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Report title: Discuss the significance of HER 2 receptor expression in breast cancer tissue and critically evaluate the two main laboratory methods employed in its detection.

Human epidermal growth factor receptor 2 (HER2) is a proto-oncogene which codes for a transmembrane glycoprotein (p185), this means it is a gene which has the potential to cause cancer, and it is a tyrosine kinase on the membrane. In roughly 25% of breast cancers it is active and overexpressed, it allows the cells to proliferate and stops the cells from carrying out apoptosis (which is needed as to destroy cells that are dead/dying/insured) without these apoptosis signals the cells can keep multiplying and mutating 2. This receptor promotes breast cancer, it results in a higher proliferation rate, it metastasizes faster, has a greater tumour burden and patients with HER2 have a worse prognosis5, the HER 2 receptor allows for a target in treatment through 2 main methods; immunohistochemistry and fluorescent in situ hybridisation. HER2 represents a good target for therapy because it is important in breast cancer cells, it is available as it is a receptor on the cell surface, and it is also expressed very little in normal /other cells and highly expressed in breast cancer 7.
Overexpression of HER-2 is strongly associated with a worse prognosis and life expectancy. HER2+ve tumours are both resistant to endocrine chemotherapy and normal chemotherapy. These patients however do benefit from Herceptin which is an anti HER2 monoclonal antibodies. Her2 detection is important as the patients outcome is based off of this so must be accurate, allows information on the patients prognosis and the best, most suitable treatment, the drugs are very expensive and should be used on the patients that will respond the best to them.

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What is HER2 negative and HER2 positive breast cancer?
HER2 negative cancers have no abnormal features in the human epidermal growth factor receptor gene, HER2 receptors manage how breast cells multiply, when there becomes an issue with the gene function the breast cells grow speedily and uncontrolled 6. These HER 2 negative breast cancers don’t have this abnormality whereas HER2 positive do have this abnormality in the human epidermal growth factor 2 resulting in rapid growth, and spread 6.
What is fluorescent in situ hybridisation (FISH)?
FISH qualifies the level of gene amplification of the HER2 gene .Fluorescent in situ hybridisation involves the fixation of DNA in tissue section onto a slide, then denatures in situ to a single strand, DNA probes which are linked to fluorescent and specific to certain gene are added and hybridisation takes place between the probe and the tissue DNA. This is then viewed through a UV microscope. This method is used to visualise portions of genes, the more HER2 receptors present means there are more receptors to receive signals to allow growth of the breast cancer cells. Fluorescent in situ hybridisation is made up of 4 main steps, the first step being denaturation with heat of chemical, this is necessary for hydrogen bonds to be able to form between the probe and the target in the next step which is hybridisation. This hybridisation step involves mixing together the probe and the target gene sequence. The probe will bind to the complimentary sequence on the chromosome. The next step in the FISH process is probe detection, if the probe is already fluorescent it is possible to detect the site of complimentary hybridisation directly however if not an additional step is required to visualise the hybridised probe. This can be done using a fluorescent microscope to detect the hybrids between the probe and target, the final step in FISH process is microscopic analysis 89
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HER2 negative HER2 positive
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Immunohistochemistry (IHC)
IHC is used to measure the level of overexpression in the HER2 receptor .if you split the word up, “immune” means the use of antibodies and “histo” refers to the study of tissue. IHC uses antibodies to detect specific antigens within a tissue, the antibodies bind with the antigen resulting in an antibody-antigen complex. In this method polyclonal or monoclonal antibodies can be used, polyclonal antibodies are more sensitive whereas monoclonal are more specific, polyclonal also produce background stain and monoclonal don’t, polyclonal are also cheaper than monoclonal 1. IHC is accepted technology and is inexpensive, it has subjective scoring and is semi qualitative, and the protein target may be affected by tissue processing. IHC has 4 main steps, the first being fixation, which is to keep everything fixed and stuck in place. Antigen retrieval is next followed by blocking and then finally antibody labelling and visualization. Tissue fixation is done as to maintain tissue structure and integrity. The main method used is formalin fixed and paraffin embedded (FFPE) method. The next step is antigen retrieval, if the FFPE method is being used it can be useful to carry out pre-treatment with antigen retrieval agents. These break down the formalin induced antigen cross linking which exposes the epitopes on the antigen or antibody binding. Heat and enzyme retrieval are both used with heat induces epitope retrieval being the most used, proteolytic enzymes can also be used. The next step in the IHC process is blocking. Blocking is essential to block endogenous material as this can result in a false positive staining. 2 factors need to be taken into consideration, the first being that you will need to block any inactive endogenous enzymes in tissue which could activate substrates that may be used in the process to see the antibody binding. Another factor is to block endogenous antibodies that could cross react leading to high background staining in sample. The final step in the IHC process is antibody labelling and visualisation. This step involves staining the sample so it can now be visualised. It can be done by indirect detection or direct detection. In indirect detection a secondary antibody is used which is bound to a label, this secondary antibody binds to the primary antibody during the staining process converting non coloured substrate to coloured product. A direct method is quicker and easier, which also reduces the chance of error as it has less steps where mistakes can be made. The label is attached to the primary antibody which means only one incubation step is required and one wash step. This method is more cost effective and less time wasting than indirect. 10
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Comparison
IHC is used to measure the level of overexpression in the HER2 receptor whereas FISH qualifies the level of gene amplification of the HER2 gene. They are both used in diagnostic testing of HER2 status. Fish is less commonly used as not as available compared to ICH, so ICH is carried out and then FISH if the ICH doesn’t evidently show if the cells are HER2 positive or negative 3. FISH is also more accurate than ICH, FISH is of high sensitivity and specificity and is also fast at performing the tests 1. ICH however can show a lot of detail. FISH does have some disadvantages, one being that is requires a costly fluorescent microscope which is equipped with multi band pass fluorescent filters and that the fluorescent fades so fast that it doesn’t leave a record. IHC is more commonly used as it is cheaper and easier to run and keep, it also allows a clear image 4.
Pros Cons
IHC Cheaper than FISH
Easier to run and maintain Less specific and sensitive
Fixation issues
Epitope specific
Reproducibility
FISH Accurate
High sensitivity
High specificity
Reproducibility
Cell localisation
Costly
Requires expensive equipment
More expensive to run and maintain
Doesn’t leave permanent record
First of all the tumour sample undergoes IHC testing which will be graded either 0 and 1+ which are negative, 2+ which is weekly positive and 3+ which is positive. If it is graded 2+ it undergoes ISH do determine if it is negative meaning it is non amplified or positive which means the HER2 receptor is amplified. If it is scored 3+ it is sent to the oncologist and it will be considered it the patient is suitable for treatment with Herceptin. 5
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Explanation why there are 2 techniques that are used in conjunction with each other. I.e. why does one technique not suffice?
IHC isn’t always 100% accurate at dictating the HER2 status, this then requires FISH testing to make a final decision as to whether the patient is HER2 negative or HER2 positive. This is done as it is essential for treatment of the patient that the HER2 status is correct as treatment and prognosis is dependent on this. If just IHC was carried out if there was any results that came back inconclusive the HER2 status would not be discovered. Thus IHC and FISH are used together to make an official diagnosis. 11
A discussion of what factors govern the use of one technique over the other and the circumstances where the use of one technique would be inappropriate.

IHC is used more commonly than FISH as it is cheaper and easier to do, FISH testing is costly and requires expensive equipment. However if the IHC comes back with a value of 2+ further testing using FISH is necessary to discover if it is HER2 positive or negative. If a value of 2+ from the IHC testing came back, using only one technique would be inappropriate as it wouldn’t be a clear result if the HER2 receptor is amplified or non-amplified. This could result in patients getting the wrong treatment which could significantly affect treatment and prognosis. If an IHC result comes back as 2+ or 3+ further testing with FISH should be undergone to determine the HER2 status as chromosome 17 polysomy can be misjudged as HER2 overexpression using IHC which can affect the patient’s treatment. IHC method should be used firstly as a screening method to determine the HER2 status and then FISH should be used in addition to detect any results from the IHC testing that may be false negatives, addition FISH testing should be used especially in tumours with high malignancy. 11
A conclusion, summarising the topic and personal opinion.

In conclusion the HER2 receptor is significant in breast cancer diagnosis and prognosis, it is essential that it is known if the tissue is HER2 negative or HER2 positive so the correct treatment can be given to the patient to give the best prognosis possible. The two tests used for evaluating HER2 status work well but need to be used together if the IHC result comes back as inconclusive so the HER2 status can be decided correctly. FISH testing is expensive and hard to carry out so I believe it should be used to test any tissues that come back as 2+ or 3+ from the IHC method but firstly IHC should be used as a screening method as if the tissue is 0 or 1+ then FISH won’t be required and no money will be wasted carrying out FISH testing on tissues that don’t require it.
Reference
1 HYPERLINK “http://www.enzolifesciences.com/science-center/technotes/2017/april/what-are-the-differences-between-IHC-and-ISH-and-how-do-they-help-in-the-fight-against-cancer?/” http://www.enzolifesciences.com/science-center/technotes/2017/april/what-are-the-differences-between-IHC-and-ISH-and-how-do-they-help-in-the-fight-against-cancer?/
2 HYPERLINK “https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242418/” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242418/
3 HYPERLINK “https://www.breastcancer.org/symptoms/testing/types/fish” https://www.breastcancer.org/symptoms/testing/types/fish
4 HYPERLINK “https://wjso.biomedcentral.com/articles/10.1186/1477-7819-7-83” https://wjso.biomedcentral.com/articles/10.1186/1477-7819-7-83
5 HYPERLINK “http://singaporesocietypathology.weebly.com/uploads/3/8/6/7/3867136/prof_tsongalis_lecture_9_-_her2_fish_and_ihc.pdf” http://singaporesocietypathology.weebly.com/uploads/3/8/6/7/3867136/prof_tsongalis_lecture_9_-_her2_fish_and_ihc.pdf
6 HYPERLINK “https://www.reference.com/health/her2-negative-breast-cancer-431dbd8b3e63195d” https://www.reference.com/health/her2-negative-breast-cancer-431dbd8b3e63195d
7https://www.nature.com/articles/3880288 8 https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-3279 https://www.researchgate.net/figure/Basic-steps-of-fluorescent-in-situ-hybridization-technique-If-the-probes-are-labeled_fig1_26841281110 https://bitesizebio.com/20929/getting-started-with-immunohistochemistry/11 http://www.advances.umed.wroc.pl/pdf/2015/24/5/899.pdf?fbclid=IwAR29A75yeoDVYccUUsL20fLCdHOVGwzzE2ltfie1jSAjPAfpPeRRmIGmbo812 https://www.leicabiosystems.com/ihc-ish-fish/companion-diagnostics/theranostics/products/leica-oracle-her2-bond-ihc-system-row-breast-gastric/13 http://maximjourney.blogspot.com/2012_10_01_archive.html

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