Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths of women in the United States and Europe and ranks as the second most common type of gynecological malignancy. Most cases are diagnosed in advanced stages and although the response rates to platinum-based chemotherapy are high, the majority of patients nevertheless have poor survival rates. Although the reasons for these poor outcomes are likely to be multifactorial, one particular area of interest has recently focused on hematogenous tumor cell dissemination that has been shown to originate from disseminated tumor cells (DTCs) in the bone marrow (BM) and circulating tumor cells (CTCs) in the blood. Here, we demonstrate that the negative prognostic impact of CTCs and DTCs arise from specific cellular phenotypes and are associated with platinum-resistance and stem cell-associated proteins.

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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
Classicalprognostic 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 researchable to
find predictive markers for:
• Progression-free survival (PFS)
• Overall survival (OS)
• Platinum resistance (PR)
What kind of biomaterial is the best
to 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
Disseminatedtumor 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
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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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4
5

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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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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% PositivityRate
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
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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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4
5

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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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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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Kuhlmannet al., Clin Cem 2014
N=143 patients
Prognostic significance of CTCs in ovarian cancer
Using AdnaTest OvarianCancer

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Kuhlmannet 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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Kuhlmannet al., Clin Cem 2014
Prognostic significance of ERCC1+CTCs

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Kuhlmannet 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 andpoor 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
expressedin 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
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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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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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2
3
4
5

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Establish a multimarker genpanel to explore the heterogenous
CTC-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
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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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Agenda
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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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Questions?
For up-to-date licensing information and product-specific disclaimers,see the respective QIAGENkit
handbook or user manual. QIAGENkit handbooks and user manuals are available at
www.QIAGEN.com or can be requested from QIAGENTechnical Services or your local distributor.