PURPOSE: Multicolor flow cytometry (MFC) is widely available, fast and has an easy-to perform approach for finding neuroblastoma (NB) cells among normal bone marrow (BM) hematopoietic cells. Aim of the study was to investigate prognostic significance of initial MFC tumor cells' detection in BM of children with NB.
METHODS: 51 patients (24 boys and 27 girls) aged from 6 days to 15 years (median age 1 year 3 months) with NB were included in the study. BM samples at the time of diagnosis were obtained from 2 to 5 aspiration sites per patient. CD45(-)CD56(+)CD81(+)GD2(+)-cells were evaluated by MFC.
RESULTS: NB cells were detected in BM by FC more frequently compared to conventional cytomorphology (49.0% and 29.4% patients, respectively, р = 0.043). Patients with NB cells detected in BM by MFC had significantly worse event-free survival and cumulative incidence of relapse/progression [0.24(0.08) and 0.60(0.10), respectively] compared to children with negative result of immunophenotyping [0.85(0.07) and 0.12(0.06), respectively, p < 0.001 in both cases]. BM involvement detection by MFC maintained its prognostic significance in various patients groups. In multivariate analysis, immunophenotyping proved to be an independent prognostic factor when analyzed jointly with other NB risk factors. In 42 patients BM involvement was also studied by RQ-PCR for PHOX2B and TH genes expression. Within groups of patients divided by RQ-PCR positivity, MFC-positivity retained prognostic significance.
CONCLUSIONS: Thus flow cytometric BM involvement detection has very strong prognostic impact even stronger than RQ-PCR. It could be used in combination with other parameters for the treatment strategy choice in patients with NB.

Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively

BACKGROUND: Neuroblastoma is the most common pediatric extracranial solid tumor. Germline pathogenic variants in ALK and PHOX2B, as well as other cancer predisposition genes, are increasingly implicated in the pathogenesis of neuroblastic tumors. A challenge for clinicians is the identification of children with neuroblastoma who require genetics evaluation for underlying cancer predisposition syndromes (CPS).
PROCEDURE: We developed a decisional algorithm (MIPOGG) to identify which patients with neuroblastic tumors have an increased likelihood of an underlying CPS. This algorithm, comprising 11 Yes/No questions, evaluates features in the tumor, personal and family history that are suggestive of an underlying CPS. We assessed the algorithm's performance in a retrospective cohort.
RESULTS: Two hundred and nine of 278 consecutive patients with neuroblastic tumors at The Hospital for Sick Children (2007-2016) had sufficient clinical data for retrospective application of the decisional algorithm. Fifty-one of 209 patients had been referred to genetics for CPS evaluation; 6/51 had a genetic or clinical confirmation of a CPS. The algorithm correctly identified all six children (Beckwith-Wiedemann (n = 2), Fanconi anemia, RB1, PHOX2B, chromosome duplication involving ALK) as requiring a genetic evaluation by using clinical features present at diagnosis. The level of agreement between the algorithm and physicians was 83.9%, with 15 more patients identified by the algorithm than by physicians as requiring a genetics referral.
CONCLUSIONS: This decisional algorithm appropriately detected all patients who, following genetic evaluation, were confirmed to have a CPS and may improve the detection of CPS in patients with neuroblastic tumors compared with current practice.

BACKGROUND: Detection of bone marrow (BM) involvement in patients with neuroblastoma is crucial for staging and defining prognosis. Furthermore, the persistence of residual tumor cells in the BM is associated with an unfavorable outcome.
METHODS: Expression of PHOX2B, TH, ELAVL4, and B4GALNT1 (GD2-synthase) was analyzed by quantitative polymerase chain reaction in neuroblastoma cell lines, control BM samples, and in BM samples from patients. The threshold level of expression for each gene was established through receiver operator characteristic analysis and used to determine the diagnostic test performance. The prognostic significance of BM involvement was assessed by survival rates calculations. The median of follow-up time was 36.1 months.
RESULTS: Neither PHOX2B nor TH expression was detected in control BM, while expression of ELAVL4 was found in 20 (76.9%) and GD2-synthase in 15 (57.7%) of 26 samples. The overall correct predictive value for TH, ELAVL4, and GD2-synthase, based on thresholds levels, was 0.952, 0.828, and 0.767, respectively, whereas the overall correct predictive value for PHOX2B was 0.994. The PHOX2B/TH expression in diagnostic BM of patients with neuroblastoma corresponded with a decreased survival rate (P < 0.001) in the total cohort and in different risk groups. Predominance of normalized expression of PHOX2B over TH > 1.68 in the diagnostic BM samples demonstrated an adverse prognostic effect (P = 0.006). Persistence of PHOX2B/TH expression in the BM during and after induction chemotherapy resulted in dismal outcome (P = 0.022 and P = 0.012).
CONCLUSION: PHOX2B and TH are the most optimal markers for detection of BM involvement, allowing identification of high-risk patients. Predominance of PHOX2B expression over TH has a strong adverse prognostic impact.

AIMS: The PHOX2B gene regulates neuronal maturation in the brain stem nuclei associated with cardiorespiratory function and in the autonomic sympathetic and enteric nervous system. PHOX2B expression is a reliable immunomarker for peripheral neuroblastic tumours; however, no systematic evaluation of central nervous system (CNS) embryonal tumours was included in the studies. We encountered two cases in which the differential diagnosis included neuroblastoma and CNS embryonal tumour, and we hypothesised that PHOX2B immunostain would be helpful in establishing the diagnosis.
METHODS AND RESULTS: PHOX2B immunostain was performed on 29 paediatric cases, with adequate controls: one retroperitoneal embryonal tumour in a child with retinoblastoma (index 1), one posterior fossa embryonal tumour in a child with a neuroblastoma (index 2), seven medulloblastomas, four atypical teratoid/rhabdoid tumours (ATRT), four retinoblastomas, six pineoblastomas, four embryonal tumours with multilayered rosettes (ETMR) and two CNS embryonal tumours, not elsewhere classified. Cell lineage immunomarkers (GFAP, OLIG2, synaptophysin, NeuN, CRX, PGP 9.5), immunosurrogates for molecular alterations (beta-catenin, INI1, Lin-28), array CGH and OncoPanel were performed as needed. Medulloblastomas, ATRTs, ETMRs, retinoblastomas and CNS embryonal tumours not elsewhere classified were essentially negative for PHOX2B. Two of six pineoblastomas had significant PHOX2B expression, while the rest were negative. Index 1 was negative for PHOX2B and PGP 9.5 and positive for CRX, consistent with retinoblastoma. Index 2 had diffuse PHOX2B expression, MYCN amplification and no copy number changes of medulloblastoma, in keeping with neuroblastoma.
CONCLUSION: PHOX2B antibody is helpful in distinguishing between peripheral neuroblastic and CNS embryonal tumours, which are immunonegative, with the caveat that a subset of pineoblastomas has significant expression.

Neuroblastoma is a cancer of neural crest stem cell (NCSC) lineage. Signaling pathways that regulate NCSC differentiation have been implicated in neuroblastoma tumorigenesis. This is exemplified by MYCN oncogene targets that balance proliferation, differentiation, and cell death similarly in normal NCSC and in high-risk neuroblastoma. Our previous work discovered a survival mechanism by which MYCN-amplified neuroblastoma circumvents cell death by upregulating components of the error-prone non-canonical alternative nonhomologous end-joining (alt-NHEJ) DNA repair pathway. Similar to proliferating stem cells, high-risk neuroblastoma cells have enhanced DNA repair capacity, overcoming DNA damage with higher repair efficiency than somatic cells. Adequate DNA maintenance is required for lineage protection as stem cells proliferate and during tumor progression to overcome oncogene-induced replication stress. On this basis, we hypothesized that alt-NHEJ overexpression in neuroblastoma is a cancer cell survival mechanism that originates from DNA repair systems of NCSC, the presumed progenitor cell of origin. A human NCSC model was generated in which inducible MYCN triggered an immortalized phenotype capable of forming metastatic neuroectodermal tumors in mice, resembling human neuroblastoma. Critical alt-NHEJ components (DNA Ligase III, DNA Ligase I, and Poly [ADP-ribose polymerase 1]) were highly expressed in normal early NCSC, and decreased as cells became terminally differentiated. Constitutive MYCN expression maintained high alt-NHEJ protein expression, preserving the expression pattern of the immature neural phenotype. siRNA knockdown of alt-NHEJ components reversed MYCN effects on NCSC proliferation, invasion, and migration. DNA Ligase III, Ligase I, and PARP1 silencing significantly decreased neuroblastoma markers expression (TH, Phox2b, and TRKB). These results utilized the first human NCSC model of neuroblastoma to uncover an important link between MYCN and alt-NHEJ expression in developmental tumor initiation, setting precedence to investigate alt-NHEJ repair mechanics in neuroblastoma DNA maintenance.

Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

Retinoblastoma (RB) is the most common intraocular malignancy in childhood. Approximately 40% of retinoblastomas are hereditary and due to germline mutations in the

Until recently, the genetic basis of neuroblastoma, a heterogeneous neoplasm arising from the developing sympathetic nervous system, remained undefined. The discovery of gain-of-function mutations in the ALK receptor tyrosine kinase gene as the major cause of familial neuroblastoma led to the discovery of identical somatic mutations and rapid advancement of ALK as a tractable therapeutic target. Inactivating mutations in a master regulator of neural crest development, PHOX2B, have also been identified in a subset of familial neuroblastomas. Other high penetrance susceptibility alleles likely exist, but together these heritable mutations account for less than 10% of neuroblastoma cases. A genome-wide association study of a large neuroblastoma cohort identified common and rare polymorphisms highly associated with the disease. Ongoing resequencing efforts aim to further define the genetic landscape of neuroblastoma.

Neuroblastoma is one of the most common cancers in infancy, arising from the neuroblasts during embryonic development. This cancer is difficult to treat and resistance to chemotherapy is often found; therefore, clinical trials of novel therapeutic approaches, such as targeted-cancer signaling, could be an alternative for a better treatment. WNT signaling plays significant roles in the survival, proliferation, and differentiation of human neuroblastoma. In this report, WNT signaling of a malignant human neuroblastoma cell line, SH-SY5Y cells, was inhibited by XAV939, a specific inhibitor of the Tankyrase enzyme. XAV939 treatment led to the reduction of β-catenin within the cells, confirming its inhibitory effect of WNT. The inhibition of WNT signaling by XAV939 did not affect cell morphology, survival, and proliferation; however, the differentiation and sensitivity to anticancer drugs of human neuroblastoma cells were altered. The treatment of XAV939 resulted in the downregulation of mature neuronal markers, including β-tubulin III, PHOX2A, and PHOX2B, whereas neural progenitor markers (PAX6, TFAP2α, and SLUG) were upregulated. In addition, the combination of XAV939 significantly enhanced the sensitivity of SH-SY5Y and IMR-32 cells to doxorubicin in both 2D and 3D culture systems. Microarray gene expression profiling suggested numbers of candidate target genes of WNT inhibition by XAV939, in particular, p21, p53, ubiquitin C, ZBED8, MDM2, CASP3, and FZD1, and this explained the enhanced sensitivity of SH-SY5Y cells to doxorubicin. Altogether, these results proposed that the altered differentiation of human malignant neuroblastoma cells by inhibiting WNT signaling sensitized the cells to anticancer drugs. This approach could thus serve as an effective treatment option for aggressive brain malignancy.

PHOX2B and its paralogue gene PHOX2A are two homeodomain proteins in the network regulating the development of autonomic ganglia that have been associated with the pathogenesis of neuroblastoma (NB), because of their over-expression in different NB cell lines and tumour samples. We used the SK-N-BE(2)C cell line to show that all-trans retinoic acid (ATRA), a drug that is widely used to inhibit growth and induce differentiation in NBs, regulates both PHOX2A and PHOX2B expression, albeit by means of different mechanisms: it up-regulates PHOX2A and down-regulates PHOX2B. Both mechanisms act at transcriptional level, but prolonged ATRA treatment selectively degrades the PHOX2A protein, whereas the corresponding mRNA remains up-regulated. Further, we show that PHOX2A is capable of modulating PHOX2B expression, but this mechanism is not involved in the PHOX2B down-regulation induced by retinoic acid. Our findings demonstrate that PHOX2A expression is finely controlled during retinoic acid differentiation and this, together with PHOX2B down-regulation, reinforces the idea that they may be useful biomarkers for NB staging, prognosis and treatment decision making.

Paired like homeobox 2B (PHOX2B) is a minimal residual disease (MRD) marker of neuroblastoma. The presence of MRD, also referred to as micro-metastases, is a powerful marker of poor prognosis in neuroblastoma. Lung metastasis is considered a terminal event in neuroblastoma. Lung micro-metastatic neuroblastoma (MicroNB) cells show high expression levels of PHOX2B and possess a less malignant and metastatic phenotype than lung macro metastatic neuroblastoma (MacroNB) cells, which hardly express PHOX2B. In vitro assays showed that PHOX2B knockdown in MicroNB cells did not affect cell viability; however it decreased the migratory capacity of the MicroNB-shPHOX2B cells. An orthotopic inoculation of MicroNB-shPHOX2B cells into the adrenal gland of nude mice resulted in significantly larger primary tumors and a heavier micro-metastatic load in the lungs and bone-marrow, than when control cells were inoculated. PHOX2B expression was found to be regulated by methylation. The PHOX2B promoter in MacroNB cells is significantly more methylated than in MicroNB cells. Demethylation assays using 5-azacytidine demonstrated that methylation can indeed inhibit PHOX2B transcription in MacroNB cells. These pre-clinical data strongly suggest that PHOX2B functions as a suppressor of neuroblastoma progression.

Neuroblastoma (NB) is a rare childhood cancer of the peripheral sympathetic nervous system and accounts for approximately 10% of all pediatric tumors. Heterozygous PHOX2B mutations have been found in association with NB development in familial, sporadic and syndromic cases. In addition, the PHOX2B gene is widely over-expressed both in tumor samples and NB cell lines. Post-transcriptional gene regulation is known to be involved in mRNA stability and, in NB, microRNAs (miRNAs) seem to be responsible for altered expression of genes driving differentiation, apoptosis, and migration. To assess the possible impact of post-transcriptional regulation in NB cell lines, we have focused on the PHOX2B mRNA stability by both in silico analysis and functional studies on its 3'untranslated region (3'UTR). PHOX2B gene expression has resulted under post-transcriptional control, as suggested by: i) instability of PHOX2B mRNA, demonstrated by short mRNA half-life levels in both IMR32 and LAN-1 cell lines, ii) role of the PHOX2B-3'UTR, confirmed by the activity of proper reporter constructs, and iii) miRNA-204, shown to enhance the PHOX2B 3'UTR mediated down-regulation of the reporter construct activity. Finally, miRNA-204 has resulted to decrease the stability of the PHOX2B mRNA at different extents in the presence of different SNP rs1063611 alleles. Therefore, post-transcriptional down-regulation of the PHOX2B gene takes place in NB cell lines and miRNA-204 participates in such a 3'UTR mediated control.

PHOX2B is a transcription factor involved in the regulation of neurogenesis and in the correct differentiation of the autonomic nervous system. The pathogenetic role of PHOX2B in neuroblastoma (NB) is supported by mutations in familial, sporadic and syndromic cases of NB and overexpression of PHOX2B and its target ALK in tumor samples and NB cell lines. Starting from these observations, we have performed in vitro drug screening approaches targeting PHOX2B overexpression as a potential pharmacological means in NB. In particular, in order to identify molecules able to decrease PHOX2B expression, we have evaluated the effects of 70 compounds in IMR-32 cell line stably expressing the luciferase gene under the control of the PHOX2B promoter. Curcumin, SAHA and trichostatin A showed to down-regulate the PHOX2B promoter activity which resulted in a decrease of both protein and mRNA expressions. In addition, we have observed that curcumin acts by interfering with PBX-1/MEIS-1, NF-κB and AP-1 complexes, in this work demonstrated for the first time to regulate the transcription of the PHOX2B gene. Finally, combined drug treatments showed successful effects in down-regulating the expression of both PHOX2B and its target ALK genes, thus supporting the notion of the effectiveness of molecule combination in tumor therapy.

Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system which accounts for 8-10% of pediatric cancers. It is characterized by a broad spectrum of clinical behaviors from spontaneous regression to fatal outcome despite aggressive therapies. Considerable progress has been made recently in the germline and somatic genetic characterization of patients and tumors. Indeed, predisposition genes that account for a significant proportion of familial and syndromic cases have been identified and genome-wide association studies have retrieved a number of susceptibility loci. In addition, genome-wide sequencing, copy-number and expression studies have been conducted on tumors and have detected important gene modifications, profiles and signatures that have strong implications for the therapeutic stratification of patients. The identification of major players in NB oncogenesis, including MYCN, ALK, PHOX2B and LIN28B, has enabled the development of new animal models. Our review focuses on these recent advances, on the insights they provide on the mechanisms involved in NB development and their applications for the clinical management of patients.

The neurocristopathy syndrome occurs because of a germline mutation of the paired-like homeobox 2b (PHOX2B) gene at 4p12, a neurogenesis regulator gene. The result is abnormal neural crest cell development resulting in congenital central hypoventilation syndrome, Hirschsprung disease, and neuroblastoma (NB), which is often multifocal and disseminated in its presentation. Previously, such widespread disease was regarded as highly aggressive and treated either with palliative intent or, conversely, with very intense, high-dose chemotherapy. We now present a patient who had neurocristopathy syndrome who had multifocal NB associated with an underlying germline PHOX2B mutation. He was treated conservatively with surgery and low-dose chemotherapy. After treatment he had extensive residual disease that has continued to mature despite no further treatment. A literature review identified 26 similar patients presenting with multifocal NB as part of the neurocristopathy syndrome. In all cases the NB behaved in an indolent manner with no deaths from tumor reported when patients received appropriate treatment. These provocative findings suggest for the first time that children who have neurocristopathy-associated NB should be treated conservatively, despite the aggressive appearance of their disease.

Bone marrow (BM) involvement in neuroblastoma patients is commonly detected by cytomorphology and associated with poor outcome. Molecular techniques, flow cytometry and immunocytochemistry were offered to detect low number of tumor cells in BM due to high value of analytical sensitivity, while prognostic significance of results, obtained with these methods is unclear. PHOX2B and/or TH genes expression was selected as molecular marker of BM involvement. It was determined in 411 BM samples obtained from 75 neuroblastoma patients. 263 BM samples were taken at the time of primary diagnosis, 80 during treatment and 68 before autologous stem cells (ASC) apheresis. Prognostic significance of BM involvement was defined using 5-year (in some groups 4-year) overall (OS), event free (EFS) and progression free (PFS) survival. 24 patients (32.0%) were positive for PHOX2B and/or TH expression in the BM at the time of primary diagnosis. They had decreased survival rates: EFS achieved 0.49+/-0.12, OS - 0.57+/-0.12, PFS - 0.54+/-0.12, comparing with 0.75+/-0.07, 0.80+/-0.07 and 0.77+/-0.07, respectively, in patients with negative BM, p=0.014, p=0.029 and p=0.033. The trend to decreased OS and PFS was detected in case of minimal residual disease presence at the end of the induction chemotherapy (OS and PFS both are 0.22+/-0.19 vs. 0.70+/-0.18 and 0.43+/-0.22, correspondingly, p=0.121, p=0.130). Detection of PHOX2B and/or TH genes expression in the BM before ASC harvesting led to significant decreasing of EFS and OS (0.00 vs. 0.59+/-0.14 and 0.75+/-0.13, respectively, p=0.021 and p=0.016).

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.

Heterozygous germline mutations in PHOX2B, a transcriptional regulator of sympathetic neuronal differentiation, predispose to diseases of the sympathetic nervous system, including neuroblastoma and congenital central hypoventilation syndrome (CCHS). Although the PHOX2B variants in CCHS largely involve expansions of the second polyalanine repeat within the C-terminus of the protein, those associated with neuroblastic tumors are nearly always frameshift and truncation mutations. To test the hypothesis that the neuroblastoma-associated variants exert their effects through loss or gain of protein-protein interactions, we performed a large-scale yeast two-hybrid screen using both wild-type (WT) and six different mutant PHOX2B proteins against over 10 000 human genes. The neuronal calcium sensor protein HPCAL1 (VILIP-3) exhibited strong binding to WT PHOX2B and a CCHS-associated polyalanine expansion mutant but only weakly or not at all to neuroblastoma-associated frameshift and truncation variants. We demonstrate that both WT PHOX2B and the neuroblastoma-associated R100L missense and the CCHS-associated alanine expansion variants induce nuclear translocation of HPCAL1 in a Ca(2+)-independent manner, while the neuroblastoma-associated 676delG frameshift and K155X truncation mutants impair subcellular localization of HPCAL1, causing it to remain in the cytoplasm. HPCAL1 did not appreciably influence the ability of WT PHOX2B to transactivate the DBH promoter, nor did it alter the decreased transactivation potential of PHOX2B variants in 293T cells. Abrogation of the PHOX2B-HPCAL1 interaction by shRNA knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B led to impaired neurite outgrowth with transcriptional profiles indicative of inhibited sympathetic neuronal differentiation. Our results suggest that certain PHOX2B variants associated with neuroblastoma pathogenesis, because of their inability to bind to key interacting proteins such as HPCAL1, may predispose to this malignancy by impeding the differentiation of immature sympathetic neurons.

Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b(+), ascl1(+), elavl3(-) cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.

Minimal residual disease (MRD) is derived from tumor-initiating cells (TICs) and is responsible for tumor relapse. Neuroblastoma is characterized by extreme tumor heterogeneity, and more than half of high-risk patients experience tumor relapse. To overcome tumor heterogeneity and achieve more sensitive detection of MRD, several sets of real-time RT-PCR markers have been reported for MRD monitoring in neuroblastoma patients from different centers. However, these markers vary across centers and are still being validated. In the present study, we validated the ability of 14 commonly used real-time RT-PCR markers to detect MRD based on their expression in neuroblastoma TICs, and we developed a novel MRD detection protocol, which scored the samples as MRD-positive when the expression of one of the 11 real-time RT-PCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) exceeded the normal range. By using this protocol, we prospectively monitored MRD in 73 bone marrow (BM), 12 peripheral blood stem cell and 8 peripheral blood samples from 14 neuroblastoma patients treated at a single center. We scored 100, 56, 56 and 57% BM cytology-positive, elevated vanillylmandelic acid (VMA), elevated homovanillic acid (HVA) and elevated neuron-specific enolase (NSE) samples as MRD-positive, respectively. MRD was also positive in 48, 45, 46 and 43% of the BM cytology-negative and normal VMA, normal HVA and normal NSE samples, respectively. These results suggest that the present MRD detection protocol based on the expression of a set of 11 real-time RT-PCR markers in neuroblastoma TICs achieves sensitive MRD monitoring in neuroblastoma patients.

Neuroblastoma is a genetically and clinically heterogeneous tumor of childhood, arising from precursor cells of the sympathetic nervous system. It is still a challenging cancer for pediatric oncology, as some tumors will spontaneously regress, while others will become refractory to all forms of therapy. The clinical course of this disease is greatly influenced by both patient age and the genetic abnormalities that occur within the tumors. MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)) amplification and loss of chromosome 11q heterozygosity have been known to be indicative of poor prognosis. In this article, we review how mutations and structural alterations in specific genes contribute to inheritable predisposition to neuroblastoma and/or to aggressive disease pathogenesis, as well as implications for diagnosis and therapy. These genes include PHOX2B (paired-like homeobox 2b), ALK (anaplastic lymphoma receptor tyrosine kinase), and ATRX (alpha thalassemia/mental retardation syndrome X-linked).

The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse Phox2b locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine β-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.

To identify novel hypermethylated genes in colorectal cancer (CRC) and to test their potential application in CRC early diagnosis, a genome-wide screening of 57,723 CpG dinucleotides covering 4,010 genes was performed using MIRA-based microarrays in paired DNA samples extracted from 3 fresh frozen CRC tissues and their matching non-cancer tissues from 3 CRC patients undergoing curative surgery. Candidate hypermethylated genes screened by MIRA-based microarrays were further validated in independent CRC samples. A total of 297 CpG dinucleotides covering 211 genes were found to be hypermethylated in CRC tissues. From these 211 candidate methylated genes, three novel hypermethylated genes with more than four probes positive were picked up for validation. Direct bisulfite sequencing revealed that methylations occurred at multiple CpG sites of these three genes in cancer tissues, especially for PHOX2B and FGF12. Combined bisulfite restriction analysis showed that these three genes were methylated in cancer samples but not in non-cancer samples. We also compared the methylation levels of these three novel hypermethylated genes with those of vimentin and SEPT9, well-known hypermethylated genes in CRC, and found that methylated PHOX2B, FGF12 and GAD2 were better than methylated vimentin and SEPT9 in differentiating CRC cancer tissue from non-cancer tissue. Significant enrichment analysis of GO terms of the hypermethylated genes showed that a high proportion of hypermethylated genes in cancer tissues are involved in the regulation of transcription. In conclusion, we found a set of novel hypermethylated genes in CRC, which may have potential to be used as biomarkers for the early diagnosis of CRC.

BACKGROUND: The congenital central hypoventilation syndrome (CCHS) or Ondine's curse is a rare autosomal dominant disease, characterized by disorders of the autonomic nervous system, with abnormal ventilatory responses to hypercapnia and hypoxia. PHOX2B has been identified as the major gene causing CCHS. It results from polyalanine repeat expansion mutations. It typically presents in the newborn period but some cases have been described in adults (late onset CCHS) reflecting the variable penetrance of PHOX2B mutations.
CASE REPORT: A 48 year-old woman presented, after ovarian cyst surgery, with severe hypoventilation requiring intubation. Arterial blood gases revealed a PaO2 of 6.6kPa (50mmHg), a PaCO2 of 10kPa (80mmHg) and a pH of 7.22. The past medical history revealed nocturnal symptoms for a few years. These included apnoeas, fitful sleep and awakening with headaches. Physical examination, pulmonary function tests, lung tomography and magnetic resonance imaging of the brainstem were all normal. Polysomnography revealed numerous central and obstructive apnoeas and hypopnoeas, with severe hypoxaemia and hypercapnia. Hypoxic and hypercapnic stimulation tests showed no adaptation of the ventilatory responses. Genetic analysis showed a heterozygous five alanine expansion mutation of the 20-residue polyalanine tract in exon 3 of the PHOX2B gene.
CONCLUSION: The diagnosis of late onset CCHS should be considered in patients with unexplained hypoventilation, and physiological evaluation should be undertaken to document the abnormal ventilatory responses. The presence of a PHOX2B mutation confirms the diagnosis.

In neuroblastoma (NB) patients, minimal residual disease (MRD) can be detected by real-time quantitative PCR (qPCR) using NB-specific target genes, such as PHOX2B and TH. However, it is unknown whether the mRNA levels of these targets vary either during treatment or at relapse. If marker genes are not stably expressed, estimation of MRD levels in bone marrow (BM) or peripheral blood will be hampered. We studied the stability of a panel of qPCR markers in primary tumors at diagnosis compared with i) paired metastasis (n = 7), ii) treated (n = 10), and iii) relapse (n = 6) tumors. We also compared relative expression of the targets in iv) primary tumors and BM at diagnosis (n = 17), v) BM and peripheral blood at diagnosis (n = 20), vi) BM at diagnosis and during treatment (n = 26), and vii) BM from different puncture sides (n = 110). Especially at diagnosis, PCR target expression is quite stable. Accurate quantification is possible when expression level can be related to the primary tumor; however, PCR target expression can alter on treatment and at relapse. If the median value of relative expression of a panel of PCR targets is used, most variations due to treatment and outgrowth of subclones level out, allowing for reliable application and quantification of MRD-PCR targets in NB patients.

The bone marrow (BM) TH, ELAVL4 and GD2 genes expression was evaluated in 331 samples from 57 different stage neuroblastoma (NB) patients, 26 BM samples from patients without NB and samples from 2 NB cell lines (IMR-32, Kelly) by real-time PCR. BM samples were considered NB-positive if PHOX2B expression was found or tumor cells were detected in BM smears. TH expression was not revealed in normal BM and was significantly lower in NB-negative samples. Expression of PHOX2B, TH and GD2 remained stable throughout NB treatment, while ELAVL4 expression was down-modulated. ROC-analysis revealed similar initial and follow-up values of TH and PHOX2B in NB patients' bone marrow making it possible to be used for disease detection and monitoring. The test prediction value was 0.994 and 0.952, respectively. The additional test for TH didn't increase the test effectiveness in comparison with PHOX2B test. ELAVL4 and GD2 assessment didn't add diagnostic value for BM involvement monitoring in NB patients.

Neuroblastic tumours may occur in a predisposition context. Two main genes are involved: PHOX2B, observed in familial cases and frequently associated with other neurocristopathies (Ondine's and Hirschsprung's disease); and ALK, mostly in familial tumours. We have assessed the frequency of mutations of these two genes in patients with a presumable higher risk of predisposition. We sequenced both genes in 26 perinatal cases (prebirth and <1 month of age, among which 10 were multifocal), 16 multifocal postnatal (>1 month) cases, 3 pairs of affected relatives and 8 patients with multiple malignancies. The whole coding sequences of the two genes were analysed in tumour and/or constitutional DNAs. We found three ALK germline mutations, all in a context of multifocal tumours. Two mutations (T1151R and R1192P) were inherited and shared by several unaffected patients, thus illustrating an incomplete penetrance. Younger age at tumour onset did not seem to offer a relevant selection criterion for ALK analyses. Conversely, multifocal tumours might be the most to benefit from the genetic screening. Finally, no PHOX2B germline mutation was found in this series. In conclusion, ALK deleterious mutations are rare events in patients with a high probability of predisposition. Other predisposing genes remain to be discovered.

BACKGROUND: Perturbation of DNA methylation is frequent in cancers and has emerged as an important mechanism involved in tumorigenesis. To determine how DNA methylation is modified in the genome of primary glioma, we used Methyl-DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays to identify differentially DNA methylation sequences between primary glioma and normal brain tissue samples.
METHODS: MeDIP-chip technology was used to investigate the whole-genome differential methylation patterns in glioma and normal brain tissues. Subsequently, the promoter methylation status of eight candidate genes was validated in 40 glioma samples and 4 cell lines by Sequenom's MassARRAY system. Then, the epigenetically regulated expression of these genes and the potential mechanisms were examined by chromatin immunoprecipitation and quantitative real-time PCR.
RESULTS: A total of 524 hypermethylated and 104 hypomethylated regions were identified in glioma. Among them, 216 hypermethylated and 60 hypomethylated regions were mapped to the promoters of known genes related to a variety of important cellular processes. Eight promoter-hypermethylated genes (ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3, and SST) were confirmed in primary glioma and cell lines. Aberrant promoter methylation and changed histone modifications were associated with their reduced expression in glioma. In addition, we found loss of heterozygosity (LOH) at the miR-185 locus located in the 22q11.2 in glioma and induction of miR-185 over-expression reduced global DNA methylation and induced the expression of the promoter-hypermethylated genes in glioma cells by directly targeting the DNA methyltransferases 1.
CONCLUSION: These comprehensive data may provide new insights into the epigenetic pathogenesis of human gliomas.