Publications by authors named "Yuichi Shiraishi"

148 Publications

Combined landscape of single-nucleotide variants and copy number alterations in clonal hematopoiesis.

Nat Med 2021 Jul 8;27(7):1239-1249. Epub 2021 Jul 8.

Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Clonal hematopoiesis (CH) in apparently healthy individuals is implicated in the development of hematological malignancies (HM) and cardiovascular diseases. Previous studies of CH analyzed either single-nucleotide variants and indels (SNVs/indels) or copy number alterations (CNAs), but not both. Here, using a combination of targeted sequencing of 23 CH-related genes and array-based CNA detection of blood-derived DNA, we have delineated the landscape of CH-related SNVs/indels and CNAs in 11,234 individuals without HM from the BioBank Japan cohort, including 672 individuals with subsequent HM development, and studied the effects of these somatic alterations on mortality from HM and cardiovascular disease, as well as on hematological and cardiovascular phenotypes. The total number of both types of CH-related lesions and their clone size positively correlated with blood count abnormalities and mortality from HM. CH-related SNVs/indels and CNAs exhibited statistically significant co-occurrence in the same individuals. In particular, co-occurrence of SNVs/indels and CNAs affecting DNMT3A, TET2, JAK2 and TP53 resulted in biallelic alterations of these genes and was associated with higher HM mortality. Co-occurrence of SNVs/indels and CNAs also modulated risks for cardiovascular mortality. These findings highlight the importance of detecting both SNVs/indels and CNAs in the evaluation of CH.
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http://dx.doi.org/10.1038/s41591-021-01411-9DOI Listing
July 2021

Molecular classification and diagnostics of upper urinary tract urothelial carcinoma.

Cancer Cell 2021 Jun;39(6):793-809.e8

Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Upper urinary tract urothelial carcinoma (UTUC) is one of the common urothelial cancers. Its molecular pathogenesis, however, is poorly understood, with no useful biomarkers available for accurate diagnosis and molecular classification. Through an integrated genetic study involving 199 UTUC samples, we delineate the landscape of genetic alterations in UTUC enabling genetic/molecular classification. According to the mutational status of TP53, MDM2, RAS, and FGFR3, UTUC is classified into five subtypes having discrete profiles of gene expression, tumor location/histology, and clinical outcome, which is largely recapitulated in an independent UTUC cohort. Sequencing of urine sediment-derived DNA has a high diagnostic value for UTUC with 82.2% sensitivity and 100% specificity. These results provide a solid basis for better diagnosis and management of UTUC.
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http://dx.doi.org/10.1016/j.ccell.2021.05.008DOI Listing
June 2021

Clonal evolution and clinical implications of genetic abnormalities in blastic transformation of chronic myeloid leukaemia.

Nat Commun 2021 05 14;12(1):2833. Epub 2021 May 14.

Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML.
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http://dx.doi.org/10.1038/s41467-021-23097-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121838PMC
May 2021

Clinical significance of RAS pathway alterations in pediatric acute myeloid leukemia.

Haematologica 2021 Mar 18. Epub 2021 Mar 18.

Department of Hematology/Oncology, Gunma Children's Medical Center, Gunma, Japan; Institute of Physiology and Medicine, Jobu University, Gunma.

RAS pathway alterations have been implicated in the pathogenesis of various hematological malignancies. However, their clinical relevance in pediatric acute myeloid leukemia (AML) is not well characterized. We analyzed the frequency, clinical significance, and prognostic relevance of RAS pathway alterations in 328 pediatric patients with de novo AML. RAS pathway alterations were detected in 80 (24.4%) out of 328 patients: NF1 (n = 7, 2.1%), PTPN11 (n = 15, 4.6%), CBL (n = 6, 1.8%), NRAS (n = 44, 13.4%), KRAS (n = 12, 3.7%). Most of these alterations were mutually exclusive and were also mutually exclusive with other aberrations of signal transduction pathways such as FLT3-ITD (p = 0.001) and KIT mutation (p = 0.004). NF1 alterations were frequently detected in patients with complex karyotype (p = 0.031) and were found to be independent predictors of poor overall survival (OS) in multivariate analysis (p = 0.007). At least four of seven patients with NF1 alterations had bi-allelic inactivation. NRAS mutations were frequently observed in patients with CBFB-MYH11 and were independent predictors of favorable outcomes in multivariate analysis [OS, p = 0.023; event-free survival (EFS), p = 0.037]. Patients with PTPN11 mutations more frequently received stem cell transplantation (p = 0.035) and showed poor EFS than patients without PTPN11 mutations (p = 0.013). Detailed analysis of RAS pathway alterations may enable a more accurate prognostic stratification of pediatric AML and may provide novel therapeutic molecular targets related to this signal transduction pathway.
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http://dx.doi.org/10.3324/haematol.2020.269431DOI Listing
March 2021

Frequent genetic alterations in immune checkpoint-related genes in intravascular large B-cell lymphoma.

Blood 2021 Mar;137(11):1491-1502

Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Intravascular large B-cell lymphoma (IVLBCL) is a unique type of extranodal lymphoma characterized by selective growth of tumor cells in small vessels without lymphadenopathy. Greater understanding of the molecular pathogenesis of IVLBCL is hampered by the paucity of lymphoma cells in biopsy specimens, creating a limitation in obtaining sufficient tumor materials. To uncover the genetic landscape of IVLBCL, we performed whole-exome sequencing (WES) of 21 patients with IVLBCL using plasma-derived cell-free DNA (cfDNA) (n = 18), patient-derived xenograft tumors (n = 4), and tumor DNA from bone marrow (BM) mononuclear cells (n = 2). The concentration of cfDNA in IVLBCL was significantly higher than that in diffuse large B-cell lymphoma (DLBCL) (P < .0001) and healthy donors (P = .0053), allowing us to perform WES; most mutations detected in BM tumor DNA were successfully captured in cfDNA and xenograft. IVLBCL showed a high frequency of genetic lesions characteristic of activated B-cell-type DLBCL, with the former showing conspicuously higher frequencies (compared with nodal DLBCL) of mutations in MYD88 (57%), CD79B (67%), SETD1B (57%), and HLA-B (57%). We also found that 8 IVLBCL (38%) harbored rearrangements of programmed cell death 1 ligand 1 and 2 (PD-L1/PD-L2) involving the 3' untranslated region; such rearrangements are implicated in immune evasion via PD-L1/PD-L2 overexpression. Our data demonstrate the utility of cfDNA and imply important roles for immune evasion in IVLBCL pathogenesis and PD-1/PD-L1/PD-L2 blockade in therapeutics for IVLBCL.
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http://dx.doi.org/10.1182/blood.2020007245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7976508PMC
March 2021

Unbiased Detection of Driver Mutations in Extramammary Paget Disease.

Clin Cancer Res 2021 Mar 15;27(6):1756-1765. Epub 2020 Dec 15.

Department of Pathology and Tumor Biology, Kyoto University, Kyoto, Japan.

Purpose: Extramammary Paget disease (EMPD) is an uncommon skin malignancy whose genetic alterations are poorly characterized. Previous reports identified mutations in chromatin remodeling genes and . In order to unambiguously determine driver mutations in EMPD, we analyzed 87 EMPD samples using exome sequencing in combination with targeted sequencing.

Experimental Design: First, we analyzed 37 EMPD samples that were surgically resected using whole-exome sequencing. Based on several analysis, we built a custom capture panel of putative driver genes and analyzed 50 additional formalin-fixed, paraffin-embedded samples using target sequencing. expression was evaluated by HER2 immunohisotochemistry. Select samples were further analyzed by fluorescence hybridization.

Results: A median of 92 mutations/sample was identified in exome analysis. A union of driver detection algorithms identified , and as likely driver mutations. Copy-number alteration analysis showed regions spanning as recurrently deleted, and as recurrently amplified. , and amplification/mutation showed tendency toward mutual exclusivity. Copy-number alteration load was associated with likelihood to recur. Mutational signatures were dominated by aging and APOBEC activation and lacked evidence of ultraviolet radiation. HER2 IHC/fluorescence analysis validated amplification but was underpowered to detect mutations. Tumor heterogeneity in terms of amplification status was observed in some cases.

Conclusions: Our comprehensive, unbiased analysis shows EMPD is characterized by alterations involving the PI3K-AKT pathway. EMPD is distinct from other skin cancers in both molecular pathways altered and etiology behind mutagenesis.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3205DOI Listing
March 2021

Acquisition of monosomy 7 and a RUNX1 mutation in Pearson syndrome.

Pediatr Blood Cancer 2021 02 16;68(2):e28799. Epub 2020 Nov 16.

Department of Pediatrics, St. Luke's International Hospital, Tokyo, Japan.

Pearson syndrome (PS) is a very rare and often fatal multisystem disease caused by deletions in mitochondrial DNA that result in sideroblastic anemia, vacuolization of marrow precursors, and pancreatic dysfunction. Spontaneous recovery from anemia is often observed within several years of diagnosis. We present the case of a 4-month-old male diagnosed with PS who experienced prolonged severe pancytopenia preceding the emergence of monosomy 7. Whole-exome sequencing identified two somatic mutations, including RUNX1 p.S100F that was previously reported as associated with myeloid malignancies. The molecular defects associated with PS may have the potential to progress to advanced myelodysplastic syndrome .
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http://dx.doi.org/10.1002/pbc.28799DOI Listing
February 2021

Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Mol Cell 2020 12 3;80(6):996-1012.e9. Epub 2020 Nov 3.

Department of Innovative Medical Science, Tokai University School of Medicine, Isehara, Japan.

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.
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http://dx.doi.org/10.1016/j.molcel.2020.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758861PMC
December 2020

Landscape of driver mutations and their clinical impacts in pediatric B-cell precursor acute lymphoblastic leukemia.

Blood Adv 2020 10;4(20):5165-5173

Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan.

Recent genetic studies using high-throughput sequencing have disclosed genetic alterations in B-cell precursor acute lymphoblastic leukemia (B-ALL). However, their effects on clinical outcomes have not been fully investigated. To address this, we comprehensively examined genetic alterations and their prognostic impact in a large series of pediatric B-ALL cases. We performed targeted capture sequencing in a total of 1003 pediatric patients with B-ALL from 2 Japanese cohorts. Transcriptome sequencing (n = 116) and/or array-based gene expression analysis (n = 120) were also performed in 203 (84%) of 243 patients who were not categorized into any disease subgroup by panel sequencing or routine reverse transcription polymerase chain reaction analysis for major fusions in B-ALL. Our panel sequencing identified novel recurrent mutations in 2 genes (CCND3 and CIC), and both had positive correlations with ETV6-RUNX1 and hypodiploid ALL, respectively. In addition, positive correlations were also newly reported between TCF3-PBX1 ALL with PHF6 mutations. In multivariate Cox proportional hazards regression models for overall survival, TP53 mutation/deletion, hypodiploid, and MEF2D fusions were selected in both cohorts. For TP53 mutations, the negative effect on overall survival was confirmed in an independent external cohort (n = 466). TP53 mutation was frequently found in IGH-DUX4 (5 of 57 [9%]) ALL, with 4 cases having 17p LOH and negatively affecting overall survival therein, whereas TP53 mutation was not associated with poor outcomes among NCI (National Cancer Institute) standard risk (SR) patients. A conventional treatment approach might be enough, and further treatment intensification might not be necessary, for patients with TP53 mutations if they are categorized into NCI SR.
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http://dx.doi.org/10.1182/bloodadvances.2019001307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594377PMC
October 2020

Genetic and clinical landscape of breast cancers with germline BRCA1/2 variants.

Commun Biol 2020 10 16;3(1):578. Epub 2020 Oct 16.

Department of Pathology and Tumor Biology, Kyoto University, Kyoto, Japan.

The genetic and clinical characteristics of breast tumors with germline variants, including their association with biallelic inactivation through loss-of-heterozygosity (LOH) and second somatic mutations, remain elusive. We analyzed germline variants of 11 breast cancer susceptibility genes for 1,995 Japanese breast cancer patients, and identified 101 (5.1%) pathogenic variants, including 62 BRCA2 and 15 BRCA1 mutations. Genetic analysis of 64 BRCA1/2-mutated tumors including TCGA dataset tumors, revealed an association of biallelic inactivation with more extensive deletions, copy neutral LOH, gain with LOH and younger onset. Strikingly, TP53 and RB1 mutations were frequently observed in BRCA1- (94%) and BRCA2- (9.7%) mutated tumors with biallelic inactivation. Inactivation of TP53 and RB1 together with BRCA1 and BRCA2, respectively, involved LOH of chromosomes 17 and 13. Notably, BRCA1/2 tumors without biallelic inactivation were indistinguishable from those without germline variants. Our study highlights the heterogeneity and unique clonal selection pattern in breast cancers with germline variants.
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http://dx.doi.org/10.1038/s42003-020-01301-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567851PMC
October 2020

Comprehensive genetic analysis of pediatric germ cell tumors identifies potential drug targets.

Commun Biol 2020 09 30;3(1):544. Epub 2020 Sep 30.

Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

To elucidate the molecular pathogenesis of pediatric germ cell tumors (GCTs), we performed DNA methylation array analysis, whole transcriptome sequencing, targeted capture sequencing, and single-nucleotide polymorphism array analysis using 51 GCT samples (25 female, 26 male), including 6 germinomas, 2 embryonal carcinomas, 4 immature teratomas, 3 mature teratomas, 30 yolk sac tumors, and 6 mixed germ cell tumors. Among the 51 samples, 11 were from infants, 23 were from young children, and 17 were from those aged ≥10 years. Sixteen of the 51 samples developed in the extragonadal regions. Germinomas showed upregulation of pluripotent genes and global hypomethylation. Pluripotent genes were also highly expressed in embryonal carcinomas. These genes may play essential roles in embryonal carcinomas given that their binding sites are hypomethylated. Yolk sac tumors exhibited overexpression of endodermal genes, such as GATA6 and FOXA2, the binding sites of which were hypomethylated. Interestingly, infant yolk sac tumors had different DNA methylation patterns from those observed in older children. Teratomas had higher expression of ectodermal genes, suggesting a tridermal nature. Based on our results, we suggest that KIT, TNFRSF8, and ERBB4 may be suitable targets for the treatment of germinoma, embryonal carcinomas, and yolk sac tumors, respectively.
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http://dx.doi.org/10.1038/s42003-020-01267-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528104PMC
September 2020

VAV1 mutations contribute to development of T-cell neoplasms in mice.

Blood 2020 12;136(26):3018-3032

Department of Hematology, Comprehensive Human Biosciences, and.

Activating mutations in the Vav guanine nucleotide exchange factor 1 (VAV1) gene are reported in various subtypes of mature T-cell neoplasms (TCNs). However, oncogenic activities associated with VAV1 mutations in TCNs remain unclear. To define them, we established transgenic mice expressing VAV1 mutants cloned from human TCNs. Although we observed no tumors in these mice for up to a year, tumors did develop in comparably aged mice on a p53-null background (p53-/-VAV1-Tg), and p53-/-VAV1-Tg mice died with shorter latencies than did p53-null (p53-/-) mice. Notably, various TCNs with tendency of maturation developed in p53-/-VAV1-Tg mice, whereas p53-/- mice exhibited only immature TCNs. Mature TCNs in p53-/-VAV1-Tg mice mimicked a subtype of human peripheral T-cell lymphoma (PTCL-GATA3) and exhibited features of type 2 T helper (Th2) cells. Phenotypes seen following transplantation of either p53-/-VAV1 or p53-/- tumor cells into nude mice were comparable, indicating cell-autonomous tumor-initiating capacity. Whole-transcriptome analysis showed enrichment of multiple Myc-related pathways in TCNs from p53-/-VAV1-Tg mice relative to p53-/- or wild-type T cells. Remarkably, amplification of the Myc locus was found recurrently in TCNs of p53-/-VAV1-Tg mice. Finally, treatment of nude mice transplanted with p53-/-VAV1-Tg tumor cells with JQ1, a bromodomain inhibitor that targets the Myc pathway, prolonged survival of mice. We conclude that VAV1 mutations function in malignant transformation of T cells in vivo and that VAV1-mutant-expressing mice could provide an efficient tool for screening new therapeutic targets in TCNs harboring these mutations.
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http://dx.doi.org/10.1182/blood.2020006513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770564PMC
December 2020

Fusion partner-specific mutation profiles and KRAS mutations as adverse prognostic factors in MLL-rearranged AML.

Blood Adv 2020 10;4(19):4623-4631

Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Mixed-lineage leukemia (MLL) gene rearrangements are among the most frequent chromosomal abnormalities in acute myeloid leukemia (AML). MLL fusion patterns are associated with the patient's prognosis; however, their relationship with driver mutations is unclear. We conducted sequence analyses of 338 genes in pediatric patients with MLL-rearranged (MLL-r) AML (n = 56; JPLSG AML-05 study) alongside data from the TARGET study's pediatric cohorts with MLL-r AML (n = 104), non-MLL-r AML (n = 581), and adult MLL-r AML (n = 81). KRAS mutations were most frequent in pediatric patients with high-risk MLL fusions (MLL-MLLLT10, MLL-MLLT4, and MLL-MLLT1). Pediatric patients with MLL-r AML (n = 160) and a KRAS mutation (KRAS-MT) had a significantly worse prognosis than those without a KRAS mutation (KRAS-WT) (5-year event-free survival [EFS]: 51.8% vs 18.3%, P < .0001; 5-year overall survival [OS]: 67.3% vs 44.3%, P = .003). The adverse prognostic impact of KRAS mutations was confirmed in adult MLL-r AML. KRAS mutations were associated with adverse prognoses in pediatric patients with both high-risk (MLLT10+MLLT4+MLLT1; n = 60) and intermediate-to-low-risk (MLLT3+ELL+others; n = 100) MLL fusions. The prognosis did not differ significantly between patients with non-MLL-r AML with KRAS-WT or KRAS-MT. Multivariate analysis showed the presence of a KRAS mutation to be an independent prognostic factor for EFS (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.35-3.59; P = .002) and OS (HR, 1.85; 95% CI, 1.01-3.31; P = .045) in MLL-r AML. The mutation is a distinct adverse prognostic factor in MLL-r AML, regardless of risk subgroup, and is potentially useful for accurate treatment stratification. This trial was registered at the UMIN (University Hospital Medical Information Network) Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm) as #UMIN000000511.
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http://dx.doi.org/10.1182/bloodadvances.2020002457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556160PMC
October 2020

Frequent mutations in HLA and related genes in extranodal NK/T cell lymphomas.

Leuk Lymphoma 2021 01 23;62(1):95-103. Epub 2020 Sep 23.

Department of Medicine, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Bangkok, Thailand.

Extranodal NK/T cell lymphomas (ENKTCLs) are aggressive Epstein-Barr virus-associated T/NK neoplasms that predominantly affect Asians. To explore the causative somatic events, we conducted a comprehensive genetic analysis of 19 ENKTCL patients by whole-genome ( = 2), whole-exome ( = 16), and targeted sequencing ( = 15). Commonly deregulated gene pathways in ENKTCLs included epigenetic modifiers (58%, 11/19) followed by human leukocyte antigens (HLAs) and related genes including , , and (32%, 6/19), and JAK-STAT pathway (26%, 5/19). Conspicuously, loss-of-function mutations in were recurrently identified in ENKTCLs (16%, 3/19). HLA protein expression was examined by immunohistochemistry in 16 patients and lower expression was associated with advanced stages at presentation ( = .007). In conclusion, the defective antigen presenting pathway is common and related to disease progression, suggesting immune escape as a pathogenic mechanism of ENKTCLs.
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http://dx.doi.org/10.1080/10428194.2020.1821011DOI Listing
January 2021

Long-read sequencing for non-small-cell lung cancer genomes.

Genome Res 2020 09 4;30(9):1243-1257. Epub 2020 Sep 4.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan.

Here, we report the application of a long-read sequencer, PromethION, for analyzing human cancer genomes. We first conducted whole-genome sequencing on lung cancer cell lines. We found that it is possible to genotype known cancerous mutations, such as point mutations. We also found that long-read sequencing is particularly useful for precisely identifying and characterizing structural aberrations, such as large deletions, gene fusions, and other chromosomal rearrangements. In addition, we identified several medium-sized structural aberrations consisting of complex combinations of local duplications, inversions, and microdeletions. These complex mutations occurred even in key cancer-related genes, such as , , , and The biological relevance of those mutations was further revealed by epigenome, transcriptome, and protein analyses of the affected signaling pathways. Such structural aberrations were also found in clinical lung adenocarcinoma specimens. Those structural aberrations were unlikely to be reliably detected by conventional short-read sequencing. Therefore, long-read sequencing may contribute to understanding the molecular etiology of patients for whom causative cancerous mutations remain unknown and therapeutic strategies are elusive.
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http://dx.doi.org/10.1101/gr.261941.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545141PMC
September 2020

Integrated multiomics analysis of hepatoblastoma unravels its heterogeneity and provides novel druggable targets.

NPJ Precis Oncol 2020 7;4:20. Epub 2020 Jul 7.

Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Although hepatoblastoma is the most common pediatric liver cancer, its genetic heterogeneity and therapeutic targets are not well elucidated. Therefore, we conducted a multiomics analysis, including mutatome, DNA methylome, and transcriptome analyses, of 59 hepatoblastoma samples. Based on DNA methylation patterns, hepatoblastoma was classified into three clusters exhibiting remarkable correlation with clinical, histological, and genetic features. Cluster F was largely composed of cases with fetal histology and good outcomes, whereas clusters E1 and E2 corresponded primarily to embryonal/combined histology and poor outcomes. E1 and E2, albeit distinguishable by different patient age distributions, were genetically characterized by hypermethylation of the HNF4A/CEBPA-binding regions, fetal liver-like expression patterns, upregulation of the cell cycle pathway, and overexpression of and . Inhibition of and in hepatoblastoma cells induced chemosensitization and growth suppression, respectively. Our results provide a comprehensive description of the molecular basis of hepatoblastoma and rational therapeutic strategies for high-risk cases.
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http://dx.doi.org/10.1038/s41698-020-0125-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341754PMC
July 2020

Clinical utility of target capture-based panel sequencing in hematological malignancies: A multicenter feasibility study.

Cancer Sci 2020 Sep 17;111(9):3367-3378. Epub 2020 Jul 17.

Department of Hematology, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.

Although next-generation sequencing-based panel testing is well practiced in the field of cancer medicine for the identification of target molecules in solid tumors, the clinical utility and clinical issues surrounding panel testing in hematological malignancies have yet to be fully evaluated. We conducted a multicenter prospective clinical sequencing study to verify the feasibility of a panel test for hematological tumors, including acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma. Out of 96 eligible patients, 79 patients (82%) showed potentially actionable findings, based on the clinical sequencing assays. We identified that genetic alterations with a strong clinical significance were found at a higher frequency in terms of diagnosis (n = 60; 63%) and prognosis (n = 61; 64%) than in terms of therapy (n = 8; 8%). Three patients who harbored a germline mutation in either DDX41 (n = 2) or BRCA2 (n = 1) were provided with genetic counseling. At 6 mo after sequencing, clinical actions based on the diagnostic (n = 5) or prognostic (n = 3) findings were reported, but no patients were enrolled in a clinical trial or received targeted therapies based on the sequencing results. These results suggest that panel testing for hematological malignancies would be feasible given the availability of useful diagnostic and prognostic information. This study is registered with the UMIN Clinical Trial Registry (UMIN000029879, multiple myeloma; UMIN000031343, adult acute myeloid leukemia; UMIN000033144, diffuse large B-cell lymphoma; and UMIN000034243, childhood leukemia).
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http://dx.doi.org/10.1111/cas.14552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469806PMC
September 2020

Diagnostic utility of integrated analysis of exome and transcriptome: Successful diagnosis of Au-Kline syndrome in a patient with submucous cleft palate, scaphocephaly, and intellectual disabilities.

Mol Genet Genomic Med 2020 09 26;8(9):e1364. Epub 2020 Jun 26.

Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan.

Background: A weakness of exome analysis lies in inability to characterize aberrant splicing other than those involving consensus donor-acceptor sequence. To overcome this limitation, we developed a novel analytic method SAVNet that combines transcriptome and exome analysis which enabled the successful detection of carriers of splicing variants in the disease-causing genes of autosomal recessive disorders within a normal cohort. However, the clinical utility of the SAVNet analysis in delineating splicing defects in patients without a diagnosis has yet to be documented.

Method: We performed SAVNet analysis using the integrated analysis of exome and transcriptome analysis from the peripheral blood of the patient. The patient is an undiagnosed Japanese female patient with submucous cleft palate, scaphocephaly and intellectual disability with no words at 8 years of age. Dysmorphic features included a long face, a short palpebral fissure, thick lips with an open month, premaxillary hypoplasia, a depressed nasal bridge, and satyr ears.

Result: A SAVNet analysis showed that a heterozygous intronic variant located at the -10 position of exon 5 of the HNRNPK gene on chromosome 9 created a new splice acceptor sequence "ag" and led to the incorporation of 9 intronic nucleotides into the coding sequence. The mutant protein would have three extra amino acid residues, Leu-Leu-Gln, inserted within the critical KH domain. The patient was diagnosed as having recently delineated Au-Kline syndrome, which is characterized by cleft palate, craniosynostosis, and intellectual disability.

Conclusion: The successful molecular diagnosis of the presently reported patient illustrates the diagnostic utility of the SAVNet analysis as an innovative way of implementing an integrated exome-transcriptome analysis in clinical settings.
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http://dx.doi.org/10.1002/mgg3.1364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503209PMC
September 2020

Landscape and function of multiple mutations within individual oncogenes.

Nature 2020 06 8;582(7810):95-99. Epub 2020 Apr 8.

Division of Molecular Oncology, National Cancer Center Research Institute, Tokyo, Japan.

Sporadic reports have described cancer cases in which multiple driver mutations (MMs) occur in the same oncogene. However, the overall landscape and relevance of MMs remain elusive. Here we carried out a pan-cancer analysis of 60,954 cancer samples, and identified 14 pan-cancer and 6 cancer-type-specific oncogenes in which MMs occur more frequently than expected: 9% of samples with at least one mutation in these genes harboured MMs. In various oncogenes, MMs are preferentially present in cis and show markedly different mutational patterns compared with single mutations in terms of type (missense mutations versus in-frame indels), position and amino-acid substitution, suggesting a cis-acting effect on mutational selection. MMs show an overrepresentation of functionally weak, infrequent mutations, which confer enhanced oncogenicity in combination. Cells with MMs in the PIK3CA and NOTCH1 genes exhibit stronger dependencies on the mutated genes themselves, enhanced downstream signalling activation and/or greater sensitivity to inhibitory drugs than those with single mutations. Together oncogenic MMs are a relatively common driver event, providing the underlying mechanism for clonal selection of suboptimal mutations that are individually rare but collectively account for a substantial proportion of oncogenic mutations.
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http://dx.doi.org/10.1038/s41586-020-2175-2DOI Listing
June 2020

Genomic analysis of multiple myeloma using targeted capture sequencing in the Japanese cohort.

Br J Haematol 2020 12 9;191(5):755-763. Epub 2020 May 9.

Department of Hematology and Oncology, Nagoya City University Graduate School of Medical Sciences, Japan.

Previous genomic studies have revealed the genomic landscape of myeloma cells. Although some of the genomic abnormalities shown are believed to be correlated to the molecular pathogenesis of multiple myeloma and/or clinical outcome, these correlations are not fully understood. The aim of this study is to elucidate the correlation between genomic abnormalities and clinical characteristics by targeted capture sequencing in the Japanese multiple myeloma cohort. We analysed 154 patients with newly diagnosed multiple myeloma. The analysis revealed that the study cohort consisted of a less frequent hyperdiploid subtype (37·0%) with relatively high frequencies of KRAS mutation (36·4%) and IGH-CCND1 translocation (26·6%) compared with previous reports. Moreover, our targeted capture sequencing strategy was able to detect rare IGH-associated chromosomal translocations, such as IGH-CCND2 and IGH-MAFA. Interestingly, all 10 patients harboured MAX mutations accompanied by 14q23 deletion. The patients with del(17p) exhibited an unfavourable clinical outcome, and the presence of KRAS mutation was associated with shorter survival in patients with multiple myeloma, harbouring IGH-CCND1. Thus, our study provides a detailed landscape of genomic abnormalities, which may have potential clinical application for patients with multiple myeloma.
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http://dx.doi.org/10.1111/bjh.16720DOI Listing
December 2020

Comprehensive analysis of indels in whole-genome microsatellite regions and microsatellite instability across 21 cancer types.

Genome Res 2020 Mar 24. Epub 2020 Mar 24.

Laboratory for Cancer Genomics, RIKEN Center for Integrative Medical Sciences, Tokyo 230-0045, Japan.

Microsatellites are repeats of 1- to 6-bp units, and approximately 10 million microsatellites have been identified across the human genome. Microsatellites are vulnerable to DNA mismatch errors and have thus been used to detect cancers with mismatch repair deficiency. To reveal the mutational landscape of microsatellite repeat regions at the genome level, we analyzed approximately 20.1 billion microsatellites in 2717 whole genomes of pan-cancer samples across 21 tissue types. First, we developed a new insertion and deletion caller (MIMcall) that takes into consideration the error patterns of different types of microsatellites. Among the 2717 pan-cancer samples, our analysis identified 31 samples, including colorectal, uterus, and stomach cancers, with a higher proportion of mutated microsatellite (≥0.03), which we defined as microsatellite instability (MSI) cancers of genome-wide level. Next, we found 20 highly mutated microsatellites that can be used to detect MSI cancers with high sensitivity. Third, we found that replication timing and DNA shape were significantly associated with mutation rates of microsatellites. Last, analysis of mutations in mismatch repair genes showed that somatic SNVs and short indels had larger functional impacts than germline mutations and structural variations. Our analysis provides a comprehensive picture of mutations in the microsatellite regions and reveals possible causes of mutations, as well as provides a useful marker set for MSI detection.
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http://dx.doi.org/10.1101/gr.255026.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111525PMC
March 2020

Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status.

Blood Adv 2020 03;4(5):845-854

Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.
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http://dx.doi.org/10.1182/bloodadvances.2019001324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065477PMC
March 2020

Identification of Novel CD74-NRG2α Fusion From Comprehensive Profiling of Lung Adenocarcinoma in Japanese Never or Light Smokers.

J Thorac Oncol 2020 06 6;15(6):948-961. Epub 2020 Feb 6.

Division of Cellular Signaling, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan.

Introduction: Studies are yet to characterize the differences in molecular profiles of lung adenocarcinoma (LUAD) among divergent ethnic groups. Herein, we conducted comprehensive molecular profiling of LUAD in never or light smokers from Asia to discover novel targetable mutations and prognostic biomarkers of this distinct disease entity.

Methods: We analyzed 996 cases of Japanese LUAD and performed whole-exome sequencing and RNA-seq in 125 cases of Japanese LUAD negative for the driver oncogenes defined by conventional laboratory testing. We also investigated the clinical and pathologic characteristics among the 996 cases.

Results: Driver oncogenes were identified in 88 cases (70.4%) with specific hotspot mutations differing from those in The Cancer Genome Atlas study. Two actionable novel fusions of FGFR2 and NRG2α were also identified. Clustering on the basis of mRNA expression profiles, but not genetic mutational ones, could predict patient prognosis. The risk score generated by the expression of a three-gene set was a strong prognostic marker for overall survival and progression-free survival in our cohort, and was further validated using The Cancer Genome Atlas cohort. Among the 996 cases, each driver alteration is distributed across all histologic subtypes. Adenocarcinoma in situ was identified to harbor driver mutations, suggesting that these alterations are early events in the pathogenesis of LUAD. ERBB2 mutations were over-represented in young adults.

Conclusions: This study indicates the value of applying gene expression profiling for predicting the prognosis after a surgical operation, and that the identification of actionable mutations is important for optimizing targeted drugs in Japanese LUAD.
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http://dx.doi.org/10.1016/j.jtho.2020.01.021DOI Listing
June 2020

Genomic basis for RNA alterations in cancer.

Nature 2020 02 5;578(7793):129-136. Epub 2020 Feb 5.

BGI-Shenzhen, Shenzhen, China.

Transcript alterations often result from somatic changes in cancer genomes. Various forms of RNA alterations have been described in cancer, including overexpression, altered splicing and gene fusions; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed 'bridged' fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.
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http://dx.doi.org/10.1038/s41586-020-1970-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054216PMC
February 2020

Single-cell analysis based dissection of clonality in myelofibrosis.

Nat Commun 2020 01 7;11(1):73. Epub 2020 Jan 7.

Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Hematology, Oncology, and Tumor Immunology, Berlin, Germany.

Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.
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http://dx.doi.org/10.1038/s41467-019-13892-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946829PMC
January 2020

Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Nature 2020 01 18;577(7789):260-265. Epub 2019 Dec 18.

Laboratory of DNA Information Analysis, Human Genome Centre, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.
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http://dx.doi.org/10.1038/s41586-019-1856-1DOI Listing
January 2020

Invariant patterns of clonal succession determine specific clinical features of myelodysplastic syndromes.

Nat Commun 2019 11 26;10(1):5386. Epub 2019 Nov 26.

Department of Translational Hematology & Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA.

Myelodysplastic syndromes (MDS) arise in older adults through stepwise acquisitions of multiple somatic mutations. Here, analyzing 1809 MDS patients, we infer clonal architecture by using a stringent, the single-cell sequencing validated PyClone bioanalytic pipeline, and assess the position of the mutations within the clonal architecture. All 3,971 mutations are grouped based on their rank in the deduced clonal hierarchy (dominant and secondary). We evaluated how they affect the resultant morphology, progression, survival and response to therapies. Mutations of SF3B1, U2AF1, and TP53 are more likely to be dominant, those of ASXL1, CBL, and KRAS are secondary. Among distinct combinations of dominant/secondary mutations we identified 37 significant relationships, of which 12 affect clinical phenotypes, 5 cooperatively associate with poor prognosis. They also predict response to hypomethylating therapies. The clonal hierarchy has distinct ranking and the resultant invariant combinations of dominant/secondary mutations yield novel insights into the specific clinical phenotype of MDS.
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http://dx.doi.org/10.1038/s41467-019-13001-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879617PMC
November 2019
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