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

diagnosis
single biomarker assessment prognosis
direct therapy

prognosis
serial biomarker assessments direct therapy
prediction
 Improving Outcomes

• the enumeration and characterization of

circulating tumor cells (CTCs) are useful in
the clinical setting

diagnosis
identification of CTC
prognosis

prognosis
alterations in CTC levels
prediction

diagnosis
CTC phenotype and genotype 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
density gradient centrifugation
enrichment filtration by size
immunomagnetic labeling

detection

Alix-Panabieres et al. Clin Cancer Res 2008. 14:5013.

quantitation of growth factor expression

characterization 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

Anti-
Anti -
Y EpCAM Y CD45-APC
Ferrofluid
EpCAM CD45
Nucleus Nucleus
DAPI DAPI

CK
Y

Anti-
CK-PE

Circulating Tumor Cell Leukocyte

Immunomagnetic Labeling and Immunofluorescent Identification of

Cells
 Immunomagnetic Capture

composite CD45 control cytokeratin DAPI

intact tumor cells

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%
 Prevalence of CTCs
% patients at or above the CTC threshold

75
71%
70 healthy volunteers (n = 145)
65 61% benign diseases (n = 200)
60 58%
55% cancer, baseline (n = 177)
55 53%
49% cancer, first follow-Up (n = 163)
50 47% 46%
45%
45 42%
40%
40 38% 38%
35% 36%
34% 34% 34%
35 32%
30% 29%
30 28% 27%
26%
24% 23% 23% 23% 23%
25 22% 21% 21% 21% 21%
20%
20 16%
15%
15 13% 13%

10 8%
8%
5 6% 3%
1% 1%
0

1000
8

100
10

13

50
11

12

14

15

20

30

40
9
1

CTC threshold (per 7.5 mL blood)

 IMC-001
• 177 patients (223 total)
• metastatic breast cancer
• measurable disease

imaging

blood

0 1 2 3 4 5 6

time points
 IMC-001: CTCs at Baseline

100% 100% CTCs / 7.5mL Median OS in

at Baseline N (%) Months (95% C.I.)
Survival

90% CTCs / 7.5mL Median PFS in 90% <5 CTC 89 (50%) 21.9 (20.1 to 28.6)
at Baseline N (%) Months (95% C.I.) >5 CTC 88 (50%) 10.9 ( 7.0 to 15.2)
Survival

80% <5 CTC 89 (50%) 7.0 (5.6 to 8.9) 80%

>5 CTC 88 (50%) 2.7 (2.1 to 4.4) Cox Hazards Ratio = 2.3581
70% chi-square = 19.54
Free

70%
Cox Hazards Ratio = 1.8523

%Probability of Survival
(p-value < 0.0001)
Progression Free

Logrank
60% chi-square = 14.44
60% p < 0.0001
Progression

(p-value = 0.0001)
7.0 Months 21.9 Months
50% 50%
2.7 10.9
Months
40% 40% Months

30% 30%
of of
%Probability

20% 20%
Probability

10% Logrank p = 0.0001 10%

0% 0%
0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
Time from Baseline (Months) Time from Baseline (Months)

n = 177 (Hayes et al. Clin Cancer Res 2006. 12:4218.)

 IMC-001: CTCs at 1st Follow-Up

p value < 0.001 p value < 0.001

Cox Hazards Ratio = 2.4842 Cox Hazards Ratio = 5.4537

n = 177 (Cristofanilli et al. N Engl J Med 2004. 351:781.)

 IMC-001: Changes in CTC
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)
% probability of PFS

70%

60% ~7.0 months

p value < 0.0001
50% Cox Hazards Ratio = 1.6600

40% ~7.6 months

30%

20% ~2.1 months

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 18 21 24
cycles
 LCCC: CTCs and Imaging
Treatment at
Time of CTC OR 95% CI p-value
Result
Overall 6.3 (3.2, 13) <0.001 CTCs Drawn at the Time of Radiographic Imaging

Chemotherapy 6.3 (2.9, 14) <0.001

Endocrine 8.9 (2.2, 35) 0.002

Treatment at
Time of CTC OR 95% CI p-value
Result
Overall 3.1 (1.6, 5.8) 0.001 CTCs Drawn 3-5 Weeks Prior to Radiographic Imaging

Chemotherapy 2.7 (1.2, 6.3) 0.02

Endocrine 5.2 (1.2, 23) 0.03

Treatment at
Time of CTC OR 95% CI p-value
Result
Overall 4.9 (2.2, 118) <0.001 CTCs Drawn 7-9 Weeks Prior to Radiographic Imaging

Chemotherapy 6.9 (3.0, 16) <0.001

Endocrine 2.9 (0.6, 14) 0.2
(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

Arm A
first chemotherapy dose
monitor for PFS & OS
CTC <5 CTC ≥5
eligible for other first-

R
line chemotherapy trials

Arm B Arm C1 Arm C2

maintain first-line maintain first-line switch to
chemotherapy chemotherapy alternate
SWOG S0500
until progression until progression chemotherapy

target n = 120
 Prospective Validation:
The Liquid Biopsy
composite leukocyte control cytokeratin nucleus

HER2 and SE17

FISH Probes
 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 12 14 16
weeks
 ASCO Tumor Marker Guidelines

• Circulating tumor cell assays as markers

for breast cancer

(Harris et al.J Clin Oncol 2007.33:5287.)

THANK YOU