MAST BOLOGNA, 25-26 OCTOBER, 2016 DEPArray User Meeting HER2 - PowerPoint PPT Presentation

MAST  BOLOGNA, 25-26 OCTOBER, 2016

DEPArray™ User Meeting HER2 expression and amplification Anja Brouwer MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression vs amplification HER2 expression HER2 amplification on Tissue in Tissue HER2 expression HER2 amplification on CTCs in CTCs MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression on CTCs HER2 expression HER2 amplification on Tissue in Tissue HER2 expression HER2 amplification on CTCs in CTCs MAST  BOLOGNA, 25-26 OCTOBER, 2016

Enrichment and Sorting of CTC CellSearch Anti-EpCAM CTC Anti-cytokeratin WBC Anti-CD45 DEPArray MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 on Cell line samples Ce Cell ll lin line sample les Cell line ER HER2 IHC HER2 FISH MDA-MB-436 - 0 normal Cell line model for HER2 MCF-7 + 0 normal analysis on CellSearch. 32.6 .617 Cells of 7 cell cell lin lines es. BT20 - 1+ normal KPL-4 - positive amplified SKBR3 - 3+ amplified Subik 2010, Breast Cancer, Riethdorf 2010, Clin Cancer Res SUM190 - positive amplified Ai 2013, Cell Signal, Klopp 2010, PloS ONE, MDA-IBC3 - positive unknown Fernandez, 2013, Breast Cancer Res Treat Model for HER2 expression Scoring with CellSearch and DEPArray Parameters MAST  BOLOGNA, 25-26 OCTOBER, 2016

Riethdorf CCR 2010, PMID: 20406831 MAST  BOLOGNA, 25-26 OCTOBER, 2016 7

HER2 on CellSearch Download all images of Cell lines - 33.500 images of single tumor cells (clusters excluded) MAST  BOLOGNA, 25-26 OCTOBER, 2016

Workflow Image J MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 intensity-bgSub per Cell line MAST  BOLOGNA, 25-26 OCTOBER, 2016 10

Prediction model MAST  BOLOGNA, 25-26 OCTOBER, 2016

Model for HER2 with training & testing group Specificity: 1244/(46+1244) = 0.96 Sensitivity: 1495/(1495+88) = 0.94 Accuracy: 2739/2873 = 0.95 MAST  BOLOGNA, 25-26 OCTOBER, 2016 12

CK HER CD 45 HER2 on DEPArray BF DAPI PE FITC APC MCF-7 (0+) • Optimization and standardizing FITC exposure time BT-20 (1+) • DEPArray of 7 spiked, CellSearch enriched cell line samples KPL-4 (2+) • 39 parameters • Model for HER2 prediction IBC-3 (3+) MAST  BOLOGNA, 25-26 OCTOBER, 2016

Reproducibility MAST  BOLOGNA, 25-26 OCTOBER, 2016

Size vs HER2 IF MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 intensity per Cell line MAST  BOLOGNA, 25-26 OCTOBER, 2016

Model for HER2 with training & testing group MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 on CTCs Total 85 (100%) Patie tient Sample les 01/2010 – 03/2015: de novo MBC 29 (34%) Primary tumor CellSearch HER2 positive 22 (26%) 120 120 samples (>5 CTCs) of 85 patients ER positive 60 (71%) 32.6 .617 CTCs range 5 – 8734 CTCs Unknown HER2/ER status 12 (14%) Metastasis DEPArray HER2 positive 5 (6%) 4 samples (>200 CTCs) of 4 patients ER positive 29 (34%) 2.0 .032 CTCs range 53 – 1005 CTCs Unknown HER2/ER status 52 (61%) MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 scoring of 4 patients on DEPArray MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 scoring of 4 patients on DEPArray MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 scoring of 4 patients on DEPArray MAST  BOLOGNA, 25-26 OCTOBER, 2016

Conclusion 1 HER2 expression on CTCs • CellSearch • Model on Cell lines does not work for patient samples. • Modelling based on patient training and testing cohort. • DEPArray • Model on Cell lines is very sensitive and specific and works for patient samples. • Not all patients are eligible due to low CTC count. MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression: CTCs vs Tissue HER2 expression HER2 amplification on Tissue in Tissue HER2 expression HER2 amplification on CTCs in CTCs MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 status of 82 patients • 58 patients where HER2- - 1 patient changed to HER2+ Primary HER2 negative HER2+ (IHC 3+ or FISH+) • 24 patients where HER2+ HER2 negative Mets - 2 patients changed to HER2- HER2 positive y y MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 model on HER2- patient samples MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 model on HER2+ patient samples MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression and amplification in CTCs HER2 expression HER2 amplification on Tissue in Tissue HER2 expression HER2 amplification on CTCs in CTCs MAST  BOLOGNA, 25-26 OCTOBER, 2016

DEPArray → FISH • Sorting of CTCs in HER2 + and – pools based on cut-off from cell line model • Soring of WBCs from patients • Cytospin, fixation, DAKO FISH MAST  BOLOGNA, 25-26 OCTOBER, 2016

FISH SKBR-3 WBC MAST  BOLOGNA, 25-26 OCTOBER, 2016 29

FISH – to be continued • Perform DEPArray and FISH on more patient samples • Perform IF-FISH on cytospins of CellSearch enriched samples is currently being performed MAST  BOLOGNA, 25-26 OCTOBER, 2016

FISH – to be continued • Perform FISH on áll tissue samples • FFPE protocol on DEPArray HER2 expression HER2 amplification on Tissue in Tissue HER2 expression HER2 amplification on CTCs in CTCs MAST  BOLOGNA, 25-26 OCTOBER, 2016

Acknowledgments Thanks to everyone who supported this research. Thanks to all patients who are willing to participate in our studies CORE Clinical Trials Oncology Dr. L. Dirix Dr. H. Dubois Prof. S. Van Laere Anke Seytsma Prof. dr. M Peeters All research nurses Dr. P. Vermeulen Dr. R. Salgado Bram De Laere Pieter-Jan van Dam Anja Brouwer Charlotte Rypens Christophe Van Berckelaer Steffi Oeyen Christel Verbruggen Katrien Coen MAST  BOLOGNA, 25-26 OCTOBER, 2016