Pathology Phase I

Front 1

Describe the macroscopic and microscopic appearance of DCIS.

Back 1

Macroscopic
- may cause extensive lesions throughout the breast, may be multifocal
- characterised by densie fibrosis and inflammation

Microscopic
- Comedocarcinoma - characterised by solid sheets of pelomorphic cells with high grade hyperchromatic nuclei and areas of central necrosis, the membranes commonly calcify and are detected on MMG as clusters of linear branching microcalcifications
- Cribriform - intraepithelial spaces are evenly distributed and regular in shape
- Solid - compeltely fills involved spaces
- Papillary - grows into spaces that lack a normal myoepithelial layer
- Micropapillary - bulbous protrusions with fibrovascular core, arranged in complex intraductal patterns

Front 2

Describe the Microscopic appearnces of prostate cancer.

Back 2

Gleason score grades adenocarcinoma of the prostate from the level of glandular differentiation.
Adenocarcinoma lacks a basal layer in compared to benign glandular tissue.
Grade 1 is composed of well differentiated glands, round nuclei with cells within a well circumscribed nodule.
Grade 5 demonstrates no glandular structures and cells are arranged in cords, nests and sheets.
The remaining grades are inbetween these two extremes

Front 3

Describe the microscopic appearance of Invasive ductal carcinoma.

Back 3

Range from well to poorly differentiated appearance based on the modified Bloom-Richardson-Elston Grading (Nottingham system) which scores tubule formation, mitoses and nuclei appearance.
Microscopic calcifications may also be present.
Macroscopically the lesion appears irregular, with a firm appearance with microscopic calcifications.

Front 4

Describe the microscopic appearance of Invasive lobular carcinoma of the breast.

Back 4

Dyscohesive cells arranged in single file or in loose clusters or sheets.
Signet ring cells producing mucin may also be present

Front 5

Describe the differences between benign and malignant tumours.

Back 5

Differentiation
Nuclear atypia/pleiomorphism, including hyperchromatic and chromatin clumping
Nuclear:cytoplasmic ratio
Mitoses
Polarity
Invasion/metastases

Front 6

Describe the basic steps of gene expression.

Back 6

Transcription
Post transcriptional modification
mRNA export
Translation
Post Translation modification
Protein folding
Protein export

Front 7

What are the steps of transcription.

Back 7

Pre-initiation: pre-initiation complex binds to core promoter (i.e. TATA box). Complex is made of transcirption factors, RNA polymerase and activators/repressors
2. Intiation: initiation complex finalised with further binding and stabilisation of complex including TF's, RNA pol
3. Promoter Clearance. RNA polymerase must clear promoter prior to transcription
4. Elongation. DNA template frame is read and complementary mRNA strand produced
5. Termination. Complex process in eukaryotes which is poorly understood.

Front 8

What are the steps of translation?

Back 8

1. Activation. tRNA binds correct amino acid that codon codes for.
2. Initiation. Ribosome facilitates binding of correct amino acid.
3. Elongation
4. Termination. Stop codon.

Front 9

What is the role of the Endoplasmic reticulum?

Back 9

2 types, 3 subtypes.
Rough ER - protein synthesis.
Smooth ER - synthesize steroid and lipids; metabolise steroids and carbohydrates, regulate caclium concentration, drug metabolism and attachment of membrane receptors
Sarcoplasmic reticulum - is a type of smooth ER which solely regulates caclium levels in smooth and striated muscle

Front 10

What is the role of the Golgi apparatus?

Back 10

Forms part of the endonuclear membrane system. Packages macromolecules prior to the secretion.

Front 11

What are the steps in RT-PCR.

Back 11

1. Reverse transcriptase
- pairs of primers (complementary to cDNA) are transtribed to cDNA using the enzyme reverse transciptase
- can either by one step (in same tube) or two step (different tubes) to PCR using temperature of 40-50 degrees
2. Denaturing. Protein is heating to 95 degrees so that the strands can separate and primers can bind at lower temperatures. Temperature is lowered until annealing temperature reached.
3. Amplification. Taq polymerase (thermostable) is used to amplify.

Types of PCR
Conventional - uses agarose gel electrophoresis and nucleic acid staining
Real time - flouroscent phosphor reporter molecule used; thermal cycler has a sluorescent detection threshold, which is reached a fter a number of cycles proportional to the concentration of DNA in the original sample

Front 12

Describe the initiator-promoter sequence of carcinogenesis.

Back 12

Initiator - irreversible (if not repaired and passed on) DNA damage, but which it self is not enough to cause carcinogenesis i.e. cyclophosphamide, benzopyrene
Promoter - not carcinogenic itself, but can cause increased proliferation of cells. i.e. oestrogens

Front 13

How does HPV cause carcinogenesis.

Back 13

Complex.
HPV DNA is incorporated into host genome.
HPV genes are transcribed which includes the proteins E6 and E7.
E6 is belived to inhibit p53, and hence promote cell survival and cell cycle progression.
E7 is believed to inhbit p53 as well as the CDK inhibitors, p21 and p27, as well as sequester Rb protein leaving E2F to transcribe S phase proteins (promoting cell cycle progression as G1/s phase). It may also directly activate cyclins.

Front 14

How is EBV implicated in carcinogenesis?

Back 14

EBV is incorporated in host genome and transcribed proteins believed to mediate oncogenic potential. It is most strongly implicated in NPC, African Burkitt's, Other lymphomas (including NK and T cell lymphomas).
LMP-1 - inhibits apoptosis by activating BCL2 and promtoes B cell survival and proliferation by NF-KB and JAK-stat pathway (acting as a constitutively active CD40 receptor, requiring no helper T cell signal)
EBNA - mimics notch receptor.

Front 15

List viruses with known oncogenic potential.

Back 15

HPV
EBV
HTLV-1
Polyoma virus
HCV/HBV
HHV-8

Front 16

What is the mechanism of HCV/HBV induced carcinogenesis?

Back 16

Believed to be as a result of chronic inflammation.
However there are oncoproteins coded for in the viral genome, but there has been no consistent findings to support their role.

Front 17

What is sole bacteria linked to malignant transformation and what cancers does it cause?

Back 17

Helicobacter pylori.
Gastric adenocarcinoma and Gastric lymphoma.
Believed to be as a result of chronic inflammation, although the genome does code for stome potentially oncogenic proteins.

Front 18

What are the hallmarks of cancer?

Back 18

Initially 6 (following DNA damage and failure of repair)
1. Self sufficiency in growth signals
2. Insensitivity to growth inhibitory signals
3. Evasion of apoptosis
4. Invasion/Metastasis
5. Sustained angiogenesis
6. Limitless replicative potential
in 2010, the following were added
7. Deranged metabolism
8. Inflammation
9. Evasion of immune system
10. Unstable DNA

Front 19

What are the steps of malignant invasion?

Back 19

1. Loosening of cell-cell contacts
- E-cadherin mediated, connected to catenin which is connected to cytoskeleton, loss of connection usually results in apoptosis
2. Degradation of ECM
- Matrix metalloproteinases are the most important. Degrade matrix, but also release ECM-bound growth factors such as VEGF
Ameboid migration results when tumours squeeze through spaces in ECM which is much quicker, rather than cutting throught the ECM
3. Attachment to Novel ECM components
4. Locomotion
- complex interaction of binding to ECM elements and contraction of the actin cytoskeleton elements

Front 20

What are the steps in the metastatic cascade?

Back 20

1. Malignant transformation
2. Clonal expansion and growth
3. Angiogenesis
4. Attachment and invasion through the BM and ECM
5. Extravasation
6. Tumour emboli and interaction with lymphoid cells and platelets
7. Extravasation/binding to cellular receptors
8. Tumour deposit
9. Angiogenesis
10. Growth

Front 21

What is cetuximab.

Back 21

Monoclonal antibody which attaches to EGFR (HER1, erbB1) receptor preventing dimerisation.

Front 22

How does trastuzumab work?

Back 22

Humanised monoclonal antibody which binds to extracellular HER2 receptor (there is no known natural occuring ligand for this receptor).
Activation prevents dimerisation of the HER2 receptor (in overexpression no signal is required and the HER2 homodimerises).
The mechansim of activation is believed to be:
1. Inhibition of growth/self sufficiency signals by preventing dimerisation and TK cytoplasmic activity
2. Immune reponse to the Fc component of herceptin may mediate some effects

Front 23

How does HER2 work?

Back 23

HER2 has no known naturally occuring ligand.
HER2 dimerises without members of the EGFR family (HER1,3 and 4). Resulting in cytoplasmic TK activity. This results in progrowth signals via the MAPK pathway (RAS-RAF-MEK-ERK) to the nucleus and also cell survival signals via the PI3K/AKT pathway.
In breast cancer HER2 may be overexpressed which results in self dimerisation without (no ligand) and activation of its cytoplasmic TK domain.

Front 24

What is the RAS gene?

Back 24

The RAS gene encodes for the RAS protein which is one of the most common mutations in human malignancy.
There are three variants HRAS (bladder), NRAS (haematological) KRAS (pancreatic, colon) associated with human malignancy.
RAS is bound to membrane (and undergoes posttranslation modification during this anchoring) to farnesyl transferase.
RAS protein is a small protein GTPase linked to a membrane bound signalling protein (such as EGFR) and connected via bridging protein. Unless activated it is in the 'off state' where it is bound to GDP. When the extracellular ligand binds to the receptor (which in the case of EGF dimerises) this causes activation of the RAS which via GEF becomes bound to GTP. This also binding of GAP and RAF.
RAF (MARP3K) is part of the MAPK pathway which activates MEK (MAP2K) and ERK (MAPK) which translocates to the nucleus to activate transcription factors.

Front 25

What is BRAF?

Back 25

BRAF is a member of the RAF family (which include RAF-1, A-RAF, C-RAF) and it is a kinase associated with the MAPK pathway.
The most commmon mutiation is of V600E, followed by V600K. It has been heavily associated with melanoma particularly in younger patients with truncal melanomas, particularly in the absencve of chronic skin damage.

Front 26

What syndrome are changes in the APC gene observed in?

Back 26

Familial Adenomatous polyposis.
Develop thousands of polyps in the large bowel, of which one will invariably develop into malignancy.
Both copies of teh APC gene must be mutated.
NB in comparison to HNPCC
APC is part of the WNT singalling pathway, involved in cell adhesion and polarity. WNT signals through frizzled receptors and stimulates sever

Front 27

What syndrome are changes in the APC gene observed in?

Back 27

Familial Adenomatous polyposis.
Develop thousands of polyps in the large bowel, of which one will invariably develop into malignancy.
Both copies of teh APC gene must be mutated.
NB in comparison to HNPCC
APC is part of the WNT singalling pathway, involved in cell adhesion and polarity. WNT signals through frizzled receptors and stimulates sever

Front 28

How do tumour cells evade apoptosis?

Back 28

1. Extrinsic
- alter receptor in a number of ways i.e. decreased affinity for FasL
2. Intrinsic
- change intrinsic balance between pro (Bax/Bak) and anti (BCL2, BCL-XL) apoptotic molecules

Best example is off t14:18 translocation in B lymphomas where BCL2 locus is translocated and result is overexpression and protection from apoptosis
p53 mutations

Front 29

What are telomeres

Back 29

Telomeres are the protective caps on the ends of chromsomes, they consist of long arrays of repeated TTAGGG that range in length from 1.5 to 150kb.
They have been termed the aging moleculear clock as they shorten with each division and are thought to lead to about 40-60 possible divisions for somatic cells.
Stem cells counteract this by regenerating the repeats after each division with teh enzyme telomerase, a reverse trnasciptase.
The proposed mechanism of cell death following this is p53 dependent and p53 deficient cells undergo accelerated tumorigenesis, presumably from increased chromosomeal instability and rearrangments and gene amplifications.

Front 30

List 5 DNA repair defects or genomic instability syndromes which result in increased risk of malignancy.

Back 30

1. HNPCC
- mismatch repair; at least 4 genes, MSH2, MLH1 most significant
2. Xeroderma Pigmentosa
- defect in NER
3. ATM
4. Fanconi Anaemia
- complex interactions, 13 genes which code, defect in HR, as protein complex relocates to nucleus to modulate repair (in particular BRCA function)
5. Bloom syndrome
- loss of BLM gene which codes for a helicase
6. BRCA 1 and 2 mutations.

Front 31

Describe the function of BRCA1 and clincial effects.

Back 31

BRCA1 has a number of functions, with its associated with BARD1, RAD51, CHK1 and BRCA2 including
- involved in HR
- cell cycle checkpoints (in particular G2 and S phase checkpoints)
- activation of DNA damage response

Clinical effects
Increased risk of breast and ovarian cancer in females. Also shown increased risk of cervix and uterine cancer.
Males - increased risk of prostate and breast.
Increased risk of pancreatic cancer.

Front 32

Describe the function of BRCA1 and clincial effects.

Back 32

BRCA2 is involved in HR pathway of dsDNA repair.
It binds to RAD51, a recombinase and promotes its assembly onto the ssDNA strand
May also participate in S checkpoint activation.

Male - breast and prostate
Female - breast and ovarian.
Also implicated in melanoma, pancreas and stomach (colon cancer has not been consistently shown )

Front 33

What are the steps of angiogenesis?

Back 33

1. Increased permeability (NO/VEGF)
2. BM degradation
3. Migration of endothelial cells towards stimulus
4. Proliferation of endothelial cells
5. Maturation of endothelial cells
6. Recruitment of periendothelial cells

Front 34

What important molecules are involved in the angiogenic switch?

Back 34

Pro
- HIF
- VEGF
- VEGFR2
- NOtch signalling
- bFGF

Anti
- p53
- VHL

Front 35

What is bevacizumab?

Back 35

A humanized monoclonal antibody that reconzes VEGF-A isoforms preventing its binding to the VEGFR.

Front 36

How does a cell achieve limitless replicative potential? What are the mechanisms?

Back 36

Normal somatic cells have about 60-70 doublings before entering senescence as a result of progressive telomerase shortening. Tumours needs to counteract this.
As cells lose their telomeres they enter a period of high genomic instability, where numerous mutations may accumulate.

2 methods:
1. Telomerase activation
- seen in almost all human tumours. Increase length of telomeres which are TTAGGG repeats (normally 1.5 to 150kb long)
2. Alternative lengthening of telomerase
- likely depends on DNA recominbation

Front 37

What is the underlying defect in HNPCC?

Back 37

defect in mismatch repair genes. Most commonly MSH2 and MLH1
microsatellite instability result in regular repeated appearance of microsatellite DNA lesions

Front 38

What is Bloom syndrome?

Back 38

Genetic disorder where the undelrying deficit is in the BLM gene which codes for a helicase.

Front 39

What cancers are assoicated with BRCA1?

Back 39

Male - breast and prosate
Female - breast, ovary, cervix and uterus
Both - pancreas

Front 40

What cancers are associated with BRCA2?

Back 40

Male - breast and prosate
Female - breast, ovary,
Both - pancreas, gall bladder, stomach and melanoma

Front 41

How does a tumour interact with the stromal microenvironment?

Back 41

Complex interplay of chemical messenging involving inflammatory cells, ECM and stored growth factors within the matrix.
- inflammatory cells secrete pro surivival and progrowth cytokines
- marcophages may be involved in the development of metastases in a non understood method
Fibroblasts - grwoth factors, desmoplasia
ECM - stored matrix compoentns that provmeote growth and angiogenesis, as well as growth factors

Front 42

What are two methods of gene amplification.

Back 42

1. Double minutes - extrachromosomal DNA which harbours oncogenes
2. Homogenously staning regions which are increased intrachromsomal expression of genes. A secgment of a chromosome may be amplified or copied many times

Front 43

How doe chromosomal rearrangements result in malignancy?

Back 43

1. Translocations and inversion
-formation of novel gene products with progrwth function i.e. T9:22 BCR:ABL wihcih has constitutive tyrosine kinase activity
- overexpression as a result of translocation of regulatory elements i.e. t8:14 in Burkitt's (myc overexpression)

2. Deletions
- loss of genetic material with loss of function i.e. TSG

Front 44

What are epigenetic changes? List the processes that lead this change of gene expression.

Back 44

Epigenetic changes are reversible, heritable changes in gene expression, but occur without mutation in the underlying DNA sequence.

Examples:
1. DNA methylation
- genetic instability is characterised by hypomethylation
- methylation of the core promoter of a TSG results in decreased transcription
2. Genomic imprinting/reversal
3. Histone acetylation/methylation
- acetylation results in increased transcription
4. Non coding RNA
- miRNAs mediate post transicptional gene silencing, and have been implicated in teh expression of oncogenes and the repression of TSG