TARGET’s Study of Neuroblastoma
Neuroblastoma (NBL) is a cancer that arises from immature nerve cells of the sympathetic nervous system, primarily affecting infants and children. It can have a devastating impact on patients and their families. Neuroblastoma accounts for ~12% of childhood cancer mortality; those between 18 months and 5 years are affected most severely. Furthermore, current NBL treatment involves harsh chemoradiotherapies that generally leave surviving children with lifelong side effects.
The discovery of novel therapeutic targets in NBL could not only improve the outcomes of high-risk patients, but could also reduce the burden of sustained complications for surviving patients. Learn more about neuroblastoma and current treatment strategies.
TARGET’s Neuroblastoma (NBL) Project
Therapeutically Applicable Research to Generate Effective Treatments (TARGET) investigators are analyzing tumors from pediatric patients to identify biomarkers that correlate with poor clinical outcome and new therapeutic approaches to treat childhood 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 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 third 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.
Tissues and clinical data used for the TARGET NBL project were obtained from patients enrolled on biology studies and clinical trials managed through 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)
Some sequence mutations identified in the discovery cohort, along with some previously published variants, were further analyzed in an additional 500 cases known as the validation cohort. 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.
TARGET Neuroblastoma Models
TARGET also produced the following models, generated in the lab of Dr. Patrick Reynolds, Texas Tech University Health Sciences Center. They all have EBV Immortalized Normal samples.
TARGET Neuroblastoma Cell Line and Patient-Derived Xenograft Models
||The pretreatment primary tumor consists of a single dominant clone that shares many mutations with the matched pretreatment 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 subclonal 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 arisen in culture or have been an undetected low frequency subclone in the primary that was sensitivity to treatment.|
||Cell lines from the same pretreatment tumor (tissue and effusion) share many mutations and a common subclone (61% cancer cell fraction). The cell line from the matched posttreatment tumor has a dramatic increase in somatic mutation burden, although many of these are found in subclonal populations; one related to the shared pretreatment (160/162 shared) and a second completely unique to the posttreatment case.|
||Cell lines established before and after treatment share a common core set of somatic mutations shared across clonal and subclonal populations. However, each of these populations have hundreds of additional unique mutations, suggesting significant divergence and possible selection of subclonal populations coexisting at similar cancer cell fractions in each cell line. Based on our current data, we cannot verify whether two subclonal populations coexist in each line without further analysis (e.g., via single-cell sequencing).|
||Cell line and mouse xenograft derived from the same postmortem blood sample share a 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, though these require verification by orthogonal methods.|
||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 versus 910) as well as an additional subclone supported by 6 mutations. The higher-level subclonal 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 versus 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.|