1. Circulating Tumor Cells and Their Prognostic and Predictive Value in Breast Cancer Massimo Cristofanilli, M.D., F.A.C.P. Professor and Chairman Medical Oncology G. Morris Dorrance Jr. Endowed Chair in Medical Oncology VIII Simposio Internacional GEICAM A Coruña 31/03-01/04 2011

2. Introduction Prognostic and predictive value of CTCs enumeration (CellSearch®) in MBC Clinical utility of CTCs monitoring Molecular characterization of CTCs and CTCs targeted therapies

3. CellSearch Technology A novel concept in oncology or simply another marker?

4. CellSearch  Technology

5. CellTracks ® Technology Standardized Enumeration of CTC

6. Allard, W. J. et al. Clin Cancer Res 2004;10:6897-6904 Circulating Tumor Cells (CTCs) in Advanced Epithelial Malignancies

7. CTCs in MBC

8. Prognostic Value of Baseline CTC Counts Progression-Free Survival Cristofanilli M, et al. N Engl J Med 2004;351:781-791. % 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%)

9. Prognostic Value of Baseline CTC Counts Overall Survival Cristofanilli M, et al. N Engl J Med 2004;351:781-791. % Probability of 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%)

10. CTCs and outcome during the Course of Therapy Progression Free SurvivalOverall Survival Hayes, DF, Clin Cancer Res. 2006,12:4218-24.

11. CTCs, Imaging and Prognosis Budd, G. T. et al. Clin Cancer Res 2006;12:6403-6409

12. CTCs Functional Imaging And Prognosis De Giorgi et al, J Clin Oncol, 2009 B 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 0246810121416182022242628303234363840 Probability of Overall Survival Time from Midtherapy Draw in Months Curve Logrank Comparison p-Value* 1 vs.2 1 3 1 4 2 3 2 4 3 4 0.0018 <0.0001 0.1073 0.2244 0.6441 Midtherapy Median OS in 1 <5 CR/PR/SD 59 (58%) ----- (25.6 to ------) 2 <5 PD 22 (21%) 14.7 (7.3 to -------) 3>5 CR/PR/SD4 (4%) 4.9 (1.5 to -------) 4 >5 PD 17 (17%) 9.8 (5.3 to -------) Group CTC FDG-PET/CT N (%) Months (95% C.I.) *p-values not adjusted for multiple hypothesis tests 1 2 3 4

13. Validation Prognostic Value Baseline CTCs in newly diagnosed MBC Dawood S et Cancer, 113(9):2422-30, 2008

15. CTCs and metastatic disease sites

16. Circulating tumor cell (CTC) count relationship with bone metastases. De Giorgi U et al. Ann Oncol 2009;21:33-39 ©

17. Ability of Site(s) of Metastasis to Predict Overall Survival in 195 Metastatic Breast Cancer Patients Probability of Overall Survival

18. Circulating tumor cells as predictor of metastatic disease spread in patients with breast cancer M. Giuliano 1,2¥, A. Giordano 1,2, B.C. Handy 1, N. T. Ueno 1, E. Andreopoulou 1, R.H. Alvarez 1, J.M. Reuben 1, V. Valero 1, G.N. Hortobagyi 1, M. Cristofanilli 3 1. M.D. Anderson Cancer Center, Houston, TX, US. 2. University of Naples Federico II, Italy. 3. Fox Chase Cancer Center, Philadelphia, PA, US. ¥ Currently at Baylor College of Medicine, Houston, TX, US.

19. To demonstrate correlations between CTC detection, metastatic site development and and prognosis. We hypothesized that baseline detection of ≥ 5 CTCs before starting systemic treatment could predict the development of new metastatic sites, when progression of disease occurs. Study Aims

20. Group Overall N (%) CTCs < 5CTCs ≥ 5 P Progression in pre- existing site(s) N (%) Development of new metastatic site(s) N (%) Progression in pre- existing site(s) N (%) Development of new metastatic site(s) N (%) Overall population 408 (100) 191 (78.3)53 (21.7)112 (68.3)52 (31.7).028 Bone metastases without visceral involvement 93 (22.8)35 (76.1)11 (23.9)26 (55.3)21 (44.7).049 Bone with or without visceral metastases 280 (68.6) 113 (79.6)29 (20.4)94 (68.1)44 (31.9).03 No bone involvement 128 (31.4) 78 (76.5)24 (24.5)18 (69.2)8 (30.8).455 Only visceral metastases 84 (20.6)53 (79.1)14 (20.9)11 (64.7)6 (35.3).219 Rates of development of new metastatic sites according to baseline CTC value, with stratification based on original site of disease

21. Lymph nodes/soft tissues Bone Visceral organs CTCs < 5 N= 53 CTCs ≥ 5 n= 52 Paerson Chi-Square P=.047 Organ distribution of new metastases in patients who developed new metastatic sites (n= 105), stratified by baseline CTC value. Patients with CTCs ≥ 5 developed significantly more metastases in visceral organs, with less metastases in other organs (lymph nodes/soft tissues, bone) compared to those with CTCs < 5. Fifteen patients out of 52 (28.8%) with CTCs ≥ 5 developed new brain metastases, compared with 7 out of 53 (13.2%) with CTCs < 5 (Fisher’s Exact test P=.058). %

22. CTCs < 5 n= 191 CTCs ≥ 5 n= 112 CTCs < 5 n= 69 CTCs ≥ 5 n= 35 P=.001 A. Type of disease progression in the original metastatic sites (increase in size vs new lesion) in patients who did not develop new metastatic sites (n= 303). B. Rates of development of new metastatic lesions (either in pre-existing sites or at new sites) in patients with oligometastatic disease (pre- existing metastases limited to a single organ) n= 104. A Dimensional increase of pre-existing lesion(s) alone Development of new metastatic lesion(s) B P=.031

23. CTCs, molecular subtypes and therapies and metastatic disease sites

24. HR+/HER2- (N= 290)HR+/HER2+ (N= 49) Triple Negative (N= 124)HR-/HER2+ (N= 51) ≥ 5 CTCs (n=128) ~ 20.5 months < 5 CTCs (n=162) ~ 34 months < 5 CTCs (n=33) ~ 19.7 months ≥ 5 CTCs (n=18) ~ 23 months < 5 CTCs (n=82) ~ 17.8 months ≥ 5 CTCs (n=42) ~ 11.1 months < 5 CTCs (n=32) N/A ≥ 5 CTCs (N=17) N/A Log Rank P <.001* Log Rank P =.064 Log Rank P =.899Log Rank P =.01*

25. PFS HER2+ Patients (N=40) receiving anti-HER2 treatment* Log Rank P =.839 < 5 CTCs (n=26) ~ 15.9 months ≥ 5 CTCs (n=14) ~ 16.1 months CTCs in HER2+ Patients (N=29) before and after 1 ST line anti-HER2 treatment HER2 positive Patients (chemo-naïve) * Trastuzumab/Lapatinib

26. Cox Regression Analysis Covariates Hazard Ratio (95% CI) P CTCs ≥5 vs <5 1.978 (1.471-2.660) <0.001* ER Positive vs Negative.485 (.328-.717) <0.001* PR Positive vs Negative.944 (.632-1.411) 0.78 Her2/neu Positive vs Negative.610 (.420-.885) 0.009* Visceral Metastasis vs Other 1.751 (1.183-2.592) 0.005* # Metastatic Sites (1 vs 2 vs ≥ 3) 1.517 (1.207-1.907) <0.001*

27. Clinical value of circulating tumor cells (CTcs) in first-line metastatic breast cancer patients according to type of treatment and immunohistochemical molecular subtype A. Giordano 1,2, M. Giuliano 1,2, B.C. Handy 1, N. T. Ueno 1, E. Andreopoulou 1, R.H. Alvarez 1, J.M. Reuben 1, V. Valero 1, G.N. Hortobagyi 1, M. Cristofanilli 3 1. M.D. Anderson Cancer Center, Houston, TX, US. 2. University of Naples Federico II, Italy. 3. Fox Chase Cancer Center, Philadelphia, PA, US.

28. To evaluate the differential effect of various types of 1 st line therapies (hormonal therapy, HTx; chemotherapy, CTx; anti- HER2 therapy; CTx plus Bevacizumab; mono-chemotherapy) on CTC enumeration (post-treatment CTC). We compared CTC modifications within immunohistochemical (IHC) defined subtypes of breast cancer at radiological documented progression of disease (progression CTC). Study Aims

29. CTCs evaluation in 1 st line MBC patients (N = 235) Baseline CTC value: assay taken within 30 days before starting therapy; Post-treatment CTC value: the lowest CTC value after starting therapy; Progression CTC value: assay taken within 30 days before documented radiological progression of disease (PD). First-line Treatment for MBC Basaline CTC Post-treatment CTC Progression CTC PD 235 (100%)161 (69%)105 (59%) N = 179 N = 235

30. HTx (n = 25) P =.68 Anti-HER2 (n = 30) P <.0001 Poli-CTx (n = 59) P <.0001 Mono-CTx (n = 16) P <.0001 Bevacizumab + CTx (n= 31) Overall (n = 161) P <.0001 Paired baseline and post-treatment CTC value according type of treatment

31. Time-related clinical trend of CTC in immunohistochemical (IHC) subtypes of breast cancer Baseline CTC Post- Treatment CTC Progression CTC Baseline CTC Post- Treatment CTC Progression CTC Baseline CTC Post- Treatment CTC Progression CTC Baseline CTC Post- Treatment CTC Progression CTC Overall (n = 87) P <.05 HR+/HER2- (n = 47) P <.05 HER2+ (n = 11) P <.05 ns TNBC (n = 29) P <.05

32. San Antonio Breast Cancer Symposium - Cancer Therapy and Research Center at UT Health Science Center – December 8-12, 2010 Prognostic & predictive value of Circulating Tumor Cells (CTC) in metastatic breast cancer patients treated by 1 st line chemotherapy Jean-Yves Pierga 1,2,Thomas Bachelot 3, Suzette Delaloge 4, Etienne Brain 5, Mario Campone 6, Bernard Asselain 1, Claire Mathiot 1, David Hajage 1, François-Clément Bidard 1 1 Institut Curie, Paris, France 2 Université Paris Descartes, Paris, France 3 Centre Léon Bérard, Lyon, France 4 Institut Gustave Roussy, Villejuif, France 5 Institut Curie, Saint Cloud, France 6 Centre René Gauducheau, Nantes, France IC 2006-04 study

33. Main objective To test if CTC decrease at the 2 nd cycle of chemotherapy (C2) is associated with PFS and OS (threshold ≥5 CTC /7.5ml)  N= 216 evaluable patients required Secondary objectives Prognostic value of CTC at baseline (PFS, OS) at clinical and imaging evaluation (C3/C4) Comparison with serum tumor markers (CA 15.3, LDH & CEA) RECIST response criteria To challenge the ≥5 CTC threshold CTC HER2 expression (not shown) CTC at radiological progression (not shown) IC 2006-04 study (NCT00898014)

34. Inclusion criteria Metastatic breast cancer patients prior to the first line of chemotherapy with evaluable or mesurable disease (prior hormonal treatment was allowed) Study Design Cycle 1 Cycle 2 Cycle 3 Cycle 4 OR CTC Markers Biology Imaging (RECIST) progression OR 1 st line CTC results were not disclosed to clinicians 5 French Cancer Centers

35. Median follow-up: 15 months Progression-Free Survival Progressions: N=162 (61%) Median PFS: 11 months Overall Survival Deaths: N= 57 (21%) Median OS: not reached First line chemotherapy regimen Taxanes (81%) Anthracyclines (11%) 5-fluorouracil (9%) Vinorelbine (3%) Targeted therapy Anti-HER2 therapies (17%) Bevacizumab (47%) 267 patients were included (June 2007 – Sept 2009) IC 2006-04 study (NCT00898014)

36. CTC detection at baseline & patients characteristics Patients characteristics’N (%)% with ≥ 5CTCP value TOTAL26744%- Premenopausal113 (43%)45% Post menopausal148 (57%)43%0.78 PS=0123(48%)29% PS >0132 (52%)57%<0.0001 Grade 1-2132 (52%)43% Grade 3123 (48%)45%0.64 HR+ / HER2-159 (62%)46% HER2+45 (17%)34%0.36 Triple neg.54 (21%)46% Liver metastasis -156 (60%)39% Liver metastasis +93 (40%)58%0.03 Bone metastasis -109 (42%)28 % Bone metastasis +150 (58%)56%<0.0001 <3 met. sites155 (59%)39% ≥3 met. sites106 (41%)52%0.03

37. CTC detection at baseline: correlation with serum markers Serum markerN (%)% with ≥ 5CTCP value TOTAL26744%- CA15.3 ≤N88 (36%)26% CA15.3 >N159 (64%)53%<0.0001 CEA ≤N103 (49%)35% CEA >N109 (51%)49%0.03 LDH ≤N121 (55%)25% LDH >N99 (45%)67%<0.0001 ALP ≤N170 (71%)36% ALP >N71 (29%)65%<0.0001

38. Baseline CTC count was associated with PFS p<0.0001

39. Baseline CTC count was associated with OS p<0.0001

40. Baseline prognostic factors: PFS Multivariate analysis Prognostic factors RRCI(0.95)P value CTC <51.00 CTC ≥51.901.2 - 2.80.002 CEA ≤N1.00 CEA >N2.021.2 - 3.10.002 HR+ and/or HER2+ 1.00 Triple neg.4.162.4 - 7.0<0.0001 PS = 01.00 PS > 02.221.4 - 3.40.0002

41. Baseline prognostic factors: OS Multivariate analysis Prognostic factors RRCI(0.95)P value HR+ and/or HER2+ 1.00 Triple negative 4.312.0 - 8.80.0001 PS = 01.00 PS > 02.671.1 - 6.50.02 CTC <51.00 CTC ≥52.411.1 - 5.30.02

42. CTC changes between baseline & before C2 Progression-Free Survival Main study endpoints: PFS p=0.04; OS p=0.01 p<0.0001 Overall Survival p <0.0001

43. CTC changes differed according to treatment Nb pts with ≥5 CTC at baseline % with ≥5 CTC before C2 % with ≥ 5 CTC before C3/4 Chemo. aloneN= 3947%38% Chemo. + bevacizumab N= 6036%23% Chemo. + anti-HER2 therapy N= 1517%0% Among patients with ≥5CTC at baseline, a stronger CTC decrease was observed with targeted therapies

44. Molecular Characterization of CTCs Moving forward

45. GeparQuinto – Decision Tree HER2-positive? HER2+: Trastuzumab Lapatinib no HER2-:Bevacizumab Everolimus (RAD001) yes Predictive factors for these new treatment are required… CTC ? CEC ?

46. HER2 expression in CTC and corresponding primary breast tumours Primary tumors CTCHER2-negativeHER2-positive Before NT After 4 cycles Before surgery Before NT After 4 cycles Before surgery Analyzed CTC- positive cases (n) 5231 1955 HER2-negative (0 or 1+) 37 (71.1%) 21 (67.7%) 25 (80.6%) 6 (31.6%) 3 (60%) HER2 equivocal (2+) 8 (15.4%) 7 (22.6%) 1 (3.2%) 3 (15.8%) 2 (40%) HER2-strongly positive (3+) 7 (13.5%) 3 (9.7%) 5 (16.1%) 10 (52.6%) 00 * No CTC with strong HER2 expression (3+) after Herceptin or Lapatinib treatment 00 * Discrepancy in HER2 status between primary tumors and CTC 7 (13.5%) 3 (9.7%) 5 (16.1%) 6 (31.6%) 3 (60%) 3 (60%)

47. FISH analysis of Circulating Tumor Cells

48. Correlation between HER2 overexpression and HER2 amplification HER2: 1+ HER2: 2+ HER2: 3+ Non amplified Non amplified Amplified Meng et al. Proc Natl Acad Sci U S A. 2004;101:9393-8. CK-FITC HER2- TRChr 17 / HER2

49. FISH & Circulating Tumor Cells Breast Cancer A CTC isolated from a patient with metastatic breast cancer. After counting the sample was dried and fixed inside the chamber and later assayed using the Repeat-free HER2 / SE 17 FISH kit. Cytokeratin-PE & DAPI HER2 SE17 CK-PEDAPI (nucleus)

50. Blood drawn at baseline prior to chemotherapy Arm A (51 pts) HER-2 negative Primary and/or mets Arm B (25 pts) HER-2 positive Primary and/or mets CTCs-HER2 CTC ≥ 5 Blood drawn 3 weeks after first chemotherapy dose

51. Blood drawn at baseline prior to chemotherapy HER-2 negative disease Arm A Standard Therapy + Trastuzumab Arm B Standard Therapy alone CTCs-Targeted Therapy CTC +/HER2+ Randomization Primary_PFS Secondary-OS CTCs

52. DNA,methylated genes and CTCs Van der Auwera I, et al. Br J Cancer. 2009;100(8):1277-86.

53. DNA,methylated genes and CTCs

54. Schematic of the MagSweeper process. Talasaz A H et al. PNAS 2009;106:3970-3975 The MagSweeper

55. Functionality of isolated cells Ameri et al, BJC 2010

56. Single cell samples vs Pooled sample Scatter plot of gene expression levels of whole transcriptome of each individual cell (FCC7, FCC8, FCC9) vs profile of pool of 3 cells (FCC7-9 AVG)

57. Gene Concordance FCC7 FCC8FCC9 39384680 4885 1029 544 509 1559 1788 13261548 FCC7 FCC8 FCC9 Pool 2939 2409 3097 1946 1529 1583 4967 Concordance of the genes detected from each individual cell Concordance of the genes detected from each individual cell and pool of cells

58. Unassisted Clustering H2O Cell type O Cell type S Clearly breast cancer cells cluster together and separately from other type of CTCs

59. AdnaTest

60. Comparison between circulating and disseminated tumor cells in breast cancer BM neg (%) BM pos (%) Total (%) Blood neg175 (78) 50 (22)225 (100) Blood pos 28 (67) 14 (33)42 (100) Total203 (76) 64 (24)267 (100) *p=0.11 2 Fehm T et al. Breast Cancer Res 11(4):R59., 2009

61. Comparison between circulating tumor cells and primary tumor in breast cancer

62. Expression of ALDH1 and EMT markers in metastatic breast cancer patients undergoing chemo-, hormonal– or trastuzumab therapy Aktas B, et al SABC 2008

63. HER2 levels in tumor-initiating cells of carcinoma cell lines Magnifico A et al. Clin Cancer Res 2009;15:2010-2021

64. Role of Notch signaling on HER2 expression of tumor- initiating cells Magnifico A et al. Clin Cancer Res 2009;15:2010-2021

65. Progression of Cancer EMT/MET play important role during metastasis (The biology of Cancer (Garland Sciences 2007) Epithelial-Mesenchymal Transition (EMT) Mesenchymal-Epithelial Transition (MET) E-cadherin  -catenin  -catenin EpCAM Vimentin Fibronectin N-cadherin E-cad - CK+/- EpCAM –

66. The epithelial-mesenchymal transition generates cells with properties of stem cells Mani SA et al, Cell 133(4):704-15,2008

67. Heterogeneity of CTC Mego et al. Int J Cancer. 2011

68. Heterogeneity of CTC Mego, M., Mani S. A. & Cristofanilli, M. (2010), Nat. Rev. Clin. Oncol.

69. Conclusions  CTCs in patients with MBC An independent prognostic factor Should be used for risk stratification and therapeutic monitoring Phenotypic analysis of CTCs suggests an heterogeneous but independent phenotype Evaluation of other technology platforms for molecular characterization  Future Prospective studies: Further characterize CTC phenotype, genotype (stem cells markers, HER-2) Prospective multicenter studies to validate the concept of CTCs targeted therapies

70. Acknowledgments MD Anderson Cancer Center Fox Chase Cancer Center James M Reuben, PhD Kathy R Alpaugh, PhD Naoto T Ueno, MD, PhD Ramona Swaby, MD Anthony Lucci, MD Kevin Johnson, PhD Savitri Krishnamurthy, MD Zhaomei Mu, MD Sendurai Mani, PhD Catherine A Bingham, PhD Balraj Singh, PhD Wendy Woodward, MD, PhD Herbert Fritsche, PhD Yang, Li-Ying, PhD