Circulating Tumor Cell
Technology:
A New Paradigm for the
Management of Patients with
Metastatic Carcinoma
David Kindelberger, MD
Divisions of Cytopathology and Women’s and Perinatal
Pathology,
Department of Pathology,
Brigham and Women’s Hospital
Financial Disclosures
• Dr. Kindelberger has no relationships to
commercial interests relating to the
content of this presentation.
Outline
• Introduction to CTC technology
• Current paradigm for using CTCs to
manage cancer patients
• Personalized medicine with lung cancer as
a model
• Emerging roles for CTCs and molecular
pathology in managing cancer patients
Tumor Metastasis
• Metastatic disease is primary cause of
death in most cancer pts.
• Tumor metastasis involves a series of
discrete steps
– Invasion of surrounding tissue
– Survival and arrest in bloodstream
– Colonization
Tumor Metastasis
Once in circulation, cells must
1. Survive—harsh environment
-shear forces
-lack of substratum
-immune cells
2. Attach
3. Extravasate
Nature Medicine 12,2006
New Models of Tumor Metastasis
Klein, Science 321, September, 2008
CTC
• 1869 Australian Medical Journal: A Case of Cancer in
•
•
which Cells Similar to Those in the Tumors were Seen in
the Blood After Death 1869;14:146.
1955 Acta Chiurgica Scandinavia: Cancer Cells
Circulating in the Blood: a Clinical Study on the
Occurrence of Cancer Cells in the Peripheral Blood and in
Venous Blood Draining the Tumor Area at Operation
1955;201:1.
1976 American Journal of Medicine: Carcinocythemia:
An Acute Leukemia-like Picture Due to Metastatic
Carcinoma Cells 1976;60:273.
Isolation of CTC from Peripheral
Blood
• Two Key Issues:
– Enrichment of epithelial/tumor cells from RBC
& WBC
– Characterization to distinguish
• Tumor cells from blood components
• Tumor cells from normal cells
Isolation of CTC from Peripheral
Blood
Enrichment Methods:
Filtration
Density gradient
Immunomagnetic
Isolation of CTC from Peripheral
Blood
Isolation of CTC from Peripheral
Blood
Immunomagnetic Selection of CTC
Isolation of CTC from Peripheral
Blood
• Two Key Issues:
– Enrichment of epithelial/tumor cells from RBC
& WBC
– Characterization to distinguish
• Tumor cells from blood components
• Tumor cells from normal cells
Immunomagnetic Selection of CTC
Distinguishing CTC after
Enrichment
Labeling of CTC and Blood Cells
Anti-EpCAM
Ferrofluid
EpCAM
Anti-EpCAM
Ferrofluid
CD45
EpCAM
Nucleus
DAPI
HER2
CK
CK
AntiCK-PE
Circulating Tumor Cell
AntiHER2PE-Cy7
Nucleus
DAPI
Y
Nucleus
DAPI
AntiCK-PE
HER2+*
Circulating Tumor Cell
Leukocyte
Anti CD45-APC
Magnetic Cell Presentation
Analysis
Cartridge
Trajectory of magnetically
labeled objects
Analysis of Enriched CTC
• Semi-automated fluorescence
microscope
• Automatically scans a
complete reaction cartridge in
about 10 minutes
• Software algorithm identifies
CTC candidates
• Cell images are presented in a
gallery format for confirmation
as CTC by a technician or
pathologist
CellSpotter Analyzer
Analysis of Enriched CTC
Composite Leukocyte
Intact Tumor Cells
Control
Cytokeratin Nucleus
Generalizability
Clinical Cancer Research 10, 2004
Generalizability
Clinical Cancer Research 10, 2004
The Current CTC Paradigm for
Metastatic Carcinoma
Treatment Efficacy in Metastatic
Breast Cancer
Monitoring Metastatic Disease
• Multicenter, prospective trial
• Inclusion criteria:
– Progressive metastatic breast cancer
– All beginning new systemic therapy
– All with measurable disease
– All with ECOG (Eastern Cooperative Oncology
Group) performance status of 0-2 (no to
moderate symptoms)
NEJM 351, 2004
Number of CTC Before New Therapy
Predicts Progression Free Survival and
Overall Survival
Number of CTC at First Follow Up Predicts
Progression Free Survival and Overall
Survival
Number of CTC at First Follow Up Predicts
Progression Free Survival and Overall
Survival
Conclusions
• The levels of baseline CTC are independent
•
•
prognostic markers of outcomes (both
progression free survival and overall survival)
Elevated levels of CTC at First Follow-Up predict
both short progression free survival and overall
survival—may indicate that pt. is receiving futile
therapy.
CTC levels give reliable estimates of disease
progression much earlier than with traditional
imaging methods (3-4 weeks vs. 8-12 weeks)
Lung Cancer As a Model for
Personalized Medicine
Lung Cancer-Overview
• NSCLC is most common cause of cancer-
related deaths in West.
• 50% of pts. present with mets. AND 40%
present with locally advanced disease.
• For pts. with advanced cancers,
chemotherapy is mainstay of treatment.
• Median survival is 8-9 months.
The EGFR Story
• By IHC, EGFR is detected in between 40 and
•
80% of NSCLC.
Using the analogy of c-KIT in GIST, companies
developed 2 small molecule EGFR inhibitors
– Gefitinib (Tarceva)
– Irlotinib (Iressa)
• Specific subsets of patients showed significant
survival increases (double)
Characteristics of Pts. Likely to
Respond to EGFR Inhibitors
Group
Response Rate
(%)
P value
Women vs. Men
19 vs. 3
.001
Japanese vs. White
27.5 vs. 10
.0023
Adeno vs. others
13 vs. 4
.046
Never-smoker vs.
others
36 vs. 8
<.001
Types of EGFR Mutations in
Responders
Action of EGFR Inhibitors
Cell Growth
Apoptosis
Resistance to EGFR Inhibitors
Cell Growth
EGFR Mutation Analysis using
Circulating Tumor Cells
NEJM, 2008
A Short Digression
Variations on a CTC Theme
Nature 2007;450: 1235
Variations on a CTC Theme
Nature 2007;450: 1235
Now Back to our Story…
Direct Sequencing of EGFR from
Isolated CTCs
Resistance to EGFR Inhibitors
Cell Growth
Cell Growth
FISH on Lung CTCs
Green is CEP 7, Red is EGFR, Blue is MET
Polysomy/polyploidy
CTC FISH Data Analysis
CTC FISH Data Analysis
The New Paradigm
• With a single blood draw, one can
– Confirm EGFR, MET, other oncogene
amplification
– Acquire material for direct sequencing of
EGFR
– Enumerate baseline CTC levels to use as
monitor for efficacy of selected therapy
– Patients may never need to have a biopsy/Cmed
Why Cytology?
Clinical Cancer Research 10, 2004
Future Directions
• Filtration Systems for isolation of CTCs
– Allows for assessment of non-epithelial
tumors (Melanoma, etc)
• Fiberoptic Array Scanning Technology
(FAST) following direct isolation
– Gentle procedure allowing for little
disruption of cytomorphologic features
Future Directions
• Filtration Systems for isolation of CTCs
– Allows for assessment of non-epithelial
tumors (Melanoma, etc)
• Fiberoptic Array Scanning Technology
(FAST) following direct isolation
– Gentle procedure allowing for little
disruption of cytomorphologic features
Future Directions
• Filtration Systems for isolation of CTCs
– Allows for assessment of non-epithelial
tumors (Melanoma, etc)
• Fiberoptic Array Scanning Technology
(FAST) following direct isolation
– Gentle procedure allowing for little
disruption of cytomorphologic features
Archives of Pathology and Laboratory Medicine, Sept. 2009
Archives of Pathology and Laboratory Medicine, Sept. 2009
Ongoing Studies
• Combination Drug Therapies in Metastatic
Breast Cancer with Hal Burstein
– Enumerating CTCs during treatment with
Herceptin/Avastin/Vinorelbine (HAV)
• Combination Drug Therapies in Metastatic
Breast Cancer with Gerburg Wulf (BIDMC)
– Enumerating CTCs during treatment with
Fulvestrant and Lapitinib
Ongoing Studies
• Molecular Characterization of Breast
Cancer with Ian Krop
– Comparison of primary tumor with CTCs with
mets.
Ongoing Studies
• Molecular Characterization of Breast
Cancer with Ian Krop
– Comparison of primary tumor with CTCs with
mets.
• Utility of CTCs as Early Predictors of
Recurrence in Metastatic Ovarian
Carcinoma with Ursula Matulonis
– Enumeration of CTCs to be used in
combination with MMP levels in urine.
Ongoing Studies
• Utility of CTC as a predictor of response to
therapy in patients with Metastatic
Squamous Cell Carcinoma from the Cervix
with Michael Birrer
• CTCs in patients with Metastatic Prostate
Carcinoma with Glen Bubley (BIDMC)