Circulating Tumor Cell

in Cancer Medicine

Jhingook Kim, MD Samsung Medical Center

Sungkyunkwan University, School of Medicine

Circulating Tumor Cell

Steps leading to cancer metastasis

Cancer Letters 2007. 253:180.-204

Circulating Tumor Cell

The Rosetta Stone of Metastasis/Cancer Research

4

History of Circulating Tumor cells

CTCs were observed for the first time in 1869 in the blood of a man with

metastatic cancer by Thomas Ashworth, who postulated that “cells identical

with those of the cancer itself being seen in the blood may tend to throw some

light upon the mode of origin of multiple tumors existing in the same person”.

A thorough comparison of the morphology of the circulating cells to tumor cells

from different lesions led Ashworth to conclude that “One thing is certain, that if

they [CTC] came from an existing cancer structure, they must have passed

through the greater part of the circulatory system to have arrived at the internal

saphena vein of the sound leg”.

Searching for cancer cell in the blood

Cancer cells = Epithelial cell origin

∴ Epithelial cell in the blood = CTC

The access for the cancer cells

Searching for CTC

CellSearch™ System

Defining a Tumor Cell 1. Positive for CK-PE (Cytokeratin)

• Morphology: Round to oval; sometimes elongated • At least 4 µm in size (should fit in the white square inside the measuring box) • In-tact (the cell & all edges are in-tact)

2. Positive for DAPI (Nucleus): • Must have a nucleus • Should fit in the cytoplasm • Should be at least 50% inside the cytoplasm

3. Negative for Blank Channel: • Good, Bright cell in Ctrl column = Control Positive • < 3 spots = Control Negative

NOTE: All patient sample tumor cells should be negative in Control Column

4. Negative for CD45/APC (Leukocyte cytoplasm): • The tumor cells should be negative for CD45/APC

5. Composite: TUMOR CELL

COMP CD45-APC CK-PE DAPI

CK-PE DAPI COMP

CD45-APC

CK-PE

or

Molecular Analysis

MagNestTM

Epithelial Cell Kit

EpCAM-based selection

CellSearch™ System

Progression-Free Survival Pr

obab

ility

of

Prog

ress

ion-

Free

Sur

viva

l

Time From Baseline (Months) 0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

Log rank P = .0001

7.0 months 2.7

months

≥ 5 CTCs/7.5 mL n = 87 (49%)

< 5 CTCs/7.5 mL n = 90 (51%)

Cristofanilli M N Engl J Med 2004

Clinical Studies on CTC

Prognostic Value of Baseline CTC Counts

Blood drawn at baseline prior to first-line chemotherapy

Arm B

Maintain first-line chemotherapy until progression

Arm C1

Maintain first-line chemotherapy until progression

Arm C2

Switch to alternate chemotherapy

Arm A

Monitor for PFS and OS

Eligible for other first-line chemotherapy trials R

CTC ≥ 5

Blood drawn 3 weeks after first chemotherapy dose

CTC ≥ 5

SWOG S0500

Clinical Studies on CTC

Treatment decision based on CTC Counts

N Engl J Med 2008;359:366-77 Nature 2007 ;450(7173):1235-9.

Circulating Tumor Cell

Enrichment, Detection and Characterization

Klaus Pantel, Nature Reviews Cancer 8, 329-340 (May 2008)

Fibre-optic array scanning technology

Company Technology

Veridex Cellsearch system

EpCAM coated beads based positive selectio using magnetic beads followed by staining and image analysis

Biocept Laboratories CEE® Microfluidics

Biocept's microfluidic channel is coated with Streptavidin . CTCs are captured using an antibody cocktail, which includes EpCAM, that is added to cells prior to capture.

Screencell size-based filtration 6.5 um and 5.5 um porous membranes for fixed and unfixed cells respectively

Clearbridge Biomedics Clear Cell FX system

microfluidic biochips able to effectively detect and isolate wholly-intact CTCs from small quantities of patient blood samples. The CTChips® contains thousands of crescent shaped microstructures that can isolate CTCs without antibody or magnetic beads

Epic Biosciences PBMCs are layered on glass slide and imaaged using a high definition scanner

Adnagen combination of antibodies on magnetic beads to enrich cells followed by nested PCR

Fluxion Biosystems EpCAM based positive selection

Available systems for CTCs (1)

Company Technology

Miltenyi Biotech EpCAM magnetic bead based selection

EPISPOT

Apocell dielectrophoretic flow field fractionation. uses electrical property differentiation between normal blood cells and tumor cells

Fluidigm single cell analysis platform

Maintrac Cells from EDTA-blood were subjected to erythrocyte lysis, isolated by centrifugation, and incubated with a fluorescence-labeled antibody against. EpCAM. The numbers of vital cells were counted via fluorescence microscopy

TelomeScan TelomeScan system does not use EpCAM enrichment. TelomeScan is viral diagnostic reagent to detect viable CTCs including lung cancer. Currently Oncolys BioPharma Inc. in Tokyo is conducting various feasibility studies to detect viable CTCs from 7.5 mL blood and differenciate EMT-CTC and CSC

Cytotrack CytoTrack detects CTCs without enrichment (such as EpCAM) and without bias on size (such as filtration). CytoTrack has a capacity to scan 100 million cells in just one minute. CytoTrack is commercial available.

ODOSA (One-Drop, One-Step Assay for Circulating Tumor Cell Detection)

The invention is a cell-specific, intracellularly-activated, one step assay for the detection of circulating tumor cells (CTC) in a single drop of blood. The invention is based on cell-specific aptamers linked to fluorophores and quenchers. Lysosomal degradation leads to release of quenching and switches on a strong fluorescent signal in CTCs.

Available systems for CTCs (2)

Company Technology

Onqity EpCAM coated microposts arranged in a geometric pattern to creat a size gradient. end result is a compbination of EpCAM and size enrichment

Apocell dielectrophoretic flow field fractionation. uses electrical property differentiation between normal blood cells and tumor cells

Parsortix uses size and deformability using a wier-type step filter

Creatv Microtech precision manufactured size-filter

Genetx ariol automated imaging

Grenier Bio one -oncoquick

ficoll gradient tube with filter to keep the separated

Silicon Biosystems electronic chip to further purify coarsely enriched CTCs

FAST Laser scanning of all blood cells

Cynvenio Biosystems high throughput microfluidic sheath flow isolation technology by ferrofluid with cell staining plus downstream DNA analysis via next generation sequencing and/or quantitative allele-specific PCR (Q-PCR)

Aviva Biosciences combination of size-based RBC depletion and WBC depletion

Available systems for CTCs (3)

Most promising CTC technologies

(from opinion poll conducted by World CTC-Berlin 2013)

Circulating Tumor Cell

CTC platform

CTC characterization 방법 개발 필요성

Positive Selection Negative Selection CTC isolation

principle Anti-EpCAM mAb-based Size filtration & CD45 depletion

Enrichment Removal of leukocyte: Trapping of CTC

Removal of leukocyte: trapping of CTC

lost CTC CTC with low expression of EpCAM Small sized CTC

Sensitivity Low High

Purity High Low

Untethered CTC Nearly impossible: CTC-mAb-microbead in situ staining after capture

Possible, but not always: CTC alone

Further EMT analysis Disadvantage Advantage

Molecular analysis Advantage Disadvantage: low purity

Merge_RBC_5min_40X Merge_Plasma_5min_40X

WBC

CTC RBC

CTC+debris

SMC-KAIST Collaboration

Microfluidic CTC Capture system

0

20

40

60

80

100

50 100# of spike cells

captu

re e

ffic

iency

(%

)

Lung cancer cell lines - GFP 발현 폐암 세포주: H358-GFP, A549-GFP, H460-GFP - Non-GFP 폐암 세포주: H1975, PC-9

SMC-KAIST platform을 이용한 spike test

성능지표 Spiked cell number in Blood

Input cell number 50개 100개

Capture efficiency 80.2±4.19% 80.7±11.3%

Recovery rate 63.0±9.16% 64.5±11.3%

Journal of Chromatograph A, 2007;1162:154-161

Negative Selection by Physical Properties Cytogen ™ System

Cytogen platform을 이용한 spike test

Spiking cell line: GFP-H358 lung cancer cell line

Spiking planned

GFP-H358 cells Spiked

(실제 넣은 수)

GFP-H358 cell count

(recovery rate)

Mean value of recovery rate

10 개

9 개 9 개 (100.0%)

96.7±5.8% 10 개 9 개 (90.0%)

11 개 11 개 (100.0%)

30 개

36 개 29 개 (80.5%)

82.8±2.9% 33 개 27 개 (81.8%)

36 개 31 개 (86.0%)

100 개

88 개 64 개 (73.0%)

82.0±7.9% 93 개 82 개 (88.0%)

92 개 78 개 (85.0%)

Negative Selection by Physical Properties Comparison of Platforms

ScreenCell RareCell Cytogen KAIST

Samples Stomach ca (1) Lung ca (1) Normal Control (1)

Stomach ca (1) Lung ca (1) Normal Control (1)

Colon ca (1) Lung ca (1) Normal Control (1)

Lung ca (1) CBD ca (1)

Volume 3ml 10ml 3ml 5ml

Results No CTCs No CTCs Colon ca (8/5ml) Lung ca (3/5ml) Normal (0/5ml)

Lung ca (15/5ml) CBD ca (8/5ml)

Confidential

24시간 후 재채혈 72시간 후 재채혈

CTC count for the same patient did not change at two different time points.

Studies on CTC

(1) Technical or platform development –related

(2) Carcinogenesis and metastasis mechanism-related

(3) Clinical translational research

Clinical studies using CTC technologies

(1) correlation with tumor extent (such as stage)

(2) prognosis or treatment-response

(3) screening role

(4) source of the liquid biopsy or serial biopsy

Clinical studies using CTC technologies (1)

Diseases • Characterization of Circulating Tumor Cells (CTC) in Melanoma • Circulating Tumor Cells for Hepatocellular Carcinoma • Detection of CTCs in Patients Undergoing Surgery for Stage IV Colorectal Cancer • Circulating Tumor Cells (CTCs) From Metastatic Breast Cancer • Circulating Tumor Cells (CTCs) as a Blood-based Tumor Marker in Patients With Small

Cell Lung Cancer (SCLC) Characterization • Characterization of Circulating Tumor Cells Captured by c-MET (CTC-MET) • Characterization of Circulating Tumor Cells (CTC) From Patients With Metastatic

Breast Cancer Using the CTCEndocrine Therapy Index • Feasibility of the Research for Mutation of K-ras and EGFR in CTCs From Metastatic

Non Small Cells Bronchial Carcinomas Screening • Circulating Tumor Cells (CTCs): A Potential Screening Test for Clinically Undetectable

Breast Carcinoma

Clinical studies using CTC technologies (2)

Prognosis/tx-response • Prognostic Value of the Levels of CTCs in Peripheral Blood in Patients With

Prostate Cancer at High Risk (Clinical Stages IIB-III) Treated Radically With Radiotherapy and Hormone Therapy

• Dynamic Circulating Tumor Cell (CTC) Changes During the Chemotherapy in NSCLC

• DETECT III - A Multicenter, Phase III Study to Compare Standard Therapy +/- Lapatinib in HER2-ve MBC-Patients With HER2+ve CTCs

• Influence of BRAF and PIK3K Status on the Efficacy of 5-Fluorouracil/Leucovorin /Oxaliplatin (FOLFIRI) Plus Bevacizumab or Cetuximab in Patients With RAS Wild-type Metastatic Colorectal Carcinoma and < 3 Circulating Tumor Cells (CTC)

• Blood Levels of Tumor Cells in Predicting Response in Patients Receiving First-Line Chemotherapy for Stage IV Breast Cancer

• Prognostic and Dynamic Change of CTC Enumeration in Advanced NSCLC With Chemotherapy and Targeted Therapy

The representative immunofluorescence images at different stages.

0

5

10

15

20

25

30

35

CTC

count

I II III IV

Stages

P<0.001

P=0.022 P=0.035

CTC enumeration depends on pathologic stages.

5ml blood

“C” platform

Enriched CTCs

total N=82 Cytogen에서 PDE staining 결과 count된 수/5ml blood 1. Stage I=1.22±1.51 CTC 2. Stage II=1.81±1.74 CTC 3. Stage III=1.85±1.95 CTC 4. Stage IV=8.00±9.95 CTC

(n=22)

(n=5)

(n=28)

(n=25)

(n=11)

Characteristics of Lung Cancer Patients

(N=82)

Gender Histological cell type Pathologic stage

Tumor location

ADC (n=60)

Male (n=51)

Female (n=31)

SCC (n=20)

others (n=2)

Stage I (n=37)

Stage II (n=16)

Stage III (n=13)

Stage IV (n=16)

Chemotherapy (n=17)

Surgery (n=65)

Wedge resection (n=6)

Segmentectomy (n=4)

Lobectomy (n=52)

Pnemonectomy (n=3)

Circulating tumor cells (CTCs) count according to the characteristics of patients.

Blood drawn at baseline prior to first-line chemotherapy

Arm B

Maintain first-line chemotherapy until progression

Arm C1

Maintain first-line chemotherapy until progression

Arm C2

Switch to alternate chemotherapy

Arm A

Monitor for PFS and OS

Eligible for other first-line chemotherapy trials R

CTC ≥ 5

Blood drawn 3 weeks after first chemotherapy dose

CTC ≥ 5

SWOG S0500

Clinical Studies on CTC

Treatment decision based on CTC Counts

Fig. Overall survival (OS) and progression-free survival (PFS) in patients with metastatic breast cancer for whom therapy failed to reduce circulating tumor cells(CTCs) within 3 weeks of starting first-line chemotherapy; patients were randomly assigned to continue receiving the initial chemotherapy or to switch to an alternative chemotherapy

J Clin Oncol 2014

Circulating Epithelial Cells in Patients with Benign Lung Diseases

Non-tuberculous mycoplasma infection (F/46) Actinomycosis-related Hemoptysis (M/59)

Circulating Epithelial Cells in Patients with Benign Colon Diseases

Klaus Pantel, Clinical Chemistry, 2012, 58:936

38 JCO, 2011

Frequency of molecular markers expressed in circulating tumor cells of breast cancer patients

Breast Cancer Research 2011, 13:228

CTC escape from EpCAM-based detection due to epithelial to mesenchymal transition (EMT).

BMC Cancer 2012, 12:178

FACS analysis for EpCAM expression

In vivo mouse model

Mobilisation of Circulating Tumour Cells into the peripheral circulation during radiotherapy

Michael MacManus, Olga Martin Roger Martin, David Ball, Ashley Cox, Robin Anderson, Judy Doherty, Patricia Daly,

Daphne Jacobs, Prue Russell

WCLC 2013

γ-H2AX-based CTC detection: proof of principle with “spiked” blood FACS analysis of blood “spiked” with ~3% of tumor cells

Lymphocytes

Tumour cells

No IR 24 hrs post-10 Gy

No IR IR Pre-sorted FACS-isolated

WCLC 2013

CK positive CTC numbers (including CTCs in CTMs) per 10 ml blood in cytospins of 17 NSCLC patients

Cellsearch are results in Red

WCLC 2013

Acta Oncologica, 2011, 50:700

OncoQuick

The comparison of the detection rates of EpCAM (–) and EpCAM (+) CTCs between OncoQuick® plus and MACS microbead®

Acta Oncologica, 2011, 50:700

EpCAM+ bead

OncoQuick plus

The comparison of the detection rates of EpCAM (–) and EpCAM (+) CTCs between OncoQuick® plus and MACS microbead®

Searching for cancer cell in the blood

Cancer cells = Epithelial cell origin

∴ Epithelial cell in the blood = CTC

The access for the cancer cells

Searching for CTC

Circulating Tumor Cell

CTC characterization 기술 개발 필요성

Q. What is a CTC specific marker? Q. Are cells from EpCAM-based positive selection real CTCs? Q. Will the CTCs develop into metastatic cancer over time? Nobody couldn’t answer those.

CTC identification in advanced lung cancer patients

DAB-EpCAM + Hematoxylin staining ~200 CTC 관찰

Lymphocytes

Red blood cell

WBC + SW480 48/64

Normal WBC 61/100

FISH Analysis (WBC vs Cancer Cell)

*SW480 : colon cancer cell line

For prognostic/predictive marker by count Is it reliable ? 1) technique 2) biology For the source of cancer cell (real-time biopsy) Is it sufficient?

The access for the cancer cells

CTC, For What?

Daniel A. Haber et. al. JCB 2011

cfDNA (circulating cell-free DNA)

cfDNA (circulating cell-free DNA)

cfDNA (circulating cell-free DNA)

Is the CTC obsolete?

No evidence of predictive role

Not reliable tool for liquid biopsy

Summary

1. Research on/with CTC can be a key in cancer research (basic/clinical).

2. Anti-EpCAM based CTC capture have shown clinical significance in some cancers.

3. However, we yet cannot say that we detect all bad cells and/or all detected cells are bad.

4. Therefore, circulating tumor cell research invites many researchers for capture, identification, characterization and application studies in various cancers.