Project 1: Identification of the Hereditary Neuroblastoma Predisposition Gene

Like all human cancers, a small percentage of neuroblastomas are inherited. We have had a program for over 15 years to collect specimens from these rare families, as well as other children presumed to have genetic susceptibility to develop this disease (1-4). We previously showed that mutations in the homeobox gene PHOX2B occur in complex cases of sporadic and hereditary neuroblastoma that co-occur with other diseases of neural crest tissues such as Hirschsprung disease and congenital central hypoventilation syndrome (5, 6). Through an international collaborative effort, we have recently completed a high resolution whole genome scan with 6000 SNPs, and discovered a locus on chromosome 2p that is highly likely to contain the familial neuroblastomagene. Because this is a region that is frequently gained somatically, we hypothesize that it will be an oncogene. We recently discovered germline mutations segregating with neuroblastoma pedigrees in one such gene, and these data will be reported at the American Association for Cancer Research Meeting in April 2008. Future work will focus on characterizing the mutation spectrum of this recently identified gene in familial and sporadic cases, understanding the functional consequences of these mutations, and developing strategies for diagnostics and therapeutics.

1. Maris JM, Brodeur GM. Genetics. In: Cheung N-KV, Cohn SL, editors. Neuroblastoma. Berlin, Heidelberg, New York: Springer; 2005. p. 21-6.

2. Maris JM, Chatten J, Meadows AT, Biegel JA, Brodeur GM. Familial neuroblastoma: A three generation pedigree and a further association with Hirschsprung disease. Medical and Pediatric Oncology. 1997;28(1):1-5.

3. Maris JM, Kyemba SM, Rebbeck TR, White PS, Sulman EP, Jensen SJ, et al. Familial predisposition to neuroblastoma does not map to chromosome band 1p36. Cancer Res. 1996;56(15):3421-5.

4. Maris JM, Weiss MJ, Mosse Y, Hii G, Guo C, White PS, et al. Evidence for a hereditary neuroblastoma predisposition locus at chromosome 16p12-13. Cancer Res. 2002 Nov 15;62(22):6651-8.

5. Mosse YP, Laudenslager M, Khazi D, Carlisle AJ, Winter CL, Rappaport E, et al. Germline PHOX2B Mutation in Hereditary Neuroblastoma. Am J Hum Genet. 2004 Oct;75(4):727-30.

6. Raabe EH, Laudenslager M, Winter C, Wasserman N, Cole K, LaQuaglia M, et al. Prevalence and functional consequence of PHOX2B mutations in neuroblastoma. Oncogene. 2008 Jan 17;27(4):469-76.

Project 2: The Genetic Basis of Neuroblastoma Tumorigenesis: A Genome-wide association study (GWAS) approach

Project 1 is focused on discovering the rare mutations that provide a very high likelihood for developing neuroblastoma. Like breast cancer and other human malignancies, these mutations are likely to account for only a small fraction of all cases. Thus, we hypothesize that common variations in the human genome can also influence susceptibility to develop neuroblastoma. We therefore are engaged in a GWAS enabled efforts through the Children's Oncology Group (www.childrensoncologygroup.org) to collect thousands of DNA samples from neuroblastoma patients. We ultimately will genotype 5000 neuroblastoma cases at over 550,000 single nucleotide polymorphisms, and compare these data to a larger group of children without cancer collected as part of the Center for Applied Genomics (link for Center for Applied Genomics) ongoing program to genotype over 100,000 children from the Children's Hospital of Philadelphia. We recently completed an interim analysis that provides critical proof-of-concept. After genotyping the first 1032 subjects, we identified a highly significant association signal that has now been replicated in three separate cohorts (30). In addition, we have discovered another strong association signal within a known Tumor Suppressor Gene when one limits the analytic cohort to children with the high-risk form of the disease. Finally, instead of looking at genotypes, we also performed an analysis restricted to copy number variation in the human genome. We discovered several strong association signals, two of which show robust replication (some of the others may require additional cases and controls, and these are being added). Ongoing work is focused on understanding the underlying genetic alterations that are driving these association signals while also building up the case-series to discover additional loci.

External data links: GWAS Phase 1 Discovery case-series:
(http://www.ncbi.nlm.nih.gov/sites/entrez?db=gap)

30. Maris JM, Mosse, Y. P., Bradfield, J. P., Hou, C., et al. A Genome-wide Association Study Identifies a Susceptibility Locus to Clinically Aggressive Neuroblastoma at 6p22 New England Journal of Medicine. 2008 May 7.

Project 3: Significance of Genetic Alterations in Neuroblastoma

Neuroblastoma is a disease that has very diverse clinical features, with about a third of cases being extremely benign and easily cured, while about half of cases are very aggressive with cure rates below 40%. Our lab has had a long-standing interest in defining the genetic correlates of this clinical behavior (7-13). This project focuses on genome-wide approaches to discover recurrent somatically acquired alterations in tumors obtained at diagnosis through the Children's Oncology Group. Project 4-6 have evolved out of these efforts as we have shown that there are patterns at the DNA and RNA levels that are strong predictors of phenotype. The current focus of this project is to develop a chip-based molecular diagnostic assay to reliably predict neuroblastoma clinical course (14). We will utilize a SNP-based system, and plan for this to be the assay that will be done on all newly diagnosed neuroblastoma patients in the COG that will allow for individualization of treatment planning.

External data links: 101 samples studied on the Affymetrix U95A expression and BAC array platform: (http://www.ncbi.nlm.nih.gov/geo/)

7. Mosse YP, Diskin SJ, Wasserman N, Rinaldi K, Attiyeh EF, Cole K, et al. Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression. Genes Chromosomes Cancer. 2007 Oct;46(10):936-49.

8. Wang Q, Diskin S, Rappaport E, Attiyeh E, Mosse Y, Shue D, et al. Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number. Cancer Res. 2006 Jun 15;66(12):6050-62.

9. Mosse YP, Greshock J, Margolin A, Naylor T, Cole K, Khazi D, et al. High-resolution detection and mapping of genomic DNA alterations in neuroblastoma. Genes Chromosomes Cancer. 2005 Aug;43(4):390-403.

10. Maris JM. The biologic basis for neuroblastoma heterogeneity and risk stratification. Curr Opin Pediatr. 2005 Feb;17(1):7-13.

11. Attiyeh EF, London WB, Mosse YP, Wang Q, Winter C, Khazi D, et al. Chromosome 1p and 11q deletions and outcome in neuroblastoma. N Engl J Med. 2005 Nov 24;353(21):2243-53.

12. Maris JM, Weiss MJ, Guo C, Gerbing RB, Stram DO, White PS, 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. Journal of Clinical Oncology. 2000;18(9):1888-99.

13. Guo C, White PS, Weiss MJ, Hogarty MD, Thompson PM, Stram DO, et al. Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas. Oncogene. 1999;18:4948-57.

14. Maris JM, Hii G, Gelfand CA, Varde S, White PS, Rappaport E, et al. Region-specific detection of neuroblastoma loss of heterozygosity at multiple loci simultaneously using a SNP-based tag-array platform. Genome Res. 2005 Aug;15(8):1168-76.

Project 4: Genomics and Epigenomics of Human Neuroblastoma

One of the more common recurrent alterations in high-risk neuroblastomas is deletion of chromosome 3p (7, 9). This project is designed to discover the Tumor Suppressor Genes located at 3p that are inactivated during the malignant evolution of these tumors. Regions of interest are mapped using high-resolution array based methodologies and highly annotated tumor samples. Genes within these regions are subjected to resequencing to identify putative inactivating alterations. In addition, we have strong preliminary evidence that several genes in our regions of interest may be functionally inactivated via epigenetic mechanisms such as hypermethylation. Thus, ongoing and future work will focus on integration of genomic and epigenomic data sets, both focused on 3p as well as genome-wide.

7. Mosse YP, Diskin SJ, Wasserman N, Rinaldi K, Attiyeh EF, Cole K, et al. Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression. Genes Chromosomes Cancer. 2007 Oct;46(10):936-49.

9. Mosse YP, Greshock J, Margolin A, Naylor T, Cole K, Khazi D, et al. High-resolution detection and mapping of genomic DNA alterations in neuroblastoma. Genes Chromosomes Cancer. 2005 Aug;43(4):390-403.

Project 5: Discovery of Chromosome Arm 11q Tumor Suppressor Genes in Neuroblastoma

We recently showed that deletion of the long arm of chromosome 11 is a very powerful biomarker for outcome in newly diagnosed neuroblastoma patients (11). This has now been incorporated into a prospective clinical trial in the Children's Oncology Group focused on children with intermediate-risk neuroblastoma (those with a clinical course most difficult to predict). This project now focuses on discovering the Tumor Suppressor Genes that are targeted by these chromosomal deletions. We have mapped regions of interest using high density SNP arrays, and hypothesize that several genes are likely involved since the fast majority of the deletions are very large. Current work is focused on resequencing lead candidates as well as functional replacement of full length cDNAs of these candidates into relevant cell line models.

11. Attiyeh EF, London WB, Mosse YP, Wang Q, Winter C, Khazi D, et al. Chromosome 1p and 11q deletions and outcome in neuroblastoma. N Engl J Med. 2005 Nov 24;353(21):2243-53.

Project 6: The Therapeutically Applicable Research to Generate Effective Treatments (NBL-TARGET) Initiative

This multi-institutional collaborative project is designed to use cancer genomics and resequencing efforts to discover neuroblastoma oncogenes and tumor suppressors that can be leveraged therapeutically. In collaboration with the NCI Cancer Genome Atlas project (http://cancergenome.nih.gov/), investigators at CHOP, the Children's Hospital Los Angeles, the Oncogenomics Branch of the NCI and the Children's Oncology Group have identified over 400 highly annotated and carefully selected diagnostic human neuroblastoma samples. Each sample will be assayed using a 550K SNP platform for copy number alterations and LOH, and also on an exon-based expression array. The aggressive timeline will result in a minimum of 100 candidate genes identified from regions of interest emerging through an integrative analysis for complete resequencing of coding regions.

External data links: Not yet available.

Project 7: Discovery of novel therapeutic strategies for human neuroblastoma

The Children's Hospital of Philadelphia holds a Program Project Grant focused on determining the underlying biology of human neuroblastoma for the purpose of improving therapy. Dr. Garrett Brodeur is the overall PI, and his project focuses on the role of neurotrophin receptors and their ligands in neuroblastomas. A phase 1 clinical trial through the NANT consortium (www.nant.org) with a targeted inhibitor of this pathway is ongoing. Dr. Michael Hogarty leads a project focused on understanding how the MYCN oncogene leads to a more aggressive clinical phenotype, and how cancer cells adapt to highly abnormal expression of the MYCN protein. Dr. Peter Adamson's lab is determining how best to utilize retinoids in neuroblastoma therapy, and how best to integrate these compounds with other chemotherapies. The Maris lab has a project focused on antiangiogenic strategies for neuroblastoma therapy. The major goal is to understand which pro- and anti-angiogenic genes are dysregulated during neuroblastoma initiation and progression, and how to utilize this knowledge therapeutically (8, 15-19). We utilize mouse models and human tumor samples to study changes in gene expression associated with tumor growth and metastasis. The lab is also responsible for the animal and pathology core, and Dr. Bruce Pawel leads efforts in advanced immunohistochemistry and in the development, use and interpretation of a large neuroblastoma tumor microarray. More recently, we have applied our model systems to discover other therapeutically relevant drug targets in other biologically relevant pathways.

8. Wang Q, Diskin S, Rappaport E, Attiyeh E, Mosse Y, Shue D, et al. Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number. Cancer Res. 2006 Jun 15;66(12):6050-62.

15. Shusterman S, Grupp SA, Barr R, Carpentieri D, Zhao H, Maris JM. The angiogenesis inhibitor TNP-470 effectively inhibits human neuroblastoma xenograft growth, especially in the setting of subclinical disease. Clinical Cancer Research. 2001;7(4):977-84.

16. Stern JW, Fang J, Shusterman S, Pierson G, Barr R, Pawel B, et al. Angiogenesis inhibitor TNP-470 during bone marrow transplant: safety in a preclinical model. Clinical Cancer Research. 2001;7(4):1026-32.

17. Accorsi R, Morowitz MJ, Charron M, Maris JM. Pinhole imaging of 131I-metaiodobenzylguanidine (131I-MIBG) in an animal model of neuroblastoma. Pediatr Radiol. 2003 Oct;33(10):688-92.

18. Wang J, Sheppard GS, Lou P, Kawai M, BaMaung N, Erickson SA, et al. Tumor suppression by a rationally designed reversible inhibitor of methionine aminopeptidase-2. Cancer Res. 2003 Nov 15;63(22):7861-9.

19. Morowitz MJ, Barr R, Wang Q, King R, Rhodin N, Pawel B, et al. Methionine aminopeptidase 2 inhibition is an effective treatment strategy for neuroblastoma in preclinical models. Clin Cancer Res. 2005 Apr 1;11(7):2680-5.

Project 8: Pediatric Preclinical Testing Program

The Pediatric Preclinical Testing Program (PPTP) is an initiative supported by the National Cancer Institute (NCI) to identify novel therapeutic agents that may have significant activity against childhood cancers. The PPTP has established panels of childhood cancer xenograft and cell lines to use for in vivo and in vitro testing. These include tumors of the kidney, sarcomas (rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma), neuroblastoma, brain tumors (glioblastoma, ependymoma, and medulloblastoma), and rhabdoid tumors (CNS and renal). There is also a detailed molecular profiling of each xenograft to help in prioritization and follow-up studies (manuscript in press). The Maris lab is responsible for the neuroblastoma xenograft panel, and we screen on average one drug per month in six or more xenograft models. This program has been highly efficient, and anti-tumor activity against neuroblastoma in these models for a variety of agents is now known (20-29). Potential active agents are being prioritized for Phase 1 clinical trials in the COG and NANT.

20. Smith MA, Morton CL, Phelps DA, Kolb EA, Lock R, Carol H, et al. Stage 1 testing and pharmacodynamic evaluation of the HSP90 inhibitor alvespimycin (17-DMAG, KOS-1022) by the pediatric preclinical testing program. Pediatr Blood Cancer. 2008 Feb 7.

21. Maris JM, Courtright J, Houghton PJ, Morton CL, Gorlick R, Kolb EA, et al. Initial testing of the VEGFR inhibitor AZD2171 by the pediatric preclinical testing program. Pediatr Blood Cancer. 2008 Mar;50(3):581-7.

22. Kolb EA, Gorlick R, Houghton PJ, Morton CL, Lock R, Carol H, et al. Initial testing (stage 1) of a monoclonal antibody (SCH 717454) against the IGF-1 receptor by the pediatric preclinical testing program. Pediatr Blood Cancer. 2008 Feb 7.

23. Houghton PJ, Morton CL, Kolb EA, Lock R, Carol H, Reynolds CP, et al. Initial testing (stage 1) of the proteasome inhibitor bortezomib by the pediatric preclinical testing program. Pediatr Blood Cancer. 2008 Jan;50(1):37-45.

24. Tajbakhsh M, Houghton PJ, Morton CL, Kolb EA, Gorlick R, Maris JM, et al. Initial testing of cisplatin by the pediatric preclinical testing program. Pediatr Blood Cancer. 2007 Jun 6.

25. Lock R, Carol H, Houghton PJ, Morton CL, Kolb EA, Gorlick R, et al. Initial testing (stage 1) of the BH3 mimetic ABT-263 by the pediatric preclinical testing program. Pediatr Blood Cancer. 2007 Dec 17.

26. Kolb EA, Gorlick R, Houghton PJ, Morton CL, Lock RB, Tajbakhsh M, et al. Initial testing of dasatinib by the pediatric preclinical testing program. Pediatr Blood Cancer. 2007 Oct 3.

27. Houghton PJ, Morton CL, Tucker C, Payne D, Favours E, Cole C, et al. The pediatric preclinical testing program: Description of models and early testing results. Pediatr Blood Cancer. 2007 Dec;49(7):928-40.

28. Houghton PJ, Morton CL, Kolb EA, Lock R, Carol H, Reynolds CP, et al. Initial testing (stage 1) of the proteasome inhibitor bortezomib by the pediatric preclinical testing program. Pediatr Blood Cancer. 2007 Apr 9.

29. Houghton PJ, Morton CL, Kolb EA, Gorlick R, Lock R, Carol H, et al. Initial testing (stage 1) of the mTOR inhibitor rapamycin by the pediatric preclinical testing program. Pediatr Blood Cancer. 2007 Jul 16.

Project 9: Targeted Therapy for Neuroblastoma

Investigators at GlaxoSmithKline have developed a research consortium of laboratory investigators seeking to leverage tumor genomics information to identify therapeutic targets. Like the PPTP, we then perform comprehensive preclinical testing of pharmacologic modulators of putative targets both for proof-of-concept as well as garnering the preclinical data necessary to support clinical development. This collaboration has led to the identification of CENPE as differentially overexpressed protein in the most aggressive subset of neuroblastomas, and pharmacologic inhibitors show broad and potent cytotoxicity in vitro and in vivo (Wood, AACR 2008).

Project 10: Translational Genomics of microRNAs in Neuroblastoma

Despite thirty years of knowledge of recurrent genomic abnormalities in neuroblastoma, no bona-fide neuroblastoma Tumor Suppressor Genes have been identified. The discovery of a new class of regulatory non coding RNAs called microRNAs (miRNAs) are attractive candidate neuroblastoma oncogenes and Tumor Suppressor Genes because of their role in normal embryonic development and in cancer. We recently showed the miR-34 family of microRNAs are likely tumor suppressor in neuroblastoma, and that replacement can induce cell death. We therefore seek to extend these findings by 1) Identifying neuroblastoma oncomirs through an integrative genomic approach, 2) Characterizing the tumor suppressor mechanism of the miR-34 family (and other identified oncomirs) in neuroblastoma and 3) Demonstrating preclinical therapeutic efficacy of miR-34a replacement in vivo. The discoveries made from this work may have broader application to cancer biology as many of the genomic alterations found in neuroblastoma are also found in other pediatric and adult solid tumors.