Identification of CASZ1 NES reveals potential mechanisms for loss of CASZ1 tumor suppressor activity in neuroblastoma.

As a transcription factor, localization to the nucleus and the recruitment of cofactors to regulate gene transcription is essential. Nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex binding are required for the zinc-finger transcription factor CASZ1 to function as a neuroblastoma (NB) tumor suppressor. However, the critical amino acids (AAs) that are required for CASZ1 interaction with NuRD complex and the regulation of CASZ1 subcellular localization have not been characterized. Through alanine scanning, immunofluorescence cell staining and co-immunoprecipitation, we define a critical region at the CASZ1 N terminus (AAs 23-40) that mediates the CASZ1b nuclear localization and NuRD interaction. Furthermore, we identified a nuclear export signal (NES) at the N terminus (AAs 176-192) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a chromosomal maintenance 1-dependent manner. An analysis of CASZ1 protein expression in a primary NB tissue microarray shows that high nuclear CASZ1 staining is detected in tumor samples from NB patients with good prognosis. In contrast, cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining is found in tumor samples from patients with poor prognosis. These findings provide insight into mechanisms by which CASZ1 regulates transcription, and suggests that regulation of CASZ1 subcellular localization may impact its function in normal development and pathologic conditions such as NB tumorigenesis.

Augmented expression of MYC and/or MYCN protein defines highly aggressive MYC-driven neuroblastoma: a Children's Oncology Group study.

BACKGROUND: MYCN amplification with subsequent MYCN protein overexpression is a powerful indicator of poor prognosis of neuroblastoma patients. Little is known regarding the prognostic significance of the homologous MYC protein expression in neuroblastoma. METHODS: Immunostaining for MYCN and MYC protein was performed on 357 undifferentiated/poorly differentiated neuroblastomas. Results were analysed with other prognostic markers. RESULTS: Sixty-seven (19%) tumours were MYCN(+), 38 (11%) were MYC(+), and one(0.3%) had both proteins(+). MYCN(+) tumours and MYC(+) tumours were more likely diagnosed in children>18months with stage4-disease. MYCN(+) tumours were associated with amplified MYCN, Unfavourable Histology (UH), and High-MKI (Mitosis-Karyorrhexis Index). MYC(+) tumours were also frequently UH but not associated with MYCN amplification, and more likely to have low-/intermediate-MKI. Favourable Histology patients without MYC/MYCN expressions exhibited the best survival (N=167, 89.7±5.5% 3-year EFS, 97.0±3.2% 3-year OS), followed by UH patients without MYC/MYCN expressions (N=84, 63.1±13.6% 3-year EFS, 83.5±9.4% 3-year OS). MYCN(+)patients and MYC(+)patients had similar and significantly low (P<0.0001) survivals (46.2±12.0% 3-year EFS, 63.2±12.1% 3-year OS and 43.4±23.1% 3-year EFS, 63.5±19.2% 3-year OS, respectively). Notably, the prognostic impact imparted by MYC expression was independent from other markers. CONCLUSIONS: In this series, ∼30% of neuroblastomas had augmented MYCN or MYC expression with dismal survivals. Prospective study of MYC/MYCN protein expression signature as a new biomarker for high-risk neuroblastomas should be conducted.

Integrating cell-based and clinical genome-wide studies to identify genetic variants contributing to treatment failure in neuroblastoma patients.

High-risk neuroblastoma is an aggressive malignancy, with high rates of treatment failure. We evaluated genetic variants associated with in vitro sensitivity to two derivatives of cyclophosphamide for association with clinical response in a separate replication cohort of neuroblastoma patients (n = 2,709). To determine sensitivity, lymphoblastoid cell lines (LCLs) were exposed to increasing concentrations of 4-hydroperoxycyclophosphamide (4HC; n = 422) and phosphoramide mustard (PM; n = 428). Genome-wide association studies were performed to identify single-nucleotide polymorphisms (SNPs) associated with sensitivity to 4HC and PM. SNPs consistently associated with LCL sensitivity were analyzed for associations with event-free survival (EFS) in patients. Two linked SNPs, rs9908694 and rs1453560, were found to be associated with (i) sensitivity to PM in LCLs across populations and (ii) EFS in all patients (P = 0.01) and within the high-risk subset (P = 0.05). Our study highlights the value of cell-based models to identify candidate variants that may predict response to treatment in patients with cancer.

A pilot study of tandem high-dose chemotherapy with stem cell rescue as consolidation for high-risk neuroblastoma: Children's Oncology Group study ANBL00P1.

Increasing treatment intensity has improved outcomes for children with neuroblastoma. We performed a pilot study in the Children's Oncology Group to assess the feasibility and toxicity of a tandem myeloablative regimen without TBI supported by autologous CD34-selected peripheral blood stem cells. Forty-one patients with high-risk neuroblastoma were enrolled; eight patients did not receive any myeloablative consolidation procedure and seven received only one. Two patients out of 41 (4.9%) experienced transplant-related mortality. CD34 selection was discontinued after subjects were enrolled due to serious viral illness. From the time of study enrollment, the overall 3-year EFS and OS were 44.8 ± 9.6% and 59.2 ± 9.2% (N=41). These results demonstrate that tandem transplantation in the cooperative group setting is feasible and support a randomized comparison of single vs tandem myeloablative consolidation with PBSC support for high-risk neuroblastoma.

Segmental chromosomal alterations have prognostic impact in neuroblastoma: a report from the INRG project.

BACKGROUND: In the INRG dataset, the hypothesis that any segmental chromosomal alteration might be of prognostic impact in neuroblastoma without MYCN amplification (MNA) was tested. METHODS: The presence of any segmental chromosomal alteration (chromosome 1p deletion, 11q deletion and/or chromosome 17q gain) defined a segmental genomic profile. Only tumours with a confirmed unaltered status for all three chromosome arms were considered as having no segmental chromosomal alterations. RESULTS: Among the 8800 patients in the INRG database, a genomic type could be attributed for 505 patients without MNA: 397 cases had a segmental genomic type, whereas 108 cases had an absence of any segmental alteration. A segmental genomic type was more frequent in patients >18 months and in stage 4 disease (P<0.0001). In univariate analysis, 11q deletion, 17q gain and a segmental genomic type were associated with a poorer event-free survival (EFS) (P<0.0001, P=0.0002 and P<0.0001, respectively). In multivariate analysis modelling EFS, the parameters age, stage and a segmental genomic type were retained in the model, whereas the individual genetic markers were not (P<0.0001 and RR=2.56; P=0.0002 and RR=1.8; P=0.01 and RR=1.7, respectively). CONCLUSION: A segmental genomic profile, rather than the single genetic markers, adds prognostic information to the clinical markers age and stage in neuroblastoma patients without MNA, underlining the importance of pangenomic studies.

International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee.

Neuroblastoma serves as a paradigm for utilising tumour genomic data for determining patient prognosis and treatment allocation. However, before the establishment of the International Neuroblastoma Risk Group (INRG) Task Force in 2004, international consensus on markers, methodology, and data interpretation did not exist, compromising the reliability of decisive genetic markers and inhibiting translational research efforts. The objectives of the INRG Biology Committee were to identify highly prognostic genetic aberrations to be included in the new INRG risk classification schema and to develop precise definitions, decisive biomarkers, and technique standardisation. The review of the INRG database (n=8800 patients) by the INRG Task Force finally enabled the identification of the most significant neuroblastoma biomarkers. In addition, the Biology Committee compared the standard operating procedures of different cooperative groups to arrive at international consensus for methodology, nomenclature, and future directions. Consensus was reached to include MYCN status, 11q23 allelic status, and ploidy in the INRG classification system on the basis of an evidence-based review of the INRG database. Standardised operating procedures for analysing these genetic factors were adopted, and criteria for proper nomenclature were developed. Neuroblastoma treatment planning is highly dependant on tumour cell genomic features, and it is likely that a comprehensive panel of DNA-based biomarkers will be used in future risk assignment algorithms applying genome-wide techniques. Consensus on methodology and interpretation is essential for uniform INRG classification and will greatly facilitate international and cooperative clinical and translational research studies.

Prevalence and functional consequence of PHOX2B mutations in neuroblastoma.

PHOX2B is a homeodomain-containing protein that is involved in the development of the peripheral nervous system and is the major disease gene for the rare congenital breathing disorder congenital central hypoventilation syndrome (CCHS). Germline PHOX2B alterations were also recently discovered in neuroblastoma cases with CCHS and/or Hirschsprung disease, but a comprehensive survey for mutational frequency and functional consequence has not been performed. We therefore studied a large panel of hereditary neuroblastomas to understand the frequency and functional effects of PHOX2B mutations. Three of 47 individuals with presumed genetic predisposition to neuroblastoma showed a germline PHOX2B mutation (6.4%). Mutations were also discovered in 2 of 30 human neuroblastoma-derived cell lines, but none of 86 primary tumors from patients with sporadically occurring neuroblastoma. The vast majority of primary tumors showed abundant PHOX2B mRNA expression relative to the remainder of the transcriptome. Consistent with its role as an important neurodevelopmental gene, forced overexpression of wild-type PHOX2B in neuroblastoma cell lines suppressed cell proliferation and synergized with all-trans retinoic acid to promote differentiation. Patient-derived mutant PHOX2B constructs retained the ability to suppress cellular proliferation, but were not able to promote differentiation or activate expression of a known PHOX2B target gene in vitro. These findings show that PHOX2B alterations are a rare cause of hereditary neuroblastoma, but disruption of this neurodevelopmental pathway can interfere with transcription-dependent terminal differentiation. These data also suggest that the genetics of neuroblastoma initiation are complex, and highlight genes involved in normal noradrenergic development as candidate predisposition genes.

Deregulated Wnt/beta-catenin program in high-risk neuroblastomas without MYCN amplification.

Neuroblastoma (NB) is a frequently lethal tumor of childhood. MYCN amplification accounts for the aggressive phenotype in a subset while the majority have no consistently identified molecular aberration but frequently express MYC at high levels. We hypothesized that activated Wnt/beta-catenin (CTNNB1) signaling might account for this as MYC is a beta-catenin transcriptional target and multiple embryonal and neural crest malignancies have oncogenic alterations in this pathway. NB cell lines without MYCN amplification express higher levels of MYC and beta-catenin (with aberrant nuclear localization) than MYCN-amplified cell lines. Evidence for aberrant beta-catenin-TCF transcriptional activity was demonstrated using expression profiles from 73 primary NBs. Findings included increased WNT ligands (WNT1, WNT6, WNT7A, WNT10B), DVL1 and TCF7 expression in high-risk NBs without MYCN amplification, consistent with canonical beta-catenin signaling. More directly, Patterns of Gene Expression and Gene Set Enrichment Analyses demonstrated beta-catenin target genes (for example, MYC, PPARD, NRCAM, CD44, TCF7) as coordinately upregulated in high-risk NBs without MYCN amplification in comparison to high-risk MYCN-amplified or intermediate-risk NBs, supporting pathway activation in this subset. Thus, high-risk NBs without MYCN amplification may deregulate MYC and other oncogenic genes via altered beta-catenin signaling providing a potential candidate pathway for therapeutic inhibition.

Evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children's Oncology Group.

PURPOSE: In the Children's Oncology Group, risk group assignment for neuroblastoma is critical for therapeutic decisions, and patients are stratified by International Neuroblastoma Staging System stage, MYCN status, ploidy, Shimada histopathology, and diagnosis age. Age less than 365 days has been associated with favorable outcome, but recent studies suggest that older age cutoff may improve prognostic precision. METHODS: To identify the optimal age cutoff, we retrospectively analyzed data from the Pediatric Oncology Group biology study 9047 and Children's Cancer Group studies 321p1-p4, 3881, 3891, and B973 on 3,666 patients (1986 to 2001) with documented ages and follow-up data. Twenty-seven separate analyses, one for each different age cutoff (adjusting for MYCN and stage), tested age influence on outcome. The cutoff that maximized outcome difference between younger and older patients was selected. RESULTS: Thirty-seven percent of patients were younger than 365 days, and 64% were > or = 365 days old (4-year event-free survival [EFS] rate +/- SE: 83% +/- 1% [n = 1,339] and 45% +/- 1% [n = 2,327], respectively; P < .0001). Graphical analyses revealed the continuous nature of the prognostic contribution of age to outcome. The optimal 460-day cutoff we selected maximized the outcome difference between younger and older patients. Forty-three percent were younger than 460 days, and 57% were > or = 460 days old (4-year EFS rate +/- SE: 82% +/- 1% [n = 1,589] and 42% +/- 1% [n = 2,077], respectively; P < .0001). Using a 460-day cutoff (assuming stage 4, MYCN-amplified patients remain high-risk), 5% of patients (365 to 460 days: 4-year EFS 92% +/- 3%; n = 135) fell into a lower risk group. CONCLUSION: The prognostic contribution of age to outcome is continuous in nature. Within clinically relevant risk stratification, statistical support exists for an age cutoff of 460 days.

Biological aspects of neuroblastomas identified by mass screening in Quebec.

BACKGROUND: Neuroblastoma has several characteristics that suggest that preclinical diagnosis might improve outcome. Therefore, the Quebec Neuroblastoma Screening Project was undertaken from 1989 to 1994 to examine infants at 3 weeks and 6 months by measuring urinary catecholamine metabolites. PROCEDURE: Over the 5-yr period, 45 tumors were detected by screening, 20 were identified clinically prior to the third week, and 64 were identified clinically at a later time. We analyzed available tumors for Shimada histopathology, tumor ploidy, MYCN copy number and serum ferritin. RESULTS: Of the tumors detected by screening, only 2 of 45 tested had unfavorable histology, 2 of 45 had diploid or tetraploid DNA content, 0 of 43 had MYCN amplification, and 4 of 44 had elevated serum ferritin. All of these patients are alive and well. The 20 patients detected prior to the 3-week screen had similar biological characteristics. In contrast, of the patients detected clinically after 3 weeks of age, 19 of 51 testedhad unfavorable histology, 25 of 66 had diploid or tetraploid tumors, 12 of 56 had MYCN amplification, and 14 of 54 had elevated ferritin. CONCLUSIONS: The difference between the screened and clinically detected cases was highly significant for each biological variable. Preliminary data on other biological variables, such as neurotrophin expression and allelic loss on 1 p in these patients are consistent with the above findings. These data suggest that mass screening for neuroblastoma at or before 6 months of age detects almost exclusively tumors that have favorable biological characteristics, many of which might have regressed spontaneously. Thus, continued mass screening for neuroblastoma at 6 months is unlikely to accomplish its intended goal, and should probably be discontinued.

Allelic deletion at chromosome bands 11q14-23 is common in neuroblastoma.

BACKGROUND: Neuroblastoma tumorigenesis may involve the differential inactivation of multiple tumor suppressor genes. Recent data have suggested that a neuroblastoma suppressor gene may be located on the long arm of chromosome 11 (11q). PROCEDURE: We therefore analyzed 295 primary neuroblastomas from a representative group of patients for loss of heterozygosity (LOH) at 25 polymorphic markers spanning 11q. RESULTS: LOH was observed in 129 primary neuroblastomas (44%), and a common region of LOH mapped to 11q14-23. No correlation was found between 11q LOH and adverse prognostic variables, but a strong inverse relationship between 11q LOH and MYCN amplification (P < 0.001) was observed. There was no difference in overall survival when patients were stratified by 11q LOH status. However, 11q LOH was associated with a decreased overall survival probability when patients whose tumors had a single copy of MYCN were analyzed separately (P = 0.008). CONCLUSION: These data support the hypothesis that a tumor suppressor gene mapping within 11q14-23 is frequently inactivated during the malignant evolution of neuroblastoma.

Loss of heterozygosity for chromosome 14q in neuroblastoma.

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.

Comprehensive analysis of chromosome 1p deletions in neuroblastoma.

BACKGROUND: Chromosome 1p deletions are common in advanced neuroblastomas, but the biological and clinical implications of this clonal rearrangement remain controversial. Previous studies of chromosome 1p loss of heterozygosity (LOH) have been limited by analyses of relatively small number of tumors derived from heterogeneously assessed and treated patient populations. Therefore, a strictly representative cohort of 288 Children's Cancer Group neuroblastoma patients treated on the most recent phase III therapeutic trials was identified. PROCEDURE: Primary tumors from these patients were analyzed for LOH at precisely mapped and highly informative 1p polymorphic loci located from 1p32 to 1p36.3 by multiplex PCR. RESULTS: Ninety-three primary tumor specimens (32%) had LOH at multiple 1p36 marker loci. All 1p deletions overlapped the previously determined smallest region of overlap (SRO). One tumor had a small terminal deletion completely within 1p36.3, allowing for further refinement of the 1p36 SRO. We found no evidence to support an additional, nonoverlapping region of LOH within 1p32-36. We confirmed the strong correlation of 1p36 LOH with MYCN amplification (P < 0.001), advanced disease stage (P < 0.001), and decreased both 3-year event-free survival and overall survival probabilities (P< 0.001). When stratified for MYCN amplification status or entered into a multivariate analysis, 1p36 LOH remained predictive for decreased event-free survival, but not overall survival probability. CONCLUSIONS: These data support the hypothesis that inactivation of a tumor suppressor gene within 1p36.3 is associated with an increased risk for disease relapse.

Detailed molecular analysis of 1p36 in neuroblastoma.

BACKGROUND: Several lines of evidence es tablish that chromosome band 1p36 is frequently deleted in neuroblastoma primary tumors and cell lines, suggesting that a tumor suppressor gene within this region is involved in the development of this tumor. PROCEDURE: We analyzed the status of 1p36 in primary neuroblastomas and cell lines to define the region of consistent rearrangement. RESULTS: Loss of heterozygosity (LOH) studies of primary neuro blastomas identified allelic loss in 135 of 503 tumors (27%), with the smallest region of overlap (SRO) defined distal to D15214 (1p36.3). No homozygous deletions were detected at 120 loci mapping to 1p36.1-p36.3 in a panel of 46 neuroblastoma cell lines. A recently identified patient with neuroblastoma was found to have a constitutional deletion within 1p36.2-p36.3, and this deletion, when combined with the LOH results, defined a smaller SRO of one megabase within 1p36.3. We constructed a comprehensive integrated map of chromosome 1 containing 11,000 markers and large-insert clones, a high-resolution radiation hybrid (RH) map of 1p36, and a P1-artificial chromosome (PAC) contig spanning the SRO, to further characterize the region of interest. Over 768 kb (75%) of the SRO has been sequenced to completion. Further analysis of distal 1p identified 113 transcripts localizing to 1p36, 21 of which were mapped within the SRO. CONCLUSION: This analysis will identify suitable positional candidate transcripts for mutational screening and subsequent identification of the 1p36.3 neuroblastoma suppressor gene.

Analysis of genomic imprinting at 1p35-36 in neuroblastoma.

BACKGROUND: Deletion of the distal short arm of chromosome 1 occurs in 25-35% of primary neuroblastomas, and a putative tumor suppressor gene has been mapped to a consensus region of deletion at 1p36.2-36.3. Indirect evidence suggests the presence of an imprinted neuroblastoma suppressor gene within this region, as well as an additional nonimprinted, proximal suppressor gene, inactivation of which correlates with MYCN amplification. PROCEDURE: To test this hypothesis, we performed 1p loss of heterozygosity (LOH) studies on a series of neuroblastomas for which parental DNA had been collected. PCR-formatted polymorphic markers were used to determine the size of the 1p deletion and the parental origin of the deleted 1p homologue. RESULTS: Twenty-six neuroblastomas with 1p LOH were evaluated. Twenty-four had MYCN amplification, and of these, 15 demonstrated loss of the paternally inherited 1p. Two neuroblastomas with a single copy of MYCN were evaluated and both had deletion of the paternally inherited 1p, with one case exhibiting a small terminal deletion. In addition, we have reviewed 49 previously reported neuroblastomas where 1p LOH data and the parental origin of the deleted lp homologue were available. CONCLUSIONS: Analyzed together, these 75 neuroblastomas demonstrate random deletion of parental 1p homologues (P = 0.30). Further, tumors with smaller deletions (breakpoints distal to D1S201 or D1S7) showed a random loss of the parental 1p homologues (P = 0.59), contrary to the expected preferential maternal 1p deletion if an imprinted suppressor gene mapped to this region. However, 19 tumors with 1p LOH and single copy MYCN had deletion of the maternal 1p homologue preferentially (P = 0.02), which does not exclude the possibility that loss of an imprinted suppressor gene plays a role in this subset.

The angiogenesis inhibitor tnp-470 effectively inhibits human neuroblastoma xenograft growth, especially in the setting of subclinical disease.

Tumor vascularity is highly correlated with disease outcome in neuroblastoma. Thus, novel therapeutics that target the vascular endothelium are candidates for incorporation into clinical trials. We therefore examined the effect of TNP-470 on human neuroblastoma growth in mouse models reflecting both clinically evident and minimal disease. Mice were inoculated s.c. or by tail vein injection with 10(7) human neuroblastoma-derived CHP-134 cells and treated with TNP-470 (100 mg/kg/dose s.c. three times a week or by continuous infusion) or saline. Treatment was given as a single agent in established xenografts, 10 days after 450 mg/kg of cyclophosphamide, or 12 h after tumor inoculation. Tumor growth rate was markedly inhibited in mice receiving TNP-470 administered alone both s.c. and by continuous infusion with a treatment to control ratio (T:C) at day 16 of 0.3 (P < 0.001) and a T:C at day 30 of 0.4 (P = 0.029) for each dosing method, respectively. TNP-470 also significantly inhibited tumor growth when administered following cyclophosphamide (T:C at day 30 = 0.2, P < 0.001) and inhibited disease establishment when given shortly after xenograft inoculation (T:C at day 30 = 0.1, P < 0.001) or tail vein injection. TNP-470 was shown to directly inhibit angiogenesis by Matrigel assay (P =.010) and to increase the apoptotic index in treated tumors. These data show that TNP-470 is a potent inhibitor of human neuroblastoma growth rate and tumorigenicity. We speculate that TNP-470 may be a useful adjuvant therapy for high-risk neuroblastoma patients, particularly when used in settings of minimal disease status.

Homozygous deletion of CDKN2A (p16INK4a/p14ARF) but not within 1p36 or at other tumor suppressor loci in neuroblastoma.

Loss of heterozygosity of several specific genomic regions is frequently observed in neuroblastoma tumors and cell lines, but homozygous deletion (HD) is rare, and no neuroblastoma tumor suppressor gene (TSG) has yet been identified. We performed a systematic search for HD, indicative of a disrupted TSG, in a panel of 46 neuroblastoma cell lines. An initial search focused on a well-characterized consensus region of hemizygous deletion at 1p36.3, which occurs in 35% of primary neuroblastomas. Each cell line was screened with 162 1p36 markers, for a resolution of 13 kb within the consensus 1p36.3 deletion region and 350 kb throughout the remainder of 1p36. No HDs were detected. This approach was expanded to survey 21 known TSGs, specifically targeting intragenic regions frequently inactivated in other malignancies. HD was detected only at the CDKN2A (p16INK4a/p14ARF) gene at 9p21 and was observed in 4 of 46 cell lines. The observed region of HD included all exons of both CDKN2A and the closely linked CDKN2B (p15INK4b) gene for cell lines LA-N-6 and CHLA-174, all exons of CDKN2A but none of CDKN2B for CHLA-179, and only 104 bp within CDKN2A exon 2 for CHLA-101. All four deletions are predicted to inactivate the coding regions of both p16INK4a and p14ARF. HD was observed in corresponding primary tumor samples for CHLA-101 and CHLA-174 but was not present in constitutional samples. These results suggest that for neuroblastoma, large HDs do not occur within 1p36, most known TSGs are not homozygously deleted, and biallelic inactivation of CDKN2A may contribute to tumorigenicity in a subset of cases.

Identification of a 1-megabase consensus region of deletion at 1p36.3 in primary neuroblastomas.

BACKGROUND: Deletion of the distal short arm of chromosome 1 occurs frequently in neuroblastoma. In addition, neuroblastoma has been described in children with constitutional deletions within 1p36, supporting the existence of one or more neuroblastoma suppressor genes within this region. PROCEDURE: We have pursued a 1p36 tumor suppressor gene identification strategy that has included deletion mapping of 566 primary neuroblastomas and 46 neuroblastoma-derived cell lines, and have determined the parental origin of the deleted 1p homologue in 44 cases to determine whether there is evidence for genomic imprinting within this region. RESULTS AND CONCLUSIONS: We have identified a 1-Mb consensus region of deletion within 1p36.3 defined by primary tumor deletions, constructed a physical map of the region that is being sequenced to completion, and have identified and prioritized candidate genes within this region for further analyses.

Localization of a hereditary neuroblastoma predisposition gene to 16p12-p13.

BACKGROUND: Hereditary predisposition to develop neuroblastoma segregates as an autosomal dominant Mendelian trait. PROCEDURE: We have performed linkage analysis on 10 families with neuroblastoma to localize a hereditary neuroblastoma predisposition gene (HNB1). RESULTS: A single genomic interval at chromosome bands 16p12-p13 was consistent with linkage (lod = 3.46), and identification of informative recombinants defined a 25.9-cM critical region between D16S748 and D16S3068. Loss of heterozygosity was identified in 5/12 familial (42%) and 55/259 nonfamilial (21%) neuroblastomas at multiple 16p polymorphic loci. A 12.8-cM smallest region of overlap of deletions was identified within the interval defined by linkage analysis (tel-D16S764-D16S412-cen). CONCLUSIONS: Taken together, these data suggest that HNB1 is located at 16p12-p13 and that inactivation of this gene may contribute to the pathogenesis of nonfamilial neuroblastomas.

Deletion of 11q23 is a frequent event in the evolution of MYCN single-copy high-risk neuroblastomas.

BACKGROUND: Deletions of the long arm of chromosome 11 are frequently identified in human neuroblastomas. PROCEDURE: We screened 394 primary neuroblastomas and 52 tumor-derived cell lines with a panel of 11q and 11p polymorphic markers to determine the frequency of chromosome 11 allelic deletion, and to differentiate partial deletions of chromosome 11q (unb[11q] LOH) from whole chromosome loss. RESULTS: Allelic deletion occurred most frequently at cytogenetic band 11q23 and was detected in 161 primary neuroblastomas (41%) and 18 cell lines (35%). Eighty-seven tumors (22%) had unb[11q] LOH with a heterogeneous distribution of deletion breakpoints. Unb[11q] LOH was highly correlated with age > 1 year at diagnosis (P = 0.008), stage 4 disease (P = 0.001), unfavorable Shimada histopathology (P < 0.001), and assignment to a high-risk therapeutic protocol (P < 0.001), and was inversely correlated with MYCN amplification (P = 0.018). Patients whose tumors showed unb[11q] LOH were less likely to survive (P < 0.001), but there was only a trend towards an independent prognostic influence in multivariate analyses. CONCLUSIONS: Thus, structural rearrangements resulting in unb[11q] LOH commonly occur during the malignant evolution of high-risk neuroblastomas with single-copy MYCN.