5731 results for "canc*"

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• Robust detection of translocations in lymphoma FFPE samples using Targeted Locus Capture-based sequencing

  Chromosomal translocations with immunoglobin (IG) loci are the classic drivers in a large subset of B-cell lymphomas. Detection of these translocations is important for confirmation of diagnosis and for prognosis and therapy decisions. Currently, molecular diagnosis of translocations in lymphomas is not addressed well by Next Generation Sequencing (NGS). The standard method for detection of translocations is Fluorescence In Situ Hybridization (FISH), which is labor-intensive, and can be difficult to interpret. There is a need for a robust technology that can be standardized. Targeted Locus Capture (TLC) selectively enriches and sequences entire genes based on the crosslinking of physically proximal sequences, and thereby enables complete sequencing of genes of interest, including detection of large structural variants. Because the technology is based on the crosslinking and fragmenting of DNA, it has particular advantages in the analysis of Formalin-Fixed, Paraffin-Embedded (FFPE) samples, in which DNA is inherently crosslinked and fragmented. In order to validate the FFPE-TLC technology as a novel approach for translocation detection in lymphoma samples, we have developed a panel assay containing genes with frequent translocations (MYC, BCL2, BCL6, IG loci). With this assay we have analyzed >140 lymphoma and control FFPE samples of variable input amounts and qualities that had previously been analyzed with FISH, and a subset also with standard targeted NGS. Good concordance with FISH results was observed for both translocation positive and negative samples. In 10 cases for which FFPE-TLC detected a relevant fusion and FISH had been called negative, discordance could be explained by higher sensitivity of FFPE-TLC or by inconclusive FISH results. In a specific case, FFPE-TLC detected a small-distance rearrangement on chromosome 3 that caused a BCL6 fusion but led to insufficient and therefore undetectable break-apart with FISH. Secondly, the FFPE-TLC approach was tested on a set of 19 B-cell lymphoma FFPE samples that had previously been analyzed using standard targeted NGS and FISH and was enriched for discordant results between these methods. FFPE-TLC-based NGS enables more robust translocation calling as the detection relies on broad sequencing coverage across the translocation partner rather than on breakpoint sequences only. In 3 cases, FFPE-TLC could proof false negative calls in standard targeted NGS due to breakpoints located in regions difficult to capture or to sequence. In 1 case, standard targeted NGS had made a false positive call on a breakpoint sequence that was shown to be caused by a small insertion rather than a genuine translocation. This study shows that FFPE-TLC promises to be a robust alternative for FISH analysis and standard targeted NGS procedures in lymphoma diagnostics and possibly in other cancers with frequent structural variants. The FFPE-TLC approach enables a single, DNA-based NGS test detecting both small mutations and translocations.

  Study EGAS00001004760

• Identification of 19 novel loci reveals gene regulatory mechanisms determining susceptibility to testicular germ cell tumour

  Testicular germ cell tumour (TGCT) is the most common cancer in young men1,2, and is characterised by strong inherited genetic risk factors3. Here we have undertaken large-scale genome wide association study (GWAS) for TGCT, encompassing ~7,500 cases and ~23,000 controls, through the Oncoarray consortium. We identified 19 novel loci, approximately doubling the number of known TGCT risk loci to 44 (P<5x10-8)4-14 and conduct deep, high-throughput functional annotation of all risk loci. We establish a network of physical interactions for all risk SNPs to candidate casual genes in 3D space, using high-throughput chromosome conformation capture techniques (HiC) in TGCT cells. Firstly, functional evidence reveals widespread disruption of developmental transcriptional regulators, consistent with failed primordial germ cell differentiation as an initiating step in TGCT oncogenesis15. We secondly observe multiple risk loci associated with defective microtubule assembly, compatible with the high level of aneuploidy observed in TGCTs, suggesting gross chromosomal instability. Finally KIT-MAPK signalling features as a recurrently dysregulated pathway. In summary our findings substantially increase the number of known TGCT risk alleles, and provides a functional basis for disease susceptibility.

  Study EGAS00001001836

• Genomics-based personalized oncology in cancer of unknown primary (CUP, project H021)

  To perform a comprehensive genomic characterization of 70 patients suffering from cancer of unknown primary (CUP) we used whole-exome, whole-genome, transcriptome and methylome analysis. We detected a substantial mutational heterogeneity with genes most commonly affected by SNVs, indels and fusions being TP53, TTN, MUC16, ABCA13, COL6A3, KRAS, LRP1B, XIRP2 and CSMD3. The most common fusion involved FGFR2, the most common focal deletion affected CDKN2A. A molecular tumor board recommended genomics-based therapies in 56/70 (80%) patients which were applied in 20/56 (35.7%) cases. Entity predictions based on transcriptome and methylome data could be made in up to 62/70 (88.6%) cases but were conclusive in only 16/48 (33.3%) cases. Germline analysis revealed 6 (likely) pathogenic mutations in 5 patients. Recommended therapies translated into a mean PFS2/1 ratio of 3.61 (median=2.25) with a median PFS1 of 89 days (n=17) compared to a median PFS2 of 182.5 days (n=20). Our data emphasize the clinical benefit of comprehensive genetic approaches in diagnostic and therapeutic management and underline the need for innovative, mechanism-based clinical trials in this heterogeneous group of diseases.

  Study EGAS00001004786

• Paediatric_CNS_tumour_autopsy_DNA

  Investigation of early embryonic mutations can reveal the earliest stages of normal tissue development and the origins of paediatric cancers. Through widespread sampling of tumour, adjacent normal and distant normal samples at autopsy and whole genome sequencing, we aim to establish the point at which lethal paediatic brain tumours diverge from normal development. With many such tumours associated with germline predisposition mutations, we will also be able to investigate the tissue-specific effects these mutations induce.

  Study EGAS00001004771

• Analysis of the Elements Involved in the Enrichment of a Panel of Genomic Regions by Nanopore Sequencing Using Adaptive Sampling

  DNA sequencing using nanopore technologies with the affordable MinION device is useful for the identification and characterization of structural variants, long haplotypes, sequencing of repetitive regions and identification of epigenetic modifications. The main limitation of this approach is the low coverage obtained, which might be avoided by adaptive sampling, a computationally controlled method of enrichment for targeted genomic regions. This study dissects the factors involved in the enrichment by adaptive sampling of a panel of 18 human genome regions containing 20 genes implicated in breast cancer sequenced in 16 patients with familial breast cancer negative for NGS screening. An average coverage of 2.0x was obtained for the whole genome and 5.1x for selected regions. Sequencing time was the main factor improving coverage. The selection of long reads (>1 Kb) did not improve the enrichment. The length of the selected region, which in our study ranged from 126 to 565 Kb, did not play a significant role in enrichment. However, the region containing PMS2 showed significantly lower coverage, which could be explained by the high number of PMS2 pseudogenes (N = 14), which were also enriched. Our study shows new evidence of enrichment obtained by adaptive sampling in a panel of genomic regions and shows parameters, the relevance of sequencing time and the role of pseudogenes, that improve the enrichment yield with no library reloading or GPU use, data useful for a more efficient application of this procedure in future studies.

  Study EGAS00001007375

• DEREGULATION OF PRE-mRNA SPLICING IN MISMATCH REPAIR DEFICIENT COLORECTAL CANCER

  Study EGAS00001004863

• RNA-seq study of a Princess Margaret Cancer Centre human acute myeloid leukemia patient cohort

  The PMCC AML RNAseq dataset consists of 81 AML patient samples (clinical data in Supplemental Table 11 of manuscript), processed in two batches. These patient samples are able to engraft in the NSG (NOD.Cg PrkdcscidIl2rgtm1Wjl /SzJ) mouse model. Five patients (90543, 598, 90240, 110484, 100500) were included in both batches. Viaably frozen material from the Leukemia Tissue Bank at Princess Margaret Cancer Centre/ University Health Network were thawed by dropwise addition of X-VIVO + 50% fetal calf serum supplemented with DNase (100μg/mL final concentration, Roche). RNA was extracted from bulk peripheral blood mononuclear cells (PBMC) using the RNeasy Micro Kit (Qiagen Inc.). A paired-end 76 base-pair flow-cell lane Illumina High seq 2000 yielded an average of 240 million sequence reads aligning to genome per sample at the Genome Sciences Centre, BC Cancer Agency for cohort 1. Cohort 2 was subjected to 125 bp, paired-end RNA-sequencing on the Illumina HiSeq 2500 with an average of 50 million reads/sample at the Centre for Applied Genomics, Sick Kids Hospital.

  Study EGAS00001004792

• 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

• Organoid_Derivation_Project___GRCh38___TGS

  Generation of normal and cancer organoids for sequencing as part of the WTSI-CRUK international collaboration with NCI (in HCMI) to generate the next generation of cancer cell lines.

  Study EGAS00001007349

• sWGS of Pap test smears from healthy donors and HGSOC patients and matched tumor tissue

  Shallow whole genome sequencing was performed on Pap test smears considering at least 10 M reads per sample. The cohort consists of 77 samples from healthy women, i.e. no tumor diagnosis, and 65 samples from women with a diagnosis of high-grade serous ovarian cancer (HGSOC) taken months or years before diagnosis. 44 primary tumor samples matched to the respective HGSOC patients were sequenced in the same manner as the Pap test smears.

  Study EGAS00001007084

• Mechanism of action and resistance to Trastuzumab Deruxtecan in patients with metastatic breast cancer

  Study EGAS00001007372

• Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing

  We present Bottleneck Sequencing System (BotSeqS), a nextgeneration sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately prior to library amplification. We use BotSeqS to show age and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal tissues can vary by several orders of magnitude, depending on biologic and environmental factors. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues.

  Study EGAS00001001838

• Alveolar Rhabdomyosarcoma case report

  Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% five-year disease-free survival rate when a chromosomal fusion is present, and 40% five-year disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-year old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.

  Study EGAS00001004828

• scRNA-seq of HGSC tumor and ascites

  Immune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few notable exceptions, their identities remain largely unknown. We uncovered the immune regulatory metabolic states and metabolomes of sorted tumor and stromal, CD4+, and CD8+ cells from the tumor and ascites of patients with high-grade serous ovarian cancer (HGSC) using high-dimensional flow cytometry and metabolomics supplemented with single cell RNA sequencing. Flow cytometry revealed that tumor cells show a consistently greater uptake of glucose than T cells, but similar mitochondrial activity. Cells within the ascites and tumor had pervasive metabolite differences, with a striking enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared to ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the gene encoding the enzyme that catalyses the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Treatment of T cells with MNA resulted in an increase in T cell-mediated secretion of the tumor promoting cytokine tumor necrosis factor alpha. Thus, the TME-derived metabolite MNA contributes to an alternative and non-cell autonomous mechanism of immune modulation of T cells in HGSC. Collectively, uncovering the tumor-T cell metabolome may reveal metabolic vulnerabilities that can be exploited using T cell-based immunotherapies to treat human cancer.

  Study EGAS00001004829

• Effect_of_FAM50_knockout_on_the_transcriptome

  A knockout clone has been generated for both FAM50A and FAM50B; knockout of the other gene is then performed and the transcriptome is analysed to look at the effect of dual gene loss.

  Study EGAS00001004836

• CancerLocator: Non-Invasive Cancer Diagnosis and Tissue-of-Origin Prediction Using Methylation Profiles of Cell-Free DNA

  Background: The detection and characterization of cell-free DNA in plasma is one of the most promising new areas in cancer diagnosis. Liquid biopsy, unlike traditional tissue biopsy, has the potential to diagnose a variety of different malignancies. Results: Here we propose a probabilistic method, CancerLocator, which exploits the diagnostic potential of cell-free DNA by determining not only the presence but also the location of tumors. CancerLocator simultaneously infers the proportions and the tissue-of-origin of tumor-derived cell-free DNA in a blood sample using genome-wide DNA methylation data. We comprehensively evaluate CancerLocator with simulations and real data, and compare its performance with that of two established multi-class classification methods. We show that the predicted tumor burdens are highly consistent with the true values. In addition, when the proportion of tumor-derived DNAs in the cell-free DNAs is low, the two popular machine learning methods completely fail for cancer diagnosis, while CancerLocator successfully overcomes the challenge. CancerLocator also achieves promising results on patient plasma samples, despite the fact that the DNA methylation data from these samples has very low sequencing coverage. Conclusions: CfDNA methylation may be developed as an important approach for non-invasive early cancer diagnosis.

  Study EGAS00001002211

• Sequencing of liver cancer cell lines

  Liver cancer is a dreadful disease with limited therapeutic options. How to translate genomic findings into biomarkers and precision treatment is challenging. A large cell line-based platform was established, termed as Liver Cancer Model Repository. Heterogeneities in patients were faithfully recapitulated by 81 liver cancer cell lines at genomic and transcriptomic levels. This platform provides a rich resource and models to study cancer drivers and drug responses in liver cancer.

  Study EGAS00001002237

• Circulating Tumor DNA in Checkpoint Inhibitor treated Lung Cancer

  BackgroundImmune checkpoint inhibitors (ICI) are increasingly used to treat non-small cell lung cancer (NSCLC) and there is a critical need for novel predictive/prognostic biomarkers. We evaluated whether dynamics of circulating tumor (ctDNA) can identify NSCLC patients who benefit from single agent ICI.MethodsCirculating cell-free DNA (cfDNA) from 177 patients was analyzed prior ICI and at first treatment evaluation using a Commercial 77-gene panel. We performed de novo mutation calling and corrected for variants related clonal hematopoiesis. ctDNA changes were evaluated as predictors of tumor response estimated with PET/diagnostic CT and survival.ResultsMutations were detected in 159 of 174 (91.4%) analyzable patients. Most responders presented decreasing ctDNA levels, while non-responders had mixed molecular responses. Patients with a >50% molecular ctDNA decrease or no detectable ctDNA demonstrated improved PFS (5.9 versus 1.8 months; HR 0.49, 95%CI, 0.35-0.67, P < .001), and OS (18.9 versus 9.9 months; HR 0.41, 95%CI, 0.30-0.57, P < .001) compared to patients not achieving these endpoints. Clearance of mutations (and no detected ctDNA) had a similar effect (PFS HR 0.45, 95%CI, 0.32-0.61, P < .001; OS 0.37,95%CI, 0.27-0.52, P < .001) compared to patients having stable or additional mutations. After adjusting for clinical factors both >50% ctDNA decrease (HR 0.4, 95% CI, 0.3-0.6, P < .001) and clearance of mutations (HR 0.42, 95% CI, 0.3-0.6, P < .001) remained independent predictors for PFS and OS.ConclusionsctDNA testing reveals significant prognostic albeit not predictive information in ICI treated advanced NSCLC. This relevant information can be obtained with a commercial easy-to-implement ctDNA test that will facilitate extensive testing.

  Study EGAS00001004847

• EAC Genomic data

  Study EGAS00001004887

• Tumor HTG EdgeSeq from metastatic castrate resistant prostate cancer

  This dataset contains tumor bulk HTG EdgeSeq from patients with metastatic castrate resistant prostate cancer. RNA libraries were prepared following manufacturer's user guide (HTG Molecular Diagnostics). Each library was sequenced in HTG EdgeSeq system.

  Study EGAS00001004852

• Efficacy of nivolumab in pediatric cancers with high mutation burden and mismatch-repair deficiency

  Purpose: Checkpoint-inhibitors have limited efficacy for children with unselected solid and brain tumors. We report the first prospective pediatric trial (NCT02992964) using nivolumab exclusively for refractory non-hematological cancers harboring tumor mutation burden (TMB) ≥5 mutations/megabase (mut/Mb) and/or mismatch-repair deficiency (MMRD). Patients and methods: Twenty patients were screened, and ten ultimately included in the response cohort of whom nine had TMB >10mut/Mb (three initially eligible based on MMRD) and one patient had TMB between 5-10 mut/Mb. Results: Delayed immune responses contributed to best overall response of 50%, improving on initial objective responses (20%) and leading to 2-year overall survival (OS) of 50% (95% CI; 27, 93). Four children, including three with refractory malignant gliomas are in complete remission at a median follow-up of 37-months (range: 32.4-60), culminating in 2-year OS of 43% (95% CI; 18.2, 100). Biomarker analyses confirmed benefit in children with germline MMRD, microsatellite instability, higher activated and lower regulatory circulating T-cells. Stochastic mutation accumulation driven by underlying germline MMRD impacted the tumor microenvironment, contributing to delayed responses. No benefit was observed in the single patient with a MMR-proficient tumour and TMB 7.4 mut/Mb. Conclusions: Nivolumab resulted in durable responses and prolonged survival for the first time in a pediatric trial of refractory hypermutated cancers including malignant gliomas. Novel biomarkers identified here need to be translated rapidly to clinical care to identify children who can benefit from checkpoint-inhibitors, including for upfront management of their cancers.

  Study EGAS00001007393

• Copy numbers in resectable gastric cancer treated with surgery alone

  Purpose: Gastric cancer is a heterogeneous disease requiring stratification to recognize its underlying biology and variable clinical outcome. The Cancer Genome Atlas (TCGA) recognizes four molecular subgroups: Epstein-Barr virus positive (EBV+) and microsatellite instable (MSI) with superior survival compared to genomically stable (GS) and chromosomal instable (CIN). Since classification criteria to distinguish GS from CIN were not defined, alternative markers such as Lauren’s histopathology or CDH1/p53 immunohistochemistry are commonly applied. Here we compared survival of gastric cancer subgroups determined by four methods. Experimental Design: EBV- and microsatellite stable tumors from a total of 309 patients treated with surgery alone were assigned to subgroups by: (i) specific copy number aberrations as determined by TCGA (GS or CIN), (ii) genome instability index (GII; high or low), (iii) Lauren’s classification (diffuse or intestinal), and (iv) CDH1/p53 immunohistochemistry (IHC; aberrant CDH1, normal-, or aberrant p53 expression). Subgroups were associated with cancer-related survival (CRS). Results: Five-year CRS was 42.0% for diffuse and 49.5% for intestinal type tumors, and 57.8% for GS and 41.6% for CIN tumors. Classification by GII or CDH1/p53 IHC did not correlate with CRS. The combination of TCGA and Lauren classifications resulted in four distinct subgroups. Five-year CRS for GS-intestinal (n=24), GS-diffuse (n=57), CIN-intestinal (n=142) and CIN-diffuse (n=86) was 61.4%, 56.5%, 47.6%, and 31.5%, respectively. Conclusions: TCGA’s GS and CIN have additional prognostic value to Lauren’s diffuse and intestinal subgroups in gastric cancer. GS-intestinal, GS-diffuse, CIN-intestinal and CIN-diffuse constitute biologically distinct entities and are suggested as stratification variables for future studies.

  Study EGAS00001007394

• Mechanism of action and resistance to Trastuzumab Deruxtecan in patients with metastatic breast cancer: the DAISY trial

  Study EGAS00001006905

• Patterns of transcription factor programs and immune pathway activation define four major subtypes of SCLC with distinct therapeutic vulnerabilities

  Despite molecular and clinical heterogeneity, small cell lung cancer (SCLC) is treated as a single entity with predictably poor results. Using tumor expression data, we applied non-negative matrix factorization, which identified four SCLC subtypes defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures accompanied by an Inflamed signature (SCLC-A, N, P and I, respectively). The presence of these subtypes were then validated in tumor gene expression data from the IMpower133 clinical trial.

  Study EGAS00001004888

• Genomic Rearrangements in Pediatric Cancer

  This study contains a collection of samples used to determine the role and impact of genomic rearrangements in a pan cancer pediatric cohort.

  Study EGAS00001005312

• Accurate detection of tumor-specific fusion genes reveals strongly immunogenic personal neo-antigens

  Fusion genes arising from cancer-associated somatic mutations are a potential rich source for highly immunogenic neo-antigens. However, their exploitation as targets for personalized cancer immunotherapy is currently limited by the lack of computational tools allowing transcriptome-wide identification of unique fusion genes in an accurate and sensitive manner. Here, we present EasyFuse, a computational pipeline, to detect individual and cancer-specific fusion genes in next-generation-sequencing transcriptome data obtained from human cancer samples. Using machine learning, EasyFuse predicts personal fusion genes with high precision and sensitivity and outperforms previously described approaches as qualified by an unprecedented ground-truth dataset of >1500 verification experiments in relevant patient samples. By testing immunogenicity with autologous blood lymphocytes from cancer patients we detected pre-established CD4+ and CD8+ T cell responses for 10 of 21 (48%), and for 1 of 30 (3%) of identified fusion genes, respectively. In conclusion, we demonstrate accurate detection of cancer-specific fusion genes. The high frequency of T cell responses detected in cancer patients support the relevance of private fusion genes as neo-antigens for personalized immunotherapies, especially for tumors with low point mutation burdens.

  Study EGAS00001004877

• Targeted next-generation sequencing of 13 chordoid gliomas of the third ventricle

  Study EGAS00001002733

• Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths

  Study EGAS00001007402

• Circulating Tumor DNA Analysis Detects Minimal Residual Disease and Predicts Recurrence in Patients with Stage II Colon Cancer

  Detection of circulating tumor DNA (ctDNA) after resection of stage II colon cancer may identify those at the highest risk of recurrence and help inform adjuvant treatment decisions. We used massively-parallel-sequencing–based assays to evaluate the ability of ctDNA to detect minimal residual disease in 1046 plasma samples from a prospective cohort of 230 patients with resected stage II colon cancer. In patients not treated with adjuvant chemotherapy, ctDNA was detected post-operatively in 14 of 178 (7.9%) patients, 11 (79%) of whom had recurred at a median follow-up of 27 months; recurrence occurred in only 16 (9.8 %) of the 164 patients with negative ctDNA (HR, 18; 95% confidence interval [CI], 7.9 to 36; P<0.001). In patients treated with chemotherapy, the presence of ctDNA following completion of chemotherapy was also associated with an inferior recurrence-free survival (HR, 11; 95% CI, 1.8 to 68; P=0.001). ctDNA detection after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence.

  Study EGAS00001001839

• checup

  Sequencing data from a phase II study of nivolumab and ipilimumab in recurrent or refractory cancer of unknown primary (CheCUP trial)

  Study EGAS00001007403

• Therapeutic Targeting of Ependymoma as Informed by Oncogenic Enhancer Profiling

  Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Here, we mapped active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts. Enhancer regions revealed novel oncogenes, molecular targets, and pathways, which when subjected to small molecule inhibitor or shRNA treatment, increased survival and slowed proliferation in mouse and neurosphere patient-derived models of ependymomas. This study represents one of the largest enhancer mapping study of any cancer type to date, and provides a framework for target and drug discovery for other cancers recalcitrant to therapeutic development because of their lack of known genetic drivers.

  Study EGAS00001002696

• Genomic evolution and transcriptional changes in the evolution of prostate cancer into neuroendocrine and ductal carcinoma types

  Prostate cancer is typically of acinar adenocarcinoma type but can occasionally present as neuro-endocrine and/or ductal type carcinoma. These are associated with clinically aggressive disease and the former often arises on a background of androgen deprivation therapy, although it can al-so arise de novo. Two prostate cancer cases were sequenced by exome capture from archival tis-sue. Case 1 was de novo small cell neuroendocrine carcinoma and ductal adenocarcinoma with 3 longitudinal samples over 5 years. Case 2 was a single time point after development of treatment related neuroendocrine prostate carcinoma. Case 1 showed whole genome doubling in all sam-ples and focal amplification of AR in all samples except the first time point. Phylogenetic analysis revealed a common ancestry for the ductal and small cell carcinoma. Case 2 showed 13q loss (involving RB1) in both adenocarcinoma and small cell carcinoma regions, and 3p gain, 4p loss, 17p loss (involving TP53) in the latter. By using highly curated samples, we demonstrate for the first time that small cell neuroendocrine and ductal prostatic carcinoma can have a common an-cestry and the process of evolution over time. We highlight whole genome doubling in a patient with prostate cancer relapse, reinforcing its poor prognostic nature.

  Study EGAS00001007412

• Nanopore sequencing from liquid biopsy: analysis of copy number variations from cell-free DNA of lung cancer patients

  Study EGAS00001004975

• Clinical outcomes in ctDNA-positive urothelial carcinoma patients treated with adjuvant immunotherapy

  Minimally invasive approaches to detect residual disease after surgery are urgently needed to select patients at highest risk for metastatic relapse for additional therapies. Circulating tumour DNA (ctDNA) holds promise as a biomarker for molecular residual disease (MRD) and relapse,1-3 but its clinical value has yet to be demonstrated in a randomised clinical trial. We evaluated outcomes in post-surgical ctDNA-positive (+) patients in a randomised phase III trial of adjuvant atezolizumab versus observation. IMvigor010 enrolled 809 patients with muscle-invasive urothelial carcinoma and did not meet its primary endpoint of disease-free survival (DFS) in the intent-to-treat population. Within the study, an exploratory planned analysis of prospectively collected plasma was performed, which tested the utility of ctDNA to identify patients who may benefit from adjuvant atezolizumab treatment. ctDNA was measured at the start of therapy (cycle 1 day 1; C1D1) and at week 6 (cycle 3 day 1; C3D1), and 581 patients were evaluable for ctDNA. The prevalence of ctDNA positivity at C1D1 was 37% (n=214), and ctDNA positivity identified patients with poor prognosis (observation arm DFS HR= 6.19 (4.29, 8.91), p<0.0001). Here we show that ctDNA(+) patients had improved DFS and overall survival (OS) with atezolizumab versus observation (DFS HR= 0.56 (0.41-0.77); p=0.0003 and OS HR= 0.58 (0.4-0.86); p=0.0063). No difference in DFS or OS between arms was noted for ctDNA-negative patients. The rate of ctDNA clearance was higher with atezolizumab (18%) versus observation (4%) (p=0.0041). Transcriptomic analysis revealed that tumours from ctDNA(+) patients had higher expression of cell cycle and keratin genes. Within the ctDNA(+) patient population in the atezolizumab arm, non-relapsing patients were further enriched in prominent immune response signatures including PD-L1, IFNG, CXCL9, and high tumour mutational burden, whereas relapse was associated with angiogenesis and fibroblast-transforming growth factor-beta signatures (F-TBRS). TCGA molecular subset analysis revealed increased efficacy of atezolizumab in patients with basal-squamous tumours, consistent with underlying tumour-immune contexture. Together these findings suggest that adjuvant atezolizumab may be associated with improved outcomes compared with observation in this high-risk ctDNA(+) population. These findings, if validated in other settings, would shift approaches to post-operative cancer care.

  Study EGAS00001004997

• Genome-wide cell-free DNA biological patterns in patients with cancer

  The structure of cell-free DNA (cfDNA) is altered in the blood of cancer patients. From whole genome sequencing we retrieved the cfDNA fragment-end composition using a new software (FrEIA), as well as the cfDNA size and tumor fraction in three independent cohorts (n=925 cancer from >10 types and 321 control samples). At 95% specificity, we detect 72% of cancer samples using at least one cfDNA measure, including 64% of early-stage cancer (n=220). cfDNA detection correlates with a shorter overall (p=0.018) and recurrence-free (p=0.006) survival in patients with resectable esophageal adenocarcinoma. Integrating cfDNA measures with machine learning in an independent test set (n=396 cancer, 90 controls) achieve a detection accuracy of 82% and AUROC of 96%. In conclusion, harnessing the biological features of cfDNA can improve at no-extra cost the diagnostic performance of liquid biopsies.

  Study EGAS00001007400

• Longitudinal single-cell RNA-seq data of metastatic ovarian cancer

  Study EGAS00001005006

• Longitudinal single-cell RNA-seq data of metastatic ovarian cancer

  Study EGAS00001005010

• CancerDetector: Ultrasensitive and Non-Invasive Cancer Detection at the Resolution of Individual Reads using Cell-free DNA Methylation Sequencing Data

  The detection of tumor-derived cell-free DNA in plasma is one of the most promising directions in cancer diagnosis. The major challenge in such approach is how to identify the tiny amount of tumor DNAs out of total cell-free DNAs in blood. Here we propose an ultrasensitive cancer detection method, termed “CancerDetector”, using the DNA methylation profiles of cell-free DNAs. The key of our method is to probabilistically model the joint methylation patterns of multiple adjacent CpG sites on an individual sequencing read, in order to exploit the pervasive nature of DNA methylation for signal amplification. Therefore, CancerDetector can sensitively identify a trace amount of tumor cfDNAs in plasma, at the level of individual reads. We evaluated CancerDetector on the simulated data, and showed a high concordance of the predicted and true tumor burden. Testing CancerDetector on real plasma data demonstrated its high sensitivity and specificity in detecting tumor DNAs. In addition, the predicted tumor burden showed great consistency with tumor size and survival outcome. Note that all of those testing were performed on sequencing data at low to medium coverage (1X to 10X). Therefore, CancerDetector holds the great potential to detect cancer early and cost-effectively.

  Study EGAS00001002728

• Single-cell roadmap of immune cell responses in chronic myeloid leukemia

  To study the innate and adaptive immune responses in CML, we profiled 25 samples from CML patients including samples at diagnosis (n = 7, N = 9, where n is the number of subjects and N the number of samples if different from n) and before and after TKI-cessation with imatinib (n = 6, N = 15) with paired single-cell RNA and T cell receptor (TCR) αβ chain sequencing (scRNA+TCRαβ-seq) and compared our data to different cancers (n = 29) and healthy donors (n = 7). This dataset contains the samples from before and after TKI-cessation, please see the manuscript for info related to the other datasets.

  Study EGAS00001005044

• Stromal cell diversity associated with immune evasion in human triple‐negative breast cancer

  Study EGAS00001005061

• Subtype specific progression from DCIS to invasive breast cancer

  Breast cancer consists of at least five main molecular “intrinsic” subtypes, which are reflected in both pre-invasive and invasive disease. Although previous studies have suggested that many of the molecular features of invasive breast cancer are established early, it is unclear what mechanisms drive progression, and whether the mechanisms of progression are dependent or independent of subtype. We have generated mRNA, miRNA and DNA copy number profiles from a total of 59 in situ lesions and 85 invasive tumors, in order to comprehensively identify those genes, signaling pathways, processes, and cell types that are involved in breast cancer progression. Our work provides evidence that there are molecular features associated with disease progression that are unique to the intrinsic subtypes. We additionally establish subtype-specific signatures that are able to identify a small proportion of pre-invasive tumors with expression profiles that resemble invasive carcinoma, indicating a higher likelihood of future disease progression.

  Study EGAS00001001866

• Finding structural variation and functional consequences from primary acute myeloid leukemia cells with complex karyotype (CK-AML) at the single-cell level

  Study EGAS00001007436

• CRISPR transduction of iPS cells

  Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three non-essential proof-of-concept genes - HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.

  Study EGAS00001005102

• CUPiD, a cfDNA methylation-based tissue-of-origin classifier for Cancers of Unknown Primary

  Study EGAS00001007445

• Chemotherapy accelerates genomic aging of normal blood in children treated for cancer

  Whole genome sequencing (WGS) data obtained from clonally expanded hematopoietic stem and progenitor cells (HSPCs) from pediatric cancer patients before and after therapy exposure. In addition, WGS data of cord-blood derived HSPCs clones exposed to various chemotherapeutics and radiation was obtained.

  Study EGAS00001005141

• Multi-omics analysis of primary glioblastoma cell-lines shows recapitulation of pivotal molecular features of parental tumors

  Glioblastoma (GBM) is the deadliest primary brain cancer in adults. Emerging innovative therapies hold promise for personalized cancer treatment. Improving therapeutic options depends on research relying on relevant preclinical models. In this line we have established in the setting of the GlioTeX project (GBM and experimental therapeutics), a GBM-patient derived cell line library (GBM-PDCL). Multi-OMIC approach was used to determine the molecular landscape of PDCL and the extent to which they represent GBM tumors.

  Study EGAS00001001871

• The Genetic Landscape of Ocular Adnexa MALT Lymphoma Reveals Frequent Aberrations in NFAT and MEF2B Signaling Pathways

  A comprehensive constellation of somatic non-silent mutations and copy number (CN) variations in ocular adnexa marginal zone lymphoma (OAMZL) is unknown. By utilizing whole-exome sequencing in 69 tumors we define the genetic landscape of OAMZL. Mutations and CN changes in CABIN1 (30%), RHOA (26%), TBL1XR1 (22%), and CREBBP (17%) and inactivation of TNFAIP3 (26%) were among the most common aberrations. Candidate cancer driver genes cluster in the B-cell receptor (BCR), NFkB, NOTCH and NFAT signaling pathways. One of the most commonly altered genes is CABIN1, a calcineurin inhibitor acting as a negative regulator of the NFAT and MEF2B transcriptional activity. CABIN1 deletions enhance BCR-stimulated NFAT and MEF2B transcriptional activity, while CABIN1 mutations enhance only MEF2B transcriptional activity by impairing binding of mSin3a to CABIN1. Our data provide an unbiased identification of genetically altered genes that may play a role in the molecular pathogenesis of OAMZL and serve as therapeutic targets.

  Study EGAS00001006631

• Pediatric Papillary Thyroid Carcinoma RNA-Seq

  Papillary thyroid carcinoma (PTC) is the most common malignancy amongst adolescent and young adult women. Clinical and genomic features of PTC in children differ from those in adults. Historically, a substantial proportion (~50%) of pediatric PTC (PPTC) lack any of the common driver mutations typical of adult PTC. Thus, molecular diagnostics developed for adults may not be valid in children. We applied novel bioinformatic pipelines to RNASeq analysis of pediatric thyroid tumors to identify known and novel fusion oncogenes as drivers of tumorigenesis and to define gene expression patterns among tumors. Analysis of 38 samples resulted in identification of 16 previously reported oncogenic fusions and three novel candidate fusions. In conducting these analyses, we reduced the proportion of cancers with unknown drivers from approximately 50% to 6%. Gene expression analysis identified three distinct categories of tumors. These studies represent the first unbiased genomic approach to childhood PTC. Novel bioinformatic pipeline. GEC Identified 3 distinct classes. DICER1, PTEN segregated with benign tumors. >90% of PPTC carry identifiable ongognic driver variants, however, the landscape differs from that in adults and existing tools for tumor genotyping in adults may not be applicable to pediatric tumors. BACKGROUND: Papillary thyroid carcinoma demonstrates clinically distinct behaviour in pediatric patients when compared to adults, with increased regional and distal metastases, more frequent recurrence, yet overall highly favourable prognosis. Recent tumor genotyping has demonstrated that in pediatric tumors, ***-*** do not carry the most common genomic variants found in adult PTC, leaving a substantial proportion of driver-unknown or “dark matter” tumors. METHODS: We established a cohort of 52 pediatric PTC and performed quantitative-PCR genotyping followed in a subset by whole exome and whole transcriptome (RNASeq) sequencing using a novel, robust bioinformatic pipeline to identify oncogenic fusion transcripts. Gene expression analysis was used to derive distinct clusters. RESULTS: In 52 pediatric PTC, genotyping identified BRAFV600E(21%), NRASQ61R(2%), RET/PTC1 (21%), RET/PTC3 (17%) or PAX8-PPAR in 33 (63%) of tumors. Among the remaining 19 tumors, oncogenic variants were identified in /19 (%). The variants included SNVs in DICER1, ETV6-NTRK3, PTEN and 9 additional oncogenic fusions including two previously-undescribed fusion transcripts, *** and ***. CONCLUSIONS: The genomic landscape of PTC differs between children and adults. In children, there is a substantial fraction of tumors that are unexplained by variants typical of adult PTC. This discrepancy is explained largely by uncommon oncogenic fusions which can be identified by whole-transcriptome analysis.

  Study EGAS00001005182

• WES of Colorectal cancer organoid-stroma biobank cohort

  In colorectal cancers (CRC) the tumor microenvironment plays a key role for prognosis and therapy efficacy. Patient-derived tumor organoids (PDTOs) show enormous potential for preclinical testing, however, in purely epithelial cultures features including the ‘consensus molecular subtypes’ (CMS) are largely eradicated. To better reflect cell heterogeneity, we established the CRC organoid-stroma biobank of matched PDTOs and cancer-associated fibroblasts (CAFs) from 30 patients. Context-specific phenotyping showed that xenotransplantation or co-culture with CAFs restores the individual transcriptomic status and instructs subtype-specific stromal gene expression. Furthermore, co-culture exposed CMS4-specific resistance to Gefitinib and SN-38 and revealed prognostic gene expression signatures. Chemogenomic library screening identified patient- and therapy-dependent mechanisms of stromal resistance including MET as common target. Our results demonstrate that CRC phenotypes are encrypted in the cancer epithelium in a plastic fashion that strongly depends on the context. Consequently, CAFs are essential for faithful representation of molecular subtypes and therapy responses ex vivo.

  Study EGAS00001007301

• Towards standardized whole exome sequencing (WES) for cancer patients: lessons from a multicentric pilot study

  A growing number of druggable targets and national initiatives for precision oncology necessitate broader genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a sensitivity between 91-95% and a positive predictive value (PPV) of 82-95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Deviations were observed for cases with low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79-1, TMB: 0.97-0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.

  Study EGAS00001007363

• A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer

  Background Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. Methods We combined immunohistochemistry (IHC) with RNA-sequencing and whole genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Further, we explored transcriptional features of immune archetypes, T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions, and assessed common adult biomarkers such as neoantigen load and TMB. Results A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. Conclusions Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

  Study EGAS00001007029

• Single-cell RNA sequencing of 6 follicular lymphoma tumors

  Follicular lymphoma (FL) is an indolent cancer of mature B-cells but carries increased risk of transformation to a more aggressive histology over time. We present here comprehensive profiling both tumor and immune compartments in 6 diagnostic FL biopsies by single-cell RNA sequencing. This confirmed results from 155 FL tumors characterized by mass cytometry (CyTOF) which revealed two distinct evolutionary trajectories with disparate risk of transformation and alternate biologies.

  Study EGAS00001005257

• BCL3-rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases

  Study EGAS00001007465

• Genomic and Transcriptomic Profile of Paired Primary-Metastasis Colorectal Tumors

  The goal of this study is to identify mechanisms of metastization in a portuguese cohort of colorectal cancer. Namely, we want to identify patterns of genomic and transcriptomic diversification associated with metastasis.

  Study EGAS00001005276

• ChIP-seq in colorectal cancer and paired adjacent normal mucosa

  Chip-seq samples for 20 colorectal patients with paired adjacent normal mucosa to characterize H3K27ac and H3K4me1.

  Study EGAS00001005303

• mutational landscape of normal human breast

  The accumulation of somatic mutations in the healthy breast throughout life and pregnancy is poorly understood. Here we describe whole genome sequencing analysis of epithelial and stromal compartments from the normal breast. We aimed to study how pregnancy and age affect both the mutational burden and the clonality of the healthy breast, and the subsequent implications on breast cancer risk.

  Study EGAS00001004672

• Secondary Resistance to Anti-EGFR Therapy by Transcriptional Reprogramming in Patient-Derived Colorectal Cancer Models (hipo_B012)

  Study EGAS00001005320

• Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

  A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.

  Study EGAS00001007426

• Identification of early disease progression in ALK-rearranged lung cancer using circulating tumor DNA analysis (hipo_K34R)

  Study EGAS00001005327

• Evaluating CRISPR-based Prime Editing for cancer modeling and CFTR repair in organoids

  Study EGAS00001005358

• SmMIP-tools:a computational toolset for processing and analysis of single-molecule molecular inversion probes derived data

  Single-molecule molecular inversion probes (smMIPs) provides a modular and cost-effective platform for high-multiplex targeted next-generation sequencing (NGS). Nevertheless, translating the raw smMIP-derived sequencing data into accurate and meaningful information currently requires proficient computational skills and a large amount of computational work, prohibiting wide-scale adoption of smMIP-based technologies. To enable easy, efficient, and accurate interrogation of smMIP-derived data, we developed SmMIP-tools, a computational toolset that combines the critical analytic steps for smMIP data interpretation into a single computational pipeline. Here, we describe in detail two major components of the software. The first is a read processing tool that performs quality control steps, generates read-smMIP linkages and retrieves molecular tags. The second is an error-aware variant caller capable of detecting single nucleotide variants (SNVs) and short insertions and deletions (indels). Using a cell-line DNA dilution series and a cohort of blood cancer patients, we benchmarked SmMIP-tools and evaluated its performance against clinical sequencing reports. We anticipate that SmMIP-tools will increase accessibility to smMIP-technology, enabling cost-effective genetic research to push personalized medicine forward.

  Study EGAS00001005359

• Whole exome sequencing data of germline and two independent primary leukemias of five patients

  The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this auto-regulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

  Study EGAS00001001889

• Spatiotemporal Genomic Profiling of Intestinal Metaplasia Reveals Clonal Dynamics of Gastric Cancer Progression

  Intestinal metaplasia (IM) is a pre-malignant condition of the gastric mucosa associated with increased gastric cancer (GC) risk. We analyzed 1256 gastric samples (1152 IMs) from 692 subjects from a 10-year prospective study. We identified 26 IM driver genes in diverse pathways including chromatin regulation (ARID1A) and intestinal homeostasis (SOX9), largely occurring as subclonal events. Analysis of clonal dynamics between and within subjects, and also longitudinally across time, revealed that IM clones are likely transient but increase in size upon progression to dysplasia, with eventual transmission of genetic events to paired GCs. Single-cell and spatial profiling highlighted changes in tissue ecology and lineage heterogeneity in IM, including an intestinal stem-cell dominant cellular compartment linked to early malignancy. Expanded transcriptome profiling revealed expression-based molecular subtypes of IM, including a body-resident “pseudoantralized” subtype associated with incomplete histology, antral/intestinal cell types, ARID1A mutations, inflammation, and microbial communities normally associated with the healthy oral tract. We demonstrate that combined clinical-genomic models outperform clinical-only models in predicting IMs likely to progress. Our results raise opportunities for GC precision prevention and interception by highlighting strategies for accurately identifying IM patients at high GC risk and a role for microbial dysbiosis in IM progression.

  Study EGAS00001007067

• Identifying transcriptional programs underlying anti-EGFR small molecule response and resistance with TraCe-seq

  Genetic and non-genetic heterogeneity within cancer cell populations represents a major challenge to anti-cancer therapies. We currently lack robust methods to determine how pre-existing and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA-sequencing, we have developed TraCe-seq, a method that captures at clonal resolution the origin, fate, and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual EGFR inhibitors-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. To QC the TraCe-seq strategy, single-cell RNA-seq libraries were generated from a variety of human cancer cell lines transduced with the TraCe-seq library to validate the TraCe-seq strategy. Specifically, 5 different cell lines (PC9, MCF-10A, MDA-MB-231, NCI-H358, and NCI-H1373) were each transduced with a unique TraCe-seq barcode. The transduced cells were selected with puromycin only, dissociated to single cell suspensions, and then mixed together. The complex mixture of the 5 cell lines was profiled by 10X scRNA-seq. Furthermore, transduced NCI-H1373 cells were sorted by FACS to enrich for the top 50% of eGFP positive cells, and sorted cells were cultured briefly and used to construct scRNA-seq libraries and profiled by 10x scRNA-seq. To carry out the full TraCe-seq experiment, ~600 PC9 cells carrying unique TraCe-seq barcodes were expanded over 12 doublings to establish the barcoded population. A subset of the barcoded PC9 population was used to generate scRNA-seq libraries and profiled by 10x scRNA-seq prior to treatment to establish a baseline transcription profile for each barcoded clone. The rest of the cells were then treated for four days with 1 µM erlotinib, 1 µM GNE-069, or 1 µM GNE-104 respectively. scRNA-seq libraries were then generated form the treated cells and profiled by 10x scRNA-seq.

  Study EGAS00001005405

• Epithelioid haemangioendothelioma (EHE) case series from the Stafford Fox Rare Cancer Program

  Nine EHE cases with comprehensive histologic and molecular profiling from the Walter and Eliza Hall Institute of Medical Research Stafford Fox Rare Cancer Program were collated via nation-wide referral to the Australian Rare Cancer Portal. Molecular analysis of RNA, DNA or both was possible in seven of nine cases. Survival was highly variable and unrelated to molecular profile. DNA sequences for two cases are deposited here.

  Study EGAS00001007474

• Evolutionary analysis of primary tumors and metastatic lesions from 20 breast cancer patients (99 samples in total) using exome sequencing data.

  Metastatic breast cancers are still incurable. Characterizing their evolutionary landscape including the role of metastatic axillary lymph nodes to seed distant organ metastasis can provide rational basis for effective treatments. Here, we describe the genomic analyses of the primary tumors and metastatic lesions from 20 breast cancer patients (99 samples). Our evolutionary analyses revealed diverse spreading and seeding patterns governing tumor progression. Although linear evolution to successive metastatic sites was common, parallel evolution from the primary tumor to multiple distant sites was also evident. Metastatic spreading was frequently coupled with polyclonal seeding, where multiple metastatic subclones originated from primary tumor and/or other distant metastases. Synchronous axillary lymph node metastasis, a well-established prognosticator of breast cancer, was not involved in seeding distant metastasis, suggesting haematogenous route for cancer dissemination. Clonal evolution coincided frequently with emerging driver alterations and evolving mutational processes, notably a significant increase in apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) associated mutagenesis. Our data provide the first genomic evidence regarding the role of axillary lymph node metastasis in seeding distant organ metastasis and elucidates the evolving mutational landscape during cancer progression.

  Study EGAS00001002737

• Nucleosome footprinting in plasma cell-free DNA for diagnosis of ovarian cancer

  Fragmentation patterns of plasma cell-free DNA (cfDNA) are known to reflect nucleosome positions of cell types contributing to cfDNA. Based on cfDNA fragmentation patterns, the deviation in nucleosome footprints was quantified between diagnosed ovarian cancer patients and healthy individuals.

  Study EGAS00001005361

• Germline variants in patients with rare cancers and their implications for precision cancer medicine: experiences from the Multicenter MASTER Trial by the German Cancer Consortium (HIPO_021)

  The era of precision oncology entails an increasing amount of molecular data from tumor sequencing in research and diagnostics. In this setting, germline variant analysis may add clinical benefits for patients and family members. In this study, we evaluated germline variants from genome/exome matched tumor-blood sequencing in 1.485 advanced cancer patients within a prospective observational study carried out by the German Cancer Consortium. Included patients had cancers of rare entities (79.2%) or were under the age of 51 at first diagnosis (76.6%). Ten percent of the patients harbored pathogenic germline variants in 35 tumor predisposition genes with autosomal-dominant inheritance such as BRCA1, BRCA2, TP53, RB1, CHEK2, ATM, PALB2, SDHB etc., while 4.6% of the patients were carriers of pathogenic germline variants in 18 genes with autosomal-recessive inheritance of tumor predisposition. Based on pathogenic germline variants, 14% of the patients received genetic counselling recommendations in the molecular tumor board report. Forty five percent of pathogenic germline variants supported a molecular treatment recommendation, which was implemented for 28 patients. Furthermore, we compared our approach and results to previous studies, broadening the insight into pan cancer germline findings but also revealing differences between studies on various levels, such as cohort demographics, tumor entities, gene nominations, variant detection and variant interpretation. Our data support the importance of germline variant interpretation in precision oncology studies for patients and their families, if applicable. However, guidelines for evaluation and reporting of germline variants in the context of genetic tumor profiling need to be established and further research on the relevance of germline mutations as biomarkers for treatment recommendations is needed.

  Study EGAS00001005537

• INSPIRE - Investigator-initiated Phase 2 Study of Pembrolizumab Immunological Response in Metastatic Solid Tumors

  Study EGAS00001003280

• WGS_of_healhy_mesothelial_cells_and_primary_mesothelima_cell_lines

  Mesothelioma is a rare and aggressive cancer associated with previous exposure to asbestos. Currently there are no effective treatments for mesothelioma and majority of patients will die within a year after diagnosis. Although a large number of preclinical and clinical trials assessed the efficacy of various therapuetic modes, none of the tested molecules entered the clinic. Thus, to better understand what drives mesothelioma carcinogenesis and to identify novel targets for therapy, here we aim at performing WGS of heathy mesothelial cells and a panel of mesothelioma cells lines. Primary mesothelial cell lines were established in prof. Marciniak lab from human pleural specimens, while primary mesothelioma cell lines were obtained from MesobanK. Both models constitute low passage cell lines and therefore should closely represent genetics of the original tissue.

  Study EGAS00001005559

• ExomeSeq Neoantigen Immunogenicity Landscapes

  Immune checkpoint blockade (ICB) therapy is a cornerstone of oncologic treatment for patients with advanced stage non-small cell lung cancer (NSCLC) and other malignancies. Neoantigen immunoediting drives ICB efficacy, yet the fundamental physiochemical characteristics of neoantigens and how neoantigen immunogenicity shapes treatment response remains poorly understood. To help address these questions, a prospective clinical trial of NSCLC patients treated with nivolumab was conducted. We assessed genomic alterations in tumors from 58 patients and performed large-scale neoepitope immunogenicity analyses, before and during treatment (CheckMate153, CA209-153). Tumors were analyzed by whole-exome and transcriptome sequencing. In responding patients, loss of mutation and neoantigen burden early during therapy associated with clinical benefit. We evaluated the immunogenicity of 1,453 candidate neoantigens and identified 502 neopeptides that bound to MHC I and 196 neopeptides that were immunogenically recognized by T cells in the setting of nivolumab treatment. These T cell reactive neoantigens were differentially present in clonal populations that underwent distinctive evolutionary trajectories across responders and nonresponders. Mapping these neoantigens to tumor clonal dynamics and clinical response revealed strong selection against immunogenic neoantigen harboring clones compared to non-immunogenic clones. Using this large collection of neoantigens, we identified position specific amino acid features related to immunogenicity, which we used to develop and validate an immunogenicity score. Changes in the genomic and neoantigen immunogenicity landscapes were accompanied by temporal changes in the tumor microenvironment. Nivolumab-induced microenvironmental evolution in NSCLC shared some similarities with that in melanoma, yet critical differences in immunologic programs were apparent from comparative network analysis between tumor types. This study provides unprecedented molecular portraits of the neoantigen landscapes underlying nivolumab’s mechanism of action.

  Study EGAS00001007508

• RNASeq Neoantigen Immunogenicity Landscape

  Immune checkpoint blockade (ICB) therapy is a cornerstone of oncologic treatment for patients with advanced stage non-small cell lung cancer (NSCLC) and other malignancies. Neoantigen immunoediting drives ICB efficacy, yet the fundamental physiochemical characteristics of neoantigens and how neoantigen immunogenicity shapes treatment response remains poorly understood. To help address these questions, a prospective clinical trial of NSCLC patients treated with nivolumab was conducted. We assessed genomic alterations in tumors from 58 patients and performed large-scale neoepitope immunogenicity analyses, before and during treatment (CheckMate153, CA209-153). Tumors were analyzed by whole-exome and transcriptome sequencing. In responding patients, loss of mutation and neoantigen burden early during therapy associated with clinical benefit. We evaluated the immunogenicity of 1,453 candidate neoantigens and identified 502 neopeptides that bound to MHC I and 196 neopeptides that were immunogenically recognized by T cells in the setting of nivolumab treatment. These T cell reactive neoantigens were differentially present in clonal populations that underwent distinctive evolutionary trajectories across responders and nonresponders. Mapping these neoantigens to tumor clonal dynamics and clinical response revealed strong selection against immunogenic neoantigen harboring clones compared to non-immunogenic clones. Using this large collection of neoantigens, we identified position specific amino acid features related to immunogenicity, which we used to develop and validate an immunogenicity score. Changes in the genomic and neoantigen immunogenicity landscapes were accompanied by temporal changes in the tumor microenvironment. Nivolumab-induced microenvironmental evolution in NSCLC shared some similarities with that in melanoma, yet critical differences in immunologic programs were apparent from comparative network analysis between tumor types. This study provides unprecedented molecular portraits of the neoantigen landscapes underlying nivolumab’s mechanism of action.

  Study EGAS00001007509

• Circulating tumor cells Exome sequencing from breast cancer

  Single cell technologies allow interrogating the extent of tumor heterogeneity, providing novel insights into tumor evolution and treatment resistance mechanisms. Here, we investigated the level of genetic heterogeneity among circulating tumor cells (CTCs) at the single nucleotide variation and copy number aberration (CNA) levels and compared it to synchronous matched primary tumor/metastatic lesion from 3 metastatic breast cancer patients with triple-negative (TNBC), HER2-positive (HER2+) and estrogen-receptor-positive (ER+) tumors. Whole exome sequencing was performed on 3 synchronous bulk tumor tissue and 21 single/pooled CTC samples. When comparing bulk tissue with CTCs from the same patient, we found similar CNA profiles and the same patient-specific driver mutations for the HER2+ and TNBC tumors. Different CNA profiles and driver mutations were found for the ER+ tumor, with two distinct clones being identified in the CTCs, one harboring an activating ESR1 mutation, the other one bearing a TP53 mutation. These data suggest that tumor tissue and CTCs provide in a subset of patients complementary clinically relevant information to map tumor heterogeneity and monitor tumor evolution. Further validation is needed.

  Study EGAS00001005228

• Genomic evolution and transcriptional changes in the evolution of prostate cancer into neuroendocrine and ductal carcinoma types (RNAseq)

  Prostate cancer is typically of acinar adenocarcinoma type but can occasionally present as neuro-endocrine and/or ductal type carcinoma. These are associated with clinically aggressive disease and the former often arises on a background of androgen deprivation therapy, although it can al-so arise de novo. Two prostate cancer cases were sequenced by exome capture from archival tis-sue. Case 1 was de novo small cell neuroendocrine carcinoma and ductal adenocarcinoma with 3 longitudinal samples over 5 years. Case 2 was a single time point after development of treatment related neuroendocrine prostate carcinoma. Case 1 showed whole genome doubling in all sam-ples and focal amplification of AR in all samples except the first time point. Phylogenetic analysis revealed a common ancestry for the ductal and small cell carcinoma. Case 2 showed 13q loss (involving RB1) in both adenocarcinoma and small cell carcinoma regions, and 3p gain, 4p loss, 17p loss (involving TP53) in the latter. By using highly curated samples, we demonstrate for the first time that small cell neuroendocrine and ductal prostatic carcinoma can have a common an-cestry and the process of evolution over time. We highlight whole genome doubling in a patient with prostate cancer relapse, reinforcing its poor prognostic nature.

  Study EGAS00001007428

• Clinical and genetic factors associated with tumor response to neoadjuvant (chemo)radiotherapy, survival and recurrence risk in rectal cancer

  Rectal cancer poses challenges in preoperative treatment response, with up to 30% achieving a complete response (CR). Personalized treatment relies on accurate identification of responders at diagnosis. This study aimed to unravel CR determinants, overall survival (OS), and time to recurrence (TTR) using clinical and targeted sequencing data. Analyzing 402 patients undergoing preoperative treatment, tumor stage, size, and treatment emerged as robust response predictors. CR rates were higher in smaller, early-stage, and intensively treated tumors. Targeted sequencing analyzed 216 cases, while 120 patients provided hotspot mutation data. KRAS mutation dramatically reduced CR odds by over 50% (odds ratio [OR]=0.3 in the targeted sequencing and OR=0.4 hotspot cohorts, respectively). In contrast, SMAD4 and SYNE1 mutations were associated with higher CR rates (OR=6.0 and 6.8, respectively). Favorable OS was linked to younger age, CR, and low baseline carcinoembryonic antigen levels. Notably, CR and an APC mutation increased TTR, while a BRAF mutation negatively affected TTR. Beyond tumor burden, SMAD4 and SYNE1 mutations significantly influenced CR. KRAS mutations independently correlated with radiotherapy resistance, and BRAF mutations heightened recurrence risk. Intriguingly, nonresponding tumors with initially small sizes carried a higher risk of recurrence. These findings offer insights into rectal cancer treatment response, guiding personalized therapeutic strategies. By uncovering factors impacting CR, OS, and TTR, this study underscores the importance of tailored approaches for rectal cancer patients. These findings, based on extensive analysis and mutation data, pave the way for personalized interventions, optimizing outcomes in the challenges of rectal cancer preoperative treatment.

  Study EGAS00001007501

• ZNF703 is a common Luminal B breast cancer oncogene that differentially regulates luminal and basal progenitors in human mammary epithelium.

  The telomeric amplicon at 8p12 is common in oestrogen receptor-positive (ER+) breast cancers. Array-CGH and expression analyses of 1172 primary breast tumours revealed that ZNF703 was the single gene within the minimal amplicon and was amplified predominantly in the Luminal B subtype. Amplification was shown to correlate with increased gene and protein expression and was associated with a distinct expression signature and poor clinical outcome. ZNF703 transformed NIH 3T3 fibroblasts, behaving as a classical oncogene, and regulated proliferation in human luminal breast cancer cell lines and immortalized human mammary epithelial cells. Manipulation of ZNF703 expression in the luminal MCF7 cell line modified the effects of TGFβ on proliferation. Overexpression of ZNF703 in normal human breast epithelial cells enhanced the frequency of in vitro colony-forming cells from luminal progenitors. Taken together, these data strongly point to ZNF703 as a novel oncogene in Luminal B breast cancer.

  Study EGAS00000000082

• METABRIC

  Solid tumors are complex tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Normal cell contamination can dilute cancer cell information and tissue architecture is generally not reflected in molecular assays. To address these challenges, we developed a computational approach based on standard Haematoxylin and Eosin-stained sections and demonstrated its power in a discovery cohort of 323 breast tumors and an independent validation cohort of 241 tumors. First, to deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy-number profiles between samples. Second, we demonstrated that a predictor for survival integrating image-based and molecular features significantly outperforms classifiers based on single data types. Third, we described and validated a novel, independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative methods refine and complement molecular assays of tumor samples and could benefit all large-scale cancer studies.First, to deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy-number profiles between samples. Second, we demonstrated that a predictor for survival integrating image-based and molecular features significantly outperforms classifiers based on single data types. Third, we described and validated a novel, independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative methods refine and complement molecular assays of tumor samples and could benefit all large-scale cancer studies.

  Study EGAS00000000098

• METABRIC miRNA landscape

  MicroRNAs (miRNAs) are key players in cancer pathogenesis1. They display differential expression across breast cancer subtypes, and play oncogenic and tumor-suppressive roles2-6. A systematic study of the global miRNA landscape in breast cancer and the factors shaping it has not been undertaken. Here we report the results of profiling miRNA expression in 1,302 breast tumors with detailed clinical annotation and long-term follow-up, and for which matching genomic and mRNA expression data were available7. This provides a comprehensive view of the quantity, distribution and variation of the miRNA population and dissects how much genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture. The key clinical parameters and cellular pathways related to the miRNA landscape are identified, exemplified by context-dependent interaction with cell adhesion and Wnt signaling. Strikingly, in breast cancer miRNAs appear to act as modulators of mRNA-mRNA interactions rather than molecular switches. We demonstrate an important modulatory role for miRNAs in the biology of breast tumors devoid of somatic copy number aberrations, a common subtype where immune response is prominent. Remarkably, prognostic miRNA signatures derived in this subtype are consistently prognostic across several other subtypes and validate in external cohorts. These findings represent a new framework to study the biology of miRNA in human cancer.

  Study EGAS00000000122

• NOWAC blood-based breast cancer case-control study

  Tumor-host interactions extend beyond the local microenvironment and cancer development largely depends on the ability of malignant cells to hijack and exploit the normal physiological processes of the host. Although abnormalities in a host’s systemic immunity are associated with increased cancer susceptibility, the functional interplay between tumor cells and circulating immune cells in regulating tumorigenic responses is unclear. We employed the Norwegian Women and Cancer study, a large prospective population-based cohort study, to identify gene expression changes in blood cells that provide a robust and reproducible diagnostic signal specific to breast cancer patients. We further show that circulating blood cells in breast cancer patients are enriched in genes involved in systemic immunosuppression and the motility, metabolism, growth, and proliferation of immune cells. By mining of the cancer-associated blood transcriptome, we identified immune mediators or biomarkers that could permit early detection of breast cancer and open avenues to novel targeted immunotherapies.

  Study EGAS00000000134

• Identification_and_functional_validation_of_driver_mutations_in_colorectal_cancer

  Targeted exome sequencing of human colorectal cancers for the identification of novel cancer drivers.

  Study EGAS00001000044

• Analysis_of_genomic_integrity_of_disease_corrected_human_induced_pluripotent_stem_cells_by_exome_sequencing

  Induced pluripotent stem (iPS) cells hold great promise for autologous cell transplantation. In order to apply this therapy to monogenic disorder, disease-causing mutations must be corrected prior to transplantation. We generated iPS cells from patients with alpha-1 antitrypsin deficiency, which is caused by homozygous non-synonymous mutation in the SERPINA1 gene. In vitro differentiation of patient-derived iPS cells to hepatocytes showed recapitulation of the disease phenotype. We corrected the mutations by gene targeting using zinc finger nuclease (ZFN) and piggyBac transposon technologies. Our mutation correction is highly accurate. There is no exogenous sequence left behind at the targeted site. The corrected iPS cell-derived hepatocytes were indistinguishable from healthy hepatocyte, suggesting that the corrected gene was fully functional. The genomic integrity was also analyzed by CGH and SNP array, showing no genomic alterations. These are important points to ensure clinical safety as well as functional restoration of the corrected gene. One remaining question is if these corrected iPS cells are clinically safe at the single base-pair level. Recent publications suggested that cancer-related mutations are significantly accumulated during iPS cell propagation. We aim to assess the clinical safety of the corrected iPS cells by using exome sequencing. Outcome from this project would bring iPS cell technology one step closer to clinical applications.

  Study EGAS00001000055

• Whole Exome Sequencing of Localized Prostate Cancer Patients

  We performed whole-exome sequencing on localized prostate cancer patients to validate tumor content as part of a single-cell RNA sequencing study of localized prostate cancer patients.

  Study EGAS00001005685

• Screening_for_human_epigenetic_variation_at_CpG_islands

  Many human characteristics, including susceptibility to disease, are determined genetically. An unexplored alternative to such genetic determination concerns epigenetic mechanisms such as DNA methylation. CpG islands (CGIs) are generally constitutively hypomethylated, however there are circumstances in which they become heavily methylated and, when coincident with a gene promoter, this invariably causes transcriptional silencing. CGI methylation occurs in normal tissues during processes such as X-inactivation, but abnormal patterns of methylation have also been implicated in disease. The vast majority of evidence relates to cancer, where silencing of multiple genes in this way appears to be a causal contributor to the cancer state. To address the role and extent of CGI methylation in ‘normal’ and diseased cells we applied MBD-affinity purification in conjunction with next generation sequencing in a panel of human brain autopsy samples. These samples represent a panel of individuals as well as specific brain regions and neurological pathologies.

  Study EGAS00001000074

• Screening_for_abnormal_CGI_methylation_in_primary_colorectal_tumours

  A common hallmark of neoplastic cells is abnormal acquisition of CpG island (CGI) methylation and the silencing of associated gene promoters. To determine the extent and distribution of CGI methylation in colorectal cancer, we solexa sequenced MBD affinity purified DNA prepared from five colorectal tumours and matched colon mucosa from the same individuals.

  Study EGAS00001000076

• DNA methylation and Panel sequencing for pancreatic neuroendocrine carcinomas (PanNECs) and pancreatic neuroendocrine tumors (PanNETs)

  Study EGAS00001005731

• Mult-omics Palbociclib Resistance Study in HR+/HER2– Metastatic Breast Cancer

  Resistance to CDK4/6 inhibitors and endocrine therapy (ET) are common and poorly understood since they have been real game changer in patients with HR-positive and HER2-negative MBC. A comprehensive genomic and transcriptomic analysis of pre-treatment and post-treatment tumors from patients treated with palbociclib plus ET identified novel markers associated with poor prognosis such as genomic scar features caused by homologous repair deficiency (HRD), estrogen response signatures, and four unique prognostic clusters with distinct molecular features. Tumors with TP53 mutations co-occurring with a unique HRD-high cluster responded poorly. By comparing genomic and transciptomic profiles of paired samples, tumors were found to be further enriched in HRD genomic scars and many had switched to aggressive molecular subtypes. Furthermore, we identified high frequencies of acquired genomic alterations upon disease progression in RB1, ESR1, PTEN, and KMT2C. Our findings provide new insight into potential predictive biomarkers that could be targeted to overcome resistance. (NCT03401359)

  Study EGAS00001005736

• Breast Cancer PDTX Encyclopaedia

  Molecular data from the collection of PDTX breast cancer models described in Bruna et al, 2016. Cell. It includes whole exome sequencing, shallow whole genome sequencing, expression arrays and reduced bisulfite representation sequencing (RRBS).

  Study EGAS00001001913

• Soft tissue sarcoma sequencing data

  Study EGAS00001006356

• early-Duodenal Cancer sequencing study

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  Study EGAS00001006357

• Treated and control Patient Derived Xenografts of colorectal cancer (CRC) samples

  Study EGAS00001006973

• Genetic and immune landscape evolution defines subtypes of MMR deficient colorectal cancer

  Mismatch repair deficient colorectal cancers have high mutation loads and many respond to immune checkpoint-inhibitors. We investigated how genetic and immune landscapes co-evolve in these tumors. All cases had high truncal mutation loads. Driver aberrations showed a clear hierarchy despite pervasive intratumor heterogeneity: Those in WNT/βCatenin, mitogen-activated protein kinase and TGFβ receptor family genes were almost always truncal. Immune evasion drivers were predominantly subclonal and showed parallel evolution. Pan-tumor evolution, subclonal evolution, and evolutionary stasis of genetic immune evasion drivers defined three MMRd CRC subtypes with distinct T-cell infiltrates. These immune evasion drivers have been implicated in checkpoint-inhibitor resistance. Clonality and subtype assessments are hence critical for predictive immunotherapy biomarker development. Cancer cell PD-L1 expression was conditional on loss of the intestinal homeobox transcription factor CDX2. This explains infrequent PD-L1 expression by cancer cells and likely contributes to the high recurrence risk of MMRd CRCs with impaired CDX2 expression.

  Study EGAS00001005769

• 'Targeted High Throughput Sequencing in Clinical Cancer Settings: Formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity'

  "Background: Massively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings. Methods: Using identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV) detection in samples of formalin-fixed paraffin embedded (FFPE) tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity.Results: We show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance. Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations, but is of minor importance for the detection of somatic variations.Conclusions: The application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies taken, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - on a long-range - result in a more reliable prediction of individual cancer therapies."

  Study EGAS00001000136

• Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency

  Whole-exome sequencing (Exome-Seq) has been successfully applied in several recent studies. We here performed Exome-Seq of 15 pancreatic tumor-normal pairs. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56 folds. This study identified a total of 1,517 somatic mutations and validated 934 of them by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among tumors. The diversity of mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-Seq revealed intensive genomic instability in a tumor with MLH1 homozygous deletion, indicated by a dramatically elevated mutation rate of somatic substitutions, small insertions/deletions (indels), as well as indels presented at microsatellites. Notably, we detected MLH1 allelic loss in 4 tumors. MLH1 expression was decreased by nearly half in these cases. While they were negative in the conventional microsatellite instability assay, they showed a 10.5-fold increase in the mutation rate of somatic indels, e.g. truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas (RCCs) and confirmed MLH1 haploinsufficiency. We observed a significant increase in the mutation rate of somatic indels in affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1 and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the mutation rate of somatic indels, could drive the development and progression of sporadic cancers.

  Study EGAS00001000149

• Exome Sequencing of Gastric Cancer

  GASTRIC cancer (GC) is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account only for small subsets of the disease. We performed exome sequencing of 22 GCs and identified mutated genes and pathway alterations previously unreported: genes involved in chromatin modification were commonly mutated. A downstream validation study confirmed frequent inactivating mutations and/or protein deficiency of the SWI/SNF chromatin remodeling complex gene ARID1A in 83% of GCs with microsatellite instability, 73% of GCs with Epstein-Barr virus (EBV) infection, and 11% of non-EBV microsatellite stable GCs. The mutation spectrum of ARID1A differs between molecular subtypes and the mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results revealed the genomic landscape and highlighted the importance of chromatin remodeling in the molecular taxonomy of GCs.

  Study EGAS00001000153

• Complete Genomics paired end sequencing; Ovarian cancer

  We sequenced the genomes from a monozygotic twin discordant for schizophrenia and a tumor-normal pair of an ovarian cancer patient. Using whole-genome twin data to discriminate between correctly identified single nucleotide variants (SNVs) and errors a strategy for the accurate detection of SNVs was developed. By applying stringent sequencing quality measures, excluding error-prone regions and selecting SNVs identified by different mapping and variation calling algorithms, error rates were ~37-fold reduced. This enabled us to identify the first discordant SNVs in monozygotic twins using whole-genome sequencing. In addition, by showing that novel SNVs are highly enriched in errors, accurate estimates of the number of novel and rare SNVs occurring in unrelated Caucasian individuals were obtained. Finally, somatic mutations in coding and regulatory sequences were reliably identified in the highly rearranged ovarian tumor. Overall, our data demonstrate that strategies to reduce error rates in whole-genomes are required for disease gene discovery.

  Study EGAS00001000158

• Whole genome sequencing of matched primary and metastatic acral melanomas

  Study EGAS00001000169

• Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma

  Study EGAS00001000226

• Accurate mutation detection in leukemia by re-sequencing a cancer gene set

  Study EGAS00001000268

• The evolutionary dynamics of human colorectal cancer using single-gland spatial multi-omic profiling of DNA, RNA and chromatin.

  Study EGAS00001005827

• Integrative analysis of small cell lung cancer

  Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype. We conducted integrated analysis of genome sequencing, transcriptome, and copy number analysis and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million basepairs. Evidence for inactivation of TP53 and RB1 was found in all sequenced cases. Furthermore, we identified recurrent mutations in CREBBP, EP300, and MLL, observed mutations in PTEN, in SLIT2, and EPHA7, as well as focal amplifications of the FGFR1 locus. As a major feature of SCLC we found mutation in histone modifying genes, observed genome alterations that are therapeutically tractable, and provide a framework for identifying biologically relevant genes in the context of a high mutation rate. Peifer et al., 2012, Nature Genetics.

  Study EGAS00001000299