Stefan Gräf

The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ∼40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.

An integrated resource for genome-wide identification and analysis of human tissue-specific differentially methylated regions (tDMRs)

We report a novel resource (methylation profiles of DNA, or mPod) for human genome-wide tissue-specific DNA methylation profiles. mPod consists of three fully integrated parts, genome-wide DNA methylation reference profiles of 13 normal somatic tissues, placenta, sperm, and an immortalized cell line, a visualization tool that has been integrated with the Ensembl genome browser and a new algorithm for the analysis of immunoprecipitation-based DNA methylation profiles. We demonstrate the utility of our resource by identifying the first comprehensive genome-wide set of tissue-specific differentially methylated regions (tDMRs) that may play a role in cellular identity and the regulation of tissue-specific genome function. We also discuss the implications of our findings with respect to the regulatory potential of regions with varied CpG density, gene expression, transcription factor motifs, gene ontology, and correlation with other epigenetic marks such as histone modifications.

The shaping and functional consequences of the microRNA landscape in breast cancer.

MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA–mRNA interactions rather than as on–off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.

Quantitative Image Analysis of Cellular Heterogeneity in Breast Tumors Complements Genomic Profiling

Image analysis of breast cancer tissue improves and complements genomic data to predict patient survival. Digitizing Pathology for Genomics The tumor microenvironment is a complex milieu that includes not only the cancer cells but also the stromal cells, immune cells, and even normal, healthy cells. Molecular analysis of tumor tissue is therefore a challenging task because all this “extra” genomic information can muddle the results. Conversely, biopsy tissue staining can provide a spatial and cellular readout (architecture and content), but it is mostly qualitative information. In response, Yuan and colleagues have developed a quantitative, computational approach to pathology. When combined with molecular analyses, the authors were able to uncover new knowledge about breast tumor biology and, in turn, predict patient survival. Yuan et al. first collected histopathology images, gene expression data, and DNA copy number variation data for 564 breast cancer patients. Using a portion of the images (the “discovery set”), they developed an image processing approach that automatically classified cells as cancer, lymphocyte, or stroma on the basis of their size and shape. This approach was validated on the remaining samples, and any errors in this analysis were digitally corrected before obtaining a plot of tumor cellular heterogeneity. With exact knowledge of the tumor’s cellular composition, the authors were able to correct copy number data to more accurately reflect HER2 status compared with uncorrected data. Yuan and colleagues combined their digital pathology with genomic information to devise an integrated predictor of survival for estrogen receptor (ER)–negative patients. Higher number of infiltrating lymphocytes (immune cells) as quantified by their image analysis platform were found in a subset of patients with better clinical outcome than the rest of ER-negative patients, and this outcome difference was significantly enhanced with the addition of gene expression. The quantitative and objective nature of this integrated predictor could benefit diagnosis and prognosis in many areas of cancer by using the rich combination of tumor cellular content and genomic data. Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin–stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. 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. We next devised a predictor for survival in estrogen receptor–negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.

Pulmonary Arterial Hypertension: A Current Perspective on Established and Emerging Molecular Genetic Defects

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II‐like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next‐generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus‐specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.

BMPR2 mutations and survival in pulmonary arterial hypertension: an individual participant data meta-analysis

Summary Background Mutations in the gene encoding the bone morphogenetic protein receptor type II (BMPR2) are the commonest genetic cause of pulmonary arterial hypertension (PAH). However, the effect of BMPR2 mutations on clinical phenotype and outcomes remains uncertain. Methods We analysed individual participant data of 1550 patients with idiopathic, heritable, and anorexigen-associated PAH from eight cohorts that had been systematically tested for BMPR2 mutations. The primary outcome was the composite of death or lung transplantation. All-cause mortality was the secondary outcome. Hazard ratios (HRs) for death or transplantation and all-cause mortality associated with the presence of BMPR2 mutation were calculated using Cox proportional hazards models stratified by cohort. Findings Overall, 448 (29%) of 1550 patients had a BMPR2 mutation. Mutation carriers were younger at diagnosis (mean age 35·4 [SD 14·8] vs 42·0 [17·8] years), had a higher mean pulmonary artery pressure (60·5 [13·8] vs 56·4 [15·3] mm Hg) and pulmonary vascular resistance (16·6 [8·3] vs 12·9 [8·3] Wood units), and lower cardiac index (2·11 [0·69] vs 2·51 [0·92] L/min per m2; all p<0·0001). Patients with BMPR2 mutations were less likely to respond to acute vasodilator testing (3% [10 of 380] vs 16% [147 of 907]; p<0·0001). Among the 1164 individuals with available survival data, age-adjusted and sex-adjusted HRs comparing BMPR2 mutation carriers with non-carriers were 1·42 (95% CI 1·15–1·75; p=0·0011) for the composite of death or lung transplantation and 1·27 (1·00–1·60; p=0·046) for all-cause mortality. These HRs were attenuated after adjustment for potential mediators including pulmonary vascular resistance, cardiac index, and vasoreactivity. HRs for death or transplantation and all-cause mortality associated with BMPR2 mutation were similar in men and women, but higher in patients with a younger age at diagnosis (p=0·0030 for death or transplantation, p=0·011 for all-cause mortality). Interpretation Patients with PAH and BMPR2 mutations present at a younger age with more severe disease, and are at increased risk of death, and death or transplantation, compared with those without BMPR2 mutations. Funding Cambridge NIHR Biomedical Research Centre, Medical Research Council, British Heart Foundation, Assistance Publique-Hôpitaux de Paris, INSERM, Université Paris-Sud, Intermountain Research and Medical Foundation, Vanderbilt University, National Center for Advancing Translational Sciences, National Institutes of Health, National Natural Science Foundation of China, and Beijing Natural Science Foundation.

From alpaca to zebrafish: Hammerhead ribozymes wherever you look

The hammerhead ribozyme was originally discovered in subviral plant pathogens and was subsequently also found in a few other genomic locations. Using a secondary structure-based descriptor, we have searched publicly accessible sequence databases for new examples of type III hammerhead ribozymes. The more than 60,000 entries fulfilling the descriptor were filtered with respect to folding and stability parameters that were experimentally validated. This resulted in a set of 284 unique motifs, of which 124 represent database entries of known hammerhead ribozymes from subviral plant pathogens and A. thaliana. The remainder are 160 novel ribozyme candidates in 50 different eukaryotic genomes. With a few exceptions, the ribozymes were found either in repetitive DNA sequences or in introns of protein coding genes. Our data, which is complementary to a study by De la Peña and García-Robles in 2010, indicate that the hammerhead is the most abundant small endonucleolytic ribozyme, which, in view of no sequence conservation beyond the essential nucleotides, likely has evolved independently in different organisms.

HyPaLib: a database of RNAs and RNA structural elements defined by hybrid patterns

The database, called HyPaLib (for Hybrid Pattern Library), contains annotated structural elements characteristic for certain classes of structural and/or functional RNAs. These elements are described in a language specifically designed for this purpose. The language allows convenient specification of hybrid patterns, i.e. motifs consisting of sequence features and structural elements together with sequence similarity and thermodynamic constraints. We are currently developing software tools that allow a user to search sequence databases for any pattern in HyPaLib, thus providing functionality which is similar to PROSITE, but dedicated to the more complex patterns in RNA sequences. HyPaLib is available at http://bibiserv. techfak.uni-bielefeld.de/HyPa/.

Plasma Metabolomics Implicates Modified Transfer RNAs and Altered Bioenergetics in the Outcomes of Pulmonary Arterial Hypertension.

Background: Pulmonary arterial hypertension (PAH) is a heterogeneous disorder with high mortality. Methods: We conducted a comprehensive study of plasma metabolites using ultraperformance liquid chromatography mass spectrometry to identify patients at high risk of early death, to identify patients who respond well to treatment, and to provide novel molecular insights into disease pathogenesis. Results: Fifty-three circulating metabolites distinguished well-phenotyped patients with idiopathic or heritable PAH (n=365) from healthy control subjects (n=121) after correction for multiple testing (P<7.3e-5) and confounding factors, including drug therapy, and renal and hepatic impairment. A subset of 20 of 53 metabolites also discriminated patients with PAH from disease control subjects (symptomatic patients without pulmonary hypertension, n=139). Sixty-two metabolites were prognostic in PAH, with 36 of 62 independent of established prognostic markers. Increased levels of tRNA-specific modified nucleosides (N2,N2-dimethylguanosine, N1-methylinosine), tricarboxylic acid cycle intermediates (malate, fumarate), glutamate, fatty acid acylcarnitines, tryptophan, and polyamine metabolites and decreased levels of steroids, sphingomyelins, and phosphatidylcholines distinguished patients from control subjects. The largest differences correlated with increased risk of death, and correction of several metabolites over time was associated with a better outcome. Patients who responded to calcium channel blocker therapy had metabolic profiles similar to those of healthy control subjects. Conclusions: Metabolic profiles in PAH are strongly related to survival and should be considered part of the deep phenotypic characterization of this disease. Our results support the investigation of targeted therapeutic strategies that seek to address the alterations in translational regulation and energy metabolism that characterize these patients.