“Learning curve” group= 10 pts with mBrCa (ER+ or ER-) who are beginning a new therapy ~5 pts with ≥5CTC/7.5 ml Group 1 5 CTC evaluable pts* with ER+ mBrCa.

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Presentation transcript:

“Learning curve” group= 10 pts with mBrCa (ER+ or ER-) who are beginning a new therapy ~5 pts with ≥5CTC/7.5 ml Group 1 5 CTC evaluable pts* with ER+ mBrCa who are beginning a new therapy:  If CTC-ETI has been successfully generated in 5/5 pts  If CTC-ETI has been successfully generated in ≤ 4/5 pts Proceed to multi-institutional, multi-blood draw pilot study Stage 1 Stage 2 Group 2 5 additional CTC evaluable pts* with ER+ mBrCa who are beginning a new therapy. Combining the findings from group 1+2:  If CTC-ETI has been successfully generated in 6/10 pts  If CTC-ETI has been successfully generated in 4-5/10 pts  If CTC-ETI has been successfully generated in ≤3/10 pts  STOP and reconsider methods Group 3 5 CTC additional evaluable pts* with ER+ mBrCa who are beginning a new therapy. Combining the findings from group (total sample size 15 CTC-evaluable pts):  If CTC-ETI has been successfully generated in ≥9/15 pts  If CTC-ETI has been successfully generated in ≤8/15 pts  STOP and reconsider methods * ≥5CTC/7.5ml WB Figure S1. Clinical trial study design

MDA-MB-231 MCF-7* Sk-Br-3MDA-MB-231MCF-7 BT-474 MDA-MB-231MCF-7*MDA-453 BT-474 MCF-7 Sk-Br-3 MDA-MB-231 MCF-7* Figure S2. Examples of biomarker staining of cultured human breast cancer cell lines after spike and capture from normal human blood using the CellSearch® system. A semi-quantitative scale was established based on relative expression of each biomarker: negative (0), weak (1+), intermediate (2+), and high (3+). *hormone depleted condition. ER BCL-2 HER-2 Ki Staining Intensity

Fig. S3. Demonstration of appropriate antibody staining of cultured human breast cancer cells with known positive and negative biomarker expression after spike and capture from normal human blood using the CellSearch ® System. A: ER staining for MCF-7 and Sk-Br-3. B: BCL- 2 staining for MCF-7 and Sk- Br-3. C: HER2 staining for Sk-Br-3 and MCF-7. D: Ki-67 staining for MCF-7.

% ER pos CTC 86% 0% Composite DAPI Cytokeratin-FITC ER-PE CD45-APC A. Hormone depleted MCF-7 +Anti-ER Ab + no Ab Sk-Br-3 +Anti-ER Ab + no Ab

% BCL-2 pos CTC 84% 0% Composite DAPI Cytokeratin-FITC BCL2-PE CD45-APC B. MCF-7 +Anti-BCL-2 Ab + no Ab Sk-Br-3 +Anti-BCL-2 Ab + no Ab

% HER2 pos CTC 100% 0% Composite DAPI Cytokeratin-FITC HER2-PE CD45-APC C. MCF-7 +Anti-HER2 Ab + no Ab Sk-Br-3 +Anti-HER2 Ab + no Ab

% Ki67 pos CTC 42% 0% Composite DAPI Cytokeratin-FITC Ki67-PE CD45-APC D. MCF-7 +Anti-Ki67 Ab + no Ab

Figure S4. Composite “CTC-Bio_Score” for different cell lines. A: Hormone deplete MCF-7 cells (Luminal-type). B: MDA-MB-231 ( Basal-like type). C: Sk-Br-3 (HER2 positive) Composite DAPI Cytokeratin-FITC Marker-PE CD45-APC Anti-ER Anti-BCL-2 Anti-HER2 Anti-Ki67 A. >10% % Pos >10% 1% >10% Assigned Bio-Points Total CTC-ETI Bio-Score 3

Composite DAPI Cytokeratin-FITC Marker-PE CD45-APC Anti-ER Anti-BCL-2 Anti-HER2 Anti-Ki67 B. 0% % Pos 1-10% 0%0% >10% Assigned Bio-Points Total CTC-ETI Bio-Score 9

Composite DAPI Cytokeratin-FITC Marker-PE CD45-APC Anti-ER Anti-BCL-2 Anti-HER2 Anti-Ki67 C. 0% % Pos 0% >10% Assigned Bio-Points Total CTC-ETI Bio-Score 12

Composite DAPI Cytokeratin-FITC Marker CD45-APC No nuclear positivity (0) Strong nuclear positivity (2+; 3+) Weak nuclear positivity (1+) Figure S5. Heterogeneity of ER expression within the cultured MCF-7 human breast cancer cell line. Cell line Staining MCF-7* + ER PE Ab15%18%67%0% Cells were cultured in hormone depleted media for 48 hours, harvested, and spiked into 7.5 ml of normal human blood. The blood was then processed using CellSearch® and stained for DAPI, CK, CD45, and ER. Examples of strong (2+, 3+), weak (1+), and no staining (0) are displayed. Apparent staining in the bottom panel is due to high gain analysis by the automated fluorescent microscopy, as indicated by very high background.

Average CTC/7.5 ml WB Coefficient of variation Figure. S6. Coefficient of variation of CTC-enumeration by mean CTC count.

ER staining Primary Tumor (IHC >95%), 200X ER staining in CTC % 50% 11% 36% IF Score % Cells 53% ER positive 47% ER negative Figure. S7. Heterogeneity between primary tissue and CTC for patient #4. The CTC galleries displayed were selected from over 1,000 images to illustrate CTC-ER heterogeneity. Composite DAPI Cytokeratin-FITC ER-PE CD45-APC 3+ 2+