Research

Goals of the Initiative

TARGET researchers utilized various sequencing and array-based methods to examine the genomes, transcriptomes, and for some diseases epigenomes of select childhood cancers. This “multi-omic” approach generated comprehensive profiles of molecular alterations for each cancer type. Alterations are changes in DNA or RNA, such as rearrangements in chromosome structure or variations in gene expression, respectively. Through computational analyses and assays to validate biological function, TARGET researchers predict which alterations disrupt the function of a gene or pathway and promote cancer growth, progression, and/or survival. Researchers identify candidate therapeutic targets and/or prognostic markers from the cancer-associated alterations.

To learn about individual sequencing and array-based methods used in TARGET research, visit Methods below.

Discovery 

TARGET project teams have characterized “discovery” cohorts of patient cases to identify molecular alterations of the transcriptome, genome, and epigenome in various pediatric cancer subtypes. Each project team independently selected their patient cohorts based upon characteristics of the disease or cancer subtype*. A comprehensive genomic profile of each patient case was generated using nucleic acids from tumor tissue taken at the time of diagnosis and case-matched “normal” tissue. Whenever available, case-matched tissues from relapsed or treatment-resistant tumors were also characterized by the same methods. Robust clinical data were obtained for each case studied in TARGET. All tissues used meet strict scientific, technical, and ethical requirements.

Verification

TARGET project teams employed multiple technologies to confirm the presence of mutations found in tumor tissue. The use of diverse sequencing approaches (e.g. mRNA-seq, whole exome or whole genome sequencing) to analyze discovery samples provided confirmation of somatic mutation(s) observed in a patient case and affirmed the quality of each data type generated. Individual project teams also analyzed a subset of their discovery cases using the method(s) employed for validation*.   

Validation

TARGET project teams used a separate sequencing method applied to an independent cohort of patients to validate at the gene level most candidate mutations found in the TARGET discovery data. Validation confirmed the population prevalence of mutations. Within each disease, characterizing a separate cohort more broadly representative of the disease population further allowed the project teams to estimate the frequency of somatic variants in a given cancer subtype.

*Note: Users can find details regarding discovery, verification, and validation by clicking on the project page for the disease of interest and reading the ‘experimental approaches’ section.

Methods

Each project team employed a large subset of the methods below. Visit the TARGET Project Experimental Methods page for detailed protocols and to learn which methods apply for each individual project.

Genome-scale Characterization

• Gene expression profiling: determines patterns of all genes transcribed
• Copy number analysis: determines structural changes of chromosomes, such as copy number alterations (chromosome region gains and losses) including the loss of heterozygosity and translocations
• DNA methylation status: determines patterns of DNA (cytosine) methylation on chromosomes
• miRNA profiling: determines expression patterns of small regulatory molecules called microRNAs (miRNAs)

Sequencing

Sanger Method

• Targeted Sequencing: DNA sequencing of specific genes or areas of the genome
  • Kinome Sequencing: DNA sequencing of genes encoding kinases

Next-generation Method

• Whole Genome Sequencing: provides the DNA sequence of the genome
• Whole Exome Sequencing: provides DNA sequences of exons
• Transcriptome Sequencing (RNA-seq): provides sequences from transcribed RNAs
  • mRNA Sequencing (mRNA-seq): generates sequences of messenger RNAs (mRNAs)
  • miRNA Sequencing (miRNA-seq): generates sequences of small RNA molecules, such as microRNAs(miRNAs)
• Bisulfite sequencing: identifies pattern of methylation of individual cytosines in DNA within a defined area of the genome (i.e. epigenome)
• ChIP sequencing: identifies protein interactions with DNA of the genome