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1.  Signatures of mutational processes in human cancer 
Alexandrov, Ludmil B. | Nik-Zainal, Serena | Wedge, David C. | Aparicio, Samuel A.J.R. | Behjati, Sam | Biankin, Andrew V. | Bignell, Graham R. | Bolli, Niccolo | Borg, Ake | Børresen-Dale, Anne-Lise | Boyault, Sandrine | Burkhardt, Birgit | Butler, Adam P. | Caldas, Carlos | Davies, Helen R. | Desmedt, Christine | Eils, Roland | Eyfjörd, Jórunn Erla | Foekens, John A. | Greaves, Mel | Hosoda, Fumie | Hutter, Barbara | Ilicic, Tomislav | Imbeaud, Sandrine | Imielinsk, Marcin | Jäger, Natalie | Jones, David T.W. | Jones, David | Knappskog, Stian | Kool, Marcel | Lakhani, Sunil R. | López-Otín, Carlos | Martin, Sancha | Munshi, Nikhil C. | Nakamura, Hiromi | Northcott, Paul A. | Pajic, Marina | Papaemmanuil, Elli | Paradiso, Angelo | Pearson, John V. | Puente, Xose S. | Raine, Keiran | Ramakrishna, Manasa | Richardson, Andrea L. | Richter, Julia | Rosenstiel, Philip | Schlesner, Matthias | Schumacher, Ton N. | Span, Paul N. | Teague, Jon W. | Totoki, Yasushi | Tutt, Andrew N.J. | Valdés-Mas, Rafael | van Buuren, Marit M. | van ’t Veer, Laura | Vincent-Salomon, Anne | Waddell, Nicola | Yates, Lucy R. | Zucman-Rossi, Jessica | Futreal, P. Andrew | McDermott, Ultan | Lichter, Peter | Meyerson, Matthew | Grimmond, Sean M. | Siebert, Reiner | Campo, Elías | Shibata, Tatsuhiro | Pfister, Stefan M. | Campbell, Peter J. | Stratton, Michael R.
Nature  2013;500(7463):415-421.
All cancers are caused by somatic mutations. However, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here, we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, kataegis, is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer with potential implications for understanding of cancer etiology, prevention and therapy.
doi:10.1038/nature12477
PMCID: PMC3776390  PMID: 23945592
2.  The Affordable Care Act and Stroke 
doi:10.1161/STROKEAHA.114.005315
PMCID: PMC4116441  PMID: 24984748
Medicaid; Medicare; access to care; stroke; stroke in young adults
3.  Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma 
Northcott, Paul A | Lee, Catherine | Zichner, Thomas | Stütz, Adrian M | Erkek, Serap | Kawauchi, Daisuke | Shih, David JH | Hovestadt, Volker | Zapatka, Marc | Sturm, Dominik | Jones, David TW | Kool, Marcel | Remke, Marc | Cavalli, Florence | Zuyderduyn, Scott | Bader, Gary | VandenBerg, Scott | Esparza, Lourdes Adriana | Ryzhova, Marina | Wang, Wei | Wittmann, Andrea | Stark, Sebastian | Sieber, Laura | Seker-Cin, Huriye | Linke, Linda | Kratochwil, Fabian | Jäger, Natalie | Buchhalter, Ivo | Imbusch, Charles D | Zipprich, Gideon | Raeder, Benjamin | Schmidt, Sabine | Diessl, Nicolle | Wolf, Stephan | Wiemann, Stefan | Brors, Benedikt | Lawerenz, Chris | Eils, Jürgen | Warnatz, Hans-Jörg | Risch, Thomas | Yaspo, Marie-Laure | Weber, Ursula D | Bartholomae, Cynthia C | von Kalle, Christof | Turányi, Eszter | Hauser, Peter | Sanden, Emma | Darabi, Anna | Siesjö, Peter | Sterba, Jaroslav | Zitterbart, Karel | Sumerauer, David | van Sluis, Peter | Versteeg, Rogier | Volckmann, Richard | Koster, Jan | Schuhmann, Martin U | Ebinger, Martin | Grimes, H. Leighton | Robinson, Giles W | Gajjar, Amar | Mynarek, Martin | von Hoff, Katja | Rutkowski, Stefan | Pietsch, Torsten | Scheurlen, Wolfram | Felsberg, Jörg | Reifenberger, Guido | Kulozik, Andreas E | von Deimlmg, Andreas | Witt, Olaf | Eils, Roland | Gilbertson, Richard J | Korshunov, Andrey | Taylor, Michael D | Lichter, Peter | Korbel, Jan O | Wechsler-Reya, Robert J | Pfister, Stefan M
Nature  2014;511(7510):428-434.
Summary Paragraph
Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation, and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoural heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and Group 4 subgroup medulloblastomas account for the majority of paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to Groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family protooncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1/GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate ‘enhancer hijacking’ as an efficient mechanism driving oncogene activation in a childhood cancer.
doi:10.1038/nature13379
PMCID: PMC4201514  PMID: 25043047
4.  Genome Sequencing of SHH Medulloblastoma Predicts Genotype-Related Response to Smoothened Inhibition 
Kool, Marcel | Jones, David T.W. | Jäger, Natalie | Northcott, Paul A. | Pugh, Trevor J. | Hovestadt, Volker | Piro, Rosario M. | Esparza, L. Adriana | Markant, Shirley L. | Remke, Marc | Milde, Till | Bourdeaut, Franck | Ryzhova, Marina | Sturm, Dominik | Pfaff, Elke | Stark, Sebastian | Hutter, Sonja | Şeker-Cin, Huriye | Johann, Pascal | Bender, Sebastian | Schmidt, Christin | Rausch, Tobias | Shih, David | Reimand, Jüri | Sieber, Laura | Wittmann, Andrea | Linke, Linda | Witt, Hendrik | Weber, Ursula D. | Zapatka, Marc | König, Rainer | Beroukhim, Rameen | Bergthold, Guillaume | van Sluis, Peter | Volckmann, Richard | Koster, Jan | Versteeg, Rogier | Schmidt, Sabine | Wolf, Stephan | Lawerenz, Chris | Bartholomae, Cynthia C. | von Kalle, Christof | Unterberg, Andreas | Herold-Mende, Christel | Hofer, Silvia | Kulozik, Andreas E. | von Deimling, Andreas | Scheurlen, Wolfram | Felsberg, Jörg | Reifenberger, Guido | Hasselblatt, Martin | Crawford, John R. | Grant, Gerald A. | Jabado, Nada | Perry, Arie | Cowdrey, Cynthia | Croul, Sydney | Zadeh, Gelareh | Korbel, Jan O. | Doz, Francois | Delattre, Olivier | Bader, Gary D. | McCabe, Martin G. | Collins, V. Peter | Kieran, Mark W. | Cho, Yoon-Jae | Pomeroy, Scott L. | Witt, Olaf | Brors, Benedikt | Taylor, Michael D. | Schüller, Ulrich | Korshunov, Andrey | Eils, Roland | Wechsler-Reya, Robert J. | Lichter, Peter | Pfister, Stefan M.
Cancer cell  2014;25(3):393-405.
Summary
Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.
doi:10.1016/j.ccr.2014.02.004
PMCID: PMC4493053  PMID: 24651015
5.  Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling 
Nature Communications  2015;6:7391.
In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.
Gene transfer is a powerful technique to investigate the mechanistic basis of tumorigenesis. Here Zuckermann et al. adapt CRISPR/Cas9 genome editing to target potential oncogenes somatically in vivo, establishing a fast and convenient system for validating novel genetic candidates.
doi:10.1038/ncomms8391
PMCID: PMC4467376  PMID: 26067104
6.  NOVEL MUTATIONS WIDEN THE PHENOTYPIC SPECTRUM OF SLOW SKELETAL/β-CARDIAC MYOSIN (MYH7) DISTAL MYOPATHY 
Human mutation  2014;35(7):868-879.
Laing early onset distal myopathy and myosin storage myopathy are caused by mutations of slow skeletal/β-cardiac myosin heavy chain encoded by the gene MYH7, as is a common form of familial hypertrophic/dilated cardiomyopathy. The mechanisms by which different phenotypes are produced by mutations in MYH7, even in the same region of the gene, are not known. To explore the clinical spectrum and pathobiology we screened the MYH7 gene in 88 patients from 21 previously unpublished families presenting with distal or generalised skeletal muscle weakness, with or without cardiac involvement. Twelve novel mutations have been identified in thirteen families. In one of these families the grandfather of the proband was found to be a mosaic for the MYH7 mutation. In eight cases de novo mutation appeared to have occurred, which was proven in three. The presenting complaint was footdrop, sometimes leading to delayed walking or tripping, in members of 17 families (81%), with other presentations including cardiomyopathy in infancy, generalised floppiness and scoliosis. Cardiac involvement as well as skeletal muscle weakness was identified in 9 of 21 families. Spinal involvement such as scoliosis or rigidity was identified in 12 (57%). This report widens the clinical and pathological phenotypes, and the genetics of MYH7 mutations leading to skeletal muscle diseases.
doi:10.1002/humu.22553
PMCID: PMC4112555  PMID: 24664454
MYH7; Laing distal myopathy; MPD1
7.  NEXT-GENERATION NEUROPATHOLOGY - IMPROVING DIAGNOSTIC ACCURACY FOR BRAIN TUMORS USING DNA METHYLATION ARRAY-BASED MOLECULAR PROFILING 
Neuro-Oncology  2014;16(Suppl 3):iii4.
BACKGROUND: The current World Health Organisation (WHO) classification of central nervous system tumors comprises over 100 entities. Most of these are defined by purely histological criteria, with varying and sometimes overlapping spectra. Histological diagnosis is often challenging, however, especially in cases with limited or non-representative biopsy material. Thus, molecular technologies that can complement standard pathology testing have the potential to greatly enhance diagnostic precision and improve clinical decision-making. DNA methylation profiling, acting as a 'fingerprint' of cellular origin and molecular alterations, is one such promising technology. METHODS: We have assembled a reference dataset of more than 2,000 methylation profiles using the Illumina HumanMethylation450 (450k) array, currently representing over 50 brain tumor entities or subgroups. The array platform is suitable for both frozen and paraffin-embedded material, with minimal DNA input required. Each new diagnostic case receives an entity prediction with an associated probability score as a confidence measure. Genome-wide copy number profiles (e.g. for scoring 1p/19q loss or gene amplifications) and target gene methylation data (e.g. MGMT) generated from the array provide important additional information. RESULTS: In addition to the reference cohort, more than 500 diagnostic samples from Heidelberg University Hospital and external institutions have been processed. Approximately 5-10% of cases displayed a discrepancy between histological and molecular diagnoses. Careful re-examination of these often resulted in refinement of the original diagnosis, and improved patient care.Furthermore, samples collected for the reference cohort have led to significant improvements in our understanding of the biology of several tumor types, including the identification of further subgroups for several entities and associations with recurrent copy number changes and/or mutations. CONCLUSIONS: Our understanding of the molecular alterations underlying brain tumors has grown enormously in recent years, and it is crucial that this is translated into the clinic promptly. DNA methylation profiling is one tool with the potential to become an important part of the diagnostic armoury of neuropathologists. This relatively inexpensive and robust method is well suited to complement standard histopathologic techniques and improve diagnostic accuracy, thereby optimising patient management. We are currently expanding our pipeline to include additional diagnostic centres, allowing for further refinement and validation as well as broader international access. SECONDARY CATEGORY: Tumor Biology.
doi:10.1093/neuonc/nou206.13
PMCID: PMC4144477
8.  PROGNOSTIC AND PREDICTIVE BIOMARKER-BASED SUBGROUPS IN THE NOA-04 TRIAL 
Neuro-Oncology  2014;16(Suppl 3):iii4.
BACKGROUND: The WHO classification, based on morphological criteria, may be increasingly supplemented with defined molecular aberrations. These might help to resolve the discrepancy between classification and clinical outcome. Molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, mutations and (consequently) loss of expression of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, improve prognostication and may even guide treatment decisions in patients with anaplastic gliomas. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) allow the determination of large-scale methylation profiles and genome-wide DNA copy number changes, enabling molecular subgrouping of tumors. In addition, algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion and MGMT promoter methylation using this assay. METHODS: In the biomarker cohort of the NOA-04 trial, the diagnostic and prognostic performance of these molecular markers using single tests, HM450 data and HM450-based algorithms has been investigated to propose biological subgroups, which reflect outcomes and potentially influence treatment decisions. RESULTS: Loss of ATRX expression was detected in 45% of anaplastic astrocytomas (AA), 27% of anaplastic oligoastrocytomas (AOA) and 10% of anaplastic oligodendrogliomas (AO). It was mostly restricted to IDH mutant tumors and almost mutually exclusive with 1p/19q co-deletion. In tumors with IDH1 mutation, MGMT promoter methylation was associated with prolonged progression-free survival (PFS) with chemotherapy or radiotherapy (RT), and thus prognostic. In tumors without IDH1 mutation, MGMT promoter methylation was associated with increased PFS in patients treated with chemotherapy, too, but not in those who received RT alone as the first-line treatment, and is thus chemotherapy-predictive. Comparisons of single assays and HM450-based algorithms revealed a high concordance for IDH and 1p/19q status. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of cases as conventional methylation-specific PCR, with 87/91 G-CIMP-positive tumors predicted as MGMT promoter-methylated. CONCLUSIONS: ATRX loss is a hallmark and favorable prognosticator of astrocytic tumors allowing a better definition of the clinically and morphologically mixed group of AOA. MGMT promoter methylation is a predictive biomarker for benefit from alkylating agent chemotherapy in patients with IDH1-wildtype, but not IDH1-mutant malignant gliomas of WHO grades III/IV. Combined IDH1/ MGMT assessment may help to individualize clinical decision making in neurooncology. G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and associated with prognosis in the NOA-04 trial. HM450 arrays allowed clustering of anaplastic gliomas into relevant subgroups. SECONDARY CATEGORY: Tumor Biology.
doi:10.1093/neuonc/nou206.14
PMCID: PMC4144478
9.  MOLECULAR (RE-)CLASSIFICATION OF CNS-PRIMITIVE NEUROECTODERMAL TUMORS 
Neuro-Oncology  2014;16(Suppl 3):iii23.
BACKGROUND: According to the current WHO classification of CNS tumors, childhood CNS primitive neuro-ectodermal tumors (CNS-PNETs; WHO °IV) are poorly differentiated embryonal tumors with early onset and aggressive clinical behavior. Histological diagnosis can be complicated by morphological heterogeneity and divergent differentiation. Recent studies suggest the existence of molecular subgroups of CNS-PNETs sharing biological characteristics with other childhood CNS tumors. Here, we aimed at a comprehensive molecular characterization of CNS-PNETs and compared our results to profiles of other brain tumor entities in order to define the biological nature of tumors diagnosed as CNS-PNETs. METHODS: A collection of 211 fresh-frozen or paraffin-embedded tumor samples with an institutional diagnosis “CNS-PNET” was profiled for genome-wide DNA methylation patterns and copy-number alterations, complemented by transcriptomic profiling of a subset (n = 71). (Epi-)genetic profiles of CNS-PNETs were compared to those of >3000 other childhood brain tumors including embryonal, astrocytic, and ependymal entities, and their respective molecular subgroups. We screened selected groups of tumors for recurrent mutations and expression of established molecular markers. RESULTS: DNA methylation and gene expression profiles showed a clear segregation of pediatric brain tumors by histological entities and molecular subgroups. Interestingly, CNS-PNET profiles showed a significant overlap with various well-defined entities, including AT/RT, ETMR, high-grade glioma, medulloblastoma, and ependymoma. When screening CNS-PNETs with profiles highly resembling other entities, hallmark genetic alterations of these, such as amplification of 19q13.42, mutations in IDH1 or H3F3A, or mutations/deletions of the SMARCB1 locus, were frequently detected. Also, established protein markers, such as INI-1, LIN28A, and OLIG2, confirmed the reclassification of these CNS-PNETs. Strikingly, a subset (∼25%) of CNS-PNETs which could not be reclassified segregated into 3-4 distinct molecular subgroups, each with its own characteristic pattern of copy-number aberrations, DNA-methylation and gene expression. Currently, whole genome and RNA-sequencing of these distinct subgroups of CNS-PNETs is ongoing to reveal their underlying genetics. CONCLUSIONS: The correct classification of CNS-PNETs remains challenging. Based on the detection of recurrent genetic aberrations, many cases can be reliably re-classified, indicating that a significant proportion of CNS-PNETs may comprise a variety of other tumor subtypes. These findings suggest that the use of established and novel subgroups markers is needed in order to assist the histopathological evaluation of these tumors. In addition, we have identified a number of true CNS-PNET subtypes and are currently analyzing them in more detail in order to elucidate the genetics of these distinct groups. SECONDARY CATEGORY: Tumor Biology.
doi:10.1093/neuonc/nou208.3
PMCID: PMC4144575
10.  INFORMING TREATMENT DECISIONS IN RELAPSED PEDIATRIC MALIGNANCIES USING NEXT-GENERATION DIAGNOSTICS 
Neuro-Oncology  2014;16(Suppl 3):iii24.
BACKGROUND: Although childhood malignancies have become curable in about 75% of cases due to empirically developed multi-modal therapeutic concepts applied in nation-wide collaborative trials, for children with a relapse, cure remains the exception. In the framework of the ICGC project PedBrain many new potentially druggable genetic lesions have been identified. However, it will not be feasible to conduct traditional phase I trials for all these new drugs in these overall rare entities. To still have our young patients participating in the recent advances in molecular targeted drug treatment, we initiated a novel innovative way of introducing these drugs in a clinical setting based on an individualized molecular rationale, a concept called INFORM (INdividualized therapy For Relapsed Malignancies in childhood). METHODS: Exome- and low-coverage whole-genome sequencing, RNA sequencing, gene expression profiling, DNA methylation profiling, bioinformatic prediction of drug targets and compound selection are carried out aiming at a turnaround time of 4 weeks or less after re-biopsy of the tumor. In the current feasibility phase, drug targets and prioritization are offered to the treating physician in the local hospital for individual treatment decisions. RESULTS: Ten patients were recruited to the INFORM-pilot study by now. For all but two, druggable targets have been identified. The first two patients with early follow-up MRIs after 6 weeks had stable disease (medulloblastoma) and 50% tumor volume reduction (myofibroblastc tumor), respectively. In an additional case with pontine glioma, molecular diagnostics significantly contributed to the establishment of an unambiguous diagnosis. CONCLUSIONS: This is the first population-based study using next-generation sequencing technologies to guide treatment decisions in a clinical setting. In addition to this advance in “next-generation” clinical oncology, INFORM will also reveal the largest comprehensive molecular datasets of relapsed tumors to date, and since primary tumor material from the same patient will also be analyzed whenever available, will likely identify key biological properties of relapsing malignancies and recurrent mechanisms of drug resistance across entities. SECONDARY CATEGORY: Pediatrics.
doi:10.1093/neuonc/nou208.6
PMCID: PMC4144608
11.  PROGNOSTIC SIGNIFICANCE OF CLINICAL, HISTOPATHOLOGICAL, AND MOLECULAR CHARACTERISTICS OF MEDULLOBLASTOMAS IN THE PROSPECTIVE HIT2000 MULTICENTER CLINICAL TRIAL COHORT 
Neuro-Oncology  2014;16(Suppl 3):iii24-iii25.
BACKGROUND: This study aimed to prospectively evaluate clinical, histopathological and molecular variables for outcome prediction in medulloblastoma patients. METHODS: Patients from the HIT2000 cooperative clinical trial were prospectively enrolled based on the availability of sufficient tumor material and complete clinical information. This revealed a cohort of 184 patients (median age 7.6 years), which was randomly split at a 2:1 ratio into a training (n = 127), and a validation (n = 57) dataset. All samples were subjected to thorough histopathological investigation, CTNNB1 mutation analysis, quantitative PCR, MLPA and FISH analyses for cytogenetic variables, and methylome analysis. RESULTS: By univariable analysis, clinical factors (M-stage), histopathological variables (large cell component, endothelial proliferation, synaptophysin pattern), and molecular features (chromosome 6q status, MYC amplification, TOP2A copy-number, subgrouping) were found to be prognostic. Molecular consensus subgrouping (WNT, SHH, Group 3, Group 4) was validated as an independent feature to stratify patients into different risk groups. When comparing methods for the identification of WNT-driven medulloblastoma, this study identified CTNNB1 sequencing and methylation profiling to most reliably identify these patients. After removing patients with particularly favorable (CTNNB1 mutation, extensive nodularity) or unfavorable (MYC amplification) markers, a risk score for the remaining “intermediate molecular risk” population dependent on age, M-stage, pattern of synaptophysin expression, and MYCN copy-number status was identified and validated, with speckled synaptophysin expression indicating worse outcome. CONCLUSIONS: Methylation subgrouping and CTNNB1 mutation status represent robust tools for the risk-stratification of medulloblastoma. A simple clinico-pathological risk score for “intermediate molecular risk” patients was identified, which deserves further validation. SECONDARY CATEGORY: Pediatrics.
doi:10.1093/neuonc/nou208.7
PMCID: PMC4144619
12.  Loss of BRMS1 Promotes a Mesenchymal Phenotype through NF-κB-Dependent Regulation of Twist1 
Molecular and Cellular Biology  2014;35(1):303-317.
Breast cancer metastasis suppressor 1 (BRMS1) is downregulated in non-small cell lung cancer (NSCLC), and its reduction correlates with disease progression. Herein, we investigate the mechanisms through which loss of the BRMS1 gene contributes to epithelial-to-mesenchymal transition (EMT). Using a short hairpin RNA (shRNA) system, we show that loss of BRMS1 promotes basal and transforming growth factor beta-induced EMT in NSCLC cells. NSCLC cells expressing BRMS1 shRNAs (BRMS1 knockdown [BRMS1KD]) display mesenchymal characteristics, including enhanced cell migration and differential regulation of the EMT markers. Mesenchymal phenotypes observed in BRMS1KD cells are dependent on RelA/p65, the transcriptionally active subunit of nuclear factor kappa B (NF-κB). In addition, chromatin immunoprecipitation analysis demonstrates that loss of BRMS1 increases Twist1 promoter occupancy of RelA/p65 K310—a key histone modification associated with increased transcription. Knockdown of Twist1 results in reversal of BRMS1KD-mediated EMT phenotypic changes. Moreover, in our animal model, BRMS1KD/Twist1KD double knockdown cells were less efficient in establishing lung tumors than BRMS1KD cells. Collectively, this study demonstrates that loss of BRMS1 promotes malignant phenotypes that are dependent on NF-κB-dependent regulation of Twist1. These observations offer fresh insight into the mechanisms through which BRMS1 regulates the development of metastases in NSCLC.
doi:10.1128/MCB.00869-14
PMCID: PMC4295387  PMID: 25368381
13.  The Basal Transcription Complex Component TAF3 Transduces Changes in Nuclear Phosphoinositides into Transcriptional Output 
Molecular Cell  2015;58(3):453-467.
Summary
Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.
Graphical Abstract
Highlights
•PIP4K2B regulates nuclear PI5P and myogenic gene expression during differentiation•A screen identifies 17 of 32 PHD fingers interacting with phosphoinositides•The basal transcription component TAF3 interacts strongly with phosphoinositides•TAF3 transduces changes in nuclear phosphoinositides into transcriptional output
Bultsma et al. show that the basal transcriptional complex protein TAF3 directly binds phosphoinositides and transduces changes in nuclear phosphoinositides into differential transcriptional output that affects myoblast differentiation. The lipid kinase PIP4K2B, phosphoinositides, and TAF3 form a conserved nuclear signaling pathway that selectively regulates transcription.
doi:10.1016/j.molcel.2015.03.009
PMCID: PMC4429956  PMID: 25866244
14.  Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity 
Science translational medicine  2014;6(261):261ra151.
Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intra-pleurally administered CAR T cells vastly out-performed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. Following intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced anti-tumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4+ T cell activation associated with a higher intra-tumoral CD4/CD8 cell ratios and CD28-dependent CD4+ T cell-mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication or persistence. The remarkable ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through “regional distribution centers.” Based on these results, we are opening a phase I clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies.
doi:10.1126/scitranslmed.3010162
PMCID: PMC4373413  PMID: 25378643
16.  Thirty-Day Mortality Underestimates the Risk of Early Death after Major Resections for Thoracic Malignancies 
The Annals of thoracic surgery  2014;98(5):1769-1775.
Background
Operative mortality rates are of great interest to surgeons, patients, policy makers, and payers as a universal metric for quality assessment. Thirty-day mortality and discharge mortality have been presumed to capture procedure-related mortality. However, many patients die after the 30-day mark or are transferred to other facilities or to home and die there, leading to the underreporting of surgery-related deaths. We hypothesized that a longer period of observation would address these concerns and provide a more accurate measure of operative mortality.
Methods
We performed a retrospective review of prospectively maintained institutional databases of patients undergoing resection for lung cancer, esophageal cancer, and mesothelioma.
Results
From 1999 to 2012, 7646 surgical resections were performed: 6119 for lung cancer, 1258 for esophageal cancer, and 269 for mesothelioma. Among the different cancers and across operations, the additional mortality from day 31 to 90 (1.4% [95% CI, 1.2% to 1.8%]; n=111) was similar to that by day 30 (1.2% [95% CI, 1.0% to 1.5%; n=95), resulting in overall 90-day mortality (2.7% [95% CI, 2.3% to 3.1%; n=206) that was more than double 30-day mortality. Respiratory failure, sepsis, and cardiac events were the leading causes of death after thoracic resection.
Conclusions
Among patients who have undergone surgery for thoracic malignancies, considerable mortality attributable to surgery occurs beyond the first 30 days after surgery, as well as after hospital discharge. As cancer surgery constitutes a large portion of general thoracic surgery, we recommend national databases consider the inclusion of 90-day mortality in their data collection.
doi:10.1016/j.athoracsur.2014.06.024
PMCID: PMC4410352  PMID: 25200731
Mortality; Outcomes; Thoracic; Lung Cancer; Esophageal Cancer; Mesothelioma
17.  Second Primary Lung Cancers: Smokers v. Nonsmokers after Resection of Stage I Lung Adenocarcinoma 
The Annals of thoracic surgery  2014;98(3):968-974.
Purpose
Smokers have a higher risk of developing non-small cell lung cancer (NSCLC) than nonsmokers, but the relative risk of developing second primary lung cancer (SPLC) is unclear. Determining the risk of SPLC in smokers versus nonsmokers after treatment of an initial cancer may help guide recommendations for long-term surveillance.
Methods
Patients who underwent resection for stage I adenocarcinoma were identified from a prospectively maintained institutional database. Patients with other histologies, synchronous lesions, or who received neoadjuvant or adjuvant therapy were excluded. SPLC were identified based on Martini criteria.
Results
From 1995 to 2012, 2151 patients underwent resection for stage I adenocarcinoma (308 never-smokers [14%] and 1843 ever-smokers [86%]). Thirty never-smokers (9.9%) and 145 ever-smokers (7.8%) developed SPLC. SPLC was detected by surveillance computed tomography scan in the majority of patients (161; 92%). In total, 87% of never-smokers and 83% of ever-smokers had stage I SPLC. There was no significant difference in the cumulative incidence of SPLC between never-smokers and ever-smokers (p=0.18) in a competing-risks analysis. The cumulative incidence at 10 years was 20.3% for never-smokers and 18.2% for ever-smokers.
Conclusions
Although smokers have a greater risk of developing NSCLC, the risk of developing a second primary cancer after resection of stage I lung cancer is comparable between smokers and never smokers. The majority of these second primary cancers are detectable at a curable stage. Ongoing postoperative surveillance should be recommended for all patients regardless of smoking status.
doi:10.1016/j.athoracsur.2014.04.098
PMCID: PMC4410355  PMID: 25038021
Metachronous; Lung; Never; Smoking; Recurrences
18.  Position of Totally Thoracoscopic Surgical Ablation in the Treatment of Atrial Fibrillation: An Alternative Method of Conduction Testing 
Recent advances in surgical techniques and understanding of the pathophysiology of atrial fibrillation has led to the development of a less invasive thoracoscopic surgical treatment including video-assisted bilateral pulmonary vein isolation using bipolar radiofrequency ablation clamps. More recently, the same operation became possible via a totally thoracoscopic approach.
In this paper we describe technical aspects of the thoracoscopic approach to surgical treatment of AF and discuss its features, benefits and limitations. Furthermore, we present a new alternative technique of conduction testing using endoscopic multi-electrode recording catheters.
An alternative electrophysiological mapping strategy involves a multi-electrode recording catheter designed primarily for percutaneous endocardial electrophysiologic mapping procedure. According to our initial experience, the recordings obtained from the multi-electrode catheters positioned around the pulmonary veins are more accurate than the recordings obtained from the multifunctional ablation and pacing pen.
The totally thoracoscopic surgical ablation approach is a feasible and efficient treatment strategy for atrial fibrillation. The conduction testing can be easily and rapidly performed using a multifunctional pen or multi-electrode recording catheter.
doi:10.12659/MSMBR.894239
PMCID: PMC4418280  PMID: 25904211
Arrhythmias, Cardiac; Cardiac Surgical Procedures; Endoscopy
19.  The Effectiveness of Different Interventions to Promote Poison Prevention Behaviours in Households with Children: A Network Meta-Analysis 
PLoS ONE  2015;10(4):e0121122.
Background
There is evidence from 2 previous meta-analyses that interventions to promote poison prevention behaviours are effective in increasing a range of poison prevention practices in households with children. The published meta-analyses compared any intervention against a “usual care or no intervention” which potentially limits the usefulness of the analysis to decision makers. We aim to use network meta-analysis to simultaneously evaluate the effectiveness of different interventions to increase prevalence of safe storage of i) Medicines only, ii) Other household products only, iii) Poisons (both medicines and non-medicines), iv) Poisonous plants; and v) Possession of poison control centre (PCC) telephone number in households with children.
Methods
Data on the effectiveness of poison prevention interventions was extracted from primary studies identified in 2 newly-undertaken systematic reviews. Effect estimates were pooled across studies using a random effects network meta-analysis model.
Results
28 of the 47 primary studies identified were included in the analysis. Compared to usual care intervention, the intervention with education and low cost/free equipment elements was most effective in promoting safe storage of medicines (odds ratio 2.51, 95% credible interval 1.01 to 6.00) while interventions with education, low cost/free equipment, home safety inspection and fitting components were most effective in promoting safe storage of other household products (2.52, 1.12 to 7.13), safe storage of poisons (11.10, 1.60 to 141.50) and possession of PCC number (38.82, 2.19 to 687.10). No one intervention package was more effective than the others in promoting safe storage of poisonous plants.
Conclusion
The most effective interventions varied by poison prevention practice, but education alone was not the most effective intervention for any poison prevention practice. Commissioners and providers of poison prevention interventions should tailor the interventions they commission or provide to the poison prevention practices they wish to promote.
Highlights
Network meta-analysis is useful for comparing multiple injury-prevention interventions.
More intensive poison prevention interventions were more effective than education alone.
Education and low cost/free equipment was most effective in promoting safe storage of medicines.
Education, low cost/free equipment, home safety inspection and fitting was most effective in promoting safe storage of household products and poisons.
Education, low cost/free equipment and home inspection were most effective in promoting possession of a poison control centre number.
None of the intervention packages was more effective than the others in promoting safe storage of poisonous plants.
doi:10.1371/journal.pone.0121122
PMCID: PMC4404249  PMID: 25894385
20.  Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster 
Nature structural biology  2003;10(9):694-700.
Summary
We have solved the high-resolution crystal structure of the Drosophila melanogaster alcohol-binding protein LUSH in the complexes it forms with a series of short chain n-alcohols. LUSH is the first known non-enzyme protein with a defined in vivo alcohol-binding function. The structure of LUSH reveals a set of molecular interactions that define a specific alcohol-binding site. A group of amino acids, Thr57, Ser52 and Thr48 form a network of concerted hydrogen bonds between the protein and the alcohol that provide a structural motif to increase alcohol binding affinity at this site. This motif appears to be conserved in a number of mammalian ligand-gated ion channels that are directly implicated in the pharmacological effects of alcohol. Further, these sequences are found in regions of ion-channels that are known to infer alcohol sensitivity. We suggest the alcohol-binding site in LUSH represents a general model for alcohol-binding sites in proteins.
doi:10.1038/nsb960
PMCID: PMC4397894  PMID: 12881720
21.  Activation of Pheromone-Sensitive Neurons Is Mediated by Conformational Activation of Pheromone-Binding Protein 
Cell  2008;133(7):1255-1265.
Summary
Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-biing protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSHD118A, produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSHD118A reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants.
doi:10.1016/j.cell.2008.04.046
PMCID: PMC4397981  PMID: 18585358
22.  Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities 
Biochemistry  2010;49(29):6136-6142.
Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as critical for optimal binding of ethanol. A set of truncated models based on the structure of the LUSH–butanol complex were constructed for the wild-type and mutant (T57S, S52A, and T57A) proteins in complexes with a series of n-alcohols and for the apoprotein bound to water and for the ligand-free protein. Using both gas-phase calculations and continuum solvation model calculations, we found that the widely used DFT model, B3LYP, failed to reproduce the experimentally observed trend of increasing binding affinity with the increasing length of the alkyl chain in the alcohol. In contrast, the recently developed M05-2X DFT model successfully reproduced this subtle trend. Analysis of the results indicated that multiple factors contribute to the differences in alcohol binding affinity: the H-bonding with Thr57 and Ser52 (4–5 kcal/mol per H-bond), the desolvation contribution (4–6 kcal/mol for alcohols and 8–10 kcal/mol for water), and the other noncovalent interaction (1.2 kcal/mol per CH2 group of the alcohol alkyl chain). These results reveal the outstanding potential for using the M05-2X model in calculations of protein–substrate complexes where noncovalent interactions are important.
doi:10.1021/bi100540k
PMCID: PMC4396708  PMID: 20550105
23.  1H, 13C and 15N NMR Assignments of the C1A and C1B Subdomains of PKC-delta 
Biomolecular NMR assignments  2010;5(2):125-129.
The Protein Kinase C family of enzymes is a group of serine/threonine kinases that play central roles in cell-cycle regulation, development and cancer. A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators.
PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα [1, 2], PKCε [3] and PKCδ [4, 5] contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC. Here we present the 1H, 15N and 13C NMR chemical shift assignments for the Rattus norvegicus PKCδ C1A and C1B proteins.
doi:10.1007/s12104-010-9283-0
PMCID: PMC4396712  PMID: 21132404
Protein Kinase C Delta; C1A domain; C1B domain; diacyl glycerol; Phorbol ester binding
24.  Strategic Assessment of Fisheries Independent Monitoring Programs in the Gulf of Mexico 
PLoS ONE  2015;10(4):e0120929.
This study evaluates information produced from 14 fisheries independent monitoring programs (FIM) in the Gulf of Mexico. We consider the uniqueness of information from each program and its usefulness in estimating fisheries management indices. Biomass values of 35 functional groups are extracted from an operating model (Ecospace) with a method that replicates the patterns of historic FIM samplings. Observation error is added to these data in order to create a set of pseudo data that replicate the type and quality of information obtained from FIM programs. The pseudo data were put into a separate fishery assessment model (Pella-Tomlinson) to determine management indices of each functional group (maximum sustainable yield (MSY), biomass at MSY, and fishing mortality at MSY). These indices are compared against values in Ecospace, and against previously published single-species stock assessments. We also evaluate the full suite of information derived from FIM within an ecosystem context, considering whether functional roles are over- or under-sampled, and whether sampling effort is proportional to the value of fish stocks. Results reveal that model derived fishery indices closely matched published indices for the majority of the functional groups, economic and ecological evaluation suggests that several piscivorous functional groups are under-sampled include forage base species that are likely to indirectly support fisheries for piscivores, and sampling efforts are not proportional to the value of some fish stocks. Following ecological modelling we performed statistical analyses on historic FIM catch data to identify optimal species-specific sampling months and gear-types that can be used to refine future FIM sampling efforts.
doi:10.1371/journal.pone.0120929
PMCID: PMC4383601  PMID: 25835742
25.  Abnormalities of AMPK Activation and Glucose Uptake in Cultured Skeletal Muscle Cells from Individuals with Chronic Fatigue Syndrome 
PLoS ONE  2015;10(4):e0122982.
Background
Post exertional muscle fatigue is a key feature in Chronic Fatigue Syndrome (CFS). Abnormalities of skeletal muscle function have been identified in some but not all patients with CFS. To try to limit potential confounders that might contribute to this clinical heterogeneity, we developed a novel in vitro system that allows comparison of AMP kinase (AMPK) activation and metabolic responses to exercise in cultured skeletal muscle cells from CFS patients and control subjects.
Methods
Skeletal muscle cell cultures were established from 10 subjects with CFS and 7 age-matched controls, subjected to electrical pulse stimulation (EPS) for up to 24h and examined for changes associated with exercise.
Results
In the basal state, CFS cultures showed increased myogenin expression but decreased IL6 secretion during differentiation compared with control cultures. Control cultures subjected to 16h EPS showed a significant increase in both AMPK phosphorylation and glucose uptake compared with unstimulated cells. In contrast, CFS cultures showed no increase in AMPK phosphorylation or glucose uptake after 16h EPS. However, glucose uptake remained responsive to insulin in the CFS cells pointing to an exercise-related defect. IL6 secretion in response to EPS was significantly reduced in CFS compared with control cultures at all time points measured.
Conclusion
EPS is an effective model for eliciting muscle contraction and the metabolic changes associated with exercise in cultured skeletal muscle cells. We found four main differences in cultured skeletal muscle cells from subjects with CFS; increased myogenin expression in the basal state, impaired activation of AMPK, impaired stimulation of glucose uptake and diminished release of IL6. The retention of these differences in cultured muscle cells from CFS subjects points to a genetic/epigenetic mechanism, and provides a system to identify novel therapeutic targets.
doi:10.1371/journal.pone.0122982
PMCID: PMC4383615  PMID: 25836975

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