Aiko Sato

Genomic landscape of liposarcoma

Liposarcoma (LPS) is the most common type of soft tissue sarcoma accounting for 20% of all adult sarcomas. Due to absence of clinically effective treatment options in inoperable situations and resistance to chemotherapeutics, a critical need exists to identify novel therapeutic targets. We analyzed LPS genomic landscape using SNP arrays, whole exome sequencing and targeted exome sequencing to uncover the genomic information for development of specific anti-cancer targets. SNP array analysis indicated known amplified genes (MDM2, CDK4, HMGA2) and important novel genes (UAP1, MIR557, LAMA4, CPM, IGF2, ERBB3, IGF1R). Carboxypeptidase M (CPM), recurrently amplified gene in well-differentiated/de-differentiated LPS was noted as a putative oncogene involved in the EGFR pathway. Notable deletions were found at chromosome 1p (RUNX3, ARID1A), chromosome 11q (ATM, CHEK1) and chromosome 13q14.2 (MIR15A, MIR16-1). Significantly and recurrently mutated genes (false discovery rate < 0.05) included PLEC (27%), MXRA5 (21%), FAT3 (24%), NF1 (20%), MDC1 (10%), TP53 (7%) and CHEK2 (6%). Further, in vitro and in vivo functional studies provided evidence for the tumor suppressor role for Neurofibromin 1 (NF1) gene in different subtypes of LPS. Pathway analysis of recurrent mutations demonstrated signaling through MAPK, JAK-STAT, Wnt, ErbB, axon guidance, apoptosis, DNA damage repair and cell cycle pathways were involved in liposarcomagenesis. Interestingly, we also found mutational and copy number heterogeneity within a primary LPS tumor signifying the importance of multi-region sequencing for cancer-genome guided therapy. In summary, these findings provide insight into the genomic complexity of LPS and highlight potential druggable pathways for targeted therapeutic approach.

Diminished nuclear RNA decay upon Salmonella infection upregulates antibacterial noncoding RNAs

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity‐related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.

Abstract 2229: Whole exome sequencing reveals the landscape of gene mutations and evolution in low-grade glioma

Low grade gliomas (LGGs) account for approximately half of all gliomas. Although LGGs typically show slower tumor progression and generally better clinical outcomes than high-grade gliomas, their clinical course is invariably indolent and most patients ultimately succumb to death. In contrast to high-grade tumors, our knowledge about the genetic lesions and clonal evolution in LGG is still incomplete. So, to obtain a complete registry of gene mutations involved in LGG pathogenesis and their role in clonal evolution, we performed whole exome sequencing (WES) of paired tumor/normal DNA from 54 cases with LGG. Clonal evolution in LGG was investigated using paired primary/relapsed tumor specimens from 9 cases as well as multiple tumor specimens (median 5) from 4cases. Major mutational targets detected in WES included not only previously known mutational genes, including IDH1/2, TP53, ATRX, CIC, FUBP1 and NOTCH1, as well as multiple components of the PI3K pathway and the SWI/SNF complex. Multi-sampling analysis revealed regional and special heterogeneity of LGG. According to the observed variant allele frequencies (VAFs), mutations of IDH1/2 and 1p19q co-deletion were thought to exist in the major clone, representing truncal mutations in most cases., In contrast, mutations in TP53, ATRX, CIC and FUBP1 were more often identified in one or more phylogenic branches in different subclones and involved in parallel evolution, where different mutations of the same genes were found at different time points in different locations. We further performed deep-sequencing of common mutational targets identified by WES and SNP array analysis among a large cohort of 327 LGG cases. As previously reported, mutations in IDH1/2 were found in 78.6%. 1p19q co-deletion (43.1%), and TP53 mutations (34.6%) with or without ATRX mutations were mutually exclusive with common IDH mutations. VAFs of coexisting IDH1/2 and 1p19q co-deletion were approximately the same, whereas mutations in other genes tended to show lower VAFs than those of IDH1/2. Combined, our findings revealed two major, mutually exclusive patterns in clonal evolution in LGG; in some cases IDH1/2 mutations and 1p19q co-deletions were trunchal events, followed by CIC, FUBP1, and other mutations in subsequent phylogenic branches. In other cases, predated by the founder IDH1/2 mutations, mutations in TP53 and ATRX seemed to predominate tumor populations after. Citation Format: Hiromichi Suzuki, Atsushi Natsume, Yusuke Sato, Yuichi Shiraishi, Yusuke Shiozawa, Kenichi Yoshida, Yasunobu Nagata, Aiko Sato, Kazuya Motomura, Masazumi Fujii, Masashi Sanada, Satoru Miyano, Toshihiko Wakabayashi, Seishi Ogawa. Whole exome sequencing reveals the landscape of gene mutations and evolution in low-grade glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2229. doi:10.1158/1538-7445.AM2014-2229

Abstract 3184: Integrative analysis of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer. The most frequent genetic event in the evolution of ccRCC is inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Recent studies have revealed frequent mutations of PBRM1 as well as other epigenetic regulators including BAP1, SETD2 and KDM5C, but their impact on therapeutics remains unclear. In this study, to obtain a better understanding of molecular pathogenesis of ccRCC, we performed an integrated genetic study of ccRCC including whole exome sequencing, copy number analysis as well as transcriptome and methylome analysis. A total of 106 paired specimens were analyzed by massively parallel sequencing of whole exomes (Agilent SureSelect, Illumina HiSeq2000) and SNP array-based copy number analysis (Affymetrix 250K array) as well as gene expression (Agilent Human Gene Expression 4x44k v2) and methylation (Illumina Infinium 450K) profiling and RNA sequencing (Illumina HiSeq2000). On average, 48.8 somatic mutations per sample were identified in whole exome analysis, in which VHL mutations were most frequent. PBRM1, BAP1 and SETD2 were also recurrently mutated and were further analyzed in 240 cases together with an additional 80 genes involved in chromatin regulation. PBRM1 mutations were found in 42% of the cases, while BAP1 and SETD2 were mutated in 12% and 10%, respectively. BAP1 mutations correlated with poor prognosis and SETD2 mutation was associated with the risk for metastatic diseases. Pathway analysis revealed frequent mutations of genes involved in mRNA processing. Among them, mutations of genes involved in 3’ splice site recognition, which were frequently mutated in MDS, were rare. Most of them were involved in release of intron, 3’-end processing or export to cytoplasm. In expression analysis, tumors were clustered into two clusters known as ccA and ccB, in which the ccA type was characterized by overexpressed genes involved in angiogenesis, whereas expression of genes in cell cycle regulators were a prominent feature in the ccB type tumors. In methylome analysis, 15 samples were clustered into hypermethylated subtype where all cases were included in the ccB type and were associated with poor prognosis. Homeobox genes were differently methylated in hypermethylated subtype which indicate deregulation of polycomb mediated gene silencing induce high-grade ccRCC. RUNX1 and SRPX2 were differently methylated between ccA type and ccB type, which may affect the differences of expression profile between two subtypes. In RNA sequencing, known fusion gene involving TFE3 and NONO was detected in single case. In total, 34 fusion genes were detected in 23 samples, although no recurrent fusion genes have been identified. Our results indicate that genomic analyses are useful for classification, prognostic prediction and development of treatment strategy in ccRCC. Citation Format: Yusuke Sato, Shigekatsu Maekawa, Yusuke Okuno, Yuichi Shiraishi, Aiko Sato, Genta Nagae, Teppei Shimamura, Yasunobu Nagata, Kenichi Yoshida, Masashi Sanada, Haruki Kume, Hiroyuki Aburatani, Satoru Miyano, Yukio Homma, Seishi Ogawa. Integrative analysis of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3184. doi:10.1158/1538-7445.AM2013-3184

Abstract 2019: Whole exome analysis reveals spectrum of gene mutations in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell neoplasm, which is etiologically associated with human T-lymphotropic virus type I (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize T-cells, there is a long latency period of ∼50 years prior to the onset of ATL, suggesting that HTLV-1 infection alone is not sufficient for the development of ATL, but additional acquired genetic hits that occur in immortalized T-cells during the later life are essential for its pathogenesis. However, little has been known about the gene mutations commonly involved in ATL pathogenesis. So, in order to understand the genetic basis of ATL, we performed whole exome sequencing of paired-normal DNA from 24 patients with different ATL subtypes, including acute (N=13), chronic (N=3), lymphoma (N=8) types. With a mean coverage of 96, 83% of the target sequences were analyzed at more than 20 depth on average. The mutation rate of 108 (44-207) per sample was significantly higher than that in acute myeloid leukemia (7.3-13), myelodysplastic syndromes (9.2) and chronic lymphocytic leukemia (11.5). What immediately drew our attention were the recurrent mutations involving TET2 mutations. They are found in a wide variety of myeloid malignancies in high frequency and implicated in their pathogenesis. The TET families of proteins are thought to be involved in the epigenetic regulation of gene expression through catalyzing conversion of 5’-methyl cytosine to 5’-hydroxymethyl cytosine, which are supposed to be further converted to unmethylated cytosine. One of the recent interests in TET2 mutations was the recent report of frequent TET2 mutations in peripheral T cell neoplasms, including angioimmunoblastic T-cell lymphomas(AITL), peripheral T-cell lymphomas not otherwise specified(PTCL-NOS), as well as other B cell neoplasms. In addition, 145 samples of ATL were screened for mutations in TET2 and other epigenetic regulators commonly mutated in myeloid malignancies, including DNMT3A, IDH1/2 using target deep sequencing. In total, 17 TET2 mutations were identified in 14 (9.6%) out of the 145 ATL samples. Less frequent mutations of IDH2 and DNMT3A were also identified. Different subtypes of ATL were almost evenly affected with 6 out of 47 acute, 3 out of 36 chronic and 5 out of 46 lymphoma types having TET2 mutations. In most evaluable cases, the TET2 mutations harbored a major tumor population, while the mutations in two cases only involved a minor population and was suggested to represent a relatively late event during clonal evolution. Combined with the recent studies reporting similar AITL and PTCL-NOS, our finding suggested a common role of deregulated epigenetic machinery in the development of mature T-cell neoplasms including ATL. Citation Format: Yasunobu Nagata, Yusuke Okuno, Aiko Sato, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Kenichi Yoshida, Masashi Sanada, Ken Ishiyama, Shuichi Miyawaki, Akira Kitanaka, Kazuya Shimoda, Satoru Miyano, Seishi Ogawa. Whole exome analysis reveals spectrum of gene mutations in adult T-cell leukemia/lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2019. doi:10.1158/1538-7445.AM2013-2019

Abstract 2092: Integrated genetic analysis of clear cell renal cell carcionoma

Renal cell carcinoma (RCC) is the most common form of adult kidney cancer and accounts for 2-3% of all adult malignancies, in which clear cell carcinoma accounts for more than 80% of the cases. As for the pathogenesis of clear cell RCC, inactivation of the VHL gene has been reported in 80% of clear cell RCC and more recently, frequent mutations of epigenetic regulators, including PBRM1, SETD2, KDM5C and UTX, have been demonstrated through high-throughput mutation studies, including PBRM1 has been demonstrated in ∼40% of the cases. Nevertheless, probably, our knowledge of the full spectrum of gene mutations in RCC is still incomplete. In this study, to obtain a better understanding of molecular pathogenesis of clear cell RCC, we performed an integrated genetic study of clear cell RCC, where a total of 93 paired specimens were analyzed by massively parallel sequencing of SureSelect (Agilent)-enriched whole exomes, SNP array-based copy number analysis (Affymetrix), as well as gene expression profiling (Agilent). In whole exome sequencing, 42 somatic mutations per sample were identified on average, which involved not only previously reported genes, but also a number of novel gene targets. Among these, mutations of genes involved in chromatin regulation or histone modification were preferentially found in advanced cases. To understand whole picture of gene mutations of epigenetic mechanism in clear cell RCC, mutation analysis of 85 genes involved in chromatin regulation or histone modification were performed in 180 cases using multiplexed barcode sequencing. A total of 201 somatic mutations were validated and 74% cases had at least one somatic mutation. PBRM1 mutations were found in 43% cases and SETD2 were mutated in 10% of cases. When comparing clinical picture with mutation status, SETD2 mutation was associated with the risk of metastasis, while PBRM1 mutations had no impact on prognosis. Our results showed that in clear cell RCC, multiple component of complexes involved in epigenetic regulation undergo gene mutations, confirming that deregulated epigenetic apparatus play important roles in pathogenesis of clear cell RCC. In this meeting, we will present the result of our integrated genetic analysis of RCC and discuss the genetic basis of RCC in terms of copy number alterations, gene mutations, as well as gene expression profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2092. doi:1538-7445.AM2012-2092

Abstract 5122: Integral view of copy number alteration and commonly targeted genes in MDS found a new aspect of correlation and interrelationship with mutated components of the RNA splicing machinery

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic myeloid neoplasms showing a predisposition to acute myeloid leukemia (AML). The recent discovery of novel pathway mutations of the RNA splicing machinery provided a new insight into the pathogenesis of MDS. These splicing pathway mutations are highly prevalent among all MDS subtypes, accounting for 45 to 85% of the cases. However, the frequency of mutations shows substantial variations among disease subtypes, the genetic/biological basis of which has not been clarified. In addition, the impact of splicing pathway mutations on prognosis has been poorly defined. To explore the genetic basis for these differences, we analyzed genome-wide copy number lesions and the spectrum of gene mutations that may coexist with splicing pathway mutations in a set of 283 cases with myelodysplasia, using SNP array karyotyping and target sequencing of common gene targets in MDS, including TET2 and EZH2. The effects of the splicing pathway mutations on clinical outcomes were evaluated together with those of these accompanying genetic lesions. Splicing pathway mutations were identified in 160 (57%) among 8 components of the splicing machinery, which occurred in a mutually exclusive manner. SNP array karyotyping revealed 138 cases (49%) showing copy number alterations, in which 7q- and/or 5q- were the most frequent abnormalities. Interestingly, the splicing pathway mutations were found at a significantly lower frequency among patients with 7q- and/or 5q- (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5122. doi:1538-7445.AM2012-5122