Circulating Tumor Cell Technology: A New Paradigm for the Management of Patients with Metastatic Carcinoma

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Circulating Tumor Cell TechnologyA New Paradigm
for the Management of Patients with Metastatic
Carcinoma

• David Kindelberger, MD
• Divisions of Cytopathology and Womens and
  Perinatal Pathology,
• Department of Pathology,
• Brigham and Womens Hospital

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Financial Disclosures

• Dr. Kindelberger has no relationships to
  commercial interests relating to the content of
  this presentation.

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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

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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

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Tumor Metastasis

• Once in circulation, cells must
• 1. Surviveharsh environment
• -shear forces
• -lack of substratum
• -immune cells
• 2. Attach
• 3. Extravasate

Nature Medicine 12,2006
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New Models of Tumor Metastasis
Klein, Science 321, September, 2008
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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
  186914146.
• 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 19552011.
• 1976 American Journal of Medicine
  Carcinocythemia An Acute Leukemia-like Picture
  Due to Metastatic Carcinoma Cells 197660273.

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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

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Isolation of CTC from Peripheral Blood

• Enrichment Methods
• Filtration
• Density gradient
• Immunomagnetic

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Isolation of CTC from Peripheral Blood
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Isolation of CTC from Peripheral Blood
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Immunomagnetic Selection of CTC
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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

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Immunomagnetic Selection of CTC
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Distinguishing CTC after Enrichment
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Labeling of CTC and Blood Cells
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Magnetic Cell Presentation
Analysis Cartridge
Trajectory of magnetically labeled objects
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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

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Analysis of Enriched CTC
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Generalizability
Clinical Cancer Research 10, 2004
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Generalizability
Clinical Cancer Research 10, 2004
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The Current CTC Paradigm for Metastatic Carcinoma
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Treatment Efficacy in Metastatic Breast Cancer
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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
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Number of CTC Before New Therapy Predicts
Progression Free Survival and Overall Survival
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Number of CTC at First Follow Up Predicts
Progression Free Survival and Overall Survival
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Number of CTC at First Follow Up Predicts
Progression Free Survival and Overall Survival
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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
  survivalmay 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)

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Lung Cancer As a Model for Personalized Medicine
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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.

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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)

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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 lt.001
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Types of EGFR Mutations in Responders
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Action of EGFR Inhibitors
Apoptosis
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Resistance to EGFR Inhibitors
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EGFR Mutation Analysis using Circulating Tumor
Cells
NEJM, 2008
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A Short Digression
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Variations on a CTC Theme
Nature 2007450 1235
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Variations on a CTC Theme
Nature 2007450 1235
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Now Back to our Story
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Direct Sequencing of EGFR from Isolated CTCs
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Resistance to EGFR Inhibitors
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FISH on Lung CTCs
Green is CEP 7, Red is EGFR, Blue is MET
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Polysomy/polyploidy
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CTC FISH Data Analysis
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CTC FISH Data Analysis
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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/C-med

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Why Cytology?
Clinical Cancer Research 10, 2004
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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

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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

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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

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Archives of Pathology and Laboratory Medicine,
Sept. 2009
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Archives of Pathology and Laboratory Medicine,
Sept. 2009
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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

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Ongoing Studies

• Molecular Characterization of Breast Cancer with
  Ian Krop
• Comparison of primary tumor with CTCs with mets.

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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.

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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)