Epigenetics and circulating biomarkers

Question 1

Integrated genomic analysis (4)

Answer 1

DNA methylation
DNA sequence
Copy number analysis
Rna expression

Question 2

Promoter and methylation

Answer 2

Promoter hypermethylation is associated with reduced transcription

Question 3

GENE body and methylation

Answer 3

GENE body hypermethylation is associated with increased transcription

Question 4

Epigenetic dysregulation in CRC

Answer 4

Global DNA methylation changes
CIMP
SEPT9

Question 5

CIMP

Answer 5

Present in 20% of CRC
Negative prognostic marker
Affects response to treatment- 5-FU CIMP high has better response

Question 6

How is SEPT9 methylation detected?

Answer 6

Circulating DNAm biomarker

Question 7

SEPT 9- why is it circulating?

Answer 7

In early stages of CRC, SEPT9 is hypermethylated and released into circulation by apoptotic cells

Question 8

What is SEPT 9 (protein)

Answer 8

SEPTIN 9 is a GTP binding protein

It is a scaffolding protein - structural support

Question 9

Physiological role of SEPT9

Answer 9

Actin dynamics, angiogenesis, motility, proliferation

Question 10

SEPT9 in CRC

Answer 10

Underexpressed in CRC

Question 11

5 challenges of using cfDNA biomarkers

Answer 11

Mutated cfDNA diluted by normal cfDNA (allele frequency)

Duration of marker in circulation

Needs stable methylation pattern and stable levels in circulation

Mutation detection cannot identify tumour location

Requires adequate sensitivity and specificity

Question 12

What are miRNAs

Answer 12

Small non coding RNA

Function in RNA silencing and post transcriptional regulation of gene expression

Question 13

What is the mechanism of miRNAs? 4

Answer 13

Complementary base pairing with mRNA leading to:

Cleavage of mRNA strand into 2 pieces
Destabilisation of mRNA through shortening of the poly(A) tail

Less efficient translation of mRNA into proteins by ribosomes

Question 14

4 types of circulating biomarkers

Answer 14

Circulating tumour cells
Cell feee DNA
MicroRNAs
Exosomes

Question 15

3 ways to methodologically isolate CTCs:

Answer 15

Antibody based capture
Physical characteristics
CD45 depletion and culture

Question 16

How to capture CTCs with antibodies?

Answer 16

EPCAM AND MUC1

Question 17

How to capture CTCs by their physical characteristics

Answer 17

Size filtration
Flow dynamics
Membrane capacitance

Question 18

What is CellSearch?

Answer 18

EPCAM based selection

Question 19

How to capture cells with EPCAM?

Answer 19

Use ferrofluid conjugated EPCAM Ab

Question 20

How to capture CTCs by CK?

Answer 20

PE-conjugated CK antibody

Question 21

How to capture by CD45?

Answer 21

APC Conjugated CD45 Ab

Question 22

What are the therapeutic options for neuroendocrine tumours? 5

Answer 22

Somatostatin analogies
Chemo
Targeted radiotherapy
TACE 
Radioembolisation

Question 23

Applications of CTCs:

5

Answer 23

See response to treatment 
Drug target expression
Pharmacodynamic marker
Genetic heterogeneity
Biology of metastases

Question 24

Steps for whole genome amplification of DNA from a single CTC? 7

Answer 24

Proteinase k digests single cell
Preannealing of adaptors
Ligation
Removal of unligated small primers
Filling reaction at 3'
Primary PCR amplification

Question 25

Concentration of cell free DNA in blood

Answer 25

1000-2000 copies of genome per ml

Question 26

Cell free DNA half life

Answer 26

1 hour

Question 27

Cell free DNA pro and con

Answer 27

Good response marker | Tumour must be of a certain size

Question 28

Optimal ctDNA collection

Answer 28

If blood is collected into EDTA tubes it should be centrifuges within a short time frame Alternatively use a stabilisation tube

Question 29

Top 5 cancers with ctDNA:

Answer 29

``` Bladder Colorectal Gastric Ovarian Pancreatic ```

Question 30

2 ways to use ctDNA

Answer 30

Detecting and tracking single mutations Sequencing ctDNA to detect mutations

Question 31

Why sequence a tumour scone from plasma?

Answer 31

Can't always get to tumour tissue | To understand tumour relapse

Question 32

6 ways to use ctDNA as a biomarker

Answer 32

``` Early detection Molecular stratification Treatment response Residual disease Tumour evolution Differing treatment response ```

Question 33

What biofluids can cancer DNA be detected in?

Answer 33

``` Blood plasma Csf Cyst fluid Urine Stool ```

Question 34

Cancer DNA detection in oesophageal/ gastric cancer:

Answer 34

Cystosponges

Question 35

What cancer is already being stratified by ctDNA

Answer 35

Lung

Question 36

Ctc > ctDNA (4)

Answer 36

Validated prognostic marker Heterogeneity at single cell level Look at rna and protein expression Study cancer biology

Question 37

CtDNA>CTCs

Answer 37

More sensitive Bigger dynamic range Cheaper