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

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.

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

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  1. 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

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

  3. 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

  4. 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

  5. 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

  6. New Models of Tumor Metastasis Klein, Science 321, September, 2008

  7. 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.

  8. 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

  9. Isolation of CTC from Peripheral Blood Enrichment Methods: Filtration Density gradient Immunomagnetic

  10. Isolation of CTC from Peripheral Blood

  11. Isolation of CTC from Peripheral Blood

  12. Immunomagnetic Selection of CTC

  13. 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

  14. Immunomagnetic Selection of CTC

  15. Distinguishing CTC after Enrichment

  16. Anti-EpCAM Ferrofluid Anti-EpCAM Ferrofluid Y Y Y Anti -CD45-APC EpCAM CD45 EpCAM Nucleus Nucleus Nucleus DAPI DAPI DAPI Y Anti-HER2-PE-Cy7 HER2 Y CK Y CK Anti-CK-PE Anti-CK-PE Circulating Tumor Cell HER2+*Circulating Tumor Cell Leukocyte Labeling of CTC and Blood Cells

  17. Magnetic Cell Presentation Analysis Cartridge Trajectory of magnetically labeled objects

  18. CellSpotter Analyzer 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

  19. Composite Leukocyte Control Cytokeratin Nucleus Intact Tumor Cells Analysis of Enriched CTC

  20. Generalizability Clinical Cancer Research 10, 2004

  21. Generalizability Clinical Cancer Research 10, 2004

  22. The Current CTC Paradigm for Metastatic Carcinoma

  23. Treatment Efficacy in Metastatic Breast Cancer

  24. 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

  25. Number of CTC Before New Therapy Predicts Progression Free Survival and Overall Survival

  26. Number of CTC at First Follow Up Predicts Progression Free Survival and Overall Survival

  27. Number of CTC at First Follow Up Predicts Progression Free Survival and Overall Survival

  28. 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)

  29. Lung Cancer As a Model for Personalized Medicine

  30. 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.

  31. 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)

  32. Characteristics of Pts. Likely to Respond to EGFR Inhibitors

  33. Types of EGFR Mutations in Responders

  34. Cell Growth Action of EGFR Inhibitors Apoptosis

  35. Cell Growth Resistance to EGFR Inhibitors

  36. EGFR Mutation Analysis using Circulating Tumor Cells NEJM, 2008

  37. A Short Digression

  38. Variations on a CTC Theme Nature 2007;450: 1235

  39. Variations on a CTC Theme Nature 2007;450: 1235

  40. Now Back to our Story…

  41. Direct Sequencing of EGFR from Isolated CTCs

  42. Cell Growth Cell Growth Resistance to EGFR Inhibitors

  43. FISH on Lung CTCs Green is CEP 7, Red is EGFR, Blue is MET

  44. Polysomy/polyploidy

  45. CTC FISH Data Analysis

  46. CTC FISH Data Analysis

  47. 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

  48. Why Cytology? Clinical Cancer Research 10, 2004

  49. 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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