by Ishika Mundhra and Gautami Patil
T.Y.B.Sc Department of Biotechnology DES Fergusson College, Pune. For BTH3507
Blog Category: Cancer Biology
Some breast cancer cells make too many copies of (overexpress) a particular gene known as HER2. The HER2 gene makes a protein known as a HER2 receptor. HER2 receptors are like ears, or antennae, on the surface of all cells. These HER2 receptors receive signals that stimulate the cell to grow and multiply. But breast cancer cells with too many HER2 receptors can pick up too many growth signals. This causes them to start growing and multiplying too much and too fast. Breast cancer cells that overexpress the HER2 gene are said to be HER2-positive.
About 1 in 5 breast and stomach cancers are HER2 positive.
Diagnostic tests
There are several tests used to find out if breast cancer is HER2-positive. Two of the most common tests are: FISH & IHC
IHC (ImmunoHistoChemistry)
The IHC test uses a chemical dye to stain the HER2 proteins. IHC measures the amount of HER2 protein present. The test gives a score of 0 to 3+ that measures the amount of HER2 proteins on the surface of cells in a breast cancer tissue sample. If the score is 0 to 1+, it’s considered HER2-negative. If the score is 2+, it's considered borderline. A score of 3+ is considered HER2-positive.
If the IHC test results are borderline, it’s likely that a FISH test will be done on a sample of the cancer tissue to determine if the cancer is HER2-positive.
This technique uses Ab-Ag interactions on given mass of tissue, the Ag here are biomarkers.
The tissue is first cut very finely using histome. It is then fixated on the slide using formalin.
There are two ways to come about this process.
1. We can attach fluorophore to the antibody, when the Ab-Ag interaction takes place the fluorophore bound Ab emitting colour can be observed under the fluorescence microscope confirming interaction.
2. We can attach an enzyme substrate which will be chromogenic to the Ab which on enzymatic reaction produces colour, i.e. when the substrate is changed to product. The colour change gives us the information of Ab-Ag interaction.
We can first use Primary Ab specific to Ag and later add secondary Ab attached with either chromogenic substance or a flourophore, this secondary Ab will be complementary to the Primary Ab. The fluorophore or chromogenic substances bind to the fc region of the Ab which is a constant, The paratope of Antibody binds to epitope of the antigen, these regions are highly specific and hence 1º antibodies are used.
One example of chromogenic substance can be avidin-biotin complex (ABC) staining.
Biotin can conjugate to both secondary Ab and HRP which is a peroxidase. Biotin attached to 2º Ab attaches to 1º Ab. Avidin protein then binds to biotinylated Ab. HRP conjugated to biotin then binds to a free space in avidin molecule. HRP catalyses the reaction between DAB and hydrogen peroxidase. The product of this reaction gives brown DAB, water and gas. This brown DAB stains the tissue and location specific Ag
can be visualised.
FISH (Fluorescence In Situ Hybridization)
The FISH test uses special labels that attach to the HER2 proteins. The special labels have chemicals added to them so they change color and glow in the dark when they attach to the HER2 positive
It is a cytogenic method for visualising locations on a chromosome. The fluorescent probe is complementary to specific sites of the chromosome. The assay detects any chromosomal abnormalities. It tells us if the chromosomal location is mutated or not.
This test is the most accurate, but it is more expensive and takes longer to return results. This is why an IHC test is usually the first test done to see if a cancer is HER2-positive. With the FISH test, you get a score of either positive or negative (some hospitals call a negative test result “zero”).
1st step in FISH is fixation of cells with formaldehyde based fixative which causes extensive protein to protein and protein to nucleic acid cross- links. Next step includes addition of probes. The probes need to be complementary to the region of interest. It can either be done by using short RNA probe or short double stranded DNA probe. Using DNase enzyme we make random nicks on the DNA. Special nucleotide attached with fluorescent protein(marker), ligase then seals these nicks with these nucleotide. Using PCR technique these probes can be amplified.
Then, we denature the probe which is introduced in the cell of interest. Chromosomal target DNA is also to be denatured. This process of denaturation can be achieved by heating the cell at approximately 95ºC, after heating it is cooled down to allow the probe to bind with the target site of DNA. All the probes that do not bind to the specific sequence of DNA are washed away by hybridisation method.
Its results can be analysed under fluorescence microscope. If probes which are made specifically for a DNA sequence do not show any fluorescence under the microscope tells us that the DNA sequence is deleted i.e mutations have occurred.
Herceptin (Trastuzumab)
Herceptin is used to treat breast cancers that are HER2-positive. It is a humanized monoclonal antibody that interferes with the HER2 receptor.
The drug binds to a domain of the extracellular segment of the HER2. This process prevents dimerization, causing cell arrest during the G1 phase. Some of the therapeutic effect may also be due to downregulation of HER2. These mechanisms cause disruption of receptor dimerization, which reduces signaling pathways and results in cell-cycle arrest.
References
• Trastuzumab (herceptin): Pharmacology Vignette; J. J. Gemette & S.K Mukherji http://www.ajnr.org/content/ajnr/32/8/1373.full.pdf
• Phosphorylation/Cytoplasmic Localization of p21Cip1/WAF1 Is Associated with HER2/neu Overexpression and Provides a Novel Combination Predictor for Poor Prognosis in Breast Cancer Patients
Weiya Xia, Jin-Shing Chen, Xian Zhou, Pei-Rong Sun, Dung-Fang Lee, Yong Liao, Binhua P. Zhou and Mien-Chie Hung
Clin Cancer Res June 1 2004 (10) (11) 3815-3824; DOI: 10.1158/1078-0432.CCR-03-0527
• Chao WR, Lee MY, Ruan A, et al. Assessment of HER2 Status Using Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) Techniques in Mucinous Epithelial Ovarian Cancer: A Comprehensive Comparison between ToGA Biopsy Method and ToGA Surgical Specimen Method. PLoS One. 2015;10(11):e0142135. Published 2015 Nov 13. doi:10.1371/journal.pone.0142135
• https://youtu.be/zO70nrWcAyk