HER2 analysis and treatment in breast cancer: Do we have to change - PowerPoint PPT Presentation

HER2 analysis and treatment in breast cancer: Do we have to change our strategy? Sabine Kasimir-Bauer Lisa König Department of Gynecology & Obstetrics University Hospital Essen Germany Director: Prof. Dr. med Rainer Kimmig

Targeted Therapy in Breast Cancer Specific ?? What does that mean with regard to HER2

Target: Primary Tumor What we finally treat is Minimal Therapy decision according to Residual Disease (MRD), Reflected predictive markers on the primary by DTCs and CTCs! tumor.

Diagnostic tool: Dako-score Non-malignant cell Tumor Cell HER2 is overexpressed in 25% of breast cancer cases

More specific diagnostic tool: FISH analysis > 4 gene copies HER2-pos Anti-HER2-treatment for patients with:  DAKO-Score: 3+  DAKO-Score: 2+ (FISH-positive)

Problem HER2-negative primary tumor HER2-positive DTCs/CTCs

Explanation?

Why is this finding so important for Breast Cancer Patients? What is the prognostic role of single tumor cells? • single DTCs • „Dormancy“ • Resistent against chemotherapy Modifiziert nach Pantel et al., Nat. Rev Clin. Oncol 2009 • Micrometastases in a „Steady State“ Modified according to Pantel et al., 2009

Tumor cells can be detected at every time point of the disease!

DTCs: Prognostic Significance 2014 N=3141 Patients First Diagnosis 2001-2013

HER2 on DTCs In n=89 Patients with primary breast cancer: Concordance between HER2 on the primary tumor and DTCs : 68.5%, p=0.021 Primary tumor=HER2-neg / DTCs =HER2- positive 23%

HER2 on DTCs N=10 Patients 6 /10: HER2-negative primary tumor

CTCs: Prognostic Significance N=3173 Patients

HER2 on CTCs N=431 patients

Purpose of our study Using the DEPArray:  We want to identify HER2-positive tumor cells in the primary tumor of breast cancer patients 1. Method validation for FDA Approval 2. Analysis in tumor tissue and DTCs of our own patients

DTCs: Detection Method Dection of cytokeratin-pos cells Bone marrow aspiration Density gradient centrifugation 25% ARIOL-SL 50

DTCs: Therapeutic options - Bisphosphonates N=525 Patients, First Diagnosis 2004-2009 5-year OS: 92% (483/525 Pat) Relapses: 12% (65/525 Pat)

Characterisation of CTCs EpCAM MUC-1 HER2 ER PR EMT markers ALDH1 Kasimir-Bauer et al., 2015, SABCS

Multimarker qPCR in CTCs of Triple-negative Breast Cancer Patients Kasimir-Bauer et al., 2015, SABCS

Comparison of HER2-Status HER2-amplified DTCs? HER2-positive CTCs HER2-pos cells? Analysis of 50µm tissue Who else will be eligible for HER2-targeted therapy? 5x10e6/vial Refreshment in culture Pre-enrichment DEPArray

HER2 analysis in BC FFPE tissue Method establishement and validation for FDA approval • FFPE sample preparation • DEPArray sorting and recovery • HER2 FISH Method establishment start: April 2016 Method established: September 2016 • How many DEPArray runs? • 25 • How many HER2 FISHs? • 26

HER2 analysis in BC FFPE tissue 1 BC FFPE sample preparation and staining DEPArray sorting and cell recovery 2 HER2 FISH analysis 3

aim! essential! Single cell suspension Deparaffinization Fluorescent staining Cytokeratine – AF488 Antigen retrieval Vimentin – AF647 Dissociation DAPI Tumor cells Stromal cells BC FFPE sample • CK+ • CK - 50µm curl • Vim- • Vim+ • DAPI • DAPI

essential! Single cell suspension Deparaffinization Fluorescent staining Cytokeratine – AF488 Antigen retrieval Vimentin – AF647 Dissociation DAPI Tumor cells Stromal cells BC FFPE sample • CK+ • CK - 50µm curl • Vim- • Vim+ • DAPI • DAPI Tissue characteristics • Cancer type  Tumor vs. Stroma • Age • Fixative • Time of fixation • Tissue storage • Sample size: tumor or biopsy? • Sample thickness

essential! Single cell suspension Deparaffinization Fluorescent staining Cytokeratine – AF488 Antigen retrieval Vimentin – AF647 Dissociation DAPI Tumor cells Stromal cells BC FFPE sample • CK+ • CK - 50µm curl • Vim- • Vim+ • DAPI • DAPI Tissue characteristics Dissociation • Cancer type  Tumor vs. Stroma Under-dissociated • • Age Cell clumps Compromises DEPArray • Fixative run and downstream • Over-dissociated Time of fixation FISH • • Tissue storage Cell lysis • • Sample size: tumor or biopsy? Release of bare nuclei • Sample thickness

Dissociation Sample Cell count time S1 64 mins 680.000 cells/ml S2 85 mins 1.18 E6 cells/ml S3 108 mins 810.000 cells/ml ≈ S4 78 mins 810.000 cells/ml S5 80 mins 3.08 E6 cells/ml S6 79 mins 2.40 E6 cells/ml S7 80 mins 520.000 cells/ml S8 66 mins 720.000 cells/ml

Dissociation Sample Cell count time S1 64 mins 680.000 cells/ml S2 85 mins 1.18 E6 cells/ml 108 mins 810.000 cells/ml ≈ S3/ S4 78 mins 810.000 cells/ml 80 mins 3.08 E6 cells/ml S5/ S6 79 mins 2.40 E6 cells/ml 80 mins 520.000 cells/ml S7/ S8 66 mins 720.000 cells/ml Tissue characteristics Dissociation • Cancer type  Tumor vs. Stroma Under-dissociated • • Age Cell clumps • Fixative • Time of fixation Over-dissociated • • Tissue storage Cell lysis • • Sample size: tumor or biopsy? Release of bare nuclei • Sample thickness Curl dependent !

Dissociated cells: Trypan Blue staining, 10x magnification F-stained cells: Trypan Blue staining, 10x magnification Cells in SB115: Trypan Blue staining, 10x magnification

Chip Scan settings - 100% main chamber scan - FFPE routing mode Vim+ CK- diploid stroma cells cells in cage All cells Integral intensity DAPI Cytokeratin-AF488 CK+ Vim- diploid tumor cells hyperploid tumor cells In Cage Vimentin-AF647 Integral intensity DAPI

Be precise • Intact cells • Properly stained • NO • Doublets • Cells next to clusters • Double positives • Cutted cells

Vim+ CK- CK+ Vim- diploid tumor cells diploid stroma cells hyperploid tumor cells Integral intensity DAPI Integral intensity DAPI DAPI BF CK Vim DAPI BF CK Vim DAPI/ Vim DAPI/ CK cell recovery

Cytospin HER2 FISH Tumor cells • CK+ • Vim- • DAPI HER2 252 = 44 = 5.7 CEP17

Get all recovered cells on slide Cytospin HER2 FISH Tumor cells • CK+ • Vim- • DAPI Optimal FISH procedure • Different to convential HER2 FISH on tissue samples • Washbuffer: temperature and time • Pepsin digestion: yes or no? • Denaturation: temperature and time

Overview, 10x magnification (Overlay Cep17/ HER2/ DAPI, A. Gerber) CEP17, 63x magnification DAPI, 63x magnification HER2, 63x magnification

CEP17 DAPI HER2

CEP17 DAPI HER2

HER2 analysis in BC FFPE tissue Dissociation 1 BC FFPE sample preparation and staining DEPArray sorting and cell recovery Cell selection 2 Optimal procedure for HER2 FISH analysis 3 FISH signal count

Future steps HER2-positive CTCs HER2-pos cells? Analysis of 50µm tissue • Can „pathologically“ we find HER2 negative patients, being HER2 positive in DEPArray processing? • Does the HER2 status of the primary tumor match with the HER2 status on CTCs? • Who else will be eligible for HER2- targeted therapy?

HER2 analysis in DTCs Method establishement still in progress • DTC refreshment in culture • DTC Pre-enrichment using Parsortix technology • DEPArray sorting and recovery • HER2 FISH Method establishment start: October 2016 Method established: ??? • Spike-in experiments: SkBr3 in healthy donor EDTA blood samples • Recovery rate of Parsortix? • Leucocyte background? • Refreshment protocol for cryopreserved SkBr3/DTCs • Staining for DEPArray?

Future steps HER2-positive CTCs HER2-amplified DTCs? • Can we refresh, pre-enrich and recover pure DTC fractions using Parsortix and DEPArray? • Does the HER2 status on DTCs match with the HER2 status of the primary tumor and CTCs? • Who else will be eligible for HER2-targeted therapy? Pre-enrichment

HER2 Prof. Dr. med. Rainer Kimmig Lab Team Lisa König Dr. med. Oliver Hoffmann Dr. med. Ann-Kathrin Bittner