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The Presence and Persistence of Resistant and Stem Cell-Like Tumor Cells as a Major Problem in Ovarian Cancer

Prof. Sabine Kasimir-Bauer

Department of Gynecology and Obstetrics

University Hospital Essen

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Welcome!

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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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Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

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Agenda

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Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

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Initial situation in ovarian cancer

Classical prognostic factors:• Tumor size

• Grading

• Histological subtype

• Tumor rest after surgery

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Residual tumor rest after surgery is the only influenceable prognostic factor in ovarian cancer.

Prognostic factors

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Influence of residual tumor rest after surgery

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Treatment strategies include:• Primary surgery aiming at macroscopic complete tumor resection

• Subsequent platinum- and paclitaxel-based chemotherapy

• Additional or combined treatment with Avastin (anti-VEGF), Olaparib (PARP inhibitor)

Biggest problem in ovarian cancer: Platinum resistance!

15-20% of patients do not respond to platinum-based chemotherapy which can only be assessed in the follow-up of the disease!

Treatment strategies

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No suitable marker to predict prognosis and platinum resistance!

Is translational research able tofind predictive markers for:

• Progression-free survival (PFS)

• Overall survival (OS)

• Platinum resistance (PR)

What kind of biomaterial is the bestto look for predictive markers?

• Primary tumor, blood, bone marrow?

• What are the right markers?

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Primary tumor

Circulating tumor cells in blood (CTCs)

Circulating biomarkers in blood• Circulating tumor DNA

• Circulating microRNAs

• Other

Disseminated tumor cells in the bone Marrow (DTCs)

Biomaterial

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Do we have reliable methods for selection and detection of DTCs and CTCs?

Are these cells of prognostic significance?

What are the characteristics of these cells?

Important questions to ask

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Agenda

12

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

1

2

3

4

5

Sample to Insight

Agenda

13

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

1

2

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4

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BM aspiration Density centrifugation,isolation of mononuclear cells

Immunocytochemistry for the detection of cytokeratin-positive cells (DTCs)

ARIOP SL-50 for the evaluation of DTCs

25% Positivity Rate

Selection and detection of DTCs in bone marrow

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Prognostic relevance of DTCs at primary diagnosis

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In a multivariate analysis, DTCs were an independent prognostic factor for PFS and OS, but not for PR!

Primary ovarian cancer patients(n = 495)

DTCs in the literature

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Is the negative prognostic impact of DTCs related to:

• DTC persistence after platinum-based chemotherapy

• to a cellular phenotype, being associated with stem cell character

Negative prognosis impact of DTCs

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Analysis of BM before and after chemotherapy

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PFS OS

Analysis of BM before and after chemotherapy (n = 79 patients)

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Analysis of BM before and after chemotherapy (n = 79 patients)

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Lin-28 positive / cytokeratin positive BM cells after therapy

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Comparable results were obtained for the Stem cell marker SOX-2.

Lin-28 positive / cytokeratin negative BM cells after therapy

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1. DTCs present after therapy express stem cell-associated proteins

2. DTCs with stem cell-associated proteins were also detected (in some cases) after, but not before therapy, which might explain negative outcomes of patients who changed from DTC negative to DTC positive

3. We also detected CK positive / LIN-28 positive (SOX-2 positive) as well as CK negative / LIN-28 positive (SOX-2 positive) cells in all patients

From tumor cells that had undergone epithelial-mesenchymal transition?

Additional therapeutic regimens are necessary to eliminate these cells.

Key messages

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Agenda

24

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

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3

4

5

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Agenda

25

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

1

2

3

4

5

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More than 40 different methods for CTC selection are available.

Aside from the prognostic relevance of CTC counts, predictive markers are needed.

Characterization of CTCs is essential to use CTCs as a liquid biopsy for targeted therapy.

Selection and detection of circulating tumor cells (CTCs)

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Selection / detection methods:

Immunomagnetic cell enrichment • Tumor cell enrichment using magnetic particles

conjugated with an antibodymixture

mRNA isolation and RT • mRNA stabilization, mRNA isolation with oligo(dT)

magnetic beads and cDNA synthesis by reverse transcription

Multiplex PCR • Expression analysis of tumor associate markers

Selection and detection of circulating tumor cells (CTCs)

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Selection and detection of circulating tumor cells (CTCs)

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QIAGEN solutions for CTC detection

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Prognostic significance of CTCs in Ovarian Cancer

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Aktas et al., Gynecol Oncol, 2011

Prognostic significance of CTCs in ovarian cancer

Using AdnaTest

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Kuhlmann et al., Clin Cem 2014

N=143 patients

Prognostic significance of CTCs in ovarian cancer

Using AdnaTest OvarianCancer

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Kuhlmann et al., Clin Cem 2014

Prognostic significance of CTCs in ovarian cancer

Using AdnaTest OvarianCancer

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We evaluated the prognostic significance of ERCC1-positive CTCs (ERCC1+CTC)

Positive for at least one of the transcripts EpCAM, MUC-1, Ca 12-5.

Positive for ERCC1-transcripts.

ERCC1: excision-repair cross-complementing rodent repair deficiency complementation group 1 nuclease

Hypothesis: Negative prognostic impact of CTCs may arise from a cellular phenotype, being associated with platinum-resistance.

Example experiment

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Kuhlmann et al., Clin Cem 2014

Prognostic significance of ERCC1+CTCs

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Kuhlmann et al., Clin Cem 2014

AdnaTest OvarianCancer ERCC1+ predicts platinum failure

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The presence of ERCC1+CTCs, a potentially platinum-resistant CTC-subgroup, is an independent predictive biomarker for primary platinum-resistance and poor prognosis of ovarian cancer.

Summary

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How does the additional assessment of ERCC1-transcripts influence overall CTC-detection rate?

Performing paired pre- and post-therapeutic blood analysis, we inquired whether ERCC1+CTC dynamics mirror response to platinum-based chemotherapy.

ERCC1+CTCs as a potential diagnostic tool

ERCC1+CTCs as a potential diagnostic tool for monitoring response to platinum-based chemotherapy and for predicting post-therapeutic outcome of ovarian cancer.

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CTC-positive: at least one of the transcripts EpCAM, MUC-1, Ca 12-5 is expressed in the sample (AdnaTest OvarianCancer). ERCC1 was analyzed seperately.

Chebouti et al., 2016 AACR New Orleans

Ovarian cancer patients pre- and post-therapy (n = 65)

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ERCC1+CTCs

Chebouti et al., 2016 AACR New Orleans

Ovarian cancer patients pre- and post-therapy (n = 65)

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A

C

B

D

Chebouti et al., 2016 AACR New Orleans

Prognostic significance of CTCs and ERCC1+CTCs

With regard to PFS, OS.

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Additional assessment of ERCC1-transcripts expands the phenotypic spectrum of CTC-detection and defines an additional highly overlapping fraction of ERCC1-expressing CTCs, which are potentially selected by platinum-based chemotherapy.

We suggest that, beside their capacity to predict platinum-resistance at primary diagnosis, ERCC1+CTCs could additionally be useful as a surrogate for monitoring response to platinum-based chemotherapy and to assess post-therapeutic outcome of ovarian cancer.

Summary

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Agenda

43

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

1

2

3

4

5

Sample to Insight

Agenda

44

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

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2

3

4

5

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Establish a multimarker genpanel to explore the heterogenousCTC-population in ovarian cancer!

These genes should include:• Epithelial marker

• Stem cell marker

• Marker that indicates epithelial mesenchymal transition (EMT)

Future goals

Recently published, regarding breast cancer:

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Agenda

46

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

1

2

3

4

5

Sample to Insight

Agenda

47

Introduction to and overview of ovarian cancer

Disseminating tumor cells (DTCs)

Circulating tumor cells (CTCs)

Future goals

Questions

1

2

3

4

5

Sample to Insight48

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Questions?

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.