1. When will the diagnosis of Circulating tumor cells (CTC) help to patients? Martin Pešta, Ondřej Topolčan Department of Internal Medicine II, Faculty of Medicine in Pilsen, Charles University in Prague, Czech Republic

2. What we know today ● Circulating tumor cells (CTC) are present in peripheral blood of many patients with advanced stage of cancer ● The number of CTC is in relation to prognosis of cancer ● Reducing number of CTCs after chemotherapy is in relation to response to treatment ● It is possible to perform various phenotypic analysis (proteins and nucleic acids) of isolated CTC

3. What should bring assessment of CTC to patients ● Detection of CTC would help determine: - early detection of cancer relapse (therapy monitoring) - the choice of cancer therapy  The patient wants to know how he's doing  The doctor wants to know what treatment to use

4. By detection of CTC can be obtained information: ● Identification number (enumeration) of circulating tumor cells ● Identification of phenotype of CTC

5. ● A re circulating tumor cells - biomarker on their own? or ● Are circulating tumor cells the substrate to determine biomarker / s? CTC

7. Incomplete definition of CTC by the FDA

8. Epithelial-mesenchymal transition (EMT) ● The dynamic process of transformation of epithelial cells into mesenchymal through a special transcription program ● During the EMT is transformed phenotype of cells - The release of intercellular connections - Change in expression of surface receptors - Reorganization of the cytoskeleton - Significantly increases cell motility - Loss cadherin expression and the ability of cells to survive in the absence of extracellular matrix components (support antiapoptotic activity) ● In the process of EMT are produced cells with properties of stem cells: - EMT by producing of cancer stem cell retains the ability to self-renewal for the development of metastases

9. Tumor stem cells (CSC) ● Origin and development of CSC is a multifactorial process - starting cells for development CSC are tissue-specific stem cells - a series of genetic and epigenetic changes activate signaling pathways managing the process of self-renewal, transformation and tumor dedifferentiation ● Model CSC - tumor activity and growth are controlled by only a small part (less than 1%) of tumor cells with the ability to self- renewal, resistance to apoptosis and increased ability to repair DNA

10. Determination of CTC phenotype Number of CTC Analysis of chromosomes (Her2, TMPRSS-ETS) Analysis of DNA Analysis of RNA expression CTC phenotype - protein (Her2, EGFR, AR)

11. CTC diagnostic is suitable only for the diagnostics of certain cancer

12. Current CTC isolation methods according to achieve the level of testing for clinical use prepared by:Mark Carle Connelly, Ph.D. Veridex, LLC – Ortho Clinical Diagnostics

13. Epic HD-CTC (EPIC, CA USA) ● "pathological approach" - blood smear on a slide – microscope evaluation ● Marking CK +, CD45-, DAPI + ● "accepted pathologists' ● Central collection, detection in laboratory, determination to 24h ● Benefits: - detection CTC doublets to large clusters CTC - You can add additional antibodies

15. Microchannel platform (Biocept, USA) ● Blood pressure flows through the chip with antibodies, in which are CTC captured

16. Microchannel platform (Biocept, USA) ● Visualization and enumeration of CTCs in the plate ● Following the analysis of CTC phenotype (eg, cytogenetics, molecular biology)

17. CTC detection using Photoacoustic flow cytometry ● Photoacoustic flow cytometry is used to detection CTC sound reverberation (echo) from tumor cells ● Photoacoustic flow cytometry monitors cells circulating in vivo ● It is currently being developed for the detection of circulating melanoma tumor cells (CMC) ● Sound reflection is made ​​ possible by the presence of melanin in cells The method is developedbe John A. Viator, Ph.D. Departments of Biological Engineering and Dermatology University of Missouri, Columbia http://kolbe.missouri.edu/Welcome.html

18. CTC detection using Photoacoustic flow cytometry

19. Problems associated with the clinical application of CTC ● Have all detected CTC the same metastatic potential? ● To what extent is the determination of CTC reproducibile? ● Conduct of clinical trials is necessary ● Each platform provides different results

20. Conclussion ● Answering and resolving these problems allows correct interpretation of results and subsequent diagnostic CTC use in treating cancer patients. ● Already existing diagnostic results CTC particularly in patients with breast cancer has shown to be beneficial. Understanding the biology of CTC should provide a more accurate choice of treatment, cure or prolongation of life.

21. Thank you for your attention!