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Flashcards in Investigation of Disease - Cancer Investigation Deck (112)
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What is the lifetime risk of cancer for people born since 1960?



What is cancer?

Disease caused by uncontrolled cell division of normal body cells


What is the definition of a tumour and neoplasia?

Tumour - swelling of a part of the body generally caused by abnormal cell growth
Neoplasia - new growth
Term can be used interchangably


What are the main 2 types of cancer?

1) Benign
2) Malignant - a malignant neoplasm is termed cancer


Describe the difference between a benign and malignant tumour in terms of the following; size, border, differentiation, growth rate, mitotic figures (dividing cells), cell death, invasion and metastasis

B - small
M - large
B - well defined
M - ill defined
B - resembles tissue of origin
M - Variable
Growth rate:
B - Slow
M - Rapid
Mitotic figures:
B - Rare
M - Common
Cell death:
B - No
M - Yes
B - No
M - Yes
B - No
M - Yes


What is the difference between cancer detection and diagnosis?

Detection: The action or process of identifying the presence of cancer in the absence of signs or symptoms
Diagnosis: The action or process of identifying the presence of cancer from its signs or symptoms


How is early detection of cancer achieved?



What are the 3 types of cancer screened for in adults in England, how are they performed and what age groups are tested?

1) Cervical screening
- Check health of cervical cells
- Offered every 3 years to women aged 26-49
- Every 5 years for women aged 50-64
2) Breast cancer screening
- Uses a mammogram (x ray) to spot cancer
- Offered to women aged 50 - 70
- NHS extending programme - trail in screening aged 47 - 53
3) Bowel cancer screening
- 2 types:
> Home testing kit (Faecal occult blood (FOB) test) offered to men and women aged 60-74
> Bowel scope screening -offered to men and women 55+, being introduced in England
- uses small camera on end of flexible tube


Define the terms histopathology, cytopathology and molecular pathology

Histopathlogy - the microscopic study of diseased tissue
Cytopathology - The microscopic study of diseased cells
Molecular pathology - the study of molecules in a disease state


What are the 4 main stages in histo-, cyto- and molecular pathology?

1) Specimen collection
2) Sample preservation
- formalin fixation etc
3) Sample processing e.g dissection, microtomy etc
4) Sample analysis


What are the 6 types of patient biopsy?

- Cell scraping
- punch biopsy
- endoscopy
- needle biopsy
- surgical biopsy
- surgical resection


What is cell scraping?

Taking a small sample of cells and observing them under the light microscope for the presence of disease


What are the advantages of cell scraping over a tissue biopsy?

- Easier to get
- Causes less discomfort to patient
- Less likely to result in serious complications
- Costs less


What type of cytology is cell scraping?

Exfoliative cytology


For what 2 cancers would you use scrapes?

- Skin cells for melanoma
- Cervical cells for cervical cancer


What is punch biopsy??

Involves taking a small disc of full thickness skin using a special punch biopsy instrument with a circular blade


When would a punch biopsy be used?

For diagnosing skin diseases such as cancer


What is the procedure of a punch biopsy?

1) Skin numbed with local anaesthetic
2)Instrument rotates into dermis
3) Instrument removed - sample either removed with instrument or with forceps
4) Samples over 3mm will need 1 or 2 stitches
4) 5mm or larger prefered for histopathology


What is endoscopy?

Looking inside the body to check for growths or abnormal looking areas


What is the endoscopy procedure?

Uses an endoscope - flexible tube with camera, light and sampling equipment on the end
Tissue sample can be taken


What are the 4 main types of endoscopy?

1) Oseophageal and upper GI tract endoscopy - look at oesophagus, stomach and duodenum
2) Bronchoscopy - look and trachea and bronchi
3) Sigmoidoscopy - lower bowel
4) Colonoscopy - look at colon


What is fine needle aspiration (FNA) (needle biopsy)

Involves using a thin, hollow needle to remove samples of tissue fragments or cells in fluid from an organ of the body or lump found under the skin


What is the FNA procedure?

- Tip of 22 or 23 gauge needle attached to a 20 cc syringe is inserted into the mass
- Plunger withdrawn
- Needle removed - aspirate is expelled on one or more glass microscope slides and smeared


What cancers can FNAs be used to diagnose?

- Breast
- Thyroid
- Lung
- Pancreatic


What is core needle biopsy (needle biopsy)

Uses a larger needle than FNA (11 to 16 gauge)
Used to extract a cyclinder of tissue, which provides more sample for analysis


What is the core needle biopsy procedure?

- Often skin is numbed with LA and small incision made
- Needle inserted 3-6 times into palpable (detected by touch) lesion to obtain cores
- If lesion is not palpable then imaging technique used to guide operator
- The include; Ultrasound, MRI or stereostatic radiography


What types of cancer can core needle biopsy be used to diagnose?

- Liver
- Prostate
- Breast


What is surgical biopsy, what are the 2 types and what does each diagnose?

Surgical biopsy is a surgical procedure to remove tissue samples
1) Incisional biopsy
Removes sample of diseased tissue/tumour
- Breast
- Skin
- Soft tissue cancers - sarcomas (GI tract, gynecological)
2) Excisional biopsy
Removes all of diseased tissue - lump often with some healthy tissue
- Breast
- Skin
- Prostate
LA usually used
If tumour is in chest or abdomen, GA used


What is resection?

Resection is medical term for surgically removing part or all of a tissue, structure or organ


What are the common types of resection?

- Lung
- Liver
- Small bowel - removal of small parts of small intestine
- Brain


What are the 4 types of lung resection?

1) Wedge resection
- Removes small portion of lobe
2) Segment resection
- Removes larger portion of lobe
3) Lobectomy
- Removes entire lobe
4) Pneumonectomy
- Removes entire lung


What are the 2 types of cervical cancer?

1) Endocervical carcinoma
- Caused by abnormal growth of glandular tissue in cervix (cervical glandular neoplasia)
2) Squamous cell carcinoma of cervix (most common - 70-80%)
- Caused by abnormal growth of skin like epithelial cells of cervix (cervical intraepithelial carcinoma)


What causes cervical cancer?

Change in cellular DNA by human papilloma virus
- Over 100 types
- Spread by sexual intercourse mainly
- Over 99% of women who get cervical cancer have been infected with HPV


What is used to extract cells from the cervix?

A surgical broom


Who developed the Pap stain and what 3 solutions are involved?

Developed by George Papanicolaou
1) Haematoxilyn (violet):
- Stains cell nuclei
2) OG6 (orange):
Acid stain - reacts with keratin - often found in abnormal squamous epithelial cells of cervix
3) Eosin azure (EA, light blue)
- Acid stain made of eosin and light green dyes
- Usually suffixed by a number to denote proportions of constituent dyes e.g EA-36, EA-50
- EA-50 most common
- Stains cytoplasm of cells various shade of pink or green depending on cellular conditions e.g pH


What is dyskaryosis?

Morphological abnormality of the nucleus
- usually a result of HPV infection


What are the 3 stages of dyskaryosis?



Define immunocytochemistry and immunohistochemistry

- Method by which antibodies are used to detect proteins (or other molecules) in cells
- Performed of intact cells that have had most of their surrounding matrix removed
- Method by which antibodies are used to detect proteins (or other molecules) in the cells of a tissue
- Performed on sections of tissue


What is an anitbody?

Proteins produced by B lymphocytes in vertebrate animals in response to a foreign molecule or organism


What is an antigen?

Any target molecule that is recognised by and bound by an antibody
- Can be proteins, lipid, sugars and even small organic molecules


What group of proteins do antibodies belong to and which is the most common?

Immunoglobulins (Igs)
- comprised of IgG, IgA, IgM, IgD and IgE
- IgG most common


What is the structure of an antibody?

- 2 identical light chains
- 2 identical heavy chains - each int interrupted in the middle by a hinge
- 2 antigen binding sites
L and H chains held together by disulphide bonds


What is the name of the region of an antigen that interacts with the antibody?



What 2 regions does each light and heavy chain have?

A constant (c) region - chains
A variable (v) region - antigen binding
The variable regions of the L and H chains combine to for the antigen binding site


The diverse range of variable regions of an antibody result in how many different types?



What bonds form between an antibody and an epitope and what are the implications of this on the reaction?

- Non covalent
- Involves hydrophobic interactions and hydrogen bonding
- Reactions are reversible


What are the key types of lympocytes in the adaptive immune reponse and what do they do?

B cells
- recognise soluble foreign molecules and secrete antibodies, which bind to foreign molecules, form aggregates and then are engulfed by macrophages
Cytotoxic T cells - recgonise foreign or infected cells and lyse them
Helper T cells
- control the responses of T and B cells


How does a B cell recognise an antigen and secrete new antibodies?

- Surface of each B cell is covered with on type of membrane bound antibody
- 10^7 different types of membrane bound antibody so 10^7 different types of B cell
- Antigen binds to membrane bound antibody
- B cell stimulated to proliferate and differentiate into plasma cell that secretes large amount of same antibody


What does immunogenicity mean?

The ability of a molecule to raise an adaptive immune response
Antigens used in antibody generation must be immunogenic


What is the process of B cell activation?

- Antigen binds to B cell surface antibody
- B cell engulfs and digests antigen
- Displays antigen fragments bound to unique MHC molecules
- combination of antigen and MHC attracts a mature, matching T cell
- Cytokines secreted by T cell help B cell to multiply into antibody producing plasma cells


What 3 things make a good immonogen?

1) Must be an antigen - bind to cell surface antibodies
2) Must be degradable one taken into B cell
3) Degraded fragments must be big enough so that they have at least one binding site that can be recognised by the MHC molecules and the T cell receptor
(antigen needs to be at least 3-5kDa in size)


How are polyclonal antibodies produced?

- Injecting an animal (usually a rabbit, mouse, sheep or goat) with an immunogen
- Repeated injections of same immunogen at intervals of several weeks stimulate antigen specific B cells to produce antibodies into blood
- If immunogen is large then will have multiple different epitopes
- Will therefore activate more than one type of B cell
- Take blood - blood will contain antibodies secreted from more than one type of plasma cell = Polyclonal antibodies


What is the difference between polyclonal and monoclonal antibodies?

Polyclonal - Antibodies from more than one type of plasma cell, which therfore each bind to a different epitode on an antigen
Monoclonal - Antibodies from one type of plasma cell and therefore only bind one epitope


How are monoclonal antibodies generated?

- Inject mouse with immunogenic antigen
- Immune response, production of antibodies
- Isolate spleen - contains large amount of plasma cells
- Plasma cells are fused with multiple myeloma cells (type of B cell tumour) that are immortal
- Fused by adding poly ethylene glycol (PEG) to the cell mixture, which fuses their plasma membranes
- Fused cell is called a hybridoma
- HAT medium will kill unfused myeloma cells but will allow fused cells to grow
- One plasma cell fuses with one myeloma cell - so hybridoma only secretes one antibody - MONOCLONAL


What are the advantages and disadvantages of polyclonal antibodies?

- Inexpensive to produce
- Low tech and skills required for production
- Production time is short
- Multiple epitopes provides more robust detection
- Batch to Batch variability
- Can produce non specific antibodies that give background signals
- Multiple epitopes make it important to check immunogen for any cross reactivity


What are the advantages and disadvantages of monoclonal antibodies?

- Less likely to cross react
- No non specific antibodies
- Highly reproducable results - less batch to batch variation
- Renewable supply of antibody - immortal hybridoma
- High technology/training required
- Time scale is long
- Antibodies may be too specific


Why do proteins make good sources for immunogens?

- Large enough
- Multiple antibody binding sites (epitopes)


What are the 2 main sources of immunogen antigen protein?

1) Protein isolated from a natural source - bovine brain, human spleen
2) Protein overexpressed in a recombinant system
- E.coli
- Yeast
- Insect cells


How are synthetic peptides used as antigens?

Advantage - doesn't require you to actually have the proteins
- Peptide synthesisier can produce peptide of around 12-20 AA long
- Peptide is antigenic but not immunogenic
- Therefore it is cross linked with an immunogenic carrier protein (large and soluble)


What are the common types of carrier protein used in peptide immunogen formation?

- Bovine serum albumin (BSA)
- Keyhole Limpet Haemocyanin (KLH)


Antibodies tend to interact with the outside of proteins which have what property?



What is the name of the computer programme that can predict the hydrophilic and hydrophobic regions of a protein?

Kyte and Doolittle
Useful for selecting a suitable immunogenic peptide


What is an antibody titre?

Measurement of how much antibody an organism has produced that recognises a particular epitope


What method is used to measure the antibody titre?

Enzyme-Linked Immuno-Sorbant Assay (ELISA)


How is the ELISA performed?

- Coat a plate with solution of the immungen
- Wash
- Add dilutions of the (rabbit) serum to be tested to the plate and leave ~2hrs so peptide binds
- wash
- Add a diluted solution of a (anti-rabbit) secondry antibody coupled to an enzyme eg HRP
- Wash
- Add colour development substrate (HRP reacts with ABTS to give a green coloured product)
- Plate added to plate reader which measures Abs


How are antibodies tested to check if they are specific to one antigen?

Western blotting using cell lysates that either
a) Express endogenous protein antigen
b) are made from cells transfected with the cDNA of the antigen, to overexpress the protein
c) mix recombinant protein into cell lysate

Western blot of cells transfected with or without the protein antigen:
A) Only transfected cells give signal, correct molecular weight. Specific antibody
B) Correct band but also other bands detected. Non specific antibody - needs purifying


In what 2 ways can all the antibodies in a serum (one you want and those you dont) be purified?

1) Precipitation with ammonium sulphate
2) Affinity chromatography using Protein A- Sepharose
- Protein A is a protein produced by bacteria that can bind immunoglobulins (antibodies)
- eluted at low pH


How can a specific antibody be purified?

Using affinity chromatography wither with immobilised purified antigen from natural source or using immobilised synthetic peptide an then elution at low pH


What is the common name for a substrate of an enzyme tagged antibody?

Chromogenic substrate or a chromogen


What are the 2 most common enzymes used in immunostaining?

Horseradish peroxidase and alkaline phosphatase


How does HRP work?

Carries out the oxidation of molecules using hydrogen peroxide (H2O2) as a co factor


What are the 2 most common chromogens for HRP and what colours do they produce?

1) 3,3 diaminobenzidine (DAB, colourless) - turns brown
2) 3-amino-9-ethylcarbazole (AEC) - turns red


How does alkaline phosphatase work?

Removes phosphate from many molecules including nucleotides and proteins


What is the chromogen of AP?

New fuchsin - converted to a pink/red product


What is the main disadvantage of HRP and AP in immunostaining?

Must be chemically blocked or quenched before labelling so that false positive staining is eliminated


What is direct immunostaining?

- One step
- Labelled antibody binds directly to the antigen
- Label may be enzyme or fluorescent tag eg. fluorescein


What is the main advantage and disadvantage of direct immunostaining?

Procedure is short and quick
Insensitive due to little signal amplification
- Rarely used


What is 2 step indirect immunostaining?

- Unlabeled primary antibody reacts with antigen
- Labeled secondary antibody reacts with primary antibody
- Secondary antibody must be agaisnt IgG of animal species in which primary antibody was raised
Eg. If primary antibody is rabbit, secondary must be (for example) sheep anti-rabbit IgG antibody


What is the main advantage of 2 step indirect immunostaining?

- More sensitive as signal amplification via several secondary antibodies react with different sites of primary antibody


What are the 2 3-step indirect methods of immunostaining?

1) Peroxidase-anti-peroxidase (PAP) method
2) Avidin-Biotin Complex (ABC)


What is the peroxidase-anti-preoxidase method procedure?

Same as 2 step methos but a complec of HRP and an antibody raised agaisnt the peroxidase itself
- good sensitivity due to signal amplification on 2nd and 3rd antibody


What is the avidin-biotin complex procedure?

- Primary antibody binds to antigen
- Secondary antibody labelled with biotin is bound to primary antibody
- Biotin is vitamin B cofactor
- Protein avidin has high affinity for biotin - 4 biotin binding sites per avidin
- Third layer is either Avidin labelled with HRP or biotin labelled with HRP together with Avidin
One disadvanatge is that biotin is found in human tissue, may need to be a blocking step with avidin


What is the polymer based method of indirect immunostaining?

- Use of a polymer back bone (e.g. dextran) that permits multiple antibodies and enzymes to be attached


What is the major advantage and potential disadvantage of polymer based methods?

Does not use biotin so avoids need for blocking steps
Potential disadvantage:
Certain epitopes may be restricted due to steric hindrance


How is the extent of staining scored in a histopathology lab?

Negative, +, ++ and +++
0, 1, 2 and 3


What are carcinomas, sarcomas, mesotheliomas, melanomas, leukaemias and lymphomas?

- Cancer of cells of epithelial origin e.g. skin or cells that line internal organs
- Subtypes include adenocarcinomas - derived from glandular epithelium e.g. GI tract or breast
- Connective tissue cancers e.g muscle
Mesothelioma cells - cover outer surface of most internal organsm e.g mesothelium of lung from asbestos exposure
Cancers of melanocytes in skin
Cancers of white blood cells
Cancers of lymphatic system e.g. lymph nodes


What is ductal carcinoma in situ (DCIS)?

Most common type of non invasive breast cancer
- A proliferation of
malignant epithelial cells that have not breached the myoepthithelial layer of the ductolobular system


What is performed to decide whether a breast tumour is invasive or not?

Immunostaining is performed using a panel of antibodies to conform the presence of an intact layer of myoepithelial cells - which indicates that the cancer is DCIS


How are oestrogen receptors in the breast related to breast cancer?

- Binding of oestrogen to oestrogen receptor causes the ER to translocate from cytoplasm to nucleus, where is binds to DNA and transcribes genes important for cell proliferation
- Can be overexpressed in breast cancer


What drug is used to prevent the activity of the ER in breast cell proliferation?

Tamoxifen - binds to oestrogen receptors and blocks activity of ER


How is immunostaining used to determine whether DCIS patients should be prescribed with tamoxifen?

Immunostaining of the ER to determine whether it is the cause


How is ductal carcinoma in situ (DCIS) treated?

- If DCIS cells have oestrogen receptors then tamoxifen prescribed
- Area of DCIS removed, with border of healthy tissue around it
> called wide local excision
- Radiotherapy common post surgery
- Some women choose to have entire breast removed (Mastectomy)


What is Human Epidermal Growth Factor 2 and what is its relation to breast cancer?

Protein found on surface of many cells
- Usually high in cancer
- ~20% of all breast cancers express high HER2
- Breast cancers that express HER2 tend to be more aggressive


How is HER2 treated?

- The HER2 test to see if its high
- Herceptin - monoclonal antibody that can bind to and block activity of HER2


What is molecular pathology and what does it aim to investigate?

- Study of molecules in the diseased state
Aims to investigate:
- Changes in the sequence, structure or amount of DNA - changes protein
- Increase or decrease in the amount of RNA - related to expression


How can molecular pathology be used in disease prevention?

Tests that look for inherited genetic disease allow for preventive measures:
E.g. people wth family history of colon cancer can be tested for presence of inherited mutations in genes such as APC
- Individuals with family history of breast and/or ovarian cancer can be tested for mutations in the BRCA1 and BRCA2 genes


How can molecular pathology be used in diagnosis and treatment?

Molecular testing to diagnose certain forms of cancer and then predict their response to certain drugs
- 'personalised medicine'
- e.g. HER2 testing in breast and gastric cancer
- BRAF testing in melanoma
- BCR-ABL testing in chronic myeloid leukaemia (CML)


Why does a APC gene mutation often cause colon cancer?

APC gene is known as a tumour suppressor gene
- Mutated APC gene causes mutated adenomatous polyposis coli (APC) protein


Describe the meaning of a substituation, insertion, deletion, amplification and translocation mutation

Substitution (point mutation) - exchanges one base for another
Insertion - extra base pair inserted
Deletion - section of DNA is deleted, usually single bp
Amplification - An increase in the number of copies of a gene
Translocation - A chromosome or chromosome segment is interchanged with another whole or partial chromosome


What is melanoma and what is its prevalence in the UK?

Cancer of melanocytes - pigment cells found in the dermis of the skin
~ 13,300 people diagnosed each year in the UK
6th most common cancer in UK


What gene can commonly cause melanomas?

BRAF gene - BRAF is a kinase protein that regulates proliferation of melanocytes


What % of melanomas have a point mutation in the BRAF gene?



What occurs to the BRAF protein and what is the corresponding nucleotide change?

Valine 600 is converted to glutamic acid - causes hyperactivation of BRAF which drives melanocyte proliferation
Wild type DNA = GTG = valine
Mutant DNA = GAG = glutamic acid


What is the name of the drug used in melanomas and how does it work?

- Vemurafenib ( commercial name = Zelboraf)
- Blocks activity of mutant BRAF kinase


How is the mutation of the BRAF gene tested using PCR?

- Detects changes from GTG to GAG
- 2 primers added
- 95 degrees - hybridisation
- 50 degrees - annealing of primers
- 72 degrees - optimum for DNA polymerase
- 1 primer will be designed to only bind to GAG mutant and not GTG wt - only mutant DNA amplified


How is the mutation of the BRAF gene tested using real time PCR?

- olignonucleotide called TagMan Probe is added to reaction mixture including enzyme, DNA and 2 primers
- TagMan probe has a fluorescent dye attached at one end and another molecule at the other end that quenches the dye signal when they are in close proximity (atm, no fluorescence)
- Probe anneals to mutant BRAF sequence between the 2 primers
- Probe sits in path of polymerase
- When polymerase reaches probe, 4' exonuclease activity vleaves probe releasing dye (fluorescent signal)
- Can be measured by fluorimeter
- Fluorescent signal = mutant BRAF


How is Human epidermal growth factor receptor 2 (HER2) tested for?

- First test is detection of protein by IHC (immunostaining)
- If test is unclear e.g. 2+ staining, then a test will be performed to see if the HER2 gene is amplified (flourescent in situ hybridisation)
- Overexpression of the HER2 protein is often due to having multiple copies of the HER2 gene


How is fluorescent in situ hybridisation performed to test for HER2 genes?

- DNA denatured
- Single strands hybridised with DNA probe with chemical tag e.g digoxigenin (dig)
- anti-dig antibodies linked with fluorophor bound to tag
- Gene located using epifluorescent microscopy


What is chronic myeloid leukaemia (CML) and what causes it?

- Cancer of the white blood cells in which the granulocyte white cells are cancerous
- 95% of CML patients have abnormality called Philadelphia chromosome
- Caused by translocation of part of chromosome 9 to chromosome 22
- Causes fusion of c-abl gene on C9 with c-Bcr gene on C22


What is the product of the fused c-abl c-Bcr gene?

- Production of fusion protein called p210 Bcr-Abl
- Has deregulated Abl tyrosine kinase activity
- Protein causes hyperproliferation of granuloctye white blood cells


What drug can be used to treat CML and how does it work?

- Imatinib (commercial name Glivec)
- blocks activity of p210 Bcr-Abl protein


How is bcr-abl detected?

Originally detected by Geimsa staining of chromosomes
- Often used in combination with FISH or RT-PCR where primers have been designed to detect translocation