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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.

Structural Variations across 71 WGS CCAs.

Summary of CCA Alterations.

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.

DNA methylation is an important epigenetic modification that is widely conserved across animal genomes. It is widely accepted that DNA methylation patterns can change in a context-dependent manner, including in response to changing environmental parameters. However, this phenomenon has not been analyzed in animal livestock yet, where it holds major potential for biomarker development. Building on the previous identification of population-specific DNA methylation in clonal marbled crayfish, we have now generated numerous base-resolution methylomes to analyze location-specific DNA methylation patterns. We also describe the time-dependent conversion of epigenetic signatures upon transfer from one environment to another. We further demonstrate production system-specific methylation signatures in shrimp, river-specific signatures in salmon and farm-specific signatures in chicken. Together, our findings provide a detailed resource for epigenetic variation in animal livestock and suggest the possibility for origin tracing of animal products by epigenetic fingerprinting.

Breast fibroepithelial lesions (FELs) encompass the common fibroadenoma (FA) and relatively rare phyllodes tumour (PT); the latter entity is usually classified as benign, borderline or malignant. Intratumoural heterogeneity is frequently present in these tumours, making accurate histologic evaluation challenging. Despite their rarity, PTs are an important clinical problem due to their propensity for recurrence and, in the case of malignant PT, metastasis. Surgical excision is the mainstay of management. Recent work has uncovered myriad genetic alterations in breast FELs. In this study, exome sequencing was performed on seven cases of morphologically heterogeneous breast FELs, including FAs, PTs of all grades, and a case of metaplastic spindle cell carcinoma arising in PT, in order to elucidate their intratumoural genetic repertoire. Gene mutations identified encompassed cell signalling, tumour suppressor, DNA repair and cell cycle regulating pathways. Mutations common to multiple tumour regions generally showed higher variant allele frequency. Frequent mutations included MED12, TP53, RARA and PIK3CA. Histological observations of increased cellular density and pleomorphism correlated with mutational burden. Phylogenetic analyses revealed disparate pathways of possible tumour progression. In summary, histological heterogeneity correlated with genetic changes in breast FELs.

BackgroundKnown collectively as breast fibroepithelial lesions (FELs), the common fibroadenomas (FAs) and the rarer phyllodes tumors (PTs) are a heterogenous group of biphasic neoplasms. Owing to limited tissue availability, inter-observer variability, overlapping histological features and heterogeneity of these lesions, diagnosing them accurately on core biopsies is challenging. As the choice management option depends on the histological diagnosis; a novel 16-gene panel assay was developed to improve the accuracy of preoperative diagnosis on core biopsy specimens.MethodsUsing this 16-gene panel, targeted amplicon-based sequencing was performed on 275 formalin-fixed, paraffin-embedded (FFPE) breast FEL specimens, archived at the Singapore General Hospital, from 2008 to 2012.ResultsIn total, 167 FAs, 24 benign, 14 borderline and 6 malignant PTs, were profiled. Compared to FAs, PTs had significantly higher mutation rates in theTERTpromoter (p RARA(p FLNA,RB1andTP53(p = 0.002, 0.020 and 0.018, respectively). In addition to a higher mutational count (p TERTpromoter (p p = 0.001, A multivariate logistic regression model was built using these as variables and a predictive scoring system was developed. It classifies a FEL at low or high risk (score p = 0.945) and is significant in predicting PTs (p ConclusionThis novel study demonstrates the ability to extract DNA of sufficient quality and quantity for targeted sequencing from FFPE breast core biopsy specimens, along with their successful characterization and profiling using our customized 16-gene panel. Prospective work includes validating the utility of this promising 16-gene panel assay as an adjunctive diagnostic tool in clinical practice.

Epitheliotropic intestinal T-cell lymphoma (EITL, also known as type II enteropathy-associated T-cell lymphoma) is an aggressive intestinal disease with poor prognosis and its molecular alterations have not been comprehensively characterized. We aimed to identify actionable easy-to-screen alterations that would allow better diagnostics and/or treatment of this deadly disease. By performing whole-exome sequencing of four EITL tumor-normal pairs, followed by amplicon deep sequencing of 42 tumor samples, frequent alterations of the JAK-STAT and G-protein-coupled receptor (GPCR) signaling pathways were discovered in a large portion of samples. Specifically, STAT5B was mutated in a remarkable 63% of cases, JAK3 in 35% and GNAI2 in 24%, with the majority occurring at known activating hotspots in key functional domains. Moreover, STAT5B locus carried copy-neutral loss of heterozygosity resulting in the duplication of the mutant copy, suggesting the importance of mutant STAT5B dosage for the development of EITL. Dysregulation of the JAK-STAT and GPCR pathways was also supported by gene expression profiling and further verified in patient tumor samples. In vitro overexpression of GNAI2 mutants led to the upregulation of pERK1/2, a member of MEK-ERK pathway. Notably, inhibitors of both JAK-STAT and MEK-ERK pathways effectively reduced viability of patient-derived primary EITL cells, indicating potential therapeutic strategies for this neoplasm with no effective treatment currently available.

Natural-killer/T-cell lymphoma (NKTL) is a rare subset of non-Hodgkin lymphoma that demonstrates a unique geographic distribution, with higher prevalence in Asia compared to the West1. While cure remains achievable in early-stage disease, the prognosis for advanced-stage NKTL is dismal2. Recently, some progress has been made in uncovering the molecular pathogenesis of NKTL. In a genome-wide association study, strong correlations between HLA-DPB1 single-nucleotide polymorphisms and NKTL susceptibility were discovered, implicating altered antigen processing and presentation to CD4-positive T-lymphocytes in this Epstein-Barr virus (EBV)-associated malignancy3. Next generation sequencing also revealed recurrent somatic mutations such as TP53, JAK3, STAT3 and DDX3X in NKTL4,5. In this paper, we report a pair of male siblings from a non-consanguineous Chinese family who were diagnosed with NKTL, and provide initial evidence for novel recessive germline mutations in FAM160A1 identified through next-generation sequencing. The index patient was 35 years old, when he presented with nasal blockage in March 2013. 18-FDG-PET/CT imaging revealed an 18-FDG-avid nasal mass infiltrating into the palate, as well as enlarged cervical lymph nodes. Biopsy of the mass showed abnormal lymphoid cells positive for CD56 by immunohistochemistry (IHC) as well as EBV-encoded RNA (EBER) by in-situ hybridization. He was diagnosed with stage IIA extranodal NKTL, nasal type, and treated with 4 cycles of bortezomib-GIFOX (gemcitabine, ifosfamide and oxaliplatin) as part of a clinical trial followed by radiotherapy to the nasal region. He had primary-refractory disease and was further treated with 4 cycles of SMILE (dexamethasone, methotrexate, ifosfamide, l-asparaginase, and etoposide) followed by high-dose chemotherapy and autologous stem cell transplantation. He progressed and received ruxolitinib off-label, followed by RAD001 (mTOR inhibitor) and LBH589B (histone deacetylase inhibitor) as part of another clinical trial. He then had radiotherapy to an ulcerating penile lesion before he died of progressive disease 27 months after diagnosis (Supplementary Table 1). His younger brother was 18 years old, when diagnosed with NKTL affecting the nasal floor in 1998 following a bout of epistaxis. He received 6 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) with high-dose methotrexate and went into complete remission. Three years later he was diagnosed with chronic myeloid leukemia, for which he received hydroxyurea. In 2004, he had a relapse of NKTL and received ESHAP (etoposide, prednisolone, cytarabine and cisplatin), total body irradiation and allogeneic bone marrow transplant. He died of transplant-related complications 6 years from diagnosis. These siblings have an older brother unaffected by any hematological malignancy. Their father died of a non-malignant condition in his fifties and their mother remains well at the age of 68 years. Among their first-degree relatives, none were known to be inflicted with hematologic malignancies (Fig. ​(Fig.11). Open in a separate window Fig. 1 Clinical characterization of brothers with familial NKTL. a 18-FDG-PET/CT image depicting a large nasal mass infiltrating into the palate, of which biopsy showed abnormal lymphoid cells positive for Epstein-Barr virus encoded RNA (EBER) by in situ hybridization. b, c Inheritance modelling identified homozygous germline mutations of FAM160A1 c.2827 C > T in both affected brothers, heterozygous carriage in their mother and paternal aunt, and homozygous wildtype in their healthy older brother. d, e The non-synonymous substitution at FAM160A1 c.2827 C > T results in an amino-acid alteration from arginine to cysteine (p.R943C). Patterns and frequencies of known mutations in other cancer types are shown

Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL. ASTAR (Agency for Sci., Tech. and Research, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore)

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.

The impact of different carcinogenic exposures on the specific patterns of somatic mutation in human tumors remains unclear. To address this issue, we profiled 209 cholangiocarcinomas (CCAs) from Asia and Europe, including 108 cases caused by infection with the liver fluke Opisthorchis viverrini and 101 cases caused by non-O. viverrini-related etiologies. Whole-exome sequencing (n = 15) and prevalence screening (n = 194) identified recurrent somatic mutations in BAP1 and ARID1A, neither of which, to our knowledge, has previously been reported to be mutated in CCA. Comparisons between intrahepatic O. viverrini-related and non-O. viverrini-related CCAs demonstrated statistically significant differences in mutation patterns: BAP1, IDH1 and IDH2 were more frequently mutated in non-O. viverrini CCAs, whereas TP53 mutations showed the reciprocal pattern. Functional studies demonstrated tumor suppressive functions for BAP1 and ARID1A, establishing the role of chromatin modulators in CCA pathogenesis. These findings indicate that different causative etiologies may induce distinct somatic alterations, even within the same tumor type.

Trithorax-like group complex containing KDM6A acts antagonistically to Polycomb-repressive complex 2 (PRC2) containing EZH2 in maintaining the dynamics of the repression and activation of gene expression through H3K27 methylation. In urothelial bladder carcinoma, KDM6A (a H3K27 demethylase) is frequently mutated, but its functional consequences and therapeutic targetability remain unknown. About 70% of KDM6A mutations resulted in a total loss of expression and a consequent loss of demethylase function in this cancer type. Further transcriptome analysis found multiple deregulated pathways, especially PRC2/EZH2, in KDM6A-mutated urothelial bladder carcinoma. Chromatin immunoprecipitation sequencing analysis revealed enrichment of H3K27me3 at specific loci in KDM6A-null cells, including PRC2/EZH2 and their downstream targets. Consequently, we targeted EZH2 (an H3K27 methylase) and demonstrated that KDM6A-null urothelial bladder carcinoma cell lines were sensitive to EZH2 inhibition. Loss- and gain-of-function assays confirmed that cells with loss of KDM6A are vulnerable to EZH2. IGFBP3, a direct KDM6A/EZH2/H3K27me3 target, was up-regulated by EZH2 inhibition and contributed to the observed EZH2-dependent growth suppression in KDM6A-null cell lines. EZH2 inhibition delayed tumor onset in KDM6A-null cells and caused regression of KDM6A-null bladder tumors in both patient-derived and cell line xenograft models. In summary, our study demonstrates that inactivating mutations of KDM6A, which are common in urothelial bladder carcinoma, are potentially targetable by inhibiting EZH2.

7549 Background: Natural killer/T-cell lymphoma (NKTL) is a rare type of non-Hodgkin lymphoma that occurs more frequently in East Asia and Latin America and is associated with Epstein–Barr virus infection. Recent whole-exome sequencing studies in NKTL have reported recurrent somatic mutations in genes associated with JAK-STAT pathway, however the role of aberrant JAK-STAT signaling in tumor immune escape through PD-L1 regulation is unclear. Methods: To determine the prevalence of JAK-STAT pathway alteration in NKTL, we performed targeted sequencing of 188 genes associated with JAK-STAT pathway in 109 NKTL (22 Singapore cases, 79 China cases and 8 cell lines). Single nucleotide variants and micro-indels were called using Freebayes and candidate variants annotated using ANNOVAR. Ba/F3 model system was used to test the transformation capacity of identified variants. Cell lines were evaluated for PD-L1 expression by immunoblotting and flow cytometry. Tissue microarrays were examined for p-STAT3 and PD-L1 expression by immunohistochemistry. Results: We identified a total of 284 non-synonymous somatic mutations candidates in 114 genes, including 243 missense, 10 nonsense, 4 splice-site and 27 indel mutations. Recurrent mutations were most frequently located in STAT3 (25/109 cases, 23%) followed by TP53 (16/109 cases, 16%) and JAK3 (8/109 cases, 7%). A total of 18 STAT3 variants were identified including known hotspot mutations and novel mutations in the SH2, coiled coil and DNA-binding domains. Characterization of novel E616K mutant residing in the SH2 domain showed that E616K conferred IL3 independent growth to Ba/F3 cells, increased STAT3 phosphorylation and PD-L1 expression. Consistent with these findings, PD-L1 was over expressed in cell lines harboring STAT3 mutations. A positive correlation between PD-L1 and p-STAT3 expression was also observed in tumor tissue (R = 0.51, P = 0.02). Conclusions: We characterized a novel activating STAT3 mutant and demonstrated its ability to drive PD-L1 expression, which may promote tumor evasion from the antitumor immune response. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising and novel therapeutic approach for NKTL in the future.

Introduction: Natural-killer/T-cell lymphoma (NKTL) is a rare subset of non-Hodgkin's lymphoma that is more prevalent in Asia than in the West. We report a case of two brothers who were diagnosed with NKTL and died of their disease at 23 and 37 years old respectively, and further investigations performed to determine the underlying genetic basis to their disease. Methods Both patients were recruited into the Singapore lymphoma study where retrieval of their clinical data and biospecimens had been approved by the Singhealth instititutional review board. Their clinical courses are described using information from electronic medical records. For the older brother we extracted tumor DNA from a patient-derived xenograft (PDX), and DNA from a buccal swab and peripheral blood (PB) sample. In the younger brother DNA was extracted from normal and tumor tissue from archived formalin fixed paraffin embedded (FFPE) samples. DNA from the unaffected mother, older brother and paternal aunt were obtained from PB and buccal swab samples. We performed whole exome sequencing (WES) on the younger affected brother's tumor, the older affected brother's, mother's and unaffected brother's blood sample. Subsequent analysis of variants took into account autosomal recessive (AR), X-linked recessive (XR) and autosomal dominant (AD) inheritance models. Candidate genes are validated using Sanger sequencing. Microarray on the older affected brother and 12 sporadic NKTL cases were analyzed. Results The older affected brother was 35 years old when he presented with left nasal blockage in March 2013. He was diagnosed with stage IIA NKTL and treated with 4 cycles of bortezomib-GIFOX as part of a clinical trial followed by radiotherapy to the nasal region. He had primary refractory disease and was further treated with 4 cycles of SMILE followed by high dose chemotherapy and autologous stem cell transplantation (HDC/ASCT). He progressed and received off-label ruxolitinib, and everolimus/HDAC inhibitor as part of a clinical trial. He then had radiotherapy to an ulcerating penile lesion before he died of progressive disease 27months after diagnosis. The younger affected brother was 18 years old when he was diagnosed with NKTL in 1998. He received 6 cycles of CHOP with intravenous high-dose methotrexate and intrathecal methotrexate and went into remission. Three years later, he was diagnosed with chronic myeloid leukemia and 6 years later, he had a relapse of NKTL for which he received ESHAP, TBI and allogeneic bone marrow transplant. He died of transplant related complication 6 years after diagnosis. They both have an older unaffected brother. Their father died of non-malignant condition in his fifties and their mother remains well. They have no other relatives with hematologic malignancy. We have identified several candidate variants and genes on WES that are being validated and functional studies are ongoing. GEP shows 234 genes that are significantly upregulated in the affected brother's tumor compared to 12 sporadic NKTL tumors including p53 and 12 genes that were significantly downregulated including CTLA4 and virus entry pathways. Conclusion: We describe a case of two brothers with a rare lymphoma and the ongoing efforts in elucidating the genetic basis of their disease. To the best of our knowledge this is the first study of its kind to be presented. Disclosures No relevant conflicts of interest to declare.

Regulatory regions maintain nucleosome-depleted, open chromatin status but simultaneously require the presence of nucleosomes for specific histone modifications. It remains unclear how these can be achieved for proper regulatory function. Here we demonstrate that nucleosomes positioned within accessible chromatin regions near the boundaries provide platforms for histone modifications while preventing the occlusion of regulatory elements. These boundary nucleosomes were particularly enriched for active or poised regulatory marks in human, such as histone acetylations, H3K4 methylations, H3K9me3, H3K79me2, and H4K20me1. Additionally, we found that based on a genome-wide profiling of ∼100 recombinant yeast strains, the location of open chromatin borders tends to vary mostly within 150 bp upon genetic perturbation whereas this positional variation increases in proportion to the sequence preferences of the underlying DNA for nucleosome formation. More than 40% of the local boundary shifts were associated with genetic variation in cis- or trans-acting factors. A sizeable fraction of the identified genetic factors was also associated with nearby gene expression, which was correlated with the distance between the transcription start site (tss) and the boundary that faces the tss. Taken together, the variation in the width of accessible chromatin regions may arise in conjunction with the modulation of the boundary nucleosomes by post-translational modifications or by chromatin regulators and in association with the activity of nearby gene transcription., Author Summary Open chromatin formation and regulation are intimately coupled with nucleosome remodelling and modification. Regulatory regions such as promoters and enhancers maintain nucleosome-free, open chromatin states whilst at the same time the presence of nucleosomes is required for specific histone modifications. In this work, we carried out detailed analyses of our data of open chromatin maps for ∼100 different yeast strains and whole-genome nucleosome occupancy along with the public data of open chromatin and nucleosome positioning in human generated in the ENCODE project. We observed nucleosomes positioned within accessible chromatin regions near their boundaries. These boundary nucleosomes appeared to carry various histone methylations without hampering the binding of DNA regulators and sequence preferences for these nucleosomes were associated with variation in the width of accessible chromatin. The end positions of open chromatin domains, particularly with high intrinsic preferences for nucleosome formation, were more flexible than the middle point, changing mostly within 150 bp upon genetic perturbation. By using quantitative trait loci (QTL) mapping, we identified genetic variants that are associated with the variation in the width of open chromatin and examined its relationship with nearby gene expression.