Search Results - EGA European Genome-Phenome Archive

A_study_of_the_genetic_basis_of_evation_by_Acute_Myeloid_Leukaemia_of_Graft_vs_Leukaemia_effects_after_allogeneic_bone_marrow_transplantation

Acute myeloid leukaemia (AML) is an aggressive and molecularly diverse disease with a poor overall survival of 20-25%. With an annual incidence of 2.9 per 100,000, AML is currently the commonest myeloid malignancy in Europe, yet the two main therapeutic options for this disease, anthracyclines and purine analogues, have remained unchanged for over 20 years. Currently patients are stratified at diagnosis according to a series of clinicopathological parameters (e.g. age, white cell count and presence/absence of previous clonal haematological disease) and molecular markers (e.g. chromosomal translocations/deletions, aneuploidy and mutations in genes such as FLT3 and NPM1). Patients with adverse prognostic features, whose prognosis is particularly poor (e.g. <15% long-term survival) are offered treatment with allogeneic bone marrow transplantation (allo-BMT) if a sibling or unrelated donor is available. This can significantly improve survival (e.g. up to 40% long-term survival in some contexts), albeit at the expense of significant toxicity and transplant-related mortality (TRM). Allo-BMT is thought to work in part by allowing the delivery of large doses of chemotherapy followed by haemopoietic "rescue" with donor haemopoietic stem cells (haemopoietic failure would otherwise ensue). However, potentially the most potent effect of allo-BMT is the cytotoxic effect of donor lymphocytes against AML blasts, a phenomenon known as graft-vs-leukaemia (GVL) effect. Increasingly, transplants using reduced chemotherapy intensity (mini-allografts) are being used that partially circumvent the toxicity from chemotherapy and rely on GVL to effect cure. Nevertheless, AML relapse after allo-BMT still occurs at a significant rate of up to 80% depending on the type of transplant. There is accumulating evidence that genetic events in residual leukaemic cells enable them to evade immunodetection and therefore survive the GVL effect and expand to cause relapse. The most striking example of this is the loss of HLA antigens after transplants in which donor and recipient are not fully HLA-matched. In these cases, the leukaemia "deletes" the genomic region containing the disparate HLA antigen which was preferentially targeted as "foreign" by the GVL effect. However, the genetic basis of immune evasion in the majority of transplants, which are fully HLA matched, is not known. One possibility is that loss of genes coding for antigens outside the HLA locus but which are also targets of GVL may operate, alternatively genetic events that affect processes downstream of immunological cytotoxicity may be responsible.The identification of genetic events that mediate immune evasion would not only facilitate the understanding of this process but can help plan therapeutic interventions that improve the outcomes of allogeneic transplantation for AML and other disorders. We intend to study this by conducting exome sequencing on 6 cases of AMLs from patients that attend my clinic at Addenbrooke's hospital and have relapsed after allogeneic transplantation. Samples from AML diagnosis, remission/normal and AML relapse (total n=18) will be studied to identify somatic mutations in the primary AML and those acquired by the relapsed clone. The 18 samples will also be studied by array CGH to detect regions of genomic amplification or deletion.

Study EGAS00001000145

The BC Cancer Agency's Personalized Onco-Genomics Project

The BC Cancer Agency's Personalized Onco-Genomics (POG) Project involves molecular and clinical characterization of individual cancer patients to guide their treatment. The project aims to use whole genome and transcriptome data together with patient information to match treatments to cancer driver alterations and support clinical decision-making. Incorporating genomic data into decision-making should enable more rational development of treatment plans, facilitate enrollment into appropriate clinical trials and potentially identify less toxic and more effective therapeutic options.

Study EGAS00001001159

Characterization of individual foci of multicentric/multifocal breast cancer using targeted next generation sequencing

Multifocality or multicentricity in breast cancer may be defined as the presence of two or more tumor foci within a single quadrant of the breast or within different quadrants of the same breast, respectively. This original classification of the breast cancer as multicentric or multifocal was based on the assumption that cancers arising in the same quadrant were more likely to arise from the same ductal structures than those occurring in separate areas of the breast. The problem with these definitions is that the ?quadrants? of the breast are arbitrary external designations, as no internal boundaries do exist. This project will therefore focus both on synchronous multifocal and multicentric tumors. The incidence of multifocal and multicentric breast cancers was reported to be between 13 and 75% depending on the definition used, the extent of the pathologic sampling of the breast and whether in situ disease is considered evidence of multicentricity (1). Although this incidence is variable, those figures show that it is a frequent phenomenon. Multiple (multifocal/multicentric) breast carcinomas, especially when occurring in the same breast, represent a real challenge for both pathologists and clinicians in terms of identifying the cellular origin and the best therapeutic management of the cancer. Multifocality or multicentricity has been associated with a number of more aggressive features including an increased rate of regional lymph node metastases and adverse patient outcome when compared with unifocal tumors (2-3), and a possible increased risk of local recurrence following breast conserving surgery (4). For the moment, the literature is divided on whether there is a corresponding impact on survival outcomes. Today, the current convention to stage and to treat multifocal and multicentric tumors is the classical tumor-node-metastasis (TNM) staging guidelines with which tumor size is assessed by the largest tumor focus without taking other foci of disease into consideration. If some papers, as the recent one from Lynch and colleagues, support the current staging convention (3), others, however, as Boyages et al. suggested that aggregate size and not the size of the largest lesion should be considered in order to refine the prognostic assessment of those tumors (5). On the top of that, the question whether multifocal/multicentric carcinomas are due to the spread of a single carcinoma throughout the breast or is due to multiple carcinomas arising simultaneously has been a matter of debate. Some studies suggested that multifocal breast cancer may result from either intramammary spread from a single primary tumor or multiple synchronous primary tumors; whereas others suggest that multiple breast carcinomas always arise from the same clone (6-8). Recently, Pietri and colleagues analyzed the biological characterization of a series of 113 multifocal/multicentric breast cancers (8) which were diagnosed over a 5-year period. The expression of estrogen (ER) and progesterone (PgR) receptors, Ki-67 proliferative index, expression of HER2 and tumor grading were prospectively determined in each tumor focus, and mismatches among foci were recorded. Mismatches in ER status were present in 5 (4.4%) cases and PgR in 18 (15.9%) cases. Mismatches in tumor grading were present in 21 cases (18.6%), proliferative index (Ki-67) in 17 (15%) cases and HER2 status in 11 (9.7%) cases. Interestingly, this heterogeneity among foci has led to 14 (12.4%) patients receiving different adjuvant treatments compared with what would have been indicated if we had only taken into account the biologic status of the primary tumor. This study therefore showed that differences in biological characteristics of multifocal/multicentric lesions play a crucial role in the adjuvant treatment decision making process. In this study, we will concentrate on a larger series of patients with multifocal invasive ductal breast cancer lesions. We aim at: 1. Evaluating the incidence of multifocality according to the different breast cancer molecular subtypes (ER-/HER2-, HER2+, ER+/HER2-). 2. Evaluating the incidence of multifocality in patients with hereditary breast cancer disease (presence of germline BRCA1 or BRCA2 mutations). Moreover, we would like to investigate if multifocal lesions with BRCA1 or BRCA2 mutations exhibit a characteristic combination of substitution mutation signatures and a distinctive profile of deletions as demonstrated recently by Nik-Zainal and colleagues (9). 3. Correlating multifocality with clinical information in order to define its influence on patients? survival (DFS and OS). 4. Carrying high coverage targeted gene sequencing of driver cancer genes and genes whose mutation is of therapeutic importance in order to compare clinically-relevant genetic differences between several multifocal breast cancer lesions. 5. Evaluating the impact of the distance between the different lesions on the clinical outcome but also on the genetic differences. 6. Comparing gene expression patterns between several multifocal breast cancer lesions and correlate them with the results of the targeted genes screen. 7. Characterizing the genomic and transcriptomic status of cancer related genes in metastatic lesions (local recurrence, positive lymph node or distant metastatic sites) from the same multifocal invasive ductal breast cancer patients in order to evaluate the consequence of genomic and transcriptomic heterogeneity of multifocal lesions on metastatic lesions. Multiple (multifocal/multicentric) breast carcinomas, especially when occurring in the same breast, represent a real challenge for both pathologists and clinicians in terms of identifying the cellular origin and the best therapeutic choice. This project has the potential to identify genetic/transcriptomic differences existing between several lesions constituting multifocal breast cancers, which in the routine clinical practice are usually considered to be homogeneous among them. We foresee validating significant results in a larger series of patients and this, in turn, could have a remarkable impact on the treatment and clinical management of multifocal breast cancers. Indeed, we hope to provide some evidence whether or not each focus matters in multifocal and multicentric breast cancer to define the adequate therapeutic approach, especially in the context of targeted therapies. The work to be done at Sanger will be target gene screen pooling of 1400 samples.

Dataset EGAD00001000624

Genomic Profiling Reveals Spatial Intra-tumour Heterogeneity in Follicular Lymphoma

Follicular lymphoma (FL) is an incurable indolent non-Hodgkin’s lymphoma (NHL) that behaves heterogeneously and is synonymous with the reciprocal translocation t(14;18)(q32;q21), leading to aberrant constitutive over-expression of BCL2 [ref]. High risk groups with poor overall survival include patients that transform to a high grade lymphoma, most commonly diffuse large B cell lymphoma (DLBCL) or progress early (within 2 years) of receiving treatment. Recently several studies have shed light on the diverse genomic landscape of FL, with frequently mutated genes including those involved in chromatin remodelling, (CREBBP, KMT2D, EZH2, EP300), immune modulation (TNFRSF14), JAK-STAT (STAT6, SOCS1), NF-ĸB (CARD11, MYD88, TNFAIP3) and mTORC1 signalling (RRAGC) [ref]. To date, the majority of these studies have either focussed on inter-patient genetic heterogeneity or established the extent of clonal heterogeneity as a patient’s tumour evolves over time. Furthermore, these temporal studies have alluded to the existence of a tumour-repopulating population (referred to as the common progenitor cell (CPC)) that evades treatment and acts as a reservoir, seeding each subsequent relapse and transformation event. Although these previous studies have illuminated the longitudinal clonal dynamics that occur in FL, our understanding of the degree of spatial or intra-tumour heterogeneity (ITH) that exists within an individual patient thus far remains limited. Earlier studies have predominantly focused on differences in cytological grade and immunophenotype between spatially separated lymph nodes and bone marrow, the most common site of extra-nodal involvement in FL, with genomic profiling restricted to examining somatic hypermutation patterns in IgVH genes [ref]. In contrast next generation sequencing has been increasingly utilised in solid organ malignancies to derive comprehensive genomic profiles from spatially separated sites. The seminal study by Gerlinger et al revealed profound differences in the genetic make-up between individual primary and metastatic sites of renal cell carcinoma [ref]. Significant heterogeneity has since been demonstrated both between and within lesions in the same patient in lung, pancreas and breast cancer with presumed driver mutations distributed within the “branches” of the evolutionary phylogenetic tree, suggesting that a single biopsy is incapable of capturing the full genomic heterogeneity of an individual’s malignancy [ref]. This geographical heterogeneity not only adds to the diverse pool of tumour subclones that may contribute to drug resistance mechanisms but importantly, presents a major obstacle for precision-based approaches focussed on targeting specific lesions within a single biopsy. In FL, the exponential increase in clinical trials using novel agents such as EZH2, PI3K and BTK inhibitors reflects this shift in cancer care and along with the development of molecular prognostic scores such as the m7-FLIPI highlights the clinical need to accurately define genomic alterations with clinical relevance. As the majority of FL patients manifest disseminated tumour involvement, we sought to uncover the extent and clinical importance of spatial heterogeneity in FL by comprehensively genetically profiling 22 synchronously removed spatially separated biopsies from 9 patients. Using a combination of whole exome and targeted deep sequencing, our results inferred the complex subclonal architecture within these tumours and the mutational differences between anatomical sites, demonstrating cases with significant intrinsic genetic diversity.

Study EGAS00001002492

Transcriptomic analysis of liver CD8+ T cells

Sorted single CD8+T cells expressing CD14 from human liver for SMARTSeq2. Livers processed: Kucykowicz et al STAR Prot 2022:pubmed.ncbi.nlm.nih.gov/35516846/ Published: Pallett et al Nature 2022 Tissue CD14+CD8+T-cells are reprogrammed by myeloid cells and modulated by LPS The liver is bathed in bacterial products including LPS transported from the intestinal portal vasculature, but maintains a state of tolerance exploited by persistent pathogens and tumours1,2,3,4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+T-cells compartmentalised in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins; CD14+CD8+T-cells preferentially accumulate within the donor pool in liver allografts, amongst hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+T-cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10/IL-2 production directly ex vivo, and an enhanced capacity to rapidly produce antiviral/anti-tumour cytokines upon TCR engagement. This CD14+CD8+T-cell profile can be recapitulated by acquisition of membrane proteins, including the LPS receptor complex, from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T-cells in vitro. CD14+CD8+T-cells express integrins and chemokine receptors favouring interactions with the local stroma, which can promote their induction through CXCL12. LPS can also increase the frequency of CD14+CD8+T-cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+T-cells with immunomodulatory capacity. For the analysis of the single cell transcriptome two intrahepatic perfusate samples were isolated and frozen at -80 'C for storage prior to transportation to Newcastle, UK. Once thawed in pre-warmed HBSS containing 0.001% DNaseI, intrahepatic leukocytes were sorted by FACS to index classical mononuclear phagocytes (MNP) defined as Singlets, CD3-CD56-CD19-CD14hiHLA-DR+ or CD14± CD8+T-cells as follows: Singlets, CD3+CD56-CD19-CD4-CD8+V7.2- and split in to CD14± based on an isotype control. Single cells were sorted into 96-well skirted LoBind plates (Eppendorf) containing 5 µL lysis buffer (TCL buffer [Qiagen], supplemented with 1% b-mercaptoethanol [Sigma Aldrich; Merck]) on a BD Bioscience FACSFusion. The sorted plates were subsequently immediately sealed, centrifuged, and placed on dry ice to flash-freeze the cell lysate and stored at -80 'C. A modified SMART-seq2 protocol57 was performed on the single flow cytometry sorted-cells as previously described58. After cDNA generation, libraries were prepared (384 cells per library) using the Illumina Nextera XT kit (Illumina). Each library was sequenced to achieve a minimum depth of 1-2 million raw reads per cell using an Illumina HiSeq 4000 using v. 4 SBS chemistry to generate 75-bp paired-end reads.

Study EGAS00001006885

Covid19 WGS variants analysis

483 samples were collected for WGS and aligned with GRCh38 human genome. The variants were called using GATK (Sentieon v. 201808.03) in GVCF and VCF format.

Dataset EGAD00001010138

GCAT| WGS BAM V1

This dataset include BAM files of 808 samples from GCAT cohort. Technology used HiSeq 4000, read length 150 bp, inner mate distance 300 bp. For each sample the paired -ends are generated in separated files. Each FASTQ is splitted in multiple LANEs and grouped by the Multiplex index.

Dataset EGAD00001008202

Irish Covid19 WGS Raw Reads

Dataset contains 483 Irish origin Individuals with Covid19. paired end sequencing has been performed For WGS and later processed by using sention v 201808.03.

Dataset EGAD00001010186

Effect of ETS2 modulation on chromatin accessibility and enhancer activity in inflammatory (TPP) macrophages

This dataset consists of H3K27ac ChIP-seq and ATAC-seq in primary human macrophages following modulation of ETS2 expression. There are three datasets: H3K27ac ChIP-seq in ETS2-edited and unedited inflammatory (TPP) macrophages, H3K27ac ChIP-seq in ETS2-overexpressing and control M0 macrophages, and ATAC-seq in ETS2-edited and unedited inflammatory macrophages. For editing experiments, monocytes were transfected with CRISPR-Cas9 ribonucleoproteins containing a gRNA that was validated to edit ETS2, or a non-targeting control gRNA. Inflammatory macrophage differentiation was 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). For overexpression experiments, controlled overexpression of ETS2 mRNA or control mRNA (an equivalent amount of mRNA encoding the reverse complement of ETS2 – thereby controlling for the quantity, length and purine/pyrimidine composition of the transfected RNA but with a transcript that would not be translated) was induced in resting, non-activated (M0) macrophages by transfecting 500ng mRNA. To minimise non-specific activation due to the transfected RNA, in vitro transcription was performed using co-transcriptional capping (to minimise uncapped products), and incorporating modified, minimally immunogenic nucleotides (replacing uridine with N1-methyl-pseudouridine and cytidine with methylcytidine). After 18 hours, transfected cells were were activated with low dose LPS and harvested 6 hours later. For H3K27ac ChIP-seq, cells were harvested, 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 on an Illumina NovaSeq 6000. Raw data are provided as raw and aligned sequencing reads from H3K27ac-bound DNA and the input chromatin. ATAC-seq in TPP macrophages was performed using the Omni-ATAC protocol (Corces et al 2017) with the following modifications: cell number was increased to 75,000 cells, cell lysis time was increased to 5 minutes; volume of Tn5 transposase in the transposition mixture was doubled; duration of the transposition step was extended to 40 minutes. Sequencing libraries were prepared by PCR with the NEBNext Ultra II Q5Master Mix (NEB) as described previously (Corces et al 2017). Amplified libraries were cleaned using AMPure XP beads (Beckman Coulter). Sequencing reads were trimmed using TrimGalore! (Phred score 24), filtered to remove reads < 20 bp and aligned to the human genome (hg19) using Burrows-Wheeler Aligner (BWA). Aligned reads were converted to BAM files, sorted and indexed using SAMtools (Li et al, 2009). PCR duplicates and unmapped reads were removed, and the resulting BAM files were re-sorted and indexed. Raw data are provided as 100 bp paired-end Illumina reads from n = 3 donors for each experiment. BAM files are also provided.

Study EGAS50000000109

Targeted_sequencing_of_genes_recurrently_mutated_in_AML

Background Massively parallel sequencing technology has transformed cancer genomics. It is now feasible, in a clinically relevant time-frame, for a clinically manageable cost, to screen DNA from patient tumours for mutations essentially genome-wide. The challenge for personalised medicine will be to increase the sample size to thousands or tens of thousands of well-characterised cases in order to attain sufficient statistical power to stratify patients accurately across the complexity and genomic heterogeneity expected for most of the common tumour types. Currently, whole genome sequencing on this scale is not feasible, and targeted sequencing of relevant portions of the genome will be required. Pilot data We have developed protocols for large-scale, multiplexed sequencing of 100-200 genes in thousands of samples. Essentially, using robotic technology, genomic DNA from the cancer specimen is processed into sequencing libraries with unique DNA barcodes, thereby allowing sequencing reads to be attributed to the sample they derive from. Currently, these sequencing libraries can be generated in a 96-well format using fully automated protocols, and we are exploring methods to expand this to a 384-well format. The sequencing libraries are pooled and hybridized to custom sets of RNA baits representing the genomic regions of interest. Sequencing of the pulled-down libraries is done in pools of 48-96 samples per lane of an Illumina Hi-Seq. This protocol is already implemented at the Sanger Institute. We have published proof that somatic mutations in novel cancer genes can be identified from exome-wide sequencing. In unpublished pilot data, we have established the feasibility of robotic library production, custom pull-down, and multiplexed sequencing of barcoded libraries for 100 known myeloid cancer genes across 760 myelodysplasia samples. Highlights of the data thus far analysed reveal that the coverage is remarkably even between samples; when 96 samples are run, average coverage per lane of sequencing is ~250, with 90-95% of targeted exons covered by >25 reads; known mutations can be discovered in the data set; and the protocol is amenable to whole genome amplified DNA. The bioinformatic algorithms for identification of substitutions and indels in pull-down data are well-established; we have pilot data proving that copy number changes, LOH and genomic rearrangements in specific regions of interest can also be identified by tiling of baits across the relevant loci. Proposal We propose to apply this methodology to 10000 samples from patients with AML enrolled in clinical trials over the last 10-20 years. Oncogenic point mutations and potentially genomic rearrangements will be identified, and linked to clinical outcome data, with a view to undertaking the following sorts of analyses: • Identification of co-occurrence, mutual exclusivity and clusters of driver mutations. • Correlation of prognosis with driver mutations and potentially gene-gene interactions • Exploration of genomic markers of drug response Ultimately, we would like to be in a position to release the mutation data together with matched clinical outcome data to genuine medical researchers via a controlled access approach, possibly within the COSMIC framework (www.sanger.ac.uk/genetics/CGP/cosmic/). The vision here is to generate a portal whereby a clinician faced with an AML patient and his / her mutational profile can obtain a ‘personalised’ prediction of outcome, together with a fair assessment of the uncertainty of the estimate. With a sufficient sample size, there would also be the potential to develop decision support algorithms for therapeutic choices based on such data.

Study EGAS00001000408

Small molecule inhibitors of LOXL synergize with 5-AZA to restore erythropoiesis in myeloid neoplasms

Objectives: Bone marrow (BM) stroma and microenvironmental factors in the BM milieu of myeloid neoplasms are increasingly being recognized as novel perspective therapeutic targets. Lysyl oxidases (LOX/LOXL) are crucial enzymes that cross-link collagen and contribute to the deposition of aberrant stiff extracellular matrix (ECM) in the BM of patients with myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). Here, we studied whether inhibition of LOX/LOXL enzymes produced by BM-derived MSCs favors the differentiation of MDS/MPN hematopoietic stem cell and progenitor cells (HPSCs) and enhances the effects of 5-azacytidine (5-AZA). Methods: Primary co-cultures of MSCs and HPSCs were established from n=31 MDS/MPN patients and treated with the pan-LOX/LOXL inhibitor PXS-5505, 5-AZA or the combination of both for 4 days. At the end of co-culture, HSPC differentiation was assessed by CFU assays and flow cytometry. In some of the experiments, MSCs monolayers were pre-treated or not with PXS-5505 for 6 days and decellularized using 20 mM ammonia. Subsequently, autologous HSPCs were cultured on ECM matrices in the presence of 5-AZA or PXS-5505 + 5-AZA combination with or without inhibitors of integrin signaling such as blebbistatin, Y-27632 and αvβ3-integrin blocking antibodies. Patient-derived xenografts (PDX) were established in NSG mice based on intrafemoral co-injection of HSPCs and autologous MSCs. PDX were treated with 3 cycles of 5-AZA (75 mg/m2), PXS-5505 (30 mg/kg on alternate days for the duration of 5-AZA treatment) or combination of both. Results: MSCs of MDS and MPN patients displayed significant overexpression of LOX/LOXL transcripts as compared to healthy age-matched controls. Remarkably, LOX/LOXL inhibition in MSCs/HSPCs co-cultures increased erythroid differentiation of patient HSPCs that had suboptimal or no response to 5-AZA alone. From a total of n=31 advanced stage MDS/MPN patients, n=11 (35.5%) responded to the combination treatment, which was evidenced by a significant increase of CD235a+CD45- erythroid cell production compared to 5-AZA/PXS/untreated arms (p<0.05). Of note, combination treatment with 5-AZA + PXS-5505 facilitated differentiation of CD235+ erythroid progenitors from HSPCs sub-clones with low mutational VAFs. The presence of MSCs was necessary for the effects of the combination treatment and required direct cell contacts between MSCs and HSPCs. Moreover, PXS-5505-mediated alterations of the ECM composition alone were sufficient to induce erythroid differentiation. Inhibition of integrin-mediated signaling using blebbistatin, Y-27632 and αvβ3-integrin antibodies completely abrogated the effects of PXS-5505 induced erythroid differentiation. In vivo, the combination of PXS-5505 + 5-AZA favored erythroid differentiation of HSPCs and confirmed the in-vitro results. The combination treatment of 5-AZA + PXS-5505 induced the greatest reduction of disease burden as assessed by decrease of spleen indexes, total engraftment rates, BM fibrosis and mutational VAF profiles. Conclusions: The combination of 5-AZA + LOX/LOXL inhibitor PXS-5505 in MDS and MPN synergistically restores erythropoietic differentiation of primary HSPCs. The effect is dependent of contact interaction between HSPCs and niche components, such as MSCs and MSCs-derived ECM. Our data propose a strong rationale for a BM niche targeted combination treatment with 5-AZA + PXS-5505 in transfusion dependent MDS and MPN, which will be assessed in forthcoming clinical trials.

Study EGAS00001006174

Prognostic and therapeutic significance of leukemia subtypes and minimal residual disease measurements in pediatric acute lymphoblastic leukemia treated with contemporary risk-directed trial: a cohort study

Background Genomic analyses to identify leukemia subtypes and refinement of minimal residual disease (MRD) determination are two recent advances that would have substantial impact on the management of children with acute lymphoblastic leukemia (ALL). However, the combined application of these two approaches to determine the prognostic and therapeutic significance of MRD measurement and full spectrum of leukemia subtypes in the context of contemporary risk-directed therapy has not been studied comprehensively.MethodsBetween October 29, 2007 and March 26, 2017, 598 consecutive children with newly diagnosed ALL were enrolled in a clinical trial at St. Jude Children’s Research Hospital. Patients were provisional classified according to the presenting clinical and laboratory features including immunophenotype and cytogenetics. Patients with ETV6-RUNX1 and high hyperdiploid B-progenitor ALL were provisionally included in the low-risk groups; those with TCF3-PBX1, hypodiploidy and T-cell ALL in the standard (intermediate)-risk group; and patients with BCR-ABL1, KMT2A and early T-cell precursor ALL in the high-risk group. Final risk assignment was based primarily on MRD levels measured by flow cytometry on Day 15 (≥1%) and Day 42 (<0.01% or ≥1%) of remission induction. Using genomic classification based on cytogenetics and genomic analysis (single nucleotide polymorphism array analysis and transcriptome, exome, and genome sequencing), patients were classified into 17 subtypes. The primary aim was to determine the event-free survival, overall survival and cumulative risk of relapse according to specific leukemia subtypes and MRD levels on Days 8, 15 and 42 of remission induction. This clinical trial was registered at ClinicalTrials.gov, number NCT00549848.FindingsPatients with ETV6-RUNX1, high hyperdiploidy and DUX4-rearranged B-ALL all received low- or standard-risk therapy, and had the highest 5-year event-free survival rates: 98.4% (95% CI, 95.9-100), 95.3% (91.2-99.4), and 95.0% (84.2-100), respectively. All 142 patients with Day 8 MRD<0.01%, except two with KMT2A-rearranged ALL and one with TCF3-PBX1, were in continuous complete remission. The TCF3-PBX1, PAX5alt, T-cell, early T-cell precursor, iAMP21 and hypodiploid ALL had intermediate outcome with 5-year event-free survival rates ranging from 80.0% (39.4-100) to 88.2% (71.7-100). Patients with KMT2A-rearranged, BCR-ABL1, BCR-ABL1-like and ETV6-RUNX1-like ALL had the worst outcomes, with 5-year event-free survival rates between 64.1% (43.9-84.3) and 76.2% (51.9-100), despite most patients receiving standard- or high-risk therapy. Treatment intensification based on MRD and genotype improved outcome. Relapse did not occur in any of the patients with ETV6-RUNX1, DUX4-rearranged, TCF3-PBX1, iAMP21, ZNF384-rearranged, hypodiploid, or BCR-ABL1-like B-ALL who had day 15 MRD≥1% and received standard-risk therapy. However, day 42 MRD<0.01% did not preclude relapse in patients with intermediate-risk or unfavorable subtypes including TCF3-PBX1, PAX5alt, T-cell, iAMP21, BCR-ABL1, BCR-ABL1-like, ETV6-RUNX1-like, KMT2A-rearranged, and MEF2D-rearranged ALL, despite intensified treatment in the standard- or high-risk arms. InterpretationContemporary risk-directed treatment based on genotypes and MRD can cure virtually all patients with ETV6-RUNX1, hyperdiploid>50 and DUX4-rearranged ALL. While intensified treatment improve outcome for patients with high MRD during induction, substantial proportions of patients with intermediate-risk or unfavorable subtypes would still relapse even achieving negative MRD status at the end of induction and require novel therapeutics to improve outcome.

Study EGAS00001004739

Exome sequencing reveals pathogenic variants in known and novel candidate genes for severe sperm motility disorders

STUDY QUESTION: What are the causative genetic variants in patients with male infertility due to severe sperm motility disorders? SUMMARY ANSWER: We identified high confidence disease-causing variants in multiple genes previously associated with severe sperm motility disorders in 9 out of 21 patients (43%). We also identified variants in novel candidate genes in 8 additional patients (38%). WHAT IS KNOWN ALREADY: Severe sperm motility disorders are a form of male infertility characterised by vital, but immotile sperm often in combination with a spectrum of structural abnormalities of the sperm flagellum. Examples of this spectrum of disorders include dysplasia of the fibrous sheath / multiple morphological abnormalities of the sperm flagella (DFS-MMAF) and severe forms of asthenozoospermia associated to non-specific structural defects. These disorders frequently recur in families and may include chronic respiratory disease. As such, genetic causes are suspected and many genes have recently been reported. Currently, depending on the clinical sub-categorisation, up to 50% of causality in patients with severe sperm motility disorders can be explained by pathogenic variants in at least 21 known genes. STUDY DESIGN, SIZE, DURATION: We performed exome sequencing in patients with severe sperm motility disorders from two different clinics, aiming to identify the underlying genetic defects and establishing a diagnostic yield in both. PARTICIPANTS/MATERIALS, SETTING, METHOD: Two groups of infertile men, one from Argentina (n= 9) and one from Australia (n=12), with clinically defined sperm motility disorders were included. All patients in the Argentine cohort were diagnosed with DFS-MMAF, based on transmission electron microscopy. Exome sequencing was performed in all 21 patients and variants with an allele frequency of <1% in the gnomAD population were prioritised and interpreted. MAIN RESULTS AND ROLE OF CHANCE: In 9/21 patients (43%), we identified pathogenic variants in known sperm motility disorder genes: CFAP43 (3 patients); CFAP44 (2 patients), QRICH2 (2 patients), DNAH1 (1 patient) and DNAH6 (1 patient). The diagnostic rate did not differ markedly between the Argentinian and the Australian cohort (44% and 42%, respectively). Furthermore, we identified patients with variants in the novel candidate sperm motility genes CFAP58, DNAH12, DRC1, MDC1, PACRG, SSPL2C and TPTE2. One patient presented with two possibly damaging variants in four candidate genes and it remains unclear which variants were responsible for the severe sperm motility defect in this patient. LIMITATIONS, REASONS FOR CAUTION: In this study, we described patients with potential bi-allelic variants in known and novel candidate genes for severe sperm motility disorders. Due to unavailability of parental DNA, we have not assessed de novo or maternally inherited dominant variants and could not phase the mutations to establish in all cases that the mutations occur on both alleles. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirm an important role for five known genes for sperm motility and we demonstrate that exome sequencing is an effective method to diagnose patients with severe sperm motility defects (9/21 diagnosed; 43%). Furthermore, our analysis revealed seven novel candidate genes for severe sperm motility disorders. Genome-wide sequencing of additional patient cohorts and re-analysis of exome data of currently unsolved cases may reveal additional variants in these novel candidate genes.

Study EGAS00001005018

Recurrent DNMT3B gene rearrangements are associated with unfavorable outcome in dicentric (9;20)-positive pediatric BCP-ALL

The dicentric chromosome dic(9;20)(p11∼13;q11), occurs in 2% of pediatric acute lymphoblastic leukemia (ALL) cases. However, the prognostic significance of the dic(9;20) alterations and the mechanism in which these alterations drive leukemogenesis remains elusive. Our study describes the demographic, clinical, prognostic, and molecular characteristics of the dic(9;20)-positive ALL in a cohort of 57 pediatric BCP-ALL patients. Targeted RNA-seq and Array CGH analysis unraveled heterogeneous breakpoints on chromosomes 9 and 20 and frequent deletions of the IKZF1 gene. Together with the intrinsic susceptibility for deletions in CDKN2A, CDKN2B, and PAX5 genes in the dic(9;20)-positive ALL, these patients are eligible for treatment escalation in the AIEOP-BFM ALL 2017 clinical study. Survival analysis revealed poor 5-year event-free survival (pEFS) of 69% in dic(9;20)-positive ALL cases, compared to BCP-ALL patients enrolled in modern treatment studies. Strikingly, we identified six dic(9;20)-positive cases with rearrangements involving the DNMT3B gene, and poor 5-year pEFS of 25% (SE = 20%), compared to 79% (SE = 9%) for the remaining cohort (P = 0.011). Despite the loss of the methyltransferase domain of the DNMT3B gene in most cases with DNMT3B rearrangements, we did not observe major changes in the global methylation patterns. Differential gene expression analysis of DNMT3B-rearranged ALL unraveled a dysregulation of genes involved in hematopoiesis, lymphocyte maturation, and the PI3K/AKT signaling pathway. After confirmation in an independent cohort, DNMT3B-rearranged ALL cases should be considered high-risk for relapse and treated accordingly. We provide clinical data sets of Array CGH, targeted RNA-seq, total RNA-seq, whole genome bisulfite (WGBS) and whole genome DNA sequencing (WGS) obtained from bone marrow (BM) or peripheral blood (PB) mononuclear cells of 57 pediatric patients with dicentric chromosome dic(9;20) positive Acute lymphocytic leukemia (ALL), from which in 6 cases DNMT3B gene rearrangement was identified. This data is complemented by total RNA-seq and WGBS of samples from 4 additional ALL patients with a t(12;21) translocation and ETV6-RUNX1 gene fusion. DNA was isolated from BM or PB lymphocytes using Qiagen QIAamp DNA Blood Midi Kit to perform i) Array CGH of 58 dic(9;20) positive samples by hybridizing 500ng DNA using a Agilent 400K SurePrint G3 Custom CGH Human Genome Microarray (e-Array design 84704) ii) WGBS of 6 DNMT3B rearrangement positive samples and 4 ETV6-RUNX1 positive samples using Tecan TrueMethyl oxBS-Seq module for library preparation and Illumina NovaSeq 6000 platform to run 2x151 cycles iii) WGS of DNMT3B rearrangement positive samples using Illumina Lotus DNA Library Prep Kit followed by sequencing running 2x160 cycles on an Illumina NovaSeq 6000 platform. RNA was isolated from PB lymphocytes using the PerkinElmer Chemagic 360 instrument, followed by i) targeted RNA-seq of 56 dic(9;20) positive samples prepared using Illumina TruSight RNA Pan-Cancer Panel and sequenced on an Illumina MiSeq platform running 2x75 cycles ii) total RNA-seq of 6 DNMT3B rearrangement positive samples and 4 ETV6-RUNX1 positive samples utilizing TruSeq Stranded Total RNA Library Prep Gold kit and running 2x100 cycles on an Illumina NovaSeq 6000 platform.

Study EGAS00001007383

The British Autozygosity Populations BioResource (2022-04-26)

This data set contains whole exome sequences of individuals from 8086 (mostly British Pakistani/British Bangladeshi, mostly self-reported parentally related) individuals from the following studies: 1. 5236 British Pakistani/British Bangladeshi adults from East London Genes & Health, now known as Genes & Health 2. 2624 British South Asian mothers from Born in Bradford (mostly Pakistani) 3. 1061 British South Asian adults from Birmingham (mostly Pakistani) This dataset contains all the exome sequence data available for this study on 2022-04-26

Dataset EGAD00001008736

Cellular indexing of transcriptomes and epitopes sequencing of peritoneal fluid from patients with achalasia

The data published here contains cellular indexing of transcriptomes and epitopes sequencing (CITEseq) using the oligo-tagged TotalSeq™-B Human Universal Cocktail, V1.0 from BioLegend on peritoneal fluid (PF) from patients with achalasia. Sequencing was performed in a paired-ended fashion on the NovaSeq6000. Below an overview of the epitopes to oligotags as obtained from BioLegend: DNA_ID Description clone Sequence Ensembl ID B0006 anti-human CD86 IT2.2 GTCTTTGTCAGTGCA ENSG00000114013 B0007 anti-human CD274 (B7-H1, PD-L1) 29E.2A3 GTTGTCCGACAATAC ENSG00000120217 B0020 anti-human CD270 (HVEM, TR2) 122 TGATAGAAACAGACC ENSG00000157873 B0023 anti-human CD155 (PVR) SKII.4 ATCACATCGTTGCCA ENSG00000073008 B0024 anti-human CD112 (Nectin-2) TX31 AACCTTCCGTCTAAG ENSG00000130202 B0026 anti-human CD47 CC2C6 GCATTCTGTCACCTA ENSG00000196776 B0029 anti-human CD48 BJ40 CTACGACGTAGAAGA ENSG00000117091 B0031 anti-human CD40 5C3 CTCAGATGGAGTATG ENSG00000101017 B0032 anti-human CD154 24-31 GCTAGATAGATGCAA ENSG00000102245 B0033 anti-human CD52 HI186 CTTTGTACGAGCAAA ENSG00000169442 B0034 anti-human CD3 UCHT1 CTCATTGTAACTCCT ENSG00000167286 B0046 anti-human CD8 SK1 GCGCAACTTGATGAT ENSG00000153563 B0047 anti-human CD56 (NCAM) 5.1H11 TCCTTTCCTGATAGG ENSG00000149294 B0050 anti-human CD19 HIB19 CTGGGCAATTACTCG ENSG00000177455 B0052 anti-human CD33 P67.6 TAACTCAGGGCCTAT ENSG00000105383 B0053 anti-human CD11c S-HCL-3 TACGCCTATAACTTG ENSG00000140678 B0058 anti-human HLA-A,B,C W6/32 TATGCGAGGCTTATC ENSG00000206503 B0063 anti-human CD45RA HI100 TCAATCCTTCCGCTT ENSG00000081237 B0064 anti-human CD123 6H6 CTTCACTCTGTCAGG ENSG00000185291 B0066 anti-human CD7 CD7-6B7 TGGATTCCCGGACTT ENSG00000173762 B0068 anti-human CD105 43A3 ATCGTCGAGAGCTAG ENSG00000106991 B0070 anti-human/mouse CD49f GoH3 TTCCGAGGATGATCT ENSG00000091409 B0071 anti-human CD194 (CCR4) L291H4 AGCTTACCTGCACGA ENSG00000183813 B0072 anti-human CD4 RPA-T4 TGTTCCCGCTCAACT ENSG00000010610 B0073 anti-mouse/human CD44 IM7 TGGCTTCAGGTCCTA ENSG00000026508 B0081 anti-human CD14 M5E2 TCTCAGACCTCCGTA ENSG00000170458 B0083 anti-human CD16 3G8 AAGTTCACTCTTTGC ENSG00000203747 B0085 anti-human CD25 BC96 TTTGTCCTGTACGCC ENSG00000134460 B0087 anti-human CD45RO UCHL1 CTCCGAATCATGTTG ENSG00000081237 B0088 anti-human CD279 (PD-1) EH12.2H7 ACAGCGCCGTATTTA ENSG00000188389 B0089 anti-human TIGIT (VSTM3) A15153G TTGCTTACCGCCAGA ENSG00000181847 B0090 Mouse IgG1, κ isotype MOPC-21 GCCGGACGACATTAA B0091 Mouse IgG2a, κ isotype MOPC-173 CTCCTACCTAAACTG B0092 Mouse IgG2b, κ isotype MPC-11 ATATGTATCACGCGA B0095 Rat IgG2b, κ isotype RTK4530 GATTCTTGACGACCT B0100 anti-human CD20 2H7 TTCTGGGTCCCTAGA ENSG00000156738 B0101 anti-human CD335 (NKp46) 9E2 ACAATTTGAACAGCG ENSG00000189430 B0124 anti-human CD31 WM59 ACCTTTATGCCACGG ENSG00000261371 B0127 anti-Human Podoplanin NC-08 GGTTACTCGTTGTGT ENSG00000162493 B0134 anti-human CD146 P1H12 CCTTGGATAACATCA ENSG00000076706 B0136 anti-human IgM MHM-88 TAGCGAGCCCGTATA ENSG00000211899 B0138 anti-human CD5 UCHT2 CATTAACGGGATGCC ENSG00000110448 B0141 anti-human CD195 (CCR5) J418F1 CCAAAGTAAGAGCCA ENSG00000160791 B0142 anti-human CD32 FUN-2 GCTTCCGAATTACCG ENSG00000143226 B0143 anti-human CD196 (CCR6) G034E3 GATCCCTTTGTCACT ENSG00000112486 B0144 anti-human CD185 (CXCR5) J252D4 AATTCAACCGTCGCC ENSG00000160683 B0145 anti-human CD103 (Integrin αE) Ber-ACT8 GACCTCATTGTGAAT ENSG00000083457 B0146 anti-human CD69 FN50 GTCTCTTGGCTTAAA ENSG00000110848 B0147 anti-human CD62L DREG-56 GTCCCTGCAACTTGA ENSG00000188404 B0149 anti-human CD161 HP-3G10 GTACGCAGTCCTTCT ENSG00000111796 B0151 anti-human CD152 (CTLA-4) BNI3 ATGGTTCACGTAATC ENSG00000163599 B0152 anti-human CD223 (LAG-3) 11C3C65 CATTTGTCTGCCGGT ENSG00000089692 B0153 anti-human KLRG1 (MAFA) SA231A2 CTTATTTCCTGCCCT ENSG00000139187 B0154 anti-human CD27 O323 GCACTCCTGCATGTA ENSG00000139193 B0155 anti-human CD107a (LAMP-1) H4A3 CAGCCCACTGCAATA ENSG00000185896 B0156 anti-human CD95 (Fas) DX2 CCAGCTCATTAGAGC ENSG00000026103 B0158 anti-human CD134 (OX40) Ber-ACT35 (ACT35) AACCCACCGTTGTTA ENSG00000186827 B0159 anti-human HLA-DR L243 AATAGCGAGCAAGTA ENSG00000204287 B0160 anti-human CD1c L161 GAGCTACTTCACTCG ENSG00000158481 B0161 anti-human CD11b ICRF44 GACAAGTGATCTGCA ENSG00000169896 B0162 anti-human CD64 10.1 AAGTATGCCCTACGA ENSG00000150337 B0163 anti-human CD141 (Thrombomodulin) M80 GGATAACCGCGCTTT ENSG00000178726 B0164 anti-human CD1d 51.1 TCGAGTCGCTTATCA ENSG00000158473 B0165 anti-human CD314 (NKG2D) 1D11 CGTGTTTGTTCCTCA ENSG00000213809 B0167 anti-human CD35 E11 ACTTCCGTCGATCTT ENSG00000203710 B0168 anti-human CD57 Recombinant QA17A04 AACTCCCTATGGAGG ENSG00000109956 B0170 anti-human CD272 (BTLA) MIH26 GTTATTGGACTAAGG ENSG00000186265 B0171 anti-human/mouse/rat CD278 (ICOS) C398.4A CGCGCACCCATTAAA ENSG00000163600 B0174 anti-human CD58 (LFA-3) TS2/9 GTTCCTATGGACGAC ENSG00000116815 B0176 anti-human CD39 A1 TTACCTGGTATCCGT ENSG00000138185 B0179 anti-human CX3CR1 K0124E1 AGTATCGTCTCTGGG ENSG00000168329 B0180 anti-human CD24 ML5 AGATTCCTTCGTGTT ENSG00000272398 B0181 anti-human CD21 Bu32 AACCTAGTAGTTCGG ENSG00000117322 B0185 anti-human CD11a TS2/4 TATATCCTTGTGAGC ENSG00000005844 B0187 anti-human CD79b (Igβ) CB3-1 ATTCTTCAACCGAAG ENSG00000007312 B0189 anti-human CD244 (2B4) C1.7 TCGCTTGGATGGTAG ENSG00000122223 B0206 anti-human CD169 7-239 TACTCAGCGTGTTTG ENSG00000088827 B0214 anti-human/mouse integrin β7 FIB504 TCCTTGGATGTACCG ENSG00000139626 B0215 anti-human CD268 (BAFF-R) 11C1 CGAAGTCGATCCGTA ENSG00000159958 B0216 anti-human CD42b HIP1 TCCTAGTACCGAAGT ENSG00000203618 B0217 anti-human CD54 HA58 CTGATAGACTTGAGT ENSG00000090339 B0218 anti-human CD62P (P-Selectin) AK4 CCTTCCGTATCCCTT ENSG00000174175 B0219 anti-human CD119 (IFN-γ R α chain) GIR-208 TGTGTATTCCCTTGT ENSG00000027697 B0224 anti-human TCR α/β IP26 CGTAACGTAGAGCGA B0236 Rat IgG1, κ isotype RTK2071 ATCAGATGCCCTCAT B0238 Rat IgG2a, κ Isotype RTK2758 AAGTCAGGTTCGTTT B0242 anti-human CD192 (CCR2) K036C2 GAGTTCCCTTACCTG ENSG00000121807 B0246 anti-human CD122 (IL-2Rβ) TU27 TCATTTCCTCCGATT ENSG00000100385 B0352 anti-human FcεRIα AER-37 (CRA-1) CTCGTTTCCGTATCG ENSG00000179639 B0353 anti-human CD41 HIP8 ACGTTGTGGCCTTGT ENSG00000005961 B0355 anti-human CD137 (4-1BB) 4B4-1 CAGTAAGTTCGGGAC ENSG00000049249 B0358 anti-human CD163 GHI/61 GCTTCTCCTTCCTTA ENSG00000177575 B0359 anti-human CD83 HB15e CCACTCATTTCCGGT ENSG00000112149 B0363 anti-human CD124 (IL-4Rα) G077F6 CCGTCCTGATAGATG ENSG00000077238 B0364 anti-human CD13 WM15 TTTCAACGCCCTTTC ENSG00000166825 B0367 anti-human CD2 TS1/8 TACGATTTGTCAGGG ENSG00000116824 B0368 anti-human CD226 (DNAM-1) 11A8 TCTCAGTGTTTGTGG ENSG00000150637 B0369 anti-human CD29 TS2/16 GTATTCCCTCAGTCA ENSG00000150093 B0370 anti-human CD303 (BDCA-2) 201A GAGATGTCCGAATTT ENSG00000198178 B0371 anti-human CD49b P1E6-C5 GCTTTCTTCAGTATG ENSG00000164171 B0373 anti-human CD81 (TAPA-1) 5A6 GTATCCTTCCTTGGC ENSG00000110651 B0384 anti-human IgD IA6-2 CAGTCTCCGTAGAGT ENSG00000211898 B0385 anti-human CD18 TS1/18 TATTGGGACACTTCT ENSG00000160255 B0386 anti-human CD28 CD28.2 TGAGAACGACCCTAA ENSG00000178562 B0389 anti-human CD38 HIT2 TGTACCCGCTTGTGA ENSG00000004468 B0390 anti-human CD127 (IL-7Rα) A019D5 GTGTGTTGTCCTATG ENSG00000168685 B0391 anti-human CD45 HI30 TGCAATTACCCGGAT ENSG00000081237 B0393 anti-human CD22 S-HCL-1 GGGTTGTTGTCTTTG ENSG00000012124 B0394 anti-human CD71 CY1G4 CCGTGTTCCTCATTA ENSG00000072274 B0396 anti-human CD26 BA5b GGTGGCTAGATAATG ENSG00000197635 B0398 anti-human CD115 (CSF-1R) 9-4D2-1E4 AATCACGGTCCTTGT ENSG00000182578 B0404 anti-human CD63 H5C6 GAGATGTCTGCAACT ENSG00000135404 B0406 anti-human CD304 (Neuropilin-1) 12C2 GGACTAAGTTTCGTT ENSG00000099250 B0407 anti-human CD36 5-271 TTCTTTGCCTTGCCA ENSG00000135218 B0408 anti-human CD172a (SIRPα) 15-414 CGTGTTTAACTTGAG ENSG00000198053 B0419 anti-human CD72 3F3 CAGTCGTGGTAGATA ENSG00000137101 B0420 anti-human CD158 (KIR2DL1/S1/S3/S5) HP-MA4 TATCAACCAACGCTT ENSG00000125498 B0446 anti-human CD93 VIMD2 GCGCTACTTCCTTGA ENSG00000125810 B0575 anti-human CD49a TS2/7 ACTGATGGACTCAGA ENSG00000213949 B0576 anti-human CD49d 9F10 CCATTCAACTTCCGG ENSG00000115232 B0577 anti-human CD73 (Ecto-5'-nucleotidase) AD2 CAGTTCCTCAGTTCG ENSG00000135318 B0579 anti-human CD9 HI9a GAGTCACCAATCTGC ENSG00000010278 B0581 anti-human TCR Vα7.2 3C10 TACGAGCAGTATTCA B0582 anti-human TCR Vδ2 B6 TCAGTCAGATGGTAT B0591 anti-human LOX-1 15C4 ACCCTTTACCGAATA ENSG00000173391 B0592 anti-human CD158b (KIR2DL2/L3, NKAT2) DX27 GACCCGTAGTTTGAT ENSG00000243772 B0599 anti-human CD158e1 (KIR3DL1, NKB1) DX9 GGACGCTTTCCTTGA ENSG00000167633 B0822 anti-human CD142 NY2 CACTGCCGTCGATTA ENSG00000117525 B0830 anti-human CD319 (CRACC) 162.1 AGTATGCCATGTCTT ENSG00000026751 B0864 anti-human CD352 (NTB-A) NT-7 AGTTTCCACTCAGGC ENSG00000162739 B0867 anti-human CD94 DX22 CTTTCCGGTCCTACA ENSG00000134539 B0871 anti-human CD162 KPL-1 ATATGTCAGAGCACC ENSG00000110876 B0896 anti-human CD85j (ILT2) GHI/75 CCTTGTGAGGCTATG ENSG00000104972 B0897 anti-human CD23 EBVCS-5 TCTGTATAACCGTCT ENSG00000104921 B0902 anti-human CD328 (Siglec-7) 6-434 CTTAGCATTTCACTG ENSG00000168995 B0918 anti-human HLA-E 3D12 GAGTCGAGAAATCAT ENSG00000204592 B0920 anti-human CD82 ASL-24 TCCCACTTCCGCTTT ENSG00000085117 B0944 anti-human CD101 (BB27) BB27 CTACTTCCCTGTCAA ENSG00000134256 B1046 anti-human CD88 (C5aR) S5/1 GCCGCATGAGAAACA ENSG00000197405 B1052 anti-human CD224 KF29 CTGATGAGATGTCAG ENSG00000100031

Dataset EGAD50000000252

Korea Epigenome Project(KEP), Korea National Research Institute of Health(KNIH)

The KEP was launched in 2012 to produce 50 epigenomic sets on Korean chronic diseases related target cells with the participation on Internationsl Human Epigenome Consortium(IHEC) of which focus is constructing 1000 reference epigenome map on 250 human cell type. The objective of the KEP was to provide a comprehensive epigenomic map of Korean chronic disease-related target tissues. Single cells were isolated from the pancreas, fat and kidney. Islet, ductal, acinar, and beta cells from the pancreas were purified into a single homogenous cell type using flow-assisted cell sorting technique. Adipocytes and pre-adipocytes were purified from fat, using Percoll gradient centrifugation technique. Mesangial, distal tubule, proximal tubule, podocytes, and collecting duct cells from the kidney were collected using mechanical sieving and immunostaining methods. Each cell type was derived from normal tissue or those with diabetes, obesity, or chronic kidney disease. Sixty reference epigenome datasets of pancreatic, fat, kidney cells were produced. Cell types such as the islets, and beta from the pancreas, adipocytes, pre-adipocytes from fat tissue, podocyte, mesangial, tubules from kidney were used for primary target to produce a Korean metabolic reference epigenome. And the bisulfite converted-whole genome sequencing, Infinium 450k DNA methylation bead array, mRNA, miRNA-Sequencing were performed to produce a comprehensive epigenomic map. The aim of roadmap reports is preparation of Long-term and Mid-term Technology Roadmaps for Korean Human Epigenome Project towards a competitive execution of International Epigenome Project and successful expansion of the research outcomes. Roadmap reports reviewed on recent technological development of DNA methylation and histone modification analysis, emerging new trends in epigenome medicine, plans for hardware and software analysis systems aimed to epigenome analysis, medical expert opinion on target cells and isolation scheme, bioinformatics expert opinion on analytical tools, new ideas for a putative epigenome studies, trends in single cell epigenome analysis. In conclusion, roadmap report suggested several projects for the future KNIH epigenome research in collaboration with epigenome research group. These are including refocusing the goals of KNIH-driven research in identification of disease mechanism, development of new studies on GWAS-EWA, higher priority on the epigenomic studies of human primary cells, establishment of collaborative relationship with tissue banking centers, establishment of mapping centers and data analysis portals, establishment of Korean Epigenome Research Consortium, establishment of steering committee governing both research and funding, launching of additional funding for epigenomic researches.

Study EGAS00001001774

panALL exome data set

Exome sequencing of panALL exome data set, total of 1948 samples

Dataset EGAD00001008193

Short-term fasting before living kidney donation has an immune-modulatory effect

Background: Short-Term Fasting (STF) is an intervention reducing the intake of calories, without causing undernutrition or micronutrient-related malnutrition. It aims to systemically improve resilience against acute stress. Several (pre-)clinical studies have suggested protective effects of STF, marking the systemic effects STF can induce in respect to surgery and ischemia-reperfusion injury. In addition, STF also affects the number of circulating immune cells. We aim to determine the effect of STF on the abundance and phenotype of different immune cell populations. Methods: Thirty participants were randomly selected from the FAST clinical trial, including living kidneys donors, randomised to a STF-diet or control arm. In an observational cohort sub-study we prospectively included 30 patients who donated blood samples repeatedly during study runtime. Using flow cytometry analyses, immune cell phenotyping was performed on peripheral blood mononuclear cells. Three panels were designed to investigate the presence and activation status of peripheral T cells, B cells, dendritic cells (DCs) and myeloid cells. Results: Eight participants were excluded due to sample constraints. Baseline characteristics showed no significant differences, except for fasting duration. Weight changes were minimal and non-significant across different time intervals, with slight trends towards long-term weight loss pre-surgery. Glucose, insulin, and β-hydroxybutyrate levels differed significantly between groups, reflecting adherence to the fasting diet. Flow cytometry analysis revealed no baseline differences between groups, with high variability within each group. We observed significant changes in immune cell populations due to fasting, particularly in B cells, T cells, and DCs. Discussion: In this study, we found that STF changes the levels and phenotype of immune cells, reducing abundance and activation of T cells and regulatory T cells, increased presence of (naïve) B cells, and elevation of type 1 conventional DCs. Further research should focus on the clinical implications of the changes in immune cell populations and significance of these observed immunological changes. Background: Short-Term Fasting (STF) is an intervention reducing the intake of calories, without causing undernutrition or micronutrient-related malnutrition. It aims to systemically improve resilience against acute stress. Several (pre-)clinical studies have suggested protective effects of STF, marking the systemic effects STF can induce in respect to surgery and ischemia-reperfusion injury. In addition, STF also affects the number of circulating immune cells. We aim to determine the effect of STF on the abundance and phenotype of different immune cell populations. Methods: Thirty participants were randomly selected from the FAST clinical trial, including living kidneys donors, randomised to a STF-diet or control arm. In an observational cohort sub-study we prospectively included 30 patients who donated blood samples repeatedly during study runtime. Using flow cytometry analyses, immune cell phenotyping was performed on peripheral blood mononuclear cells. Three panels were designed to investigate the presence and activation status of peripheral T cells, B cells, dendritic cells (DCs) and myeloid cells. Results: Eight participants were excluded due to sample constraints. Baseline characteristics showed no significant differences, except for fasting duration. Weight changes were minimal and non-significant across different time intervals, with slight trends towards long-term weight loss pre-surgery. Glucose, insulin, and β-hydroxybutyrate levels differed significantly between groups, reflecting adherence to the fasting diet. Flow cytometry analysis revealed no baseline differences between groups, with high variability within each group. We observed significant changes in immune cell populations due to fasting, particularly in B cells, T cells, and DCs. Discussion: In this study, we found that STF changes the levels and phenotype of immune cells, reducing abundance and activation of T cells and regulatory T cells, increased presence of (naïve) B cells, and elevation of type 1 conventional DCs. Further research should focus on the clinical implications of the changes in immune cell populations and significance of these observed immunological changes.

Study EGAS00001008034

Clonal dynamics of normal haematopoiesis across the human lifespan

Age-related loss of function in the human haematopoietic system is well documented, manifesting as reduced regenerative capacity, age-related cytopenias and immune dysfunction. However, the cellular and population level changes that underpin both this functional decline and the increased risk of clonal haematopoiesis and blood cancer in the elderly remain elusive. Here we performed whole genome sequencing on >3350 single haematopoietic stem cell / multipotent progenitors (HSC/MPP) derived colonies across 10 haematologically normal subjects aged 0 to 81. We found that HSC/MPPs accumulated 17 single nucleotide variants per year post birth and had a reduction in telomere length of 50bp per year throughout young adult life. We reconstructed phylogenies of the sampled HSC/MPPs to interrogate changes in clonal dynamics through life. Haematopoiesis in adults aged less than 65 was predominantly polyclonal, with few known driver mutations. In contrast, individuals aged over 75 displayed a profound change in clonal structure, with frequent clonal expansions, many unexplained by known driver mutations. The ratio of non-synonymous to synonymous mutations revealed widespread positive selection, estimating around 1000 driver mutations in the dataset (10-fold more than the number of known drivers). We identified novel genes ZNF318 and HIST2H3D as being under positive selection, despite not being enriched in myeloid malignancies. Our data show that HSC clonal dynamics is more complex than previously thought. One implication is that by old age, the majority of HSCs carry at least one of a number of largely undescribed driver mutations, which may underlie aspects of their functional decline.

Dataset EGAD00001007851

SABE_1171_WGS_BAM

Whole-genome sequencing BAM files of a census-based elderly cohort of Brazilians (n=1171)

Dataset EGAD00001008640

LBC1936 fastq files

1075 members of the LBC1936 were sequenced using the Illumina HiSeq X platform. This dataset contains the paired fastq files.

Dataset EGAD00001008774

CD_prognosis_UKB

Crohn's disease DNA samples genotyped using UK Biobank Axiom array

Dataset EGAD00010001155

Institut Curie Neuroblastoma Whole Genome Sequencing Diagnosis Relapse

Neuroblastoma, a clinically heterogeneous pediatric cancer, is characterized by distinct genomic profiles but few recurrent mutations. As neuroblastoma is expected to have high degree of genetic heterogeneity, study of neuroblastoma's clonal evolution with deep coverage whole-genome sequencing of diagnosis and relapse samples will lead to a better understanding of the molecular events associated with relapse. Samples were included in this study if sufficient DNA from constitutional, diagnosis and relapse tumors was available for WGS. Whole genome sequencing was performed on trios (constitutional, diagnose and relapse DNA) from eight patients using Illumina Hi-seq2500 leading to paired-ends (PE) 90x90 for 6 of them and 100x100 for two. Expected coverage for sample NB0175 100x100bp was 30X for tumor and constitutional samples. For the seven other patients expected coverage was 80X for tumor samples with PE 100x100, 100X in the other tumor samples and 50X for all constitutional samples (see table 1). Following alignment with BWA (Li et al., Oxford J, 2009 Jul) allowing up to 4% of mismatches, bam files were cleaned up according to the Genome Analysis Toolkit (GATK) recommendations (Van der Auwera et al., Current Protocols in Bioinformatics, 2013, picard-1.45, GenomeAnalysisTK-2.2-16). Variant calling was performed in parallel using 3 variant callers: GenomeAnalysisTK-2.2-16, Samtools-0.1.18 and MuTect-1.1.4 (McKenna et al., Genome Res, 2010; Li et al., Oxford J, 2009 Aug; Cibulskis et al., Nature, 2013). Annovar-v2012-10-23 with cosmic-v64 and dbsnp-v137 were used for the annotation and RefSeq for the structural annotation. For GATK and Samtools, single nucleotide variants (SNVs) with a quality under 30, a depth of coverage under 6 or with less than 2 reads supporting the variant were filter out. MuTect with parameters following GATK and Samtools thresholds have been used to filter our irrelevant variants. .SNVs within and around exons of coding genes overlapping splice sites.. Then,variants reported in more than 1% of the population in the 1000 genomes (1000gAprl_2012) or Exome Sequencing Project (ESP6500) have been discarded in order to filter polymorphisms. Finally, synonymous variants were filtered out. MuTect focuses on somatic by filtering with constitutional sample. Mpileup comparison between constitutional and somatic DNAs allowed us to focus also on tumor specific SNVs with GATK and Samtools. Finally, every SNV called by our pipeline and also supported in any constitutional samples were filtered our in order to prevent putative constitutional DNA coverage deficiency. Then we analyzed CNVs (copy number variants) with HMMcopy-v0.1.1 (Gavin et al., Genome Res, 2012) and control-FREEC-v6.7 (Boeva et al., Bioinformatics 2011) with a respective window of 2000bp and 1000 bp, and auto-correction of normal contamination of tumor samples for Control-FREEC. Finally we explored Structural variants (SVs) including deletions, inversions, tandem duplications and translocations using DELLY-v0.5.5 with standard parameters (Rausch et al., Oxford J, 2012). In tumors, at least 10 supporting reads were required to make a call and 5 supporting reads for the sample NB0175 with a coverage of only 40X (see table 2). To predict SVs in constitutional samples for subsequent somatic filtering, only 2 supporting reads were required in order not to miss one. To identify somatic events, all the SVs in each normal sample were first flanked by 500 bp in both directions and any SVs called in a tumor sample which was in the combined flanked regions of respective normal sample was removed (see graph 1). Deletions with more than 5 genes impacted or larger than 1Mb and inversions or tandem duplications covering more than 4 genes, were removed. We focused on exonic and splicing events for deletions, inversions, and tandem duplications. For translocation, we keep all SVs that occurred in intronic, exonic, 5'UTR, upstream or splicing regions. Bioinformatics detection of variations with Deep sequencing approach Once PE reads merged and adaptors trimmed by SeqPrep with default parameters, merged reads were aligned via the BWA (Li H. and Durbin R. 2009 PMID 19451168) allowing up to 1 differences in the 22-base-long seeds and reporting only unique alignments. Only reads having a mapping quality 20 or more have been further analysed. Variant calling software was not used, since we aimed to predict variations at low frequencies, observed in less than 1% of reads. Such variants require a custom approach. Using DepthOfCoverage functions of the Genome Analysis Toolkit (GATK) v2.13.2 (McKenna A, et al., 2010 Genome Research PMID: 20644199), we focused on high quality coverage of bases A, C, G and T at the targeted variant position. Depth of coverage of each base following a mapping quality higher than 20 and a base quality higher than 10 have been taken into account in order to focus only on high quality data. Aiming to determine the background level of variability at the studied regions, 10 control samples were included in the analysis. The same approach and filtering criteria have been applied as introduced above over the entire amplicons. In order to highlight variants, for each sample the frequencies of each bases at each amplicon position were then compared to those observed in the set of controls. Statistical analyses were performed with the R statistical software (http://www.R-project.org). Fisher’s exact two-sided tests with a Bonferroni correction were performed to compare percentages of bases between the data sets, i.e. for a given base between a case and the controls. Finally, significant variations were filtered-in once (i) a significant increase in the percentage of avariant base and (ii) a significant decrease in the percentage of it's reference base following our p.values criteria was observed (p.val < 0.05).

Study EGAS00001001184

WGS samples for study EGAS00001001623

Dataset EGAD00001001856

Characterization_of_individual_foci_of_multicentric_multifocal_breast_cancer_using_targeted_next_generation_sequencing

Multifocality or multicentricity in breast cancer may be defined as the presence of two or more tumor foci within a single quadrant of the breast or within different quadrants of the same breast, respectively. This original classification of the breast cancer as multicentric or multifocal was based on the assumption that cancers arising in the same quadrant were more likely to arise from the same ductal structures than those occurring in separate areas of the breast. The problem with these definitions is that the “quadrants” of the breast are arbitrary external designations, as no internal boundaries do exist. This project will therefore focus both on synchronous multifocal and multicentric tumors. The incidence of multifocal and multicentric breast cancers was reported to be between 13 and 75% depending on the definition used, the extent of the pathologic sampling of the breast and whether in situ disease is considered evidence of multicentricity (1). Although this incidence is variable, those figures show that it is a frequent phenomenon. Multiple (multifocal/multicentric) breast carcinomas, especially when occurring in the same breast, represent a real challenge for both pathologists and clinicians in terms of identifying the cellular origin and the best therapeutic management of the cancer. Multifocality or multicentricity has been associated with a number of more aggressive features including an increased rate of regional lymph node metastases and adverse patient outcome when compared with unifocal tumors (2-3), and a possible increased risk of local recurrence following breast conserving surgery (4). For the moment, the literature is divided on whether there is a corresponding impact on survival outcomes. Today, the current convention to stage and to treat multifocal and multicentric tumors is the classical tumor-node-metastasis (TNM) staging guidelines with which tumor size is assessed by the largest tumor focus without taking other foci of disease into consideration. If some papers, as the recent one from Lynch and colleagues, support the current staging convention (3), others, however, as Boyages et al. suggested that aggregate size and not the size of the largest lesion should be considered in order to refine the prognostic assessment of those tumors (5). On the top of that, the question whether multifocal/multicentric carcinomas are due to the spread of a single carcinoma throughout the breast or is due to multiple carcinomas arising simultaneously has been a matter of debate. Some studies suggested that multifocal breast cancer may result from either intramammary spread from a single primary tumor or multiple synchronous primary tumors; whereas others suggest that multiple breast carcinomas always arise from the same clone (6-8). Recently, Pietri and colleagues analyzed the biological characterization of a series of 113 multifocal/multicentric breast cancers (8) which were diagnosed over a 5-year period. The expression of estrogen (ER) and progesterone (PgR) receptors, Ki-67 proliferative index, expression of HER2 and tumor grading were prospectively determined in each tumor focus, and mismatches among foci were recorded. Mismatches in ER status were present in 5 (4.4%) cases and PgR in 18 (15.9%) cases. Mismatches in tumor grading were present in 21 cases (18.6%), proliferative index (Ki-67) in 17 (15%) cases and HER2 status in 11 (9.7%) cases. Interestingly, this heterogeneity among foci has led to 14 (12.4%) patients receiving different adjuvant treatments compared with what would have been indicated if we had only taken into account the biologic status of the primary tumor. This study therefore showed that differences in biological characteristics of multifocal/multicentric lesions play a crucial role in the adjuvant treatment decision making process. In this study, we will concentrate on a larger series of patients with multifocal invasive ductal breast cancer lesions. We aim at: 1. Evaluating the incidence of multifocality according to the different breast cancer molecular subtypes (ER-/HER2-, HER2+, ER+/HER2-). 2. Evaluating the incidence of multifocality in patients with hereditary breast cancer disease (presence of germline BRCA1 or BRCA2 mutations). Moreover, we would like to investigate if multifocal lesions with BRCA1 or BRCA2 mutations exhibit a characteristic combination of substitution mutation signatures and a distinctive profile of deletions as demonstrated recently by Nik-Zainal and colleagues (9). 3. Correlating multifocality with clinical information in order to define its influence on patients’ survival (DFS and OS). 4. Carrying high coverage targeted gene sequencing of driver cancer genes and genes whose mutation is of therapeutic importance in order to compare clinically-relevant genetic differences between several multifocal breast cancer lesions. 5. Evaluating the impact of the distance between the different lesions on the clinical outcome but also on the genetic differences. 6. Comparing gene expression patterns between several multifocal breast cancer lesions and correlate them with the results of the targeted genes screen. 7. Characterizing the genomic and transcriptomic status of cancer related genes in metastatic lesions (local recurrence, positive lymph node or distant metastatic sites) from the same multifocal invasive ductal breast cancer patients in order to evaluate the consequence of genomic and transcriptomic heterogeneity of multifocal lesions on metastatic lesions. Multiple (multifocal/multicentric) breast carcinomas, especially when occurring in the same breast, represent a real challenge for both pathologists and clinicians in terms of identifying the cellular origin and the best therapeutic choice. This project has the potential to identify genetic/transcriptomic differences existing between several lesions constituting multifocal breast cancers, which in the routine clinical practice are usually considered to be homogeneous among them. We foresee validating significant results in a larger series of patients and this, in turn, could have a remarkable impact on the treatment and clinical management of multifocal breast cancers. Indeed, we hope to provide some evidence whether or not each focus matters in multifocal and multicentric breast cancer to define the adequate therapeutic approach, especially in the context of targeted therapies. The work to be done at Sanger will be target gene screen pooling of 1400 samples.

Study EGAS00001000407

BC Cancer, part of the Provincial Health Services Authority, Technology Development Office, Data Access Committee (PHSA TDO DAC)

Access to data generated by BCCA is made available by completing the data access agreement for review by the data access committee and will be granted to qualified investigators for appropriate use.

Dac EGAC00000000011

GCAT| WGS FASTQ V1

This dataset include FASTQ files of 808 samples from GCAT cohort. Technology used HiSeq 4000, read length 150 bp, inner mate distance 300 bp. For each sample the paired -ends are generated in separated files. Each FASTQ is splitted in multiple LANEs and grouped by the Multiplex index.

Dataset EGAD00001008201

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