Cytopath of Cancer

Question 1

How many types of cancer are there?

Answer 1

• Over 200 types

Question 2

Briefly describe the cellular basis of cancer

Answer 2

• cancer arises from a loss of normal growth control
• disruption can result from;
  1. uncontrolled cell growth
  2. loss of a cell’s ability to undergo apoptosis

Question 3

What are the 6 hallmarks of Cancer ?

Answer 3

1. Evasion of apoptosis
2. Growth signal autonomy
3. Evasion of growth inhibitory signals
4. Angiogenesis
5. Unlimited replicative potential
6. Invasion & Metastasis

Question 4

Describe Cancer at a molecular level

Answer 4

• most cancers are sporadic -> arising from normal adult somatic cells
• carcinogenic insult can initiate cascade of genetic changes
• clonal disease (mono&poly clonal)

Question 5

Define Karyotype

Answer 5

• visual representation of individual’s complete set of chromosomes, ordered & arranged by size & shape
  1-24

Question 6

What is the function of banding on Chromosomes?

Answer 6

• binding differentiates regions along the mitotic chromosomes

Question 7

Why is chromosomal location important?

Answer 7

• Gene Amplification -> large regions amplified-oncogene
• Gene deletion -> large deletions-tumour suppressor gene

Question 8

Describe Retinoblastoma Tumours

Answer 8

• rare childhood eye tumour
• 1:20,000 children ages 0-8 yr old
• radiation or removal
• Familial form appears in children whose parent who also suffered & was cured

Question 9

Describe the Rb genes involvement in Retinoblastoma

Answer 9

• Wild-type normal parents
• cancer requires 2 somatic mutations; to inactivate both copies
• Children who inherited Rb mutation -> only one hit required

Question 10

Describe some mutations which can affect Tumour Suppressor Genes

Answer 10

• large deletion & mutation
• large deletion & methylation
• Haploinsufficiency -> deletion or mutation

Question 11

Describe Burkitt’s Lymphoma

Answer 11

• Translocation of the protocol-oncogene (c-myc) from 1 chromosome to another
• transcription factor

Question 12

Describe Fluorescence in situ Hybridisation (FISH)

Answer 12

• Lab technique which is used to visualise where a particular gene or DNA sequence is located within a person’s genome

Question 13

What does Fluoresence in situ Hybridisation enable clinical scientists to do?

Answer 13

• scientists can check for specific chromosomal alterations which may cause a genetic condition

Question 14

What cells are needed for FISH ?

Answer 14

• Samples of blood, chorionic villi or other materials containing cells
• dividing or non-dividing cells

Question 15

Describe the use of DNA probes in FISH

Answer 15

• FISH uses a fluorescently labelled hybridisation probe which will attach to the exact part of the DNA to be visualised
• hybridisation probe is a short fragment of DNA that has fluorescent dye attached

Question 16

What does SKY refer to ?

Answer 16

• refers to hybridisation of 24 differentially labelled, chromosome painting probes
• this allows simultaneous visualisation of all human chromosomes using spectral imaging

Question 17

Describe how the MCF-7 Cell Line was derived

Answer 17

• MCF-7 = breast cancer cell line
• derived from a 69 yr old caucasian woman
• nodules in chest wall were removed, a pleural effusion was discovered –> MCF-7 derived from here

Question 18

Describe the original karyotype of MCF-7

Answer 18

• cell line displayed 85 chromosomes, compared to the typical 46

Question 19

What is a microtome ?

Answer 19

• a device which cuts extremely thin slices of material

Question 20

What is the purpose of fixation?

Answer 20

• preserves tissues permanently in as life-like a state as possible
• fixing agents include = aldehydes, alcohols, mercurials

Question 21

What is the typical thickness of a tissue sample?

Answer 21

• 2-4 micrometers
• cold wax improves thin sections though freezing can cause cracking

Question 22

Describe Giemsa Staining

Answer 22

• usually used for staining blood & bone marrow smears
• nuclei = dark-blue/ violet
• cytoplasm = pale blue
• erythrocytes = pale pink

Question 23

Describe OCT Embedding

Answer 23

• Optimal Cutting Temperature medium
• tissue embedded in OCT
• Flash freezing
• DNA, RNA & Protein integrity maintained
• allows sectioning of frozen tissue
• sensitive for bublles

Question 24

What are the advantages of Laser Capture Microdissection ?

Answer 24

• avoidance of surrounding tissue (normal)
• works on even the tiniest tumours
• allows collection of all tumour cells in one tube

Question 25

What further testing can be carried out on captured cells ?

Answer 25

- DNA Isolation = PCR, microarrays - RNA Isolation = cDNA conversion & gene expression analysis - Whole genome amplification = limited number of cells, even just one -> regneration of entire genome

Question 26

What are some advantages of tissue microarrays ?

Answer 26

- real data compared to cell lines & other models - high-throughput technology - testing of a candidate biomarker/drug using thousands of tumour samples

Question 27

How can tissue samples be collected?

Answer 27

- Biopsy - Surgery - Autopsy

Question 28

What regulates tissue collection & sampling ?

Answer 28

Human Tissue Act (HTA, 2004)

Question 29

What are some advantages of Tissue Microarrays ?

Answer 29

- Tissue in diagnostic blocks can be associated with clinical data, including response to treatment, clincial outcome & diagnosis - Uni hospitals can provide tumour tissue microarrays for drug companies

Question 30

Describe how Tissue Microarrays are prepared

Answer 30

- paraffin embedded in tissue block - 1 slide needs to be H&E stained - array mould - paraffin heated & melted to liquid - stylet device