Supplementary appendix

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1 Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Bidard F-C, Peeters DJ, Fehm T, et al. Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol 2014; published online March S (14)

2 Supplementary Table 1: Studies included (*): these studies were multicentric. For some studies, median follow-up could not be calculated («NC») with the reversed Kaplan-Meier methods since too many events occurred. Assessment of PFS data: «RECIST» indicates central review of all imaging studies by the coordinating institution of that study; signifies investigator-reported outcomes according to the respective study protocols and/or local clinical practice. References for published studies are below: 1 Peeters DJE, Van den Eynden GG, van Dam P-J, et al. Circulating tumour cells in the central and the peripheral venous compartment in patients with metastatic breast cancer. Br J Cancer 2011; 104: Peeters DJE, van Dam PJ, van den Eynden GGM et al. Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes. Br J Cancer Consoli F, Grisanti S, Amoroso V, et al. Circulating tumor cells as predictors of prognosis in metastatic breast cancer: clinical application outside a clinical trial. Tumori 2011; 97: Ignatiadis M, Rothé F, Chaboteaux C, et al. HER2-positive circulating tumor cells in breast cancer. PLoS ONE 2011; 6: e Dawson S-J, Tsui DWY, Murtaza M, et al. Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer. N Engl J Med doi: /nejmoa Androulakis N, Agelaki S, Perraki M, et al. Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer. Br J Cancer 2012; 106: Martín M, Custodio S, de Las Casas M-LM, et al. Circulating tumor cells following first chemotherapy cycle: an early and strong predictor of outcome in patients with metastatic breast cancer. Oncologist 2013; 18: Munzone E, Botteri E, Sandri MT, et al. Prognostic value of circulating tumor cells according to immunohistochemically defined molecular subtypes in advanced breast cancer. Clin Breast Cancer 2012; 12: Bidard F-C, Mathiot C, Degeorges A, et al. Clinical value of circulating endothelial cells and circulating tumor cells in metastatic breast cancer patients treated first line with bevacizumab and chemotherapy. Ann Oncol 2010; 21: Pierga J-Y, Hajage D, Bachelot T, et al. High independent prognostic and predictive value of circulating tumor cells compared with serum tumor markers in a large prospective trial in first-line chemotherapy for metastatic breast cancer patients. Ann Oncol 2012; 23: Pierga J-Y, Bidard F-C, Cropet C, et al. Circulating tumor cells and brain metastasis outcome in patients with HER2-positive breast cancer: the LANDSCAPE trial. Ann Oncol doi: /annonc/mdt Hartkopf AD, Wagner P, Wallwiener D, Fehm T, Rothmund R. Changing levels of circulating tumor cells in monitoring chemotherapy response in patients with metastatic breast cancer. Anticancer Res 2011; 31: Müller V, Riethdorf S, Rack B, et al. Prognostic impact of circulating tumor cells assessed with the CellSearch System TM and AdnaTest Breast TM in metastatic breast cancer patients: the DETECT study. Breast Cancer Res 2012; 14: R118. 1

3 Center Antwerp (BE) (*) Barcelona (SP) Brescia (IT) Brussels (BE) Cambridge (UK) Cremona (IT) Heraklion (GR) London (UK) Madrid H12O (SP) Madrid SC (SP) Milan (IT) Padova (IT) Pamplona (SP) Paris (FR) (*) Paris (FR) (*) Paris (FR) (*) Roma (IT) Tübingen (DE) Tübingen (DE) (*) Valencia (SP) N pts (%) 243 (12 5%) 24 (1 2%) 104 (5 3%) 19 (1 0%) 70 (3 6%) 85 (4 4%) 141 (7 3%) 81 (4 2%) 36 (1 9%) 82 (4 2%) 203 (10 3%) 31 (1 6%) 27 (1 4%) 63 (3 2%) 260 (13 4%) 41 (2 1%) 66 (3 4%) 45 (2 3%) 168 (8 6%) 155 (8 0%) Study Peeters ,2 & unpublished CTC data available Baseline / Week 3-5 / Week 6-9 CA 15.3 data available Baseline / Week 3-5 / Week 6-9 CEA data available Baseline / Week 3-5 / Week /46/34 237/106/109 24/9/3 Unpublished 24/0/0 20/4/1 19/4/1 Consoli, & unpublished 104/14/9 60/12/10 52/10/9 Ignatiadis, /0/0 8/4/1 0/0/0 Dawson, & unpublished 70/10/22 1/1/0 1/1/0 Unpublished 85/84/84 85/85/85 85/85/85 Androulakis, & unpublished 141/0/0 0/0/0 0/0/0 Unpublished 81/12/3 52/14/1 1/0/0 Unpublished 36/24/5 28/22/6 19/17/3 Martin, & unpublished 82/62/9 32/26/4 33/25/4 Munzone, /97/38 183/89/36 179/81/36 Unpublished 31/2/2 22/ 2/0 21/2/0 Unpublished 27/0/0 19/3/6 6/1/1 Bidard, /0/40 51/0/38 0/0/0 Pierga, /193/81 221/191/67 192/164/45 Pierga, /36/0 0/0/0 0/0/0 Unpublished 66/2/16 27/ 6/9 28/7/8 Hartkopf, /0/5 44/0/5 44/0/5 Müller /0/0 55/0/3 35/0/3 Unpublished 155/90/84 154/91/83 154/90/83 PFS data events/ available (median follow-up) Assessment 190/243 (28 months) 16/24 (NC) 88/104 (36 months) 15/19 (NC) 60/70 (15 months) 63/85 (22 months) 118/141 (42 months) 71/81 (19 months) 8/36 (2 months) 68/82 (45 months) 203/203 (NC) 24/31 (38 months) 18/27 (33 months) 26/63 (10 months) 205/260 (23 months) RECIST 40/41 (NC) RECIST 50/66 (17 months) 0/0-115/168 (16 months) 129/155 (39 months) OS data events/ available (median follow-up) 121/243 (30 months) 14/24 (40 months) 57/104 (24 months) 14/19 (NC) 45/70 (17 months) 28/85 (19 months) 68/141 (37 months) 69/81 (61 months) 0/36 (2 months) 40/82 (46 months) 158/203 (55 months) 24/31 (44 months) 11/27 (17 months) 12/63 (10 months) 55/260 (17 months) 24/41 (22 months) 32/66 (18 months) 18/45 (16 months) 52/168 (15 months) 87/155 (23 months) 2

4 Supplementary Tables 2A-C: Clinical pathological prognostic models HR+: hormone positive; HER2+: HER2 positive; TNeg: triple negative; chemot: chemotherapy; hormonet: hormone therapy; PS: performance status; CNS: central nervous system. Synchronous metastases: patient with de novo stage IV breast cancer at diagnosis. As defined in Cristofanilli et al 5, visceral metastasis included any of the following sites: lung, pleura, liver, adrenal gland, brain, kidney, pancreas and/or peritoneal involvement. Supplementary Table 2A: Unadjusted hazard ratio (HR) for progression-free survival (PFS) Prognostic factor tested Tumor subtype (HR+/HER2+/TNeg) Tumor grade (I/II-III) N previous chemot lines (0/1/ 2) N previous hormonet lines (0/ 1) PS (continuous) Age (<65/ 65 years) Synchronous metastases Metastasis-free interval (0-3/>3 years) Visceral metastasis Number of metastatic sites (<3/ 3) Metastatic site: liver Metastatic site: locoregional Metastatic site: CNS Metastatic site: lung Metastatic site: bone Metastatic site: soft tissue N events / N assessable 1475/1835 HR [95%CI] 0 88 [ ] 1 47 [ ] 1210/ [ ] 1457/ [ ] 2 32 [ ] 1367/ [ ] 1265/ [ ] 1499/ [ ] 1490/ [ ] 1490/ [ ] 1491/ [ ] 1491/ [ ] 1491/ [ ] 1491/ [ ] 1491/ [ ] 1490/ [ ] 1491/ [ ] 1491/ [ ] 3

5 Supplementary Table 2B: Unadjusted hazard ratio (HR) for overall survival (OS) Prognostic factor tested Tumor subtype (HR+/HER2+/TNeg) Tumor grade (I/II-III) N events / N assessable 910/1880 N previous chemot lines (0/1/ 2) 883/1820 HR [95%CI] 0 85 [ ] 1 73 [ ] 737/ [ ] 1 89 [ ] 2 75 [ ] N previous hormonet lines (0/ 1) 837/ [ ] PS (continuous) Age (<65/ 65 years) Synchronous metastases Metastasis-free interval (0-3/>3 years) Visceral metastasis Number of metastatic sites (<3/ 3) Metastatic site: liver Metastatic site: locoregional Metastatic site: CNS Metastatic site: lung Metastatic site: bone Metastatic site: soft tissue 756/ [ ] 927/ [ ] 921/ [ ] 921/ [ ] 924/ [ ] 920/ [ ] 924/ [ ] 924/ [ ] 924/ [ ] 972/ [ ] 924/ [ ] 924/ [ ] 4

6 Supplementary Table 2C: final clinical pathological prognostic models for PFS and OS. PFS N=1189 pts OS N=1501 pts Prognostic factor multivariate HR P value multivariate HR P value Primary tumor characteristics Tumor subtype (HR+/HER2+/TNeg) 0 90 [ ] 1 79 [ ] 0 25 < [ ] 1 76 [ ] < < Tumor grade (I/II-III) 1 35 [ ] 0 02 Previous treatments N previous chemot lines (0/1/ 2) 1 36 [ ] 2 00 [ ] < [ ] 2 31 [ ] < < N previous hormonet lines (0/ 1) 1 33 [ ] General condition PS (continuous) Age (<65/ 65 years) 1 56 [ ] < [ ] < [ ] Metastases characteristics Synchronous metastases Metastasis-free interval (0-3/>3 years) Visceral metastasis 0 77 [ ] [ ] [ ] Metastatic site: liver Metastatic site: CNS 1 29 [ ] [ ] < [ ] <

7 Supplementary Tables 3A-B: subgroup analyses at week 3-5 HR: hazard ratio. The reported number of patients starting a treatment regimen that includes anti-her2 drug is lower than the reported number of patients with HER2-positive breast cancers. This discrepancy is partly due to patients with known tumor subtype but unknown treatment regimen; Among the 74 HER2-positive breast cancer patients with no anti-her2 treatments and known treatment line, 40 of them (54%) were starting a third (or more) line of treatment. 6

8 Supplementary Table 3A: Univariate Cox regression of OS on CTC positivity status at week 3-5. Modality Number of Patients Number of events HR for 5 CTC 95% CI Interaction test (p value) Performance status at inclusion 0 07 Full model or Tumor Subtype 0 32 Full model HER HR Triple Negative Number of metastatic sites 0 97 Full model sites < 3 sites Metastatic sites 0 19 Full model Bone only Any CNS metastasis Other Number of previous treatment lines (hormone therapy or chemotherapy) 0 47 Full model or more Treatment initiated: including chemotherapy 0 25 Full model Yes No Treatment initiated: including hormone therapy 0 34 Full model Yes No Treatment initiated: including any targeted therapy 0 48 Full model Yes No Treatment initiated: including bevacizumab 0 54 Full model Yes No Treatment initiated: including anti-her2 targeted therapy 0 20 Full model Yes No

9 Supplementary Table 3B: Univariate Cox regression of PFS on CTC positivity status at week 3-5. Modality Number of Patients Number of events HR for 5 CTC 95% CI Interaction test (p value) Performance status at inclusion 0 32 Full model or NA Tumor Subtype 0 93 Full model HER HR Triple Negative Number of metastatic sites 0 50 Full model sites < 3 sites Metastatic sites 0 15 Full model Bone only Any CNS metastasis Other Number of previous treatment lines (hormone therapy or chemotherapy) 0 47 Full model or more Treatment initiated: including chemotherapy 0 15 Full model Yes No Treatment initiated: including hormone therapy 0 04 Full model Yes No Treatment initiated: including any targeted therapy 0 20 Full model Yes No Treatment initiated: including bevacizumab 0 47 Full model Yes No Treatment initiated: including anti-her2 targeted therapy 0 42 Full model Yes No

10 Supplementary Table 4: Evaluation of added prognostic information of CTC and serum markers at baseline and during treatment ULNV: upper limit of normal value. CTC BL : CTC count at baseline; CA15-3 BL : CA 15-3 level at baseline; CEA BL : CEA level at baseline. CTC 3-5 : CTC count at 3 to 5 weeks; CA : CA 15-3 level at 3 to 5 weeks; CEA 3-5 : CEA level at 3 to 5 weeks. Model 1 Model 1 average c-index Model 2 Model 2 average c-index Average c-index increase (Model 2- Model1) Average increase (chi square) Degree of freedom Likelihood ratio test p value Progression-Free Survival, CA15-3 at baseline (N= 914 patients) CP CP ( or > ULNV) CP CP (splines) CP+CTC BL CP+CTC BL ( or > ULNV) CP+CTC BL CP+CTC BL (splines) [-0 01;-0 003] [-0 012;0 003] [ ;0 001] [-0 01;0 001] 2 65 [0 006;8 7] 7 90 [2 33;16 5] 0 95 [2 33;16 5] 4 7 [0 67;12 7] Overall Survival, CA15-3 at baseline (N= 1124 patients) CP CP ( or > ULNV) CP CP (splines) CP+CTC BL CP+CTC BL ( or > ULNV) CP+CTC BL CP+CTC BL (splines) Progression-Free Survival, CEA at baseline (N= 593 patients) CP CP ( or > ULNV) CP CP (splines) CP+CTC BL CP+CTC BL ( or > ULNV) CP+CTC BL CP+CTC BL (splines) Overall Survival, CEA at baseline (N= 754 patients) CP CP ( or > ULNV) CP CP (splines) [-0 016;0 006] [-0 012;0 006] [-0 007;0 001] [-0 01;0 001] [-0 012;-0 014] [-0 015;0 008] [ ;0 008] [-0 013;0 003] [-0 014;0 020] [-0.012;0 011] 3 94 [0 16;10 8] 9 46 [3 13;18 9] 0 89 [0 002;4 3] 4 4 [0 98;11 7] 4 17 [0 034;12 7] 4 60 [0 67;10 8] 2 38 [0 007;9 8] 2 9 [0 34;8 6] 7 16 [0 82;16 9] 6 03 [1 56;13 2]

11 CP+CTC BL CP+CTC BL ( or > ULNV) [-0 015;0 007] 4 13 [0 06;12 1] CP+CTC BL CP+CTC BL (splines) [-0 013;0 004] 3 97 [0 65;10 1] Progression-Free Survival, CA15-3 changes at weeks 3-5 (N= 357 patients) CP+CTC BL CP+CTC BL +CA [-0 03;0 007] 2 79 [0 11;8 3] CP+CTC BL +CTC CP+CTC BL +CTC 3-5 +CA15-3 +CA [-0 026;0 007] 2 67 [0 96;8 3] Overall Survival, CA15-3 changes at weeks 3-5 (N= 436 patients) CP+CTC BL CP+CTC BL +CA [-0 03;0 0012] 4 82 [0 34;12 3] CP+CTC BL +CTC CP+CTC BL +CTC 3-5 +CA [-0 031;0 012] 4 03 [0 36;10 5] Progression-Free Survival, CEA changes at weeks 3-5 (N= 289 patients) CP+CTC BL CP+CTC BL +CEA [-0 029;0 011] 2 07 [0 04;7 1] CP+CTC BL +CTC CP+CTC BL +CTC 3-5 +CEA [-0 028;0 011] 1 75 [0 05;6 5] Overall Survival, CEA changes at weeks 3-5 (N= 367 patients) CP+CTC BL CP+CTC BL +CEA [-0 03;0 0018] 5 32 [0 59;13 4] CP+CTC BL +CTC CP+CTC BL +CTC 3-5 +CEA [-0 03;0 014] 5 15 [0 51;12 7] Progression-Free Survival, CA15-3 changes at weeks 6-8 (N= 215 patients) CP+CTC BL CP+CTC BL +CA [-0 034;0 021] 1 80 [0 06;5 9] CP+CTC BL +CTC CP+CTC BL +CTC 6-8 +CA [-0 036;0 017] 2 04 [0 05;7 2] Overall Survival, CA15-3 changes at weeks 6-8 (N= 290 patients) CP+CTC BL CP+CTC BL +CA [-0 045;0 024] 3 41 [0 19;9 2] CP+CTC BL +CTC CP+CTC BL +CTC 6-8 +CA [-0 05;0 019] 3 15 [0 12;9 5] Progression-Free Survival, CEA changes at weeks 6-8 (N= 170 patients) Not enough events Overall Survival, CEA changes at weeks 6-8 (N= 217 patients) 10

12 CP+CTC BL CP+CTC BL +CEA [-0 038;0 0015] 1 26 [0 002;5 2] CP+CTC BL +CTC CP+CTC BL +CTC 6-8 +CEA [-0 034;0 009] 1 08 [0 002;4 5]

13 Supplementary Figure 1: Patients repartition Center (Country); Number of individual data contributed; percentage within the study population (19444 patients) 12

14 Supplementary Figures 2A-B: Hazard ratio as a function of baseline CTC Supplementary Figure 2A: Logarithm of hazard ratio as a function of baseline CTC count for progression-free survival, and 95% confidence interval. Supplementary Figure 2B: Logarithm of hazard ratio as a function of baseline CTC count for overall survival, and 95% confidence interval. 13

15 Supplementary Figures 3: Survival according to CTC count changes at 6-8 weeks Supplementary Figure 3A: Progression-Free Survival PFS Cohort Patients Events Median PFS (months) Decrease: >=5 - < [7.6;10.8] Increase: <5 - >= [3.7;12.8] Stable neg.: <5 - < [9.0;14.5] Stable pos.: >=5 - >= [4.2; 7.0] Months Decrease: >=5 - < Increase: <5 - >= Stable neg.: <5 - < Stable pos.: >=5 - >=5 Number at risk 14

16 Supplementary Figure 3B: Overall Survival 1.0 OS Cohort Patients Events Median OS (months) Decrease: >=5 - < [17.0;35.9] Increase: <5 - >= [11.3; NA] Stable neg.: <5 - < [31.3; NA] Stable pos.: >=5 - >= [11.6;17.8] Months Decrease: >=5 - < Increase: <5 - >= Stable neg.: <5 - < Stable pos.: >=5 - >=5 Number at risk 15

17 Supplementary material 1: study protocol EPAC: European pooled analysis of circulating tumor cells studies in metastatic breast cancer Draft v. 2.2 (last revised 01/17/2013) STUDY SECRETARIAT Dr François-Clément Bidard, MD PhD, Institut Curie, FR Stefan Michiels, PhD, Institut Jules Bordet, BE Dr Michail Ignatiadis, MD PhD, Institut Jules Bordet, BE Pr Klaus Pantel, MD PhD, Hamburg-Eppendorf University, GE Pr Jean-Yves Pierga, MD PhD, Institut Curie, FR 1. BACKGROUND Circulating tumor cells (CTCs) represent tumor cells deriving from the primary or secondary (i.e. metastases) tumor sites that can be identified and measured in the blood of patients. Recent technical advances have rendered it easier to reproducibly and repeatedly sample this population of cells with a high degree of accuracy, and these cells represent now an attractive biomarker. CTCs are now generally defined as nucleated cells lacking CD45 and expressing epithelial cytokeratins. Although multiple commercially available methods for isolating CTCs exist, the CellSearch is the only FDA approved system for clinical use with reproducible results across many different laboratories. Briefly, CTCs expressing the epithelial cell adhesion molecule (Ep-Cam) are immunomagnetically enriched and stained with 4,2-diamidino-2-phenylindole dihydrochloride (DAPI) (+), cytokeratin 8,18,19(+) and CD45. Since 2004 and the development of the CellSearch system, several studies have focused on the clinical utility of CTCs count, and especially its predictive and prognostic capability in metastatic breast cancer patients. In the first large, multicentric, prospective study, one hundred and seventy-seven patients with measurable disease had CTCs tested prior to beginning a new treatment regimen for progressive metastatic disease, followed by repeat assessment at first follow-up visit approximately 4 weeks later. This landmark study prospectively identified a CTCs cut-off level of 5 cells per 7.5 ml of blood to be a reliable identifier of patients at higher risk for disease progression and decreased survival from metastatic breast cancer. Additional CTCs assessment in this same patient cohort at essentially monthly intervals following first follow-up also confirmed improved PFS and OS for patients with <5 CTCs at any subsequent time point. Moreover, combining CTCs counts before and after treatment with one cycle of chemotherapy, it was shown that a decline of CTCs <5 cells was associated with a better prognosis and correlated with a response to the chemotherapy regimen. In the United States, this observation has led to the FDA approval for using CTCs as a tool for managing chemotherapy in MBCs. Since then, several prospective and retrospective studies have confirmed the cut-off level of 5 cells as a bad prognosis factor, independently of the choice of the treatment and of the hormone receptor and HER2/neu status. A pooled analysis has been presented at the 2011 ASCO meeting: it took into account published reports before 2010 and some non-published data from American centers. This pooled analysis included the individual data of 841 patients, of which 764 were included in the final multivariate analysis for overall survival. Among these patients, only 143 (18%) were treated in Europe. This pooled analysis confirmed the interest of CTC as a prognostic marker at baseline, but did not assess the changes under treatment or the comparison with serum tumor markers. The objective of this collaborative analysis is to use the vast amount of data currently available (published or unpublished) in the 51 European centers which are using the CellSearch platform to assess several clinically relevant issues in the current management of metastatic breast cancer patients. 2. OBJECTIVES 2.1 First objective (in relation to mandatory data) To evaluate the prognostic value (before treatment) of baseline CTCs count (/7.5ml) by the CellSearch method in metastatic breast cancer, on progression free-survival (PFS), and on Overall Survival (OS). 2.2 Secondary objectives (in relation to non-mandatory data) 16

18 To evaluate the prognostic value of early (between weeks 3 and 5) and late (between weeks 6 and 9) variations of CTCs count on PFS and OS. To study the correlation between CTCs count and patients clinico-biological characteristics. To check the relevance of the currently proposed threshold of 5 CTCs, for the prognosis and for the early variations during treatment. To compare the clinical validity of CTC changes during treatment, with regards to serum tumor marker changes. To study the CTC prognostic impact in subgroup analyses (according to patient and tumor baseline characteristics; to the number of lines previously received; to the kind of therapy initiated) To establish an easy-to-apply prognostic model for survival of individual patients, based on CTCs. To validate the online prognostic nomogram This study will also allow to report patient cohorts that will not be published otherwise (due to lack of power, lack of time ) and to develop collaborative links between European CTC centers. Also, this database may be used for other objectives that may become relevant in the future (non pre-planned analyses), after approval by the participating centers. 3. STUDY ELIGIBILITY AND IDENTIFICATION 3.1 Trial eligibility criteria Inclusion criteria All study, published or not, answering to the following criteria, will be included in the analysis: - Studies conducted in metastatic breast cancer patients - Studies with data of PFS and/or OS available (retrospectively or prospectively assessed) - Study declared and approved by an adequate ethical committee - Study with CTC count by the Cellsearch method at baseline (before the start of the treatment) - Study conducted in a European center. - Patients accrual initiated in or after 01/ Closed to patient accrual in or before 07/2012 Exclusion criteria - Study in which CTC count was taken into account by clinicians and impacted the patient management 3.2 Study search strategy The aim of the search procedure is to contact all centres with published studies and all centers which potentially have unpublished data. For this purpose, the main approach will be to contact directly investigators at the 51 centers that are using the CellSearch technique in Europe in December These centers will be asked to report any other potentially relevant study they are aware of. To ensure no study is missing, we will identify studies by searching Medline, using mapped terms circulating tumor cells, circulating tumor cell, CellSearch and the exploded MeSH term breast neoplasms, Neoplastic Cells, Circulating. Conference proceedings will be investigated via online websites (American Society of Clinical Oncology San Antonio Breast Conference European Society for Medical Oncology prospective clinical trial registers (clinicaltrial.gov) and scanning the reference lists of key studies and review articles. The search period for published literature is 2004 (based on the first reported study of CTC Cellsearch ) to the end of December No language restrictions will be applied. 3.3 Studies identified Studies identified as of March, 2012 are shown in the appendix 2. It is expected that numerous studies are still unreported. 4. CONTACT WITH AUTHORS AND DATA REQUEST 4.1 Contacts with Authors 17

19 The secretariat of this project will invite principal investigators of all eligible studies to participate in the study. These investigators will be requested to provide individual patient data. If there are duplications of data, we will use the most current data. 4.2 Data items to be requested from principal investigators Individual data & outcome The following data items should be collected in an Excell spreadsheet for all individual patients included in all studies included in this pooled-analysis (note that the codes are provided for the sake of simplicity; any other codes may be used). Baseline patient characteristics A: Trial ID B: Institution ID C: Patient ID D: Patient age E: Tumor estrogen receptor status F: Tumor progresterone receptor status G: Tumor HER2 status H: Tumor grade I: Metastasis-free interval (time from primary tumor diagnosis to metastasis diagnosis, in months) J: Date of first metastatic relapse K: Number of previous chemotherapy lines for metastatic disease before inclusion L: Number of hormone therapy lines for metastatic disease before inclusion Location of metastatic sites at inclusion M: Locoregional N: Lung, O: Liver P: Bone Q: Central nervous system (brain & leptomeningeal mets) R: Soft Tissue S: Other T: Unknown U: Performance status at inclusion V: Date of inclusion (baseline CTC count) W: Result of baseline CTC count (numerical result) Type of the new line of treatment X: hormone therapy Y: chemotherapy Z: anti-her2 targeted therapy AA: bevacizumab AB: other targeted therapy AC: unknown. Baseline counts AD: Date of CEA assessment at inclusion, if any AE: Result of CEA at inclusion, if any AF: local upper limit of normal value for CEA AG: Date of CA 15-3 assessment at inclusion, if any AH: Result of CA 15-3 at inclusion, if any AI: local upper limit of normal value for CA 15-3 AJ: Date of CA assessment at inclusion, if any AK: Result of CA at inclusion, if any AL: local upper limit of normal value for CA nd count during treatment, if any AM: Date of 2 nd CTC count AN: Results of 2 nd CTC count 18

20 AO: Date of 2 nd CEA assessment, if any AP: Result of 2 nd CEA, if any AQ: local upper limit of normal value for CEA AR: Date of 2 nd CA 15-3 assessment, if any AS: Result of 2 nd CA 15-3, if any AT: local upper limit of normal value for CA 15-3 AU: Date of 2 nd CA assessment, if any AV: Result of 2 nd CA 27-29, if any AW: local upper limit of normal value for CA rd count during treatment, if any AX: Date of 2 nd CTC count AY: Results of 2 nd CTC count AZ: Date of 2 nd CEA assessment, if any BA: Result of 2 nd CEA, if any BB: local upper limit of normal value for CEA BC: Date of 2 nd CA 15-3 assessment, if any BD: Result of 2 nd CA 15-3, if any BE: local upper limit of normal value for CA 15-3 BF: Date of 2 nd CA assessment, if any BG: Result of 2 nd CA 27-29, if any BH: local upper limit of normal value for CA Survival endpoints BI: date of tumor progression (write 1 in BJ column) or last follow-up if the patient had no progression (write 0 in BJ column) BJ: progression indicator: see BI BK: date of death (write 1 in BL column) or last follow-up if the patient was still alive (write 0 in BL column) BL: death indicator: see BK Study data The following data items should be collected for all studies to allow for the assessment of differences between those studies: 1. Trial name 2. Country where the trial was conducted 3. The definition of tumor progression in the trial Data can be sent in confidence to the secretariat where they will be held secure: Stefan Michiels, PhD Breast Cancer Translational Research Laboratory Institut Jules Bordet, Bld de Waterloo 125 B-1000 Brussels, Belgium stefan.michiels@bordet.be; stefan.michiels@igr.fr 5. STATISTICAL METHODS Endpoints definition Overall survival (OS) is defined as time from randomisation to death whatever the cause. Patients still alive at the last visit will be censored at the date of last follow-up. Progression-free survival (PFS) is defined as the time from randomisation to the first event: clinical or radiological progression, whichever comes first or death from any cause. Patients without documented evidence of an event will be censored at the date of last follow-up. Cox proportional hazards models will be constructed for PFS and OS. Added prognostic value of the candidate biomarkers (CTCs or serum markers) to the established clinico-biological will be assessed through the use of likelihood ratio and specific measures of predictive accuracy (explained variation or c- index). Splines will be fitted to the candidate biomarkers to assess their prognostic effect when modelling 19

21 the continuous biomarkers. Repeated candidate biomarkers during treatment will be analysed using the landmark method. Statistical plan 1) Description of population characteristics : a. Overall descriptive table b. Association of population characteristics with baseline CTC positivity ( 5 CTCs) and with CTC as continuous variable (Fisher exact tests and Mann- Whitney tests) 2) Survival analyses, always stratified by center/study. a. Univariate analyses of population characteristics, baseline CTC and baseline serum markers, CTC and serum markers as time-dependent variables for progression free survival and overall survival. Kaplan-Meier curves will be constructed. b. Development of a clinicopathological model (CP) with all baseline characteristics except CTCs and serum markers, using a forward selection strategy. Variables will be used as much as possible as continuous variables in the Cox models, the functional form will be verified by fitting cubic splines with 2 degrees of freedom. c. Crossvalidation strategy for constructing the baseline models (only if the factors below have shown a significant univariate effect) CP + CTC, CP + CEA, CP+ CA15-3, CP + CTC + CEA, CP + CTC + CA15-3, The entire series will be randomly divided 500 times into a training and validation series, the average increase in likelihood ratio statistic (chi-squared value) and the average increase in c-index will be reported (an the average c- indices for the CP model and the other models), both together with 95% confidence intervals based on the percentiles of the 500 resamples. The reference models for calculating increases are the CP and CP+ CTC models. d. Concordance index (c-index) of the MDACC / Curie nomogram for PFS/OS prediction at baseline (after having excluded the IC study from the data). If possible, the nomogram estimates will be provided by Fox Chase Cancer Center statisticians. e. Same as 1c but with inclusion of all changes of CTC and serum during treatment. f. Compare c-indices of a multivariate model that includes early CTC changes (baseline to week 2-5) and late (baseline to week 6-9) CTC changes for PFS and OS. 3) If external cohorts are available, establishment and validation of a prognostic nomogram taking into account CTC changes. 6. WORKING PARTIES Two groups with specific functions have been created: 1) the Secretariat 2) the Steering Committee 20

22 The Secretariat is in charge of the coordination of the study. It is responsible for and in charge of checking, processing and analyzing the data. Finally, the Secretariat is responsible for preparing reports and publications. Membership to the Steering Committee will be granted to each CTC centre with respect to the number of patients included by each centre (summing patients in case of multiple studies conducted in a same centre): - less ( ) than 18 patients : no member - from 19 to 50 patients : 1 member - from 51 to 100 patients : 2 members - from 101 to 200 : 3 members - over 200 patients : 4 members These members will be designated by the principal investigator of each study. This may include the data manager(s), statistician(s) and all other significant contributors to this project (research fellows included). 7. HANDLING AND CONFIDENTIALITY OF DATA Data will be provided by principal investigators of studies in confidence to Institut Jules Bordet, where they will be kept secure during verification. These data will be used solely for the purpose of this study, and will not be shared with other parties. The statistical analyses will be performed at Institut Jules Bordet, Institut Gustave Roussy and IDDI, and will be shared with the working parties mentioned above. 8. PUBLICATION POLICY A manuscript summarizing the results of the pooled analysis will be prepared for submission to and publication by a peer-reviewed scientific journal. The manuscript will be prepared by the Secretariat and submitted to the Steering Committee for review and agreement. All authors will be given sufficient time to provide comments and attempts will be made to come to mutual agreement. Any publication arising from this project will be made on behalf of the pooled analysis Steering Committee. None of the parties involved in this project has any financial interest in its results. Appendix 1: CellSearch european centers (confidential) Appendix 2: Identified potential studies In grey, studies for which patients survival was not reported Study Name (if any) First Author Reference Year Country ~ Nber of patients ATHENA Bidard Ann Oncol 2010 FR 67 - Munzone SABCS PD IT 187 (1) LANDSCAPE Pierga SABCS P FR 41 IC Pierga Ann Oncol 2011 FR Hartkopf Anticancer Res 2011 GE 58 - Consoli Tumori 2011 IT 93 DETECT Muller SABCS P GE 254 CirCe01 (part 1) Bidard not reported yet FR 58 TOTAL ~

23 - Riethdorf Clin Cancer Res 2007 GE 92 - Peeters SABCS P NE 30 - Sieuwerts Clin Cancer Res 2011 NE 50 AVALUZ Manso SABCS P SP 37 TOTAL ~ 1234 (1) This report included the previously published cohort published by F Nolé in Ann Oncol 2008 (N=80). 22