Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma

Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature

11q deletion or ALK activity curbs DLG2 expression to maintain an undifferentiated state in neuroblastoma

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature'' that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, high-lighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas

High frequency of subclonal ALK mutations in high risk neuroblastoma patients. A SIOPEN study

Introduction: In neuroblastoma (NB), activating ALK receptor tyrosine kinase point mutations are detected in 8–10% at diagnosis using conventional sequencing. To determine the potential occurrence and the prognostic impact of ALK mutations in a series of high risk NB patients we studied ALK variation frequencies using targeted deep sequencing in samples of patients enrolled in the SIOPEN HR-NBL01 stud

Integración de la genómica en la modelización de los tumores neuroblásticos = Integration of genomics in neuroblastic tumors modeling

*Introducción: El neuroblastoma es un tumor maligno embrionario del sistema nervioso simpático con una gran heterogeneidad en la presentación clínica, morfológica y genética y en otras características biológicas. La estratificación de grupos de riesgo pretratamiento se basa en los siguientes factores: edad, estadio, histopatológica del tumor, estado del oncogén MYCN, integridad del brazo cromosómico 11q y ploidía. Por otra parte, los cambios numéricos y estructurales detectados en el perfil genético se han incluido recientemente en la estratificación terapéutica de los pacientes con bajo riesgo a recaer o morir. El amplio espectro clínico de la enfermedad va desde pacientes con neuroblastoma en estadio 1, 2, 3 y 4S, generalmente caracterizados por su pronóstico favorable, a pacientes con tumores en estadio 4 con una mala evolución de la enfermedad, clasificados como pacientes de alto riesgo. Por otra parte se han descrito cohortes de neuroblastoma con unas características clínicas y biológicas inusuales y con baja prevalencia. Típicamente el neuroblastoma se diagnostica en la época neonatal y en bebés menores de un año y generalmente el oncogén MYCN se encuentra amplificado en aproximadamente el 25% de todos los casos. El diagnóstico en pacientes mayores de 10 años, la coexistencia de la amplificación de MYCN y deleción del brazo 11q o la amplificación heterogénea de MYCN son fenómenos infrecuentes. En los neuroblastomas de alto riesgo y en los raros grupos mencionados se necesitan de forma urgente investigaciones dirigidas a la identificación de nuevos enfoques terapéuticos para lograr una mejora de la evolución de la enfermedad. Una mejor caracterización de los diferentes grupos pronóstico y la identificación de nuevas dianas terapéuticas en el tumor y células del estroma, y en los elementos de la matriz extracelular dará lugar a terapias más adecuadas. Hasta la fecha son pocos los estudios que han determinado de forma precisa la frecuencia y detallado las alteraciones genéticas en neuroblastomas de pacientes adolescentes y adultos, o en neuroblastomas con amplificación atípica de MYCN y por tanto, están lejos de aportar estrategias tangibles para el cuidado de los pacientes. Sin embargo, ampliar esta información es crucial para identificar nuevos enfoques terapéuticos mediante la minería de datos. *Objetivos generales: En esta tesis perseguimos describir las características biológicas de tres grupos infrecuentes de tumores neuroblásticos identificando las alteraciones genéticas de cada grupo con el fin último de facilitar grandes estudios multicéntricos que puedan lograr una terapia más precisa de los pacientes. *Objetivos específicos: - Buscar patrones dependientes de la edad avanzanda en el neuroblastoma, asociados con características biológicas particulares, en una cohorte de pacientes mayores de 10 años al diagnóstico. El estudio está centrado en: 1) Determinar los patrones de alteraciones cromosómicas segmentarias mediante la técnica de micromatrices de polimorfismos de un único nucleótido. 2) Investigar la infiltración de las células del sistema inmune en el tejido tumoral mediante la expresión inmunohistoquímica de marcadores celulares (CD4, CD8, CD20, CD11b, CD11c y CD68). 3) Evaluar la expresión de la proteína ATRX mediante inmunohistoquímica. - Determinar el perfil genético de tumores neuroblásticos con dos alteraciones genéticas agresivas: la amplificación del oncogén MYCN y deleción de la región cromosómica 11q, poniendo énfasis en: 1) Identificar las alteraciones cromosómicas segmentarias y definir sus puntos de ruptura cromosómica. 2) Definir las regiones más pequeñas de solapamiento de las alteraciones segmentarias. 3) Describir la heterogeneidad intratumoral de los cambios cromosómicos presentes en este grupo tumoral. - Describir las características genéticas de los tumores neuroblásticos con amplificación heterogénea del oncogén MYCN. La descripción genética se basa en: 1) Identificación de los patrones de alteraciones cromosómicas segmentarias y de los puntos de ruptura cromosómica. 2) Comparación de los perfiles genómicos y de determinados puntos de ruptura cromosómica entre los tumores con amplificación heterogénea de MYCN y los subgrupos genéticos agresivos de neuroblastoma (con y sin amplificación homogénea de MYCN y deleción de 11q). *Metodología: El presente trabajo de tesis doctoral es un compendio de tres artículos científicos, en los que la candidata es la primera autora o primera coautora, que tratan sobre tumores neuroblásticos infrecuentes. En el texto del compendio de tesis hemos usado el término ultra-raro para referirnos al conjunto de tumores neuroblásticos caracterizados. Los artículos que constituyen el compendio son los siguientes: 1. Genetic instability and intratumoral heterogeneity in neuroblastoma with MYCN amplification plus 11q deletion. Eva Villamón,*, Ana P. Berbegall,*, Marta Piqueras, Irene Tadeo, Victoria Castel, Anna Djos, Tommy Martinsson, Samuel Navarro, and Rosa Noguera. * Equal contribution. PLoS One, 2013;8(1): e53740. 2. Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration. Ana P. Berbegall, Eva Villamón, Irene Tadeo, Tommy Martinsson, Adela Cañete, Victoria Castel, Samuel Navarro and Rosa Noguera. Neoplasia, 2014; 16(6): 471-480. 3. Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma with aggressive genetic profile neuroblastic tumors. Ana P. Berbegall, Eva Villamón, Marta Piqueras, Irene Tadeo, Anna Djos, Peter Ambros, Tommy Martinsson, Inge Ambros, Adela Cañete, Victoria Castel, Samuel Navarro and Rosa Noguera. Oncogene, 2015; Aceptado para su publicación, Abril 2015. Como se razona a continuación, los artículos reúnen tres desafíos en la investigación del neuroblastoma: 1. Centrarse en una cohorte minoritaria de pacientes afectos de neuroblastoma. Dentro de la enfermedad neuroblástica, considerada como una entidad rara (ORPHA635, http://www.orpha.net/), existen grupos clínica y biológicamente ultra-raros, en los que es necesaria una mayor investigación. La clínica de los pacientes con tumores ultra-raros es problemática, el tratamiento en estos pacientes con mucha frecuencia supone un dilema y todo el proceso de la enfermedad se vuelve más complejo. El escaso avance experimentado en el conocimiento de estos tumores no ha contribuido a la mejora de la supervivencia ni al desarrollo de tratamientos más adecuados. Por lo tanto, a pesar de la infrecuencia de estos tumores su investigación posee un gran valor. 2. Proponer una gestión más adecuada de las muestras. Dado que los grupos ultra-raros de neuroblastoma han sido a menudo excluidos o considerados como parte de otros grandes grupos de neuroblastomas con características clínicas y biológicas distintas a estos, la determinación de sus características biológicas se ha visto comprometida. Además, la escasez de la muestra tumoral disponible para realizar los estudios de investigación dificulta consensuar el papel de los análisis genéticos en la estratificación de riesgo terapéutica. Es crucial resaltar la importancia de la recogida metódica de la mayor cantidad de tejido tumoral posible para ser analizado secuencialmente por hibridación in situ fluorescente y por técnicas pangenómicas para determinar la presencia de heterogeneidad genética intratumoral. Considerar a estos tumores como entidades especiales con protocolos específicos para el manejo de las muestras beneficiaría su diagnóstico e investigación. 3. Considerar la utilidad potencial de los datos. La pretensión final del trabajo de tesis es contribuir a la recogida de datos y modelización del neuroblastoma. La descripción genética de estas cohortes facilitará la consecución de grandes estudios multicéntricos que son necesarios para coordinar proyectos clínicos. La ausencia de consenso diagnóstico respecto a los grupos de edad avanzada, una relativa falta de atención a los casos de neuroblastoma con amplificación atípica de MYCN, la escasez de la muestra tumoral y la heterogeneidad genética intratumoral son limitaciones que se pueden superar en el marco de los estudios multicéntricos almacenando adecuadamente los datos. Los tumores neuroblásticos ultra-raros debido a sus características biológicas singulares también pueden servir como modelos que proporcionarían conocimiento preciso sobre la patogénesis del neuroblastoma. *Conclusiones: Los estudios presentados son de relevancia para nuevos enfoques terapéuticos específicos y para generar estrategias de colaboración que permitan desarrollar propuestas de ensayos clínicos adecuadas.*Background: Neuroblastoma is an embryonic malignancy of the sympathetic nervous system with a remarkable heterogeneity in clinical, histological, genetic and in other biological features. The pre-treatment risk group stratification is based on the following factors: age, stage, tumor histology, MYCN gene status, chromosome 11q integrity and ploidy. Recently, numerical and segmental chromosome aberrations detected in the genetic profile have been included in the treatment stratification of patients with a low-risk to relapse or die. The wide clinical spectrum of the disease ranges from neuroblastoma patients with stage 1, 2, 3 and 4S usually characterized by favorable prognosis, to advanced stage 4 with an adverse outcome and classified as high-risk neuroblastoma. Besides these groups, neuroblastoma cohorts characterized by unusual clinical and biological features have been described with low prevalence. Typically, neuroblastoma is diagnosed during the neonatal period and infancy, and finding that the MYCN oncogene is usually amplified in approximately 25% of all cases. Diagnosis in ages over 10 years, coexistence of MYCN amplification plus 11q chromosome deletion or heterogeneous MYCN amplification are very infrequent events. In high-risk neuroblastoma and in the above-mentioned rare groups, research efforts focusing on the identification of new therapeutic approaches are urgently needed to improve outcome. A better characterization of the different prognostic groups and the identification of therapeutic targets in the tumor and stroma cells, as well as in the extracellular matrix elements would lead to the identification of more specific treatments. To date, few studies have determined the precise frequency or detailed the genetic alterations of samples derived from adolescent and adult life neuroblastomas, or from tumors with atypical presence of MYCN amplification, and therefore almost do not constitute actionable goals for patient care. However, their accurate characterization is crucial for the identification of new therapeutic approaches through data mining studies. *General objectives: In this thesis research, we pursue to describe the biological features of three ultra-rare cohorts of neuroblastic tumors, searching for any genetic features specific to each group, with the ultimate goal of facilitating large multicenter studies that could lead to the implementation of a more accurate management and care of the patients. *Specifics objectives: - To search for older-age-dependent patterns in neuroblastoma associated with particular biological characteristics in a cohort of patients older than 10 years at diagnosis. Our actions concentrated on: 1). Exploring the patterns of segmental chromosome aberrations by performing the Single Nucleotide Polymorphisms array technique. 2). Investigating the presence of immune cells tumor infiltration by immunohistochemistry expression of cell markers (CD4, CD8, CD20, CD11b, CD11c and CD68). 3). Evaluating the expression of the ATRX protein by immunohistochemistry. - To characterize the genetic profile of uncommon neuroblastic tumors harboring two chromosomal aggressive features, MYCN amplification and 11q deletion. Emphasis was placed on: 1). Identification of the segmental chromosome aberrations present and mapping their chromosomal breakpoints. 2). Defining the smallest region of the segmental chromosomal alterations. 3). Describing the intratumoral heterogeneity of the structural chromosome changes present in this tumor cohort. - To demarcate the genetic characteristics of unusual heterogeneous MYCN amplified neuroblastomas. Our approach consisted of: 1). Identification of the patterns of segmental chromosome aberrations and mapping of the chromosomal breakpoints. 2). Comparison between their genomic profiles and certain breakpoints with those from aggressive genetic subgroups of neuroblastoma (homogeneous MYCN amplified tumors and non-MYCN amplified with 11q deletion). *Methodology: The present doctoral thesis research is presented as a compendium of three publications, of which the candidate is the first or co-first author, dealing with ultra-rare neuroblastic tumors; henceforth in this text of the compendium we use the term ultra-rare neuroblastoma to refer to all these characterized tumors. The articles of the compendium are the following: 1. Genetic instability and intratumoral heterogeneity in neuroblastoma with MYCN amplification plus 11q deletion. Eva Villamón,*, Ana P. Berbegall,*, Marta Piqueras, Irene Tadeo, Victoria Castel, Anna Djos, Tommy Martinsson, Samuel Navarro, and Rosa Noguera. * Equal contribution. PLoS One, 2013; 8(1): e53740. 2. Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration. Ana P. Berbegall, Eva Villamón, Irene Tadeo, Tommy Martinsson, Adela Cañete, Victoria Castel, Samuel Navarro and Rosa Noguera. Neoplasia, 2014; 16(6): 471-480. 3. Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma with aggressive genetic profile neuroblastic tumors. Ana P. Berbegall, Eva Villamón, Marta Piqueras, Irene Tadeo, Anna Djos, Peter Ambros, Tommy Martinsson, Inge Ambros, Adela Cañete, Victoria Castel, Samuel Navarro and Rosa Noguera. Oncogene, 2015; Accepted for publication, April_2015. As outlined below, the articles unify three research challenges in neuroblastoma: 1. Focus on minor cohorts of neuroblastoma patients. Within neuroblastoma, considered as a rare disease (ORPHA635, http://www.orpha.net/), there are clinical and biological ultra-rare neuroblastoma groups in which further research is necessary. Ultra-rare neuroblastoma patients’ care is problematic, therapeutic decision-making very frequently poses a dilemma and dealing with the disease process becomes more complicated. The scant knowledge of these tumors has led to a failure in patient survival improvement or in the development of better treatments. Therefore, although uncommon tumors their research is worthy and worthwhile. 2. Propose an accurate management of the samples. Since ultra-rare groups of neuroblastoma have often been excluded or considered as part of larger groups of neuroblastoma with dissimilar clinical and biological characteristics, delineation of their biology has been compromised. The scarcity of tumor material available for research studies makes it difficult to achieve a consensus on the role of genetic analyses for establishing a therapeutic risk stratification. It is crucial to stress the importance of a methodical collection of as much tumor tissue as possible to be sequentially analyzed by Fluorescence In Situ Hybridization and pangenomic techniques to ascertain the presence of the genetic intratumoral heterogeneity. Considering these tumors as special entities with specific standard protocols for sample management would benefit their diagnosis and research. 3. Consider potential utility of the data. The final challenge of the thesis work is to contribute to the data collection and modeling of neuroblastic tumors. The genetic description of these cohorts will facilitate the consecution of large multi-center studies needed to coordinate clinical projects. The lack of a diagnostic consensus referent to older age neuroblastoma, the relative lack of attention to ultra-rare neuroblastoma cases with MYCN amplification, the sample scarcity and genetic intratumoral heterogeneity are limitations that can be overcome in the frame of multicenter studies with adequate databases. Ultra-rare neuroblastomas, because of their singular biological features, can also serve as models that could provide accurate knowledge concerning neuroblastoma pathogenesis. *Conclusions: The studies presented are of relevance for new targeted treatment approaches and to generate collaborative strategies to develop proposals for appropriate clinical trials proposals

1p36 deletion results in a decrease in glycosaminoglycans which is associated with aggressiveness in neuroblastic tumors

Despite our deep understanding of neuroblastic tumors, some patients still suffer treatment failure, so pre-treatment risk stratification still requires improvement and the search for new therapeutic targets must continue. Here we correlated prognostic clinical and biological features of neuroblastic tumors with the density of extracellular matrix glycosaminoglycans (the main components of the extracellular matrix ‘ground substance’), in nearly 400 primary samples. We also studied the relationship between the density of extracellular matrix glycosaminoglycans and the expression of B3GALT6, an enzyme required for their synthesis. We associated a decrease in glycosaminoglycans with neuroblastomas that were histopathologically poorly-differentiated or undifferentiated, as well as with metastatic disease, and 1p36 deleted tumors. This decrease in glycosaminoglycans was also related to abnormal nuclear B3GALT6 expression in neuroblastic cells. These findings point towards the importance of the ground substance in the aggressiveness of neuroblastic tumors, which should therefore be considered when developing novel therapies for treating neuroblastomas

Intra-Tumour Genetic Heterogeneity and Prognosis in High-Risk Neuroblastoma

Spatial ITH is defined by genomic and biological variations within a tumour acquired by tumour cell evolution under diverse microenvironments, and its role in NB patient prognosis is understudied. In this work, we applied pangenomic techniques to detect chromosomal aberrations in at least two different areas of each tumour and/or in simultaneously obtained solid and liquid biopsies, detecting ITH in the genomic profile of almost 40% of HR-NB. ITH was better detected when comparing one or more tumour pieces and liquid biopsy (50%) than between different tumour pieces (21%). Interestingly, we found that patients with ITH analysed by pangenomic techniques had a significantly better survival rate that those with non-heterogeneous tumours, especially in cases without MYCN amplification. Moreover, all patients in the studied cohort with high ITH (defined as 50% or more genomic aberration differences between areas of a tumour or simultaneously obtained samples) survived after 48 months. These results clearly support analysing at least two solid tumour areas (separately or mixed) and liquid samples to provide more accurate genomic diagnosis, prognosis and therapy options in HR-NB

Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration

Neuroblastoma (NB) is a common malignancy in children but rarely occurs during adolescence or adulthood. This subgroup is characterized by an indolent disease course, almost uniformly fatal, yet little is known about the biologic characteristics. The aim of this study was to identify differential features regarding DNA copy number alterations, α-thalassemia/mental retardation syndrome X-linked (ATRX) protein expression, and the presence of tumor-associated inflammatory cells. Thirty-one NB patients older than 10 years who were included in the Spanish NB Registry were considered for the current study; seven young and middle-aged adult patients (range 18-60 years) formed part of the cohort. We performed single nucleotide polymorphism arrays, immunohistochemistry for immune markers (CD4, CD8, CD20, CD11b, CD11c, and CD68), and ATRX protein expression. Assorted genetic profiles were found with a predominant presence of a segmental chromosome aberration (SCA) profile. Preadolescent and adolescent NB tumors showed a higher number of SCA, including 17q gain and 11q deletion. There was also a marked infiltration of immune cells, mainly high and heterogeneous, in young and middle-aged adult tumors. ATRX negative expression was present in the tumors. The characteristics of preadolescent, adolescent, young adult, and middle-aged adult NB tumors are different, not only from childhood NB tumors but also from each other. Similar examinations of a larger number of such tumor tissues from cooperative groups should lead to a better older age–dependent tumor pattern and to innovative, individual risk-adapted therapeutic approaches for these patients

Vascular patterns provide therapeutic targets in aggressive neuroblastic tumors.

Angiogenesis is essential for tumor growth and metastasis, nevertheless, in NB, results between different studies on angiogenesis have yielded contradictory results. An image analysis tool was developed to characterize the density, size and shape of total blood vessels and vascular segments in 458 primary neuroblastic tumors contained in tissue microarrays. The results were correlated with clinical and biological features of known prognostic value and with risk of progression to establish histological vascular patterns associated with different degrees of malignancy. Total blood vessels were larger, more abundant and more irregularly-shaped in tumors of patients with associated poor prognostic factors than in the favorable cohort. Tumor capillaries were less abundant and sinusoids more abundant in the patient cohort with unfavorable prognostic factors. Additionally, size of post-capillaries & metarterioles as well as higher sinusoid density can be included as predictive factors for survival. These patterns may therefore help to provide more accurate pre-treatment risk stratification, and could provide candidate targets for novel therapies

NeuPAT: an intranet database supporting translational research in neuroblastic tumors will be published in Computers in Biology and Medicine

Translational research in oncology is directed mainly towards establishing a better risk stratification and searching for appropriate therapeutic targets. This research generates a tremendous amount of complex clinical and biological data needing speedy and effective management. The authors describe the design, implementation and early experiences of a computer-aided system for the integration and management of data for neuroblastoma patients. NeuPAT facilitates clinical and translational research, minimizes the workload in consolidating the information, reduces errors and increases correlation of data through extensive coding. This design can also be applied to other tumor types.This work was supported by RD06/0020/0102 and PI10/0015 (ISCIII & ERDF); 396/2009 (FAECC).We are grateful to Mr.D. Harrison for assistance with English language editing.Villamon Ribate, E.; Piqueras, M.; Meseguer Anastasio, JE.; Blanquer Espert, I.; Berbegall Beltrán, AP.; Tadeo Cervera, I.; Hernández García, V.... (2013). NeuPAT: an intranet database supporting translational research in neuroblastic tumors will be published in Computers in Biology and Medicine. Computers in Biology and Medicine. 43(3):219-228. doi:10.1016/j.compbiomed.2012.11.011S21922843