1. CirculatingTumor Cells: Toward a clinical benefit? Giuseppe Naso MD, PhD, Associated Professor of Medical Oncology Director of Traslational Oncology Paola Gazzaniga, MD, PhD, University Researcher in Traslational Oncology Sapienza Università di Roma

2. Metastatic Breast Cancer MBC is a heterogeneous condition Treatment is palliative Current clinical, laboratory, and radiological methods inadequate for –Assessment of prognosis –Definition of biology –Measurement of efficacy/treatment benefit

3. Prognostic and Predictive Value of CTCs in MBC Hypothesis Measurement of CTCs in MBC may: –Identify aggressive disease (prognostic value) –Provide early determination of treatment efficacy/benefit (predictive value)

5. Progression-Free Survival Probability of Progression-Free Survival Time From Baseline (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 Log rank P =.0001 7.0 months 2.7 months 5 CTCs/7.5 mL n = 87 (49%) < 5 CTCs/7.5 mL n = 90 (51%) Cristofanilli M, et al. N Engl J Med. 2004;351:781-791. Prognostic Value of Baseline CTC Counts

6. Overall Survival Time From Baseline (Months) 0 2 46 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 Log rank P <.0001 21.9 months 10.9 months 5 CTCs/7.5 mL n = 87 (49%) < 5 CTCs/7.5 mL n = 90 (51%) Cristofanilli M, et al. N Engl J Med. 2004;351:781-791. Prognostic Value of Baseline CTC Counts Probability of Progression-Free Survival

7. 64% 43% < 5 CTCs n = 90 (51%) Cristofanilli M, et al. ASCO 2005. Abstract 524. Probability of Death and Baseline CTC Counts Time From Baseline (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 29% 5 CTCs n = 87 (49%) 53% 12% 19% Probability of Death

8. Change in CTC Count During Therapy Predicts Overall Survival Cristofanilli M, et al. ASCO 2005. Abstract 524. 4 1vs4P <.0001 4> 5 CTCs at all time points 39 (22%) 2 1vs2P =.3188 2vs4 P <.0001 2> 5 at baseline and < 5 CTC at last draw38 (21%) 3 1vs3P =.0014 3vs4P =.0051 2vs3P =.0397 3 5 CTC at last draw 17 (10%) Time From Baseline (Months) 0 2 4 6 8 1012 14161822242628 30 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 1 1<5 CTCs at all time points 83 (47%) Probability of Survival

9. CTC Significance in MBC: Prognostic and Predictive Values Prognosis strongest independent factor –In newly diagnosed disease (first-line treatment) –Over the course of the disease (second-, third-, or higher- line treatment) Prediction predict treatment efficacy at 3-4 weeks May represent a new standard of care

10. 1.Una elevata conta delle CTC (>5/7.5 ml) è associata a ridotta sopravvivenza globale (10 vs 22 mesi) e ridotto intervallo libero da malattia (2 vs 8 mesi) 2. La conta delle CTC è fattore prognostico più affidabile delle indagini radiologiche e dello stato recettoriale nei k mammari 3. 1/3 delle pazienti con k mammario HER-2 negativo hanno CTC HER-2 positive

11. 1.La risposta alla terapia adiuvante è associata ad una riduzione del numero di CTC 2. La presenza di CTC 4 settimane dopo la chirurgia correla con un ridotto intervallo libero da malattia e con ridotta sopravvivenza globale

18. Human equilibrative nucleoside transporter 1 Deoxycytidine kinase (dCK)

25. CK8 Spiked MCF7 cells in 10 ml of blood

29. PRIMERSEQUENCE 53Annealing (t°C) SIZE bp GAPDHupstreamACATGTTCCAATATGATTCC60161 downstreamTGGACTCCACGACGTACTCAG CD 45upstreamCAGCGCTTCCAGAAGGGCTC60 downstreamCCGTGCAGCTCTACGAGAGG CK-8 upstreamACTGAGATCTCAGAGATCAA 60 downstreamAATACTCATGTTCTGCATCC MRP 1upstreamCTGACAAGCTAGACCATGAATGT60353 downstreamTCACACCAAGCCGGCGTCTTT MRP 2upstreamGTGATGGTGCTTGTAATCCCA60277 downstreamTGACCACAGATACCAGGACTG MRP 4upstreamGGATCCAAGAACTGATGAGTTAAT65358 downstreamTCACAGTGCTGTCTCGAAAATAG MRP 5 upstreamCTAGAGAGACTGTGGCAAGAAGAGC68332 downstreamAAATGCCATGGTTAGGATGGC MRP 7upstreamTAGGCACTGACTCTGAACGG60160 DownstreamTTGTTGACGGGTACCAGCAG dCKupstreamTGCAGGGAAGTCAACATT52146 downstreamTCCCACCATTTTTCTGAG hENT 1upstreamGCTTGAAGGACCCGGGGAGC67502 downstreamTGGAGAAGGCAAAGGCAGCCA Sensibilità: bassa espressione MRP Elevata espressione hENT/dCK Resistenza: elevata espressione MRP Bassa espressione hENT/dCK