Integrative clinical transcriptome analysis reveals TMPRSS2-ERG dependency of prognostic biomarkers in prostate adenocarcinoma.

In prostate adenocarcinoma (PCa), distinction between indolent and aggressive disease is challenging. Around 50% of PCa are characterized by TMPRSS2-ERG (T2E)-fusion oncoproteins defining two molecular subtypes (T2E-positive/negative). However, current prognostic tests do not differ between both molecular subtypes, which might affect outcome prediction. To investigate gene-signatures associated with metastasis in T2E-positive and T2E-negative PCa independently, we integrated tumor transcriptomes and clinicopathological data of two cohorts (total n = 783), and analyzed metastasis-associated gene-signatures regarding the T2E-status. Here, we show that the prognostic value of biomarkers in PCa critically depends on the T2E-status. Using gene-set enrichment analyses, we uncovered that metastatic T2E-positive and T2E-negative PCa are characterized by distinct gene-signatures. In addition, by testing genes shared by several functional gene-signatures for their association with event-free survival in a validation cohort (n = 272), we identified five genes (ASPN, BGN, COL1A1, RRM2 and TYMS)-three of which are included in commercially available prognostic tests-whose high expression was significantly associated with worse outcome exclusively in T2E-negative PCa. Among these genes, RRM2 and TYMS were validated by immunohistochemistry in another validation cohort (n = 135), and several of them proved to add prognostic information to current clinicopathological predictors, such as Gleason score, exclusively for T2E-negative patients. No prognostic biomarkers were identified exclusively for T2E-positive tumors. Collectively, our study discovers that the T2E-status, which is per se not a strong prognostic biomarker, crucially determines the prognostic value of other biomarkers. Our data suggest that the molecular subtype needs to be considered when applying prognostic biomarkers for outcome prediction in PCa.

Functional genomics identifies AMPD2 as a new prognostic marker for undifferentiated pleomorphic sarcoma.

Soft-tissue sarcomas are rare, heterogeneous, and often aggressive mesenchymal cancers. Many of them are associated with poor outcome, partially because biomarkers that can identify high-risk patients are lacking. Studies on sarcomas are often limited by small sample-sizes rendering the identification of biomarkers difficult when focusing on individual cohorts. However, the increasing number of publicly available 'omics' data opens inroads to overcome this obstacle. Here, we combine transcriptome analyses, immunohistochemistry, and functional assays to show that high adenosine monophosphate deaminase 2 (AMPD2) is a robust prognostic biomarker for worse outcome in undifferentiated pleomorphic sarcoma (UPS). Gene expression and survival data for UPS from two independent studies were subjected to survival association-testing. Genes, whose high expression was significantly correlated with worse outcome in both cohorts, were considered as biomarker candidates. The best candidate, AMPD2, was validated in a tissue microarray. Analysis of DNA copy-number data and matched transcriptomes indicated that high AMPD2 expression is significantly correlated with gains at the AMPD2 locus. Gene set enrichment analyses of AMPD2 co-expressed genes in both transcriptome datasets suggested that AMPD2-high UPS are enriched in tumorigenic signatures. Consistently, knockdown of AMPD2 by RNA interference in an UPS cell line inhibited proliferation in vitro and tumorigenicity in vivo. Collectively, we provide evidence that AMPD2 may serve as a biomarker for outcome prediction in UPS. Our study exemplifies how the integration of 'omics' data, immunohistochemistry, and functional experiments can identify novel biomarkers even in a rare sarcoma, which may serve as a blueprint for biomarker identification for other rare cancers.

Prenatal Diagnosis of Fryns Syndrome through Identification of Two Novel Splice Variants in the PIGN Gene-A Case Series.

Fryns syndrome (FS) is a multiple congenital anomaly syndrome with different multisystemic malformations. These include congenital diaphragmatic hernia, pulmonary hypoplasia, and craniofacial dysmorphic features in combination with malformations of the central nervous system such as agenesis of the corpus callosum, cerebellar hypoplasia, and enlarged ventricles. We present a non-consanguineous northern European family with two recurrent cases of FS: a boy with multiple congenital malformations who died at the age of 2.5 months and a female fetus with a complex developmental disorder with similar features in a following pregnancy. Quad whole exome analysis revealed two likely splicing-affecting disease-causing mutations in the PIGN gene: a synonymous mutation c.2619G>A, p.(Leu873=) in the last nucleotide of exon 29 and a 30 bp-deletion c.996_1023+2del (NM_176787.5) protruding into intron 12, with both mutations in trans configuration in the affected patients. Exon skipping resulting from these two variants was confirmed via RNA sequencing. Our molecular and clinical findings identified compound heterozygosity for two novel splice-affecting variants as the underlying pathomechanism for the development of FS in two patients.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Western Blot Analysis in Ewing Sarcoma.

Western blot is an experimental method used to analyze protein expression. In Ewing sarcoma, as in many other diseases, Western blot provides information about the level of protein expression in different cell conditions, in comparison with other tissues or upon induced molecular changes. Based on the specific pattern of protein expression of the tissue, as well as on the characteristics of the protein of interest, the antibodies and protocol of Western blot may be modified according to different specifications. Here we describe some of these peculiarities in frame of Ewing sarcoma field.

Therapeutic targeting of the PLK1-PRC1-axis triggers cell death in genomically silent childhood cancer.

Chromosomal instability (CIN) is a hallmark of cancer1. Yet, many childhood cancers, such as Ewing sarcoma (EwS), feature remarkably 'silent' genomes with minimal CIN2. Here, we show in the EwS model how uncoupling of mitosis and cytokinesis via targeting protein regulator of cytokinesis 1 (PRC1) or its activating polo-like kinase 1 (PLK1) can be employed to induce fatal genomic instability and tumor regression. We find that the EwS-specific oncogenic transcription factor EWSR1-FLI1 hijacks PRC1, which physiologically safeguards controlled cell division, through binding to a proximal enhancer-like GGAA-microsatellite, thereby promoting tumor growth and poor clinical outcome. Via integration of transcriptome-profiling and functional in vitro and in vivo experiments including CRISPR-mediated enhancer editing, we discover that high PRC1 expression creates a therapeutic vulnerability toward PLK1 inhibition that can repress even chemo-resistant EwS cells by triggering mitotic catastrophe.Collectively, our results exemplify how aberrant PRC1 activation by a dominant oncogene can confer malignancy but provide opportunities for targeted therapy, and identify PRC1 expression as an important determinant to predict the efficacy of PLK1 inhibitors being used in clinical trials.

SOX6: a double-edged sword for Ewing sarcoma.

Developmental pathways play an important role in cancer. We have recently demonstrated that the constitutive activation of the developmental transcription factor SOX6 via the fusion oncoproteinne EWSR1-FLI1 (Ewing sarcoma breakpoint region 1 - Friend leukemia virus integration 1) contributes to the aggressive phenotype of Ewing sarcoma but on another hand provides an opportunity for targeted therapy.

Publisher Correction: Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20017-2.

Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma.

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.

Targeting the CALCB/RAMP1 axis inhibits growth of Ewing sarcoma.

Ewing sarcoma (EwS) is an aggressive cancer characterized by chromosomal translocations generating fusions of the EWSR1 gene with ETS transcription factors (in 85% FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity correlates with the number of consecutive GGAA-repeats. Herein we investigate the role of the secretory neuropeptide CALCB (calcitonin-related polypeptide ß) in EwS, which signals via the CGRP (calcitonin gene-related peptide) receptor complex, containing RAMP1 (receptor activity modifying protein 1) as crucial part for receptor specificity. Analysis of 2678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed that CALCB is specifically and highly overexpressed in EwS. Time-course knockdown experiments showed that CALCB expression is tightly linked to that of EWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that of CALCB indicated that it is co-expressed with other EWSR1-FLI1 target genes and associated with signatures involved in stemness and proliferation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for FLI1 and histone marks from EwS cell lines demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close to CALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass spectrometric analyses of EwS cell culture supernatants demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediated CALCB knockdown had no effect on proliferation and clonogenic growth of EwS cells in vitro, its long-term knockdown decreased EwS growth in vitro and in vivo. Similarly, knockdown of RAMP1 reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the RAMP1-comprising CGRP receptor reduced growth of EwS. Collectively, our findings suggest that CALCB is a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1 axis may offer a new therapeutic strategy for inhibition of EwS growth.

Cooperation of cancer drivers with regulatory germline variants shapes clinical outcomes.

Pediatric malignancies including Ewing sarcoma (EwS) feature a paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here, we demonstrate in EwS how cooperation of dominant oncogenes and regulatory germline variants determine tumor growth, patient survival and drug response. Binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2 mediating these phenotypes. Whole-genome and RNA sequencing reveals that variability at this locus is inherited via the germline and is associated with variable inter-tumoral MYBL2 expression. High MYBL2 levels sensitize EwS cells for inhibition of its upstream activating kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a putative biomarker for anti-CDK2-therapy. Collectively, we establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and highlight the importance of integrating the regulatory genome in precision medicine.

Gene expression and immunohistochemical analyses identify SOX2 as major risk factor for overall survival and relapse in Ewing sarcoma patients.

BACKGROUND: Up to 30-40% of Ewing sarcoma (EwS) patients with non-metastatic disease develop local or metastatic relapse within a time span of 2-10 years. This is in part caused by the absence of prognostic biomarkers that can identify high-risk patients and thus assign them to risk-adapted monitoring and treatment regimens. Since cancer stemness has been associated with tumour relapse and poor patient outcomes, we investigated in the current study the prognostic potential SOX2 (sex determining region Y box 2) - a major transcription factor involved in development and stemness - which was previously described to contribute to the undifferentiated phenotype of EwS. METHODS: Two independent patient cohorts, one consisting of 189 retrospectively collected EwS tumours with corresponding mRNA expression data (test-cohort) and the other consisting of 141 prospectively collected formalin-fixed and paraffin-embedded resected tumours (validation and cohort), were employed to analyse SOX2 expression levels through DNA microarrays or immunohistochemistry, respectively, and to compare them with clinical parameters and patient outcomes. Two methods were employed to test the validity of the results at both the mRNA and protein levels. FINDINGS: Both cohorts showed that only a subset of EwS patients (16-20%) expressed high SOX2 mRNA or protein levels, which significantly correlated with poor overall survival. Multivariate analyses of our validation-cohort revealed that high SOX2 expression represents a major risk-factor for poor survival (HR = 3·19; 95%CI 1·74-5·84; p < 0·01) that is independent from metastasis and other known clinical risk-factors at the time of diagnosis. Univariate analyses demonstrated that SOX2-high expression was correlated with tumour relapse (p = 0·002). The median first relapse was at 14·7 months (range: 3·5-180·7). INTERPRETATION: High SOX2 expression constitutes an independent prognostic biomarker for EwS patients with poor outcomes. This may help to identify patients with localised disease who are at high risk for tumour relapse within the first two years after diagnosis. FUNDING: The laboratory of T. G. P. Grünewald is supported by grants from the 'Verein zur Förderung von Wissenschaft und Forschung an der Medizinischen Fakultät der LMU München (WiFoMed)', by LMU Munich's Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative, the 'Mehr LEBEN für krebskranke Kinder - Bettina-Bräu-Stiftung', the Walter Schulz Foundation, the Wilhelm Sander-Foundation (2016.167.1), the Friedrich-Baur foundation, the Matthias-Lackas foundation, the Barbara & Hubertus Trettner foundation, the Dr. Leopold & Carmen Ellinger foundation, the Gert & Susanna Mayer foundation, the Deutsche Forschungsgemeinschaft (DFG 391665916), and by the German Cancer Aid (DKH-111886 and DKH-70112257). J. Li was supported by a scholarship of the China Scholarship Council (CSC), J. Musa was supported by a scholarship of the Kind-Philipp foundation, and T. L. B. Hölting by a scholarship of the German Cancer Aid. M. F. Orth and M. M. L. Knott were supported by scholarships of the German National Academic Foundation. G. Sannino was supported by a scholarship from the Fritz-Thyssen Foundation (FTF-40.15.0.030MN). The work of U. Dirksen is supported by grants from the German Cancer Aid (DKH-108128, DKH-70112018, and DKH-70113419), the ERA-Net-TRANSCAN consortium (project number 01KT1310), and Euro Ewing Consortium (EEC, project number EU-FP7 602,856), both funded under the European Commission Seventh Framework Program FP7-HEALTH (http://cordis.europa.eu/), the Barbara & Hubertus Trettner foundation, and the Gert & Susanna Mayer foundation. G. Hardiman was supported by grants from the National Science Foundation (SC EPSCoR) and National Institutes of Health (U01-DA045300). The laboratory of J. Alonso was supported by Instituto de Salud Carlos III (PI12/00816; PI16CIII/00026); Asociación Pablo Ugarte (TPY-M 1149/13; TRPV 205/18), ASION (TVP 141/17), Fundación Sonrisa de Alex & Todos somos Iván (TVP 1324/15).

Robust diagnosis of Ewing sarcoma by immunohistochemical detection of super-enhancer-driven EWSR1-ETS targets.

Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity. Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care.

Systematic identification of cancer-specific MHC-binding peptides with RAVEN.

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Immunogenic peptides encoded by cancer-specific genes (CSGs) may enable targeted immunotherapy, even of oligo-mutated cancers, which lack neo-antigens generated by protein-coding missense mutations. Here, we describe an algorithm and user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues) that automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from transcriptome data. We applied RAVEN to a dataset assembled from 2,678 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on oligo-mutated pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs, and identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated in cancer cell lines and in a comprehensive tissue-microarray. Subsequently, RAVEN identified likely immunogenic CSG-encoded peptides by predicting their affinity to MHCs and excluded sequence identity to abundantly expressed proteins by interrogating the UniProt protein-database. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed binding-kinetics like a very immunogenic influenza control peptide. Collectively, we provide an exquisitely curated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer types, and a freely available software (https://github.com/JSGerke/RAVENsoftware) to easily apply our algorithm to any gene expression dataset. We anticipate that our peptide libraries and software constitute a rich resource to advance anti-cancer immunotherapy.

Epithelial-to-Mesenchymal and Mesenchymal-to-Epithelial Transition in Mesenchymal Tumors: A Paradox in Sarcomas?

The epithelial-to-mesenchymal transition (EMT) is a reversible process comprised of various subprograms via which epithelial cells reduce their intercellular adhesions and proliferative capacity while gaining a mesenchymal phenotype with increased migratory and invasive properties. This process has been well described in several carcinomas, which are cancers of epithelial origin, and is crucial to metastatic tumor cell dissemination and drug resistance. In contrast, the precise role of EMT-related processes in tumors originating from mesenchymal tissues, such as bone and soft-tissues sarcomas, is still largely unclear. In fact, although the existence of the EMT in sarcomas appears paradoxical because these cancers are, by definition, mesenchymal ab initio, accumulating evidence suggests that many sarcomas can undergo EMT-related processes, which may be associated with aggressive clinical behavior. These processes may be especially operative in certain sarcoma subtypes, such as carcinosarcomas displaying a biphenotypic morphology with characteristics of both mesenchymal and epithelial tumors. In this review, we discuss findings regarding the potential existence of EMT-related processes in sarcomas and propose that sarcomas can reside in a metastable state, enabling them to become either more mesenchymal or epithelial under specific conditions, which likely has important clinical implications. Cancer Res; 77(17); 4556-61. ©2017 AACR.