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

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

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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

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

DEPArray™ User Meeting

HER2 expression and amplification

Anja Brouwer

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression vs amplification

HER2 expression

  • n Tissue

HER2 amplification in Tissue HER2 expression

  • n CTCs

HER2 amplification in CTCs

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression on CTCs

HER2 expression

  • n Tissue

HER2 amplification in Tissue HER2 expression

  • n CTCs

HER2 amplification in CTCs

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

Enrichment and Sorting of CTC

CellSearch

CTC WBC

Anti-EpCAM Anti-cytokeratin Anti-CD45

DEPArray

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 on Cell line samples

Ce Cell ll lin line sample les Cell line model for HER2 analysis on CellSearch. 32.6 .617 Cells of 7 cell cell lin lines es.

Cell line ER HER2 IHC HER2 FISH MDA-MB-436

  • normal

MCF-7 + normal BT20

  • 1+

normal KPL-4

  • positive

amplified SKBR3

  • 3+

amplified SUM190

  • positive

amplified MDA-IBC3

  • positive

unknown

Subik 2010, Breast Cancer, Riethdorf 2010, Clin Cancer Res Ai 2013, Cell Signal, Klopp 2010, PloS ONE, Fernandez, 2013, Breast Cancer Res Treat

Model for HER2 expression Scoring with CellSearch and DEPArray Parameters

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

Riethdorf CCR 2010, PMID: 20406831

7

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HER2 on CellSearch

Download all images of Cell lines

  • 33.500 images of single tumor cells (clusters excluded)
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Workflow Image J

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HER2 intensity-bgSub per Cell line

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Prediction model

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Model for HER2 with training & testing group

Specificity: 1244/(46+1244) = 0.96 Sensitivity: 1495/(1495+88) = 0.94 Accuracy: 2739/2873 = 0.95

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

MCF-7 (0+) BT-20 (1+) KPL-4 (2+) IBC-3 (3+)

CK PE DAPI BF HER FITC CD45 APC

HER2 on DEPArray

  • Optimization and

standardizing FITC exposure time

  • DEPArray of 7 spiked,

CellSearch enriched cell line samples

  • 39 parameters
  • Model for HER2

prediction

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

Reproducibility

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Size vs HER2 IF

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HER2 intensity per Cell line

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

Model for HER2 with training & testing group

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 on CTCs

Patie tient Sample les 01/2010 – 03/2015: CellSearch 120 120 samples (>5 CTCs) of 85 patients 32.6 .617 CTCs range 5 – 8734 CTCs DEPArray 4 samples (>200 CTCs) of 4 patients 2.0 .032 CTCs range 53 – 1005 CTCs

Total 85 (100%) de novo MBC 29 (34%) Primary tumor HER2 positive 22 (26%) ER positive 60 (71%) Unknown HER2/ER status 12 (14%) Metastasis HER2 positive 5 (6%) ER positive 29 (34%) Unknown HER2/ER status 52 (61%)

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 scoring of 4 patients on DEPArray

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 scoring of 4 patients on DEPArray

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HER2 scoring of 4 patients on DEPArray

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

Conclusion 1

  • 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.

HER2 expression

  • n CTCs
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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression: CTCs vs Tissue

HER2 expression

  • n Tissue

HER2 amplification in Tissue HER2 expression

  • n CTCs

HER2 amplification in CTCs

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 status of 82 patients

  • 58 patients where HER2-
  • 1 patient changed to HER2+
  • 24 patients where HER2+
  • 2 patients changed to HER2-

y y Primary HER2 negative HER2+ (IHC 3+ or FISH+) Mets HER2 negative HER2 positive

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HER2 model on HER2- patient samples

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HER2 model on HER2+ patient samples

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

HER2 expression and amplification in CTCs

HER2 expression

  • n Tissue

HER2 amplification in Tissue HER2 expression

  • n CTCs

HER2 amplification in CTCs

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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
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FISH

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SKBR-3 WBC

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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

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MAST  BOLOGNA, 25-26 OCTOBER, 2016

FISH – to be continued

HER2 expression

  • n Tissue

HER2 amplification in Tissue HER2 expression

  • n CTCs

HER2 amplification in CTCs

  • Perform FISH on áll tissue samples
  • FFPE protocol on DEPArray
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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