The neuroendocrine carcinoma of the gastrointestinal system (GIS-NEC) is a rare but highly malignant neoplasm. We analyzed 115 cases using whole-genome/exome sequencing, transcriptome sequencing, DNA ...methylation assays, and/or ATAC-seq and found GIS-NECs to be genetically distinct from neuroendocrine tumors (GIS-NET) in the same location. Clear genomic differences were also evident between pancreatic NECs (Panc-NEC) and nonpancreatic GIS-NECs (Nonpanc-NEC). Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic features. Alterations in TP53 and RB1 proved common in GIS-NECs, and most Nonpanc-NECs with intact RB1 demonstrated mutually exclusive amplification of CCNE1 or MYC. Alterations of the Notch gene family were characteristic of Nonpanc-NECs. Transcription factors for neuroendocrine differentiation, especially the SOX2 gene, appeared overexpressed in most GIS-NECs due to hypermethylation of the promoter region. This first comprehensive study of genomic alterations in GIS-NECs uncovered several key biological processes underlying genesis of this very lethal form of cancer.
GIS-NECs are genetically distinct from GIS-NETs. GIS-NECs arising in different organs show similar histopathologic features and share some genomic features, but considerable differences exist between Panc-NECs and Nonpanc-NECs. In addition, Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic and epigenomic features. This article is highlighted in the In This Issue feature, p. 587.
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.
•RHOA mutations are common in ATLL and show a unique distribution compared with other T-cell lymphomas.•Depending on patients, functionally distinct RHOA mutations are clonally selected and involved in the pathogenesis of ATLL.
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.
Abstract
Background
CNS germ cell tumors (GCTs) predominantly develop in pediatric and young adult patients with variable responses to surgery, radiation, and chemotherapy. This study aimed to ...examine the complex and largely unknown pathogenesis of CNS GCTs.
Methods
We used a combined transcriptomic and methylomic approach in 84 cases and conducted an integrative analysis of the normal cells undergoing embryogenesis and testicular GCTs.
Results
Genome-wide transcriptome analysis in CNS GCTs indicated that germinoma had a transcriptomic profile representative of primitive cells during early embryogenesis with high meiosis/mitosis potentials, while nongerminomatous GCTs (NGGCTs) had differentiated phenotypes oriented toward tissue formation and organogenesis. Co-analysis with the transcriptome of human embryonic cells revealed that germinomas had expression profiles similar to those of primordial germ cells, while the expression profiles of NGGCTs were similar to those of embryonic stem cells. Some germinoma cases were characterized by extensive immune-cell infiltration and high expression of cancer-testis antigens. NGGCTs had significantly higher immune-cell infiltration, characterized by immune-suppression phenotype. CNS and testicular GCTs (TGCTs) had similar mutational profiles; TGCTs showed enhanced copy number alterations. Methylation analysis clustered germinoma/seminoma and nongerminoma/nonseminoma separately. Germinoma and seminoma were co-categorized based on the degree of the tumor microenvironment balance.
Conclusions
These results suggested that the pathophysiology of GCTs was less dependent on their site of origin and more dependent on the state of differentiation as well as on the tumor microenvironment balance. This study revealed distinct biological properties of GCTs, which will hopefully lead to future treatment development.
Recurrent fusion genes involving C11orf95, C11orf95‐RELA, have been identified only in supratentorial ependymomas among primary CNS tumors. Here, we report hitherto histopathologically unclassifiable ...high‐grade tumors, under the tentative label of “ependymoma‐like tumors with mesenchymal differentiation (ELTMDs),” harboring C11orf95‐NCOA1/2 or ‐RELA fusion. We examined the clinicopathological and molecular features in five cases of ELTMDs. Except for one adult case (50 years old), all cases were in children ranging from 1 to 2.5 years old. All patients presented with a mass lesion in the cerebral hemisphere. Histologically, all cases demonstrated a similar histology with a mixture of components. The major components were embryonal‐appearing components forming well‐delineated tumor cell nests composed of small uniform cells with high proliferative activity, and spindle‐cell mesenchymal components with a low‐ to high‐grade sarcoma‐like appearance. The embryonal‐appearing components exhibited minimal ependymal differentiation including a characteristic EMA positivity and tubular structures, but histologically did not fit with ependymoma because they lacked perivascular pseudorosettes, a histological hallmark of ependymoma, formed well‐delineated nests, and had diffuse and strong staining for CAM5.2. Molecular analysis identified C11orf95‐NCOA1, ‐NCOA2, and ‐RELA in two, one, and two cases, respectively. t‐distributed stochastic neighbor embedding analysis of DNA methylation data from two cases with C11orf95‐NCOA1 or ‐NCOA2 and a reference set of 380 CNS tumors revealed that these two cases were clustered together and were distinct from all subgroups of ependymomas. In conclusion, although ELTMDs exhibited morphological and genetic associations with supratentorial ependymoma with C11orf95‐RELA, they cannot be regarded as ependymoma. Further analyses of more cases are needed to clarify their differences and similarities.
We report hitherto histopathologically unclassifiable high‐grade tumors, ELTMDs.
ELTMDs exhibited minimal ependymal differentiation and C11orf95‐NCOA1/2 or ‐RELA.
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.
Aims
Approximately 60–70% of high‐grade round‐cell sarcomas that lack the Ewing sarcoma breakpoint region 1 (
EWSR
1
) rearrangement harbour a rearrangement of the
CIC
gene, most commonly
CIC
–
DUX
4
.... Recent studies have established that
CIC
‐rearranged sarcomas constitute a distinct group characterized by recognizable histology and immunoprofiles, such as positivity for
ETV
4 and
WT
1 and negativity for
NKX
2.2. Although these sarcomas are diagnosed increasingly in practice by fluorescence
in‐situ
hybridization (
FISH
) with
CIC
break‐apart probes, the optimal modality to diagnose these sarcomas has not been determined. In this study, we describe four round‐cell sarcomas that showed false‐negative results by
CIC
break‐apart
FISH
assays.
Methods and results
These sarcomas showed characteristic histology of
CIC
‐rearranged sarcomas, and all were immunohistochemically positive for
ETV
4 and
WT
1 and negative for
NKX
2.2. Although
FISH
showed non‐atypical negative signals for
CIC
rearrangement, high‐throughput
RNA
sequencing identified
CIC
–
DUX
4
and its fusion breakpoint in all cases. Their clinical and histological findings, as well as fusion points determined by
RNA
sequencing, did not differ significantly from those of nine
FISH
‐positive
CIC
–
DUX
4
sarcoma cases. We estimated that the
FISH
false‐negative rate for
CIC
‐rearranged sarcomas was 14%. Although neither histology nor immunoprofiles (e.g.
ETV
4 and
WT
1) are entirely sensitive or specific for
CIC
‐rearranged sarcomas, the observation that these four cases were identified successfully by such phenotypes suggested their practical utility.
Conclusions
CIC
break‐apart
FISH
assays missed a significant minority of
CIC
–
DUX
4
sarcomas, and full awareness of typical morphology and judicious immunohistochemical work‐ups, including analyses of
ETV
4 and
WT
1, should complement diagnostic assessment.
Abstract
As a research group participating in the International Human Epigenome Consortium, we have performed whole-genome bisulfite sequencing (WGBS) using post-bisulfite adaptor tagging, chromatin ...immunoprecipitation (ChIP)-sequencing (seq), RNA-seq and whole-genome sequencing (WGS) using normal hepatocytes purified from partial hepatectomy specimens of 6 Japanese patients without hepatitis virus infection, chronic liver disease or hepatocellular carcinoma.
DNA methylation profiles such as low CpG methylation levels in the region 200 bp upstream from the transcription start site (TSS200), the first coding exon and CpG island were obtained. CHH methylation was observed more frequently on the anti-sense strands than on the sense strands in the 5’ untranslated region (UTR), first intron, gene body and 3’ UTR. non-CpG methylation was inversely correlated with gene expression levels.
Personal differentially methylated regions (pDMRs) were observed less frequently in TSS200, the first coding exon, TSS1500, and CpG island where CpG methylation levels were low, indicating that the regions important for expression regulation may be protected from personal variations of CpG methylation. Histone modification profiles of pDMRs differed considerably among samples. pDMRs were observed around the TSSs of genes whose expression levels are reportedly regulated by CpG methylation, such as LRP1B, RASGRF2 and TFF1. pDMRs were located more frequently in the vicinity of loci showing genetic variation (single-nucleotide variations SNVs and/or insertions/deletions indels) than on all autosomes. Although further study is needed to clarify the molecular background of such genome-epigenome interaction, we speculate that SNVs and indels may affect the binding of non-coding RNAs and/or protein complexes that induce histone modification or CpG methylation alterations around the SNV and indel loci. MetaCore pathway analysis revealed that genes showing both genetic (SNVs and indels) and epigenetic (pDMRs) variations in multiple samples were significantly accumulated in signaling pathways participating in hepatocyte function and disease susceptibility.
Our data suggest that genetic variations may induce epigenetic variations and generate individual differences in the phenotypes of normal hepatocytes and/or determine disease susceptibility through variations in expression. After accumulation of numerous reference epigenome profile data in the IHEC database, comparison between IHEC data for normal cells and cancer cells may facilitate the accurate identification of cancer-specific epigenome profiles, which would be indispensable to the development of biomarkers and molecular targeted therapies.
Citation Format: Eri Arai, Fumihito Miura, Yasushi Totoki, Satoshi Yamashita, Ying Tian, Masahiro Gotoh, Hidenori Ojima, Hiroyuki Nakagawa, Yoriko Takahashi, Hiromi Nakamura, Natsuko Hama, Mamoru Kato, Hiroshi Kimura, Yutaka Suzuki, Takashi Ito, Tatsuhiro Shibata, Yae Kanai. Epigenome landscape of human normal purified hepatocytes: analysis by the International Human Epigenome Consortium (IHEC). abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4517.
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