A New Mutation of the p53 Gene in Human Neuroblastoma, Not Correlated with N-myc Amplification

N-myc gene amplification and/or loss of heterozygosity of chromosome 1 (LOH lp) are important criteria for prognosis and progression in human neuroblastoma (NB). Despite the high incidence of alterations of the p53 gene in human cancers, very few p53 mutations have been reported in NB. The objective of our study was to search for p53 mutations in NB and their correlation with N-myc amplification and clinical or pathologic parameters. We analyzed 14 selected cases of NB from the Spanish Protocol N-II-92. We found a missense mutation in codon 248 CGG to GGG (Arg/Gly) in one case of stage 4 NB with no N-myc amplification. Our results confirm the low incidence of p53 gene mutation in neuroblas…

Study of Proliferation and Apoptosis in Neuroblastoma. Their Relation with Other Prognostic Factors

Abstract Background Our objective was to study the proliferation and apoptotic process in 111 cases of neuroblastoma (NB) and to seek their relationship with other prognostic factors and survival. Methods Immunohistochemistry following ABC peroxidase was carried out for PCNA, Ki-67, bcl-2, and p53 proteins. Apoptosis analysis was performed with in situ detection of chromosomal breakdown. Molecular detection of DNA ladders by electrophoresis and amplification of MYCN was studied with PCR and Southern blot. Statistical study was performed with Pearson χ 2 and Kruskal-Wallis tests and Cox regression. Results Our results indicate that proliferative factors PCNA and Ki-67 were correlated to each…

Alternative lengthening of telomeres--an enhanced chromosomal instability in aggressive non-MYCN amplified and telomere elongated neuroblastomas

Telomere length alterations are known to cause genomic instability and influence clinical course in several tumor types, but have been little investigated in neuroblastoma (NB), one of the most common childhood tumors. In the present study, telomere-dependent chromosomal instability and telomere length were determined in six NB cell lines and fifty tumor biopsies. The alternative lengthening of telomeres (ALT) pathway was assayed by scoring ALT-associated promyelocytic leukemia (PML) bodies (APBs). We found a reduced probability of overall survival for tumors with increased telomere length compared to cases with reduced or unchanged telomere length. In non-MYCN amplified tumors, a reduced o…

miR‐200c and phospho‐AKT as prognostic factors and mediators of osteosarcoma progression and lung metastasis

Lung metastasis is the major cause of death in osteosarcoma patients. However, molecular mechanisms underlying this metastasis remain poorly understood. To identify key molecules related with pulmonary metastasis of pediatric osteosarcomas, we analyzed high-throughput miRNA expression in a cohort of 11 primary tumors and 15 lung metastases. Results were further validated with an independent cohort of 10 primary tumors and 6 metastases. In parallel, we performed immunohistochemical analysis of activated signaling pathways in 36 primary osteosarcomas. Only phospho-AKT associated with lower overall survival in primary tumors, supporting its role in osteosarcoma progression. CTNNB1 expression a…

Extracellular matrix composition defines an ultra-high-risk group of neuroblastoma within the high-risk patient cohort

Background: Although survival for neuroblastoma patients has dramatically improved in recent years, a substantial number of children in the high-risk subgroup still die. Methods: We aimed to define a subgroup of ultra-high-risk patients from within the high-risk cohort. We used advanced morphometric approaches to quantify and characterise blood vessels, reticulin fibre networks, collagen type I bundles, elastic fibres and glycosaminoglycans in 102 high-risk neuroblastomas specimens. The Kaplan-Meier method was used to correlate the analysed elements with survival. Results: The organisation of blood vessels and reticulin fibres in neuroblastic tumours defined an ultra-high-risk patient subgr…

Angiogenesis in neuroblastoma: relationship to survival and other prognostic factors in a cohort of neuroblastoma patients.

PURPOSE: To study angiogenesis in neuroblastoma, using morphometric and computerized image analysis, and correlate the results with survival and other prognostic factors. PATIENTS AND METHODS: Sixty-nine patients from the Spanish Cooperative Study for Neuroblastoma were studied. Tumoral angiogenesis was studied using an avidin-biotin immunoperoxidase technique with an anti-CD34 antibody. Vascular parameters (VPs) were analyzed by a computerized system. Statistical analysis was also performed. RESULTS: Sixty-six samples had adequate tumoral tissue, and their tumoral vessels were counted. Endothelial cells were more prominent in pure neuroblastomas than in maturing and more mature tumors. VP…

Determination of busulfan in human plasma using high-performance liquid chromatography with pre-column derivatization and fluorescence detection.

A rapid, sensitive and reproducible high-performance liquid chromatographic assay for busulfan in human plasma was developed. After extraction of plasma samples with acetonitrile and methylene chloride, busulfan and the internal standard [1,5-bis(methanesulfonyloxy)pentane] were derivatized with 8-mercaptoquinoline to yield fluorescent compounds which were detected with a fluorescence detector equipped with filters of 360 nm (excitation) and 425 nm (emission). Calibration graphs showed a linear correlation (r>0.9990) over the concentration range of 20-2000 ng/ml. The recovery of busulfan from plasma standards was 70+/-5%. The detection and quantification limits for busulfan in plasma sample…

Evaluación y rehabilitación neuropsicológica en oncología pediátrica.

Objetivo. El presente trabajo tiene como objetivo el estudio de los efectos neuropsicológicos a largo plazo del cáncer infantil y sus tratamientos así como plantear propuestas para la rehabilitación cognitiva. Método. El método ha consistido en una revisión sistemática de los resultados en la literatura científica acerca de los efectos de la quimioterapia y la radioterapia sobre el SNC y en las funciones cognitivas y conductuales detectados a través de estudios de neuroimagen como calcificaciones y cambios en la sustancia blanca. Presentamos, además, el protocolo de evaluación neuropsicológica ejemplificado con un diagnóstico frecuente que presenta deterioro generalizado. Proponemos un prog…

Next-Generation Sequencing Identifies Potential Actionable Targets in Paediatric Sarcomas

Background: Bone and soft-tissue sarcomas represent 13% of all paediatric malignancies. International contributions to introduce next-generation sequencing (NGS) approaches into clinical application are currently developing. We present the results from the Precision Medicine program for children with sarcomas at a reference centre. Results: Samples of 70 paediatric sarcomas were processed for histopathological analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS) with a consensus gene panel. Pathogenic alterations were reported and, if existing, targeted recommendations were translated to the clinic. Seventy paediatric patients with sarcomas…

Neuropsychological rehabilitation in children with central nervous system tumors and irradiated leukemias

Actualmente la intervención e investigación en psicooncología pediátrica se dirige hacia la evaluación de las consecuencias de la enfermedad y los efectos secundarios de los tratamientos, y a los factores predictivos tanto de desajustes como de aspectos adaptativos a la enfermedad del paciente y de su familia. Las tasas de curación alcanzadas en la actualidad, nos permiten contar con un número considerable de supervivientes de cáncer infantil, susceptibles de ser evaluados y poder determinar así las secuelas a largo plazo de la enfermedad y sus procedimientos terapéuticos. Además de incrementar considerablemente la supervivencia, también se ha mejorado la calidad de vida de los niños, media…

Hypermethylation of apoptotic genes as independent prognostic factor in neuroblastoma disease

Neuroblastoma (NB) is an embryonal tumour of neuroectodermal cells, and its prognosis is based on patient age at diagnosis, tumour stage and MYCN amplification, but it can also be classified according to their degree of methylation. Considering that epigenetic aberrations could influence patient survival, we studied the methylation status of a series of 17 genes functionally involved in different cellular pathways in patients with NB and their impact on survival. We studied 82 primary NB tumours and we used methylation-specific-PCR to perform the epigenetic analysis. We evaluated the putative association among the evidence of hypermethylation with the most important NB prognostic factors, a…

Two independent epigenetic biomarkers predict survival in neuroblastoma.

Background Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with a highly variable clinical course, ranging from spontaneous regression to life-threatening disease. Survival rates for high-risk NB patients remain disappointingly low despite multimodal treatment. Thus, there is an urgent clinical need for additional biomarkers to improve risk stratification, treatment management, and survival rates in children with aggressive NB. Results Using gene promoter methylation analysis in 48 neuroblastoma tumors with microarray technology, we found a strong association between survival and gene promoter hypermethylation (P = 0.036). Hypermethylation of 70 genes significantly …

Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma versus aggressive genetic profile neuroblastic tumors.

Intratumoral heterogeneous MYCN amplification (hetMNA) is an unusual event in neuroblastoma with unascertained biological and clinical implications. Diagnosis is based on the detection of MYCN amplification surrounded by non-amplified tumor cells by fluorescence in situ hybridization (FISH). To better define the genetic features of hetMNA tumors, we studied the Spanish cohort of neuroblastic tumors by FISH and single nucleotide polymorphism arrays. We compared hetMNA tumors with homogeneous MNA (homMNA) and nonMNA tumors with 11q deletion (nonMNA w11q-). Of 1091 primary tumors, 28 were hetMNA by FISH. Intratumoral heterogeneity of 1p, 2p, 11q and 17q was closely associated with hetMNA tumor…

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

Simple Summary Neuroblastoma (NB) is the most common extra-cranial solid paediatric cancer and is responsible for 15% of childhood cancer deaths. Patients with NB are characterized by presenting a very heterogeneous clinic (inter-tumoural heterogeneity) and also both spatial and temporal intra-tumour heterogeneity (ITH) reflected in their genetic aberrations, which may be the consequence of the coexistence of different microenvironments within the tumour. Applying pangenomic techniques to detect genomic aberrations in different biopsies (solid and liquid) of high risk NB (HR-NB) we have detected spatial ITH in a surprisingly high percentage (almost 40%) of the studied cohort. Moreover, a po…

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group

Purpose: For localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome. Patients and methods: Diagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children's Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIO…

Immunoproteomic studies on paediatric opsoclonus-myoclonus associated with neuroblastoma

We aimed to identify new cell-membrane antigens implicated in opsoclonus-myoclonus with neuroblastoma. The sera of 3 out of 14 patients showed IgG electron-microscopy immunogold reactivity on SH-SY5Y neuroblastoma cells. Immunoprecipitation experiments using rat brain synaptosomes and SH-SY5Y cells led to the identification of: (1) thirty-one nuclear/cytoplasmic proteins (including antigens HuB, HuC); (2) seven neuronal membrane proteins, including the Shaw-potassium channel Kv3.3 (KCNC3), whose genetic disruption in mice causes ataxia and generalized muscle twitching. Although cell-based assays did not demonstrate direct antigenicity, our findings point to Shaw-related subfamily of the pot…

Retinoblastoma and mosaic 13q deletion: a case report

Abstract Background Patients with 13q-syndrome are at risk of retinoblastoma when the RB1 gene, located in the chromosomal band 13q14.2, is deleted. This syndrome is frequently associated with congenital malformations and developmental delay, although these signs could be mild. Mosaic 13q-deletion patients have been previously reported in the literature; their phenotype is variable, and they may not be recognized. Case presentation Retinoblastoma diagnosed in a child with 13q-mosaicism confirmed in blood, oral mucosa, healthy retina and retinoblastoma. A second RB1 hit is present exclusively in the retinoblastoma sample (RB1 c.958C>T p.Arg320Ter). Other detected molecular events in retin…

Pharmacogenetics in Neuroblastoma: What Can Already Be Clinically Implemented and What Is Coming Next?

Pharmacogenetics is one of the cornerstones of Personalized Precision Medicine that needs to be implemented in the routine of our patients’ clinical management in order to tailor their therapies as much as possible, with the aim of maximizing efficacy and minimizing toxicity. This is of great importance, especially in pediatric cancer and even more in complex malignancies such as neuroblastoma, where the rates of therapeutic success are still below those of many other types of tumors. The studies are mainly focused on germline genetic variants and in the present review, state of the art is presented: which are the variants that have a level of evidence high enough to be implemented in the c…

A stiff extracellular matrix is associated with malignancy in peripheral neuroblastic tumors

Purpose and objective Improved prognosis for patients with peripheral neuroblastic tumors (PNB) depends on enhanced pretreatment risk stratification combined with research into new therapeutic targets. This study investigated the potential contribution of extracellular matrix (ECM) elements toward this endeavor. Methods We characterized certain elements such as reticulin fibers, collagen type I fibers, and elastic fibers by digital pathology in almost 400 untreated PNB. Results A reticular and poorly porous ECM was identified in neuroblastomas (NBs) from patients with clinical and biological features associated with poor prognosis compared with a loose and permeable matrix found in NBs of t…

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

AbstractNeuroblastoma (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 c…

Vascular patterns provide therapeutic targets in aggressive neuroblastic tumors

// Irene Tadeo 1 , Gloria Bueno 2 , Ana P. Berbegall 1 , M. Milagro Fernandez-Carrobles 2 , Victoria Castel 3 , Marcial Garcia-Rojo 4 , Samuel Navarro 1 , Rosa Noguera 1 1 Pathology Department, Medical School, University of Valencia, INCLIVA, 46010 Valencia, Spain 2 VISILAB, E.T.S. Ingenieros Industriales, University of Castilla-La Mancha, 13071 Ciudad Real, Spain 3 Pediatric Oncology Unit, University and Polytechnic Hospital La Fe, 46026 Valencia, Spain 4 Department of Pathology, Hospital de Jerez de la Frontera, 11407 Jerez de la Frontera, Cadiz, Spain Correspondence to: Rosa Noguera Salva, e-mail: rnoguera@uv.es Keywords: extracellular matrix, blood vessels, capillaries, sinusoids, neuro…

Genetic Instability and Intratumoral Heterogeneity in Neuroblastoma with MYCN Amplification Plus 11q Deletion

Background/Aim Genetic analysis in neuroblastoma has identified the profound influence of MYCN amplification and 11q deletion in patients’ prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features. Methods We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques. Results and Conclusion Thi…

Predicting outcomes for children with neuroblastoma using a multigene-expression signature: a retrospective SIOPEN/COG/GPOH study

Summary Background More accurate prognostic assessment of patients with neuroblastoma is required to better inform the choice of risk-related therapy. The aim of this study is to develop and validate a gene-expression signature to improve outcome prediction. Methods 59 genes were selected using an innovative data-mining strategy, and were profiled in the largest neuroblastoma patient series (n=579) to date using real-time quantitative PCR starting from only 20 ng of RNA. A multigene-expression signature was built using 30 training samples, tested on 313 test samples, and subsequently validated in a blind study on an independent set of 236 tumours. Findings The signature has a performance, s…

Anaplastic lymphoma kinase expression in neuroblastomas and its relationship with genetic, prognostic, and predictive factors

Prognostic value of partial genetic instability in neuroblastoma with ≤50% neuroblastic cell content

Piqueras M, Navarro S, Canete A, Castel V & Noguera R (2011) Histopathology59, 22–30 Prognostic value of partial genetic instability in neuroblastoma with ≤50% neuroblastic cell content Aims:  Better understanding of neuroblastoma genetics will improve with genome-wide techniques. However, performing these analyses in samples with <60% neuroblast cells is not adequate. We evaluated the utility of fluorescence in situ hybridization (FISH) on tissue microarrays (TMA) in detecting partial genetic instability (PGI), focusing on samples with ≤50% neuroblast cells. Methods and results:  Alterations of 11q and 17q were detected by FISH on 369 neuroblastoma samples in TMA. Status of the MYCN gene a…

MYCN gain and MYCN amplification in a stage 4S neuroblastoma.

Abstract Stage 4S neuroblastoma is a disease associated with spontaneous regression and good survival. We present a patient whose evolution has shown the variety and complexity of this disease in infants. Biologic factors, such as ploidy, MYCN copy number, loss of 1p36, and other chromosomal gains and losses were determined. A complex pattern of genetic abnormalities, such as near-diploidy, MYCN gain (2–4 copies per haploid genome) and imbalance/deletion of 1p36 was seen in the diagnostic sample. An extensive disseminated disease after a latent period of 26 months was associated with a special genetic evolution, such as tetraploidy, MYCN amplification (2:100–500 copies), 1p36 deletion, and …

Expression of type I interferon receptor and its relation with other prognostic factors in human neuroblastoma.

Expression of type I interferon receptor (IFN-R) has been found in several normal tissues and in malignant neoplasms, mainly those with epithelial differentiation. In order to analyze the immunohistochemical expression of type I IFN-R we studied 79 cases of neuroblastoma. Results of expression of type I IFN-R were statistically correlated with histopathology, stage, bcl-2 and PCNA expression, N-myc amplification and apoptosis. We found expression of type I IFN-R in 54/79 cases showing statistical correlation with bcl-2 expression (P=0.017) and favourable histopathology (P=0.015). The overexpression found in ganglion cells suggests that IFN-R could be involved in the pathway of neuroblastoma…

Pharmacogenetics implementation in the clinics: information and guidelines for germline variants.

The aim of this work was to supply an overview of the germline Pharmacogenetics that can be already implemented in the oncology clinical practice. An explanation of the three pillars considered necessary for determining which genetic polymorphisms should be used has been provided. These are PharmGKB single nucleotide polymorphism (SNP)-Drug Clinical Annotations with levels of evidence 1 or 2; the genetic information provided in the drug labels by the drug regulatory main agencies (Food and Drug Administration and European Medicines Agency, mainly); and the guidelines elaborated by international expert consortia (mainly Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacoge…

Imbalance between genomic gain and loss identifies high-risk neuroblastoma patients with worse outcomes

Survival in high-risk neuroblastoma (HR-NB) patients remains poor despite multimodal treatment. We aimed to identify HR-NB patients with worse outcomes by analyzing the genomic instability derived from segmental chromosomal aberrations. We calculated 3 genomic instability indexes for primary tumor SNP array profiles from 127 HR-NB patients: (1) Copy number aberration burden (%gainslength+%losseslength), (2) copy number load (CNL) (%gainslength-%losseslength) and (3) net genomic load (NGL) (%gainsamount-%lossesamount). Tumors were classified according to positive or negative CNL and NGL genomic subtypes. The impact of the genomic instability indexes on overall survival (OS) was assessed with…

How to minimise the effect of tumour cell content in detection of aberrant genetic markers in neuroblastoma

Background: Clinical heterogeneity reflects the complexity of genetic events associated with neuroblastoma (NB). To identify the status of all described genetic loci with possible prognostic interest, high-throughput approaches have been used, but only with tumour cell content >60%. In some tumours, necrotic, haemorrhagic and/or calcification areas influence the low amount of neuroblasts. We evaluated the effect of tumour cell content in the detection of relevant aberrant genetic markers (AGM) diagnosed by fluorescence in situ hybridisation (FISH) on tissue microarrays (TMA) in NB. Methods: Two hundred and thirty-three MYCN non-amplified primary NB included in 12 TMAs were analysed. Results…

MTHFR and VDR Polymorphisms Improve the Prognostic Value of MYCN Status on Overall Survival in Neuroblastoma Patients

Single nucleotide polymorphisms (SNPs) in Pharmacogenetics can play an important role in the outcomes of the chemotherapy treatment in Neuroblastoma, helping doctors maximize efficacy and minimize toxicity. Employing AgenaBioscience MassArray, 96 SNPs were genotyped in 95 patients looking for associations of SNP with response to induction therapy (RIT) and grade 3&ndash

Deletion of 11q in Neuroblastomas Drives Sensitivity to PARP Inhibition

AbstractPurpose: Despite advances in multimodal therapy, neuroblastomas with hemizygous deletion in chromosome 11q (20%–30%) undergo consecutive recurrences with poor outcome. We hypothesized that patients with 11q-loss may share a druggable molecular target(s) that can be exploited for a precision medicine strategy to improve treatment outcome.Experimental Design: SNP arrays were combined with next-generation sequencing (NGS) to precisely define the deleted region in 17 primary 11q-loss neuroblastomas and identify allelic variants in genes relevant for neuroblastoma etiology. We assessed PARP inhibitor olaparib in combination with other chemotherapy medications using both in vitro and in v…

Intratumoral immunosuppression profiles in 11q-deleted neuroblastomas provide new potential therapeutic targets

In this issue, Coronado et al. attempt to improve our understanding of the factors affecting the response to immunotherapy in a large subset of high‐risk neuroblastoma with hemizygous deletion of chromosome 11q. By using several computational approaches, the authors study potential transcriptional and post‐transcriptional pathways that may affect the response to immunotherapy and further be leveraged therapeutically in a biomarker‐directed fashion.

Germline Predisposition to Pediatric Cancer, from Next Generation Sequencing to Medical Care

Knowledge about genetic predisposition to pediatric cancer is constantly expanding. The categorization and clinical management of the best-known syndromes has been refined over the years. Meanwhile, new genes for pediatric cancer susceptibility are discovered every year. Our current work shares the results of genetically studying the germline of 170 pediatric patients diagnosed with cancer. Patients were prospectively recruited and studied using a custom panel, OncoNano V2. The well-categorized predisposing syndromes incidence was 9.4%. Likely pathogenic variants for predisposition to the patient’s tumor were identified in an additional 5.9% of cases. Additionally, a high number of pathogen…

Analysis of biological prognostic factors using tissue microarrays in neuroblastic tumors

Background Neuroblastic tumors (NT) are pediatric neoplasms with a heterogeneous genetic profile. They present genotypic alterations of prognostic value, the study of which is mandatory in designing therapeutic management. Tissue microarrays (TMA) from paraffin material allow the analysis of a large number of cases with minimal costs. The main purpose of the present study is to analyze specific genetic markers of neuroblastic tumors included in TMAs and determine their prognostic value. We compare the results obtained by different molecular techniques at different substrates to evaluate the feasibility of these assays. Procedure One hundred thirty-nine samples were included in four differen…

Frequency and prognostic impact of ALK amplifications and mutations in the European Neuroblastoma Study Group (SIOPEN) high-risk neuroblastoma trial (HR-NBL1)

Purpose: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. Materials and methods: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). Results: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with …