Search Results - EGA European Genome-Phenome Archive

Genome-wide identification of distinct miRNA-mRNA target regulation pairs in Non-Hodgkin lymphomas: a report from the ICGC MMML-Seq consortium

We obtained miRNA profiles by miRnome sequencing from ICGC MMML-Seq patients diagnosed with Burkitt lymphoma, diffuse large B-cell lymphoma and follicular lymphoma and provide evidence of subtype-specific miRNA expression differences. We describe differentially expressed, mutated, edited and not previously annotated miRNAs. Addditionally, by performing argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) experiments, we obtained a set of biochemically-validated miRNA binding sites and identified miRNA-mRNA interaction pairs with a negative correlation in patient RNASeq data.

Study EGAS00001001394

Gut microbiome modulates response to anti PD1 immunotherapy in metastatic melanoma patients

There is a growing appreciation of the role of the microbiome in cancer, and evidence in pre-clinical models that the gut microbiome may modulate responses to immune checkpoint blockade though this has not been well-characterized in patients. We analyzed the oral (n=86)and gut (n=43) 16S microbiome in melanoma patients on PD-1 blockade. Significant differences were noted in the diversity and composition of the gut microbiome between responders and non-responders in patients with a fecal microbiome sample, with significantly higher alpha diversity and relative abundance of Ruminococcaceae bacteria) in R. Metagenomic studies (n=25) revealed functional differences in gut bacteria in R including enrichment of anabolic pathways. Immune profiling demonstrated enhanced systemic and anti-tumor immunity in patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplant from responding patients. Together, these data have important implications for treatment with immune checkpoint blockade in cancer. The fastq files associated with this dataset are stored at ENA under the following accession numbers: PRJEB22894 (Fecal 16S), PRJEB22874 (Oral 16S), PRJEB22895 (Murine 16S), PRJEB22893 (Fecal WGS).

Study EGAS00001002698

Next-Generation Sequencing of RNA and DNA Isolated from Paired Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Samples of Human Cancer and Normal Tissue

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5 % success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80 % of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Study EGAS00001000737

Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T-cell lymphomas with hemophagocytic lymphohistiocytic syndrome

Sub-cutaneous panniculitis-like T-cell lymphomas (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation which adversely impacts survival1,2. T-cell-immunoglobulin mucin-3 (TIM-3) is a modulator of immune responses expressed on subgroups of T- and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants in highly conserved residues of TIM-3, c.245A>G(p.Y82C) and c.291A>G(p.I97M), each with specific geographic distribution. Y82C-TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while I97M-TIM-3 occurs in Caucasians. Both variants induce protein misfolding and abrogate TIM-3’s plasma-membrane expression leading to persistent immune activation, increased production of inflammatory cytokines, including TNF-α and IL-1β, promoting HLH and SPTCL. Our findings highlight HLH/SPTCL as a new genetic entity and showcase TIM-3 mutations as the first causative genetic defect in SPTCL. While TIM-3-mutant HLH/SPTCL benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

Study EGAS00001002765

Xenograft_Sequencing

Sequencing of mouse/human carcinoma xenograft samples.

Study EGAS00001000140

Targeted resequencing of ribosomal proteins in multiple myeloma patient samples.

We studied 38 multiple myeloma samples to discover mutations in ribosomal proteins. To this end, we ran a specifically designed targeted resequencing study to resequence the coding regions of all ribosomal proteins.

Study EGAS00001002405

Colorectal advanced adenomas NKI-AvL TGO COCOS series

Colorectal cancer (CRC) develops from normal epithelium, through a benign precursor lesion called adenoma, by accumulation of genetic alterations affecting oncogenes and tumour suppressor genes. About 5% of colorectal adenomas are estimated to progress to CRC. However, it is important to identify which adenomas actually carry a high-risk of progression, because these serve as intermediate endpoints for e.g. screening programs. In clinical practice, adenomas with a size of ≥10 mm, villous component and/or high-grade dysplasia, called advanced adenomas, are considered high-risk, although solid evidence for this classification is lacking. Specific DNA copy number changes are associated with adenoma-to-carcinoma progression.For this tissue dataset, we applied low-pass whole genome sequencing to 96 advanced adenomas. Advanced adenomas were classified as lesions with low-risk or high-risk of progression, according to the presence of specific DNA copy number changes (Carvalho et al, CancerPrevRes, 2018).

Study EGAS00001002952

RNAseq___Discovery_of_resistance_mechanisms_to_the_BRAF_inhibitor_vemurafenib_in_metastatic_BRAF_mutant_melanoma

We propose to biopsy 20 consented BRAF mutant melanoma patients at Addenbrooke's Hospital pre-treatment with vemurafenib and also upon the development of resistant disease, with the aim of using exome sequence and SNP6 data to identify novel sequence variants and copy number alterations that can be used to validate observed resistance mechanisms in our cell line models and also to use these models to inform as to likely candidate small molecule inhibitors to overcome resistance and that could be tested in the clinical trial setting.

Study EGAS00001000813

The evolutionary history of human colitis-associated colorectal cancer

Inflammatory bowel disease confers an increased lifetime risk of developing colorectal cancer (CRC), and colitis-associated CRC (CA-CRC) is molecularly distinct from sporadic CRC (S-CRC). Here we have dissected the evolutionary history of CA-CRC using multi-region sequencing. Exome sequencing was performed on fresh-frozen multiple regions of carcinoma, adjacent non-cancerous mucosa and blood from 12 CA-CRC patients (n=55 exomes), and key variants were validated with orthogonal methods. Genome-wide copy number profiling was performed using SNP arrays and low-pass whole genome sequencing on archival non-dysplastic mucosa (n=9), low-grade dysplasia (LGD, n=30), high-grade dysplasia (HGD, n=13), mixed LGD/HGD (n=7) and CA-CRC (n=19). Phylogenetic trees were reconstructed, and evolutionary analysis used to reveal the temporal sequence of events leading to CA-CRC. 10/12 tumors were microsatellite stable with a median mutation burden of 3.0 single nucleotide alterations (SNAs) per Mb, around 20% higher than S-CRC (2.5 SNAs/Mb), and consistent with elevated aging-associated mutational processes. Non-dysplastic mucosa had considerable mutation burden (median 47 SNAs), including mutations shared with the neighboring CA-CRC, indicating a pre-cancer mutational field. CA-CRCs were often near-triploid (42%) or near-tetraploid (21%) and phylogenetic analysis revealed that copy number alterations (CNAs) began to accrue in non-dysplastic bowel, but the LGD/HGD transition often involved a punctuated catastrophic CNA increase.

Study EGAS00001003028

Whole-genome sequencing in 14 cases and whole-exome sequencing in 90 cases of Chinese ESCC

Esophageal cancer is one of the most aggressive cancers and the sixth leading cause of cancer death worldwide1. Approximately 70% of the global esophageal cancers occur in China and over 90% histopathological forms of this disease are esophageal squamous cell carcinoma (ESCC)2-3. Currently, there are limited clinical approaches for early diagnosis and treatment for ESCC, resulting in a 10% 5-year survival rate for the patients. Meanwhile, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we show a comprehensive genomic analysis in 158 ESCC cases, as part of the International Cancer Genome Consortium (ICGC) Research Projects (http://icgc.org/icgc/cgp/72/371/1001734). We conducted whole-genome sequencing in 14 ESCC cases and whole-exome sequencing in 90 cases.

Study EGAS00001001487

Targeting the DNA Repair Pathway in Ewing Sarcoma

Ewing’s Sarcoma is a bone and soft tissue tumor that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive but survival for metastatic and recurrent disease is poor. Whole genome sequencing of 19 Ewing’s Sarcoma tumors showed that STAG2 was mutated in 10% (2/19) of the tumors and STAG2 protein was absent in 14% (13/106) tumors by immunohistochemical staining. Previous studies have shown that glioblastoma cells lacking STAG2 are more sensitive to poly-ADP ribose polymerase (PARP) inhibitors. We found that Ewing’s Sarcoma cell lines are sensitive to PARP inhibitors irrespective of STAG2 protein expression. Ewing Sarcoma cell lines are defective in double strand DNA break repair. PARP inhibitor cytotoxicity in Ewing’s Sarcoma cells was potentiated 10-1,000 fold by DNA damaging agents (irinotecan and temozolomide). To extend these studies in vivo, we developed an orthotopic Ewing’s Sarcoma mouse model and performed pharmacokinetic and pharmacodynamic studies with three different PARP inhibitors (BMN-673, olaparib and veliparib) in clinical development for pediatric cancer. Those data were used to design preclinical phase I studies to identify tolerable drug combinations for pilot efficacy testing (preclinical phase II). Based on the results of the preclinical phase I/II data, we performed a double blind, randomized, placebo controlled preclinical phase III trial with 274 mice in 15 treatment groups. Irinotecan administered in a low-dose protracted schedule optimized for pediatric patients was an effective DNA damaging agent to combine with olaparib and BMN-673 and was better tolerated than combinations with temozolomide. Combining olaparib or BMN-673 with irinotecan and temozolomide gave complete and durable responses in over 80% of the mice.

Study EGAS00001000839

Exome-wide analysis identifies three low-frequency missense variants associated with pancreatic cancer risk in Chinese populations

Germline coding variants have not been systematically investigated for pancreatic ductal adenocarcinoma (PDAC). Here, we report an exome-wide investigation using the Illumina Human Exome Beadchip with 943 PDAC cases and 3,908 controls in the Chinese population, followed by two independent replicate samples including 2,142 cases and 4,697 controls. We identify three low-frequency missense variants associated with the PDAC risk: rs34309238 in PKN1 (OR = 1.77, 95% CI: 1.48-2.12, P = 5.35 X 10-10), rs2242241 in DOK2 (OR = 1.85, 95% CI: 1.50-2.27, P = 4.34 X 10-9) and rs183117027 in APOB (OR = 2.34, 95% CI: 1.72-3.16, P = 4.21 X 10-8).

Study EGAS00001003040

Conjunctival fibrosis and the innate barriers to Chlamydia trachomatis intracellular infection: a genome wide association study

This data comes from a genome-wide association study in 1090 cases of scarring trachoma and 1531 controls from a non-selected multi-ethnic sampling of The Gambia. The data constitutes hard base calls at 1457295 features from the HumanOmni2.5-8 (Omni2.5) BeadChip. Three genotype-calling algorithms were used : Illuminus (Bioinformatics 2007, 23:2741–6), GenCall (Patent US7035740, 2006) and GenoSNP (Bioinformatics 2008, 24:2209–14). Data from each calling algorithm was filtered to retain only SNPs with a call rate ≥ 0.98. To obtain a merged set of SNPs, all genotypes that matched across call-sets were retained, whilst those that mismatched between call-sets were set to missing. Genotypes that were present in one call set and missing in others were also retained. The merged SNP set contained 1467876 SNPs, which were filtered for call rate ≥ 0.99 and Hardy-Weinberg equilibrium P value < 5 x 10-8. The case and control groups were approximately equivalent with respect to gender and ethnicity. 70% of cases and 63% of controls were female. Self-described ethnic composition of the cases was 29% Jola, 27% Mandinka, 21% Wolof, 10% Fula and 13% other/no data. In controls the composition was 25% Jola, 24% Mandinka, 21% Wolof, 8% Fula and 22% other/no data. Median age was 49 (range 32-60) in cases and 37 (range 12-52) in controls (t = -3.6019, P = 0.0003). This data set has substantial levels of population structure, which reflects extensive networks of pairwise kinship between participants. First pass tests of association in EMMAX (Nat Genet 2010, 42:348–54) led to genome-wide deflation of the test statistics (λ = 0.982 SE = 1.74e-05) that could be directly attributed to the differing age and gender distributions between cases and controls. Modelling the phenotype to adjust for age and gender successfully controlled for this and no genome-wide deviation from the null was subsequently observed (λ = 1.001, SE = 8.7 x 10-7). Neither principal components analysis (PCA) nor tests of proportional identity by state (IBS) variance in PLINK identified significant levels of within or between group genetic variance. The phenotypes submitted in the phenotypes file include both uncorrected (case/control status) and corrected (age and gender corrected case/control status) phenotypes, as well as age and self-described ethnicity.

Study EGAS00001001516

Genetic landscape of relapsed DLBCL

Diffuse large B-cell lymphoma is a heterogenous malignancy that can arise de novo or can result from histologic transformation from indolent lymphomas. Considerable sequencing effort has been placed on determining the mutations that are common at the time of diagnosis in DLBCL but little is known about the nature of relapsed disease. We have sequenced tumour and matched normal exomes of 38 rrDLBCL patients including 25 de novo DLBCLs and 13 transformed lymphomas to identify somatic mutations and copy number alterations associated with relapse.

Study EGAS00001001553

Balanced_Ependymoma

We propose to definitively characterise the somatic genetics of a number of pediatric malignant tumours including ependymoma, high grade glioma and central nervous system primitive neurectodermal tumours through generation of comprehensive catalogues of somatic mutations by high coverage genome sequencing.

Study EGAS00001000174

Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma

We conducted a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322) and assessed clinical responses and immune correlates. Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST overall response rate 73%, pathologic complete response rate 45%) but substantial toxicity (73% grade 3 treatment related adverse events), whereas treatment with nivolumab monotherapy yielded modest responses (RECIST overall response rate 25%, pathologic complete response rate 25%) and low toxicity (8% grade 3 treatment related adverse events). Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy. Mutational load was also higher in responders to therapy.

Study EGAS00001003178

Enhanced detection of circulating tumor DNA by fragment size analysis

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on sensitivity for detecting genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for non-invasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 cancer patients using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90–150 bp, and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90–150 bp improved detection of tumor DNA, with more than 2-fold median enrichment in &gt;95% of cases, and more than 4-fold enrichment in &gt; 10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with AUC&gt; 0.99 compared to AUC &lt; 0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC &gt; 0.91, compared to AUC &lt; 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection. This could reduce the required depth of cfDNA sequencing for clinical applications, earlier diagnosis and study of tumor biology.

Study EGAS00001003258

Chordoma_Extension__known_cancer_genes_

Extension analysis to pursue candidate genes of interest in chordoma

Study EGAS00001000895

Segmental_cherry_angioma_case

Cherry angiomas are common benign blood vessel growths on the skin. In this study we characterise mutational profile of a cherry angioma and adjacent skin biopsied from a patient with multiple lesions with segmental presentation.

Study EGAS00001008212

Whole genome and RNA sequencing of paediatric glioblastoma in the ICGC PedBrain project

Paediatric glioblastoma (pedGBM) is characterised by substantial intertumoural heterogeneity. In order to identify the genetic lesions underlying tumourigenesis, we analysed whole genomes and transcriptomes of pedGBMs and in vitro model systems by next generation sequencing. Novel fusions of the receptor tyrosine kinase MET lead us to investigate targeted therapy using MET inhibitors. This study is part of the International Cancer Genome Consortium PedBrain Tumor Project (ICGC PedBrain, http://www.pedbraintumor.org/).

Study EGAS00001001139

H014: HES5 mediates NOTCH signaling by interaction with AKT to drive liver carcinogenesis

Notch signaling plays a pivotal role in liver development and aberrant Notch signaling may lead to hepatocellular (HCC) or cholangiocellular carcinoma (CCA) development. Using whole exome sequencing of 54 human HCC samples, we discovered 19 somatic missense mutations in 15 different Notch pathway genes affecting 24.6% (14 of 57) of patients. Prediction of functional impact of these Notch pathway mutations revealed HES5-R31G to have high relevance. We found HES5 to be strongly activated by Notch signaling. In vitro, HES5 reduced cell migration and clonogenicity and in vivo, HES5 inhibited MYC-dependent liver tumor formation. But together with AKT, HES5 increased liver tumor formation in a mouse model. HES5 directly binds to AKT and AKT expression leads to increase HES5 protein levels. Furthermore, HES5 exhibits a negative feed-back loop by inhibiting NOTCH1 and HES1 gene expression. In contrast, HES5-R31G appears to be non-functional possibly by loss of nuclear translocation

Study EGAS00001003329

Whole genome sequencing and whole exome sequencing of mucosal melanoma

We conducted whole genome sequencing of 5 mucosal melanomas and their matched DNA from blood. We conducted whole exome sequencing of a further 5 mucosal melanomas and their matched DNA from blood.

Study EGAS00001000474

The genomic landscape of follicular and diffuse large B-cell lymphoma

As part of the ICGC, the ICGC MMML-Seq project performed whole genome sequencing and RNA sequencing of follicular and diffuse large B-cell lymphoma. Follicular and diffuse large B-cell lymphomas are the most common B-cell lymphomas in adulthood. Analyses were performed in concordance with the guidelines of the ICGC. The results define the genomic landscape of structural variants, somatic single nucleotide variants and mutational signatures in these lymphomas

Study EGAS00001002199

Whole genome sequencing of Ewings Sarcoma

Sarcomas are cancers of the bone and soft tissue often defined by their gene fusions. However, the timing, context, and processes by which these pathogenic fusions arise are unknown. We explored this in Ewing sarcoma, a cancer driven by EWSR1-ETS fusions, with very few cooperating mutations. Combining whole-genome sequencing with enhanced informatics, we found that the EWSR1-ETS fusion arose from striking rearrangement clusters in 42% of cases (52/124). Notably, these were organized in loops that universally contained the fusion at their center, while also weaving up to 18 genes together with it. We found the same pattern of rearrangements in three additional types of sarcoma. From these data, we define a new signature for sarcoma fusions that precedes other somatic changes, in the earliest replicating DNA of the genome. This dramatic, sudden process impinges on many genes – generating multiple coding changes that profoundly affect the transcriptome, with the disease-defining gene fusion at its core. These rearrangement loops emerge in an early ES clone from which both the primary tumor and the lethal relapse emerged, and then evolved in parallel until clinically detected.

Study EGAS00001003385

OAC RNASeq

Please see our related publication, 'Molecular profiling to predict immunochemotherapy outcomes in esophageal adenocarcinoma'

Study EGAS00001006468

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