Next Generation Genomic Profiling For Breast Cancer Patients

Agendia’s MammaPrint Microarray Platform

Agendia utilizes a revolutionary microarray platform that leverages the biological breakthroughs of the Human Genome Project with the latest advances made in computer science.  This methodology has advantages over previous generation tests such as FISH or PCR because, in a single analysis, it can evaluate the expression of all of the genes that may be involved in a cancer, rather than just a few. This makes the identification of cancer subtype more precise, and very flexible as our knowledge base grows.

Agendia measured the expression levels, or activity, of thousands of genes from tumor samples of a breast cancer patient cohort to identify the genes involved in breast cancer.  With the unprecedented ability to graphically visualize how all of the genes involved in breast cancer interact, Agendia’s scientists were able to identify a “genomic signature” for a breast cancer recurrence.

The MammaPrint genomic signature for breast cancer recurrence, plus the thousands of other genes Agendia analyzes on the same microarray platform, may lead to a greater understanding of how breast cancer metastasizes and potentially how to develop more individualized treatment for women with breast cancer.

How do microarrays work?   Agendia’s microarray method consists of five basic steps:

• Preparation of the sample and RNA extraction
• Hybridizing the sample on the microarray chip (oligonucleotide chip)
• Scanning the microarray chip
• Normalization
• Computer analysis of the results

Preparation of the sample: In the initial step, cDNA is synthesized from RNA by reverse transcription (transcription involves copying DNA to make RNA, so reverse transcription is generating DNA from RNA) from RNA that has been extracted from both a test and a reference sample. The sample DNA segments are labeled with fluorochromes, so that they can be detected after they combine with the microarray chip.

Combining the sample with the microarray chip: Next, the sample is applied to the microarray chip, which is a rectangular grid of spots. These spots are known as probes which are oligonucleotides representing the individual genes, and for MammaPrint, these probes are printed 9-fold.  Each spot has many copies of a particular DNA sequence. These sequences are derived from public databases of DNA sequences that were generated through the Human Genome Project, the scientific endeavor that identified virtually all of the DNA sequences in the human species.

When the sample is added to the microarray chip, a process called hybridization occurs. This means that the sample DNA segment binds (hybridizes) to the segment on the microarray chip that has the exact complimentary sequence of nucleotides (the four compounds that are the alphabet of genetics). 

Scanning the microarray chip: Once hybridization is complete, scanners are used to detect fluorescence and create a digital image that reflects where the sample DNA combined with spots on the microarray chip.

Normalization: Because raw signal intensities may vary between individual chips from many patients or experiments, individual chip intensity must be adjusted to a common standard, or normalized. For example, subtraction of background noise is a common normalization method that is applied to all samples. Normalization makes it possible to compare gene expression profiles from many patients or experiments.

Computer analysis: The final step in a microarray experiment is the computer analysis otherwise called bioinformatics. The thousands of data points that result from microarray analyses are essentially unintelligible unless they are evaluated in the context of other results. For example, the gene expression profile (microarray results) of normal and diseased tissue can be compared to identify genes that vary in their expression and also identify a pattern (profile) that may indicate a distinct class or stage of disease.