Epigenetics and circulating biomarkers Flashcards
Integrated genomic analysis (4)
DNA methylation
DNA sequence
Copy number analysis
Rna expression
Promoter and methylation
Promoter hypermethylation is associated with reduced transcription
GENE body and methylation
GENE body hypermethylation is associated with increased transcription
Epigenetic dysregulation in CRC
Global DNA methylation changes
CIMP
SEPT9
CIMP
Present in 20% of CRC
Negative prognostic marker
Affects response to treatment- 5-FU CIMP high has better response
How is SEPT9 methylation detected?
Circulating DNAm biomarker
SEPT 9- why is it circulating?
In early stages of CRC, SEPT9 is hypermethylated and released into circulation by apoptotic cells
What is SEPT 9 (protein)
SEPTIN 9 is a GTP binding protein
It is a scaffolding protein - structural support
Physiological role of SEPT9
Actin dynamics, angiogenesis, motility, proliferation
SEPT9 in CRC
Underexpressed in CRC
5 challenges of using cfDNA biomarkers
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
What are miRNAs
Small non coding RNA
Function in RNA silencing and post transcriptional regulation of gene expression
What is the mechanism of miRNAs? 4
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
4 types of circulating biomarkers
Circulating tumour cells
Cell feee DNA
MicroRNAs
Exosomes
3 ways to methodologically isolate CTCs:
Antibody based capture
Physical characteristics
CD45 depletion and culture
How to capture CTCs with antibodies?
EPCAM AND MUC1
How to capture CTCs by their physical characteristics
Size filtration
Flow dynamics
Membrane capacitance
What is CellSearch?
EPCAM based selection
How to capture cells with EPCAM?
Use ferrofluid conjugated EPCAM Ab
How to capture CTCs by CK?
PE-conjugated CK antibody
How to capture by CD45?
APC Conjugated CD45 Ab
What are the therapeutic options for neuroendocrine tumours? 5
Somatostatin analogies Chemo Targeted radiotherapy TACE Radioembolisation
Applications of CTCs:
5
See response to treatment Drug target expression Pharmacodynamic marker Genetic heterogeneity Biology of metastases
Steps for whole genome amplification of DNA from a single CTC? 7
Proteinase k digests single cell Preannealing of adaptors Ligation Removal of unligated small primers Filling reaction at 3' Primary PCR amplification
Concentration of cell free DNA in blood
1000-2000 copies of genome per ml
Cell free DNA half life
1 hour
Cell free DNA pro and con
Good response marker
Tumour must be of a certain size
Optimal ctDNA collection
If blood is collected into EDTA tubes it should be centrifuges within a short time frame
Alternatively use a stabilisation tube
Top 5 cancers with ctDNA:
Bladder Colorectal Gastric Ovarian Pancreatic
2 ways to use ctDNA
Detecting and tracking single mutations
Sequencing ctDNA to detect mutations
Why sequence a tumour scone from plasma?
Can’t always get to tumour tissue
To understand tumour relapse
6 ways to use ctDNA as a biomarker
Early detection Molecular stratification Treatment response Residual disease Tumour evolution Differing treatment response
What biofluids can cancer DNA be detected in?
Blood plasma Csf Cyst fluid Urine Stool
Cancer DNA detection in oesophageal/ gastric cancer:
Cystosponges
What cancer is already being stratified by ctDNA
Lung
Ctc > ctDNA (4)
Validated prognostic marker
Heterogeneity at single cell level
Look at rna and protein expression
Study cancer biology
CtDNA>CTCs
More sensitive
Bigger dynamic range
Cheaper
