. To characterize somatic alterations in ampullary carcinomas, we performed whole-exome sequencing and DNA copy-number analysis on 60 ampullary carcinomas resected from clinically well-characterized Japanese and American patients. We next selected 92 genes and performed targeted sequencing to validate significantly mutated genes in an additional 112 cancers. The prevalence of driver gene mutations in carcinomas with the intestinal phenotype is different from those with the pancreatobiliary phenotype. We identified a characteristic significantly mutated driver gene (ELF3) as well as previously known driver genes (TP53, KRAS, APC, and others). Functional studies demonstrated that ELF3 silencing in normal human epithelial cells enhances their motility and invasion.
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http://dx.doi.org/10.1016/j.ccell.2015.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503303PMC
February 2016

Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer.

Sci Rep 2015 Dec 16;5:18425. Epub 2015 Dec 16.

Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo 1040045, Japan.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients.
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http://dx.doi.org/10.1038/srep18425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4680882PMC
December 2015

Variegated RHOA mutations in adult T-cell leukemia/lymphoma.

Blood 2016 Feb 16;127(5):596-604. Epub 2015 Nov 16.

Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan;

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.
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http://dx.doi.org/10.1182/blood-2015-06-644948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291304PMC
February 2016