Suzanne Miller

Novel insights into the genetics of smoking behaviour, lung function, and chronic obstructive pulmonary disease (UK BiLEVE): a genetic association study in UK Biobank.

Summary Background Understanding the genetic basis of airflow obstruction and smoking behaviour is key to determining the pathophysiology of chronic obstructive pulmonary disease (COPD). We used UK Biobank data to study the genetic causes of smoking behaviour and lung health. Methods We sampled individuals of European ancestry from UK Biobank, from the middle and extremes of the forced expiratory volume in 1 s (FEV1) distribution among heavy smokers (mean 35 pack-years) and never smokers. We developed a custom array for UK Biobank to provide optimum genome-wide coverage of common and low-frequency variants, dense coverage of genomic regions already implicated in lung health and disease, and to assay rare coding variants relevant to the UK population. We investigated whether there were shared genetic causes between different phenotypes defined by extremes of FEV1. We also looked for novel variants associated with extremes of FEV1 and smoking behaviour and assessed regions of the genome that had already shown evidence for a role in lung health and disease. We set genome-wide significance at p<5 × 10−8. Findings UK Biobank participants were recruited from March 15, 2006, to July 7, 2010. Sample selection for the UK BiLEVE study started on Nov 22, 2012, and was completed on Dec 20, 2012. We selected 50 008 unique samples: 10 002 individuals with low FEV1, 10 000 with average FEV1, and 5002 with high FEV1 from each of the heavy smoker and never smoker groups. We noted a substantial sharing of genetic causes of low FEV1 between heavy smokers and never smokers (p=2·29 × 10−16) and between individuals with and without doctor-diagnosed asthma (p=6·06 × 10−11). We discovered six novel genome-wide significant signals of association with extremes of FEV1, including signals at four novel loci (KANSL1, TSEN54, TET2, and RBM19/TBX5) and independent signals at two previously reported loci (NPNT and HLA-DQB1/HLA-DQA2). These variants also showed association with COPD, including in individuals with no history of smoking. The number of copies of a 150 kb region containing the 5′ end of KANSL1, a gene that is important for epigenetic gene regulation, was associated with extremes of FEV1. We also discovered five new genome-wide significant signals for smoking behaviour, including a variant in NCAM1 (chromosome 11) and a variant on chromosome 2 (between TEX41 and PABPC1P2) that has a trans effect on expression of NCAM1 in brain tissue. Interpretation By sampling from the extremes of the lung function distribution in UK Biobank, we identified novel genetic causes of lung function and smoking behaviour. These results provide new insight into the specific mechanisms underlying airflow obstruction, COPD, and tobacco addiction, and show substantial shared genetic architecture underlying airflow obstruction across individuals, irrespective of smoking behaviour and other airway disease. Funding Medical Research Council.

Markers of survival and metastatic potential in childhood CNS primitive neuro-ectodermal brain tumours: an integrative genomic analysis

BACKGROUND Childhood CNS primitive neuro-ectodermal brain tumours (PNETs) are very aggressive brain tumours for which the molecular features and best treatment approaches are unknown. We assessed a large cohort of these rare tumours to identify molecular markers to enhance clinical management of this disease. METHODS We obtained 142 primary hemispheric CNS PNET samples from 20 institutions in nine countries and examined transcriptional profiles for a subset of 51 samples and copy number profiles for a subset of 77 samples. We used clustering, gene, and pathway enrichment analyses to identify tumour subgroups and group-specific molecular markers, and applied immunohistochemical and gene-expression analyses to validate and assess the clinical significance of the subgroup markers. FINDINGS We identified three molecular subgroups of CNS PNETs that were distinguished by primitive neural (group 1), oligoneural (group 2), and mesenchymal lineage (group 3) gene-expression signatures with differential expression of cell-lineage markers LIN28 and OLIG2. Patients with group 1 tumours were most often female (male:female ratio 0·61 for group 1 vs 1·25 for group 2 and 1·63 for group 3; p=0·043 [group 1 vs groups 2 and 3]), youngest (median age at diagnosis 2·9 years [95% CI 2·4-5·2] for group 1 vs 7·9 years [6·0-9·7] for group 2 and 5·9 years [4·9-7·8] for group 3; p=0·005), and had poorest survival (median survival 0·8 years [95% CI 0·5-1·2] in group 1, 1·8 years [1·4-2·3] in group 2 and 4·3 years [0·8-7·8] in group 3; p=0·019). Patients with group 3 tumours had the highest incidence of metastases at diagnosis (no distant metastasis:metastasis ratio 0·90 for group 3 vs 2·80 for group 1 and 5·67 for group 2; p=0·037). INTERPRETATION LIN28 and OLIG2 are promising diagnostic and prognostic molecular markers for CNS PNET that warrant further assessment in prospective clinical trials. FUNDING Canadian Institute of Health Research, Brainchild/SickKids Foundation, and the Samantha Dickson Brain Tumour Trust.

Germ-line and somatic DICER1 mutations in pineoblastoma

Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

Sixteen new lung function signals identified through 1000 Genomes Project reference panel imputation.

Lung function measures are used in the diagnosis of chronic obstructive pulmonary disease. In 38,199 European ancestry individuals, we studied genome-wide association of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and FEV1/FVC with 1000 Genomes Project (phase 1)-imputed genotypes and followed up top associations in 54,550 Europeans. We identify 14 novel loci (P<5 × 10−8) in or near ENSA, RNU5F-1, KCNS3, AK097794, ASTN2, LHX3, CCDC91, TBX3, TRIP11, RIN3, TEKT5, LTBP4, MN1 and AP1S2, and two novel signals at known loci NPNT and GPR126, providing a basis for new understanding of the genetic determinants of these traits and pulmonary diseases in which they are altered.

An investigation of WNT pathway activation and association with survival in central nervous system primitive neuroectodermal tumours (CNS PNET).

Central nervous system primitive neuroectodermal tumours (CNS PNET) are high-grade, predominantly paediatric, brain tumours. Previously they have been grouped with medulloblastomas owing to their histological similarities. The WNT/β-catenin pathway has been implicated in many tumour types, including medulloblastoma. On pathway activation β-catenin (CTNNB1) translocates to the nucleus, where it induces transcription of target genes. It is commonly upregulated in tumours by mutations in the key pathway components APC and CTNNB1. WNT/β-catenin pathway status was investigated by immunohistochemical analysis of CTNNB1 and the pathway target cyclin D1 (CCND1) in 49 CNS PNETs and 46 medulloblastomas. The mutational status of APC and CTNNB1 (β-catenin) was investigated in 33 CNS PNETs and 22 medulloblastomas. CTNNB1 nuclear localisation was seen in 36% of CNS PNETs and 27% of medulloblastomas. A significant correlation was found between CTNNB1 nuclear localisation and CCND1 levels. Mutations in CTNNB1 were identified in 4% of CNS PNETs and 20% of medulloblastomas. No mutations were identified in APC. A potential link between the level of nuclear staining and a better prognosis was identified in the CNS PNETs, suggesting that the extent of pathway activation is linked to outcome. The results suggest that the WNT/β-catenin pathway plays an important role in the pathogenesis of CNS PNETs. However, activation is not caused by mutations in CTNNB1 or APC in the majority of CNS PNET cases.

GSTCD and INTS12 regulation and expression in the human lung.

Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C-terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFβ1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD.

Loss of INI1 Protein Expression Defines a Subgroup of Aggressive Central Nervous System Primitive Neuroectodermal Tumors

Pediatric embryonal brain tumors can be difficult to classify. Atypical teratoid rhabdoid tumors (ATRT) contain rhabdoid cells, while primitive neuroectodermal tumors (PNETs) are composed of “small round blue cells.” Loss of INI1 is a common event in ATRT; therefore, we investigated if the loss of INI1 protein expression was also observed in central nervous system (CNS) PNET and pineoblastoma. A histological review of 42 CNS PNETs and six pineoblastomas was performed. INI1 expression was assessed by immunohistochemistry. Sequencing was performed on the mutational hotspots of INI1. INI1‐immunonegative tumors were further investigated using fluorescence in situ hybridization. Epithelial membrane antigen (EMA) protein expression was assessed in six CNS PNETs to further define the phenotype. Five CNS PNETs without rhabdoid cell morphology were immuno‐negative for both INI1 and EMA. Of these primary CNS PNET patients, three died <11 months postdiagnosis, which was dissimilar to the INI1‐immunopositive primary CNS PNETs where 18/24 (75%) patients were alive 1 year postdiagnosis. We have identified a small subgroup of CNS PNETs which lack INI1 protein expression, but have no evidence of rhabdoid cell morphology. INI1 protein loss may occur through mechanisms other than gene deletion. INI1 immunohistochemistry should be performed for all CNS PNET cases.

Histologically-defined central nervous system primitive neuro-ectodermal tumours (CNS-PNETs) display heterogeneous DNA methylation profiles and show relationships to other paediatric brain tumour types

To the editors: Central nervous system primitive neuro-ectodermal tumours (CNS-PNETs) are a group of rare childhood embryonal brain tumours associated with a poor prognosis (approximately 50% overall survival) and defined by a common histology according to the current consensus World Health Organisation (WHO) classification [7]. CNS-PNETs occur supratentorially and are defined by histological features shared with cerebellar PNETs (termed medulloblastomas), however the histological classification of CNS-PNET can be challenging. Individual CNS-PNETs are often reclassified as other paediatric supratentorial tumour groups, including anaplastic astrocytoma, atypical teratoid rhabdoid tumour (ATRT), anaplastic oligodendroglioma and anaplastic ependymoma, following central immunophenotypic and histological review [3, 12]. Initial studies have shown that substantial molecular heterogeneity exists within CNS-PNETs; molecular features characteristic of other cerebral brain tumour types (e.g. IDH1 mutation, CDKN2A deletion) have been detected in subsets, but unifying genomic defects have not yet been reported [4, 8, 10, 11]. The recent definition of embryonal tumours with abundant neuropil and true rosettes (ETANTR) as a discrete tumour entity occurring in very young children within the CNS-PNET group - characterised by focal amplification of 19q13.42 and dismal outcome [5, 6, 9] - suggests the existence of currently unrecognised molecular pathological variants, and a refined understanding of CNS-PNET biology could lead to their improved subclassification and the subsequent development of directed therapies. We and others have recently demonstrated the utility of DNA methylation profiling for the discovery and distinction of clinical and molecular sub-classes of brain tumour types including medulloblastomas, gliomas and ependymomas [13-15]. To investigate the potential of DNA methylation profiles to enhance the molecular classification of CNS-PNETs, we assessed 1505 CpG residues across 807 genes in a series of 29 archival CNS-PNETs using established methods [14], alongside assessment of clinical and molecular characteristics (Figure 1h). All biopsies underwent central neuropathological review according to WHO criteria [7] by a three pathologist panel (TSJ, KR and JL). Tumours representing ETANTRs, CNS-PNETs with significant glial (GFAP) or neuronal (synaptophysin) differentiation, and SMARCB1/INI1-negative tumours (by immunohistochemistry (IHC)), were excluded and not assessed, thus defining a study population of morphologically homogeneous CNS-PNETs for analysis (Figure 1a). Finally, DNA methylation profiles from 136 further paediatric brain tumours were generated contemporaneously and assessed in comparison. These included medulloblastomas of defined molecular subgroup (n=60; 15 representative examples each from the WNT (MBWNT), SHH (MBSHH), Group 3 (MBGroup3) and Group 4 (MBGroup4) [14]), alongside ependymomas (n=61; 45 posterior fossa (16 anaplastic, 29 classic; median age at diagnosis, 2.8 years), 16 supratentorial (9 anaplastic, 7 classic; median age, 6.9 years)) and cerebral high-grade gliomas (pHGG; n=15; 12 glioblastoma multiforme, 3 anaplastic astrocytoma; median age 7.1 years) with histology confirmed by central histological review (by WHO criteria [7]). Figure 1 Consensus clustering of CNS-PNETs with other molecularly and histologically-defined paediatric brain tumours does not identify a discrete CNS-PNET tumour subgroup We first undertook unsupervised clustering of the CNS-PNET tumour group based on their DNA methylation patterns using non-negative matrix factorisation (NMF [2, 14]). Three sub-groups produced the most consistent consensus clustering; the majority of tumours clustered confidently into a single large group (21/29), while the two smaller remaining groups (n≤5) were less well defined (Figure 1b). Next, we sought to compare the DNA methylation patterns observed for CNS-PNETs with those of the seven other paediatric brain tumour groups with available data. Prior to the addition of CNS-PNETs into our analysis, these tumours formed seven groups as expected (Figure 1c,d,f), representing discrete confidently-defined (average silhouette width, 0.82) groups of MBWNT, MBSHH, MBGroup3 and MBGroup4, posterior-fossa ependymomas and pHGG tumours, and a mixed tumour group containing all (n=16) supratentorial ependymomas alongside some posterior fossa ependymomas (n=9) and pHGGs (n=3). Whilst the inclusion of CNS-PNETs in the analysis yielded 8 optimal clusters (Figure 1c,e,f), the overall quality of these clusters was reduced (average silhouette width, 0.69) and CNS-PNETs did not form a single discrete group; indeed, CNS-PNETs clustered into six of the different tumour groups observed (Figure 1e,f), showing closer similarities to the other clinically and molecularly-defined paediatric brain tumour groups investigated than to each other. Finally, we made an initial assessment of relationships between the clustered CNS-PNETs and other clinical and molecular disease features (Figure 1g,h). Although numbers were limited, TP53 nuclear stabilisation was common and detected in most clusters, while TP53 mutation and MYCN amplification were rare. Most notably, both IDH1 mutations were exclusively detected in pHGG-like CNS-PNETs arising in adults [4], although no pHGG-characteristic HIST1H3B or H3F3A hotspot mutations were observed [15]. The single WNT pathway-activating CTNNB1 mutation was detected in a MBWNT-like CNS-PNET tumour. No relationships to clinical or pathological disease features were observed in this cohort (Figure 1h). In summary, our data show that despite a defining histological homogeneity using current diagnostic criteria, CNS-PNETs display highly heterogeneous DNA methylation patterns which are more commonly related to other paediatric brain tumour types than to each other. These initial findings raise important issues in the classification of CNS-PNETs and indicate their current clinical definition and grouping by common ‘PNET’ histology [7], and treatment using uniform therapeutic approaches, does not adequately address their underlying biological and clinical complexity. Moreover, our data suggest the potential of refined molecular sub-classification for the improved diagnosis and discrimination of CNS-PNET molecular variants, and to support molecularly-directed clinical trials across tumour types defined currently by clinical and pathological criteria. Despite the modest resolution of our platform, robust discrimination of recognised non-CNS-PNET tumour groups was achieved, both supporting these conclusions and highlighting the potential benefits of higher-resolution molecular investigations in expanded cohorts to validate and extend our findings. The variable DNA methylation patterns observed in CNS-PNETs are likely to represent complex factors, including cellular and developmental origins and ‘driver’ events in tumourigenesis [1]. The collection of snap-frozen tumour cohorts will now be essential to support comprehensive integrated genomic/epigenomic investigations, and comparison with transcriptomic features [11], which were not tractable in our current archival cohort. Finally, understanding the biological significance of epigenetic events in CNS-PNET and related tumour types could lead to the development of novel and/or targeted approaches for the improved therapy of these tumours.

ECM crosslinking enhances fibroblast growth and protects against matrix proteolysis in lung fibrosis

&NA; Idiopathic pulmonary fibrosis (IPF) is characterized by accumulation of extracellular matrix (ECM) proteins and fibroblast proliferation. ECM cross‐linking enzymes have been implicated in fibrotic diseases, and we hypothesized that the ECM in IPF is abnormally cross‐linked, which enhances fibroblast growth and resistance to normal ECM turnover. We used a combination of in vitro ECM preparations and in vivo assays to examine the expression of cross‐linking enzymes and the effect of their inhibitors on fibroblast growth and ECM turnover. Lysyl oxidase‐like 1 (LOXL1), LOXL2, LOXL3, and LOXL4 were expressed equally in control and IPF‐derived fibroblasts. Transglutaminase 2 was more strongly expressed in IPF fibroblasts. LOXL2‐, transglutaminase 2‐, and transglutaminase‐generated cross‐links were strongly expressed in IPF lung tissue. Fibroblasts grown on IPF ECM had higher LOXL3 protein expression and transglutaminase activity than those grown on control ECM. IPF‐derived ECM also enhanced fibroblast adhesion and proliferation compared with control ECM. Inhibition of lysyl oxidase and transglutaminase activity during ECM formation affected ECM structure as visualized by electron microscopy, and it reduced the enhanced fibroblast adhesion and proliferation of IPF ECM to control levels. Inhibition of transglutaminase, but not of lysyl oxidase, activity enhanced the turnover of ECM in vitro. In bleomycin‐treated mice, during the postinflammatory fibrotic phase, inhibition of transglutaminases was associated with a reduction in whole‐lung collagen. Our findings suggest that the ECM in IPF may enhance pathological cross‐linking, which contributes to increased fibroblast growth and resistance to normal ECM turnover to drive lung fibrosis.