This document discusses liquid biopsy, a non-invasive technique to detect tumor biomarkers shed into body fluids like blood. It can identify circulating tumor cells, circulating tumor DNA, exosomes, and newly, tumor educated platelets. Liquid biopsy offers advantages over tissue biopsy like being less invasive, allowing repeated sampling over time. While sensitivity remains a limitation, liquid biopsy shows promise for early cancer detection, monitoring treatment response and tumor evolution, and assessing tumor heterogeneity. The document reviews technologies to analyze various biomarkers and potential clinical applications of liquid biopsy.

1. Dr Kusuma K N MD,DNB

2. CONTENTS
 Introduction
 Definition
 What comes under liquid biopsy
 Indication
 Advantages
 Limitations
 Summary
 References

3. INTRODUCTION
 With high morbidity and mortality, cancer has
become a big health threat problem around the
world .
 Two main techniques of tumor diagnosis
 Medical imaging
 ultrasonic testing, X-ray , CT, MRI, PETCT and endoscope
 Pathology
 tissue biopsies, serological indicators (eg. CEA and PSA)
test and molecular pathology test (eg. FISH, RT-PCR and
NGS)

4. LIMITATIONS
 Cancer is a heterogeneous disease
 Molecular properties differ within a tumour
 Primary tumour biopsy may not reflect current disease
condition
 Therapy causes changes in tumour cells
 Biopsy
 invasive test
 High cost for procedure
 May not be feasible to do in all patients
 Impractical for periodic monitoring for progression/
recurrence

5. New diagnostic test which address these
limitation
 simple, non-invasive
 Allows early disease detection
 Allows real time evaluation of
 metastasis
 actual treatment response
 Assessment of tumour heterogeneity and monitoring
of tumour dynamics
 Can be cheaper and faster than classical biopsy
testing

6. LIQUID BIOPSY
 Non-invasive tests that detect tumor biomarkers
that are shed into the body fluid from the tumor.
 Samples included
 Blood
 Saliva
 Urine
 CSF

8. WHAT COMES UNDER LIQUID BIOPSY
Circulating
tumour cells
Circulating
tumour DNA
Exosomes
New addition :
Tumour
educated
platelets

9. CIRCULATING TUMOR CELLS (CTCS)
• Carries phenotypic and genotypic information of tumor
 1869 Ashworth : first found and named CTC
 2002 and 2003 Thiery and Steeg : found the
formation mechanism of CTC.
 2007  CTC has been recommended as new tumor
biomarker by American Society of Clinical Oncology
(ASCO)

10. SAMPLE COLLECTION
 Cell save preservative tube
 made of negatively charged materials
 contain stabilizing reagents
 preventing blood cell breakdown for a period of
about 5 days

11. TECHNOLOGIES TO DETECT AND CAPTURE
CTCS:
Biological property /
EpCAM-affinity
based:
• CellSearch® system
• AdnaTest Breast
CancerDetect
• CTC-Chip
• MACS® (magnetic
activated cell
sorting system)
• Mag Sweeper
Physical properties-
based:
• ISET (isolation by
size of epithelial
tumor cells)
• ScreenCell®
• ApoStream™
• Density gradient
centrifugation
Other methods:
• FAST (fiber-optic
array scanning
technology)
• EPISPOT (epithelial
immunospot)
• FACS (flow
cytometry)
• PRO Onc Assay

15. ADVANTAGES
 CTC Count: significantly correlate with prognosis
 Allow both phenotypic and genotypic analysis
 Potential relation with the tumor progression and
metastasis
 Various technologies for CTC enrichment or isolation
 High specificity
 Allow culturing and analysis in vitro

16. DISADVANTAGES
 Low concentration
 1-10cells per 10 ml peripheral blood
 fragility of cells
 False negative and false positive results
 Cannot decrease the diagnosis bias from tumor
heterogeneity

17. Complex heterogeneity
 morphology of CTCs derived from different tumor
tissues through EMT is significantly different.
 even within only one cancer, the morphology and
amount of CTCs derived from different molecular
subtypes of solid tumors or distant sites are
distinct
 eg. prostate primary and bone metastatic cancers
 the percentage of patients whose CTCs can be
detected is also different between cancers
 colorectal, ovarian and breast cancer : 50-70%
 non-small cell lung cancer is only 30%.

18. inter- and intra-patient heterogeneity
Misses some small subclones of tumor cells like tissue
biopsy.
lead to diagnosis bias

19. CIRCULATING DNA’S
 Releases by both
healthy and tumour
cells
 Released by solid
tumor
 Have cancer-
associated genetic
mutations:
 Half life is less than 2
hrs
 Percentage of ctDNA
is very low (0.01%-1%)
Cell free DNA: cfDNA Circulating tumor DNA:
ctDNA

21.  High sensitivity
 Allow analysis of DNA
sequence and methylation,
including PCR and NGS
 Can decrease the diagnosis
bias from tumor
heterogeneity
 Timely and dynamically
monitor tumor progression
 ctDNA is unstable
 requires fast processing
 low specificity because of
cfDNA from normal tissue
 False positive and false
negative results
Advantages
limitations

23. EXOSOMES
 Small (50–100nm in diameter)
 lipid bilayer membrane vesicles of endocytic origin.
 play a central role in cell-to-cell communication

25.  Role in tumour
 Initiation
 Progression
 Metastasis
 drug-resistance
 Based on these results, researchers have
developed some exosomes targeted drugs and
tried to use exosomes for drug delivery

26.  Enrichment methods : similar to those of CTC.
 Analysis regular biological technologies, such
as electron microscope, RT-PCR, western blot,
FISH, flow cytometry and NGS

27.  Allow analysis of
DNA, RNA and
proteins from solid
tumor
 Potential relation with
the tumor drug-
resistance and
metastasis
 Lacking effective
enrichment method
Advantages Limitation

28. New addition in liquid
biopsy

29. TUMOUR EDUCATED PLATELATES
 Besides tumor cells and their products, normal
cells present in the tumor microenvironment are
also released into the blood stream.
 harbor important information about the tumor
 platelets have been studied extensively and gave
promising results

30.  interaction between blood platelets and tumor
cells
 not only affects the expression of relevant genes in
tumor cells, but also alters the RNA profile of blood
platelets
 mRNA sequencing of TEPs can be done

32. INDICATIONS
 To monitor
 residual disease
 Treatment response
 disease progression and tumor evolution (i.e. development
of tumor resistance).
 Helps to explore other options of treatment when the patient
is resistant to current therapies.
 Provide an alternative method for biopsy when
 tissue is difficult to obtain or not available
 primary site of metastatic disease is unknown.
 Limited quantity of tissue and traditional molecular
genotyping is requested.
 • Provide prognostic information.

33. ADVANTAGES
 Noninvasively take repeated tumor samples
 Fewer side effects
 Rapid testing speed
 Decreasing the diagnosis bias from tumor
heterogeneity
 Dynamically reflect the tumor progression

34. LIMITATIONS
 lack of standardization of techniques.
 sensitivity and specificity and current detection
technologies need to be improved
 sample size in most of current researches is small

35.  low concentration of CTC, ctDNA and exosomes
 difficult to detect gene mutations, amplifications
and fusions in parallel
 NGS can address these limitation
 However, cannot be used in general
 High cost
 requirement for high-quality DNA (not
characteristic of cfDNA)
 extensive data analysis requiring a dedicated
bioinformatician

36. CONCLUSION

38. REFERENCES

40.  DNA Liquid Biopsy Metastasis Test
 EGFR KRAS BRAF genes
 DNA Liquid Biopsy Lung Panel Metastasis Test
 BRAF, EGFR, ERBB2, KRAS, and PIK3CA genes

 DNA Liquid Biopsy Colon Cancer Metastasis Test
 KRAS, NRAS, EGFR, and PIK3CA genes