1. 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

2. Era of Personalized Medicine need an individualized approach to treatment to maximize benefit and minimize costneed an individualized approach to treatment to maximize benefit and minimize cost  assessment of prognosis  measurement of treatment benefit  understanding of tumor biology

3. Biomarker Strategies serial biomarker assessments single biomarker assessment prognosis direct therapy prediction diagnosisprognosis

4. Improving Outcomes the enumeration and characterization of circulating tumor cells (CTCs) are useful in the clinical settingthe enumeration and characterization of circulating tumor cells (CTCs) are useful in the clinical setting alterations in CTC levels CTC phenotype and genotype identification of CTC prognosisprediction diagnosisprognosis direct therapy diagnosisprognosis

5. (Paterlini-Brechot et al. Cancer Letters 2007. 253:180.) Origin of CTCs

6. etiologyetiology  disseminated cancer cells  cancer stem cells  bystander cells rare cells in a dormant, nonproliferative staterare cells in a dormant, nonproliferative state  unaffected by chemotherapy  unrecognized by the host immune system  difficult to isolate Origin of CTCs

7. Isolation of CTCs enrichment detection characterization density gradient centrifugation filtration by size immunomagnetic labeling quantitation of growth factor expression gene expression profiling detection of epigenetic alterations Alix-Panabieres et al. Clin Cancer Res 2008. 14:5013.

8. Detection of CTCs antibody based markersantibody based markers  cytokeratins (CK8, CK18, CK19)  epithelial membrane antigen (EMA)  epithelial cell adhesion molecule (EpCAM) nucleic acid based markersnucleic acid based markers  CK19 by RT-PCR  mammoglobin by RT-PCR  erbB2 by RT-PCR or FISH  EGFR by RT-PCR

9. Available Technologies for Isolation of CTCs

10. Circulating Tumor Cell CK Y EpCAM Nucleus DAPI Anti- CK-PE Y Anti-EpCAMFerrofluid Immunomagnetic Capture Leukocyte CD45 Nucleus DAPI Y Anti - CD45-APC Immunomagnetic Labelingand Immunofluorescent Identification of Cells Immunomagnetic Labeling and Immunofluorescent Identification of Cells

11. Immunomagnetic Capture DAPIcytokeratincontrolCD45composite intact tumor cells

12. (Zheng et al. J Chromatogr A 2007. 1162:154.) Parylene Filter Microdevice

13. (Nagrath et al. Nature 2007. 450:1235.) CTC Microchip

14. Immunomagnetic Labeling: Enumeration in MBC

15. 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%

16. Prevalence of CTCs CTC threshold (per 7.5 mL blood) % patients at or above the CTC threshold 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 1234567 8 9 101112131415 203040 50 100 1000 cancer, baseline (n = 177) 3% 16% 21% 23% 27% 34% 36% 38% 42% 45% 46% 47% 49% 53% 58% 61% 71% cancer, first follow-Up (n = 163) 1% 8% 13% 15% 20% 21% 22% 23% 24% 26% 28% 29% 30% 32% 35% 40% 55% 6% healthy volunteers (n = 145) benign diseases (n = 200) 1% 8%

17. IMC-001 time points imaging blood 0234516 177 patients (223 total)177 patients (223 total) metastatic breast cancermetastatic breast cancer measurable diseasemeasurable disease

18. (Hayes et al. Clin Cancer Res 2006. 12:4218.) IMC-001: CTCs at Baseline Probability of Progression Free Survival n = 177

19. (Cristofanilli et al. N Engl J Med 2004. 351:781.) p value < 0.001 Cox Hazards Ratio = 5.4537 p value < 0.001 Cox Hazards Ratio = 2.4842 IMC-001: CTCs at 1 st Follow-Up n = 177

20. (Cristofanilli et al. N Engl J Med 2004. 351:781.) % probability of PFS time from baseline (weeks) 0 5 10 15 20 2530 3540455560657075 80 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 50 ~7.0 months ~2.1 months ~7.6 months p value < 0.0001 Cox Hazards Ratio = 1.6600 # patients (median PFS) < 5 CTC at baseline & at 1 st follow-up 81 (30.3 weeks) decrease in CTC to < 5 at 1 st follow-up 33 (32.9 weeks) > 5 CTC at 1 st follow-up 49 (8.9 weeks) IMC-001: Changes in CTC n = 177

21. (Cristofanilli et al. J Clin Oncol 2005. 23:1420.) n = 83 IMC-001: CTCs and Imaging

22. (Budd et al. Clin Cancer Res 2006. 12:6403.) n = 138 IMC-001: CTCs and Imaging

23. (Budd et al. Clin Cancer Res 2006. 12:6403.) n = 138 IMC-001: CTCs and Imaging

24. LCCC Validation Study imaging blood 06912152124 cycles 318 74 evaluable patients74 evaluable patients metastatic breast cancermetastatic breast cancer measurable diseasemeasurable disease

25. (Liu et al. J Clin Oncol 2009. 27:5153.) Treatment at Time of CTC Result OR95% CIp-value Overall6.3(3.2, 13)<0.001 Chemotherapy6.3(2.9, 14)<0.001 Endocrine8.9(2.2, 35)0.002 CTCs Drawn at the Time of Radiographic Imaging Treatment at Time of CTC Result OR95% CIp-value Overall4.9(2.2, 118)<0.001 Chemotherapy6.9(3.0, 16)<0.001 Endocrine2.9(0.6, 14)0.2 CTCs Drawn 7-9 Weeks Prior to Radiographic Imaging Treatment at Time of CTC Result OR95% CIp-value Overall3.1(1.6, 5.8)0.001 Chemotherapy2.7(1.2, 6.3)0.02 Endocrine5.2(1.2, 23)0.03 CTCs Drawn 3-5 Weeks Prior to Radiographic Imaging LCCC: CTCs and Imaging

26. 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

27. Conclusions Conclusions

28. meaningful rate of detectionmeaningful rate of detection reliable thresholdreliable threshold correlation with clinical outcomes (validity)correlation with clinical outcomes (validity) ability to improve clinical outcomes (utility)ability to improve clinical outcomes (utility) Response/Futility Marker

29. 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 (?????)

30. blood drawn at baseline prior to first-line chemotherapy Arm B maintain first-line chemotherapy until progression Arm C1 maintain first-line chemotherapy until progression Arm C2 switch to alternate chemotherapy Arm A monitor for PFS & OS eligible for other first- line chemotherapy trials R CTC <5CTC ≥5 blood drawn three weeks after the first chemotherapy dose CTC ≥5CTC <5 target n = 120 Prospective Validation: Means to Improve Survival SWOG S0500

31. Prospective Validation: The Liquid Biopsy nucleuscytokeratincontrolleukocytecomposite HER2 and SE17 FISH Probes

32. Prospective Validation: The Liquid Biopsy CALGB 40601 blood 0468101416 weeks 212 paclitaxel and trastuzumab and lapatinib N = 400 paclitaxel and trastuzumab paclitaxel and lapatinib tissue

33. (Harris et al.J Clin Oncol 2007.33:5287.) ASCO Tumor Marker Guidelines Circulating tumor cell assays as markers for breast cancerCirculating tumor cell assays as markers for breast cancer

34. THANK YOU