Genetics of vestibular schwannoma : Genetic landscape of irradiated and radiation-naïve benign and malignant vestibular schwannoma

Background: Vestibular schwannoma (VS) is a benign intracranial neoplasm associated with reduced quality of life. Malignant peripheral nerve sheath tumor of the vestibular nerve (VN-MPNST) is the malignant counterpart, an exceedingly rare cancer associated with high mortality. The genetics underlying VS and its etiology is not well understood and the genome of irradiated VS and VN-MPNST has not been characterized. We addressed these shortcomings in this thesis. Material and methods: Tumor specimens from the Bergen neurosurgical tissue bank were subjected to a combination of whole-exome sequencing (WES), whole-genome sequencing and microarray, MLPA, transcriptome sequencing, ViroChip and Sanger sequencing. Results: A median of 14 (4-57) genes were mutated and a median of 0.17% of the autosome was affected by copy number aberrations (CNA) in VS. NF2 mutation was observed in 89%. Tumors with wildtype NF2 harbored mutations in genes linked to NF2. Novel genes and pathways identified in VS included CDC27 (11%), USP8 (7%) and axonal guidance pathway (54%). One clinically aggressive VS was identified and correlated with high mutational burden (231) and mutated RAD54L. Variant allele frequencies for both small mutations and CNAs indicated intratumoral heterogeneity. No plausible virus was associated with VS. We identified a premalignant VS characterized by large chromosomal aberrations and mutated NF2. Malignant transformation was accompanied by whole-genome doubling and mutations in GNAQ, FOXO4 and PDGFRB. VN-MPNST is characterized by gross chromosomal aberrations and homozygous loss of CDKN2A. Previous treatment with GKRS in VS and VN-MPNST did not correlate with neither specific mutations nor genome wide signatures. COSMIC mutational signature 3 contributes to VN-MPNST while signature 6 contributes to a subset of VS. Conclusion: VS is characterized by intratumoral genetic heterogeneity and relatively few mutations. We found recurrent mutations in NF2 and the axonal guidance pathway in addition to novel genes in subsets. Mutated RAD54L might correlate with a hypermutator phenotype and worse clinical course. We identified CDKN2A as a likely tumor suppressor in both premalignant VS and VN-MPNST. Premalignant VS showed signs of chromosomal instability making it prone to malignant transformation. No biomarker of radioresistance or signature of exposure to ionizing radiation was identified in neither VS nor VN-MPNST. We found no evidence of a viral etiology in VS.Doktorgradsavhandlin

CIRA annual report FY 2015/2016

Reporting period April 1, 2015-March 31, 2016

Thoracic aortic aneurysms and dissections: genetic analysis of Mendelian and complex cases

The present doctoral thesis deals with the still partially unraveled genetic component of thoracic aortic aneurysms and dissections, a frequently asymptomatic but potentially lethal condition and major cause of sudden death. Our main objective was to contribute to further elucidate the genetics behind it, from both Mendelian and complex perspectives. We analyzed single and familial, forensic and clinical mendelian cases applying either a candidate-gene or whole exome massive parallel sequencing approach, respectively. We were able to solve approximately 23% of the forensic single cases and identified two strong candidate mutations in TGFB2 and PRKG1 genes in the two non-syndromic familial cases analyzed. For the analysis of complex cases we chose a population-based approach. We selected bicuspid aortic valve patients with and without concomitant thoracic aortic dilation and faced them against general population controls. We were not able to identify any consistently significant association, though a promising one arose involving HMCN2 and calcium metabolism that should be considered in future studies. The direct clinical consequences some of these results had supported molecular diagnosis, reliable genotype-phenotype correlations, and risk stratification as important tools for clinical management of these patients and family members at risk, as well as the need of research to continue

Improving Outcomes in Prostate Cancer

The results of the STAMPEDE trial demonstrate that intensified systemic therapy with docetaxel or abiraterone added to androgen deprivation therapy (ADT) improves overall survival (OS). Treatment benefit and tolerance vary, therefore prognostic and predictive biomarkers able to inform treatment selection are required to improve patient outcomes. Through performing a systematic review and meta-analysis I contextualised the celecoxib and zoledronic acid (celecoxib-ZA) results with the aim of understanding the intriguing synergistic therapeutic effect that was only seen in metastatic disease. Secondly, I explored whether prostate-specific antigen (PSA) response, assessed after commencing ADT, and PSA nadir assessed after completion of docetaxel, are prognostic of OS. Through collaboration with industry partners, I assessed the feasibility of performing targeted nextgeneration sequencing (tNGS) using formalin-fixed paraffin embedded (FFPE) prostate tumour samples. I explored the genomic profile of mCSPC and sought prevalence data to inform the evaluation of therapies such as poly ADP ribose polymerase inhibitors (PARPi) in homologous recombination deficient (HRD) cancers. No other trials evaluating a cox-2 inhibitor with a bisphosphonate were identified. However, supported by pre-clinical data, an immunological mechanism mediated by γδ T cells is proposed to explain the observed synergy. This strengthens the need for future trials and informs parallel translational research. PSA response can be used to risk-stratify patients shortly after commencing ADT and may be useful in informing the use of docetaxel. PSA nadir, assessed after completion of docetaxel, was also prognostic of OS and may be used to identify patients who remain at high risk where additional systemic therapies e.g. abiraterone, should be evaluated. The genomic study revealed that 94% (108/115) of sequenced samples had ≥1 pathogenic mutation although individual mutation frequencies remain low and pathway aberrations often co-exist, which would necessitate hierarchical allocation if used to guide treatment. The prevalence of HRD is clinically significant (15%) however the screening burden is considerable, compounded by variable sample quality, compromising the sequencing success rate (64%). These data support the further evaluation of the combination of cox-2 inhibitors and bisphosphonates and the use of PSA-based outcomes in risk-stratification, whilst the genomic feasibility and prevalence data will inform future trial designs incorporating molecular stratification

The identification of new familial pheochromocytoma/paraganglioma genes using whole exome sequencing

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 30-04-2015Los feocromocitomas (PCC) son tumores neuroendocrinos, desarrollados a partir del tejido cromafín de la médula adrenal, que suelen causar hipertensión arterial por sobre-secreción de catecolaminas. Los paragangliomas (PGL) son PCCs, en su mayoría secretores y con un gran riesgo de malignizar, que se desarrollan a partir de paraganglios localizados fundamentalmente en la región intra-abdominal o torácica. Algunos PGLs pueden desarrollarse en la región de la cabeza y el cuello, y en esa localización suelen comportarse como masas benignas no secretoras. Los PCCs presentan una incidencia anual en población española de 2 casos por millón de habitantes y son por tanto una enfermedad rara. Tanto la secuenciación masiva del genoma completo, como la limitada a las regiones codificantes (secuenciación exómica, SE), se han convertido a lo largo de los últimos años en herramientas de gran utilidad para el descubrimiento de genes de susceptibilidad responsables de enfermedades mendelianas. De este modo, el objetivo principal de esta tesis doctoral fue la identificación de nuevos genes de susceptibilidad implicados en el desarrollo de PCC/PGL mediante el uso de la SE aplicada a 3 proyectos independientes. En el primer proyecto, se llevó a cabo la SE de tres pacientes no relacionados, con antecedentes familiares de la enfermedad y sin mutaciones en ninguno de los genes conocidos. Los pacientes fueron seleccionados como candidatos para el estudio debido a que sus correspondientes tumores presentaban perfiles de expresión muy homogéneos. El filtrado y posterior análisis de los datos de secuenciación permitió identificar mutaciones germinales patogénicas en el gen MAX en los tres pacientes. Además, la pérdida de heterocigosidad del alelo silvestre, la ausencia de proteína MAX en los tumores y el descubrimiento de otras 5 mutaciones en pacientes aquejados de la enfermedad permitió demostrar que MAX constituía un nuevo gen supresor de tumores asociado con el desarrollo de PCC hereditario. En un segundo trabajo, el estudio de una serie compuesta por más de 1500 pacientes no relacionados permitió establecer tanto la prevalencia de las mutaciones en MAX en pacientes con PCC/PGL (1,12%), como el fenotipo asociado a dichas mutaciones. Por último, con el objeto de determinar la patogenicidad de las variantes con significado desconocido halladas en el gen MAX, se llevó a cabo un estudio funcional de las mismas en células de PCC de rata (PC12) y se implementó una herramienta de predicción in silico basada en el consenso de 5 predictores. En el segundo proyecto, los candidatos a estudio mediante SE (3 tríos paciente/madre/padre) fueron seleccionados en base a la presencia de una característica fenotípica poco frecuente en pacientes con PCC/PGL: policitemia idiopática. Durante el análisis de los datos, se publicó el descubrimiento de mutaciones somáticas post-zigóticas en el gen EPAS1 en pacientes con PCC/PGL múltiple y policitemia idiopática. El análisis de EPAS1 en los correspondientes tumores de los pacientes seleccionados para la SE, reveló la presencia de mutaciones somáticas en mosaico en el gen EPAS1 en todos ellos. Además, el estudio de una serie adicional de tumores identificó mutaciones somáticas en tumores de pacientes sin policitemia. Finalmente, se identificó la ganancia de la región 2p como exclusiva de tumores con mutación en EPAS1. En el tercer proyecto, se llevó a cabo una selección de pacientes basada en la presencia de tumores múltiples (más de 5) como indicador de la existencia de una enfermedad hereditaria. Durante el filtrado de las variantes encontradas en uno de los pacientes seleccionados, se identificó una mutación en el gen MDH2, implicado en el ciclo de Krebs. La ausencia de RNAm, proteína y actividad enzimática malato deshidrogenasa, así como el diagnóstico de la enfermedad en un pariente portador de la variante permitió concluir que el gen MDH2 es un nuevo gen supresor tumoral responsable de susceptibilidad a desarrollar PCC/PGL. La identificación de este segundo gen de susceptibilidad a desarrollar PCC/PGL demuestra la eficacia de la SE en la identificación de nuevos genes responsables de enfermedades mendelianas.Pheochromocytomas (PCCs) are neuroendocrine tumors arising from the medulla of the adrenal gland that usually cause hypertension due to oversecretion of cathecolamines. On the other hand, paragangliomas (PGLs) are normally secretor tumors with high risk of malignancy that arise from the paraganglia of the intra-abdominal or thoracic regions. Some PGLs arise in the head and neck region, usually as benign, non-secretor tumors. PCC/PGL is a rare disease, with an incidence in the Spanish population of approximately 2 cases per million habitants. High-throughput techniques such as wholegenome sequencing (WGS) and whole-exome sequencing (WES) are nowadays widely applied to discover susceptibility genes involved in mendelian diseases. Thus, the main objective of this thesis was to identify new genes related to the susceptibility to develop PCC/PGL by applying WES in three independent projects. In the first project, we applied WES to three unrelated patients with a family history of disease testing negative for mutations in the major PCC/PGL susceptibility genes. The three patients were selected because they shared a common homogeneous transcriptional profile, suggesting a common underlying genetic alteration. The variant filtering process and the posterior analysis of the WES data allowed us to identify pathogenic germline mutations in the MAX (MYC associated factor X) gene in all three patients. Moreover, loss of heterozygosity of the wild type allele, loss of the protein in the tumors and the identification of MAX mutations in additional patients with the disease, together indicated that MAX constitutes a novel tumor suppressor gene. In a posterior international collaborative study, we recruited more than 1500 unrelated patients and established the prevalence of MAX mutations to be 1.12%; we also characterized the associated phenotype. Finally, we developed a functional model to determine the pathogenicity of variants of unknown significant (VUS) found in MAX. This model was implemented based on a consensus in silico prediction obtained from five algorithms available online. In the second project, the selection of three trios (patient/mother/father) was based on the presence of a very infrequent phenotypic characteristic in patients suffering for PCC/PGL: idiopathic polycythemia. While the analysis of the WES data was underway, a study was published reporting the presence of somatic post-zygotic mutations in the gene EPAS1 (HIF2A) in patients presenting multiple PCC/PGL and idiopathic polycythemia. Our posterior screening for EPAS1 mutations in the tumors from the index patients revealed the presence of somatic mutations in all of them. In addition, the analysis of an additional series of tumors identified somatic mutations in tumors from patients without polycythemia. Finally, we indentified a gain of the chromosomic region 2p exclusive to tumors harboring EPAS1 mutations. In the last WES Project, we selected patients based on the presence of multiple (more than five) tumors as phenotypic marker of hereditary disease. We identified a splice-site mutation affecting the MDH2 gene involved in the Krebs cycle. The observed absence of the messenger RNA, protein and malate dehydrogenase enzymatic activity, as well as the positive diagnosis for the disease of a family member, led us to conclude that MDH2 is a novel tumor suppressor gene implicated in susceptibility to develop PCC/PGL. The identification of this second susceptibility gene implicated in PCC/PGL development demonstrates the efficacy of WES in discovering susceptibility genes involved in Mendelian disease

Abstracts from the 50th European Society of Human Genetics Conference: Posters

International audienc

Investigation of external refrigeration systems for long term cryogenic storage Final report

Data on external refrigeration systems for space storage of cryogens for long period