Yania Yáñez

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, as well as to determine the relationship among methylation, clinical classification and survival. CASP8 hypermethylation showed association with relapse susceptibility and, TMS1 and APAF1 hypermethylation are associated with bad prognosis and showed high influence on NB overall survival. Hypermethylation of apoptotic genes has been identified as a good candidate of prognostic factor. We propose the simultaneous analysis of hypermethylation of APAF1, TMS1 and CASP8 apoptotic genes on primary NB tumour as a good prognostic factor of disease progression. Mol. Carcinog.

Targeting Neuroblastoma Stem Cells with Retinoic Acid and Proteasome Inhibitor

Background Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients. Methodology/Principal Findings To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132. Conclusions Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

Neuroblastoma in adolescents: genetic and clinical characterisation

BackgroundAge at diagnosis is an important risk factor in neuroblastoma (NB) with worse prognosis in children older than 18 months. A more indolent course with long-term relapses and fatal outcome has been described in small series of adolescents. Our objective was to describe biological factors that contribute to this particular behaviour and could be helpful in their treatment.ProcedureNB cases older than 10 years of age at diagnosis registered in the files of the Neuroblastoma Group of SEOP from 1992 to 2007 were included. Disease extension was classified according to the International Neuroblastoma Staging System (INSS). Tumour samples were studied according to the International Neuroblastoma Pathology Classification (INPC). Biological studies included MNA, 1p, 11q and 17q status and ploidy.ResultsTwenty-two patients, from 10.1 to 24.6 years old, were included. Advanced stages predominated. 14/17 patients presented unfavourable histology. None had NMA or 1p del. However, 11q del was found in 8/13 cases and 17q gain in 7/11. Overall survival (OS) and event-free survival (EFS) for the entire series at 5 years were 0.45 and 0.32, respectively. Moreover, 5-year OS and EFS for stage 4 patients were 0.33 and 0.15.ConclusionsNB in adolescents is a special subgroup characterised by high-risk prognostic features which differ from those seen in younger patients, especially in relation to genetic abnormalities. The outcome in stage 4 was worse than in younger metastatic children, outlining the need for new therapeutic approaches in this subgroup of patients. The exact cut-off to separate older patients has not yet been established and will probably be based on biology.

TH and DCX mRNAs in peripheral blood and bone marrow predict outcome in metastatic neuroblastoma patients

PurposeIn metastatic neuroblastoma (NB) patients, accurate risk stratification and disease monitoring would reduce relapse probabilities. This study aims to evaluate the independent prognostic significance of detecting tyrosine hydroxylase (TH) and doublecortin (DCX) mRNAs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral blood (PB) and bone marrow (BM) samples from metastatic NB patients.ProceduresRT-qPCR was performed on PB and BM samples from metastatic NB patients at diagnosis, post-induction therapy and at the end of treatment for TH and DCX mRNAs detection.ResultsHigh levels of TH and DCX mRNAs when detected in PB and BM at diagnosis independently predicted worse outcome in a cohort of 162 metastatic NB. In the subgroup of high-risk metastatic NB, TH mRNA detected in PB remained as independent predictor of EFS and OS at diagnosis. After the induction therapy, high levels of TH mRNA in PB and DCX mRNA in BM independently predicted poor EFS and OS. Furthermore TH mRNA when detected in BM predicted worse EFS. TH mRNA in PB samples at the end of treatment is an independent predictor of worse outcome.ConclusionTH and DCX mRNAs levels in PB and BM assessed by RT-qPCR should be considered in new pre-treatment risk stratification strategies to reliable estimate outcome differences in metastatic NB patients. In those high-risk metastatic NB, TH and DCX mRNA quantification could be used for the assessment of response to treatment and for early detection of progressive disease or relapses.

New prognostic markers in neuroblastoma.

INTRODUCTION The hallmark of neuroblastoma is its clinical and biological heterogeneity, with the likelihood of cure varying widely according to age at diagnosis, extent of disease and tumor biology. We hope this review will be useful for understanding part of the unfamiliar neuroblastoma codex. AREAS COVERED In the first part of this review, the authors summarize the currently used prognostic factors for risk-adapted therapy, with the focus on clinical management of neuroblastoma patients. In the second part, the authors discuss the evolving prognostic factors for future treatment schemes. A search of online medical research databases was undertaken focusing especially on literature published in the last six years. EXPERT OPINION Harnessing the synergy of the various forms of data, including clinical variables and biomarker profiles, would allow mathematical predictive models to be built for the individual patient, which could eventually become molecular targets of specific therapies.

TIAM1 variants improve clinical outcome in neuroblastoma

Identification of tumor driver mutations is crucial for improving clinical outcome using a personalized approach to the treatment of cancer. Neuroblastoma is a tumor of the peripheral sympathetic nervous system for which only a few driver alterations have been described including MYCN amplification and ALK mutations. We assessed 106 primary neuroblastoma tumors by next generation sequencing using a customized amplicon-based gene panel. Our results reveal that genetic variants in TIAM1 gene associate with better clinical outcome, suggesting a role for these TIAM1 variants in preventing progression of this disease. The detected variants are located within the different domains of TIAM1 that signal to the upstream regulator RAS and downstream effector molecules MYC and RAC, which are all implicated in neuroblastoma etiology and progression. Clinical outcome was improved in tumors where a TIAM1 variant was present concomitantly with either ALK mutation or MYCN amplification. Given the function of these signaling molecules in cell survival, proliferation, differentiation and neurite outgrowth, our data suggest that the TIAM1-mediated network is essential to neuroblastoma and thus, inhibiting TIAM1 reflects a rational strategy for improving therapy efficacy in neuroblastoma.

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 potassium voltage-gated channels complexed proteins as hypothetical antigenic targets.

Event‐free survival of infants and toddlers enrolled in the HR‐NBL‐1/SIOPEN trial is associated with the level of neuroblastoma mRNAs at diagnosis

The purpose of this study was to evaluate whether levels of neuroblastoma mRNAs in bone marrow and peripheral blood from stage M infants (≤12 months of age at diagnosis, MYCN amplified) and toddlers (between 12 and 18 months, any MYCN status) predict event‐free survival (EFS).

Li-Fraumeni: ¿la detección de familias aumentaría la supervivencia entre sus miembros?

Li-Fraumeni syndrome is a rare cancer predisposition syndrome with an autosomal dominant pattern of inheritance and of variable phenotypic expression associated with germline mutations in gene TP53. This disease predisposes to the development of a wide variety of malignant tumours. The most frequent tumours are soft-tissue sarcomas (rhabdomyosarcoma and others), osteosarcoma, breast cancer in premenopausal women, hypodiploid leukaemia, brain tumours (choroid plexus carcinoma, glioblastoma and medulloblastoma) and adrenocortical carcinoma. These tumours may develop at any age, including in children. The prevalence of this syndrome is not well known, as it is without a doubt underdiagnosed. Since followup of families has not proven to improve long-term survival, to date no programme has been established for detection of affected individuals. Circumstances are changing in regard to this predisposition syndrome. According to a study published by Villani et al. in 2011 and updated in 2016, it is possible to carry out a followup that could potentially increase long-term survival. We present the case of a previously healthy boy aged 2 years that visited the emergency services of his local hospital due to a convulsive seizure (complex partial seizure). The evaluation started with imaging tests and, due to the suspicion of a space-occupying lesion, he was transferred to the referral hospital. The imaging tests revealed a malignant tumour with disseminated meningeal involvement throughout the neuraxis, and a primary lesion inside the brain

Pediatric Neuroblastoma: Use of Hypermethylation of Apoptotic Genes as a Prognostic Factor

Neuroblastoma, the most common solid extracraneal tumor in childhood, develops from immature or de-differentiated neural-crest derived cells. Prognosis depends on the patient’s age at diagnosis, tumor stage and MYCN oncogene amplification. Several established molecular parameters (DNA content, allelic loss in 1p and 11q and gain of genetic material in 17q) have been introduced as prognostic indicators, however, the molecular basis of NB development and progression remains poorly understood. Epigenetic mechanisms, such as DNA hypermethylation, are important regulators of gene expression and are frequently involved in silencing tumor suppressor genes. A clinically relevant methylation profile in NB has recently been studied using different screening techniques and hypermethylation of apoptotic genes such as caspase-8, have been identified as good prognostic indicators, emphasizing the potential use of epigenetic biomarkers for prognosis purposes.