Breast cancer is the most common invasive cancer in women, and the second main cause of cancer death in women. Cancer cells are very similar to the cells of the organism from which they originated and have similar (but not identical) DNA and RNA. Which is why they are not always detected by the immune system, in particular, if it is weakened.[1] Another fact that makes breast cancer even more difficult as a disease, is that its clinically heterogeneous and patients differ widely with respect to natural history and response to treatment[1].This clinical heterogeneity is most likely due to the varying mutational spectrum and genetic differences among individuals[2]. These factors in combination, influence the expression of many genes involved in tumor growth, invasion, metastasis, and survival [2].
Adjuvant systemic therapy are treatments that are given in addition to the primary or initial therapy to maximize its effectiveness. Hemotherapy, radiation therapy, hormone therapy and targeted therapy are all forms of adjuvant systemic therapy. These treatments can save a significant number of lives[8], but many patients are subjected to unnecessary therapies with the potential of causing more harm than good [4]. Approximately 25% of all women diagnosed with breast cancer die from their disease, despite having been treated according to state-of-the-art clinical guidelines [4,7]. The present lack of criteria to help individualize breast cancer treatment indicates a need for a novel technology to develop better prognostication and therapy prediction. [4]
Many women have low risks of incurable recurrence after treatment with surgery, radiation, or hormonal therapy. Chemotherapy can reduce that risk by an additional 30%, but it also produces potentially life-threatening toxic effects in 2%–3% of otherwise healthy women. Whether chemotherapy’s added benefits justify the potential harm to low-risk patients often isn’t clear. Therefore, clinicians are turning to molecular assays for new insights. One such assay is the MammaPrint assay. The MammaPrint is a DNA microarray. DNA microarrays have been used to obtain genome-wide views of human tumor gene expression.
1.2 Micro array
A DNA micro array Is a collection of tiny DNA spots attached to a surface [9]. These spots can be used to measure the expression levels of a large numbers of genes. This technology has made possible to relate cell states to gene expression patterns to study tumors, diseases progression, cellular response to stimuli, and drug target identification. [9] It is known that complementary single stranded sequences of nucleic acids form double stranded hybrids. This property is the basis of molecular biology tools such as Northern and Southern blots, in situ hybridization and Polymerase Chain Reaction (PCR). In which, specific single stranded DNA sequences are used to probe for its complementary sequence (DNA or RNA) forming hybrids. This same idea is also used in DNA microarray technologies. [9] The aim is however not only to detect but also to measure the expression levels of thousands of genes in the same experiment. [9]
For this purpose, thousands of single-stranded sequences that are complementary to target sequences are bound, synthesized, or spotted to a glass support which is around the size of a typical microscope slide. There are mainly two types of DNA arrays depending on the type of spotted probes [9]. One use small single-stranded oligonucleotides (~22nt) synthesized in situ. The other type of arrays which are used in this project, use complementary DNA (cDNA) obtained by reverse transcription of RNA [9] Fluorescent dyes are used to label the amplified cDNAs samples to be analyzed.
The DNA array is then hybridized with the labeled sample(s) by incubating overnight, followed by washing to remove non-specific hybrids.
A laser excites the attached fluorescent dyes to produce light which is detected by a (confocal) scanner. The scanner generates a digital image from the excited microarray. The digital image is further processed by specialized software to transform the image of each spot to a numerical reading. This process is performed, first by finding the specific location and shape of each spot, followed by the integration (summation) of intensities inside the defined spot, and finally estimating the surrounding background noise. Background noise is subtracted from the integrated signal. This final reading is an integer value assumed to be proportional to the concentration of the target sequence in the sample to which the probe in the spot is directed to. [9]