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

Transcription

1 Welcome! 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 1

2 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 or can be requested from QIAGEN Technical Services or your local distributor. 2

3 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 3

4 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 4

5 Initial situation in ovarian cancer Classical prognostic factors: Tumor size Grading Histological subtype Tumor rest after surgery

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

7 Influence of residual tumor rest after surgery

8 Treatment strategies 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!

9 No suitable marker to predict prognosis and platinum resistance! Is translational research able 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?

10 Biomaterial 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)

11 Important questions to ask 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?

12 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 12

13 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 13

14 Selection and detection of DTCs in bone marrow BM aspiration Density centrifugation, isolation of mononuclear cells 25% Positivity Rate Immunocytochemistry for the detection of cytokeratin-positive cells (DTCs) ARIOP SL-50 for the evaluation of DTCs

15 Prognostic relevance of DTCs at primary diagnosis

16 DTCs in the literature In a multivariate analysis, DTCs were an independent prognostic factor for PFS and OS, but not for PR! Primary ovarian cancer patients (n = 495)

17 Negative prognosis impact of DTCs 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

18 Analysis of BM before and after chemotherapy

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

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

21 Lin-28 positive / cytokeratin positive BM cells after therapy

22 Lin-28 positive / cytokeratin negative BM cells after therapy Comparable results were obtained for the Stem cell marker SOX-2.

23 Key messages 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.

24 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 24

25 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 25

26 Selection and detection of circulating tumor cells (CTCs) 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.

27 Selection and detection of circulating tumor cells (CTCs) 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

28 Selection and detection of circulating tumor cells (CTCs)

29 QIAGEN solutions for CTC detection

30 Prognostic significance of CTCs in Ovarian Cancer

31 Prognostic significance of CTCs in ovarian cancer Using AdnaTest Aktas et al., Gynecol Oncol, 2011

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

33 Prognostic significance of CTCs in ovarian cancer Using AdnaTest OvarianCancer Kuhlmann et al., Clin Cem 2014

34 Example experiment Hypothesis: Negative prognostic impact of CTCs may arise from a cellular phenotype, being associated with platinum-resistance. We evaluated the prognostic significance of ERCC1-positive CTCs (ERCC1+CTC) Positive for at least one of the transcripts EpCAM, MUC-1, Ca Positive for ERCC1-transcripts. ERCC1: excision-repair cross-complementing rodent repair deficiency complementation group 1 nuclease

35 Prognostic significance of ERCC1 + CTCs Kuhlmann et al., Clin Cem 2014

36 AdnaTest OvarianCancer ERCC1 + predicts platinum failure Kuhlmann et al., Clin Cem 2014

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

38 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. 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.

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

40 Ovarian cancer patients pre- and post-therapy (n = 65) ERCC1+CTCs Chebouti et al., 2016 AACR New Orleans

41 Prognostic significance of CTCs and ERCC1 + CTCs A With regard to PFS, OS. B C D Chebouti et al., 2016 AACR New Orleans

42 Summary 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 posttherapeutic outcome of ovarian cancer.

43 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 43

44 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 44

45 Future goals 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) Recently published, regarding breast cancer:

46 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 46

47 Agenda 1 Introduction to and overview of ovarian cancer 2 Disseminating tumor cells (DTCs) 3 Circulating tumor cells (CTCs) 4 Future goals 5 Questions 47

48 Thank you for attending today s webinar! Questions? Contact QIAGEN Call: (NA) (EU) QIAWebinars@QIAGEN.com 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 or can be requested from QIAGEN Technical Services or your local distributor. 48