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• Genome, epigenome and RNA sequences of monozygotic twins discordant for multiple sclerosis

  Monozygotic (MZ) twins have been widely employed for dissection of the relative contributions of genetics and environment in disease. In multiple sclerosis (MS), an autoimmune demyelinating disease that commonly causes neurodegeneration and disability in young adults, disease discordance in MZ twins has been interpreted to indicate environmental importance in pathogenesis. However, genetic and epigenetic differences between MZ twins have been described, challenging the accepted experimental paradigm in disambiguating effects of nature and nurture. Here, we report the genome sequences of one MS-discordant MZ twin pair and messenger RNA (mRNA) transcriptome and epigenome sequences of CD4+ lymphocytes from three MS-discordant, MZ twin pairs. No reproducible differences were detected between co-twins among ~3.6 million single nucleotide polymorphisms (SNPs) or ~0.2 million insertion-deletion polymorphisms (indels). Nor were any reproducible differences observed between siblings of the three twin pairs in HLA haplotypes, confirmed MS-susceptibility SNPs, copy number variations, mRNA and genomic SNP and indel genotypes, or expression of ~19,000 genes in CD4+ T cells. Only two to 176 differences in methylation of ~2 million CpG sites were detected between siblings of the three twin pairs, in contrast to ~800 differences in methylation between T cells of unrelated individuals and several thousand differences between tissues or normal and cancerous tissues. In the first systematic effort to estimate sequence variation among MZ co-twins, we did not find evidence for genetic, epigenetic or transcriptome differences that explained disease discordance. These are the first female, twin and autoimmune disease genome sequences reported.

  Study phs000239

• Center for Craniofacial and Dental Genetics: Exome Sequencing of Orofacial Cleft Families

  Orofacial Clefts (OFCs) are genetically complex structural birth defects caused by genetic factors, environmental exposures, and their interactions. OFCs are the most common craniofacial anomalies in humans. Affect about 1 in 800 newborns, OFCs are also one of the most common structural birth defects worldwide. OFCs can impose a substantial personal cost resulting from the associated emotional and financial burden. On average a child undergoes 6 surgeries, spends 30 days in hospital, receives 5 years of orthodontic treatment, and participates in ongoing speech therapy, leading to an estimated total lifetime treatment cost of about $200,000. Individuals born with an OFC have higher infant mortality (especially in developing countries with limited access to care), higher mortality rates at all other stages of life, increased incidence of mental health problems, and higher risk for other disorders (notably including breast, brain, and colon cancers). Prior genetic studies have focused on the contribution of common genetic variants through genome-wide linkage and association studies. These studies have revealed a significant etiologic heterogeneity. One promising approach to dissect the etiology of OFCs into focus on subclinical phenotypic features present in the non-cleft family members of affected individuals. These subclinical features are subtle phenotypic features that may represent mild manifestations of the same risk genes; thus, their inclusion in family-based studies may clarify the inheritance patterns in OFC families. In this project, we conducted whole exome sequencing in multiplex families with both overt OFCs and subclinical phenotypes to identify high-impact genes causing OFCs.

  Study phs001675

• Rare Diseases Clinical Research Network (RDCRN) Vasculitis Clinical Research Consortium (VCRC) Longitudinal Protocol for Polyarteritis Nodosa

  The development and validation of accurate biological markers and predictors of disease activity and outcome in Polyarteritis Nodosa (PAN), a form a idiopathic vasculitis, would have a major positive impact on the clinical care of patients with this rare disease, be an important advance in the design of clinical trials and feasibility of new drug development, and provide important insight into the pathogenesis of this condition. Current measures of disease activity, including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), while helpful in selected settings, are mostly considered highly flawed and markers whose poor correlation with disease flares makes them inadequate to help guide therapy. Similarly, ESR and CRP are clearly unhelpful as diagnostic tests for vasculitis. The current state of clinical investigation for vasculitis relies heavily upon determination of disease status solely by clinical presentation and investigator opinion. Thus, discovery and validation of more precise markers that quantifiably measure activity, predict disease course, and correlate with an important biological process, would be of great importance. Knowledge to be gained from this study could potentially be highly important. Discovery of effective biomarkers of vasculitis and assessment of long term disease course and complications by the collection of clinical and diagnostic imaging studies proposed could lead to better care and less toxic treatment regimens. Furthermore, insight into the pathophysiology of vasculitis could be gained and this might lead to better treatments and improved outcomes in terms of reduction of vascular and treatment related complications. Ideally, excellent biomarkers reflect a sophisticated understanding of the pathophysiology of a disease.

  Study phs000590

• Genetic Modifiers of Syndromic Orofacial Clefts

  Reduced penetrance and variable expressivity are common, but poorly understood, features of most monogenic diseases. Genetic modifiers are possible factors to contribute to this phenotypic variability and blur the traditional distinction between monogenic and complex disease. This study focuses on the most common orofacial cleft syndrome (Van der Woude syndrome, VWS) and nonsyndromic orofacial clefts (OFCs), which intersect both in phenotype and in genetic etiology, as a model to understand reduced penetrance and variable expressivity. VWS occurs in 1 in 35,000 individuals and accounts for 2% of all OFCs. The features of VWS include an OFC and/or lower lip pits, but 15% of VWS patients present with an isolated OFC, making them indistinguishable from nonsyndromic OFC patients. Mutations in the genes IRF6 or GRHL3 cause VWS, but approximately 20% of VWS patients currently lack a molecular diagnosis. Furthermore, there are few genotype-phenotype correlations for known mutations and patient phenotypes. By contrast, GWAS of nonsyndromic OFCs have identified a number of risk factors, some of which we have shown increase risk for specific types of OFCs or act as modifiers of OFC subtypes. Whole genome sequencing will be used to identify the remaining genetic mutations for VWS, determine the relationship between those genes/regions with nonsyndromic OFCs, and identify rare and common modifiers of VWS and OFC phenotypes. These analyses will provide further insight into the genetic architecture of VWS and OFCs and will serve as a model for exploring links between other Mendelian disorders that share phenotypes with complex traits.

  Study phs002221

• Melbourne Collaborative Cohort Study DNA Methylation Studies

  This dataset contains raw Illumina 450K blood DNA methylation data from a subset of participants in the Melbourne Cohort Study (MCCS), a prospective study of 41,513 healthy adult volunteers (24,469 women) aged between 27 and 76 years when recruited between 1990 and 1994. Nested case-control studies: DNA methylation data were generated as part of cancer case-control studies nested in the MCCS. Participants were cancer-free at blood draw; blood samples were taken at baseline (1990-1994) for 97% of participants; and for 3% of participants at the second wave of follow-up (2003-2007). Eight separate studies were conducted of breast, prostate, colorectal, lung, kidney, urothelial and gastric cancer and B-cell lymphoma, for a total of > 3,500 cancer cases. Controls were individually matched to cases on age, sex, and country of birth using incidence density sampling. Additional matching on smoking history was made for the study of lung cancer. Longitudinal study: For a subset of 1,100 participants with baseline DNA methylation measures (from dried blood spots) who were selected as controls in the nested case-control studies, repeated methylation measures were made using wave 2 follow-up samples (also from dried blood spots) to study longitudinal DNA methylation changes. Project aims: 1. Nested case-control studies: To assess prospective associations between peripheral blood DNA methylation and risk of eight cancer types (breast, prostate, colorectal, lung, kidney, urothelial cell and gastric cancer, and mature B-cell neoplasms). 2. Longitudinal study: To assess associations between lifestyle changes and changes in peripheral blood DNA methylation.

  Study phs003213

• NHLBI TOPMed: Genetic Epidemiology of COPD (COPDGene)

  Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States, and the only leading cause of death that is steadily increasing in frequency. This project established a racially diverse cohort that is sufficiently large and appropriately designed for genome-wide association analysis of COPD. A total of 10,720 subjects were recruited, including control smokers and nonsmokers, definite COPD cases (GOLD Stage 2 to 4), and subjects not included in either group (GOLD 1 and PRISm). This cohort is being used for cross-sectional analysis, and long-term longitudinal follow-up visits after five years and after ten years are also being performed. The primary focus of the study is to identify the genetic risk factors that determine susceptibility for COPD and COPD-related phenotypes. Detailed phenotyping of both cases and controls, including chest CT scan assessment of emphysema and airway disease, will allow identification of genetic determinants for the heterogeneous components of the COPD syndrome. The aims for this study are: Precise phenotypic characterization of COPD subjects using computed tomography, as well as clinical and physiological measures, that will provide data to enable the broad COPD syndrome to be decomposed into clinically significant subtype; Genome-wide association studies will identify genetic determinants for COPD susceptibility that will provide insight into clinically relevant COPD subtypes; Distinct genetic determinants influence the development of emphysema and airway disease. The TOPMed analysis will include approximately 10,500 subjects with whole genome sequencing after quality control is completed. Comprehensive phenotypic data for COPDGene subjects is available through dbGaP study phs000179.

  Study phs000951

• Oncogenome of Kaposi Sarcoma

  Kaposi Sarcoma (KS) is an aggressive cancer caused by the Kaposi Sarcoma Herpesvirus (KSHV/HHV-8). Individuals with immunodeficiencies, exemplified by HIV, have an elevated risk of developing KS. However, understanding of the genetic factors contributing to KS progression is still limited. To explore potential genetic alterations in KS that could offer biological or therapeutic insights, whole exome sequencing was performed on 78 KS tumors and matching normal skin samples from 59 adults with KS (46 with HIV-associated KS and 13 with HIV-negative KS) receiving treatment at the Uganda Cancer Institute in Kampala, Uganda. Results showed a very low mutational burden in all samples except one (median = 11 mutations), which is the smallest number of mutations found across all 33 cancer types in The Cancer Genome Atlas (TCGA). No recurrent mutations were identified. Mutational signatures included impaired DNA mismatch repair and smoking. There was no evidence suggesting that multiple tumors from the same patient originated from a single clone. The number of genome copy alterations per genome was higher in tumors from individuals without HIV and those with advanced-stage disease. This suggests that lesions that take longer to develop may accumulate more alterations, although the overall number of alterations remains low compared to other cancers. These findings suggest that KS pathogenesis differs from other cancers, with KSHV viral infection and the expression of viral oncogenes being the primary drivers of carcinogenesis, rather than clonal oncogenic transformation arising from genetic alterations of cancer-related cellular genes.

  Study phs003897

• Whole-genome and transcriptome versus panel sequencing in precision oncology: A translational-clinical comparison

  Precision oncology offers new cancer treatment options. While the use of whole-genome (WGS) and transcriptome (TS) sequencing remains often confined to specific clinical programs, molecular analysis with gene panels is more common clinical practice. Methods: Twenty patients with rare or advanced tumors sequenced by WGS/TS within the DKFZ/NCT/DKTK-MASTER program from 2015 to 2020 (MASTER 1) were additionally sequenced with the TSO500 DNA and TST170 RNA Assay by Illumina. Original Molecular Tumor Board (MTB) therapy recommendations (TR) were juxtaposed with TR from NGS panel data analysis (comparison 1). To mitigate temporal differences between the two sequencings, WGS/TS was bioinformatically reanalyzed (MASTER 2), and new TR were again compared (comparison 2). Results: In comparison 1, 47.1% of the MASTER 1’s TR and 62.8% of the panel’s TR were identical, as were 45.9% of the TR of MASTER 2 and 54.9% of the panel in comparison 2. In both MASTER 1 and MASTER 2, 26.5% and 36.1% of TR, respectively, relied on Biomarkers (BM) which were not covered by the panel. Eight of the ten MTB-derived therapy implementations were also supported in the panel, the two remaining ones being based on BM absent from the panel. Conclusions: About half of the TR in WGS/TS and panel sequencing were identical. Around a third of TR issued by WGS/TS were based on BM which are not covered by the panel, leading to therapy implementations. The presented data underscores the clinical value of comprehensive molecular analyses in individuals with advanced cancers.

  Study EGAS50000000431

• Frequent post-treatment monitoring of colorectal cancer using individualized ctDNA validated by multi-regional molecular profiling

  Purpose: Circulating tumor DNA (ctDNA) analysis has been proposed as an approach for prediction of post-treatment patient outcomes. However, whether a single platform will provide optimal information in all patients or alternatively a patient-specific monitoring approach based on assessment of mutations in the primary tumor from that patient remains an urgent question. Experimental Design: We conducted multiregional sequencing of 42 specimens of 14 colorectal tumors (Stage III and more) from 12 patients, including two double cancer cases, to identify the full spectrum of mutational heterogeneity and identify aberrations that could be used to develop personalized ctDNA assays. Results: ?Founder? mutations that occur in all regions of the sample were identified in 12/14 (85.7%) tumors. Subsequent phylogenetic analysis of each tumor showed that 12/14 tumors (85.7%) carried at least one ?truncal? mutation. Most founder and truncal mutations exhibited higher variant allele frequency (VAF) than ?non-founder? and ?branch? mutations. In addition, both founder and truncal mutations were more likely to be detected as ctDNA than non-founder and branch mutations. Synchronized ctDNA dynamics of multiple mutations suggested those mutations from the same clonal origin. For 10/12 patients (83.3%) with nearly 1,000 days of post-operative observation, the validity of frequent personalized ctDNA monitoring was confirmed in terms of early relapse prediction, treatment efficacy, and non-relapse corroboration. Conclusions: Personalized ctDNA monitoring based on aberrations with a high VAF in the primary tumor site should be explored in larger prospective clinical trials to determine the full clinical validity.

  Study JGAS000243

• Case Report: Pre-Clinical Combination Targeting VEGF and PI3K in a Rare, Aggressive Mixed Endometrial Carcinoma

  We report a rare case of a young patient (VENUS 167) initially diagnosed with grade 1 endometrioid endometrial cancer, which, following endocrine treatment, presented with mixed aggressive carcinoma with three distinct histologic patterns: grade 1 endometrioid, large cell neuroendocrine, and undifferentiated carcinoma. The surgical specimen at the time of disease progression was used to establish OPTO.85, a patient-derived organoid (PDO), followed by a corresponding organoid-derived xenograft (ODX). Multi-omic analyses confirmed that OPTO.85 accurately reflected the patient’s tumor characteristics. WES analysis identified oncogenic alterations in PIK3CA, ARID1A, and CTNNB1. Further RNA-seq and ATAC-seq analyses revealed enrichment in VEGF and Wnt signaling pathways, suggesting therapeutic vulnerabilities. A high-throughput drug screen was conducted using ApexBio-Approved and Epigenetic Drug Libraries, along with Kinase Inhibitor and Tool Compound Libraries developed at the Ontario Institute of Cancer Research. The OPTO.85 PDO exhibited sensitivity to PI3K inhibitors and responsiveness to VEGF inhibition. Cediranib demonstrated synergy with BKM120, significantly reducing organoid growth. This combination also showed in vivo efficacy in the ODX model, where dual inhibitors significantly suppressed tumor growth compared to single compounds. This case exemplifies the impact of genomic profiling and patient-derived models in identifying actionable molecular changes in rare cancers with limited therapeutic options and poor prognosis. It highlights that high-throughput sequencing for individual patient tumors and generation of patient-derived models are feasible in endometrial cancer. This preclinical model may assist clinical decision and personalized therapy requiring validation in prospective studies.

  Study EGAS50000001665

• DNA methylation and the adverse metabolic outcomes of adiposity

  Overweight and obesity affect ~1.5 billion people worldwide, and are major risk factors for type-2 diabetes (T2D), cardiovascular disease and related metabolic and inflammatory disturbances.1,2 Although the mechanisms linking adiposity to its clinical sequelae are poorly understood, recent studies suggest that adiposity may influence DNA methylation,3-6 a key regulator of gene expression and molecular phenotype.7 Here we use epigenome-wide association to show that body mass index (BMI, a key measure of adiposity) is associated with widespread changes in DNA methylation (187 genetic loci at P<1x10-7, range P=9.2x10-8 to 6.0x10-46; N=10,261 samples). Genetic association analyses demonstrate that the alterations in DNA methylation are predominantly the consequence of adiposity, rather than the cause. We find the methylation loci are enriched for functional genomic features in multiple tissues (P<0.05), and show that sentinel methylation markers identify gene expression signatures at 38 loci (P<9.0x10-6, range P=5.5x10-6 to 6.1x10-35, N=1,785 samples). The methylation loci identified highlight genes involved in lipid and lipoprotein metabolism, substrate transport, and inflammatory pathways. Finally, we show that the disturbances in DNA methylation predict future type-2 diabetes (relative risk per 1SD increase in Methylation Risk Score: 2.3 [2.07-2.56]; P=1.1x10-54). Our results provide new insights into the biologic pathways influenced by adiposity, and may enable development of new strategies for prediction and prevention of type-2 diabetes and other adverse clinical consequences of obesity.

  Study EGAS00001001922

• Analysis of recent shared ancestry in a familial cohort identifies coding and noncoding autism spectrum disorder variants

  Autism spectrum disorder (ASD) is a collection of neurodevelopmental disorders characterized by deficits in social communication and restricted, repetitive patterns of behavior or interests. ASD is highly heritable, but genetically and phenotypically heterogeneous, reducing the power to identify causative genes. We performed whole genome sequencing (WGS) in an ASD cohort of 68 individuals from 22 families enriched for recent shared ancestry. We identified an average of 3.07 million variants per genome, of which an average of 112,512 were rare. We mapped runs of homozygosity (ROHs) in affected individuals and found an average genomic homozygosity of 9.65%, consistent with expectations for multiple generations of consanguineous unions. We identified potentially pathogenic rare exonic or splice site variants in 12 known (including KMT2C, SCN1A, SPTBN1, SYNE1, ZNF292) and 12 candidate (including CHD5, GRB10, PPP1R13B) ASD genes. Furthermore, we annotated noncoding variants in ROHs with brain-specific regulatory elements and identified putative disease-causing variants within brain-specific promoters and enhancers for 5 known ASD and neurodevelopmental disease genes (ACTG1, AUTS2, CTNND2, CNTNAP4, SPTBN4). We also identified copy number variants in two known ASD and neurodevelopmental disease loci in two affected individuals. In total we identified potentially etiological variants in known ASD or neurodevelopmental disease genes for ~61% (14/23) of affected individuals. We combined WGS with homozygosity mapping and regulatory element annotations to identify candidate ASD variants. Our analyses add to the growing number of ASD genes and variants and emphasize the importance of leveraging recent shared ancestry to map disease variants in complex neurodevelopmental disorders.

  Study EGAS00001006058

• High-throughput Detection of Clinically Relevant Mutations in Archived Tumor Samples By Multiplexed PCR and Next Generation Sequencing

  PURPOSE: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high content genetic profiling in archived clinical samples. EXPERIMENTAL DESIGN: We first validated MMP-seq’s technical performance in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin embedded (FFPE) clinical samples using matched fresh frozen (FF) tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 endometrial cancer patients. RESULTS: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA. CONCLUSIONS: MMP-seq provides a robust solution for comprehensive, reliable and high- throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice.

  Study EGAS00001000674

• pancreatic ductal adenocarcinoma exomes in study: Pro-immunogenic Impact of MEK inhibition combined with an anti-CD40 immunostimulatory antibody

  Cancer types with lower mutational load and a non-permissive tumor microenvironment are intrinsically resistant to immune checkpoint blockade. While the combination of cytostatic drugs and immunostimulatory antibodies constitutes an attractive concept for overcoming this refractoriness, suppression of immune cell function by cytostatic drugs may limit therapeutic efficacy. Here we show that targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) does not impair dendritic cell-mediated T-cell priming and activation. Accordingly, combining MEK inhibitors (MEKi) with agonist antibodies (Abs) targeting the immunostimulatory CD40 receptor resulted in potent synergistic anti-tumor efficacy. Detailed analysis of the mechanism of action of MEKi GDC-0623 by means of flow cytometric analysis of the tumor immune infiltrate and whole tumor transcriptomics showed that, in addition to its cytostatic impact on tumor cells, this drug exerts multiple pro-immunogenic effects, including the suppression of M2-type macrophages, myeloid derived suppressor cells and CD4+ T-regulatory cells. In addition, MEKi was found to induce tumor-cell intrinsic interferon signaling, which contributed to antigen presentation by tumor cells. Finally, the tumoridical impact of MEKi involves the activation of multiple pro-inflammatory pathways involved in immune cell effector function in the tumor microenvironment. Our data therefore indicate that the combination of MEK inhibition with agonist anti-CD40 Ab is a promising therapeutic concept, especially for the treatment of mutant Kras-driven tumors such as pancreatic ductal adenocarcinoma. The pancreatic ductal adenocarcinoma exomes were generated in the context of the Heidelberg Center for Personalized Oncology project HIPO-K28E.

  Study EGAS00001004196

• Genome-Wide Association Study of aspirin-induced PUD in a UK cohort

  Aspirin is a widely prescribed non-steroidal anti-inflammatory drug used as an effective analgesic and at low doses as anti-platelet drug for the prevention of cardiovascular diseases. However, aspirin is associated with a significant incidence of upper gastrointestinal (GI) complications, notably peptic ulceration. This study aims to identify genetic risk factors associated with the pathogenesis of aspirin-induced upper GI ulceration.A cohort of 248 patients with aspirin-induced, endoscopiclly confirmed GI ulceration and 491 controls (ulcer, without aspirin or aspirin without ulcer) were genotyped for 1.524,956 SNPs using the Illumina Omni 2.5 SNP array. A replication cohort (206 NSAID-induced ulcer cases [of which 87 were aspirin] and 309 controls) was subsequently genotyped for identified SNP association signals using Sequenom iPLEX Massarry. Logistic regression analysis of identified two SNP signal with suggestive association with aspirin-induced GI ulceration. A SNP within the EYA1 locus (rs12678747) was found to be associated with aspirin GI ulceration in both the discovery cohort (p=3.79x10-7, OR=2.06 ([1.55-2.73]) and, to a lesser extent, in the replication cohort (p=.0005, OR=1.71[1.17-2.50]. Combined analysis of both cohorts yielded a genome wide significant association (p=1.46x10-8). The rs12678747 genotype was found to be associated with significantly higher EYA1 transcript in ulcer biopsy tissue (p<0.05).Data suggest that EYA1 may represent a risk locus for aspirin-induced GI ulceration. Though it is thought that EYA1 may play a key role in mediating cell death/ survival

  Study EGAS00001002052

• Breast cancer DNA repair

  Tumors of germline BRCA1/2 mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double strand break (DSB) repair and high sensitivity to Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors. Although this therapy is expected to be effective beyond germline BRCA1/2 mutated carriers, a robust validated test to detect HRD tumors is lacking. In the present study we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh breast cancers (BrC) tissue: the RECAP test.Methods Fresh samples of 170 primary BrC were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring BRCA deficiencies, mutational signatures, tumor infiltrating lymphocytes (TILs) and microsatellite instability (MSI).Results RECAP was completed successfully in 148 out of 170 samples (87%). 24 tumors showed HRD (16%), while 6 tumors were HR intermediate (HRi) (4%). HRD was explained by BRCA deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases. Several non-BRCA deficient HRD tumors showed BRCAness mutational signatures, suggesting that they are also bona fide HRD cases. HRD tumors showed an increased incidence of high TIL counts (p=0.023) compared to HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in BrC (1%) (p=0.017).Conclusion The RECAP test is a robust assay detecting both BRCA1/2 deficient and BRCA1/2 proficient HRD tumors, suggesting that this test identifies approximately 50% more patients that may benefit from PARPi treatment than BRCA gene testing only.

  Study EGAS00001002792

• Structure and evolution of double minutes in diagnosis and relapse brain tumors

  Double minute chromosomes are extrachromosomal circular DNA fragments frequently found in brain tumors. To understand their evolution, we characterized the double minutes in paired diagnosis and relapse tumors from a pediatric high-grade glioma and four adult glioblastoma patients. We determined the full structures of the major double minutes using a novel approach combining multiple types of supporting genomic evidence. Among the double minutes identified in the pediatric tumor, only one carrying EGFR was maintained at high abundance in both samples, whereas two others were resent in only trace amounts at diagnosis but abundant at relapse, and the rest were found either in the relapse sample only or in the diagnosis sample only. For the EGFR-carrying double minutes, we found a secondary somatic deletion in all copies at relapse, after erlotinib treatment, but this was rare at diagnosis, suggesting potential resistance to the EGFR inhibitor. This mutation caused an in-frame RNA transcript to skip exon 16, a novel transcript absent in EST database and in about 700 brain tumors that we reviewed. We observed similar patterns involving longitudinal copy number shift of double minutes in another four pairs (diagnosis / relapse) of adult glioblastoma. Overall, in three of five paired tumor samples, we found that although the same oncogenes were amplified at diagnosis and relapse, they were amplified on different double minutes. Our results suggest that double minutes readily evolve, increasing tumor heterogeneity rapidly. Understanding patterns of double minute evolution can shed light on future therapeutic solutions to brain tumors carrying such variants.

  Study EGAS00001003212

• Spatial and temporal evolution of distal 10q deletion, a prognostically unfavorable event in diffuse low-grade gliomas

  Background: The disease course of patients with diffuse low-grade glioma is notoriously unpredictable. Temporal and spatially distinct samples may provide insight into the evolution of clinically relevant copy number aberrations (CNAs). The purpose of this study is to identify CNAs that are indicative of aggressive tumor behaviour and can thereby complement the prognostically favorable 1p/19q co-deletion. Results: Genome-wide, 50 base pair single-end, sequencing was performed to detect CNAs in a clinically well-characterized cohort of 98 formalin-fixed paraffin-embedded low-grade gliomas. CNAs are correlated with overall survival as an endpoint. Seventy-five additional samples from spatially distinct regions and paired recurrent tumors of the discovery cohort were analysed to interrogate the intratumoral heterogeneity and spatial evolution. Loss of 10q25.2-qter is a frequent subclonal event and significantly correlates with an unfavorable prognosis. A significant correlation is furthermore observed in a validation set of 126 and confirmation set of 184 patients. Loss of 10q25.2-qter arises in a longitudinal manner in paired recurrent tumor specimens, whereas the prognostically favorable 1p/ 19q co-deletion is the only CNA that is stable across spatial regions and recurrent tumors. Conclusions: CNAs in low-grade gliomas display extensive intratumoral heterogeneity. Distal loss of 10q is a late onset event and a marker for reduced overall survival in low-grade glioma patients. Intratumoral heterogeneity and higher frequencies of distal 10q loss in recurrences suggest this event is involved in outgrowth to the recurrent tumor.

  Study EGAS00001000643

• A unidirectional histone code in bidirectional promoters across cell types

  Recent studies indicate that thousands of genes are expressed from bidirectional promoters (BDPs). Gene regulation at BDPs is poorly understood, in particular how the cell is able to regulate them differently. Here we investigate the effect of histone Modifications in BDP regulation. In this study, we model the gene activity using different gene expression assays, such as RNA-Seq, GRO-cap, and CAGE around the BDP transcription start sites (TSSs) using different histone modifications in various cell types. We develop a new statistical approach that links histone modifications to gene expression at BDPs. It improves over previous methods, because it is able to capture spatial dependencies of histone modifications along a promoter and leads to more interpretable results. We predict a general histone code that is independent of transcript orientation, cell type, and promoter configuration. The histone code at BDPs reveals that promoters are regulated unidirectionally, such that the majority of histone marks associated with gene expression occur downstream of the gene's TSS. By contrasting associations of histone marks with steady-state levels of capped RNAs in CAGE and nascent capped RNAs in GRO-cap, we show which histone marks have a preference for initiating polymerases and actively elongating polymerases. Using single-cell data we show that the bidirectional histone signal of activating marks is often due averaging of a heterogeneous cell population. Our results have implications for other experiments where the relationship between histone modification and gene initiation or gene elongation is investigated on a genome-wide scale.

  Study EGAS00001001656

• Transcriptional profiling of ovarian tumours and cell lines

  Purpose: Endogenous retroviruses (ERVs) play a role in a variety of biological processes, including embryogenesis and cancer. DNA methyltransferase inhibitors (DNMTi)-induced ERV expression triggers interferon responses in ovarian cancer cells via the viral sensing machinery. Baseline expression of ERVs also occurs in cancer cells, though this process is poorly understood and previously unexplored in epithelial ovarian cancer (EOC). Here, the prognostic and immunomodulatory consequences of baseline ERV expression was assessed in EOC. Experimental Design: ERV expression was assessed using EOC transcriptional data from TCGA and from an independent cohort (HH), as well as from untreated or DNMTi-treated EOC cell lines. LASSO logistic regression defined an ERV expression score to predict patient prognosis. Immunohistochemistry (IHC) was conducted on the HH cohort. Combination of DNMTi treatment with γδ T cells was tested in vitro, using EOC cell lines and patient-derived tumor cells. Results: ERV expression was found to define clinically relevant subsets of EOC patients. An ERV prognostic score was successfully generated in TCGA and validated in the independent cohort. In EOC patients from this cohort, a high ERV score was associated with better survival (log-rank p=0.0009) and correlated with infiltration of CD8+PD1+ T cells (r=0.46, p=0.0001). In vitro, baseline ERV expression dictates the level of ERV induction in response to DNMTi. Manipulation of ERV expression by DNMTi resulted in improved EOC cell killing by cytotoxic immune cells. Conclusions: These findings uncover the potential for baseline ERV expression to robustly inform EOC patient prognosis, influence tumor immune infiltration and affect anti-tumor immunity.

  Study EGAS00001004814

• Transcriptomic intra-tumor heterogeneity of colorectal cancer varies depending on tumor location within the colorectum

  Background; Intra-tumor heterogeneity (ITH) of colorectal cancer (CRC) complicates molecular tumor classification, such as transcriptional subtyping. Differences in cellular states, biopsy cell composition, and tumor microenvironment may all lead to ITH. Here we analyze ITH at the transcriptomic and proteomic levels to ascertain whether subtype discordance between multiregional biopsies reflects relevant biological ITH or lack of classifier robustness. Further, we study the impact of tumor location on ITH.MethodsMultiregional biopsies from stage II and III CRC tumors were analyzed by RNA sequencing (41 biopsies, 14 tumors) and multiplex immune protein analysis (89 biopsies, 29 tumors). CRC subtyping were performed using consensus molecular subtypes (CMS), CRC intrinsic subtypes (CRIS), and TUMOR types. ITH-scores and network maps were defined to determine the origin of heterogeneity. A validation cohort was used with one biopsy per tumor (162 tumors).ResultsOverall, inter-tumor transcriptional variation exceeded ITH, and subtyping calls were frequently concordant between multiregional biopsies. Still, some tumors had high ITH and were classified discordantly. Proximal tumors showed ITH related to differences in the microenvironment, while ITH within distal tumors were cancer-cell related. This subtype discordancy reflected actual molecular ITH within the tumors. The relevance of the subtypes was reflected at protein level where several inflammation markers were significantly increased in immune related transcriptional subtypes, which was verified in an independent cohort (Wilcoxon rank sum test; p<0.05).ConclusionOur transcriptomic and proteomic analyses show that the tumor location along the colorectum influence the ITH of CRC, which again influence the concordance of subtyping.

  Study EGAS00001004668

• RaScALL: Rapid screening of RNA-seq in acute lymphoblastic leukaemia

  RNA-sequencing (RNA-seq) efforts in acute lymphoblastic leukaemia (ALL) have identified numerous prognostically significant genomic alterations which can guide diagnostic risk stratification and treatment choices when detected early. However, a full RNA-seq Bioinformatics workflow is time-consuming and costly in a clinical setting where rapid detection and accurate reporting of clinically relevant alterations are essential. To accelerate the identification of ALL-specific alterations (including gene fusions, single nucleotide variants and focal gene deletions), we developed the rapid screening tool RaScALL, capable of identifying more than 100 prognostically significant lesions directly from raw sequencing reads. RaScALL uses the k-mer based targeted detection tool km and known ALL variant information to achieve a high degree of accuracy for reporting subtype defining genomic alterations compared to standard alignment-based pipelines. Gene fusions, including difficult to detect fusions involving EPOR and DUX4, were accurately identified in 98% (164 samples) of reported cases in a 180-patient Australian study cohort and 95% (n=63) of samples in a North American validation cohort. Pathogenic sequence variants were correctly identified in 75% of tested samples, including all cases involving subtype defining variants PAX5 p.P80R (n=12) and IKZF1 p.N159Y (n=4). Accurate detection of intragenic IKZF1 deletions resulting in aberrant transcript isoforms was also detectable with 98% accuracy. Importantly, the median analysis time for detection of all targeted alterations averaged 22 minutes per sample, significantly shorter than standard alignment-based approaches, ensuring accerelated risk-stratification and therapeutic triage.

  Study EGAS00001006460

• Chronic lymphocytic leukemia IGHV somatic hypermutation detection by targeted capture next-generation sequencing

  Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene locus plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex PCR of the IGH gene followed by Sanger sequencing; however, this technique is low throughput, vulnerable to failure, and does not allow multiplexing with other molecular diagnostic assays. Here we designed and validated a DNA targeted capture approach that allows the assessment of IGHV SHM status as a sub-module of a larger next-generation sequencing (NGS) assay. We developed an end-to-end assay that takes as input raw FASTQ files and outputs a report containing IGHV SHM status and V-allele usage following the guidelines of the European Research Initiative on CLL (ERIC). We validated the approach on 35 CLL patient samples, 34 of which were previously characterized using Sanger sequencing. The targeted capture NGS panel identified the SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect common and prognostically important CLL single nucleotide variants (SNV) and insertion/deletion (indel) variants as well as copy number variants (del17p). Thus, a targeted capture approach to IGHV SHM detection can be easily integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

  Study EGAS00001006887

• A Machine Learning Tool Integrating Circulating Cell-Free DNA Methylation with Clinical Variables for Non-Invasive Diagnosis of Liver Graft Pathology

  Liver Transplant Recipients (LTRs) with elevated liver enzymes can develop graft injury due to various etiologies, such as T-cell mediated rejection (TCMR) and NASH (NASH-LT). While liver biopsy is the gold standard method for diagnosis of graft pathology, it is invasive and is associated with risks. The goal of our study was to develop a Machine Learning (ML) tool integrating clinical variables with methylation patterns on circulating DNA in plasma as a non-invasive diagnostic tool of graft injury. We generated methylation profiles of circulating DNA in a pilot study of 43 LTRs (11 NASH-LT, 19 TCMR, and 13 Control-LT), and developed an L2 multinomial logistic regression ML approach across 101 bootstrapped models to distinguish between the graft conditions. NASH was associated with distinctive methylation patterns on genes involved in fatty acid metabolism, while methylation of platelet-derived growth factors was identified in patients with TCMR. Our ML model achieved a mean multi-classification accuracy of 0.91, with mean specificity and sensitivity of 0.94 and 0.91, respectively. The model was found to be particularly adept at detecting TCMR and Control-LT, with true positive rates (TPRs) of 95% and 90%, and areas under the curve (AUROC) of 0.992 and 0.985, respectively. For NASH-LT, the models achieved a performance with a TPR of 82% and an AUROC of 0.991. These preliminary results suggest that our newly developed ML tool, leveraging cell-free DNA methylation and clinical variables, is a promising non-invasive classifier of graft pathology.

  Study EGAS00001007171

• Clinical relevance of somatic copy-number alterations in plasma circulating tumor DNA from advanced EGFR-mutated NSCLC patients treated with osimertinib

  Background: Somatic copy-number alterations (SCNAs) are associated with drug resistance in patients with advanced epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) treated with EGFR-tyrosine kinase inhibitors (TKIs). The purpose of our study was to assess the clinical relevance and development of genome-wide SCNAs in plasma circulating tumor DNA (ctDNA) under osimertinib therapy.Methods: We included 43 patients with advanced EGFR T790M-positive lung adenocarcinoma who were treated with osimertinib after progression under previous EGFR-TKI therapy. We performed genomic profiling of ctDNA in plasma samples from each patient obtained pre-osimertinib and after patients developed resistance to osimertinib. SCNAs were detected by shallow whole-genome plasma sequencing and EGFR mutations were assessed by droplet digital PCR.Results: SCNAs in resistance-related genes (rrSCNAs) were detected in 10 out of 39 (25.6%) evaluable patients before start of osimertinib. The presence of rrSCNAs in plasma before initiation of osimertinib therapy was associated with a lower response rate to osimertinib (50% versus 86%, p=0.02) and was an independent predictor for shorter progression-free survival (adjusted HR 3.26, 95% CI 1.37-7.75, p=0.008) and overall survival (adjusted HR 2.41, 95% CI 1.09-5.34, p=0.03).Conclusions: Genomic profiling of plasma ctDNA is clinically relevant and affects the efficacy and clinical outcome of osimertinib. Our approach allows comprehensive assessment of SCNAs in plasma samples of lung adenocarcinoma patients and may help to guide genotype-specific therapeutic strategies in the future.

  Study EGAS00001004539