SpecimenCTCTissue MarkerAntibodySourceAntibodySource ER monoclonal murine ER Janssen Diagnostics, LLC monoclonal murine, clone 1D5, 1:50, high pH epitope retrieval solution DAKO BCL-2 monoclonal murine monoclonal BCL-2/ (100) BD Pharmingen rabbit polyclonal antibody, 1:100, no antigen retrival, Envision+ detection system DAKO HER2 monoclonal murine Her81 Janssen Diagnostics, LLC monoclonal murine monoclonal, Clone 124, 1:100, low pH epitope retrieval solution, LSAB+ detection system DAKO Ki67monoclonal murine B56 BD Pharmingen monoclonal murine, clone MIB-1, 1:100, high pH epitope retrieval solution, LSAB+ detection system DAKO Table S1. Antibodies used for CTC and tissue studies.

Group Stop for success Continue to next group Stop for failure 150 – 4N/A 26 – 104 – 50 – 3 39 – 15N/A4 – 8 Table S2. Stage 2 stopping rules for the cumulative number of successfully assayed patients

Low (Favorable) Intermediate High (Unfavorable) Number of CTC/7.5mL (mean of 4 aliquots)<55-10> 10 CTC-Enumeration Points012 Presumed Prediction of Response to ET Favorable Intermediate Unfavorable % CTC positive Assigned Bio-Points % CTC positive Assigned Bio-Points % CTC positive Assigned Bio-Points ER>10%01-10% b 20% b 6b6b BCL-2>10%01-10%10%2 HER20%01-10%1>10%2 Ki670%01-10%1>10%2 Table S3. B. CTC-Bio-Points a a Only aliquots with CTC ≥5/7.5 ml WB evaluated b Low or negative ER expression is weighed more than BCL-2, HER2, and Ki67 due to its fundamental role in endocrine responsiveness. A. CTC-Enumeration Points based on CTC Enumeration

Average CTC Counts CTC- Enumer ation Points Marke r CTC-Bio-Points Potential CTC-ETI Score (sum of CTC Assigned and Bio- Points) Low Intermedi ate High CTC <50 N/A 0000 CTC ER BCL HER2 012 Ki CTC >102 ER BCL HER2 012 Ki C. Calculation of CTC-ETI Scores. CategoryCTC-ETI Presumed Outcome for Patient with ER Positive MBC starting new ET Presumed Clinical Action Low0-3 Favorable; respond to ET and/or indolent disease; long time to progression Treat with ET Intermediate4-6 Probably respond or moderately indolent disease; modest time to progression Treat with ET High7-14Poor; Resistant to ET, rapid progression Treat as ER negative with chemotherapy D. CTC-ETI Categories Abbreviations: CTC= circulating tumor cells; ER = estrogen receptor; ET= Endocrine Therapy; MBC = metastatic breast cancer; ET= endocrine therapy.

Table S4. CTC-ETI determination in cultured human breast cancer cell lines spiked into normal human blood and evaluated by the CellSearch ® system. Cell lines % ER+ Bio- Points % BCL-2+ Bio- Points % HER2+ Bio- Points % Ki67+ Bio- Points Total Bio- Score CTC- ETI MCF-7 (hormone depleted) >10%0 01%1>10%23LOW Sk-Br-30%602>10%2 212HIGH MDA-MB-2310%61-10%10%0>10%29HIGH

Table S5. Examples of low, intermediate, and high CTC-ETI from patient samples. A.LOW CTC-ETI CTC Enumeration CTC-Biomarker Determination # of CTC/7.5 ml WB Mean CTC/7.5 ml WB CTC- Enumeration Points % of CTC positive Assigned Bio-Points Bio- Score CTC- ETI score ER %0 2 3 LOW BCL-2560%0 HER26 0%0 Ki67812%2 Patient # 5 (Primary Tissue: ER 90% positive, HER2 negative by FISH; Metastatic tissue: ER positive, HER2 negative by FISH)* B. INTERMEDIATE CTC-ETI Patient #4 (Primary Tissue: ER ≥95% positive, HER2 0 by IHC; Metastatic tissue: insufficient tissue to evaluate ER and HER2)* CTC Enumeration CTC-Biomarker Determination # of CTC/ 7.5 ml WB Mean CTC/7.5 ml WB CTC - Enumeration Points % of CTC positive Assigned Bio- Points Bio- Score CTC- ETI score ER %0 2 4 INT BCL %0 HER2838 0%0 Ki %2 *ER and HER2 as per initial clinical laboratory using the laboratory’s criteria for positivity or negativity. CTC Enumeration CTC-Biomarker Determination # of CTC/7.5 ml WB Mean CTC/7.5 ml CTC- Enumeration Assigned Points % of CTC positive Assigned Bio- Points Bio- Score CTC- ETI score ER % HIGH BCL-21560%0 HER217 0%0 Ki671414%2 C. HIGH CTC-ETI Patient # 3 (Primary Tissue: ER 60% positive, HER2 1+/2+ by IHC; Metastatic tissue: ER 75% positive; HER2 1+ by IHC)*

Table S6. CTC-ETI for each patient. Pat. Num. Average CTC/7.5 ml Assigned CTC Points based on CTC Enumeration Assigned CTC-Bio-Points CTC-ETI CTC-ETI Category 11Screen Failure 100NA LOW 620NA LOW 740NA LOW 930NA LOW 1800NA LOW 2140NA LOW 2200NA LOW 2500NA LOW 2600NA LOW 2700NA LOW 3200NA LOW 3700NA LOW 3900NA LOW 4200NA LOW 5100NA LOW LOW LOW INT INT INT INT INT INT INT INT INT 28167HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH HIGH 108NA AF a 177NA AF a 198NA AF a 298NA AF a 1567NA TF b 2013NA TF b a AF =Analytical Failure (CTC<5 in 1 of 4 aliquots, while the other 3 aliquots had ≥5 CTC/7.5mL and the average was ≥5 CTC/7.5mL); b TF=Technical Failure (Machine failure in at least one of 4 aliquots)

Table S7. Concordance between biomarkers in primary and metastatic tissues and CTC. Kappa Score a MarkerPrimaryMetastatic ER0-0.1 BCL HER Ki a Kappa score for concordance between CTC and tissue biomarker for respective biomarker.