Neuroblastoma - Molecular Biology


Neuroblastoma tumour cells are characterised by a wide diversity of somatic genetic mutations. Some common genetic features include:
  • Amplification of the MYCN gene is one of the most established genetic prognostic factors. Amplified tumours are mostly (though not exclusively) found in children aged over 1 year at diagnosis with advanced stage disease . Other genes, such as DDX1 are often co-amplified with MYCN.
  • Deletion of material from the chromosome 1p36 region is also associated with adverse prognosis. This is thought to be a candidate region for a suppressor gene which has yet to be identified.
  • Gain of 17q material is the most frequent genetic abnormality in neuroblastoma. Unbalanced 17q gain is an adverse prognostic factor and is strongly associated with adverse clinical features, 1p deletion, and MYCN amplification.
  • Expression of TRKA in contrast is a favourable genetic feature. This is associated with low stage and age under 1 yrear at diagnosis. TRKA is frequently supressed in MYCN amplified Tumours. Other members of the TRK neurotrophine receptor gene family, TRKB and TRKC, are also implicated in neuroblastoma.
See also: Familial Neuroblastoma and Genetic Susceptibility.

See also: Neuroblastoma - clinical resources (21)

Literature Analysis

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Tag cloud generated 08 August, 2015 using data from PubMed, MeSH and CancerIndex

Mutated Genes and Abnormal Protein Expression (100)

How to use this data tableClicking on the Gene or Topic will take you to a separate more detailed page. Sort this list by clicking on a column heading e.g. 'Gene' or 'Topic'.

MYCN 2p24.3 NMYC, ODED, MODED, N-myc, bHLHe37 Amplification
-MYCN amplification in Neuroblastoma
-ABCC1 (MRP1) Overexpression in Neuroblastoma
CASP8 2q33-q34 CAP4, MACH, MCH5, FLICE, ALPS2B, Casp-8 Methylation
-CASP8 Inactivation in Neuroblastoma
NTRK2 9q22.1 TRKB, trk-B, GP145-TrkB Prognostic
-NTRK2 expression in Neuroblastoma
ALK 2p23 CD246, NBLST3 -ALK and Neuroblastoma
-ALK mutations in Familial Neuroblastoma
PHOX2B 4p12 PMX2B, NBLST2, NBPhox -PHOX2B germline mutations in familial neuroblastoma
-PHOX2B and Neuroblastoma
-PHOX2B and Monitoring of Residual Disease
CD44 11p13 IN, LHR, MC56, MDU2, MDU3, MIC4, Pgp1, CDW44, CSPG8, HCELL, HUTCH-I, ECMR-III -CD44 and Neuroblastoma
BIRC5 17q25 API4, EPR-1 Overexpression
-Survivin Expression in Neuroblastoma
NTRK3 15q25 TRKC, gp145(trkC) Prognostic
-NTRK3 expression in Neuroblastoma
TP53 17p13.1 P53, BCC7, LFS1, TRP53 -P53 and Neuroblastoma
ID2 2p25 GIG8, ID2A, ID2H, bHLHb26 -ID2 Expression in Neuroblastoma
BDNF 11p13 ANON2, BULN2 -BDNF and Neuroblastoma
DDX1 2p24 DBP-RB, UKVH5d Amplification
-DDX1 Amplification in Neuroblastoma
NME1 17q21.3 NB, AWD, NBS, GAAD, NDKA, NM23, NDPKA, NDPK-A, NM23-H1 -NME1 and Neuroblastoma
NRAS 1p13.2 NS6, CMNS, NCMS, ALPS4, N-ras, NRAS1 -NRAS and Neuroblastoma
NTRK1 1q21-q22 MTC, TRK, TRK1, TRKA, Trk-A, p140-TrkA Prognostic
-NTRK1 expression in Neuroblastoma
REST 4q12 XBR, NRSF -REST and Neuroblastoma
PTER 10p12 HPHRP, RPR-1 -PTER and Neuroblastoma
NGFR 17q21-q22 CD271, p75NTR, TNFRSF16, p75(NTR), Gp80-LNGFR -NGFR and Neuroblastoma
VEGFA 6p12 VPF, VEGF, MVCD1 -VEGFA Expression in Neuroblastoma
FGF2 4q26 BFGF, FGFB, FGF-2, HBGF-2 -FGF2 and Neuroblastoma
ASCL1 12q23.2 ASH1, HASH1, MASH1, bHLHa46 -ASCL1 and Neuroblastoma
MAX 14q23 bHLHd4 -MAX and Neuroblastoma
KIF1B 1p36.2 KLP, CMT2, CMT2A, CMT2A1, HMSNII, NBLST1 -KIF1B and Neuroblastoma
CHD5 1p36.31 CHD-5 -CHD5 and Neuroblastoma
PARK2 6q25.2-q27 PDJ, PRKN, AR-JP, LPRS2 -PARK2 and Neuroblastoma
IGF1R 15q26.3 IGFR, CD221, IGFIR, JTK13 -IGF1R Expression in Neuroblastoma
EFNB2 13q33 HTKL, EPLG5, Htk-L, LERK5 -EFNB2 expression in Neuroblastoma
ABCC1 16p13.1 MRP, ABCC, GS-X, MRP1, ABC29 Overexpression
-ABCC1 (MRP1) Overexpression in Neuroblastoma
VIP 6q25 PHM27 -VIP and Neuroblastoma
VEGFC 4q34.3 VRP, Flt4-L, LMPH1D -VEGFC Expression in Neuroblastoma
TOP1 20q12-q13.1 TOPI -TOP1 and Neuroblastoma
MAGEA1 Xq28 CT1.1, MAGE1 -MAGEA1 and Neuroblastoma
BARD1 2q34-q35 -BARD1 polymorphisms in Neuroblastoma
MAGEA3 Xq28 HIP8, HYPD, CT1.3, MAGE3, MAGEA6 -MAGEA3 and Neuroblastoma
TGFA 2p13 TFGA -TGFA Expression in Neuroblastoma
EPHB6 7q33-q35 HEP -EPHB6 and Neuroblastoma
NME2 17q21.3 PUF, NDKB, NDPKB, NM23B, NDPK-B, NM23-H2 -NME2 and Neuroblastoma
BCHE 3q26.1-q26.2 E1, CHE1, CHE2 -BCHE and Neuroblastoma
CAMTA1 1p36.31-p36.23 CANPMR -CAMTA1 and Neuroblastoma
TNFRSF10D 8p21 DCR2, CD264, TRUNDD, TRAILR4, TRAIL-R4 -TNFRSF10D and Neuroblastoma
CHAT 10q11.2 CMS6, CMS1A, CMS1A2, CHOACTASE -CHAT and Neuroblastoma
EFNB3 17p13.1 EFL6, EPLG8, LERK8 -EFNB3 Expression in Neuroblastoma
LMO1 11p15 TTG1, RBTN1, RHOM1 -LMO1 and Neuroblastoma
BIN1 2q14 AMPH2, AMPHL, SH3P9 -Reduced BIN1 expression in MYCN amplified Neuroblastoma
ATRX Xq21.1 JMS, SHS, XH2, XNP, ATR2, SFM1, RAD54, MRXHF1, RAD54L, ZNF-HX -ATRX and Neuroblastoma
DLK1 14q32 DLK, FA1, ZOG, pG2, DLK-1, PREF1, Delta1, Pref-1 -DLK1 and Neuroblastoma
TP73 1p36.3 P73 -TP73 and Neuroblastoma
CHGA 14q32 CGA -CHGA and Neuroblastoma
CDK5 7q36 PSSALRE -CDK5 and Neuroblastoma
NBL1 1p36.13 NB, DAN, NO3, DAND1, D1S1733E -NBL1 and Neuroblastoma
CNTF 11q12.2 HCNTF -CNTF and Neuroblastoma
HOXC6 12q13.3 CP25, HOX3, HOX3C, HHO.C8 -HOXC6 and Neuroblastoma
SOD1 21q22.11 ALS, SOD, ALS1, IPOA, hSod1, HEL-S-44, homodimer -SOD1 and Neuroblastoma
VEGFB 11q13 VRF, VEGFL -VEGFB Expression in Neuroblastoma
MIR107 10q23.31 MIRN107, miR-107 -MicroRNA mir-107 and Neuroblastoma
-LIN28B and Neuroblastoma
ZMYND10 3p21.3 BLU, FLU, CILD22 -ZMYND10 and Neuroblastoma
PIK3CD 1p36.2 APDS, PI3K, IMD14, p110D, P110DELTA -PIK3CD and Neuroblastoma
RBL2 16q12.2 Rb2, P130 -RBL2 and Neuroblastoma Differentiation
EPHB2 1p36.1-p35 DRT, EK5, ERK, CAPB, Hek5, PCBC, EPHT3, Tyro5 -EPHB2 Expression in Neuroblastoma
ODC1 2p25 ODC -ODC1 and Neuroblastoma
CDK9 9q34.1 TAK, C-2k, CTK1, CDC2L4, PITALRE -CDK9 and Neuroblastoma
ENO1 1p36.2 NNE, PPH, MPB1, ENO1L1 -ENO1 and Neuroblastoma
TNFRSF10C 8p22-p21 LIT, DCR1, TRID, CD263, TRAILR3, TRAIL-R3, DCR1-TNFR -TNFRSF10C and Neuroblastoma
PDGFA 7p22 PDGF1, PDGF-A -PDGFA and Neuroblastoma
P2RX7 12q24 P2X7 -P2RX7 and Neuroblastoma
MYCL 1p34.2 LMYC, L-Myc, MYCL1, bHLHe38 -MYCL and Neuroblastoma
CD81 11p15.5 S5.7, CVID6, TAPA1, TSPAN28 -CD81 and Neuroblastoma
RASSF5 1q32.1 RAPL, Maxp1, NORE1, NORE1A, NORE1B, RASSF3 Methylation
-RASSF5 methylation in neuroblastoma
HACE1 6q16.3 GWAS
-HACE1 and Neuroblastoma
EXTL1 1p36.1 EXTL -EXTL1 and Neuroblastoma
SSTR2 17q24 -SSTR2 and Neuroblastoma
CDK7 5q12.1 CAK1, HCAK, MO15, STK1, CDKN7, p39MO15 -CDK7 and Neuroblastoma
LMO4 1p22.3 -LMO4 and Neuroblastoma
TNFRSF25 1p36.2 DR3, TR3, DDR3, LARD, APO-3, TRAMP, WSL-1, WSL-LR, TNFRSF12 -TNFRSF25 and Neuroblastoma
SCFV 14 -SCFV and Neuroblastoma
LGI1 10q24 EPT, ETL1, ADLTE, ADPAEF, ADPEAF, IB1099, EPITEMPIN -LGI1 and Neuroblastoma
GAS7 17p13.1 MLL/GAS7 -GAS7 and Neuroblastoma
RASSF7 11p15.5 HRC1, HRAS1, C11orf13 Methylation
-RASSF7 methylation in neuroblastoma
POU2F2 19q13.2 OCT2, OTF2, Oct-2 -POU2F2 and Neuroblastoma
MAPKAPK2 1q32 MK2, MK-2, MAPKAP-K2 -MAPKAPK2 and Neuroblastoma
MCF2 Xq27 DBL, ARHGEF21 -MCF2 and Neuroblastoma
TERC 3q26 TR, hTR, TRC3, DKCA1, PFBMFT2, SCARNA19 -TERC Expression in Neuroblastoma
GAGE1 Xp11.23 CT4.1, GAGE-1 -GAGE1 and Neuroblastoma
ANGPT2 8p23.1 ANG2, AGPT2 -ANGPT2 Expression in Neuroblastoma
CD276 15q23-q24 B7H3, B7-H3, B7RP-2, 4Ig-B7-H3 -CD276 and Neuroblastoma
MIRLET7E 19q13.41 LET7E, let-7e, MIRNLET7E, hsa-let-7e -MicroRNA let-7e and Neuroblastoma
SRGAP3 3p25.3 WRP, MEGAP, SRGAP2, ARHGAP14 -SRGAP3 and Neuroblastoma
SEPT7 7p14.2 CDC3, CDC10, SEPT7A, NBLA02942 -SEPT7 Expression in Neuroblastoma
IL24 1q32 C49A, FISP, MDA7, MOB5, ST16, IL10B -IL24 and Neuroblastoma
EPAS1 2p21-p16 HLF, MOP2, ECYT4, HIF2A, PASD2, bHLHe73 -EPAS1 and Neuroblastoma
HOXD11 2q31.1 HOX4, HOX4F -HOXD11 and Neuroblastoma
PNN 14q21.1 DRS, DRSP, SDK3, memA -PNN and Neuroblastoma
RASSF6 4q13.3 Methylation
-RASSF6 methylation in neuroblastoma
LRRN2 1q32.1 GAC1, LRRN5, LRANK1, FIGLER7 -LRRN2 and Neuroblastoma
SSTR1 14q13 SS1R, SS1-R, SRIF-2, SS-1-R -SSTR1 Expression in Neuroblastoma
CDK12 17q12 CRK7, CRKR, CRKRS -CDK12 and Neuroblastoma
E2F3 6p22 E2F-3 -E2F3 and Neuroblastoma
PAFAH1B2 11q23 HEL-S-303 -PAFAH1B2 and Neuroblastoma
MYBL2 20q13.1 BMYB, B-MYB Prognostic
-MYBL2 and Neuroblastoma

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications

Niu H, Xu T, Wang F, et al.
[Prognostic significance of MYCN amplification in children neuroblastic tumors].
Zhonghua Bing Li Xue Za Zhi. 2015; 44(2):111-7 [PubMed] Related Publications
OBJECTIVE: To summarize the clinicopathologic features of neuroblastic tumors (NT), and to explore the prognostic significance of MYCN amplification in NT.
METHODS: The clinicopathologic data of 267 NT were reviewed. MYCN gene amplification was detected by fluorescence in situ hybridization (FISH) in 119 cases and the relationship with pathological characteristics and prognostic significance were analyzed.
RESULTS: The study included 267 cases of children NT from patients aged from 1 day to 13 years (median 27 months). The male to female ratio was 1.43. There were 38 cases (14.2%), 43 cases (16.1%), 71 cases (26.6%), and 115 cases (43.1%) of INSS stages I, II, III and IV respectively.Favorable histology group had 157 cases (59.9%); unfavorable histology group had 110 cases (40.1%).Of the 119 NT cases with MYCN FISH performed, 18 cases (15.1%) showed amplification and the signal ratio of MYCN to CEP2 was 4.08-43.29. One hundred and one cases of non-amplified MYCN included MYCN gain in 79 cases (66.3%) and MYCN negative in 22 cases (18.5%). MYCN expression showed significant difference (P = 0.000) between ages, gender, NT type and MKI, but not INPC and clinical stage (P > 0.05).Of the 18 cases with MYCN amplification, 3 were undifferentiated, and 15 poorly differentiated; 17 had high MKI and one moderate MKI. All 18 cases were in unfavorable histology group; the overall survival rate was 3/18, with an average survival time of (17.9 ± 2.4) months.Of the 101 MYCN non-amplification cases, the overall survival rate was 68.3% (69/101), with an average survival time of (29.8 ± 1.3) months. Survival analysis showed the cases with MYCN amplification had worse prognosis (P < 0.05).
CONCLUSIONS: NT were commonly diagnosed in early ages and easily to metastasize. Most of cases with favorable histology. The cases of MYCN amplification showed unfavorable histology, and the majority cases with high MKI; The patients with MYCN gene amplification had poor prognosis.

Vishniakova PA, Doronin II, Holodenko IV, et al.
[Caspases participarion in the cell death induced by GD2-specific monoclonal antibody].
Bioorg Khim. 2014 May-Jun; 40(3):305-14 [PubMed] Related Publications
The participation of the main caspases in the cytotoxic effects induced by monoclonal antibody 14G2a specific against tumor-associated ganglioside GD2 was studied in the EL-4 cells. It has been found constitutive expression ofprocaspases genes in the EL-4 cells; incubation of the cells with 14G2a antibodies didnot result in increasing of the procaspases expression. Weak enzymatic activity of caspases has been shown using fluorescent labeled substrates. At the same cell death level, activity of caspase-3 and caspase-9 in the cells incubated with 14G2a was about 7.5- and 3-fold lower than in cells after incubation with staurosporine. Pan caspase inhibitor Z-VAD-FMK, and caspase-3 inhibitor reduced the cytotoxic effects induced by 14G2a at 9-16 and 6-13%, respectively. At the same conditions, pan caspase inhibitor decreased staurosporine-induced apoptosis at 55-65%. Inhibitors of other caspases had no effect on the cell death triggered by the antibodies. Inhibition analysis demonstrated also that caspases did not involved in the cell volume decreasing and permeabilization of the cell plasma membrane, which were the first stages of anti-GD2-mAb-induced cell death in the EL-4 cells. Thus, despite the slight activation of caspases during the cell death induced by antibodies directed to GD2, they do not play a key role and do not determine the mechanism of cell death triggered through the tumor-associated ganglioside GD2.

Bell JL, Turlapati R, Liu T, et al.
IGF2BP1 harbors prognostic significance by gene gain and diverse expression in neuroblastoma.
J Clin Oncol. 2015; 33(11):1285-93 [PubMed] Related Publications
PURPOSE: Chromosomal 17q21-ter gain in neuroblastoma is both a common and prognostically significant event. The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) gene is located near the proximal edge of this region. Here, its prognostic value is evaluated in neuroblastoma.
METHODS: The mRNA expression of IGF2BP family members was first evaluated by microarray data sets. In addition, in a separate cohort of 69 tumors, IGF2BP1 gene copy number, mRNA, and protein abundance were determined and compared with clinical parameters.
RESULTS: In two independent microarray data sets, 77% to 100% of tumors had substantial IGF2BP1 mRNA levels measured. High IGF2BP1 transcript abundance was significantly associated with stage 4 tumors (P < .001) and decreased patient survival (P < .001). IGF2BP1 was also associated with MYCN gene amplification and MYCN mRNA abundance. In the 69 neuroblastoma samples, IGF2BP1 DNA copy number (increased in 84% of tumors), mRNA, and protein abundance were significantly higher in stage 4 compared with stage 1 tumors. Importantly, IGF2BP1 protein levels were associated with lower overall patient survival (P = .012) and positively correlated with MYCN mRNA, even when excluding MYCN-amplified tumors. Moreover, IGF2BP1 clearly affected MYCN expression and neuroblastoma cell survival in vitro.
CONCLUSION: In neuroblastoma, IGF2BP1 was expressed in the majority of neuroblastoma specimens analyzed and was associated with lower overall patient survival and MYCN abundance. These data demonstrate that IGF2BP1 is a potential oncogene and an independent negative prognostic factor in neuroblastoma.

Dang CV
Web of the extended Myc network captures metabolism for tumorigenesis.
Cancer Cell. 2015; 27(2):160-2 [PubMed] Related Publications
In this issue of Cancer Cell, Carroll and colleagues describe the role of MondoA, a member of the Myc-Max-Mxd-Mxl transcription factor network (termed the extended Myc network herein), in Myc-mediated alterations in cancer cell metabolism and tumorigenesis.

Carroll PA, Diolaiti D, McFerrin L, et al.
Deregulated Myc requires MondoA/Mlx for metabolic reprogramming and tumorigenesis.
Cancer Cell. 2015; 27(2):271-85 [PubMed] Article available free on PMC after 09/02/2016 Related Publications
Deregulated Myc transcriptionally reprograms cell metabolism to promote neoplasia. Here we show that oncogenic Myc requires the Myc superfamily member MondoA, a nutrient-sensing transcription factor, for tumorigenesis. Knockdown of MondoA, or its dimerization partner Mlx, blocks Myc-induced reprogramming of multiple metabolic pathways, resulting in apoptosis. Identification and knockdown of genes coregulated by Myc and MondoA have allowed us to define metabolic functions required by deregulated Myc and demonstrate a critical role for lipid biosynthesis in survival of Myc-driven cancer. Furthermore, overexpression of a subset of Myc and MondoA coregulated genes correlates with poor outcome of patients with diverse cancers. Coregulation of cancer metabolism by Myc and MondoA provides the potential for therapeutics aimed at inhibiting MondoA and its target genes.

Ruiz-Pérez MV, Medina MÁ, Urdiales JL, et al.
Polyamine metabolism is sensitive to glycolysis inhibition in human neuroblastoma cells.
J Biol Chem. 2015; 290(10):6106-19 [PubMed] Article available free on PMC after 06/03/2016 Related Publications
Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors.

Nakaguro M, Kiyonari S, Kishida S, et al.
Nucleolar protein PES1 is a marker of neuroblastoma outcome and is associated with neuroblastoma differentiation.
Cancer Sci. 2015; 106(3):237-43 [PubMed] Related Publications
Neuroblastoma (NB) is a childhood malignant tumor that arises from precursor cells of the sympathetic nervous system. Spontaneous regression is a phenomenon unique to NBs and is caused by differentiation of tumor cells. PES1 is a multifunctional protein with roles in both neural development and ribosome biogenesis. Various kinds of models have revealed the significance of PES1 in neurodevelopment. However, the roles of PES1 in NB tumorigenesis and differentiation have remained unknown. Here we show that NB cases with MYCN amplification and clinically unfavorable stage (INSS stage 4) express higher levels of PES1. High PES1 expression was associated with worse overall and relapse-free survival. In NB cell lines, PES1 knockdown suppressed tumor cell growth and induced apoptosis. This growth inhibition was associated with the expression of NB differentiation markers. However, when the differentiation of NB cell lines was induced by the use of all-trans retinoic acid, there was a corresponding decrease in PES1 expression. Pes1 expression of tumorspheres originated from MYCN transgenic mice also diminished after the induction of differentiation with growth factors. We also reanalyzed the distribution of PES1 in the nucleolus. PES1 was localized in the dense fibrillar component, but not in the granular component of nucleoli. After treatment with the DNA-damaging agent camptothecin, this distribution was dramatically changed to diffuse nucleoplasmic. These data suggest that PES1 is a marker of NB outcome, that it regulates NB cell proliferation, and is associated with NB differentiation.

Bresler SC, Weiser DA, Huwe PJ, et al.
ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma.
Cancer Cell. 2014; 26(5):682-94 [PubMed] Article available free on PMC after 10/11/2015 Related Publications
Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1,596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples--at three hot spots and 13 minor sites--and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from nononcogenic mutations and allowed robust computational prediction of their effects. The mutated variants also showed differential in vitro crizotinib sensitivities. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma and will allow tailoring of ALK-targeted therapy to specific mutations.

Selmi A, de Saint-Jean M, Jallas AC, et al.
TWIST1 is a direct transcriptional target of MYCN and MYC in neuroblastoma.
Cancer Lett. 2015; 357(1):412-8 [PubMed] Related Publications
In neuroblastoma, MYCN amplification is associated with a worse prognosis and is a criterion used in the clinic to provide intensive treatments to children even with localized disease. In correlation with MYCN amplification, upregulation of TWIST1, a transcription factor playing a crucial role in inhibition of apoptosis and differentiation, was previously reported. Clinical data set analysis of MYCN, MYC and TWIST1 expression permits us to confirm that TWIST1 expression is upregulated in MYCN amplified neuroblastoma but also in a subset of neuroblastoma harboring high expression of MYCN or MYC without gene amplification. In silico analyses reveal the presence of several MYC regulatory motifs (E-Boxes and INR) within the TWIST1 promoter. Using gel shift assay and reporter activity assays, we demonstrate that both N-Myc and c-Myc proteins can bind and activate the TWIST1 promoter. Therefore, we propose TWIST1 as a direct MYC transcriptional target.

Hackett CS, Quigley DA, Wong RA, et al.
Expression quantitative trait loci and receptor pharmacology implicate Arg1 and the GABA-A receptor as therapeutic targets in neuroblastoma.
Cell Rep. 2014; 9(3):1034-46 [PubMed] Article available free on PMC after 10/11/2015 Related Publications
The development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene-expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma and that benzodiazepines in clinical use may have potential applications for neuroblastoma therapy.

Cecen E, Altun Z, Ercetin P, et al.
Promoting effects of sanguinarine on apoptotic gene expression in human neuroblastoma cells.
Asian Pac J Cancer Prev. 2014; 15(21):9445-51 [PubMed] Related Publications
Neuroblastoma is the most common extracranial solid tumor in children. Approximately half of the affected patients are diagnosed with high-risk poor prognosis disease, and novel therapies are needed. Sanguinarine is a benzophenanthridine alkaloid which has anti-microbial, anti-oxidant and anti-inflammatory properties. The aim of this study is whether sanguinarine has in vitro apoptotic effects and which apoptotic genes might be affected in the human neuroblastoma cell lines SH-SY5Y (N-myc negative), Kelly (N-myc positive, ALK positive), and SK- N-BE(2). Cell viability was analysed with WST-1 and apoptotic cell death rates were determined using TUNEL. After RNA isolation and cDNA conversion, expression of 84 custom array genes of apoptosis was determined. Sanguinarine caused cell death in a dose dependent manner in all neuroblastoma cell lines except SK-N-BE(2) with rates of 18% in SH-SY5Y and 21% in Kelly human neuroblastoma cells. Cisplatin caused similar apoptotic cell death rates of 16% in SH-SY5Y and 23% in Kelly cells and sanguinarine-cisplatin combinations caused the same rates (18% and 20%). Sanguinarine treatment did not affect apoptototic gene expression but decreased levels of anti-apoptotic genes NOL3 and BCL2L2 in SH-SY5Y cells. Caspase and TNF related gene expression was affected by the sanguinarine-cisplatin combination in SH-SY5Y cells. The expression of regulation of apoptotic genes were increased with sanguinarine treatment in Kelly cells. From these results, we conclude that sanguinarine is a candidate agent against neuroblastoma.

Ackermann S, Kocak H, Hero B, et al.
FOXP1 inhibits cell growth and attenuates tumorigenicity of neuroblastoma.
BMC Cancer. 2014; 14:840 [PubMed] Article available free on PMC after 10/11/2015 Related Publications
BACKGROUND: Segmental genomic copy number alterations, such as loss of 11q or 3p and gain of 17q, are well established markers of poor outcome in neuroblastoma, and have been suggested to comprise tumor suppressor genes or oncogenes, respectively. The gene forkhead box P1 (FOXP1) maps to chromosome 3p14.1, a tumor suppressor locus deleted in many human cancers including neuroblastoma. FoxP1 belongs to a family of winged-helix transcription factors that are involved in processes of cellular proliferation, differentiation and neoplastic transformation.
METHODS: Microarray expression profiles of 476 neuroblastoma specimens were generated and genes differentially expressed between favorable and unfavorable neuroblastoma were identified. FOXP1 expression was correlated to clinical markers and patient outcome. To determine whether hypermethylation is involved in silencing of FOXP1, methylation analysis of the 5' region of FOXP1 in 47 neuroblastomas was performed. Furthermore, FOXP1 was re-expressed in three neuroblastoma cell lines to study the effect of FOXP1 on growth characteristics of neuroblastoma cells.
RESULTS: Low expression of FOXP1 is associated with markers of unfavorable prognosis like stage 4, age >18 months and MYCN amplification and unfavorable gene expression-based classification (P < 0.001 each). Moreover, FOXP1 expression predicts patient outcome accurately and independently from well-established prognostic markers. Array-based CGH analysis of 159 neuroblastomas revealed that heterozygous loss of the FOXP1 locus was a rare event (n = 4), but if present, was associated with low FOXP1 expression. By contrast, DNA methylation analysis in 47 neuroblastomas indicated that hypermethylation is not regularly involved in FOXP1 gene silencing. Re-expression of FoxP1 significantly impaired cell proliferation, viability and colony formation in soft agar. Furthermore, induction of FOXP1 expression led to cell cycle arrest and apoptotic cell death of neuroblastoma cells.
CONCLUSIONS: Our results suggest that down-regulation of FOXP1 expression is a common event in high-risk neuroblastoma pathogenesis and may contribute to tumor progression and unfavorable patient outcome.

Defferrari R, Mazzocco K, Ambros IM, et al.
Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.
Br J Cancer. 2015; 112(2):290-5 [PubMed] Article available free on PMC after 20/01/2016 Related Publications
BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis.
METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol.
RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018).
CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

Lindner S, Henssen A, Astrahantseff K, Schulte JH
ALK pERKs up MYCN in neuroblastoma.
Sci Signal. 2014; 7(349):pe27 [PubMed] Related Publications
The gene expressing the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) is mutated and aberrantly expressed in several cancers. The clinical efficacy of the ALK inhibitor, crizotinib, lags behind expectations for treating MYCN-amplified, ALK-mutant neuroblastoma, a deadly childhood cancer. In this issue of Science Signaling, Umapathy et al. identify the kinase extracellular signal-regulated kinase 5 (ERK5) as a central mediator that enables ALK to boost MYCN expression, and they show that inhibiting ERK5 in concert with ALK reduced neuroblastoma cell viability in vitro and in xenograft tumor models. This report has important clinical implications for the treatment of patients with neuroblastoma or other tumors that overexpress MYC(N) and harbor ALK mutations, such as non-small-cell lung cancer.

Chen S, Zhou C, Ma X, Gong L
[Abnormality of anaplastic lymphoma kinase gene and its expression in pediatric neuroblastoma].
Zhonghua Bing Li Xue Za Zhi. 2014; 43(8):541-5 [PubMed] Related Publications
OBJECTIVE: To correlate the abnormal expression of anapastic lymphoma kinase (ALK) protein with the genetic and epigenetic changes of ALK, and to analyze its clinical application in pediatric neuroblastoma.
METHODS: Three neuroblastoma (NB) cell lines (two ALK positive: SH-SY5Y and SK-N-SH, one ALK negative: SK-N-AS) and 43 paraffin-embedded NB tissues were included in the study. In both cell lines and clinical cases, immunohistochemistry was used to detect ALK protein expression; PCR and Sanger sequencing were used to detect ALK point mutation; fluorescence in situ hybridization (FISH) was used to detect ALK abnormality and bisulfite sequencing PCR (BSP) was used to detect methylation of CpG island in the promoter area of ALK.
RESULTS: The cell lines SH-SY5Y and SK-N-SH were positive for ALK expression (cytoplasm), while the SK-N-AS was negative; among the 43 cases of NB, 26 (60.5%, 26/43) were positive for ALK protein (membrane and cytoplasm), and the rest were negative. Survival analysis showed ALK protein expression was related to survival time, with ALK positive cases having shorter survival time than ALK negative cases (P = 0.020). But ALK protein expression had no association with tumor differentiation (P = 0.503), tumor sites (P = 1.000) and age of patients (P = 0.063). FISH showed ALK amplification in two cases (4.6%, 2/43), ALK gain was found in 30 cases (69.7%, 30/43), and the remaining cases had normal ALK copy (25.6%, 11/43). The presence of extra copies (amplification and gain) of ALK was associated with ALK positive protein expression (P = 0.020), but there was no association with tumor differentiation (P = 1.000), tumor sites (P = 0.775) and age of patients (P = 0.328). No point mutation was found in all three cell lines. Of the 43 NB cases, only one case (2.3%, 1/43) showed point mutation in exon 23, and was a synonymous mutation [A1200A (G4552C)]. The case was ALK negative, but the patient died two months after diagnosis. BSP analysis showed that CpG island in ALK promoter region were all unmethylated in three cell lines and 6 NB cases (including 3 ALK positive, 3 ALK negative).
CONCLUSIONS: ALK protein is expressed in most NB, and the expression indicates poor outcome. ALK expression is associated with extra copies of ALK, but there is no association with the methylation status of CpG island of ALK; the presence of extra copies of ALK is the most common genetic aberration in NB. Point mutation of ALK is rare, and may predict poor prognosis in pediatric NB.

Brodeur GM, Bagatell R
Mechanisms of neuroblastoma regression.
Nat Rev Clin Oncol. 2014; 11(12):704-13 [PubMed] Article available free on PMC after 20/01/2016 Related Publications
Recent genomic and biological studies of neuroblastoma have shed light on the dramatic heterogeneity in the clinical behaviour of this disease, which spans from spontaneous regression or differentiation in some patients, to relentless disease progression in others, despite intensive multimodality therapy. This evidence also suggests several possible mechanisms to explain the phenomena of spontaneous regression in neuroblastomas, including neurotrophin deprivation, humoral or cellular immunity, loss of telomerase activity and alterations in epigenetic regulation. A better understanding of the mechanisms of spontaneous regression might help to identify optimal therapeutic approaches for patients with these tumours. Currently, the most druggable mechanism is the delayed activation of developmentally programmed cell death regulated by the tropomyosin receptor kinase A pathway. Indeed, targeted therapy aimed at inhibiting neurotrophin receptors might be used in lieu of conventional chemotherapy or radiation in infants with biologically favourable tumours that require treatment. Alternative approaches consist of breaking immune tolerance to tumour antigens or activating neurotrophin receptor pathways to induce neuronal differentiation. These approaches are likely to be most effective against biologically favourable tumours, but they might also provide insights into treatment of biologically unfavourable tumours. We describe the different mechanisms of spontaneous neuroblastoma regression and the consequent therapeutic approaches.

Wang J, Gu S, Huang J, et al.
Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells.
BMC Cancer. 2014; 14:768 [PubMed] Article available free on PMC after 20/01/2016 Related Publications
BACKGROUND: Aberrant Hedgehog (Hh) signaling is often associated with neuroblastoma (NB), a childhood malignancy with varying clinical outcomes due to different molecular characteristics. Inhibition of Hh signaling with small molecule inhibitors, particularly with GANT-61, significantly suppresses NB growth. However, NB with MYCN amplification is less sensitive to GANT-61 than those without MYCN amplification.
METHODS: Autophagic process was examined in two MYCN amplified and two MYCN non-amplified NB cells treated with GANT-61. Subsequently, chemical and genetic approaches were applied with GANT-61 together to evaluate the role of autophagy in GANT-61 induced cell death.
RESULTS: Here we show that GANT-61 enhanced autophagy in MYCN amplified NB cells. Both an autophagic inhibitor 3-methyladenine (3-MA) and genetic disruption of ATG5 or ATG7 expression suppressed GANT-61 induced autophagy and significantly increased apoptotic cell death, whereas pre-treatment with an apoptotic inhibitor, Z-VAD-FMK, rescued GANT-61 induced cell death and had no effect on the autophagic process. In the other hand, GANT-61 barely induced autophagy in MYCN non-amplified NB cells, but overexpression of MYCN in MYCN non-amplified NB cells recapitulated GANT-61 induced autophagy seen in MYCN amplified NB cells, suggesting that the level of GANT-61 induced autophagy in NB cells is related to MYCN expression level in cells.
CONCLUSION: Aberrant Hh signaling activation as an oncogenic driver in NB renders inhibition of Hh signaling an effective measure to suppress NB growth. However, our data suggest that enhanced autophagy concomitant with Hh signaling inhibition acts as a pro-survival factor to maintain cell viability, which reduces GANT-61 efficacy. Besides, MYCN amplification is likely involved in the induction of the pro-survival autophagy. Overall, simultaneous inhibition of both Hh signaling and autophagy could be a better way to treat MYCN amplified NB.

Capasso M, Diskin S, Cimmino F, et al.
Common genetic variants in NEFL influence gene expression and neuroblastoma risk.
Cancer Res. 2014; 74(23):6913-24 [PubMed] Article available free on PMC after 01/12/2015 Related Publications
The genetic etiology of sporadic neuroblastoma is still largely obscure. In a genome-wide association study, we identified single-nucleotide polymorphisms (SNP) associated with neuroblastoma at the CASC15, BARD1, LMO1, DUSP12, HSD17B12, HACE1, and LIN28B gene loci, but these explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the multiple testing corrections. In this study, we evaluated eight additional genes selected as candidates for further study based on proven involvement in neuroblastoma differentiation. SNPs at these candidate genes were tested for association with disease susceptibility in 2,101 cases and 4,202 controls, with the associations found replicated in an independent cohort of 459 cases and 809 controls. Replicated associations were further studied for cis-effect using gene expression, transient overexpression, silencing, and cellular differentiation assays. The neurofilament gene NEFL harbored three SNPs associated with neuroblastoma (rs11994014: Pcombined = 0.0050; OR, 0.88; rs2979704: Pcombined = 0.0072; OR, 0.87; rs1059111: Pcombined = 0.0049; OR, 0.86). The protective allele of rs1059111 correlated with increased NEFL expression. Biologic investigations showed that ectopic overexpression of NEFL inhibited cell growth specifically in neuroblastoma cells carrying the protective allele. NEFL overexpression also enhanced differentiation and impaired the proliferation and anchorage-independent growth of cells with protective allele and basal NEFL expression, while impairing invasiveness and proliferation of cells homozygous for the risk genotype. Clinically, high levels of NEFL expression in primary neuroblastoma specimens were associated with better overall survival (P = 0.03; HR, 0.68). Our results show that common variants of NEFL influence neuroblastoma susceptibility and they establish that NEFL expression influences disease initiation and progression.

Wang L, Che XJ, Wang N, et al.
Regulatory network analysis of microRNAs and genes in neuroblastoma.
Asian Pac J Cancer Prev. 2014; 15(18):7645-52 [PubMed] Related Publications
Neuroblastoma (NB), the most common extracranial solid tumor, accounts for 10% of childhood cancer. To date, scientists have gained quite a lot of knowledge about microRNAs (miRNAs) and their genes in NB. Discovering inner regulation networks, however, still presents problems. Our study was focused on determining differentially-expressed miRNAs, their target genes and transcription factors (TFs) which exert profound influence on the pathogenesis of NB. Here we constructed three regulatory networks: differentially-expressed, related and global. We compared and analyzed the differences between the three networks to distinguish key pathways and significant nodes. Certain pathways demonstrated specific features. The differentially-expressed network consists of already identified differentially-expressed genes, miRNAs and their host genes. With this network, we can clearly see how pathways of differentially expressed genes, differentially expressed miRNAs and TFs affect on the progression of NB. MYCN, for example, which is a mutated gene of NB, is targeted by hsa-miR-29a and hsa-miR-34a, and regulates another eight differentially-expressed miRNAs that target genes VEGFA, BCL2, REL2 and so on. Further related genes and miRNAs were obtained to construct the related network and it was observed that a miRNA and its target gene exhibit special features. Hsa-miR-34a, for example, targets gene MYC, which regulates hsa-miR-34a in turn. This forms a self-adaption association. TFs like MYC and PTEN having six types of adjacent nodes and other classes of TFs investigated really can help to demonstrate that TFs affect pathways through expressions of significant miRNAs involved in the pathogenesis of NB. The present study providing comprehensive data partially reveals the mechanism of NB and should facilitate future studies to gain more significant and related data results for NB.

Lee S, Craig BT, Romain CV, et al.
Silencing of CDC42 inhibits neuroblastoma cell proliferation and transformation.
Cancer Lett. 2014; 355(2):210-6 [PubMed] Article available free on PMC after 28/12/2015 Related Publications
Cell division cycle 42 (CDC42), a small GTPase of the Rho-subfamily, regulates diverse cellular functions including proliferation, cytoskeletal rearrangement and even promotes malignant transformation. Here, we found that increased expression of CDC42 correlated with undifferentiated neuroblastoma as compared to a more benign phenotype. CDC42 inhibition decreased cell growth and soft agar colony formation, and increased cell death in BE(2)-C and BE(2)-M17 cell lines, but not in SK-N-AS. In addition, silencing of CDC42 decreased expression of N-myc in BE(2)-C and BE(2)-M17 cells. Our findings suggest that CDC42 may play a role in the regulation of aggressive neuroblastoma behavior.

Salcher S, Hagenbuchner J, Geiger K, et al.
C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.
Mol Cancer. 2014; 13:224 [PubMed] Article available free on PMC after 28/12/2015 Related Publications
BACKGROUND: FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.
METHODS: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.
RESULTS: We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.
CONCLUSION: The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.

Fransson S, Hansson M, Ruuth K, et al.
Intragenic anaplastic lymphoma kinase (ALK) rearrangements: translocations as a novel mechanism of ALK activation in neuroblastoma tumors.
Genes Chromosomes Cancer. 2015; 54(2):99-109 [PubMed] Related Publications
Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.

Estiar MA, Fazilaty H, Aslanabadi S, et al.
MYCN gene amplification in patients with neuroblastic tumors.
Cell Mol Biol (Noisy-le-grand). 2014; 60(3):23-8 [PubMed] Related Publications
Although neuroblastic tumors are the most prevalent solid tumors, little is known about the genetic basis underlying their progression. The prognostic role for the MYCN gene in neuroblastic tumors is irrefutable. The aim of this study is to identify the frequency of MYCN gene amplification and its relationship with clinicopathological and prognostic factors in 40 patients with neuroblastic tumors by using real-time quantitative PCR. There was significant association between the age of older than 18 months and the high number of metastasis. 83.3% of metastatic neuroblastic tumors in patients aged more than 18 months were in stage 4, while it was about 12.5% in patients aged less than 18 months. We found an amplification of MYCN in 19 out of 40 patients. Also, we found MYCN gene amplification in 64% of neuroblastoma (NB) and 8% of gangelioneuroblastoma (GNB) cases. There was a significant association between the histological type of samples with MYCN gene amplification. Neuroblastic tumors have a varied range of MYCN gene amplification depend on histopathology types. No significant associations have been found between MYCN gene amplification and tumor evaluation, CNS involvement, metastasis, stage of disease and patients outcome.

Yan B, Kuick CH, Lim M, et al.
Platform comparison for evaluation of ALK protein immunohistochemical expression, genomic copy number and hotspot mutation status in neuroblastomas.
PLoS One. 2014; 9(9):e106575 [PubMed] Article available free on PMC after 28/12/2015 Related Publications
ALK is an established causative oncogenic driver in neuroblastoma, and is likely to emerge as a routine biomarker in neuroblastoma diagnostics. At present, the optimal strategy for clinical diagnostic evaluation of ALK protein, genomic and hotspot mutation status is not well-studied. We evaluated ALK immunohistochemical (IHC) protein expression using three different antibodies (ALK1, 5A4 and D5F3 clones), ALK genomic status using single-color chromogenic in situ hybridization (CISH), and ALK hotspot mutation status using conventional Sanger sequencing and a next-generation sequencing platform (Ion Torrent Personal Genome Machine (IT-PGM)), in archival formalin-fixed, paraffin-embedded neuroblastoma samples. We found a significant difference in IHC results using the three different antibodies, with the highest percentage of positive cases seen on D5F3 immunohistochemistry. Correlation with ALK genomic and hotspot mutational status revealed that the majority of D5F3 ALK-positive cases did not possess either ALK genomic amplification or hotspot mutations. Comparison of sequencing platforms showed a perfect correlation between conventional Sanger and IT-PGM sequencing. Our findings suggest that D5F3 immunohistochemistry, single-color CISH and IT-PGM sequencing are suitable assays for evaluation of ALK status in future neuroblastoma clinical trials.

Solari V, Borriello L, Turcatel G, et al.
MYCN-dependent expression of sulfatase-2 regulates neuroblastoma cell survival.
Cancer Res. 2014; 74(21):5999-6009 [PubMed] Article available free on PMC after 28/12/2015 Related Publications
Heparan sulfate proteoglycans (HSPG) play a critical role in the interaction of tumor cells and their microenvironment. HSPG activity is dictated by sulfation patterns controlled by sulfotransferases, which add sulfate groups, and sulfatases (Sulf), which remove 6-O-sulfates. Here, we report altered expression of these enzymes in human neuroblastoma cells with higher levels of Sulf-2 expression, a specific feature of MYCN-amplified cells (MYCN-A cells) that represent a particularly aggressive subclass. Sulf-2 overexpression in neuroblastoma cells lacking MYCN amplification (MYCN-NA cells) increased their in vitro survival. Mechanistic investigations revealed evidence of a link between Sulf-2 expression and MYCN pathogenicity in vitro and in vivo. Analysis of Sulf-2 protein expression in 65 human neuroblastoma tumors demonstrated a higher level of Sulf-2 expression in MYCN-A tumors than in MYCN-NA tumors. In two different patient cohorts, we confirmed the association in expression patterns of Sulf-2 and MYCN and determined that Sulf-2 overexpression predicted poor outcomes in a nonindependent manner with MYCN. Our findings define Sulf-2 as a novel positive regulator of neuroblastoma pathogenicity that contributes to MYCN oncogenicity. Cancer Res; 74(21); 5999-6009. ©2014 AACR.

Harvey H, Piskareva O, Creevey L, et al.
Modulation of chemotherapeutic drug resistance in neuroblastoma SK-N-AS cells by the neural apoptosis inhibitory protein and miR-520f.
Int J Cancer. 2015; 136(7):1579-88 [PubMed] Related Publications
The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research.

Naiditch JA, Jie C, Lautz TB, et al.
Mesenchymal change and drug resistance in neuroblastoma.
J Surg Res. 2015; 193(1):279-88 [PubMed] Related Publications
BACKGROUND: Metastatic initiation has many phenotypic similarities to epithelial-to-mesenchymal transition, including loss of cell-cell adhesion, increased invasiveness, and increased cell mobility. We have previously demonstrated that drug resistance is associated with a metastatic phenotype in neuroblastoma (NB). The purpose of this project was to determine if the development of doxorubicin resistance is associated with characteristics of mesenchymal change in human NB cells.
MATERIALS AND METHODS: Total RNA was isolated from wild type (WT) and doxorubicin-resistant (DoxR) human NB cell lines (SK-N-SH and SK-N-BE(2)C) and analyzed using the Illumina Human HT-12 version 4 Expression BeadChip. Differentially expressed genes (DEGs) were identified. Volcano plots and heat maps were generated. Genes of interest with a fold change in expression >1.5 and an adjusted P < 0.1 were analyzed. Immunofluorescence (IF) and Western blot analysis confirmed microarray results of interest. Matrigel invasion assay and migration wounding assays were performed.
RESULTS: Volcano plots and heat maps visually demonstrated a similar pattern of DEGs in the SK-N-SH and SK-N-BE(2)C DoxR cell lines relative to their parental WT lines. Venn diagramming revealed 1594 DEGs common to both DoxR cell lines relative to their parental cell lines. Network analysis pointed to several significantly upregulated epithelial-to-mesenchymal transition pathways, through TGF-beta pathways via RhoA, PI3K, and ILK and via SMADs, as well as via notch signaling pathways. DoxR cell lines displayed a more invasive phenotype than respective WT cell lines.
CONCLUSIONS: Human SK-N-SH and SK-N-BE(2)C NB cells display characteristics of mesenchymal change via multiple pathways in the transition to a drug-resistant state.

Schleiermacher G, Janoueix-Lerosey I, Delattre O
Recent insights into the biology of neuroblastoma.
Int J Cancer. 2014; 135(10):2249-61 [PubMed] Related Publications
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.

Dreidax D, Bannert S, Henrich KO, et al.
p19-INK4d inhibits neuroblastoma cell growth, induces differentiation and is hypermethylated and downregulated in MYCN-amplified neuroblastomas.
Hum Mol Genet. 2014; 23(25):6826-37 [PubMed] Related Publications
Uncontrolled cell cycle entry, resulting from deregulated CDK-RB1-E2F pathway activity, is a crucial determinant of neuroblastoma cell malignancy. Here we identify neuroblastoma-suppressive functions of the p19-INK4d CDK inhibitor and uncover mechanisms of its repression in high-risk neuroblastomas. Reduced p19-INK4d expression was associated with poor event-free and overall survival and neuroblastoma risk factors including amplified MYCN in a set of 478 primary neuroblastomas. High MYCN expression repressed p19-INK4d mRNA and protein levels in different neuroblastoma cell models with conditional MYCN expression. MassARRAY and 450K methylation analyses of 105 primary neuroblastomas uncovered a differentially methylated region within p19-INK4d. Hypermethylation of this region was associated with reduced p19-INK4d expression. In accordance, p19-INK4d expression was activated upon treatment with the demethylating agent, 2'-deoxy-5-azacytidine, in neuroblastoma cell lines. Ectopic p19-INK4d expression decreased viability, clonogenicity and the capacity for anchorage-independent growth of neuroblastoma cells, and shifted the cell cycle towards the G1/0 phase. p19-INK4d also induced neurite-like processes and markers of neuronal differentiation. Moreover, neuroblastoma cell differentiation, induced by all-trans retinoic acid or NGF-NTRK1-signaling, activated p19-INK4d expression. Our findings pinpoint p19-INK4d as a neuroblastoma suppressor and provide evidence for MYCN-mediated repression and for epigenetic silencing of p19-INK4d by DNA hypermethylation in high-risk neuroblastomas.

Li MH, Harel M, Hla T, Ferrer F
Induction of chemokine (C-C motif) ligand 2 by sphingosine-1-phosphate signaling in neuroblastoma.
J Pediatr Surg. 2014; 49(8):1286-91 [PubMed] Article available free on PMC after 28/12/2015 Related Publications
BACKGROUND/PURPOSE: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Preliminary data derived from a human angiogenesis array in NB showed that the bioactive lipid sphingosine-1-phosphate (S1P) induced the secretion of several angiogenesis-related proteins including the important inflammatory factor chemokine (C-C motif) ligand 2 (CCL2). In the present study, we investigated the mechanism of S1P-induced CCL2 expression in NB.
METHODS: Quantitative real-time PCR and CCL2 ELISA were conducted to detect the mRNA expression and protein secretion of CCL2 in NB cells. Gain and loss of function studies were performed by using specific S1PR antagonists, adenoviral transduction and siRNA transfection. Macrophage F4/80 receptor in NB xenografts was detected by quantitative real-time PCR and immunohistochemistry staining.
RESULTS: S1P induced CCL2 mRNA expression and protein secretion in a time- and concentration-dependent manner in NB cells. Blockade of S1P2 signaling using the selective S1P2 antagonist JTE-013 inhibited S1P-induced CCL2 expression. Overexpression of S1P2 by adenoviral transduction increased CCL2 secretion while knockdown of S1P2 by siRNA transfection decreased S1P-induced CCL2 secretion in NB cells. Macrophage infiltration, as detected by F4/80 staining, was significantly decreased in JTE-013-treated NB xenografts.
CONCLUSIONS: Taken together, our data for the first time demonstrate that S1P induced the macrophage-recruiting factor CCL2 expression in NB cells via S1P2, providing new insights into the complicated functions of S1P2 in cancer.

Recurrent Chromosome Abnormalities

Selected list of common recurrent structural abnormalities

This is a highly selective list aiming to capture structural abnormalies which are frequesnt and/or significant in relation to diagnosis, prognosis, and/or characterising specific cancers. For a much more extensive list see the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer.

del(1p36) in Neuroblastoma

Enomoto H, Ozaki T, Takahashi E, et al.
Identification of human DAN gene, mapping to the putative neuroblastoma tumor suppressor locus.
Oncogene. 1994; 9(10):2785-91 [PubMed] Related Publications
The expression of DAN gene (previously designated as N03 gene) is significantly reduced in a variety of transformed rat fibroblasts, including v-src- (SR-3Y1), SV40- and v-mos-transformed 3Y1 cells, compared with that in parental 3Y1 cells. Recently, DAN gene has been shown to possess a tumor suppressive activity when it is overexpressed in SR-3Y1 cells (Ozaki & Sakiyama, 1994). To assess the involvement of DAN gene with human neoplasms, we have isolated human DAN counterpart from a normal lung cDNA library by using rat DAN cDNA as a probe, and determined its chromosomal location. Human DAN gene mapped to chromosome 1p36.11-p36.13, which is well known to show highly significant linkage with the genesis and/or progression of human neuroblastoma. Southern blot analysis on tumor DNA from 26 patients with neuroblastoma has detected three patients showing genomic rearrangement or deletion within or closely linked to the DAN gene locus. Collectively, we propose that human DAN gene is a possible candidate for a tumor suppressor gene of human neuroblastoma.

White PS, Maris JM, Beltinger C, et al.
A region of consistent deletion in neuroblastoma maps within human chromosome 1p36.2-36.3.
Proc Natl Acad Sci U S A. 1995; 92(12):5520-4 [PubMed] Free Access to Full Article Related Publications
Deletion of the short arm of human chromosome 1 is the most common cytogenetic abnormality observed in neuroblastoma. To characterize the region of consistent deletion, we performed loss of heterozygosity (LOH) studies on 122 neuroblastoma tumor samples with 30 distal chromosome 1p polymorphisms. LOH was detected in 32 of the 122 tumors (26%). A single region of LOH, marked distally by D1Z2 and proximally by D1S228, was detected in all tumors demonstrating loss. Also, cells from a patient with a constitutional deletion of 1p36, and from a neuroblastoma cell line with a small 1p36 deletion, were analyzed by fluorescence in situ hybridization. Cells from both sources had interstitial deletions of 1p36.2-36.3 which overlapped the consensus region of LOH defined by the tumors. Interstitial deletion in the constitutional case was confirmed by allelic loss studies using the panel of polymorphic markers. Four proposed candidate genes--DAN, ID3 (heir-1), CDC2L1 (p58), and TNFR2--were shown to lie outside of the consensus region of allelic loss, as defined by the above deletions. These results more precisely define the location of a neuroblastoma suppressor gene within 1p36.2-36.3, eliminating 33 centimorgans of proximal 1p36 from consideration. Furthermore, a consensus region of loss, which excludes the four leading candidate genes, was found in all tumors with 1p36 LOH.

Spieker N, Beitsma M, van Sluis P, et al.
An integrated 5-Mb physical, genetic, and radiation hybrid map of a 1p36.1 region implicated in neuroblastoma pathogenesis.
Genes Chromosomes Cancer. 2000; 27(2):143-52 [PubMed] Related Publications
Common genetic aberrations of neuroblastoma are deletions of the short arm of chromosome 1 (1p36) and MYCN amplification. Our deletion analysis of 25 tumor cell lines and 171 tumors strongly suggests that 1p harbors several tumor suppressor loci. Distinct loci are involved in MYCN single-copy versus MYCN-amplified neuroblastoma. Deletions in MYCN single-copy tumors have a shortest region of overlap (SRO) of 20 cM at 1p36.3. MYCN-amplified tumors have large deletions with an SRO of about 60 cM, from 1p36.1 to the telomere. This SRO is defined by D1S7 (1p36.1), which was the most distal locus retained. Therefore, a suppressor gene associated with MYCN-amplified tumors probably maps within a few megabases distal of D1S7. In order to map this locus, we further refined this SRO. We mapped the breakpoint of the MYCN-amplified neuroblastoma with the smallest 1p deletion between 56.6 and 57.2 cM from 1pter. Pulsed-field gel electrophoresis and radiation hybrid mapping were used to construct a 5-Mb physical map of this region. The map includes the region from 82.73 till 92.89 cR from 1pter. About half of it was isolated in P1 and PAC clones. The region harbors the genes FGR, SLC9A1, HMG17, EXTL1, AML2, RH, OP18, four ESTs, and a newly identified gene with a transcript size of approximately 7 Kb. Several of the mapped genes have a putative role in cell growth, differentiation, and morphogenesis. Genes Chromosomes Cancer 27:143-152, 2000.

Maris JM, Weiss MJ, Guo C, et al.
Loss of heterozygosity at 1p36 independently predicts for disease progression but not decreased overall survival probability in neuroblastoma patients: a Children's Cancer Group study.
J Clin Oncol. 2000; 18(9):1888-99 [PubMed] Related Publications
PURPOSE: To determine the independent prognostic significance of 1p36 loss of heterozygosity (LOH) in a representative group of neuroblastoma patients.
PATIENTS AND METHODS: Diagnostic tumor specimens from 238 patients registered onto the most recent Children's Cancer Group phase III clinical trials were assayed for LOH with 13 microsatellite polymorphic markers spanning chromosome band 1p36. Allelic status at 1p36 was correlated with other prognostic variables and disease outcome.
RESULTS: LOH at 1p36 was detected in 83 (35%) of 238 neuroblastomas. There was a correlation of 1p36 LOH with age at diagnosis greater than 1 year (P = .026), metastatic disease (P<.001), elevated serum ferritin level (P<.001), unfavorable histopathology (P<.001), and MYCN oncogene amplification (P<.001). LOH at 1p36 was associated with decreased event-free survival (EFS) and overall survival (OS) probabilities (P<.0001). For the 180 cases with single-copy MYCN, 1p36 LOH status was highly correlated with decreased EFS (P = .0002) but not OS (P = .1212). Entering 1p36 LOH into a multivariate regression model suggested a trend toward an independent association with decreased EFS (P = .0558) but not with decreased OS (P = .3687). Furthermore, allelic status at 1p36 was the only prognostic variable that was significantly associated with decreased EFS in low-risk neuroblastoma patients (P = .0148).
CONCLUSION: LOH at 1p36 is independently associated with decreased EFS, but not OS, in neuroblastoma patients. Determination of 1p36 allelic status may be useful for predicting which neuroblastoma patients with otherwise favorable clinical and biologic features are more likely to have disease progression.

del(9p) in Neuroblastoma

Takita J, Hayashi Y, Kohno T, et al.
Deletion map of chromosome 9 and p16 (CDKN2A) gene alterations in neuroblastoma.
Cancer Res. 1997; 57(5):907-12 [PubMed] Related Publications
We reported previously that loss of heterozygosity (LOH) on chromosomes 2q, 9p and 18q frequently occurs in neuroblastoma and that patients with 9p LOH in the tumors showed statistically significant association with an advanced stage of the disease and poor prognosis. To determine the role of chromosome 9 loss in neuroblastoma, we performed deletion mapping of chromosome 9 in 80 cases of neuroblastoma using 11 polymorphic microsatellite markers and a restriction fragment length porymorphism marker. LOH at one or more loci on chromosome 9 was detected in 33 of 80 cases (41%). Chromosome 9p was lost in 24 of 80 cases (32%), whereas chromosome 9q was lost in 18 of 80 cases (23%). There were two commonly deleted regions mapped to 9p21 between the D9S171 marker and the IFNB1 marker and 9q34-qter distal to the D9S176 marker. In addition, patients with LOH at 9p21 but not at 9q34-qter in the tumors showed statistically significant association with poor prognosis (P = 0.023). Because the commonly deleted regions at 9p21 includes the p16 (CDKN2A) gene, the status of the p16 gene was further examined in 80 fresh tumors and 19 cell lines of neuroblastoma. A missense mutation was detected at codon 52 in a fresh tumor. The p16 gene was not expressed in 13 of 19 cell lines (72%), and 5 of the 13 cell lines displayed methylation of the CpG island surrounding the first exon of the p16 gene. These results suggest that the p16 gene is a candidate tumor suppressor gene for neuroblastoma, and its inactivation may contribute to the progression of neuroblastoma.

Marshall B, Isidro G, Martins AG, Boavida MG
Loss of heterozygosity at chromosome 9p21 in primary neuroblastomas: evidence for two deleted regions.
Cancer Genet Cytogenet. 1997; 96(2):134-9 [PubMed] Related Publications
The genes responsible for the development of neuroblastoma following in vivo deletion or mutation are largely unknown. We have performed loss of heterozygosity studies on a series of 24 Portuguese primary neuroblastomas using 6 polymorphic markers located at chromosome 9p21 spanning the p16/MTS1/CDKN2, p15/MTS2/CDKN2B, and the interferon alpha and beta genes. Loss of heterozygosity was observed in 4 of the 24 tumors (17%), a somewhat lower percentage than a previous study that identified patients by a mass screening program. A correlation was also observed between 9p21 LOH and 1p36 LOH in our group of tumors. Two distinct regions of 9p21 deletion were observed: one located in the region adjacent to the markers D9S162 and D9S1747 and a second located centromerically of the p16 gene near the D9S171 marker. The latter region is exclusive of the p16 gene. This result suggests the presence of at least one other tumor suppressor gene at 9p21, apart from the p16 and p15 genes, which may be of importance to the development of neuroblastoma.

Gain of Chromosome 17q in Neuroblastoma

Gain of extra 17q material is the most frequent genetic abnormality in neuroblastoma. Unbalanced (partial) gain is associated with 1p deletion and MYCN amplification; in some cases 1p deletion can be caused by t(1;17) translocation.

Brinkschmidt C, Poremba C, Christiansen H, et al.
Comparative genomic hybridization and telomerase activity analysis identify two biologically different groups of 4s neuroblastomas.
Br J Cancer. 1998; 77(12):2223-9 [PubMed] Free Access to Full Article Related Publications
Chromosomal aberrations of 20 stage 4s neuroblastomas were analysed by comparative genomic hybridization (CGH). In a subset of 13/20 tumours, telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP). The CGH data were compared with the CGH results of ten stage 1 and 2 (stage 1/2) and 22 stage 3 and 4 (stage 3/4) neuroblastomas. A total of 17/20 stage 4s neuroblastomas did not progress clinically, whereas tumour progression with lethal outcome occurred in 3/20 cases. The CGH data of clinically non-progressing stage 4s tumours revealed a high rate of whole-chromosome aberrations (73.4%) with an overrepresentation of mainly chromosomes 2, 6, 7, 12, 13, 17, 18 and an underrepresentation of mainly chromosomes 3, 4, 11, 14. MYCN amplification or 1p deletion was observed in only 1/27 or 2/17 clinically non-progressing stage 4s tumours respectively, whereas all three progressive stage 4s neuroblastomas showed MYCN amplification, 1p deletion and, in 2/3 cases, distal 17q gains. Except for one case, telomerase activity was not observed in non-progressing stage 4s neuroblastomas. In contrast, 4s tumours with lethal outcome revealed elevated telomerase activity levels. Our data suggest that stage 4s neuroblastomas belong to two biologically different groups, one of which displays the genetic features of localized stage 1/2 tumours, whereas the other mimics advanced stage 3/4 neuroblastomas.

Plantaz D, Mohapatra G, Matthay KK, et al.
Gain of chromosome 17 is the most frequent abnormality detected in neuroblastoma by comparative genomic hybridization.
Am J Pathol. 1997; 150(1):81-9 [PubMed] Free Access to Full Article Related Publications
Neuroblastoma behavior is variable and outcome partially depends on genetic factors. However, tumors that lack high-risk factors such as MYCN amplification or 1p deletion may progress, possibly due to other genetic aberrations. Comparative genomic hybridization summarizes DNA copy number abnormalities in a tumor by mapping them to their positions on normal metaphase chromosomes. We analyzed 29 tumors from nearly equal proportions of children with stage I, II, III, IV, and IV-S disease by comparative genomic hybridization. We found two classes of copy number abnormalities: whole chromosome and partial chromosome. Whole chromosome losses were frequent at 11, 14, and X. The most frequent partial chromosome losses were on 1p and 11q. Gains were most frequent on chromosome 17 (72% of cases). The two patterns of gain for this chromosome were whole 17 gain and 17q gain, with 17q21-qter as a minimal common region of gain. Other common gains were on chromosomes 7, 6, and 18. High level amplifications were detected at 2p23-25 (MYCN region), at 4q33-35, and at 6p11-22. Chromosome 17q gains were associated with 1p and/or 11q deletions and advanced stage. The high frequency of chromosome 17 gain and its association with bad prognostic factors suggest an important role for this chromosome in the development of neuroblastoma.

Caron H
Allelic loss of chromosome 1 and additional chromosome 17 material are both unfavourable prognostic markers in neuroblastoma.
Med Pediatr Oncol. 1995; 24(4):215-21 [PubMed] Related Publications
In neuroblastoma, N-myc amplification and loss of heterozygosity for the short arm of chromosome 1 (LOH 1p) are common genetic abnormalities. We have recently shown that the presence of additional material of the long arm of chromosome 17 (add.17q) also occurs relatively frequently. In the present study, we analyzed a series of 55 tumors for LOH 1p, N-myc amplification and add.17q, using Southern blot analysis with polymorphic DNA probes of pairs of tumor and constitutional DNA. We determined the correlation of these parameters with clinical variables, such as age, stage, serum lactate dehydrogenase (LDH) and ferritin and also with outcome. LOH 1p occurred in 20 out of 55 cases (36%) and was found more often in stage III/IV tumors and in the older age group, although both correlations were not statistically significant. N-myc amplification was only demonstrated in 12 tumors with concomitant LOH 1p and was not present in the 35 cases without LOH 1p. Add.17q was found in 20/53 (38%) informative cases. LOH 1p was shown to be the most significant predictor of a poor outcome (P < 0.00001), independent of age and stage. LOH 1p is also of prognostic value in those cases without N-myc amplification, indicating a stronger prognostic value for LOH 1p. Add.17q was also associated with an unfavourable prognosis, although this was less significantly then with LOH 1p (P = 0.00004).

Bown N, Cotterill S, Lastowska M, et al.
Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma.
N Engl J Med. 1999; 340(25):1954-61 [PubMed] Related Publications
BACKGROUND: Gain of genetic material from chromosome arm 17q (gain of segment 17q21-qter) is the most frequent cytogenetic abnormality of neuroblastoma cells. This gain has been associated with advanced disease, patients who are > or =1 year old, deletion of chromosome arm 1p, and amplification of the N-myc oncogene, all of which predict an adverse outcome. We investigated these associations and evaluated the prognostic importance of the status of chromosome 17.
METHODS: We compiled molecular cytogenetic analyses of chromosome 17 in primary neuroblastomas in 313 patients at six European centers. Clinical and survival information were collected, along with data on 1p, N-myc, and ploidy.
RESULTS: Unbalanced gain of segment 17q21-qter was found in 53.7 percent of the tumors, whereas the chromosome was normal in 46.3 percent. The gain of 17q was characteristic of advanced tumors and of tumors in children > or =1 year of age and was strongly associated with the deletion of 1p and amplification of N-myc. No tumor showed amplification of N-myc in the absence of either deletion of 1p or gain of 17q. Gain of 17q was a significant predictive factor for adverse outcome in univariate analysis. Among the patients with this abnormality, overall survival at five years was 30.6 percent (95 percent confidence interval, 21 to 40 percent), as compared with 86.0 percent (95 percent confidence interval, 78 to 91 percent) among those with normal 17q status. in multivariate analysis, gain of 17q was the most powerful prognostic factor, followed by the presence of stage 4 disease and deletion of 1p (hazard ratios, 3.4, 2.3, and 1.9, respectively).
CONCLUSIONS: Gain of chromosome segment 17q21-qter is an important prognostic factor in children with neuroblastoma.

Abel F, Ejeskär K, Kogner P, Martinsson T
Gain of chromosome arm 17q is associated with unfavourable prognosis in neuroblastoma, but does not involve mutations in the somatostatin receptor 2(SSTR2) gene at 17q24.
Br J Cancer. 1999; 81(8):1402-9 [PubMed] Free Access to Full Article Related Publications
Deletion of chromosome arm 1p and amplification of the MYCN oncogene are well-recognized genetic alterations in neuroblastoma cells. Recently, another alteration has been reported; gain of the distal part of chromosome arm 17q. In this study 48 neuroblastoma tumours were successfully analysed for 17q status in relation to known genetic alterations. Chromosome 17 status was detected by fluorescence in situ hybridization (FISH). Thirty-one of the 48 neuroblastomas (65%) showed 17q gain, and this was significantly associated with poor prognosis. As previously reported, 17q gain was significantly associated with metastatic stage 4 neuroblastoma and more frequently detected than both deletion of chromosome arm 1p and MYCN amplification in tumours of all stages. 17q gain also showed a strong correlation to survival probability (P = 0.0009). However, the most significant correlation between 17q gain and survival probability was observed in children with low-stage tumours (stage 1, 2, 3 and 4S), with a survival probability of 100% at 5 years from diagnosis for children with tumours showing no 17q gain compared to 52.5% for those showing 17q gain (P = 0.0021). This suggests that 17q gain as a prognostic factor plays a more crucial role in low-stage tumours. Expression of the somatostatin receptor 2 (SSTR2), localized in chromosome region 17q24, has in previous studies been shown to be positively related to survival in neuroblastoma. A point mutation in the SSTR2 gene has earlier been reported in a human small-cell lung cancer. In this study, mutation screening of the SSTR2 gene in 43 neuroblastoma tumours was carried out with polymerase chain reaction-based single-stranded conformation polymorphism/heteroduplex (SSCP/HD) and DNA sequencing, and none of the tumours showed any aberrations in the SSTR2 gene. These data suggest that mutations in the SSTR2 gene are uncommon in neuroblastoma tumours and do not correlate with either the 17q gain often seen or the reason some tumours do not express SSTR2 receptors. Overall, this study indicates that gain of chromosome arm 17q is the most frequently occurring genetic alteration, and that it is associated with established prognostic factors.

Godfried MB, Veenstra M, v Sluis P, et al.
The N-myc and c-myc downstream pathways include the chromosome 17q genes nm23-H1 and nm23-H2.
Oncogene. 2002; 21(13):2097-101 [PubMed] Related Publications
Gain of chromosome 17q material is the most frequent genetic abnormality in neuroblastomas. The common region of gain is at least 375 cR large, which has precluded the identification of genes with a role in neuroblastoma pathogenesis. Neuroblastoma also frequently show amplification of the N-myc oncogene, which correlates closely with 17q gain. Both events are strong predictors of unfavorable prognosis. To identify genes that are part of the N-myc downstream pathway, we constructed SAGE libraries of an N-myc transfected and a control cell line. This identified the chromosome 17q genes nm23-H1 and nm23-H2 as being 6-10 times induced in the N-myc expressing cells. Northern and Western blot analysis confirmed this up-regulation. Time-course experiment shows that both genes are induced within 4 h after N-myc is switched on. Furthermore, we demonstrate also that c-myc can up-regulate nm23-H1 and nm23-H2 expression. Neuroblastoma tumor and cell line panels reveal a striking correlation between N-myc amplification and mRNA and protein expression of both nm23 genes. We show that the nm23 genes are located at the edge of the common region of chromosome 17q gain previously described in neuroblastoma cell lines. Our findings suggest that nm23-H1 and nm23-H2 expression is increased by 17q gain in neuroblastoma and can be further up-regulated by myc overexpression. These observations suggest a major role for nm23-H1 and nm23-H2 in tumorigenesis of unfavorable neuroblastomas.

14q Deletions in Neuroblastoma

Theobald M, Christiansen H, Schmidt A, et al.
Sublocalization of putative tumor suppressor gene loci on chromosome arm 14q in neuroblastoma.
Genes Chromosomes Cancer. 1999; 26(1):40-6 [PubMed] Related Publications
RFLP and microsatellite analysis with 23 polymorphic markers spanning the entire long arm of chromosome 14 in 108 neuroblastomas showed allelic loss in 19 out of 107 (18%) informative tumors, placing 14q among the most frequently affected chromosomal regions in neuroblastoma. One minimal deletion region could be sublocalized in 17 of 19 cases between markers D14S1 and D14S16, and a second one between markers D14S17 and D14S23 in band 14q32. Furthermore, breakpoints in bands 14q23 and 14q12 were detected. These results suggest the presence of at least two putative tumor suppressor gene loci on chromosome 14. Survival analyses revealed no prognostic impact of allelic loss of 14q in neuroblastoma. Genes Chromosomes Cancer 26:40-46, 1999.

Hoshi M, Otagiri N, Shiwaku HO, et al.
Detailed deletion mapping of chromosome band 14q32 in human neuroblastoma defines a 1.1-Mb region of common allelic loss.
Br J Cancer. 2000; 82(11):1801-7 [PubMed] Free Access to Full Article Related Publications
Neuroblastoma (NB) is a well-known malignant disease in infants, but its molecular mechanisms have not yet been fully elucidated. To investigate the genetic contribution of abnormalities on the long arm of chromosome 14 (14q) in NB, we analysed loss of heterozygosity (LOH) in 54 primary NB samples using 12 microsatellite markers on 14q32. Seventeen (31%) of 54 tumours showed LOH at one or more of the markers analysed, and the smallest common region of allelic loss was identified between D14S62 and D14S987. This region was estimated to be 1-cM long from the linkage map. Fluorescence in situ hybridization also confirmed the loss. There was no statistical correlation between LOH and any clinicopathologic features, including age, stage, amplification of MYCN and ploidy. We further constructed a contig spanning the lost region using bacterial artificial chromosome and estimated this region to be approximately 1.1-Mb by pulsed-field gel electrophoresis. Our results will contribute to cloning and characterizing the putative tumour-associated gene(s) in 14q32 in NB.

Thompson PM, Seifried BA, Kyemba SK, et al.
Loss of heterozygosity for chromosome 14q in neuroblastoma.
Med Pediatr Oncol. 2001; 36(1):28-31 [PubMed] Related Publications
BACKGROUND: Neuroblastoma is a genetically heterogeneous disease, with subsets of tumors demonstrating rearrangements of several genomic regions. Preliminary studies from several groups have identified loss of heterozygosity (LOH) for the long arm of chromosome 14 (14q) in 20-25% of primary neuroblastomas.
PROCEDURE: To determine precisely the frequency and extent of 14q deletions, we performed LOH analysis for a large series of primary neuroblastomas using a panel of 11 highly polymorphic markers.
RESULTS: LOH was detected in 83 of 372 tumors (22%). Although the majority of tumors with allelic loss demonstrated allelic loss for all informative markers, 13 cases showed LOH for only a portion of 14q. A single consensus region of deletion, which was shared by all tumors with 14q LOH, was defined within 14q23-q32 between D14S588 and the 14q telomere. Allelic loss for 14q was strongly correlated with the presence of 11q LOH (P < 0.001 ) and inversely correlated with MYCN amplification (P= 0.04).
CONCLUSIONS: LOH for 14q was evident in all clinical risk groups, indicating that this abnormality may be a universal feature of neuroblastoma tumor development. These findings suggest that a tumor suppressor gene involved in the initiation or progression of neuroblastoma is located within distal 14q.

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