TARGET investigators are analyzing tumors from pediatric patients to identify biomarkers that correlate with poor clinical outcome and/or new therapeutic approaches to treat childhood Neuroblastoma (NBL). The tissues used in this study were collected from patients enrolled in Children's Oncology Group (COG) biology studies and clinical trials.
The TARGET Neuroblastoma project team members (like other TARGET researchers) are generating data in two phases: Discovery and Validation. Visit the TARGET Research page to learn more.
The TARGET neuroblastoma (NBL) project has produced comprehensive genomic profiles of more than 200 clinically annotated patient cases within the discovery dataset. This cohort includes nearly 200 high-risk patient cases, including some who have relapsed and subsets of low-risk and/or stage 4S NBL cases (tumors that spontaneously regress without treatment). Each fully-characterized TARGET NBL case includes data from nucleic acid samples extracted from tumor and normal tissues as follows:
- Primary tumor sample collected at diagnosis
- Normal tissue sample from peripheral blood or bone marrow (case-matched)
- Relapsed tumor sample (case-matched) when available; some cases have 3rd sample (those cases are considered a “trio”)
There are a large number of additional cases, varying in risk level, with partial molecular characterization and/or sequencing data that are available to the research community.
Case Selection Criteria
Tissues and clinical data used for the TARGET NBL project were obtained from patients enrolled on biology studies and clinical trials managed through the Children’s Oncology Group (COG). Patient samples with full characterization were chosen based on the following criteria:
- Tumor cellularity of >70% in tumor specimens and tumor necrosis of <30%
- High-quality nucleic acids in amounts adequate to complete comprehensive genomic profiling
- Preference for high-risk cases (Stage 4) who have relapsed or whose tumors spontaneously regress without treatment (Stage 4S)
The TARGET NBL project team relied on a variety of platforms to obtain a fully characterized dataset of more than 200 cases. The COG NBL Statistics and Data Center provided clinical annotations and outcome data for all cases. Visit the TARGET Project Experimental Methods page for detailed information and protocols.
||COG NBL Protocols
||Affymetrix Human Exon ST Array
|Chomosome Copy Number Analyses & Loss of Heterozygosity
||Affymetrix SNP 6.0 Array
|Epigenetics (DNA Methylation)
||Illumina Infinium 450K
|Whole Genome Sequencing
||Complete Genomics Incorporated,
Illumina Genome Analyzer IIx or Hi-Seq 2000
|Whole Exome Sequencing
||Illumina Genome Analyzer II or Hi-Seq 2000
Illumina Genome Analyzer IIx or Hi-Seq 2000
Verification of Discovery Variants
The TARGET NBL project team utilized a variety of sequencing approaches to confirm candidate variants identified in the discovery sample cohort as somatic. For example, mRNA-seq results are being used to determine variants which were expressed and originally identified through whole genome or exome sequencing. These verified variants will be made available as open-access data.
Some sequence mutations identified in the discovery cohort, along with some previously published variants, were further analyzed in an additional 500 cases. The TARGET NBL project team employed targeted capture sequencing to look at the presence and frequency of alterations in 400 gene variants. This validation effort was performed in an unbiased cohort that was randomly selected from patients enrolled on a single COG protocol, which allowed for determination of the frequency of these changes across a broader spectrum of NBL subtypes.
Cell Line and Patient-derived Xenograft Models
The following models were generated in the lab of Dr. Patrick Reynolds, Texas Tech University Health Sciences Center. They all have 'EBV Immortalized Normal' samples.
||The pre-treatment primary tumor consists of a single dominant clone that shares many mutations with the matched pre-treatment cell line. Fewer mutations are shared with a cell line established upon progression. The single subclone present in the primary tumor is not evident in the derivative cell line. However, the cell line has acquired an additional sub-clonal population well supported by 75 mutations that are not evident in the match primary nor is there evidence of this clone in the cell line established upon progression. Therefore, this subclone may have arising in culture or have been an undetected low frequency subclone in the primary that was sensitivity to treatment.
- Cell line from primary tumor at diagnosis
- Cell line from pleural fluid at diagnosis
- Cell line from bone marrow after induction chemo
|Cell lines from the same pre-treatment tumor (tissue and effusion) share many mutations and a common subclone (61% cancer cell fraction). The cell line from the matched post-treatment tumor has a dramatic increase in somatic mutation burden, although many of these are found in sub-clonal populations; one related two the shared pretreatment (160/162 shared) and a second completely unique to the post-treatment case.
- Cell line from bone marrow at diagnosis
- Cell line from bone marrow at progression (heavily treated with chemo)
|Cell lines established before and after treatment share a common core set of somatic mutations shared across clonal and sub-clonal populations. However, each of these populations have hundreds of additional unique mutations, suggesting significant divergence and possible selection of sub-clonal populations co-existing at similar cancer cell fractions in each cell line. Based on our current data, we cannot verify whether two sub-clonal populations co-exist in each line without further analysis (potentially single cell sequencing).
- Cell line, post-mortem blood heavily treated with chemo
- Mouse xenograft post-mortem blood sample
|Cell line and mouse xenograft derived from the same post-mortem blood sample share the majority of mutation patterns, although the mouse xenograft lacks the higher frequency subclones found in the cell line. Possibly, the subclone at 20% cancer cell fraction in the cell line contained additional mutations that conferred a growth advantage over higher frequency population lacking the 41 additional mutations in these clones. Further genetic divergence is evident from additional singleton mutations at very low allele fractions (<1%) in each case, that require verification by orthogonal methods.
- Cell line from post-mortem blood, room air
- Cell line from post-mortem blood, 5% O2
- Xenograft from post-mortem blood
|Cell lines derived from the same primary share a large fraction of mutations, although each contains hundreds of mutations unique to the growth conditions. In TARGET-LEFT, the cell line grown under hypoxic conditions contains a greater number of mutations (956 vs 910) as well as an additional subclone supported by 6 mutations. The higher-level sub-clonal structure is consistent with a mouse xenograft derived from the same material. The mouse xenograft has incurred additional subclones, albeit with fairly weak mutational support. This pattern is reversed in TARGET-RIGHT whereby the hypoxic cell line has fewer mutations (928 vs 1005) and no low frequency subclone. The subclone in the mouse xenograft appears to be derived from a subclone common across all model organisms, although the lower cancer cell fraction suggests it is not well suited to growth in the xenograft or may have been outcompeted for growth by the clonal population.
- Cell line from post-mortem blood
- Post-mortem blood cell line, 5% O2
- Xenograft of post-mortem blood cell line
All data from the discovery and validation efforts are made available as specified in the Using TARGET Data and TARGET Publication Guidelines pages. The TARGET Data Matrix provides an overview of the data generated and described above.