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)

(Paterlini-Brechot et al. Cancer Letters 2007. 253:180.)

Origin of CTCs

Main approaches for CTC detection

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%

Beyond the enumeration: Is the CTC count relevant for development &

progression of metastases?

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: methods

What is Droplet Digital PCR?

Expensive equipment?

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

Tumor signatures in the blood, Nature Biotechnology, 32, 2014

Thank you for you attention!