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

?? What does that mean with regard to HER2 Specific

Target: Primary Tumor

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

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

• DAKO-Score: 3+
• DAKO-Score: 2+ (FISH-positive)

Anti-HER2-treatment for patients with:

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?

Modifiziert nach Pantel et al., Nat. Rev Clin. Oncol 2009

• single DTCs
• „Dormancy“
• Resistent against chemotherapy
• 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 N=3141 Patients First Diagnosis 2001-2013

2014

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

• We want to identify HER2-positive tumor cells in the primary tumor of

breast cancer patients Using the DEPArray:

• 2. Analysis in tumor tissue and DTCs of our own patients
• 1. Method validation for FDA Approval

DTCs: Detection Method

25%

Bone marrow aspiration Density gradient centrifugation Dection of cytokeratin-pos cells 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-positive CTCs

HER2-pos cells? Analysis of 50µm tissue

HER2-amplified DTCs?

5x10e6/vial Pre-enrichment DEPArray

Who else will be eligible for HER2-targeted therapy?

Refreshment in culture

HER2 analysis in BC FFPE tissue

Method establishment start: April 2016 Method established: September 2016

• How many DEPArray runs?
• 25
• How many HER2 FISHs?
• 26

Method establishement and validation for FDA approval

• FFPE sample preparation
• DEPArray sorting and recovery
• HER2 FISH

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

1 2 3

Deparaffinization Fluorescent staining Cytokeratine – AF488 Vimentin – AF647 DAPI

Single cell suspension

Tumor cells

• CK+
• Vim-
• DAPI

Stromal cells

• CK -
• Vim+
• DAPI

Antigen retrieval Dissociation BC FFPE sample 50µm curl

essential! aim!

Deparaffinization Fluorescent staining Cytokeratine – AF488 Vimentin – AF647 DAPI

Single cell suspension

Tumor cells

• CK+
• Vim-
• DAPI

Stromal cells

• CK -
• Vim+
• DAPI

Antigen retrieval Dissociation BC FFPE sample 50µm curl

essential!

Tissue characteristics

• Cancer type  Tumor vs. Stroma
• Age
• Fixative
• Time of fixation
• Tissue storage
• Sample size: tumor or biopsy?
• Sample thickness

Deparaffinization Fluorescent staining Cytokeratine – AF488 Vimentin – AF647 DAPI

Single cell suspension

Tumor cells

• CK+
• Vim-
• DAPI

Stromal cells

• CK -
• Vim+
• DAPI

Antigen retrieval Dissociation BC FFPE sample 50µm curl

essential!

Tissue characteristics

• Cancer type  Tumor vs. Stroma
• Age
• Fixative
• Time of fixation
• Tissue storage
• Sample size: tumor or biopsy?
• Sample thickness

Dissociation Under-dissociated

• Cell clumps

Over-dissociated

• Cell lysis
• Release of bare nuclei

Compromises DEPArray run and downstream FISH

Sample Dissociation time Cell count

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

≈

Sample Dissociation time Cell count

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

≈

Tissue characteristics

• Cancer type  Tumor vs. Stroma
• Age
• Fixative
• Time of fixation
• Tissue storage
• Sample size: tumor or biopsy?
• Sample thickness

Dissociation Under-dissociated

• Cell clumps

Over-dissociated

• Cell lysis
• Release of bare nuclei

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

Cytokeratin-AF488 Vimentin-AF647

cells in cage All cells

In Cage

Vim+ CK-

Integral intensity DAPI

diploid stroma cells

CK+ Vim-

Integral intensity DAPI

diploid tumor cells hyperploid tumor cells

Be precise

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

Vim+ CK-

Integral intensity DAPI

diploid stroma cells

CK+ Vim-

Integral intensity DAPI

diploid tumor cells hyperploid tumor cells

DAPI BF CK Vim

DAPI/ Vim

DAPI BF CK Vim

DAPI/ CK

cell recovery

Cytospin Tumor cells

• CK+
• Vim-
• DAPI

HER2 FISH

HER2 CEP17 252 44 = 5.7 =

Cytospin Tumor cells

• CK+
• Vim-
• DAPI

HER2 FISH

Get all recovered cells on slide 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 BC FFPE sample preparation and staining DEPArray sorting and cell recovery HER2 FISH analysis

1 2 3 Dissociation Cell selection Optimal procedure for FISH signal count

Future steps

HER2-positive CTCs

HER2-pos cells? Analysis of 50µm tissue

• Can

we find „pathologically“ 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 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?

Method establishement still in progress

• DTC refreshment in culture
• DTC Pre-enrichment using Parsortix technology
• DEPArray sorting and recovery
• HER2 FISH

Future steps

HER2-positive CTCs HER2-amplified DTCs?

Pre-enrichment

• 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?

HER2

• Prof. Dr. med. Rainer Kimmig

Lab Team

• Dr. med. Ann-Kathrin Bittner

Lisa König

• Dr. med. Oliver Hoffmann