The Molecular Analysis on Circulating Tumor Cells to Determine Prognostic and Predictive Factors in Cancer Research

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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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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

QIAGEN Hannover GmbH

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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

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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


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


Prognostic impact for disease-free survival


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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


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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


AdnaTest ColonCancer OS/DFS in KRAS wt patients under Panitumab/Cetuximab


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


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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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



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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



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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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ERCC1-positive CTCs in ovarian cancer


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AdnaTest OvarianCancer: ERCC1+ predicts platinum failure



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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ARV7-positive CTCs in prostate cancer


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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.


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Her2-positive CTCs in breast cancer


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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


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)


10%

20%

30%

40%

50%

HER2 (NR) HER2 (RS)


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


Bredemeier et al. (2014) Poster, AACR, San Diego

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...only two more slides


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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

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Questions?

Contact QIAGEN

[email protected]

Siegfried Hauch, PhD

[email protected]

Further information is also available at

www.adnagen.com


mailto:[email protected]

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The Molecular Analysis on Circulating Tumor Cells to Determine Prognostic and Predictive Factors in Cancer Research