Introduction To Techniques Flashcards
(160 cards)
1
Q
Give an overview of the process a specimen will go through in routine histopathology.
A
Specimen reception»_space;> fixation»_space;> tissue selection (dissection)»_space;> processing»_space;> embedding»_space;> microtomy»_space;> demonstration techniques»_space;> quality control»_space;> pathologist.
2
Q
What problems need to be overcome in order for the pathology department to provide microscopic material for diagnosis by pathologist and material to archive?
A
Human tissue degrades, Human tissues are mostly water, Light transmission through human tissue, Whatever you do to human tissue will affect its appearance, Cost, History and habit, Human driven systems aren't perfect.
3
Q
What takes place in specimen reception?
A
Specimen reception receives and logs samples and ensures that specimens are fully tracked.
They check materials being received into the lab are correct and ensures the paperwork matches.
Samples are designed a unique number.
4
Q
What is the purpose of specimen fixation?
A
Specimen fixation preserves human tissue.
It prevents cellular breakdown due to auto lysis and bacteria.
It preserves physical structure, chemical structure and tissue components.
5
Q
What factors influence chemical fixation?
A
Type of fixative, Composition of the tissue, The form of the fixative (liquid, gas, etc), pH, Vehicle (chemicals that allow fixative to penetrate more easily), Temperature, Duration, Volume and concentration of fixative.
6
Q
How have fixatives historically been classified?
A
Aldehydes,
Oxidising agents,
Protein-denaturing agents,
Unknown mechanisms.
7
Q
How do most fixatives act?
A
Most fixatives act on proteins within the tissues by anchoring and stabilising the proteins and thus the tissue components.
8
Q
What is a more modern way of classifying fixatives?
A
To look at the way they work on tissue themselves and which tissue elements they fix best (i.e. Proteins, fats, carbohydrates, nucleic acids) or what tissue they fix.
9
Q
What fixative fixes proteins best?
A
Formaldehyde.
10
Q
What fixative would be best for fixing fats?
A
Mercuric acid.
11
Q
What fixative would be best for fixing carbohydrates?
A
Alcohol based fixative.
12
Q
What is the best fixative for fixing nucleic acids?
A
Formaldehyde based fixatives.
13
Q
What artefacts may occur as a result of fixation?
A
Pigments (e.g. Brown formaldehyde pigment),
Volume changes (e.g. Glomerulus shrinkage),
Alteration of tissue chemistry,
Loss of tissue components.
14
Q
What is the standard fixative used in the cytology lab?
A
Formaldehyde - usually at a concentration of 4% known as formalin.
15
Q
What is one problem with using formalin as a fixative and how may this be overcome?
A
Formaldehyde based fixatives don’t penetrate very well and are most effective if used in larger volumes. Because we only use formaldehyde as a 4% formalin solution we need very large volumes.
We can however incise the tissue to penetrate better, but this does risk damaging it. We make margins using Indian ink so that we don’t lose track of the margins of excision.
16
Q
What is a good fixative to use for sperm?
A
Bouins alcohol based fixative is good for sperm and for nuclear detail.
17
Q
What type of fixative should be used for nucleic acids?
A
Alcohol based (carnoys).
18
Q
What is a good fixative to use for electron microscopy?
A
Glutaraldehyde. It isn’t as safe as formaldehyde. It makes tissue very hard and rigid and is therefore good for electron microscopy.
19
Q
Why do tissues need to be dissected in the histology department?
A
Tissue selection is required because we need smaller samples because they are easier to examine and fix. It is a very skilled process.
We physically can’t store whole specimen taken from patients.
20
Q
Who carries out tissue dissection?
A
Either a pathologist or a trained scientist carries out dissection on a ventilated bench.
21
Q
Describe the general process of tissue dissection.
A
The process of dissection is a pivotal point on the whole pathology process.
Margins are marked. This is very important in cases where the margins of the tissue are not easily defined in order to ensure that the whole of a tumour is removed.
Small pieces are put in to labelled and numbered cassettes and then put back into fixative.
The dissector will also provide a description of the sample.
Health and safety and cleanliness are very important.
22
Q
What is involved in tissue processing on the histology lab?
A
Processing is the treatment of the tissue necessary to impregnate it with a solid medium to facilitate the production of microscope slides.
Processing involves a wide range of chemicals to dehydrate the tissue and can be hazardous.
The impregnation medium of choice is usually paraffin wax with some other agents added to improve density. It has a melting range of 58-63 degrees Celsius.
1) . Complete fixation in formalin.
2) . Dehydration in increasing concentration of alcohols (avoids tissue shock).
3) . Clearing of dehydrating agent in xylene.
4) . Impregnation with wax.
5) . Embedding
23
Q
What factors may affect tissue processing?
A
Composition of tissue (density),
Agitation makes take up wax,
Temperature (increased temperature speeds up the process),
Fluid viscosity (more fluid agents are faster acting),
Vacuum can pull fluid into tissue.
24
Q
What artefacts might arise as a result of tissue processing?
A
Alteration of tissue chemistry,
Volume changes,
Loss of tissue components.
25
What are the advantages of tissue processing automation?
Tissue processing can be fully automated. This is advantages as it can take up to 23 hours manually. Automated tissue processing is carried out in a tissue processor. Automation saves man power and allow the process to be enhanced by agitation and the use of vacuum chambers. Chemicals are pumped in and out of the vacuum chamber. Molten paraffin wax is then introduced after the final xylene step.
26
What step takes place after tissue processing?
Embedding.
27
What does embedding involve?
After the processing the wax blocks need surrounding to make sure that they are fully rigid and supported. This involves an embedding machine which has a cold plate and a molten wax chamber.
The blocks are taken from the tissue processor and then placed in the molten wax in the embedding machine chamber. The samples are then removed from their cassettes and placed into an appropriate sized mould. The specimen need to be orientate correctly within the mould to ensure we can see an edge. The tissue processing cassette is then placed on top of the mould and it is topped up with wax.
28
What would be a good embedding material for electro microscopy samples?
Resin or plastic.
29
What embedding material could we use when the processing required for paraffin wax would destroy the tissue components?
Agar or gelatine as they don't require the use of alcohol.
30
What is the microtomy step achieve?
It makes the slides from the paraffin wax blocks.
31
What factors may influence the microtomy step?
Density and rigidity of impregnation and embedding medium and tissue.
The presence of solid material.
The type of microtome used (usually a rotary microtome now).
32
Describe the process of microtomy.
1) . Chill the paraffin blocks to make them more rigid ensuring nice thin sections.
2) . Place the block in the chuck. Turn the microtome handle so that the chuck moves up and down against the blade creating 2-3 um thick sections (can cut between 0.5 and 10um).
3) . The sections are then taken and floated out on a water bath and picked up on a slide.
33
When are frozen sections and what are they used for?
A frozen section is a thin slice of tissue cut from a frozen specimen. They are often used to make rapid diagnosis (e.g. in surgery).
Frozen sections may also be used when fixation and processing may destroy what you want to look at (e.g. fat assessment).
34
Describe how frozen sections may be made.
1) . Sample received into department.
2) . Tissue is selected and placed onto a cork disk with moutant.
3) . Tissue is frozen and then a slice is taken using a cryostat which is then picked up using a slide.
35
Why isn't frozen sectioning used routinely?
Frozen sectioning is not routinely used for a number of reasons.
The tissue needs freezing down to -30 degrees Celsius in some cases and it is difficult to subsequently store.
It is very useful when we need a very quick answer (20 mins) such as intraoperatively.
Frozen sectioning is backed up by normal processing to get a more storeable and useable sample.
36
What type of chemicals are organic dyes based on?
Benzene ring type chemicals.
37
List 3 types of histochemical demonstration techniques.
1) . Organic dyes
2) . Colourless stains that produce colour by interacting with tissue elements (e.g. Schiff reagent reacting with aldehyde groups to give pink colour).
3) . Metallic pigments that stick to tissue components.
38
Most stains are aqueous based. What difficulties does this pose?
Wax is hydrophobic and so the wax must be removed using xylene and then alcohol and then water before staining is able to take place.
39
What are the basic principles and interactions by which stains work?
The primary action of any chemical stain is due to a chemical or electrostatic reaction with tissue components.
40
Why do some tissue elements stain better than others?
The primary action of any chemical stain is due to a chemical or electrostatic reaction with tissue components.
Some things stain better than others because of repelling forces, the size of staining molecules, the speed of the staining reaction (the quicker the reaction the less likely we can remove it before it has reacted with all of the tissue components - i.e. we can't evict the stain from certain tissue compartments).
The strength of the bonds is why tissues stay stained, however, the stains will fade over time. They are not stable indefinitely.
41
How else can stains be classified other than by their mechanism of action?
We can also classify stains by what they demonstrate.
I.e.
Demonstration of tissue structure,
Demonstration of tissue components,
Demonstration of material that does not appear on normal tissue,
Demonstration of infectious agents.
42
What is the Masson trichrome stain?
It is a 3 stain process in which different densities of tissue stain differently.
43
What does Alcian blue demonstrate?
Alcian blue demonstrates acidic mucins.
44
What does PAS demonstrate and how does it work?
PAS demonstrates neutral mucins.
PA is used to produce aldehyde groups on mucins and then Schiffs reagent is used to react with them.
45
What can the Oil Red O stain be used to demonstrate?
The Oil Red O stain can be used to demonstrate fat on frozen section material.
46
What can the Masson Fontana stain be used for?
The Massin Fontana stain can be used to demonstrate melanin by enhancing its staining.
47
What stain can be used to demonstrate amyloid?
Congo Red stain can be used to demonstrate amyloid.
48
What does Pearle's stain demonstrate?
Pearle's stain demonstrates iron from red blood cell break down.
49
What stains may be used to demonstrate helicobacter?
Toluidine blue and silver methods.
50
Why do most labs limit their regularly performed special stains to about 20 different types even though there are hundreds available?
It keeps costs down and is more consistent.
There is a lot of redundancy in special staining methods.
51
Stains are of used on their own. True or false?
False. Staines are rarely used on their own. Timing is critical. Stains are used in conjunction with other chemical reagents to adjust the uptake and distribution of stains.
52
What is haematoxylin?
Haematoxylin is a nuclear stain. It can give considerable detail about the nuclear details of cells (i.e. doesn't simply demonstrate them).
53
What does eosin do in the H and E stain?
Eosin acts as a counter stain to demonstrate all the tissue components other than the nucleus. It also gives information about tissue structure. It can demonstrate cell membranes, fibrous tissue etc.
54
H and E staining is good at identifying cells that have large and active nuclei (i.e. rapidly dividing cells). What does this make the H and E staining procedure particularly good at examining?
Tumour cells.
55
How is the H and E stain usually carried out these days?
On automated machines that stain hundreds of slides per day. They are stained in one machine and then cover slipped in another.
56
What is the aim of quality control in the histology lab?
To ensure the ID and link between samples and patients and to ensure satisfactory test quality.
57
What 2 steps does most quality control on the histology lab consist of?
1) . Tracking of patient info.
| 2) . Microscopic examination of the slide.
58
What will happen before a slide leaves the histology department?
Before a slide leaves the department and goes out to a pathologist a scientist will assess that slide and make sure that the fixation is adequate, no damage has occurred due to microtomy, the staining itself is demonstrating the correct tissue components and equally and importantly actually identifying the tissue type is correct.
Identifying the tissue type and cross referencing with the patient record is an important quality control method.
No material will leave the histology department without quality control.
59
What is the very basic principle of IHC?
IHC uses antibodies to detect antigens in cells or tissues.
60
True or false? IHC can demonstrate the presence and localisation of antigen.
True.
61
IHC can detect as few as ____ per cell.
IHC can detect as few as 100 molecules per cell.
62
True or false? Multiple labelling can be used to detect different antigens simultaneously in IHC.
True.
63
List some of the diagnostic uses of IHC.
```
Differentiation of tumours,
Tumour sub-typing,
Confirm or refute H and E diagnosis,
Secretory products,
Lymphoma/leukaemia characterisation,
Identification of metasteses,
Prognostic markers.
```
64
What are the main 2 antibodies used for IHC? Briefly describe these antibodies.
1) . IgG - is Y shaped and has two identical binding sites for antigens.
2) . IgM - is a larger molecule and consists of 5 igG like molecules bound together to form a pentamer.
Antibodies may be polyclonal or monoclonal.
65
What are polyclonal antibodies?
Polyclonal antibodies are not specific. The antibodies raised may cross react with proteins other than specific antigen. The antibodies are not always identical and will vary depending on the animal.
66
What are monoclonal antibodies?
Monoclonal antibodies are very specific and are less cross reactive than polyclonal antibodies. They are always identical and do not vary depending on the animal.
67
What are the most common antibodies used to detect oestrogen receptor?
Clone 1DS,
Clone 6F11,
Clone SP1 (rabbit monoclonal).
68
What antibody clones are commonly used to detect progesterone receptor?
Clone PgR 636,
Clone 1A6,
Clone 16,
Clone SP2 (rabbit monoclonal).
69
What are the 2 mechanisms by which IHC can occur? Which one is preferred and why?
IHC can either be direct or indirect.
Indirect methods are preferred because they allow for amplification via labelling with multiple secondary antibodies.
70
What may secondary antibodies be couple to in order to be detectable?
1) . Secondary antibodies are often coupled to horseradish peroxidase (HRP). Substrates for this enzyme include diaminobenzidine (DAB) which gives a dark brown insoluble precipitate and aminoethylcarbozole (AEC), which gives a red precipitate.
2) . Secondary antibodies can also be coupled to alkaline phosphatase. The most popular substrate for alkaline phosphatase are Bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT), which gives an intense blue precipitate, and AEC which gives a red precipitate.
71
Why is the alkaline phosphate detection method favoured over HRP methods in tissues such as blood and bone marrow?
Because these tissues have high levels of endogenous peroxidases.
72
What markers may be utilised for cellular localisation in IHC?
Membrane markers: membrane receptors, signal transduction molecules, cell adhesion molecules.
Nuclear markers: signal transduction molecules, DNA replication molecules, cell cycle molecules.
Cytoplasmic markers: signal transduction molecules, metabolic molecules, cytoskeleton molecules.
73
What is one of the most popular methods of indirect IHC?
The Avidin-Biotin-Complex method.
74
Describe the Avidin-Biotin-Complex method of IHC.
See diagram.
Avidin is a glycoprotein that has a strong affinity for the vitamin biotin. This binding is stronger than the usual antibody binding and it is very difficult to disrupt. By linking biotin to the secondary antibody a complex can be formed that is very stable.
The ABC method allows a combination of several avidin molecules and several biotin peroxidase molecules. This allows a much greater level of amplification of signal.
Avidin can be replaced by the protein streptavidin which has similar binding properties.
75
What method has now largely superseded Avidin-Biotin-Complex methods?
Polymer-based methods.
76
How do polymer-based IHC methods work?
They use a polymer spine called Dextran with multiple antibodies and multiple enzymes (up to 70) which can be attached.
This allows much higher levels of enzyme to process substrate thus giving a stronger response to antibody.
77
What are the advantages of polymer-based IHC methods?
Increased sensitivity,
Increased specificity,
Decreased background,
Detection simplicity.
78
Describe a generalised step-by-step IHC protocol.
1) . Formalin fixed paraffin embedded sections cut at 3-5um and heated for 10 mins at 69 degrees Celsius to enhance tissue adherence.
2) . Paraffin removal via 2x5mins xylene immersion.
3) . Rehydration via 3 x immersion in graded alcohol for 2 mins followed by running tap water for 5 mins.
4) . Antigen retrieval if necessary.
5) . Block endogenous peroxidase activity for 5 mins to minimise background staining.
6) . Incubate in primary antibody for 30-60 mins.
7) . Incubate in polymer link for 30 mins.
8) . Incubate with enzyme substrate for 5 mins.
9) . Counter stain in haematoxylin for 5 mins.
10) . Dehydrate in alcohol, clear in xylene and mount.
79
Why might antigen retrieval be necessary before IHC?
One of the issues of using formalin fixed paraffin embedded material is that during the formaldehyde fixation process it can form cross links between proteins which then masks the epitopes of interest and makes it impossible for the antibodies to target the specific antigens.
A process of antigen retrieval it therefore necessary for some antibodies.
80
How might antigen retrieval be performed?
Digestion with proteolytic enzyme such as proteinase K or pepsin.
Treatment with microwave energy which is most favoured.
Exposure to heat and pressure in a pressure cooker.
Various buffers necessary such as citrate, EDTA, commercial buffers).
81
What are some of the problems and drawbacks with IHC?
Antibody concentration needs to be optimised,
Loss of antigen may occur due to autolysis,
Antigen may present at very low concentrations and be very difficult to detect,
Can get cross reactivity with antibodies,
Can get non specific binding to tissues,
Presence of endogenous peroxidases needs to be blocked.
82
List 2 examples of automated IHC systems.
```
Bondmax from Leica,
Benchmark system (Ventana).
```
83
How are IHC slides generally evaluated?
Using light microscopy.
84
What systems of evaluation may be use for evaluation of IHC slides?
In some cases a simple negative or positive answer is sufficient.
For some markers scoring systems can be used to determine the level of expression.
For example, the measurement of ER in breast cancer is a very important indicator of patient response to endocrine therapy. 70% of tumours are ER positive. We can treat with anti-oestrogens such as Tamoxifen or Fulvestrant, or oestrogen deprivation via aromatase inhibitors (aromatase converts androgen to oestrogen).
For ER we use the Allred score to allow us to understand whether or not a patient will respond to endocrine therapy. The Allred score takes into account both the proportion of cells stained and the intensity of staining. The proportion of cells stained is split on a 6 point basis (0-5) and the intensity score is split on a 4 point basis (0-3).
85
What is the Allred scoring system used for? What are the cutoff points for the Allred score?
The ER status as quantified via the Allred score after IHC allows us to define different treatment need in breast cancer.
```
0-1 = no effect of endocrine treatment
2-3 = 20% chance of endocrine treatment working
4-6 = 50% chance
7-8 = 75% chance
```
86
What treatment would you recommend for a patient that has been found to have breast cancer that is endocrine non-responsive?
Chemotherapy alone. Will not benefit from endocrine therapy.
87
What treatment would you recommend for a patient that has been found to have breast cancer that is moderately endocrine responsive (low positive)?
Endocrine therapy, less benefit from chemotherapy.
88
What treatment would you recommend for a patient that has been found to have breast cancer that is endocrine responsive?
Endocrine therapy alone. Less benefit from chemotherapy.
89
What is the primary use of IHC?
The diagnosis of tumours.
90
What kind of cancer would express AE1/AE3?
Carcinomas. These are cytokeratin stains.
91
What kind of cancer cells will express LCA?
Lymphoma.
92
What are CAM 5.2 and MNF116 markers for?
They are epithelial markers.
93
What kinds of cancers is CEA present in?
CEA is present in carcinomas, particularly GIT carcinomas.
94
What is EMA?
An epithelial tissue marker.
95
What can the CK7 marker be used for?
CK7 is a marker that is found in most ductal, glandular and transitional epithelium of the urinary tract and bile duct epithelial cells.
CK7 distinguishes between lung and breast epithelium (which both stain positive) and colon and prostate epithelium (which both stain negative).
96
What is CK20 used as a marker for?
CK20 will be negative in squamous cell carcinomas and adenocarcinomas of the breast, lung, endometrium, non-mucinous tumours of the ovary, small cell carcinomas.
CK20 will be positive in colon carcinomas.
CK20 is often used in conjunction with CK7 to distinguish colon carcinomas from ovarian, pulmonary and breast carcinomas.
97
What is IHC and what is it used for?
IHC is a technique used to localise antigen de or proteins of interest in FFPE tissue sections by the use of labelled antibodies as specific reagents through antigen-antibody interactions that are visualised by an enzyme interaction.
IHC is. Often used to provide further information for the histo pathologist to aid diagnosis and determine specific treatment plans.
98
Approximately what percentage of invasive breast carcinomas show over expression of HER-2/neu (c-erb B-2)?
20%
99
What kind of mutation is usually responsible for HER2 over expression?
It is a result of gene amplification in 93-94% of cases.
100
What is the result of HER2 gene amplification?
Gene amplification results in protein over expression and a higher growth fraction, poor disease free survival and poorer overall survival.
101
How many HER2 receptors per cell do normal non amplified cases have?
20-50k
102
How many HER2 receptors per cell are found in cases where gene amplification has occurred?
More than 2 million receptors per cell.
103
What drug can be used to target HER2?
Herceptin (trastuzamab).
104
List some indicators of increased HER2 production.
Increased gene copy number,
Increased mRNA transcription,
Increased surface receptor protein expression,
Increased release of extra cellular domain.
105
What protein assays may be used to measure HER2 expression?
Western blot or immunohistochemistry.
106
What assays can be used to examine amplification of HER2 genes at a DNA level?
FISH, RT-PCR, southern blots.
107
What assays can be used to look at mRNA expression levels?
Northern blots.
108
What method can be used to detect circulating shed HER2 receptor proteins?
ELISA.
109
What method uses fluorescence labelled (e.g. Texas red, FITC green, acridine Orange) oligonucleotide probes that attach to target sequences to detect specific regions of DNA?
FISH.
110
FISH has better reproducibility than IHC. True or false?
True.
111
What are some of the disadvantages of using FISH in comparison with IHC?
FISH has poorer tissue localisation, limited availability and a lack of laboratory standardisation.
IHC is more generally available.
112
What are some of the advantages and disadvantages of IHC over FISH?
IHC is more generally available and retains tissue architecture.
However, the results are subjective, it lacks sensitivity and there is likewise a lack of lab standardisation.
113
When interpreting IHC HER2 results should you score cytoplasmic components and in situ tumour components?
No. For HER2 IHC IT is important to score membrane staining only and only those of invasive components.
114
Describe IHC HER2 scoring.
```
0 = membrane staining in less than 10% of cells.
1+ = faint/barely perceptible part membrane staining in more than 10% of cells.
2+ = weak/moderate complete membrane staining in more than 10% of cells.
3+ = strong complete membrane staining in more than 10% of cells.
```
115
In the UK HER2 testing for breast cancer is carried out according to a 2 tier strategy. Describe this strategy.
Firstly an IHC analysis will be carried out. A score of 0 or 1+ is deemed negative and a score of 3+ is deemed positive.
A score of 2 is borderline and will be put through for FISH analysis. A FISH ratio of less than 2.0 indicates no HER2 gene amplification and a negative result. A FISH ratio of more than 2.0 indicates amplification of the HER2 gene and a positive result.
116
What is CISH?
CISH stands for chromogenic in situ hybridisation. It follows the same principle as FISH but uses chromogenic detection.
It allows direct comparison between morphological features of neoplastic cells and the presence of gene amplification.
117
What are the advantages of CISH?
It allows for direct comparison between morphological features of neoplastic cells and the presence of gene amplification.
Analysis is relatively quicker and doesn't require a fluorescence microscope.
Dual coloured CISH is a very reliable and useful method that has several advantages over FISH.
118
What can tumour gene expression profiles tell us?
They can give indications of prognosis, primary tumour sites and best suited therapies.
119
What method can potentially be used to determine the simultaneous expression of thousands of genes in any one sample?
Microarray analysis.
120
Describe the basic method for CGH for tumour profiling.
1) . Label normal RNA green and tumour RNA red.
2) . Mix normal and tumour RNA in equal proportions.
3) . Hybridise to a microarray slide with DNA probes spotted on.
4) . Scan and then measure signals.
5) . If there is equal normal and tumour RNA representing a certain gene then that spot will appear yellow, if there is over expression of the tumour RNA then it will appear red and if there is under expression of the tumour RNA then it will appear green.
121
How has gene expression profiling impacted upon our understanding of disease?
It has increased our understanding of disease.
There are hopes that it will lead to personalised therapy, tailored treatments to characteristics specific tumours and may lead to new diagnostic tests.
It has helped us increase our understanding of breast cancer classification and has led to the discovery of novel prognostic markers for breast cancer.
122
How has gene expression profiling increased our understanding of breast cancer sub types?
We now know that breast cancer is a family of diseases. This is based on over 200 studies. For example there are ER positive and ER negative breast cancers.
When looking at the prognosis of different subtypes they have different outcomes. ER positive subtypes generally have a better outcome than ER negative subtypes.
123
At a minimum how many sub types of breast cancer are there?
4 different subtypes:
1) . HER2 +
2) . Basal like or triple negative (ER-,HER2-,PR-)
3) . ER+ luminal A (higher PR expression)
4) . ER + luminal B
124
What types of breast cancer generally have a better outcome? ER+ or ER-?
ER+
125
What was the oncogene DX study?
It was a multi gene assay to predict recurrence of tamoxifen treated node negative breast cancer.
126
How many cases did the oncogene DX study look at?
675 cases of paraffin-embedded tumour tissue.
127
How many genes did the oncotype DX study examine?
RT-PCR of 21 genes was carried out (16 Cancer-related genes and 5 reference genes).
128
What was the end point of the oncotype DX study?
Distant recurrence.
129
What did the oncotype DX study use to determine risk groups?
Prospectively defined algorithm was used to determine risk groups (low, intermediate or high).
130
What was developed as a result of the oncotype DX study?
Since the oncotype DX study was published the multigene assay has been developed as a diagnostic test that quantifies the likelihood of breast cancer recurrence in women with newly diagnosed, early stage breast cancer.
In addition to predicting distant disease recurrence, also assesses the benefit from certain types of chemotherapy.
131
In what patients has the oncotype DX test been validated for use?
Breast cancer patients whose disease is:
1) . Newly diagnosed.
2) . Stage I or II.
3) . Node negative
4) . ER positive.
AND who will be treated with tamoxifen.
Those patients with early or late stage disease or those who are ER negative will not benefit from this test.
132
How does the oncogene DX test work?
The oncotype DX test analyses the expression of 21 genes from FFPE tumour specimens using qPCR. The expression of each of the 16 genes is measured in triplicate and then normalised relative to a set of 5 reference genes.
The target genes are proliferative, HER2, oestrogen and invasion genes.
The recurrence score on a scale of 1-100 is derived from the reference normalised expression measurement on four steps.
133
The oncotype DX standardised testing method has been optimised to minimise variability due to a number of factors. List some of them.
Variability introduced due to:
Tissue preparation methods (FFPE vs. Frozen sections),
Tumour block age, storage and variability in preparation,
Heterogeneity between and within FFPE blocks,
Heterogeneity with respect to enriched tumour and non tumour areas within an FFPE block.
134
What is classified as a low oncotype DX recurrence score?
Less than 18.
135
What is classified as an intermediate oncotype DX recurrence score?
18-30.
136
What is classified as a high oncotype DX recurrence score?
31 or higher.
137
What was the B-20 clinical trial?
The B20 clinical trial in the USA showed that those with a high risk oncotype DX recurrence score (31 or higher) benefited from tamoxifen in combination with chemotherapy. Those with a low or intermediate risk score did not benefit from chemotherapy in addition to tamoxifen.
Those with a high score showed 28% absolute benefit from tamoxifen and chemotherapy combined.
138
When did Genomic Health Inc. start offering the oncotype DX test?
January 2004.
139
How would you go about getting an oncotype DX test on the UK?
A tumour block is sent using an oncotype DX specimen kit to Genomic Health. Genomic Health is a CLIA-certified CAP accredited reference lab.
The turnaround time is 10-14 days and it cost £1500 per test.
140
What is included in the oncotype DX patient report?
```
Recurrence score (RS),
Average 10 tear distant recurrence rate for that recurrence score (with 95% CI),
Graph of 10 year recurrence risk as a function of recurrence score in lymph node negative ER positive tamoxifen treated patients.
```
The report is sent to the treating physician and the submitting pathologist.
141
What oncotype DX -like test is offered by agendia?
MammaPrint
142
What is MammaPrint a test for?
It is a test to determine whether the breast cancer is at high or low risk for distant metastasis.
143
Who can the MammaPrint test be carried out on?
Patients under 61 years of age with a tumour size of less than 5cm who have lymph node negative stage I and II invasive breast cancer (early stage patients).
Unlike oncotype DX patients it can be informative for both ER positive and ER negative patients and is tamoxifen independent.
144
How does the MammaPrint test work?
Unlike oncogene DX (which uses FFPE material to look at genes using RT-PCR) the MammaPrint method is a microarray technique that looks at the expression of 70 specific genes in fresh tumour and comparing it to the reference expression profile of low risk or high risk tumours.
Risk of tumour recurrence is determined according to the degree of similarity between the tumours expression profile and the reference profiles.
The test can predict survival and disease progression based on the tumour profile.
145
What is the likelihood of patients who are classified as low risk by the MammaPrint remaining metastasis free within the next 5 years? 10 years?
Low risk patients have a 95% chance of being metastasis free within the following 5 years and a 90% chance of being metastasis free within the following 10 years.
146
What is the likelihood of patients who are classified as high risk by the MammaPrint remaining metastasis free within the next 5 years? 10 years?
High risk patients have a 78% chance of being metastasis free within the following 5 years and 71% within the following 10 years.
147
What are the disadvantages of gene expression profiling for diagnostic purposes?
Due to the large amounts of control at the level of translation, the transcriptional changes may not represent protein expression.
Gene microarray studies have not confirmed protein expression analysis, no comparison with traditional classification methods.
Only small datasets have been used which increases the proportion of misclassification due to noisy discovery.
There are concerns regarding the robustness and reproducibility of screening methods for routine clinics use.
Formalin fixed tissue doesn't preserve RNA as well as frozen material.
There are differences among cohorts and platform technologies used in studies.
Heterogeneity of breast tumours.
Multiple gene sets appear to correlate with survival and hence further studies are needed before prognostic gene signatures van be reliably applied in clinical practice.
148
How have some diagnostic tests tried to overcome the disadvantages associated with gene expression profiling?
By using protein expression instead of gene profiling.
149
What is one tumour profiling test that uses protein expression profiles rather than gene expression profiles?
MammoStrat (AGI).
The proteins tested actually come from gene expression studies.
150
What is the function of the MammoStrat test?
The MammoStrat test can be used to classify individual patients as having high, moderate, or low risk of breast cancer recurrence following surgical removal of primary tumours.
151
Who is the MammoStrat test for?
Post-menopausal, node-negative, ER positive patients who receive hormonal therapy and are considering adjuvant chemotherapy.
152
How does the MammoStrat test work?
The MammoStrat test is an IHC test performed in FFPE samples.
It uses five monoclonal antibody bio markers and a diagnostic algorithm.
153
What are the bio markers used in the MammoStrat test?
1) . P53
2) . HTF9C (cell cycle control and DNS replication)
3) . CEA CAMS (aberrantly expression in some cancers)
4) . NDRG1 (expressed under conditions of hypoxia and other stresses)
5) . SLC7A5 (involved in nutrient transport)
These are measured and then the algorithm categorises them.
154
What methods does the Clarient Insight DX breast cancer profile test use? What is its purpose?
It consists of a panel of molecular marker assays performed by FISH and IHC on FFPE tumour tissue.
It measures ER, PR, BCL2, CDKN1B, EGFR and TP53 using IHC.
It measures CCDN1, HER2, MYC genes using FISH.
A risk score then predicts outcome in HR positive patients.
155
What is the limit of resolution of light microscopy?
0.2um
X1400 is the greatest magnification possible.
156
What is the effective wavelength of a transmission electron microscope?
Operating at 100kV approximately 0.004nm, which is which is three orders of magnitude smaller than that of light.
157
Why might you need to use electron microscopy over IHC in the diagnosis of tumours?
Applications in non-immunoreactive tumours, tumours with unexpected or confusing immunophenotype, and exceptionally unusual tumours.
158
What are some of the problems with IHC?
Non-immunoreactivity in some tumours, lack of antibody specificity, inconsistent staining for the same antibody in different laboratories, the use of different technical procedures and instrumentation, and uncertainty in the distinction between weak positive and negative staining - all contribute to the view that immunohistochemistry, although exceedingly important, has limitations which give rise to interpretational uncertainties, some of which can be resolved by electron microscopy.
159
What is the main down side to electron microscopy?
It is labour intensive and is not easily automated.
160
Give some examples of when electron microscopy may be useful.
Examining difficult to classify, confusing tumours of those for which IHC cannot be performed.
Ultrastructural abnormalities of diseased human muscle can be observed in most of the organelles of individual fibres and also in other components of the tissue. Can differentiate between normal and diseased muscle by confirming structures seen in light microscopy and identifying structures not seen in light microscopy such as endothelial tubules and various inclusions.
The investigation of some infections, particularly those of viral or protazoan. In virology it is an important diagnostic technique for gastroenteritis and skin infections. In addition, it is especial,guise full for the investigation of the emerging parasitic protazoan infections, particularly in immunocomprimised patients.
Renal pathology. Ultrastructural features may enable a diagnosis to be made where light micro is apparently normal, for example minimal change, thin membrane disease, hereditary nephropathy, and fibrillation and immunotactoid glomerulonephritis. In addition it can provide information to confirm or elucidate a diagnosis, as in immune complex glomerulonephritis, renal amyloidosis, dense deposit disease, and diabetes.
