ctc detection and molecular characterization – challenges and solutions

Sample to Insight

CTC Detection and Molecular Characterization – Challenges and Solutions

Dr. Siegfried Hauch

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Legal disclaimer

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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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Agenda

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AdnaTest beginning: prognostic information

1.1 The COCP principle

1.2 The EpCam problem

AdnaTest solutions

2.1 Leukocyte bias

2.2 EMT and stemness in CTCs

2.3 CTC metabolic pathways

2.4 Future solutions

Summary

Questions

1

2

3

4

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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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The AdnaTests

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The Combination of Combinations Principle (COCP)

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 an easy and straightforward 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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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Increased sensitivity in colon cancer

CS Ck8/18 Adnatest0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

Posi

tive

case

s

EpCam EGFR CEA0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Freq

ueny

in A

DN C

TC+

case

s

Targeting two EpCam variants instead of one for enrichment seems to be important, but also the choice of the PCR targets dramatically increases CTC detection sensitivity in colorectal cancer.

Raimondi et al. Cancer Biol Ther. 2014 May;15(5):496-503

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Increased sensitivity in prostate cancer

0 1-4 5-10 ≥100

2

4

6

8

10

12

14

16

Patient distribution (CellSearch)# of patients detected by AdnaTest (%)

CTC#/7.5ml blood

Posi

tive

case

s

Samoila et al. 2012 Poster at http://www.adnagen.com/m089v01_upload/Samoila_poster_2012.pdf

Prostate cancer detection also seems to benefit from targeting Her2, considering the significant number of low-EpCam cases.

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Increased sensitivity in breast cancer

EpCam Muc1 Her20%5%

10%15%20%25%30%35%40%45%50%

frequ

ency

in A

DN C

TC+

case

sCS (cutoff

2 cells)CS (cutoff

5 cells)AdnaTest

totalEpCam Muc1 Her2

0%

10%

20%

30%

40%

50%

60%

47%

36%

53%

# of

pos

itive

cas

es

Using EpCam as well as high- and low-glycosylated MUC1, target sensitivity increases 17% over the gold standard as per definition.

Andropoulou et al. ; Int J Cancer. 2012 Apr 1;130(7):1590-7

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Increased sensitivity in lung cancer

CS AdnaTest0%

5%

10%

15%

20%

25%

30%

35%

# of

pos

itive

cas

es

Pi3KCA Akt2 ALDH10%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

frequ

ency

in A

DN C

TC+

case

s

In lung cancer, the AdnaTest EMT2 provides additional antibodies against Her2 and EGFR. This doubles performance and choosing the right detection markers increases even more.

Hanssen et al. ; Nature Sci Rep. 2016 Jun 15;6:28010.

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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Agenda

15




AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Leukocyte bias

100 1000 100000

5

10

15

20

25

CD45Akt2Her2Notch1Parp1Pi3KSRC1Gapdh

Number of leukocytes

35 -

Ct

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Example: repeated specificity and sensitivity comparison

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Series 1 Series 2 Series 3 Series 4 Series 5 Series 6

CTC+ (0 spiked cells) 0/21 0/20 0/22 1/20 0/20 1/20

CTC+ (2 spiked cells) 16/20 21/22 17/21 19/20 20/20 18/20

CTC+ (5 spiked cells) 20/21 22/22 22/22 19/20 20/20 19/20

Specificity Recovery (2 cells) Recovery (5 cells)86%

88%

90%

92%

94%

96%

98%

100%

Six different AdnaTest Breast lots

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Leukocyte reduction is critical to minimize leukocyte bias

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S1 S2 S3 S4 S5 S6 S7 S8 S9 S100

5

10

15

20

25

30Leukocyte reduction using AdnaWash

AdnaWash PBS

ng/µ

l CD

45 fr

agm

ent c

once

ntra

tion

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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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The meaning of CTC

What kinds of CTCs might be relevant?

McAllister, S.S. et al. (2008) Systemic endocrine instigation of indolent tumor growth requires osteopontin. Cell 133(6), 994-1005.

Primary tumor

Tumor cell dissemination

Most of the cells die

Tumor stem cells

Metastasis at the same site or at distant organs

Dormant cells

?

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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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ER, PR and Her2 expression in CTCs in breast cancer

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Comparing ER, PR and Her2 expressions in CTCs and primary tissue

ER PR HER20

10

20

30

40

50

60

70

80

90

CTCPT

Fehm, T. Detection and characterization of circulating tumor cells in blood of primary breast cancer patients by RT-PCR and comparison to status of bone marrow disseminated cells et al., (2009) Breast Cancer Res. 11(4), R59.

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EMT and tumor stem cells in metastatic breast cancer

Epithelial-mesenchymal transition (EMT) / stem cell (SC) markers in correlation with CTCs

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.

Twist Akt2 Pi3k ALDH10%

10%

20%

30%

40%

50%

60%

70%

80%

42%

62%58%

69%

7%4% 4%

14%

CTC+ CTC-

Cor

rela

tion

with

CTC

s

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EMT and tumor stem cells 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.

EMT / SC markers in correlation with therapy response

Twist Akt2 Pi3k ALDH10%

10%

20%

30%

40%

50%

60%

70%

32%

44%41%

62%

10% 10% 10%

5%

Non-responder Responder

Clin

ical

cor

rela

tion

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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Agenda

27




AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Gene expression analysis in breast cancer

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PI3KCACD44

EPCAMPTEN

TWISTHER2KRAS

ALDH1mTORESR1AKT2

CD24L4EGFR

PGRMUC1CD45

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Frequency of gene expression

Frequency

Gen

e

Bredemeier, Kasimir-Bauer et al. (2013) Poster, AACR

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Some cases in detail

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CD

44C

D24

L4A

LDH

1E

PC

AM

MU

C1

ES

R1

EG

FRP

GR

HE

R2

AK

T2P

TEN

TWIS

Tm

TOR

PI3

KC

AK

RA

SC

D45

0123456

Expression profile Patient P

Gene

Delta

C(t)

CD

44C

D24

L4A

LDH

1E

PC

AM

MU

C1

ES

R1

EG

FRP

GR

HE

R2

AK

T2P

TEN

TWIS

Tm

TOR

PI3

KC

AK

RA

SC

D45

0123456

Expression profile Patient G

Gene

Delta

C(t)

CD

44C

D24

L4A

LDH

1E

PC

AM

MU

C1

ES

R1

EG

FRP

GR

HE

R2

AK

T2P

TEN

TWIS

Tm

TOR

PI3

KC

AK

RA

SC

D45

0123456

Expression profile Patient M

Gene

Delta

C(t)

CD

44C

D24

L4A

LDH

1E

PC

AM

MU

C1

ES

R1

EG

FRP

GR

HE

R2

AK

T2P

TEN

TWIS

Tm

TOR

PI3

KC

AK

RA

SC

D45

0123456

Expression profile Patient I

Gene

Delta

C(t)


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Patient groups

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Epithelial EMT-Like Stem-like

Patient G Patient P Patient I Patient M

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The AdnaPanel Breast: profile changes before and after therapy


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The AdnaPanel Breast: marker groups


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p-values in red indicate statistical significance

The AdnaPanel Breast: profiles help predict responsiveness


Before therapy After therapy

CTC phenotype

Epithelial-like 0.16 0.017

Receptor expression 0.47 0.88

Resistance marker 0.08 0.064

EMT-like 0.82 0.71

Stem cell-like 0.43 0.053

Genes detected in CTCs

HER2/Aurak 0.09 0.14

Her3/Aurak 0.085 0.014

EGFR/Aurak 0.014 0.98

EPCAM/Aurak 0.02 0.013

EPCAM/EGFR/Aurak 0.01 0.11

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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Agenda

35




AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Molecular classification of the primary tumor FFPE

48 genes expression testing

Preamplification of cDNA for 33 genes

K. Kolostova, S. Kasimir-Bauer et al., Cellular and Molecular Biology 37

http://www.fluidigm.com/BioMarkvideo/index.html

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ADNAtest negative CTC

ADNAtest positive CTC – primary BC

ADNAtest positive CTC – metastatic BC

FPPE tissue samples (BC)

ADNAtest negative CTC

ADNAtest positive CTC – primary BC

ADNAtest positive CTC – metastatic BC

FPPE tissue samples (BC)

K. Kolostova, S. Kasimir-Bauer et al., Cellular and Molecular Biology 37

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Agenda

38




AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Agenda

39




AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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Molecular detection of CTCs using the AdnaTest proved to be a highly sensitive and specific method.

The COCP Principle turned out to improve sensitivity and specificity for CTC detection substantially.

Molecular characterization of CTCs shows EMT and tumor stem cell-related phenotypes of CTCs and gives n indication about molecular pathways involved in metastasization and therapy resistance formation.

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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Agenda

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AdnaTest solutions

2.1 Leukocyte bias




Summary

Questions

1

2

3

4

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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]

ctc detection and molecular characterization – challenges and solutions

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