the molecular analysis on circulating tumor cells to determine prognostic and predictive factors in...
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Harnessing the High Value of Circulating Tumor Cells in Liquid BiopsySiegfried Hauch, PhDQIAGEN Hannover
1QIAGEN Hannover GmbH
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Legal disclaimer
QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available at
www.QIAGEN.com or can be requested from QIAGEN Technical
Services or your local distributor.
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The AdnaTest Principle
Introduction
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The AdnaTests
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The COCP principle
• Immunomagnetic cell enrichment ◦ Tumor cell enrichment using magnetic particles
conjugated with an antibody mixture
• mRNA isolation and RT◦ mRNA stabilization ◦ mRNA isolation with oligo(dT) magnetic beads◦ cDNA synthesis using reverse transcription
• Multiplex-PCR◦ Expression analysis of tumor-associated markers
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AdnaTest: the circulating tumor cell (CTC) revolution
Blood sampling Determination of prognostic or
predictive biomarkers (liquid
biopsy)
Turn around time: 5hCTC enrichment
using multi antibody-labelled magnetic beads
Lysis of the enriched cells
RT and multiplex PCR
Two-step procedure for the enrichment of CTCs from blood samples:
1. Immunomagnetic-capturing using a multi-antibody mix
2. Multiplex RT-PCR to determine the number of tumor-associated transcripts
• Every AdnaTest uses different antibody mixtures optimized for each tumor entity
• The method is easy and straight forward manual assay
Type AdnaTest in questions box to request further information
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Available AdnaTests
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AdnaTest Target Genes
AdnaTest BreastCancer (CE) Muc-1, Her2, EpCAM, ER/PR
AdnaTest ProstateCancer (CE) PSA, PSMa, EGFR, AR
AdnaTest ColonCancer (CE) EpCAM, EGFR, CEA
AdnaTest OvarianCancer (CE) EpCAM, Muc-1, CA-125, ERCC1
AdnaTest OvarianCancer-2 (non-CE) EpCAM, Muc-1, CA-125, ERCC1
AdnaTest EMT-1/StemCell (non-CE) Muc-1, Her2, EpCAM, ALDH1, Pi3K, Akt2, Twist
AdnaTest EMT-2/StemCell (non-CE)ALDH1, Pi3K, Akt2, Twist and choice of breast, colon, prostate or ovarian PCR
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AdnaTest beginning: prognostic information
The prognostic value of CTCs in breast, ovarian, prostate and colon cancers
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Prognostic value of AdnaTest CTC determination
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Prognostic value Reference
AdnaTest BreastCancer OS/DFS; primary and metastatic BC
Tewes, M. et al. (2009) Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies. Breast Cancer Res Treat 115(3), 581–90.
AdnaTest ColonCancer OS/DFS in KRAS wt patients under Panitumab/Cetuximab
Musella, V. et al. (2015) Circulating tumor cells as a longitudinal biomarker in patients with advanced chemorefractory, RAS-BRAF wild-type colorectal cancer receiving cetuximab or panitumumab, Int J. Cancer 137(6), 1467ff.
AdnaTest ProstateCancer DFS/OS in CRPCAntonarakis et al. (2014) AR-V7 and Resistance to Enzalutamide and Abiraterone in Prostate Cancer, NEJM 371(11), 1028ff.
AdnaTest OvarianCancer DFS/OS in ovarian cancer
Kuhlmann, J.D. et al. (2014) ERCC1-positive circulating tumor cells in the blood of ovarian cancer patients as a predictive biomarker for platinum resistance. Clin. Chem. 60(10), 1282-9.
AdnaTest EMT1/StemCell Therapy response in metastatic breast cancer
Aktas, B. et al. (2009) Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer Res. 11(4), R46.
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CTCs in breast cancer
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CTCs in primary breast cancer have a negative prognostic meaning
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0 10 20 30 40 50 60 70 80 90 1000
0.2
0.4
0.6
0.8
1
1.2
CTCs in primary Breast Cancer
CTC- CTC+
DFS (months)
Cum
ulat
ive
surv
ival
Kasimir Bauer et al. (2014) Poster, SABCS, submitted for publication
p = 0.036
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AdnaTest BreastCancer predicts therapy failure
10
Tewes, M. et al. (2009) Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies. Breast Cancer Res Treat 115(3), 581–90.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 200 400 600
Survival (days)
Cum
ulat
ive
sur
viva
l
CTC+ CTC- Cens. CTC+ Cens. CTC-
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AdnaTest OvarianCancer
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Prognostic impact for overall survival
0 10 20 30 40 50 60 70 800
0.2
0.4
0.6
0.8
1
1.2
CTCs in ovarian cancer before or after surgery
CTC- CTC+
OS (months)
Cum
ulat
ive
sur
viva
l
Kuhlmann, J.D. et al. (2014) ERCC1-positive circulating tumor cells in the blood of ovarian cancer patients as a predictive biomarker for platinum resistance. Clin. Chem. 60(10), 1282-9.
p = 0.003
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0 10 20 30 40 50 60 70 800
0.2
0.4
0.6
0.8
1
1.2
CTCs in ovarian cancer before or after surgery
CTC- CTC+
DFS (months)
Cum
ulat
ive
surv
ival
AdnaTest OvarianCancer
p = 0.0006
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Prognostic impact for disease-free survival
Kuhlmann, J.D. et al. (2014) ERCC1-positive circulating tumor cells in the blood of ovarian cancer patients as a predictive biomarker for platinum resistance. Clin. Chem. 60(10), 1282-9.
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AdnaTest ProstateCancer – prognostic impact
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Todenhöfer, T. et al. (2012) Preliminary experience on the use of the AdnaTest® System for detection of circulating tumor cells in prostate cancer patients. Anticancer Res. 32(8), 3507–13.
remission stable progress0%
10%
20%
30%
40%
50%
60%
70%
CTC- CTC before therapy CTC during therapy
Freq
uenc
y
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AdnaTest advanced: predictive information
CTCs and potential predictive information
14QIAGEN Hannover GmbH
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Prognostic value of AdnaTest CTC determination
15
Prognostic value Reference
AdnaTest BreastCancer OS/DFS; primary and metastatic BC
Tewes, M. et al. (2009) Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies. Breast Cancer Res Treat 115(3), 581–90.
AdnaTest ColonCancer OS/DFS in KRAS wt patients under Panitumab/Cetuximab
Musella, V. et al. (2015) Circulating tumor cells as a longitudinal biomarker in patients with advanced chemorefractory, RAS-BRAF wild-type colorectal cancer receiving cetuximab or panitumumab, Int J. Cancer 137(6), 1467ff.
AdnaTest ProstateCancer DFS/OS in CRPCAntonarakis et al. (2014) AR-V7 and Resistance to Enzalutamide and Abiraterone in Prostate Cancer, NEJM 371(11), 1028ff.
AdnaTest OvarianCancer DFS/OS in ovarian cancer
Kuhlmann, J.D. et al. (2014) ERCC1-positive circulating tumor cells in the blood of ovarian cancer patients as a predictive biomarker for platinum resistance. Clin. Chem. 60(10), 1282-9.
AdnaTest EMT1/StemCell Therapy response in metastatic breast cancer
Aktas, B. et al. (2009) Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer Res. 11(4), R46.
QIAGEN Hannover GmbH
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AdnaTest advanced: predictive information
KRAS-positive CTCs in colon cancer
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CTCs in colon cancer
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CTCs correlate with therapy failure for Cetuximab or Panitumumab in wt RAS/RAF patients
Musella, V. et al. (2015) Circulating tumor cells as a longitudinal biomarker in patients with advanced chemorefractory, RAS-BRAF wild-type colorectal cancer receiving cetuximab or panitumumab, Int J. Cancer 137(6), 1467ff.
QIAGEN Hannover GmbH
Sample to Insight
CTCs in colon cancer
18
CTCs correlate with therapy failure for Cetuximab or Panitumumab in wt RAS/RAF patients
Musella, V. et al. (2015) Circulating tumor cells as a longitudinal biomarker in patients with advanced chemorefractory, RAS-BRAF wild-type colorectal cancer receiving cetuximab or panitumumab, Int J. Cancer 137(6), 1467ff.
QIAGEN Hannover GmbH
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CTCs in colorectal cancer – KRAS mutational status
KRAS overexpression vs. mutation (codon12)
Kras+ Kras-
Tissue wt. 8 8
Tissue mut. 0 12
Raimondi et al. (2013) Cancer biol. Ther 85(4), 164ff.
p = 0.0005
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AdnaTest advanced: predictive information
ERCC1-positive CTCs in ovarian cancer
20QIAGEN Hannover GmbH
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AdnaTest OvarianCancer: ERCC1+ predicts platinum failure
21QIAGEN Hannover GmbH
Kuhlmann, J.D. et al. (2014) ERCC1-positive circulating tumor cells in the blood of ovarian cancer patients as a predictive biomarker for platinum resistance. Clin. Chem. 60(10), 1282-9.
Cox Regression Cox Regression DFS Log. regression platinum resistance
Independent HR 95%-CI Independent HR 95%-CI
pT <0.0005 10.5 3.7 – 30.1 * * *
pN * * * * * *
M <0.0005 3.9 1.9 – 7.9 0.001 9.3 2.6 – 33.5
G <0.0005 0.29 0.15 – 0.57 0.006 0.2 0.07 – 0.63
RTB * * * * * *
CTC (ERCC1+) 0.007 3.6 1.4 – 9.1 0.012 8.6 1.6 – 46.5
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AdnaTest advanced: predictive information
ARV7-positive CTCs in prostate cancer
22QIAGEN Hannover GmbH
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AdnaTest ProstateCancer: ARV7 predicts Enzalutamide failure
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Antonarakis et al. (2014) AR-V7 and Resistance to Enzalutamide and Abiraterone in Prostate Cancer, NEJM 371(11), 1028ff.
QIAGEN Hannover GmbH
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AdnaTest advanced: predictive information
Her2-positive CTCs in breast cancer
24QIAGEN Hannover GmbH
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Her2 in CTCs form metastatic breast cancer
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Comparing Her2 in CTCs and primary tissue
n = 60
ADN+/Histo+
ADN-/Histo-
ADN+/Histo-
ADN-/Histo+17
30
21
32
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AdnaTest BreastCancer: liquid biopsy for Her2+ metastases
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Aktas et al. (2013) SABCS, poster discussion
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Metastatic tissue CTCs
QIAGEN AdnaTest
=
≠CellSearch
p = 0.04
p = 0.9
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Study Results AdnaPanel Breast: study design
Bredemeier et al. (2014) Poster, AACR, San Diego
• Her3 • Her2 • ALDH1 • EpCAM • c-Met • Aurka • Pi3K • ERCC1 • CD45 • GAPDH • Akt2 • EGFR
AdnaPanel Breast
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Rate T0 Rate T1 Rate T20%
20%
40%
60%
80%
EPCAM (RS) EPCAM (NR)
Rate T0 Rate T1 Rate T20%
10%
20%
30%
40%
50%
HER2 (NR) HER2 (RS)
Rate T0 Rate T1 Rate T20%
20%
40%
60%
80%
Aurka (RS) Aurka (NR)
Rate T0 Rate T1 Rate T20%5%
10%15%20%25%30%35%40%45%50%
Her3 (RS) Her3 (NR)
Overexpression frequencies over all time points T0–T2
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Bredemeier et al. (2014) Poster, AACR, San Diego
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AdnaTest advanced: predictive information
...only two more slides
29QIAGEN Hannover GmbH
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• Molecular detection of CTCs using the AdnaTest proved to be a highly sensitive and specific method
• Using the AdnaTest therapy, efficacy can be determined early after beginning treatment.
• Molecular characterization of CTCs shows EMT and tumor stem cell-related phenotypes of CTCs and how they are linked to resistance to conventional therapy
• The AdnaTest is an open platform that enables an intense investigation of CTC characteristics and molecular signal transduction pathways in all major tumor entities
Summary
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Thank you for attending
31
Questions?
Contact QIAGEN
qiawebinars@qiagen.com
Siegfried Hauch, PhD
Siegfried.Hauch@qiagen.com
Further information is also available at
www.adnagen.com
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