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• Bone marrow breakout lesions act as key sites for tumor-immune cell diversification and exhaustion in multiple myeloma

  Multiple myeloma is a disease characterized by the expansion of cancerous plasma cells in the bone marrow. The bone marrow microenvironment plays a pivotal role in supporting myeloma growth and modulating tumor immunity. As the disease progresses, myeloma cells may become independent of the bone marrow environment, leading to extramedullary disease associated with poor prognosis. However, the early processes associated with bone marrow independence and its implications for disease and immune control remain poorly understood. Here, we employed comprehensive single-cell and spatial mapping to identify the disruption of the cortical bone and subsequent expansion of myeloma cells in breakout lesions as a key event in multiple myeloma pathogenesis and tumor immunity. Breakout lesions harbor an adapted niche and a unique immune microenvironment conferred by sustained immune-tumor interactions. In particular, tumor-reactive T cells with a highly exhausted phenotype, alongside unique NK cell and macrophage subtypes, specifically expanded in breakout lesions. Spatially-resolved genomic, transcriptomic and cellular analyses uncovered extensive intra-lesion heterogeneity, suggesting a divergent co-evolution of genomic variation and locally confined T cell responses in distinct subregions. Ultra-high plex imaging revealed that tumor-reactive T cells, together with other immune-regulatory cells and dendritic cells, co-expand and exhaust upon sustained tumor interactions in locally confined immune islands. In contrast, immune-regulatory macrophage and NK cell subsets operate spatially separated within myeloma cell deserts. Jointly, our analyses uncover bone marrow breakout lesions as a hotspot for tumor-immune cell interactions, diversification and exhaustion, representing a key event in myeloma pathogenesis with significant therapeutic implications.

  Study EGAS50000000304

• Confronting historical legacies of biological anthropology in South Africa—Restitution, redress and community-centered science: The Sutherland Nine

  We describe a process of restitution of nine unethically acquired human skeletons to their families, together with attempts at redress. Between 1925–1927 C.E., the skeletonised remains of nine San or Khoekhoe people, eight of them known-in-life, were removed from their graves on the farm Kruisrivier, near Sutherland in the Northern Cape Province of South Africa. They were donated to the Anatomy Department at the University of Cape Town. This was done without the knowledge or permission of their families. The donor was a medical student who removed the remains from the labourers’ cemetery on his family farm. Nearly 100 years later, the remains are being returned to their community, accompanied by a range of community-driven interdisciplinary historical, archaeological and analytical (osteobiographic, craniofacial, ancient DNA, stable isotope) studies to document, as far as possible, their lives and deaths. The restitution process began by contacting families living in the same area with the same surnames as the deceased. The restitution and redress process prioritises the descendant families’ memories, wishes and desire to understand the situation, and learn more about their ancestors. The descendant families have described the process as helping them to reconnect with their ancestors. A richer appreciation of their ancestors’ lives, gained in part from scientific analyses, culminating with reburial, is hoped to aid the descendant families and wider community in [re-]connecting with their heritage and culture, and contribute to restorative justice, reconciliation and healing while confronting a traumatic historical moment. While these nine individuals were exhumed as specimens, they will be reburied as people.

  Study EGAS50000000971

• Discovery and validation of an ancillary genomic test of malignancy for primary melanocytic tumors

  Background:Pathological diagnosis of melanocytic tumors can be difficult and prone to error. More accurate and reliable ancillary investigations than those currently available are required to improve diagnostic accuracy. Goals: (1) to generate a genomic model for distinguishing benign (nevi) from malignant (melanoma) melanocytic skin tumors, developed from a customized gene panel evaluated on a cohort of 250 nevi and melanomas. (2) To evaluate the performance of the genomic model in melanocytic tumors of uncertain diagnosis/behavior. Details: The cohort of 250 melanomas and naevi was split into a discovery set and a validation set. Our genomic model obtained an area under the curve of 95% in the discovery cohort and 96% for the validation cohort. Based on a Youden index threshold, the resulting classifier shows high specificity (>95% in the discovery and validation cohorts), with sensitivities >80% in the discovery and validation cohorts. Evaluation of the genomic model on a cohort of 110 melanocytic tumors identifies key driver mutations in agreement with the likely pathway of origin of the tumors. The probability of melanoma (p=0.02) from the genomic model was significantly associated with pathology-based estimates of malignancy for melanocytic tumors lacking pathway-defining genomic aberrations (conventional tumors). The genomic model identified 8/36 (22%) conventional tumors with unresolved malignancy to have a high probability of melanoma, with 7/8 of these cases showing negative/non-aberrant results with either fluorescent in-situ hybridization, PRAME and/or p16 immunostains. Furthermore, no evidence of malignant behavior based on the genomic model was seen in 4 conventional borderline tumors with more than 1 year follow-up. Conclusion: This genomic model can become a clinically useful ancillary tool that is highly specific for differentiating melanomas from nevi.

  Study EGAS50000000887

• Development and validation of multivariate predictors of primary endocrine resistance to tamoxifen and aromatase inhibitors in luminal breast cancer reveal drug-specific differences

  Background: Endocrine therapy is highly effective in blocking the estrogen receptor pathway in HR+/HER2- early breast cancer (EBC). However, up to 40% of patients experience relapse during or after adjuvant endocrine therapy. Here, we investigate molecular mechanisms associated with primary resistance to endocrine therapy and develop predictive models. Results: TP53 mutations were prominently associated with primary resistance to both tamoxifen (TAM) and aromatase inhibitors (AI), with AI non-responders exhibiting resistance in up to 32% of cases. Additionally, we identified distinct DNA methylation patterns in TAM and AI non-responders, with TAM non-responders showing global DNA methylation loss, associated with KRAS signaling, apical junctions and epithelial-mesenchymal transition (EMT). Conversely, we observed methylation gain in AI non-responders affecting developmental transcription factors, hypoxia and estrogen signaling. TAM or AI resistance was associated with increased methylation-inferred proportions of immune cells and decreased proportions of endothelial cells. Based on these findings and patient age, we developed the Predictive Endocrine ResistanCe Index (PERCI). PERCI stratified NR and R cases in both treatment groups and cohorts with high accuracy (ROC AUC TAM discovery 93.9%, validation 83%; AI discovery 98.6%, validation 76.9%). A simplified PERCI efficiently predicted progression-free survival in the TCGA-BRCA sub-cohort (Kaplan-Meier log-rank p-value = 0.03 between low and high PERCI groups). Conclusions: We identified genomic and epigenomic features associated with primary resistance to TMA and AI. By combining information on genomic alterations, patient age, differential methylation and tumor microenvironment (TME) composition, we developed PERCI TAM and PERCI AI as novel predictors of primary resistance, with potential additional prognostic value. Applying PERCI in a clinical setting may allow patient-specific drug selection to overcome resistance.

  Study EGAS50000001102

• 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

• Biallelic variants in the non-protein coding minor spliceosome components RNU6ATAC and RNU6ATAC cause syndromic monogenic autoimmune diabetes

  Non-protein coding genes are emerging as critical contributors to the aetiology of rare diseases, providing key insights into human biology and uncovering novel disease mechanisms. We identified 7 individuals from 4 families with early-onset diabetes (diagnosed <5 years) and immune dysregulatory features caused by biallelic variants in RNU6ATAC. RNU6ATAC encodes a small nuclear RNA (snRNA) acting as a catalytic component of the minor spliceosome, a protein-RNA complex mediating splicing of ~700 genes containing U12/minor-type introns. Variant screening of the other 64 minor spliceosome genes in 276 infants with diabetes identified 12 unrelated individuals with biallelic disease-causing variants in RNU4ATAC. Biallelic pathogenic RNU4ATAC variants are known to cause a variable spectrum of clinical features, which until now did not include diabetes. Clinically, 12/19 RNU6ATAC/RNU4ATAC patients had additional immune dysregulatory features, and 50% of patients tested were islet-autoantibody positive, strongly supporting an autoimmune aetiology for their diabetes. RNA-seq in 3 individuals with biallelic RNU6ATAC variants showed a pattern of intron retention in U12 intron-containing genes similar to that seen in RNU4ATAC-individuals (n=3), supporting a shared disease mechanism. Analysis of patients’ transcriptomic, methylation and immune data revealed impaired B cell development and maturation. We conclude that biallelic RNU6ATAC variants cause a novel syndrome of early-onset autoimmune diabetes and immune dysregulation. We further show that infancy-onset diabetes is a previously unrecognised feature of RNU4ATACopathy. Our work highlights the important role of two snRNAs critical to minor spliceosome function in immune system regulation, providing novel insights into the pathogenesis of autoimmune diabetes.

  Study EGAS50000001565

• Luminal breast epithelial cells from wildtype and BRCA mutation carriers harbor copy number alterations commonly associated with breast cancer

  Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation and evolution is not well understood. Here, using single cell DNA sequencing, we describe the landscape of CNA variants in >48,000 breast epithelial cells from women with normal or high risk of developing breast cancer. Individual cells with one or two of a specific subset of CNAs (e.g. 1q gain and 16q, 22q, 7q, and 10q loss) are detectable in almost all breast tissues and, in those from BRCA1 or BRCA2 mutations carriers, occurs prior to loss of heterozygosity (LOH) of the wildtype alleles. These CNAs, which are among the most common associated with ductal carcinoma in situ (DCIS) and malignant breast tumors, are enriched almost exclusively in luminal cells and are generally absent from basal myoepithelial cells. Allele-specific analysis of the enriched CNAs reveals that each allele was independently altered, demonstrating convergent evolution of these CNAs in an individual breast. Tissues from BRCA1 or BRCA2 mutation carriers contained a small percentage of cells with extreme aneuploidy, featuring loss of TP53, LOH of BRCA1 or BRCA2, and multiple breast cancer-associated CNAs in addition to one or more of the common CNAs in 1q, 10q or 16q. Notably, cells with intermediate levels of CNAs were not detected, arguing against a stepwise gradual accumulation of CNAs. Overall, our findings demonstrate that chromosomal alterations in normal breast epithelium including from high risk women, link early stage CNAs to clonally expanded tumor genomes .

  Study EGAS00001007716

• A proof-of-concept study of sequential treatment with the HDAC inhibitor vorinostat following BRAF and MEK inhibitors in BRAFV600 mutated melanoma

  "Purpose: Development of resistance limits the clinical benefit of BRAF and MEK inhibitors (BRAFi/MEKi) in BRAFV600 mutated melanoma. It has been shown that short-term treatment (14 days) with vorinostat was able to initiate apoptosis of the resistant tumor cells. We aimed to assess the anti-tumor activity of sequential treatment with vorinostat following BRAFi/MEKi in patients with BRAFV600 melanoma who progressed after initial response to BRAFi/MEKi. Patients and Methods: Patients with BRAFi/MEKi resistant BRAFV600 melanoma were treated with vorinostat 360 mg QD for 14 days followed by BRAFi/MEKi. The primary endpoint was an objective response rate of progressive lesions of at least 30% according to RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), safety, pharmacokinetics of vorinostat and translational molecular analyses using ctDNA and tumor biopsies. Results: Twenty-six patients with progressive BRAFi/MEKi resistant BRAFV600 mutated melanoma received treatment with vorinostat. Twenty-two patients were evaluable for response. The ORR was 9% (one complete response for 31.2 months and one partial response for 14.9 months. Median PFS and OS were 1.4 and 5.4 months, respectively. Common adverse events were fatigue (23%) and nausea (19%). ctDNA analysis showed emerging secondary mutations in NRAS and MEK in eight patients at time of BRAFi/MEKi resistance. Elimination of these mutations by vorinostat treatment was observed in three patients. Conclusions: Intermittent treatment with vorinostat in patients with resistant BRAFV600 mutated melanoma is well tolerated. Although the primary endpoint of this study was not met, durable anti-tumor responses were observed in a minority of patients (9%). "

  Study EGAS00001007709

• Toxigenic Clostridium perfringens isolated from at-risk pediatric inflammatory bowel disease patients

  With the goal of studying mucosal-associated bacteria and products detrimental to inflammatory bowel disease (IBD) patients, we directly cultured bacterial communities from mucosal biopsies and examined for detrimental traits. Focusing on red blood cell lysis, a virulence trait, communities from 5 of 9 patients exhibited hemolytic activity. Clostridium perfringens was isolated from each hemolytic community, and whole-genome sequencing confirmed presence of hemolytic toxins – alpha toxin in all 5 strains, perfringolysin O (PFO) in 4 strains, alveolysin in 2 strains – and absence of enterotoxins. In vitro cytotoxicity experiments demonstrated strain supernatants were toxic to various cell types located beneath the intestinal epithelial barrier, including endothelial, neuroblasts, and neutrophils, while impact on epithelial cells was less pronounced. These findings suggest C. perfringens may be damaging particularly when intestinal barrier integrity is compromised, such as in IBD patients. Further characterization using purified toxins and genetic insertion mutants confirmed PFO toxin was sufficient for cell toxicity. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion (DRG) neuron cells in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. Strikingly, C. perfringens was detected in biopsies of 8 of 9 pediatric patients while not in all corresponding stool samples, and in other datasets, C. perfringens was in stool samples of IBD adults (18.7-27.1%) much more frequently versus healthy (5.1%). Our findings showcase C. perfringens may have an important pathogenic impact on IBD patients. Similar to Clostridioides difficile, routine monitoring of C. perfringens and PFO toxins in patients may be beneficial.

  Study EGAS50000000334

• Cell-Free DNA Genomic and Fragmentomic Features for Early Outcome Prediction in Diffuse Large B-Cell Lymphoma

  Purpose: Diffuse large B-cell lymphoma (DLBCL) patients need an accurate and early risk stratification strategy as prompt therapy escalation may improve outcome. Patients and methods: We evaluated cell free DNA (cfDNA) genomic and fragmentomic features in 190 patients with large B-cell lymphoma from more than 40 hospitals in the Benelux using Whole Genome Sequencing (WGS). We defined a new metric called ACT score (Aberrations, Contribution of short fragments, Terminal motif analyses) by combining four cfDNA signals and evaluated its performance to predict end-of-treatment (EOT) response and survival. Results: Individual cfDNA features and ACT score were altered per EOT response after one cycle of treatment (T1). The ACT score at T1 outperformed all individual cfDNA features in predicting EOT response (AUC = 0.74). Patients with a positive ACT score had an inferior outcome compared to ACT score negative patients [progression-free survival (PFS): HR 5.0 (95% CI 3.1-8.2), log-rank test, p < 0.0001) and overall survival (OS): HR 6.5 (95% CI 3.5-12.2), log-rank test, p < 0.0001]. The 2-year PFS in ACT score positive and negative patients were 29.8% and 78.1%, respectively. The prognostic value of the ACT score is independent of the International Prognostic Index and interim imaging. Conclusions: ACT score computed from a single plasma sample collected after one cycle of treatment can predict clinical outcome. This low-cost and easy-to-interpret test does not require tissue biopsies or a priori knowledge of mutations and has the potential to aid patient selection in interventional clinical trials and risk-adapted treatment strategies.

  Study EGAS50000000412

• Bladder cancer subtyping study across 4 atezo clinical trials

  Checkpoint inhibitors targeting the PD-1/PD-L1 interaction have revolutionized cancer therapy across many indications. In urothelial carcinoma (UC), several PD-1/PD-L1 inhibitors have been approved for treatment in localized and metastatic disease. While these agents provide durable clinical benefit in a number of patients, the majority do not respond. A better understanding of the molecular mechanisms underlying response and resistance is needed to tailor existing treatments and develop new therapeutics in UC. We profiled tumors from 2803 patients from four late-stage randomized clinical trials evaluating the PD-L1 inhibitor atezolizumab in metastatic UC and muscle-invasive bladder cancer by RNA-seq, a targeted DNA panel, immunohistochemistry, and digital pathology. A machine learning algorithm identified four UC transcriptional subtypes, representing luminal desert, stromal, immune, and basal tumors. Overall survival benefit from atezolizumab over standard-of-care was observed in immune and basal tumors (45% of patients). Digital pathology showed enrichment of human interpretable features in specific molecular subtypes, and a self-supervised approach could classify molecular subtypes from H&E slides with high accuracy, suggesting that machine-based image interpretation could accelerate tumor molecular profiling. Somatic alterations showed limited capacity to identify responders. Finally, mechanisms of benefit with PD-L1 blockade appeared different in immune and basal tumors, with immune tumors benefiting from B/plasma cell enrichment, while basal tumors benefited from granulocyte enrichment. This study represents the largest integration of UC molecular and curated clinical data in randomized clinical trials to date, paving the way for prospective clinical studies tailoring treatment regimens to specific tumor molecular subtypes in UC and other indications.

  Study EGAS50000000497

• Single-cell Transcriptome Profiling of Treatment-naïve and Post-treatment Colorectal Cancer: Insights into Putative Mechanisms of Chemoresistance

  Drug resistance remains a major clinical challenge in the treatment of colorectal cancer (CRC) with conventional chemotherapy. Investigation of cancer cells and tumor microenvironment (TME) following chemotherapy is critical for unraveling the mechanisms of resistance. In this study, we analyzed scRNA-Seq data from 56 CRCs including treatment-naïve tumors (25 microsatellite stable (MSS) tumors and 7 tumors with microsatellite instability (MSI)); and tumors treated with standard chemotherapy with the known response status (18 responders and 6 progressors). We observed in treatment-naïve MSS CRC that right-sided tumors are more immunogenic with higher numbers of B cells and CD8+ T memory effector cells than left-sided tumors. MSI CRC were even more immunogenic then MSS CRC and were characterized by elevated Tregs in their TME. In the post-treatment CRC there was a high prevalence of dendritic cells (DC) in the TME in the response group. The DC-derived signature was associated with better survival in a large CRC cohort from the TCGA. We also observed two fibroblast subtypes in CRC, one of which was enriched in progressors and was associated with poor survival in a CRC-TCGA cohort. Progressors also showed elevated exhausted CD8+ T memory cells and monocytes suggesting a pro-inflammatory TME. In tumor cells in progressor group we identified specific expression of chemo-protective markers MTRNR2L1 and CDX1; and their co-expression with stemness-related marker CD24. In summary, scRNA-Seq provides a valuable information for the discovery of prognostic markers, and reveals distinct features of CRC based on location, metastasis status, MSI status and treatment response status.

  Study EGAS50000000830

• Mutational patterns and regulatory networks in epigenetic subgroups of meningioma (H033)

  DNA methylation patterns delineate clinically relevant subgroups of meningioma. We previously established the six meningioma methylation classes (MC) benign-1, 2, 3, intermediate-A, B and malignant. Here, we set out to identify subgroup-specific mutational patterns and pathway regulation. Whole-genome-sequencing was performed on 62 samples across all MCs and WHO grades from 62 patients with matched blood control, including 40 sporadic and 22 radiation-induced (Mrad) meningiomas. RNA sequencing was added for 18 cases and chromatin-immunoprecipitation for the enhancer mark H3K27ac followed by sequencing (ChIP-seq) for 16 samples. Besides the known mutations in meningioma, structural alterations were found to contribute to the spectrum of mechanisms inactivating NF2 in sporadic meningioma similar to previous reports for Mrad. Aberrations of DMD were found to be enriched in MCs with NF2 mutations and DMD was among the most differentially upregulated genes in NF2 mutant compared to NF2 wild-type cases. The mutational signature AC3 was detected in both sporadic meningioma and Mrad, but distributed across the genome in sporadic cases and enriched near genomic breakpoints in Mrad. In general, the malignant MC presented a significantly higher exposure to AC3 and higher genomic instability beyond the mutational load than the other MCs. Pathway analysis of the ChIP-seq clusters revealed that the FOXM1 is most differentially activated in high-grade cases, along with super enhancer recruitment near HOX genes and respective upregulation of expression. This data further elucidate the biological mechanisms differentiating meningiomas of different WHO grade and epigenetic subgroups and suggest leveraging the genomic instability as novel therapeutic targets.

  Study EGAS00001003481

• Whole-exome Sequencing Combined with Functional Genomics Reveals Novel Candidate Driver Cancer Genes in Endometrial Cancer

  Endometrial cancer is the most common gynecological malignancy, with more than 280,000 cases occurring annually worldwide. Although previous studies have identified important common somatic mutations in endometrial cancer, they have primarily focused on a small set of known cancer genes and have thus provided a limited view of the molecular basis underlying this disease. Here we developed an integrated systems biology approach to identifying novel cancer genes contributing to endometrial tumorigenesis. We first performed whole-exome sequencing on 13 endometrial cancers and matched normal samples, systematically identifying somatic alterations with high precision and sensitivity. We then combined bioinformatics prioritization with high-throughput screening (including both shRNA-mediated knockdown and expression of wild type and mutant constructs) in a highly sensitive cell viability assay. Our results revealed 12 potential driver cancer genes including 10 tumor suppressor candidates (ARID1A, INHBA, KMO, TTLL5, GRM8, IGFBP3, AKTIP, PHKA2, TRPS1 and WNT11) and two oncogene candidates (ERBB3 and RPS6KC1). The results in the “sensor” cell line were recapitulated by siRNA-mediated knockdown in endometrial cancer cell lines. Focusing on ARID1A, we integrated mutation profiles with functional proteomics in 222 endometrial cancer samples, demonstrating that ARID1A mutations frequently co-occur with mutations in the phosphatidylinositol 3-kinase (PI3K) pathway and are associated with PI3K pathway activation. siRNA knockdown in endometrial cancer cell lines increased AKT phosphorylation supporting ARID1A as a novel regulator of PI3K pathway activity. Our study presents the first unbiased view of somatic coding mutations in endometrial cancer and provides functional evidence for diverse driver genes and mutations in this disease.

  Study EGAS00001000318

• Distinct evolutionary trajectories of primary high grade serous ovarian cancers revealed through spatial mutational profiling

  High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to emergence of treatment-resistant sub-clones. We sought to measure the degree of genomic diversity within primary, untreated HGSC to examine the natural state of tumor evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on thirty-one spatially and temporally separated HGSC tumor specimens (six patients) including ovarian masses, distant metastases, and fallopian tube lesions. We found widespread intra-tumoral variation in mutation, copy number, and gene expression profiles, with key driver alterations in genes present in only a subset of samples (e.g. PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range: 10.2% to 91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology with common etiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating diversity arises at early stages of tumorigenesis. Our results reveal that HGSC exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug resistance mechanisms.

  Study EGAS00001000547

• MiR expression profiles of paired primary colorectal cancerand metastases by next-generation sequencing

  MicroRNAs (miRs) have been recognized as promising biomarkers. It is unknown to what extent tumor-derived miRs aredifferentially expressed between primary colorectal cancers (pCRCs) and metastatic lesions, and to what extent the expressionprofiles of tumor tissue differ from the surrounding normal tissue. Next-generation sequencing (NGS) of 220 fresh-frozen samples,including paired primary and metastatic tumor tissue and non-tumorous tissue from 38 patients, revealed expression of 2245known unique mature miRs and 515 novel candidate miRs. Unsupervised clustering of miR expression profiles of pCRC tissue withpaired metastases did not separate the two entities, whereas unsupervised clustering of miR expression profiles of pCRC withnormal colorectal mucosa demonstrated complete separation of the tumor samples from their paired normal mucosa. Twohundred and twenty-two miRs differentiated both pCRC and metastases from normal tissue samples (false discovery rate (FDR)o0.05). The highest expressed tumor-specific miRs were miR-21 and miR-92a, both previously described to be involved in CRC withpotential as circulating biomarker for early detection. Only eight miRs, 0.5% of the analysed miR transcriptome, were differentiallyexpressed between pCRC and the corresponding metastases (FDR o0.1), consisting of five known miRs (miR-320b, miR-320d,miR-3117, miR-1246 and miR-663b) and three novel candidate miRs (chr 1-2552-5p, chr 8-20656-5p and chr 10-25333-3p). Theseresults indicate that previously unrecognized candidate miRs expressed in advanced CRC were identified using NGS. In addition,miR expression profiles of pCRC and metastatic lesions are highly comparable and may be of similar predictive value for prognosisor response to treatment in patients with advanced CRC

  Study EGAS00001001127

• Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down Syndrome

  Children with Down Syndrome (DS) are prone to development of high risk B-cell precursor ALL (DS-ALL) that differs genetically from most sporadic pediatric ALLs. Increased expression of CRLF2, the receptor to thymic stromal lymphopoietin (TSLP) characterizes about half of DS-ALLs and also a subgroup of sporadic “Philadelphia-like ALLs”. To understand the pathogenesis of relapsed DS-ALL we performed integrative genomic analysis of 25 matched diagnosis remission and relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are “relapse driving”. We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2 positive DS-ALL by loss-of-function aberrations in USP9X, a deubuitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2 positive, “Ph like” ALLs. Pharmacological or genetic inhibition of USP9X as well as treatment with low-dose Ruxolitinib enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT “hyper-signaling” may be beneficial to leukemic B cell precursors. This and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

  Study EGAS00001002410

• Telomerase activation by genomic rearrangements in high-risk neuroblastoma

  Neuroblastoma is a malignant pediatric tumor of the sympathetic nervous system1. Roughly half of these tumors regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavorable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2-4. We have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n=39; low-risk, n=17) and discovered recurrent genomic rearrangements affecting a chromosomal region (5p15.22) proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumor type1,2,5. In an extended case series (n=217), TERT rearrangements defined a subgroup of high-risk tumors with particularly poor outcome. Despite the large diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumors, TERT expression was also elevated in MYCN-amplified tumors, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.22 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodeling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cells bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodeling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the center of transformation in a large fraction of these tumors.

  Study EGAS00001001308

• RNA-seq as a tool for evaluating human embryo competence

  The majority of embryos that are created through IVF do not implant. One possibility for this inefficiency is an incomplete understanding of the molecular factors affecting embryo competence. Currently, the process of selecting an embryo for uterine transfer utilizes an ad-hoc combination of morphological criteria, the kinetics of development, and genetic testing for aneuploidy. However, no single criterion can ensure selection of a viable embryo. In contrast, RNA-sequencing of embryos could yield highly dimensional data, which may provide additional insight and illuminate the discrepancies among current selection criteria. Indeed, recent advances enabling the production of RNA-sequencing (RNA-seq) libraries from single cells have facilitated the application of this technique to the study of some transcriptional events in early human development. However, these studies have not assessed the quality of their constituent embryos relative to commonly used embryological criteria. Here, we perform proof-of-principle advancement to clinical selection procedures by generating high quality RNA-seq libraries from a trophectoderm biopsy as well as the remaining whole embryo. We combine state-of-the-art embryological methods with low-input RNA-seq to develop the first transcriptome-wide approach for use in future predictive embryology studies. Specifically, we demonstrate the capacity of RNA-seq as a promising tool in preimplantation screening by showing that biopsies of an embryo can capture valuable information content available in the whole embryo from which they are derived. Furthermore, we show that this technique can be used to generate a RNA-based digital karyotype, and to develop a foundational dataset for identifying candidate competence-associated genes. Together, these data establish the foundation for a future RNA-based diagnostic in IVF.

  Study EGAS00001003667

• Whole-genome Sequencing Suggests Mechanisms for 22q11.2 deletion-associated Parkinson’s disease

  Objectives: To investigate disease risk mechanisms of early-onset Parkinson’s disease (PD) associated with the recurrent 22q11.2 deletion, a genetic risk factor for early-onset PD. Methods: In a proof-of-principle study, we used whole-genome sequencing (WGS) to investigate sequence variants in nine adults with 22q11.2DS, three with neuropathologically confirmed early-onset PD and six without PD. Adopting an approach used recently to study schizophrenia in 22q11.2DS, here we tested candidate gene-sets relevant to PD. Results: No mutations common to the cases with PD were found in the intact 22q11.2 region. While all were negative for rare mutations in a gene-set comprising PD disease-causing and risk genes, another candidate gene-set of 1000 genes functionally relevant to PD presented a nominally significant (P=0.03) enrichment of rare putatively damaging missense variants in the PD cases. Polygenic score results, based on common variants associated with PD risk, were non-significantly greater in those with PD. Conclusions: The results of this first-ever pilot study of WGS in PD suggest that the cumulative burden of genome-wide sequence variants may contribute to expression of early-onset PD in the presence of threshold-lowering dosage effects of a 22q11.2 deletion. We found no evidence that expression of PD in 22q11.2DS is mediated by a recessive locus on the intact 22q11.2 chromosome or mutations in known PD genes. These findings offer initial evidence of the potential effects of multiple within-individual rare variants on the expression of PD and the utility of next generation sequencing for studying the etiology of PD.

  Study EGAS00001002275

• Intra-tumor heterogeneity and clonal evolution patterns towards platinum-resistant high-grade serous ovarian cancer

  Although many high-grade serous ovarian carcinoma (HGSOC) patients are initially sensitive to the standard-of-care platinum-based therapy, intra-tumor heterogeneity may fuel selection towards platinum-resistant clones over subsequent lines of therapy. We performed whole-exome coupled to ultra-deep re-sequencing and copy number alteration (CNA) profiling on 31 matched diagnosis and relapse tumors from HGSOC patients treated with platinum. Genetic heterogeneity within tumor pairs varied extremely, with some recurrent tumors being identical and others completely different from the primary tumor. All primary tumors showed evidence of homologous recombination (HR)-deficiency, but none of them carried reactivating mutations in the HR pathway at relapse, also not when becoming resistant to subsequent lines of platinum. We did also not identify other mutations, CNAs or pathways that were enriched in platinum-resistant clones. However, a platinum score consisting of 13 CNAs correlated significantly with platinum-free interval (PFI) after first-line therapy in multiple cohorts of &gt;450 primary HGSOCs. A relative increase of this score in the relapsed tumor correlated with a change in PFI during subsequent therapy. Clonal evolution under platinum therapy contributes to genetic heterogeneity within tumor pairs, implicating that genetic tests linked to targeted treatment options for recurrent HGSOC need to be performed on biopsies collected at relapse. Platinum resistance of an initially platinum-sensitive HR-deficient HGSOC at relapse is not caused by reactivation of the HR pathway, suggesting that these tumors are still responsive to PARP inhibitors, instead, several chromosomal aberrations contribute to a score predictive of disease outcome under platinum therapy, both in first and subsequent lines of platinum therapy.

  Study EGAS00001001244

• UK10K NEURO IMGSAC

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches.The IMGSAC cohort is an international collection of families containing children ascertained for ASDs (autism spectrum disorders). The affected individuals are have been phenotyped, including using the ADI-R and ADOS instruments. Individuals with a past or current medical disorder of probable etiological significance or TSC have been excluded. Where possible, karyotyping has been performed on one affected individual per family to exclude Fragile X syndrome. Many of the samples have been genotyped, using the Affymetrix 10k and Illumina 1M platforms. All samples to be included in the current study are of UK origin.For further information on this cohort please contact Jeremy Parr (jeremy.parr@newcastle.ac.uk).

  Study EGAS00001000120

• Pharmacological and genomic profiling identifies NFκB-targeted treatment strategies for mantle cell lymphoma

  Mantle cell lymphoma (MCL) is an aggressive malignancy characterized by poor prognosis, underscoring the need for novel therapeutic strategies1. In this study, we employed large-scale pharmacological profiling of 16 pathway inhibitors across more than 100 hematological cell line models to identify novel therapeutic approaches. This screen identified a subset of MCL cell lines that were highly sensitive to the B-cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin (STN), which are currently in clinical trials2, 3. Sensitive MCL models exhibited chronic activation of the CARD11-BCL10-MALT1 (CBM) complex and dependency on downstream classical NFκB signaling. In contrast, insensitive cell lines displayed activation of the alternative NFκB pathway. Massively parallel transcriptome sequencing identified mutations in negative regulators of the alternative NFκB pathway in ibrutinib-insensitive cell lines. Notably, sequencing of 165 MCL patient samples revealed that components of the alternative NFκB pathway, particularly TRAF2 and BIRC3, are mutated in roughly 15% of MCL patients. Functional analyses of patient-derived BIRC3 mutant alleles indicated that these are loss-of-function mutants that constitutively activate alternative NFκB signaling. While being associated with insensitivity to ibrutinib, genetic lesions in the alternative pathway conferred dependence on NIK, a kinase that mediates signaling downstream of the TRAF2/3-BIRC2/3 complex. Thus, NIK represents a novel therapeutic target for MCL treatment, particularly for tumors refractory to BCR pathway inhibitors. Collectively, these findings reveal a pattern of mutually exclusive activation of the CBM-NFκB or alternative NIK-NFκB pathways in MCL and provide critical insights into patient stratification strategies for NFκB-pathway targeted agents.

  Study EGAS00001000622

• A genome scan for genes underlying adult body size differences between Central African hunter-gatherers and farmers.

  The evolutionary and biological bases of the Central African pygmy phenotype, a characteristic of rainforest huntergatherers defined by reduced body size compared with neighboring farmers, remain largely unknown. Here, we perform a joint investigation in Central African hunter-gatherers and farmers of adult standing height, sitting height, leg length, and body mass index (BMI), considering 358 hunter-gatherers and 169 farmers with genotypes for 153,798 SNPs. In addition to reduced standing heights, hunter-gatherers have shorter sitting heights and leg lengths and higher sitting/standing height ratios than farmers and lower BMI for males. Standing height, sitting height, and leg length are strongly correlated with inferred levels of farmer genetic ancestry, whereas BMI is only weakly correlated, perhaps reflecting greater contributions of non-genetic factors to body weight than to height. Single- and multi-marker association tests identify one region and eight genes associated with hunter-gatherer/farmer status, and 24 genes associated with the height-related traits. Many of these genes have putative functions consistent with roles in determining their associated traits and the pygmy phenotype, and they include three associated with standing height in non-Africans (PRKG1, DSCAM, MAGI2). We find evidence that European height-associated SNPs or variants in linkage disequilibrium with them contribute to standing- and sitting-height determination in Central Africans, but not to the differential status of hunter-gatherers and farmers. These findings provide new insights into the biological basis of the pygmy phenotype, and they highlight the potential of cross-population studies for exploring the genetic basis of phenotypes that vary naturally across populations. Pemberton et al. Human Genetics 2018 https://doi.org/10.1007/s00439-018-1902-3

  Study EGAS00001002975

• We evaluate the potential for routine WGS using ONT by sequencing the well-characterised reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome accompanied by severe immune dysregulation.

  Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. While current sequencing technologies have been extremely successful, their reliance on short read lengths necessarily involves some limitations: accurately assaying certain genomic regions and classes of variation can be problematic. Recent advances in throughput and cost have made WGS using the Oxford Nanopore Technologies (ONT) MinION long-read single-molecule sequencer a potential solution to these challenges. Here we evaluate the potential for routine WGS using ONT by sequencing the well-characterised reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome accompanied by severe immune dysregulation, to 82× and 30× respectively. For NA12878, we evaluated single-nucleotide variant (SNV) calls based on data from multiple base-calling algorithms. We demonstrate that phasing metrics from a novel, reference panel-free, long-read-based method can improve variant-calling performance from otherwise modest levels, resulting in a false discovery rate of 7.1% and false negative rate of 8.5%; remaining errors are concentrated near homopolymers and in regions of reduced sequencing coverage. In the clinical sample, we are able to identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they both lie on a common paternal haplotype by overlap with parental genotypes at nearby common variants. This work demonstrates that methodological innovation can substantially reduce variant-calling error rates in ONT data, and that with on-going improvements in throughput, base-calling and dedicated long-read-based SNV-calling methodology, ONT offers promise as an option for clinical WGS.

  Study EGAS00001003469