Circulating Tumor Cells
Minetta C. Liu, MD
Associate Professor of Medicine and Oncology
Director, Translational Breast Cancer Research
Lombardi Comprehensive Cancer Center
Georgetown University Medical Center
Era of Personalized Medicine
• need an individualized approach to treatment
to maximize benefit and minimize cost
assessment of prognosis
 measurement of treatment benefit
 understanding of tumor biology

Biomarker Strategies
single biomarker assessment
diagnosis
prognosis
direct therapy
serial biomarker assessments
prognosis
direct therapy
prediction
Improving Outcomes
• the enumeration and characterization of
circulating tumor cells (CTCs) are useful in
the clinical setting
identification of CTC
alterations in CTC levels
CTC phenotype and genotype
diagnosis
prognosis
prognosis
prediction
diagnosis
prognosis
direct therapy
Origin of CTCs
(Paterlini-Brechot et al. Cancer Letters 2007. 253:180.)
Origin of CTCs
• etiology



disseminated cancer cells
cancer stem cells
bystander cells
• rare cells in a dormant, nonproliferative state



unaffected by chemotherapy
unrecognized by the host immune system
difficult to isolate
Isolation of CTCs
enrichment
density gradient centrifugation
filtration by size
immunomagnetic labeling
detection
Alix-Panabieres et al. Clin Cancer Res 2008. 14:5013.
characterization
quantitation of growth factor expression
gene expression profiling
detection of epigenetic alterations
Detection of CTCs
• antibody based markers



cytokeratins (CK8, CK18, CK19)
epithelial membrane antigen (EMA)
epithelial cell adhesion molecule (EpCAM)
• nucleic acid based markers




CK19 by RT-PCR
mammoglobin by RT-PCR
erbB2 by RT-PCR or FISH
EGFR by RT-PCR
Available Technologies
for Isolation of CTCs
Immunomagnetic Capture
AntiEpCAM
Ferrofluid
Anti CD45-APC
EpCAM
Nucleus
DAPI
Nucleus
DAPI
CD45
CK
AntiCK-PE
Circulating Tumor Cell
Leukocyte
Immunomagnetic Labeling and Immunofluorescent Identification of Cells
Immunomagnetic Capture
composite
intact tumor cells
CD45
control
cytokeratin
DAPI
Parylene Filter Microdevice
(Zheng et al. J Chromatogr A 2007. 1162:154.)
CTC Microchip
(Nagrath et al. Nature 2007. 450:1235.)
Immunomagnetic Labeling:
Enumeration in MBC
Clinical Parameters
• CTCs in individuals with MBC
 detected in ~70%
 >5 per 7.5 mL blood in ~50%
• CTCs in individuals without a malignancy
 detected in <10%
 >5 per 7.5 mL blood in 0%
71%
53%
50
45
49%
47% 46%
45%
40%
40
35%
35
32%
30
30% 29%
28%
25
26%
20
42%
38% 38%
36%
34% 34% 34%
27%
24% 23% 23% 23%
23%
22% 21% 21%
21% 21%
20%
15%
15
CTC threshold (per 7.5 mL blood)
20
15
14
13
12
11
10
9
7
6
5
1%
8
3%
1%
4
0
16%
8%
3
5
8%
6%
2
10
13% 13%
1000
55
58%
55%
100
60
61%
50
65
healthy volunteers (n = 145)
benign diseases (n = 200)
cancer, baseline (n = 177)
cancer, first follow-Up (n = 163)
40
70
30
75
1
% patients at or above the CTC threshold
Prevalence of CTCs
IMC-001
• 177 patients (223 total)
• metastatic breast cancer
• measurable disease
imaging
blood
0
1
2
3
4
time points
5
6
IMC-001: CTCs at Baseline
100%
90%
CTCs / 7.5mL
at Baseline
N (%)
<5 CTC
89 (50%)
>5 CTC
88 (50%)
80%
Median PFS in
Months (95% C.I.)
7.0 (5.6 to 8.9)
2.7 (2.1 to 4.4)
70%
60%
50%
40%
7.0 Months
CTCs / 7.5mL
Median OS in
at Baseline
N (%)
Months (95% C.I.)
<5 CTC
89 (50%) 21.9 (20.1 to 28.6)
>5 CTC
88 (50%) 10.9 ( 7.0 to 15.2)
90%
%Probability of Survival
%Probability
of Progression
Free
Survival
Survival
Free
of Progression
Probability
100%
Cox Hazards Ratio = 1.8523
chi-square = 14.44
(p-value = 0.0001)
2.7
Months
30%
20%
80%
70%
Logrank
p < 0.0001
60%
Cox Hazards Ratio = 2.3581
chi-square = 19.54
(p-value < 0.0001)
21.9 Months
50%
10.9
Months
40%
30%
20%
Logrank p = 0.0001
10%
10%
0%
0%
0
5
n = 177
10
15
20
25
30
35
Time from Baseline (Months)
40
45
50
0
5
10
15
20
25
30
35
Time from Baseline (Months)
40
45
50
(Hayes et al. Clin Cancer Res 2006. 12:4218.)
IMC-001: CTCs at 1st Follow-Up
p value < 0.001
Cox Hazards Ratio = 2.4842
n = 177
p value < 0.001
Cox Hazards Ratio = 5.4537
(Cristofanilli et al. N Engl J Med 2004. 351:781.)
IMC-001: Changes in CTC
% probability of PFS
100%
# patients (median PFS)
90%
< 5 CTC at baseline & at 1st follow-up
81 (30.3 weeks)
80%
decrease in CTC to < 5 at 1st follow-up
33 (32.9 weeks)
> 5 CTC at 1st follow-up
49 (8.9 weeks)
70%
~7.0 months
60%
p value < 0.0001
Cox Hazards Ratio = 1.6600
50%
~7.6 months
40%
30%
~2.1 months
20%
10%
0%
0
5
10
15
20
25
30
35
40
45
50 55
60
65
70
75
80
time from baseline (weeks)
n = 177
(Cristofanilli et al. N Engl J Med 2004. 351:781.)
IMC-001: CTCs and Imaging
n = 83
(Cristofanilli et al. J Clin Oncol 2005. 23:1420.)
IMC-001: CTCs and Imaging
n = 138
(Budd et al. Clin Cancer Res 2006. 12:6403.)
IMC-001: CTCs and Imaging
n = 138
(Budd et al. Clin Cancer Res 2006. 12:6403.)
LCCC Validation Study
• 74 evaluable patients
• metastatic breast cancer
• measurable disease
imaging
blood
0
3
6
9
12
15
cycles
18
21
24
LCCC: CTCs and Imaging
Treatment at
Time of CTC
Result
OR
95% CI
p-value
Overall
6.3
(3.2, 13)
<0.001
Chemotherapy
6.3
(2.9, 14)
<0.001
Endocrine
8.9
(2.2, 35)
0.002
Treatment at
Time of CTC
Result
OR
95% CI
p-value
Overall
3.1
(1.6, 5.8)
0.001
Chemotherapy
2.7
(1.2, 6.3)
0.02
Endocrine
5.2
(1.2, 23)
0.03
Treatment at
Time of CTC
Result
OR
95% CI
p-value
Overall
4.9
(2.2, 118)
<0.001
Chemotherapy
6.9
(3.0, 16)
<0.001
Endocrine
2.9
(0.6, 14)
0.2
CTCs Drawn at the Time of Radiographic Imaging
CTCs Drawn 3-5 Weeks Prior to Radiographic Imaging
CTCs Drawn 7-9 Weeks Prior to Radiographic Imaging
(Liu et al. J Clin Oncol 2009. 27:5153.)
Recommendations for Use
• to assess for progression in patients with
measurable metastatic disease
• to provide a guide by which to determine the
timing of radiographic restaging studies
• to assess the feasibility and timing of drug
holidays in patients with stable disease and
intolerable drug related toxicities
Conclusions
Response/Futility Marker
• meaningful rate of detection
• reliable threshold
• correlation with clinical outcomes (validity)
• ability to improve clinical outcomes (utility)
CTCs – Present
• clinical validity
IMC-001
 LCCC study

• clinical utility
assess disease status (with imaging)
 guide the timing of radiographic studies
 guide the timing of drug holidays
 guide systemic therapy (?????)

Prospective Validation:
Means to Improve Survival
blood drawn at baseline prior to first-line chemotherapy
CTC <5
CTC ≥5
blood drawn three weeks after the
first chemotherapy dose
Arm A
monitor for PFS & OS
CTC <5
eligible for other firstline chemotherapy trials
SWOG S0500
CTC ≥5
R
Arm B
Arm C1
Arm C2
maintain first-line
chemotherapy
until progression
maintain first-line
chemotherapy
until progression
switch to
alternate
chemotherapy
target n = 120
Prospective Validation:
The Liquid Biopsy
composite
leukocyte
control
cytokeratin
HER2 and SE17
FISH Probes
nucleus
Prospective Validation:
The Liquid Biopsy
paclitaxel and trastuzumab and lapatinib
N = 400
paclitaxel and trastuzumab
paclitaxel and lapatinib
tissue
blood
CALGB 40601
0
2
4
6
8 10
weeks
12 14 16
ASCO Tumor Marker Guidelines
• Circulating tumor cell assays as markers
for breast cancer
(Harris et al.J Clin Oncol 2007.33:5287.)
THANK YOU