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Resistance to immune-checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold-, myeloid cell-, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell-subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC.

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.Significance: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

Supplementary Data from Comprehensive Genomic Profiling of Neuroendocrine Carcinomas of the Gastrointestinal System

Multivariate analysis regarding to overall survival in EHCC.

Summary of Patients and Clinical Data.

Supplementary Figure 1. Unsupervised Clustering on CCA samples. Supplementary Figure 2. Alterations Found in CCA Clusters. Supplementary Figure 3. MAP2K4 Homozygous Deletions and ERBB2 Amplifications. Supplementary Figure 4. Alterations Related to Structural Variations Found in CCAs. Supplementary Figure 5. FIREFLY Analysis of Pathways Systematically Dysregulated by Somatic Promoter Mutations that Alter Transcription Factor Binding. Supplementary Figure 6. Epigenetic Clusters and Mutation Signatures.

Multivariate analysis regarding to overall survival in IHCC.

Structural Variations across 71 WGS CCAs.

Disease free survival according to HSP90 expression

Summary of CCA Alterations.

IC50 inhibition values for NVP-AUY922

Further details of methods, in addition to the supplementary figure and table legends.

Alterations in CCA Clusters.

Coverage Statistics and List of Genes for Targeted Sequencing.

Supplemental experimental procedures, Figure S1-S16, Table S5

The transcription factor E74-like factor 3 (ELF3) is inactivated in a range of cancers, including biliary tract cancer (BTC). Here, we investigated the tumor-suppressive role of ELF3 in bile duct cells by identifying several previously unknown direct target genes of ELF3 that appear to be implicated in biliary duct carcinogenesis. ELF3 directly repressed ZEB2, a key regulator of epithelial–mesenchymal transition, and upregulated the expression of CGN, an integral element in lumen formation. Loss of ELF3 led to decreased cell–cell junctions and enhanced cell motility. ALOX5 and CXCL16 were also identified as additional direct targets of ELF3; their corresponding proteins 5-lipoxygenase and CXCL16 play a role in the immune response. Conditioned medium from cells overexpressing ELF3 significantly enhanced the migration of natural killer cells and CD8+ T cells toward the conditioned medium. Gene expression profiling for BTC expressing high levels of ELF3 revealed significant enrichment of the ELF3-related genes. In a BTC xenograft model, activation of ELF3 increased expression of ELF3-related genes, enhanced the tubular structure of the tumors, and led to a loss of vimentin. Overall, our results indicate that ELF3 is a key regulator of both epithelial integrity and immune responses in BTC.Significance:Thease finding shows that ELF3 regulates epithelial integrity and host immune responses and functions as a tumor suppressor in biliary tract cancer.

Synovial sarcoma is characterized by variable epithelial differentiation and specific SS18-SSX gene fusions. The diagnosis is primarily based on phenotype, but fusion gene detection is increasingly being considered indispensable, with SS18 break-apart fluorescence in situ hybridization (FISH) being favored in many laboratories. However, SS18 FISH assay produces negative or atypical results in a minority of cases, leaving uncertainties in diagnosis and management. Here, we analyzed this challenging subset of SS18 FISH-negative/atypical synovial sarcoma using RNA sequencing and monoclonal antibodies that recognize SS18-SSX and the SSX C-terminus. Among 99 synovial sarcoma cases that were previously subjected to SS18 break-apart FISH, eight cases were reported as negative and three cases were indeterminate, owing to atypical signal patterns. Three of these 11 tumors (two monophasic and one biphasic) harbored novel EWSR1-SSX1 fusions, were negative for SS18-SSX staining, and were positive for SSX C-terminus staining. One monophasic tumor harbored a novel MN1-SSX1 fusion, and showed negative SS18-SSX expression and positive SSX C-terminus staining. Another monophasic tumor carried an SS18L1-SSX1 fusion, and was weakly positive for SS18-SSX, while SMARCB1 expression was reduced. The presence of these novel and/or rare fusions was confirmed using RT-PCR and Sanger sequencing. EWSR1-SSX1 was further validated by EWSR1 FISH assay. The remaining six tumors (five monophasic and one biphasic) showed strong SS18-SSX expression, and RNA sequencing successfully performed in three cases identified canonical SS18-SSX2 fusions. Based on a DNA methylation-based unsupervised clustering, the tumors with EWSR1-SSX1 and SS18L1-SSX1 clustered with synovial sarcoma, while the MN1-SSX1-positive tumor was not co-clustered despite classic histology and immunoprofile. In summary, we discovered novel and rare SSX1 fusions to non-SS18 genes in synovial sarcoma. The expanded genetic landscape carries significant diagnostic implications and advances our understanding of the oncogenic mechanism.

SMARCB1 is mutated in most rhabdoid tumors (RTs) developing in the kidney (RTK) and various other organs. Focal deletions found in patients with 22q11.2 deletion syndrome show breakpoints within clusters of segmental duplications (SDs), and those in some RTs show breakpoints in the 22q11-q12 region. SDs are known to cause focal deletion mediated by non-allelic homologous recombination. The present study identified SMARCB1 alterations in all 30 RTKs, using SNP array CGH, MLPA, and sequence analyses. Twenty-eight tumors had a total of 51 breakpoints forming focal 22q deletion and/or uniparental disomy (22qUPD), and the other two had compound mutation with no breakpoints in 22q. Twenty-four (47.1%) of the 51 breakpoints were within SDs, and occurred in 16 (53.3%) of the 30 tumors. The association of breakpoints with SDs was found not only in focal deletion, but also in 22qUPD, indicating that SDs mediate the first and second hits (focal deletion) and the second hit (22qUPD) of SMARCB1 alteration. Of the 51 breakpoints, 14 were recurrent, and 10 of the 14 were within SDs, suggesting the presence of hotspots in the 22q11.2 region. One recurrent breakpoint outside SDs resided in SMARCB1, suggesting inactivation of the gene by out-of-frame fusion. The association between SDs and focal deletion has been reported in two other types of cancer. RTKs may be the third example of SD-associated tumors. Thus, the present study indicated that RTKs exploit genomic instability in the 22q11.1-11.2 SDs region, and 22qUPD caused by mitotic recombination may also be mediated by SDs. This article is protected by copyright. All rights reserved.

Two cases of pediatric lung cancer (in 23-month-old and 6-year-old boys) resulting from mother-to-infant transmission of uterine cervical tumors were incidentally detected during routine next-generation sequencing of paired samples of tumor and normal tissue. Spontaneous regression of some lesions in the first child and slow growth of the tumor mass in the second child suggested the existence of alloimmune responses against the transmitted tumors. Immune checkpoint inhibitor therapy with nivolumab led to a strong regression of all remaining tumors in the first child. (Funded by the Japan Agency for Medical Research and Development and others; TOP-GEAR UMIN Clinical Trials Registry number, UMIN000011141.).

BCORL1 encodes a transcriptional corepressor homolog to BCOR. BCORL1 rearrangements have been previously described as rare events, and among them, CREBBP-BCORL1 has been reported only in 2 cases of ossifying fibromyxoid tumors. Herein, we present the first case of diffusely infiltrating glioma with CREBBP-BCORL1 involving a 17-year-old female patient. Histologically, the tumor was composed of a diffusely infiltrative proliferation of small tumor cells with moderate cellularity showing prominent microcystic formation. DNA methylation analysis revealed that the current case and a previously reported anaplastic ependymoma with EP300-BCORL1 were clustered together in close proximity to but distinct from methylation class high-grade neuroepithelial tumor with BCOR alteration. RNA sequencing demonstrated high mRNA expression of not only BCORL1 but BCOR, and the latter was compatible with diffuse nuclear expression of BCOR detected by immunohistochemistry. Our findings suggest that central nervous system tumors with CREBBP/EP300-BCORL1 may exhibit diverse morphologies but form a distinct DNA methylation group and that BCORL1 fusion genes may lead to upregulation of both BCOR and BCORL1.

The CIC-DUX4 translocation is the most common genetic alteration of small round cell sarcomas without EWSR1 rearrangement. These "Ewing-like sarcomas" usually occur in peripheral soft tissues, and rare primary central nervous system (CNS) tumors have been described. We report a rare case of primary spinal intramedullary Ewing-like sarcoma harboring CIC-DUX4 translocation. A 23-year-old man presented with weakness in the extremities. Magnetic resonance imaging revealed a large intramedullary tumor spanning C3-C5 with heterogeneous enhancement following gadolinium administration. Histologically, most of the tumor displayed dense myeloid proliferation composed of medium- to slightly small-sized primitive cells. Postoperatively, he received local adjuvant radiation therapy without tumor progression for 10 months. Target RNA sequencing analysis revealed the CIC-DUX4 fusion gene. Methylation array analysis resulted in a diagnosis of "methylation class CNS Ewing sarcoma family tumor with CIC alteration". Although this tumor lacked characteristic histological features such as lobular structures in association with desmoplastic stroma, relatively uniform nuclei with prominent nucleoli and eosinophilic cytoplasm, which are often found in CIC-rearranged sarcomas of soft tissue, were identified. Recently, many CNS and soft tissue tumors require genetic analysis for precise diagnosis. To consider certain molecular testing, careful histological examination is essential.

With the advent of new molecular diagnostic techniques, retrieving DNA from the formalin-fixed paraffin-embedded (FFPE) tissues has become an essential yet challenging step for efficient downstream processes. Owing to low quality and quantity of DNA retrieved from the FFPE sections, the process is often impractical and needs significant improvements. Here, we established an efficient method for the purification of DNA from FFPE specimens by optimizing incubation temperature, incubation time, and the concentration of a formalin scavenger tris(hydroxymethyl)aminomethane (Tris) for reverse-crosslinking. The optimized method, named “Highly concentrated Tris-mediated DNA extraction” (HiTE), yielded three times the DNA yield per tissue slice compared with a representative DNA extraction kit. Moreover, the use of HiTE-extracted DNA increased the yield of the sequencing library three times and accordingly yielded a log higher and more reproducible sequencing library compared with that obtained using the commonly used commercial kit. The sequencing library prepared from HiTE-extracted FFPE-DNA had longer inserts and produced reads that evenly covered the reference genome. Successful application of HiTE-extracted FFPE-DNA for whole-genome and targeted gene panel sequencing indicates its practical usability.

Biliary tract cancer (BTC) is an aggressive cancer with a poor prognosis partially due to the limited success in developing novel therapies, including molecularly targeted therapies and immunotherapies. Programmed cell death-1 (PD-1) blockade therapy is less effective against BTCs, necessitating further studies to understand the detailed immunological status of the tumor microenvironment (TME) in BTC. Here, we examined the immunological status of the TME in 37 BTCs with early- to late-stage disease, especially focusing on PD-1+CD8+ T cells. PD-1+CD8+ T cells, which are reportedly associated with the clinical response to PD-1 blockade therapy, were frequently observed in early-stage BTC and decreased with disease progression. Imaging mass cytometry for representative PD-1+CD8+TIL-high and -low patients demonstrated that tumor-infiltrating PD-1+CD8+ T cells were localized adjacent to tumor cells, whereas PD-1−CD8+ T cells were detected mainly in the stroma of the TME. In a mouse model, PD-1 expression by tumor-infiltrating CD8+ T cells was higher in smaller tumors and decreased with tumor growth. Consequently, large tumors became resistant to PD-1 blockade, while small tumors containing higher numbers of PD-1+CD8+ T cells were sensitive. We propose the important role of tumor-infiltrating PD-1+CD8+ T cells in anti-tumor immunity and the potential application of PD-1 blockade therapy for early-stage BTC.

The transcription factor Nrf2 plays a crucial role in the anti-oxidative stress response, protection of DNA from injury and DNA repair mechanisms. Nrf2 activity reduces cancer initiation, but how Nrf2 affects whole-genome alterations upon carcinogenic stimulus remains unexplored. Although recent genome-wide analysis using next-generation sequencing revealed landscapes of nucleotide mutations and copy number alterations in various human cancers, genomic changes in murine cancer models have not been thoroughly examined. We elucidated the relationship between Nrf2 expression levels and whole exon mutation patterns using an ethyl-carbamate (urethane)-induced lung carcinogenesis model employing Nrf2-deficient and Keap1-kd mice, the latter of which express high levels of Nrf2. Exome analysis demonstrated that single nucleotide and trinucleotide mutation patterns and the Kras mutational signature differed significantly and were dependent on the expression level of Nrf2. The Nrf2-deficient tumors exhibited fewer copy number alterations relative to the Nrf2-wt and Keap1-kd tumors. The observed trend in genomic alterations likely prevented the Nrf2-deficient tumors from progressing into malignancy. For the first time, we present whole-exome sequencing results for chemically-induced lung tumors in the Nrf2 gain or loss of function mouse models. Our results demonstrate that different Nrf2 expression levels lead to distinct gene mutation patterns that underly different oncogenic mechanisms in each tumor genotype.

CIC-rearranged sarcoma is characterized by round cell undifferentiated histology, frequent expression of ETV4 and WT1, and aggressive behavior. A clinical encounter of a case with CIC-DUX4 fusion and ERG/CD31 co-expression prompted us to systematically investigate ERG and CD31 expression status in 30 archival cases of CIC-rearranged sarcoma. Half (15) of them showed moderate or strong ERG expression in5-100% of tumor cells, among which nine showed heterogeneous membranous CD31 reactivity, including four cases each showing diffuse or strong expression. None of them showed uniformly strong and diffuse ERG/CD31 co-expression; however, three cases were initially interpreted and treated as angiosarcoma without response. Except for smaller superficial tumor enrichment, the clinicopathological characteristics of these nine cases of ERG+/CD31+ CIC-rearranged sarcoma did not differ from those of remaining 21 cases. Five showed focal hemorrhagic clefts/cysts, mimicking vascular spaces. All tumors expressed ETV4 and/or nuclear WT1, and fusion to DUX4 was confirmed in seven cases. Four tumors examined by next-generation sequencing harbored no CIC missense mutations. Using DNA methylation profiling, one CD31+ CIC-rearranged sarcoma was clustered with CD31- CIC-rearranged sarcomas, but distant from angiosarcomas. When compared with epithelioid angiosarcomas lacking CIC rearrangements, ERG+/CD31+ CIC-rearranged sarcomas were distinguished by focal myxoid change and the entire lack of vasoformative architecture. The angiosarcomas were characterized by uniform strong expression of ERG and CD31, but none of them were found positive for ETV4 or nuclear WT1. Heterogeneous ERG/CD31 co-expression in a subset of CIC-rearranged sarcoma is a clinically relevant pitfall for angiosarcoma, as these two diseases are treated differently.

We here document a sarcoma with a recently reported MGA-NUTM1 fusion. A 49-year-old man presented with a nodule in the right lung, which grew to a giant mass in 5 years. The tumor showed uniform oval to spindle cell proliferation in a hypervascular stroma, associated with focal myxoid change and peculiar collagen deposition resembling an osteoid. The tumor showed an undifferentiated phenotype, including negativity for cytokeratin, although it was immunoreactive to BCOR and MUC4, and was initially suspected as BCOR-associated sarcoma. After complete resection, the tumor recurred in the mediastinal lymph node, and the patient died of the disease. RNA sequencing detected MGA (exon 22)-NUTM1 (exon 3), which was confirmed by reverse transcriptase-polymerase chain reaction, Sanger sequencing, fluorescence in situ hybridization, and NUT immunohistochemistry. Clinicopathological features of the present case were similar to some of the reported cases of MGA-NUTM1 sarcomas, suggesting the emergence of a distinct tumor subtype.

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.Significance: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

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 present our novel software, nanomonsv, for detecting somatic structural variations (SVs) using tumor and matched control long-read sequencing data with a single-base resolution. The current version of nanomonsv includes two detection modules, Canonical SV module, and Single breakend SV module. Using paired long-read sequencing data from three cancer and their matched lymphoblastoid lines, we demonstrate that Canonical SV module can identify somatic SVs that can be captured by short-read technologies with higher precision and recall than existing methods. In addition, we have developed a workflow to classify mobile element insertions while elucidating their in-depth properties, such as 5’ truncations, internal inversions, as well as source sites for 3’ transductions. Furthermore, Single breakend SV module enables the detection of complex SVs that can only be identified by long-reads, such as SVs involving highly-repetitive centromeric sequences, and LINE1- and virus-mediated rearrangements. In summary, our approaches applied to cancer long-read sequencing data can reveal various features of somatic SVs and will lead to a better understanding of mutational processes and functional consequences of somatic SVs.

Two recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors. In contrast to K27M mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42 for comparison with gliomas harboring K27M and no mutations in H3F3A and with G34W-mutated bone tumors. G34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs, compared to the other two glioma subgroups. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. They were notably hypermethylated in osteosarcomas with, but not without, G34W mutation. Independent component analysis revealed that G34 mutation-specific components shared a significant similarity between glioma and osteosarcoma, suggesting that G34 mutations exert characteristic methylomic effects regardless of the tumor tissue-of-origin. CRISPR/Cas9-mediated disruption of G34V-allele in KNS-42 cells led to demethylation of a subset of CGIs hypermethylated in G34R-mutated gliomas. These findings will provide a basis for elucidating epigenomic roles of G34 oncohistone in tumorigenesis.

Summary CIC- rearranged sarcoma is a new entity of undifferentiated small round cell sarcoma characterized by chimeric fusions with CIC rearrangement. We report a NUTM2A-CIC fusion sarcoma in a 43-year-old woman who died of rapidly progressive disease. Histologic analysis revealed multinodular proliferation of small round tumor cells with mild nuclear pleomorphism. The sclerotic fibrous septa separated the tumor into multiple nodules. Immunohistochemistry showed that the tumor cells were diffusely positive for vimentin, focally positive for cytokeratin, and negative for CD99 and NKX2.2. Tumor cells were also negative for ETV4, which was recently identified as a specific marker for CIC -rearranged sarcoma. High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded clinical sample unveiled a novel NUTM2A-CIC fusion between NUTM2A exon 7 and CIC exon 12, and fluorescence in situ hybridization identified CIC and NUTM2A split signals. This case shared several clinicopathological findings with previously reported CIC -rearranged cases. We recognized the tumor as a genetically distinct variant of CIC -rearranged sarcomas with a novel NUTM2A-CIC fusion.

Aims BCOR gene alteration is a genetic signature of rare subsets of sarcomas. Most BCOR-associated sarcomas thus far reported are in the pediatric population, except for uterine sarcomas. We studied seven cases of BCOR-associated non-uterine sarcomas in adult patients. Methods and results The patients were four men and three women ranging from 26 to 71 years in age. Three tumors, two of which primarily affected the kidney, showed BCOR-CCNB3. One tumor with a ZC3H7B-BCOR occurred in the chest wall, and a tumor with a novel CIITA-BCOR was found in the sinonasal tract. Two tumors in the lung and breast harbored exon 15 internal tandem duplications of BCOR, a highly unexpected observation in this age group. All seven sarcomas consisted of dense proliferations of uniform round to spindle cells with fine chromatin within vascular stroma. BCOR-CCNB3 sarcomas showed swirling fascicular growth. The tumor with the ZC3H7B-BCOR fusion showed a multinodular growth of spindle cells, and the tumors with the CIITA-BCOR fusion showed palisading of oval cells. Both tumors with BCOR internal tandem duplication demonstrated nested to palisading growth of round cells within sclerotic non-myxoid stroma. All seven sarcomas diffusely expressed BCOR and SATB2 immunohistochemically, with all three BCOR-CCNB3 sarcomas being immunopositive for CCNB3. BCOR alterations were confirmed by RNA sequencing, polymerase chain reaction, Sanger sequencing, and/or fluorescence in situ hybridization. Conclusions This study expands the clinicopathologic and molecular spectrum of BCOR-associated sarcomas, and emphasizes the importance of being aware of this entity in the differential diagnosis of adult non-uterine sarcomas.

Aim To characterise unclassifiable sarcomas by use of a combined histological and molecular approach. Methods and results Using RNA sequencing, we identified in-frame fusions involving KMT2A (MLL) in two cases. Case 1 was a 20-year-old woman with a deep soft tissue mass in the thigh. The tumour consisted of monomorphic round, epithelioid and spindle cells in a highly sclerotic background that were focally immunopositive for CD34, CD31, and ERG. Case 2 was a 30-year-old woman with a tumour that affected the femur and surrounding soft tissue. The tumour consisted of monomorphic round to spindle cells that were immunopositive for BCOR, Wilms tumour 1, and NKX2-2. Both tumours were aggressive and had metastasised to the lung; both patients died within a few years. RNA sequencing identified a YAP1 (exon 5)-KMT2A (exon 4) fusion in case 1 and a VIM (exon 4)-KMT2A (exon 2) fusion in case 2, both of which were confirmed by reverse transcription polymerase chain reaction, Sanger sequencing, and fluorescence in-situ hybridisation. The fusion protein structure was different from that in acute leukaemia, suggesting a novel oncogenic mechanism. Conclusions KMT2A fusions account for a subset of aggressive unclassifiable sarcomas in young adults. Although it is presently unclear whether these sarcomas belong to a single group, the well-established role of KMT2A fusions as drivers of acute leukaemia and a recent publication regarding identification of YAP1-KMT2A in one unclassifiable sarcoma support the significance of these fusions. Further studies on additional cases are necessary to fully understand the clinicopathological and molecular aspects of KMT2A-rearranged sarcomas.

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.

Osteosarcoma is the most common primary malignancy in bone. Patients who respond poorly to induction chemotherapy are at higher risk of adverse prognosis. The molecular basis for such poor prognosis remains unclear. We investigated miRNA expression in eight open biopsy samples to identify miRNAs predictive of response to induction chemotherapy and thus maybe used for risk stratification therapy. The samples were obtained from four patients with inferior necrosis (Huvos I/II) and four patients with superior necrosis (Huvos III/IV) following induction chemotherapy. We found six miRNAs, including miR-125b and miR-100, that were differentially expressed > 2-fold (p<0.05) in patients who respond poorly to treatment. The association between poor prognosis and the abundance of miR-125b and miR-100 was confirmed by quantitative reverse transcriptase-polymerase chain reaction in 20 additional osteosarcoma patients. Accordingly, overexpression of miR-125b and miR-100 in three osteosarcoma cell lines enhanced cell proliferation, invasiveness, and resistance to chemotherapeutic drugs such as methotrexate, doxorubicin, and cisplatin. In addition, overexpression of miR-125b blocked the ability of these chemotherapy agents to induce apoptosis. As open biopsy is routinely performed to diagnose osteosarcoma, levels of miR-125b and miR-100 in these samples may be used as basis for risk stratification therapy.

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., Myxofibrosarcoma occurs in adults and is associated with high local relapse. Here, based on exome/transcriptome sequencing and DNA methylation analysis, the authors identify driver genes and methylation clusters associated with unique combinations of mutations, outcomes, and immune cell compositions.

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., Genomic aberrations contribute to the development of cancer; however, their interdependence remains poorly understood. Here the authors analyze liver cancer samples to find correlation between epigenetic features and genetic aberrations including somatic substitutions, mutation signatures, and HBV integration sites.

The incidence of biliary tract cancer (BTC), including intrahepatic (ICC) and extrahepatic (ECC) cholangiocarcinoma and gallbladder cancer, has increased globally; however, no effective targeted molecular therapies have been approved at the present time. Here we molecularly characterized 260 BTCs and uncovered spectra of genomic alterations that included new potential therapeutic targets. Gradient spectra of mutational signatures with a higher burden of the APOBEC-associated mutation signature were observed in gallbladder cancer and ECC. Thirty-two significantly altered genes, including ELF3, were identified, and nearly 40% of cases harbored targetable genetic alterations. Gene fusions involving FGFR2 and PRKACA or PRKACB preferentially occurred in ICC and ECC, respectively, and the subtype-associated prevalence of actionable growth factor-mediated signals was noteworthy. The subgroup with the poorest prognosis had significant enrichment of hypermutated tumors and a characteristic elevation in the expression of immune checkpoint molecules. Accordingly, immune-modulating therapies might also be potentially promising options for these patients.

Aims Approximately 60–70% of high-grade round-cell sarcomas that lack the EWSR1 rearrangement harbour a rearrangement of the CIC gene, most commonly CIC-DUX4. Recent studies have established that CIC-rearranged sarcomas constitute a distinct group characterised by recognisable histology and immunoprofiles, such as positivity for ETV4 and WT1 and negativity for NKX2.2. Although these sarcomas are increasingly diagnosed in practice by fluorescence in situ hybridisation (FISH) with CIC break-apart probes, the optimal modality to diagnose these sarcomas has not been determined. In this study, we describe 4 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 ETV4 and WT1 and negative for NKX2.2. Although FISH showed non-atypical negative signals for CIC rearrangement, high-throughput RNA sequencing identified CIC-DUX4 and its fusion breakpoint in all cases. Their clinical and histological findings as well as fusion points determined by RNA sequencing did not significantly differ from those of 9 FISH-positive CIC-DUX4 sarcoma cases. We estimated that the FISH false-negative rate for CIC-rearranged sarcomas was 14%. Although neither histology nor immunoprofiles (e.g., ETV4 and WT1) are entirely sensitive or specific for CIC-rearranged sarcomas, the observation that these 4 cases were successfully identified by such phenotypes suggested their practical utility. Conclusions CIC break-apart FISH assays missed a significant minority of CIC-DUX4 sarcomas, and full awareness of typical morphology and judicious immunohistochemical workups, including analyses of ETV4 and WT1, should complement diagnostic assessment. This article is protected by copyright. All rights reserved.

Chondrosarcoma is the second most frequent malignant bone tumor. However, the etiological background of chondrosarcomagenesis remains largely unknown, along with details on molecular alterations and potential therapeutic targets. Massively parallel paired-end sequencing of whole genomes of 10 primary chondrosarcomas revealed that the process of accumulation of somatic mutations is homogeneous irrespective of the pathological subtype or the presence of IDH1 mutations, is unique among a range of cancer types, and shares significant commonalities with that of prostate cancer. Clusters of structural alterations localized within a single chromosome were observed in four cases. Combined with targeted resequencing of additional cartilaginous tumor cohorts, we identified somatic alterations of the COL2A1 gene, which encodes an essential extracellular matrix protein in chondroskeletal development, in 19.3% of chondrosarcoma and 31.7% of enchondroma cases. Epigenetic regulators (IDH1 and YEATS2) and an activin/BMP signal component (ACVR2A) were recurrently altered. Furthermore, a novel FN1-ACVR2A fusion transcript was observed in both chondrosarcoma and osteochondromatosis cases. With the characteristic accumulative process of somatic changes as a background, molecular defects in chondrogenesis and aberrant epigenetic control are primarily causative of both benign and malignant cartilaginous tumors.

Cholangiocarcinoma is an intractable cancer, with limited therapeutic options, in which the molecular mechanisms underlying tumor development remain poorly understood. Identification of a novel driver oncogene and applying it to targeted therapies for molecularly defined cancers might lead to improvements in the outcome of patients. We performed massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients without KRAS/BRAF/ROS1 alterations and identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1. In reverse-transcriptase polymerase chain reaction (RT-PCR) screening, the FGFR2 fusion was detected in nine patients with cholangiocarcinoma (9/102), exclusively in the intrahepatic subtype (9/66, 13.6%), rarely in colorectal (1/149) and hepatocellular carcinoma (1/96), and none in gastric cancer (0/212). The rearrangements were mutually exclusive with KRAS/BRAF mutations. Expression of the fusion kinases in NIH3T3 cells activated MAPK and conferred anchorage-independent growth and in vivo tumorigenesis of subcutaneous transplanted cells in immune-compromised mice. This transforming ability was attributable to its kinase activity. Treatment with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074 effectively suppressed transformation. Conclusion: FGFR2 fusions occur in 13.6% of intrahepatic cholangiocarcinoma. The expression pattern of these fusions in association with sensitivity to FGFR inhibitors warrant a new molecular classification of cholangiocarcinoma and suggest a new therapeutic approach to the disease. (Hepatology 2014;59:1427-1434)

Recent discovery of ROS1 gene fusion in a subset of lung cancers has raised clinical interest, because ROS1 fusion-positive cancers are reportedly sensitive to kinase inhibitors. To better understand these tumors, we examined 799 surgically resected non-small cell lung cancers by reverse transcriptase polymerase chain reaction and identified 15 tumors harboring ROS1 fusion transcripts (2.5% of adenocarcinomas). The most frequent fusion partner was CD74 followed by EZR. The affected patients were often younger nonsmoking female individuals, and they had overall survival rates similar to those of the ROS1 fusion-negative cancer patients. All the ROS1 fusion-positive tumors were adenocarcinomas except 1, which was an adenosquamous carcinoma. Histologic examination identified an at least focal presence of either solid growth with signet-ring cells or cribriform architecture with abundant extracellular mucus in 53% of the cases. These 2 patterns are reportedly also characteristic of anaplastic lymphoma kinase (ALK)-rearranged lung cancers, and our data suggest a phenotypic resemblance between the ROS1-rearranged and ALK-rearranged tumors. All tumors except 1 were immunoreactive to thyroid transcription factor-1. Fluorescence in situ hybridization using ROS1 break-apart probes revealed positive rearrangement signals in 23% to 93% of the tumor cells in ROS1 fusion-positive cancers, which were readily distinguished using a 15% cutoff value from 50 ROS1 fusion-negative tumors tested, which showed 0% to 6% rearrangement signals. However, this perfect test performance was achieved only when isolated 3' signals were included along with classic split signals in the definition of rearrangement positivity. Fluorescence in situ hybridization signal patterns were unrelated to 5' fusion partner genes. All ROS1 fusion-positive tumors lacked alteration of EGFR, KRAS, HER2, ALK, and RET genes.

Germ cell tumors (GCTs) are thought to arise from primordial germ cells (PGCs) that undergo epigenetic reprogramming. To explore the mechanisms of GCT formation, we analyzed single-nucleotide polymorphism array comparative genomic hybridization patterns and the methylation status of 15 tumor suppressor genes (TSGs) and differentially methylated regions (DMRs) of two imprinted genes, H19 and SNRPN, in 28 children with GCTs. Three GCTs with 25-26 segmental uniparental disomies (UPDs), heterozygous centromeric regions, and a highly methylated SNRPN DMR may have occurred through meiosis I error. Three other GCTs with whole UPD and homozygous centromeric regions of all chromosomes may have occurred through endoreduplication of a haploid set in an ovum or testis. The other 22 GCTs had heterozygous centromeric regions of all chromosomes and no or a small number of segmental or whole UPDs and may have developed from premeiotic PGCs before imprint erasure or a reestablishment of imprinting. Gain and amplification of 3p24-p22 and 20q13-q13, and loss and UPD of 1p36-p35, 4q21-q21, 5q11-q13, and 6q26-qter were found in five or more tumors. 1p36-p35 loss was frequent, and found in 19 tumors; RUNX3 residing at 1p36 was methylated in the promoter regions of 16 tumors. Two yolk sac tumors with many segmental UPDs or whole UPD of all chromosomes had gain of 20q13-q13 and loss of 1p36-p35, and seven or eight methylated TSGs. These genetic and epigenetic alterations may have caused malignant transformation because they were rarely found in teratomas with segmental or whole UPDs.

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