4. INTRODUCTION
• Cancer is a heterogenous disease.
Molecular properties differ within a tumour
Primary tumour biopsy may not reflect current
disease condition.
Therapy causes changes in tumour cells.
• Tumour releases a multitude of biomarkers into
blood.
• Liquid biopsy is a minimally invasive alternative
to surgical biopsy for tumour molecular profiling.
5. DEFINITION
• Liquid biopsy (blood based biomarkers) are
non invasive blood tests that detect:-
Circulating tumour cells(CTCs)
Fragments of tumour DNA(ctCNA)/cell free
DNA(cfDNA)
Exosomes and microvesicles
that are shed into blood from primary tumour
and from metastatic sites.
• Tumour educated platelets(TEPs)
11. • 1869 – T.R.Ashworth first described the
presence of epithelial cells in the blood of a
woman with metastatic breast cancer that were
similar in appearance to her primary tumour
cells.
• 2002 and 2003 – Thiery and Steeg : Found the
formation mechanism of CTC.
• 2007 – CTC has been recommended as new
tumour biomarker by ASCO
12. CTCs
• Represent the clones within the tumour that
has the metastatic potential.
• CTCs represent a rare cell population - less
than 10 cells/mL.
• Number of cells reflect the stage of the tumour
• Sample collection – Cell save preservative
tube
18. CTCs
ADVANTAGES
• Minimally invasive
prognostic marker
• Both phenotypic and
genotypic analysis
• Potential relation with
tumour progression and
metastasis
• High specificity
• Availability of FDA approved
method for isolation
DISADVANTAGES
• Low sensitivity in low
volume diseases
• Low concentration
• Fragility of cells
• Presence of benign
circulating epithelial cells
• Cannot decrease the
diagnosis bias from tumour
heterogeneity
19. Complex heterogeneity
• Morphology of CTCs derived from different tumour
tissues through EMT is sigificantly different.
• Even within only one cancer,the morphology and
amount of CTCs derived from different molecular
subtypes of solid tumour or distant sites are distinct.
• The percentage of patients whose CTCs can be detected
is also different between cancers.
Eg: Colorectal,ovarian and breast cancer : 50-70%
Non small cell lung cancer is only 30%
20.
21.
22.
23.
24. ctDNA
ADVANTAGES
• High sensitivity
• Allow analysis of DNA
sequence and
methylation,including PCR
and NGS
• Can decrease the diagnosis
bias from tumour
heterogeneity
• Timely and dynamically
monitortumour progression
LIMITATIONS
• ctDNA is unstable , so
requires fast processing
• Low specificity because of
cfDNA from normal tissue
• False positive and false
negative results
25.
26. • Role in tumour initiation,progression,metastasis
and drug resistance
• Based on these results,researchers have
developed some exosomes targeted drugs and
tried to use exosomes for drug delivery.
• Enrichment methods: Similar to those of CTC
• Analysis – Electron microscope,RT-PCR,Western
blot,FISH,Flow cytometry and NGS.
27. EXOSOMES
ADVANTAGES
• Allow analysis of
DNA,RNA and proteins
from solid tumour
• Potential relation with the
tumour drug-resistance and
metastasis
LIMITATIONS
• Lacking effective
enrichment method
28. TUMOUR EDUCATED PLATELETS
• Besides tumour cells and their products,normal
cells present in the tumour microenvironment
are also released into the blood stream.
• They harbour important information about the
tumour.
• Platelets have been studied extensively and
gave promising results.
29. • Interaction between blood platelets and tumour
cells:
Not only affects the expression of relevant
genes in the tumour cells,but also alters the
RNA profile of blood platelets.
• mRNA sequencing of TEPs can be done and
can be used to distinguish from platelets of
healthy individuals.
30.
31. ADVANTAGES
• Non invasively take repeated tumour samples.
• Fewer side effects
• Rapid testing speed
• Decreasing the diagnosis bias from tumour
heterogeneity.
• Dynamically reflect the tumour progression.
32. LIMITATIONS
• Lack of standardisation of techniques
• Sensitivity and specificity and current detection
technolgies need to be improved
• Low concentration of CTC,ctDNA and exosomes
• However cannot be used in general:
High cost
Requirement for high quality DNA(not
characteristic of cfDNA)
Extensive data analysis requiring a dedicated
bioinformation
33.
34. IS THIS THE END OF TISSUE BIOPSIES?
• Tissue biopsy will remain as the gold standard.
• Liquid biopsies are recommended when tissue
biopsy is difficult,or when primary site of
disease unknown
• Help patients get the right treatment.
35. REFERENCES
• Liquid biospsy in cancer patients – The hand lens
for tumour evaluation Antonio Russo,Antonio
Giordano,Christian Rolfo
• Current and future applications of liquid biopsy in
non small cell lung cancer from early to advanced
stages Nicolas Guibert,Anne Pradines et al
European respiratory Review 2020 29
• Liquid biopsy of cancer:a multiodal diagnostic
toolin clinical oncology Raffaele palmirotta et al
Ther Adv Med Oncol.2018;10
Editor's Notes
Tumour biomarkers in blood:-cell free dna
Circulating tumour cells
Exosomes and microvesicles
Cells that carry phenotypic and genotypic information of the tumour.1 million WBCs/mL Appearnce in blood- requirement for metastasis.
Low- low stage tumours
Advanced tumour- more
Detection of CTCS within a routine blood specimen provides an opportunity to monitor cancer non invasively,that is what occurs in liquid biopsy.
Made of negatively charged materials
Contain stabilizing reagents
Preventing blood cell breakdown for a period of about 5 days
Cell search system –FDA approved
Real time quantitative PCR assay
Multifluroscent immunostaining of peripheral blood mononuclear cells
It uses ferrofluid nanoparticles with antibodies against the epithelial adhesion molecule (EpCAM), thus separating epithelial cells from majority of blood cells.
Then staining of cells with specific antibodies for CK, CD45, nuclear dye and visualisation under microscopy enables the identification of CTCs.
Therapeutic management
Epithelial to mesenchymal transition
Inter and Intra – patient heterogeneity
Misses some small subclones of tumour cells like tissue biopsy
Leads to diagnosis bias
Eg 1 –prostate primary and bone metastatic tumours
Small – 30-100nm in diameter
Formed by budding of plasma membrane,lipid bilayer membrane vesicles of endocytic origin
Difficult to detect gene mutations,amplifications and fusions in parallel
NGS can address these limitations
Lung cancer.It has a powerful role in helping patients get to the right treatment. Introduction of Liquid biopsy will enable oncologists to use drugs intelligently to combat changes in individual cancers as they happen.