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• Determining genome-wide binding of ETS2 by CUT&RUN in primary human macrophages

  This dataset consists of sequencing data from an ETS2 CUT&RUN experiment in primary inflammatory (TPP) macrophages (n = 2). Pre-cultured TPP macrophages were harvested and processed immediately using the CUT&RUN Assay kit (Cell Signaling) according to the manufacturer’s instructions with the following modifications (essentially, avoiding the use of ConA-coated beads). Anti-ETS2 (ThermoFisher) or IgG control (Cell Signaling) antibodies were used for targeted digestion of chromatin. For each donor, 5x10^5 cells were pelleted, washed in Wash Buffer, and resuspended in Antibody Binding buffer. Cells were incubated with antibodies (1:100 dilution for anti-ETS2) for 2h at 4°C. After washing in Digitonin Buffer, cells were incubated with pA/G-MNase for 1h at 4°C. Cells were washed twice in Digitonin Buffer, resuspended in the same buffer and cooled for 5 minutes on ice. Calcium chloride was added to activate pA/G-MNase digestion and cells were incubated for 30 minutes at 4°C before Stop Buffer was added, and cells were incubated for 10 min at 37°C to release cleaved chromatin fragments. Supernatants were collected by centrifugation and DNA extracted using DNA Purification Buffers and Spin Columns (Cell Signaling). Library preparation was performed according to a protocols.IO protocol (dx.doi.org/10.17504/protocols.io.bagaibse) using the NEBNext Ultra II DNA Library Prep Kit. Size selection was performed using AMPure XP beads (Beckman Coulter) and fragment sizes were determined using an Agilent 2100 Bioanalyzer (High Sensitivity DNA kit). Equimolar pools of indexed libraries were sequenced on an Illumina NovaSeq 6000 (100bp PE reads). Raw data are provided as 100 bp paired-end Illumina reads.

  Dataset EGAD00001011349

• Diverse mutational landscapes in human lymphocytes

  A lymphocyte suffers many threats to its genome, including programmed mutation during differentiation, antigen-driven proliferation and residency in diverse microenvironments. After developing protocols for single-cell lymphocyte expansions, we sequenced whole genomes from 717 normal naive and memory B and T lymphocytes and hematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells – the extra mutations were mostly acquired during differentiation, with burdens higher in memory than naive lymphocytes, although T cells also had a higher rate of mutation accumulation throughout life. Off-target effects of immunological diversification accounted for most of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every one on-target IGV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than stem cells, with ~15% of deletions being attributable to off-target RAG activity. Mutational processes associated with ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory lymphocytes. The mutation burden and signatures of normal B lymphocytes were broadly comparable to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

  Dataset EGAD00001008107

• Targeted sequencing data for various human bulk tissues

  To estimate the contribution of early embryogenic cell lineages in adult tissues, we performed deep targeted sequencing on 379 bulk tissues from various organs of the five individuals (DB3, DB6, DB8, DB9, DB10). Of the 441 early embryonic mutations targeted, 411 mutations could have high-quality baits designed for them. DNA libraries were prepared by SureSelectXT Library Prep Kit (Agilent), hybridized to the appropriate capture panel, multiplexed on flow cells, and subjected to paired-end sequencing (150-bp reads) on the NovaSeq 6000 platform (Illumina) with a mean ~2,900x depth of coverage for the early mutations. Sequence reads were trimmed and mapped to the human reference genome (GRCh37) using the BWA-MEM algorithm.

  Dataset EGAD00001007704

• H3K27ac ChIP-seq in primary inflammatory macrophages from 2 minor allele homozygotes and 2 major allele homozygotes at rs2836882

  This dataset consists of H3K27ac ChIP-sequencing in inflammatory (TPP) macrophages from 2 minor allele homozygotes and 2 major allele homozygotes at rs2836882. Monocytes were positively selected from PBMC using CD14 Microbeads and inflammatory macrophage differentiation performed using conditions that model chronic inflammation (TPP): 3 days GM-CSF (50ng/mL) followed by 3 days GM-CSF, TNFa (50ng/mL), PGE2 (1mg/mL), and Pam3CSK4 (1mg/mL). After harvesting, cells were cross-linked, quenched, lysed, and sheared. Immunoprecipitation of histone-DNA complexes was performed overnight at 4C with rotation using an anti-H3K27ac antibody and the SimpleChIP Plus Sonication ChIP kit (Cell Signaling Technology). Following reverse cross-linking, 50ng of immunoprecipitated DNA or input DNA were used to prepare sequencing libraries using the iDeal Library Preparation kit (Diagenode), according to manufacturer instructions. 10 PCR cycles were used for the amplification step and size selection was not performed. The quality and molarity of all libraries was assessed using a BioAnalyzer 2100 (Agilent) and the libraries were sequenced in pools of 8, with each pool being sequenced in 2 lanes of an Illumina HiSeq2500 high output flow-cell (50bp, single-end reads). Raw data are provided as raw and aligned single-end sequencing reads from H3K27ac-bound DNA and the input chromatin. Raw reads were trimmed using Trim Galore and aligned to the reference human genome (hg19) using Burrows-Wheeler Aligner (v0.7.12) with default parameters. Aligned reads were converted to BAM files, sorted, and technical duplicates merged before indexing – all using SAMtools (v1.4). PCR duplicates were identified using Picard tools (v2.18.1) and removed together with unmapped reads using SAMtools (v1.4). The resulting BAM files were re-sorted and indexed after filtering.

  Dataset EGAD00001011351

• HGSC WGS for the study Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes

  HGSC cases in the OvCaRe and CRCHUM Tumour Banks were selected according to the following criteria: (i) were administered platinum taxane based therapy; (ii) relapsed within 12 months (365 days) or had at least longer than 4.5 years (1642.5 days) follow-up data; (iii) had at least 50% tumour content by H&E staining and expert pathology review. All cases were re-reviewed by expert pathologists to confirm the diagnosis of HGSC. Germline BRCA1 and BRCA2 was determined for all patients through hereditary cancer screening programs. The design of cases selection as a discovery cohort was engineered to amplify biological differences by selecting cases from the extremes of the outcome distribution. All HGSC tumours are primary tumour samples. Library construction and sequencing Frozen specimens with >50% tumour cellularity (based on initial slide review) were used for cryosectioning and subsequent nucleic acid extraction. Patient tumour and normal blood samples derived from primary, untreated fresh frozen tumour specimens harvested at diagnosis during standard of care debulking surgery. Germline DNA was provided from peripheral blood buffy coat on all specimens except 13 from Tokyo, where non-cancer frozen tissue was used as a germline source. DNA extraction from both matched normal (blood) and tumour samples (frozen tissue) were performed using the QIAamp Blood and Tissue DNA kit (Qiagen) and quantified using a Qbit fluorometer and reagents (high-sensitivity assay). Three lanes of Illumina HiSeq 2500 v4 chemistry for normal samples and five lanes for tumour samples were obtained. The PCR-free protocol was adopted to eliminate the PCR-induced bias and improve coverage across the genome.

  Dataset EGAD00001003268

• miRNA expression data from primary tumors, metastasis and matched normals.

  MicroRNAs (miRs) have been recognized as promising biomarkers. It is unknown to what extent tumor-derived miRs are differentially expressed between primary colorectal cancers (pCRCs) and metastatic lesions, and to what extent the expression profiles of tumor tissue differ from the surrounding normal tissue. Next-generation sequencing (NGS) of 220 fresh-frozen samples, including paired primary and metastatic tumor tissue and non-tumorous tissue from 38 patients, revealed expression of 2245 known unique mature miRs and 515 novel candidate miRs. Unsupervised clustering of miR expression profiles of pCRC tissue with paired metastases did not separate the two entities, whereas unsupervised clustering of miR expression profiles of pCRC with normal colorectal mucosa demonstrated complete separation of the tumor samples from their paired normal mucosa. Two hundred and twenty-two miRs differentiated both pCRC and metastases from normal tissue samples (false discovery rate (FDR) <0.05). The highest expressed tumor-specific miRs were miR-21 and miR-92a, both previously described to be involved in CRC with potential as circulating biomarker for early detection. Only eight miRs, 0.5% of the analysed miR transcriptome, were differentially expressed between pCRC and the corresponding metastases (FDR <0.1), consisting of five known miRs (miR-320b, miR-320d, miR-3117, miR-1246 and miR-663b) and three novel candidate miRs (chr 1-2552-5p, chr 8-20656-5p and chr 10-25333-3p). These results indicate that previously unrecognized candidate miRs expressed in advanced CRC were identified using NGS. In addition, miR expression profiles of pCRC and metastatic lesions are highly comparable and may be of similar predictive value for prognosis or response to treatment in patients with advanced CRC.

  Dataset EGAD00001001644

• Cooperative activity of BRAF F595L and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling

  Mutations that activate the RAF-MEK-ERK signaling pathway, in particular BRAFV600E, occur in many cancers, and mutant BRAF-selective inhibitors have clinical activity in these diseases. Activating BRAF alleles are usually considered to be mutually exclusive with mutant RAS, whereas inactivating mutations in the D594F595G596 motif of the BRAF activation segment can coexist with oncogenic RAS and cooperate via paradoxical MEK/ERK activation. We determined the functional consequences of a largely uncharacterized BRAF mutation, F595L, which was detected along with an HRASQ61R allele by clinical exome sequencing in a patient with histiocytic sarcoma and also occurs in epithelial cancers, melanoma, and neuroblastoma, and investigated its interaction with mutant RAS. We demonstrate that, unlike previously described DFG motif mutants, BRAFF595L is a gain-of-function variant with intermediate activity towards MEK that does not act paradoxically, but nevertheless cooperates with mutant RAS to promote oncogenic signaling. Of immediate clinical relevance, BRAFF595L shows divergent responses to different mutant BRAF-selective inhibitors, whereas signaling driven by BRAFF595L with and without mutant RAS is efficiently blocked by pan-RAF and MEK inhibitors. Mutation data from primary patient samples and cell lines show that BRAFF595L, as well as other BRAF mutations with intermediate activity, frequently coincide with mutant RAS in a broad spectrum of cancers. These data define a novel class of activating BRAF mutations that cooperate with oncogenic RAS in a non-paradoxical fashion to achieve an optimal level of MEK-ERK signaling, extend the spectrum of patients with systemic histiocytic disorders and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway, and underscore the value of comprehensive genetic profiling for understanding the signaling requirements of individual cancers.

  Dataset EGAD00001001384

• 13 WGS and 8 TCS data of ENKL patients treated with pembrolizumab.

  Whole-genome sequencing (WGS) was performed for 13 pairs of tumor-normal samples from patients diagnosed with NKTL. Genomic DNA from tumor tissue was extracted with QIAamp DNA Mini Kit. The DNA for the matching normal was obtained from blood or buccal swabs and purified by Blood and Cell Culture DNA Mini kit or E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) according to manufacturer’s instructions. The quantity and quality were assessed by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and agarose gel electrophoresis. All sequencing libraries were prepared using TruSeq Nano DNA Library Prep Kit (Illumina). Paired-end sequencing was performed on Illumina HiSeq 2000 or HiSeq X Ten as 2x101 bp or 2x151 bp, respectively. 8 NKTL FFPE specimens were screened for somatic mutations using deep targeted capture sequencing (TCS). FFPE rolls or slides were extracted using QIAamp DNA FFPE Tissue kit (QIAGEN). The FFPE genomic DNA was treated with NEBNext FFPE DNA Repair Mix and assessed by Quant-it PicoGreen dsDNA Assay Kit (Invitrogen). The library was generated from 10-200 ng DNA with SureSelectXT Low Input Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent Technologies) according to manufacturer’s instructions. RNA based probe was designed with SureDesign (Agilent Technologies) to target-capture 140 genes. Next, the captured libraries were pooled in equimolar concentration and sequenced on Illumina Novaseq 6000 platform with SP or S1 chip. Reads aligning to 40 selected genes were isolated post-alignment for this submission. Prefix used in filenames: T - Tumor samples N - Matched-Normal samples

  Dataset EGAD00001004140

• Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

  Here, we utilized single-cell and spatial mapping to interrogate the mechanisms directing tissue architecture in human lymph nodes (LN) and its disruption in nodal B cell lymphoma. Specifically, we mapped the cellular landscape of follicular lymphoma (FL; n=6) and diffuse large B cell lymphoma (DLBCL; n=8) alongside non-malignant reactive LNs (rLN; n=5) using single-cell RNA sequencing. Our data support the central role of LN-resident stromal cells in chemokine-driven lymphocyte zonation and reveal an inflammatory feedback loop fueled by tumor-reactive T cells, triggering stromal remodeling, progressive loss of homeostatic chemokine gradients and tissue organization from non-malignant to FL and DLBCL. Collectively, our results highlight the principles of LN organization and suggest how lymphoma-induced microenvironmental reprogramming drives loss of tissue organization.

  Study EGAS00001006986

• Cancer of Low survival and UnMet Need – Pathologist Initiated

  Despite significant advances in diagnostic cancer histopathology, a subset of tumors remain unable to be classified using WHO criteria. The resulting diagnostic uncertainty can result in inappropriate clinical management and negative patient outcomes. We investigated whether combining histopathology with whole genome and transcriptome sequencing (WGTS) could improve the classification of tumors that posed diagnostic dilemmas despite extensive histopathology and standard molecular work-up at a quaternary oncology center. We successfully sequenced 45 tumors from an initial set of 54 unclassified tumors (83% success rate). A confident diagnosis was made for 38/45 tumors (84%). Additionally, potential treatment targets were identified in 21/45 tumors (47%). Theoretical comparison with alternative assays demonstrated that WGTS was uniquely capable of detecting critical diagnostic findings in 15/38 tumors (39%). This work supports augmenting histopathology and standard molecular pathology with WGTS in the classification of difficult-to-diagnose tumors.

  Study EGAS00001008112