Liquid biopsy: Tumor diagnosis and
treatment monitoring in a blood test
Radmila Jankovic, PhD
Institute for Oncology and Radiology of Serbia
Term “Liquid biopsy” stands for:
Circulating tumor cells (CTCs)
Cell-free DNA (cfDNA)
Plasma
Serum
Other liquid material
Exosomes
etiology
disseminated cancer cells
cancer stem cells
rare cells in a dormant, nonproliferative state
unaffected by chemotherapy
unrecognized by the host immune system
difficult to isolate
advanced cancer has less then 10 CTCs per 7.5ml of blood
Circulating tumor cells (CTCs)
Circulating Tumor Cell
CK
EpCAM
Nucleus DAPI
Anti- CK-PE
Anti- EpCAM Ferrofluid
Immunomagnetic Capture
Leukocyte
CD45 Nucleus DAPI
Anti - CD45-APC
DAPI cytokeratin control CD45 composite
CellSearch™ System
p. 7
Individual Cell Sorting with DEPArray™
Main
chamber
Parking Recovery
1. Inject, trap and image all cells
2. Move all cells of interest into
Parking chamber
3. Move separately to Recovery
chamber and flush
Features Multiparametric image-based
sorting Single cell resolution Small cell loads Gentle on cells No a priori thresholds choose
best cells Sorting based on slow kinetics
(10-100s minutes) possible
In vivo isolation of circulating tumor cells from
peripheral blood of cancer patients
Saucedo-Zeni N et al, Int J Oncol. 2012 Oct; 41(4): 1241–1250.
Microtube surface is coated with both E-selectin and anti-
epithelial antibodies (EpCAM or PSMA)
Halloysite NanotubeProtein G
E-Selectin-Fc Chimera
Anti-EpCAM/PSMA mAb
Clinical relevance of CTCs in breast
cancer
Applications
Prognosis
Monitor Metastasis
Drug Activity
Cell culture Xenograft
Model
Approaches
Enumeration
Molecular characterization
DNA/RNA NGS Sequencing
Baseline enumeration in MBC
CTC threshold (per 7.5 ml blood)
% p
atie
nts
at
or
above
the C
TC
thre
shold
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
1
2
3
4
5
6
7 8
9
10
11
12
13
14
15
20
30
40
50
100
1000
cancer, baseline (n = 177)
3%
16%
21% 21% 23%
27%
34% 34% 34% 36%
38% 38%
42% 45% 46% 47%
49%
53%
58% 61%
71%
cancer, first follow-Up (n = 163)
1%
8%
13% 13% 15%
20% 21% 21% 22% 23% 23% 23% 24% 26%
28% 29% 30% 32%
35%
40%
55%
6%
healthy volunteers (n = 145)
benign diseases (n = 200)
1%
8%
The independent prognostic effect of CTC count on progression-free survival and
overall survival. CTC count also improves the prognostication of metastatic breast
cancer when added to full clinicopathological predictive models, whereas serum
tumour markers do not.
Beyond the enumeration: Is the CTC count relevant for therapy efficacy
monitoring?
Study confirms the prognostic significance of CTCs in patients with MBC
receiving first-line chemotherapy. For patients with persistently increased
CTCs after 21 days of first-line chemotherapy, early switching to an
alternate cytotoxic therapy was not effective in prolonging OS.
Negative study?
Beyond the enumeration: Molecular characterization of CTCs
Nat Genet 2013 Dec;45(12):1415-6. doi: 10.1038/ng.2831.
The search for ESR1 mutations in breast cancer.
Oesterreich S, Davidson NE
Term “Liquid biopsy” stands for:
Circulating tumor cells (CTC)
Cell-free DNA (cfDNA)
Plasma
Serum
Other liquid material
Exosomes
ctDNA is tumour DNA that has been shed into the bloodstream
ctDNA can be present in 0.01% - >90% of the total Cell Free DNA (cfDNA)
The amount of ctDNA is related to the tumour burden and varies between patients with different clinical presentations
What is ctDNA?
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32
Origin of Circulating Tumour DNA (ctDNA)
19
Clinical relevance of ctDNA
Applications:
Tumor genotyping:
Tissue versus liquid
biopsy
Monitoring tumor
burden, therapeutic
response and treatment
Minimal residual disease
monitoring and early
detection
PCR-based approaches
Nested real-time PCR PARE ARMS-Scorpion PCR PAP-A amplification BEAMing Microfluidic digital PCR Droplet-based digital PCR
NGS-based approaches
Targeted deep sequencing Tam-Seq Safe-Seq CAPP-Seq Ion-AmpliSeqTM
Whole exome sequencing Whole genome Sequencing
Approaches:
FFPE vs. ctDNA
FFPE Samples
Problems with quality of DNA due to fixation
Mixture of normal and tumour DNA
Some patients have no tumour sample available
The sample represents the tumour at one fixed time point
ctDNA Samples
ctDNA shed directly from tumour
Extracted from the plasma component of whole blood
Large fragment sizes possible
Small quantities extracted ~ 30ng/ 5ml plasma
Serial samples can be taken at various time points during the patient’s treatment
Isolation and processing of ctDNA
Blood sample taken Sample spun down to
isolate the plasma
Plasma is stored at -80ºc
ctDNA is
extracted from
the plasma using
the QIAamp
Circulating
Nucleic Acid or
some other
commercial kit
Applications::
Next-generation
sequencing
Quantative PCR
BEAMing
Digital PCR
Major techical advantage compared to CTC
Possible problems?
ctDNA is unstable: requires fast processing
Getting only 30ng of ctDNA per 5ml plasma extraction
The technique used must be sensitive enough to pick up the low level variants
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32
ctDNA for monitoring metastasis
This proof-of-concept analysis showed that circulating tumor DNA is an
informative, inherently specific, and highly sensitive biomarker of
metastatic breast cancer
ctDNA for monitoring drug resistance
Exome-wide analysis of circulating tumour DNA could
complement current invasive biopsy approaches to identify muta-
tions associated with acquired drug resistance in advanced cancers.
Serial analysis of cancer genomes in plasma constitutes a new para-
digm for the study of clonal evolution in human cancers.
ctDNA as biomarkers of microscopic residual disease after surgery?
Study demonstrates accurate mutation detection in tumor tissues using
ddPCR, and that ptDNA can be detected in blood before and after surgery
in patients with early-stage breast cancer. ptDNA could identifies patients
at risk for recurrence, which could guide chemotherapy decisions for
individual patients.
Daniel A. Haber, and Victor E. Velculescu Cancer
Discovery 2014;4:650-661
©2014 by American Association for Cancer Research
CTCs or ctDNA?
Complementary roles as cancer
biomarkers!
Term “Liquid biopsy” stands for:
Circulating tumor cells (CTC)
Cell-free DNA (cfDNA)
Plasma
Serum
Other liquid material
Exosomes