1. Katerina Kypraiou Detection, Characterization and Clinical Validity of CTCs in Melanoma Research Laboratory of Photobiology & Skin Cancer Department of Dermatology, University of Athens Andreas Sygros Hospital Athens, Greece

2. Cutaneous Malignant Melanoma  One of the most aggressive forms of cancer * curable by surgical excision in early stages  High tendency to metastasize  High resistance to chemotherapy  Thickness of the primary tumor  Regional lymph node status  Imaging diagnostic methods can only inform us of metastasis in an advanced stage  Detection of CTCs in the peripheral blood could screen out patients with a higher risk of relapse at the time of diagnosis Very poor overall survival Most important clinicopathological factors associated with metastasis

3. METASTASIS

4. Methods used for CMCs isolation and enrichment marker independent Density Gradient Centrifugation (DGS) (de Vries et al., 2000, Reinhold et al., 2001) - established method for the collection of CMCs using Sucrose or CsCl - Ficoll-Hypaque is commonly used for this purpose. - relies on the physical characteristics of density and size to separate mononuclear cells and CMCs. Major disadvantage is low efficiency of cell recovery which may result in loss of melanoma cells due to shearing or formation of cell aggregates at the bottom of the tube.

5. - The depletion of erythrocytes from the sample is another common and cost- efficient enrichment procedure. It involves erythrocyte lyses through the use of an NH4Cl buffer, followed by centrifugation and discarding of the supernatant (Liu et al., 2011, Kitago et al., 2009). This technique offers slightly improvement over DGC but is much easier and maintains uniformity between samples (Mitropapas et al., 2006, Nezos et al., 2011). Methods used for CMCs isolation and enrichment marker independent Red Blood Cell Lysis Buffer

6. Methods used for CMCs isolation and enrichment marker independent OncoQuick

7. Methods used for CMCs isolation and enrichment marker independent ISET

8. Immunomagnetic enrichment: - Commonly used devices: MACs microbeads and separator (Miltenyi Biotech), EasySep (StemCell Technologies) and Dynamag Separator (Invitrogen). A key advantage CMC integrity and morphological features are left intact Major disadvantage for CMCs is that heterogeneity requires multiple markers to positive select CMCs. Methods used for CMCs isolation and enrichment marker dependent Positive Immunomagnetic enrichment: Ags that characterize melanoma and its surface antigens (Liu et al, 2011). -Commonly used Abs are: - Melanoma Chondroitin Sulfate proteoglycan also known as High Molecular Weight Melanoma-Associated Ag (MCSP/HMW-MAA) (Kitago et al., 2009). -MCAM/MUC18/CD146 target cell with higher metastatic potential, associated with aggressive phenotypes and disease protection (Shih et al., 1994, Xie et al., 1997, Bardin et al., 1996). - Markers for ATP binding cassette subfamily B member 5 (ABCB5), nestin (NES), CD133, CD271 (Schatton et al., 2008,Fusi et al., 2011, Boiko et al., 2010). Multimarker immunomagnetic isolation allows the enrichment of more CMCs than the use of individual Abs, supporting the notion that there is heterogeneity in CMCs.

9. Methods used for CMCs isolation and enrichment marker dependent Negative Immunomagnetic enrichment - removes all leukocytes so that the remaining CMC population can be isolated Six studies have used negative enrichment through CD45 depletion (leukocytes) and/or CD34 depletion (endothelial cells) (Rao et al., 2011, Khoja et al., 2013, Liu et al., 2011, Georgieva et al., 2002, Fusi et al., 2011, Sakaizawa et al., 2012). Georgieva et al., found negative enrichment to be superior to positive, along with being time efficient and more practical than other isolation procedures usually combined with RT-PCR. Sakaizawa et al., stated that a consequence of CD45 depletion is a potentially low yield as a result of loosing CMCs with the leukocytes. Clawson et al., suggests that melanoma- macroplhage hybrids are lost.

10. Cell Search: Semi Automated Technology immunomagnetic enrichment with EpCAM, MSCP, MCAM. Ab staining of both CMCs (MCSP) and contaminating cells (CD45, CD35), followed by DAPI and visualization through digital fluorescent microscopy (Rao et al., 2011, Khoja et al., 2013, Khoja et al., 2014, Steen et al., 2008). DAPI positive and at least 4 microns diameter. FDA approved. Automation allows comparison between studies as it excludes many external variables. Further studies need to evaluate Cell Search specificity in CMCs. Rao et al., 2011; Khoja et al., 2013 and 2014 have showed that inclusion of more detection markers in the Veridex melanoma CTC kit will make Cell Search extremely valuable approach. Methods used for CMCs isolation and enrichment

11. CMC detection : Direct techniques Manual Immunocytometry (ICM) Can be combined with immnonomagnetic enrichment (Benez et al., 1999, Siewert et al., 2001). Immnostaining is followed by microscopy (Freeman et al., 2012, Benez et al., 1999, Siewert et al., 2001, Sakaizawa et al., 2012) or even laser capture microdissection to isolate individual cell for genotyping (Sakaizawa et al., 2012). CMCs remain intact for morphological examination. Can use negative CD45 staining to exclude leukocytes. Disadvantages: Time consuming approach, subjective morphological interpretation, CMCs can be confused with large leukocytes or circulating endothelial cells (De Giorgi et al., 2010, Khoja et al., 2014).

12. Flow Cytometry: FACS Records the fluorescent signal of ab labeled cells as they rapidly pass by a detector in a stream of individual cells (Liu et al., 2001, Fusi et al., 2011). Allows multi-parameter analysis of individual cells. Automatic sorting for further characterization. CMC detection : Direct techniques Disadvantages: Requires enriched samples or large volume. Non-specific binding may cause false positives, low sensitivity without enrichment. Aggregates give false results.

13. Photoacoustic Flow Cytometry -Irradiates cells with pulsed light and then measures ultrasonic high frequency waves produced by pigmented cells as a result of thermoelastic expansion (O’Brien et al, 2012, Weight et al, 2009). -High specificity, no markers needed. CMC detection : Direct techniques Disadvantages: Lacks clinical validation False positive results due to circulating benign nevus cells False negative results due to amelanotic cells

14. qRT-PCR- quantitative Real Time PCR -Can quantify the number of mRNA copies in a sample through the use of DNA probes that generate a measurable fluorescent signal which is subsequently quantified after each cycle in comparison to control values. -Quantitative assay. - More sensitive than other PCR-based protocol. -Practical and cost-efficient, independent of additional enrichment techniques. The inclusion of both spiked blood samples as positive controls and healthy samples as negative controls during each round of PCR further ensure assay reliability (Schittek et al., 1999, Koyanagi et al., 2005, De Vries et al., 1999) CMC detection : Indirect techniques Disadvantage: Indirect proof, does not determine exact number of CMCs.

15. Circulating Melanoma Cell Detection RT – PCR Melanocytic MarkersMelanoma Specific Less Specific for Melanoma Tumor Markers TYRMART-1/Melan-AMAGE-A1,-A3,-A6 MITFMelanotransferrin -P97PRAME PAX3GalNAcTNY-ESO-1 TRP-1MIAS100b TRP-2/DctMUC18/MCAMYKL-40 gp100

16. Circulating Melanoma Cell Detection TYR is a basic enzyme involved in the conversion of tyrosinase to melanin in process of melanin biosynthesis. is expressed in normal melanocytes, melanoma cells, brain and colon cancers and in low levels in testis and normal colon tissue. Expression of TYR in blood indicates the presence of CMCs since normal melanocytes are not present in the circulation. Although TYR has been negative in some cases has also been very helpful in many cases with patients in advanced stage melanomas (III – IV) (Tsao et al, 2001). Some commonly studied markers

17. PAX3 is a transcription factor involved in melanoblast proliferation, migration, and differentiation during development. positively regulates other CMC biomarkers (MITF, TGFb1, MCAM) (Medic et al., 2009, Medic et al., 2011). In some cases has been a useful marker of both primary malignant melanoma cells and CTCs (Blake et al, 2005, Koyanagi et al, 2005, Takeuchi et al, 2004). Interestingly was found even in melanoma cell lines that were amelanotic, where other melanocytic factors had been repressed (Vachtenheim et al., 1999). Circulating Melanoma Cell Detection Most studied Lin and Fisher, Nature 2007

18. MITF is implicated in melanocyte determination and differentiation during development and even more in regulation of melanoma proliferation (Carreira et al, 2005, Garraway et al, 2005, Carreira, et al, 2005) and invasiveness (Loercher et al, 2005). Until now MITF has been proved as a very important marker for disease progression and overall survival of melanoma patients (Koyanagi et al, 2006), especially the MITF-M isoform has been shown to be melanocyte specific (Lin et al, 2004). Moreover most melanoma markers TYR, TYRP-1, gp 100 are dependent on MITF. Circulating Melanoma Cell Detection Most studied

19. MAGE A3 (MAGE-A3) is commonly expressed by various tumours and not by healthy tissue, except male germ line and placenta (Miyashiro I et al., 2001, Takeuchi H et al., 2004, Gkalpakiotis S et al., 2010). Using RT-PCR has been proven as the most sensitive progression marker (Arenberger P. et al, 2008). Circulating Melanoma Cell Detection Some commonly studied markers

20. MART1 is specific for the melanocyte lineage, found in normal skin, uvea, retina and melanocytes but not in other normal tissues (Nezos et al., 2009). is detected at AJCC stage I between 5-20% and at stage II between 7- 30%. However the detection level at AJCC stages III and IV is between 30-76% (Keilhoz et al, 2004, Sarantou et al, 1997, Palmieri et al, 2003, Enk et al, 2004, Reynolds et al, 2003, Koyanagi et al, 2005, Mocellin et al, 2004, Curry et al, 1999). Moreover TYR and MART-1 have been found in the blood of patients with uveal melanoma and patients with liver cancer from uveal melanoma (Keilhoz et al, 2004). Although a good marker the detection levels vary due to differential gene expression between patients and even the same patient over time (Arenberger et al, 2008, Enk et al, 2004). Circulating Melanoma Cell Detection Most studied

21. Patients: Number of patients: 111-817 Multiple sampling in most studies Median follow up: 19 – 75 months Methods: whole blood or PBMCs Used RT-PCR All used tyrosinase (plus Melan A/Mart 1, MAGE3, gp-100 ) CTC detection: Detection rate of CTCs: 13-80% A >2 marker assay more sensitive than tyrosinase alone Longer follow up and multiple sampling increased positivity Clinical outcome: Different outcome measures:DFS,OS,DMFS Conflicting results on prognostic value Khoja L et al., Annals of Oncology 00:1-7,2014

24. Hashimoto et al., CTC detection at any point was associated with worse DFS and OS; Detection of more than 1 markers was an adverse prognostic marker Quanglino et al., new metastases in all patients were associated with positive CTC detection of more than 1 time points; patients with detectable CTCs during follow up had iferior time to progression and OS Khoja et al., showed that detection of more than 2 CTCs / 7.5 ml blood is a an adverse prognostic factor ; Patients receiving treatment in whom CTC number remained more than 2 CTCs during treatment had shorter median OS than those who maintained less than 2 CTCs