Research

CGCI researchers primarily use sequencing and, in some cases, other genome-based approaches to examine genomes and transcriptomes of tumors. With these in-depth analyses, they are able to detect cancer-associated alterations ranging from genomic rearrangements and copy number alterations in gene expression to single nucleotide insertion or deletion of small nucleotide mutations. By uncovering the full spectrum of alterations in tumors, CGCI researchers aim to elucidate which physiological pathways are disrupted in cancer. Finding the underlying molecular causes of cancer will, in some cases, inform better treatment strategies.

Read below to learn about CGCI’s research approaches and the type of information they provide.

Central Pathology Review

Pathological diagnosis of tumors is important to confirm disease identification. To avoid any subjectivity associated with pathology reviews, and to allow efficient resolution of discrepancies, CGCI formed Pathology Review Committees (PRCs) consisting of three board‐certified pathologists. The committees ensure that the samples meet the requirements of CGCI projects. The tissue source sites submit either a Formalin-Fixed Paraffin-Embedded (FFPE) block or an appropriate number of unstained slides of tumor samples for pathology analysis. An H&E stained slide is used for initial tissue analysis for each submitted tumor to identify the location of the tumor. Immunohistochemistry (IHC) stains are performed depending on the project and tumor subtype.

The pathology analysis can be done either individually or using Tissue Microarrays (TMAs). TMAs were used for Burkitt Lymphoma Genome Sequencing Project.

Tissue Microarrays (TMAs)

Tissue Microarrays (TMAs) consist of paraffin blocks with separate tissue cores that are assembled in array fashion to allow multiplex histological analysis. TMAs for CGCI projects involve up to three tissue cores per case. In most TMAs, the tissue cores are placed according to a map (figure below) to remove possible pathology diagnosis bias. The coordinates indicating locations of specific cores on the slide are included in slide metadata. H&E stains from preliminary pathology analysis will be made available along with TMA images.

IHC and Fluorescence in situ hybridization (FISH)

Antibody stains against the following proteins are used for respective projects as follows:

• BLGSP:  CD3, CD10, CD20, BCL2, BCL6, Ki67 and MYC (FISH analysis)
• HTMCP: 

• Lung:  p40 (optional in cases of suspected Squamous Cell Carcinoma (SCC)), TTF-1 (required for cases of suspected Adenocarcinoma)
• Cervical: p16, p63 (optional for SCC), p40 (optional for SCC), BER-EP4 (optional for adenocarcinoma), MOC 31 (optional for adenocarcinoma), B72.3 (optional for adenocarcinoma)
• DLBCL:  CD3, CD10, CD20, MUM1, BCL2, BCL6, Ki67, TP53, CD79a, EBER (RNA In situ Hybridization analysis)

Next-generation Sequencing Methods

The following next-generation sequencing methods were/are used for all BLGSP and HTMCP cases.

Whole Genome Sequencing

• Performed on both tumor and matching normal (if available) samples
• Provides the DNA sequence of the entire genome
• Is used to identify structural alterations, such as translocations and inversions, as well as insertion and deletions (indels) and single point mutations

Transcriptome Sequencing 

• Performed on tumor samples only
• Provides sequence from transcribed genes and non-coding RNAs (RNA-seq) and/or from small regulatory RNAs known as microRNAs (miRNA-seq)
• Identifies mutations in the protein-coding regions of the genome, non-coding or regulatory RNAs, and a variety of alterations, including novel gene fusions, alternatively spliced isoforms, and variations in gene expression

Targeted Sequencing

• Custom hybridization-based DNA capture protocols were used to capture and sequence targeted genes.
• Targeted sequencing libraries that featured exons of recurrently mutated genes, exons of several known diffuse large B-cell lymphoma and Burkitt Lymphoma genes were used for the pediatric BLGSP manuscript

Epigenetic Characterization Methods

Illumina Infinium MethylationEPIC BeadChip

• Provides high-throughput genome-wide DNA methylation data of 850,000 CpG islands

ChIP sequencing

• Uses combinations of immunoprecipitation and sequencing to identify various histone modifications and the chromatin state
• Provides sequences for the DNA bound proteins (e.g. transcription factors and histones) to identify their locations in the genome

See the Experimental Methods for the Burkitt Lymphoma Genome Sequencing Project page for more information on pediatric BLGSP experimental methods.

See the Experimental Methods for HIV+ Tumor Molecular Characterization Project page for more information on HTMCP-Cervical Cancer experimental methods.