CLINICAL HANDS ON PRESENTATION 14/09/2014 Marina Chatziioannou RnD - - PowerPoint PPT Presentation

CLINICAL HANDS ON PRESENTATION 14/09/2014

Marina Chatziioannou RnD department R.G.C.C. Ltd

Points of interest

• 1. CTCs isolation

− Manual or automated cell isolation − Magnetic bead epitope selection

• 2. Clinical flow cytometry

− What for? − Epitope selection − Sample handling − Data analysis

CTCs isolation

Topics

• 1. Sample arrival and handling
• 2. Manually or automated cell isolation
• 3. Magnetic bead epitope selection
• 4. Culture treatment

CTCs isolation

Manually

 Ficoll layering  Washing  Beads incubation  Magnetic CTCs isolation  Culture in FL25

Automated

 Robosep handling  Culture in FL25

Epitopes (bead selection)

Categorization

• 1. Epithelial origin
• 2. Hematological origin
• 3. Sarcomas
• 4. Non epithelial, non hematological, non

sarcoma

Epithelial origin

EpCAM i. Expressed exclusively in epithelia and epithelial-derived neoplasms.

• ii. Can be used as diagnostic marker for various

cancers.

• iii. Plays a role in tumorigenesis and metastasis
• f carcinomas, so it can also act as a

potential prognostic marker.

Hematological origin

CD45

i. Leukocyte Common Antigen (LCA). ii. Its various forms are present on all differentiated hematopoietic cells except erythrocytes and plasma cells.

• iii. It is expressed in lymphomas, B-cell chronic

lymphocytic leukemia, hairy cell leukemia, and acute nonlymphocytic leukemia.

• iv. A monoclonal antibody to CD45 is used in routine

immunohistochemistry to differentiate between histological sections from lymphomas and carcinomas.

Sarcomas

CD99 i. Ewing’s marker

• ii. Present to all sarcomas

Non epithelial, non hematological, non sarcomas

CD45 negative selection i. For types of cancer that are not included in the previous categorizations.

• ii. For example:
• Renal cancer
• SCLC
• NK lymphomas
• Neuroblastomas
• Adrenal cancer

Flow cytometry for clinical samples

Topics

• What for?
• Epitopes selected
• Sample handling
• Data analysis

What for?

• Easy, quick and reliable technique.
• Gives accurate results.
• Quick view of what to expect in the isolated

culture from the same patient.

Flow cytometry clinical epitopes

i. CD45:LCA

• ii. CD31: Endothelial cell marker
• iii. cMet: Metastatic cell marker
• iv. PanCK: Marker of cell of origin of various

tumors (depends on cytokeratis pattern)

• v. CD227: Breast, Lung cancer marker
• vi. CD63: Melanoma cancer cell marker
• vii. PSMA: Prostate cancer cell marker

Sample handling

Steps

• 1. Extracellular staining
• 2. Washing
• 3. Fixation
• 4. Washing
• 5. Permeabilization
• 6. Intracellular staining

Data analysis

• Flow cytometry data file is analyzed and

percentages of epitopes are produced.

• Results are represented as below:

References

1. Ignatiadis, M., C. Sotiriou, et al. (2012). "Minimal residual disease and circulating tumor cells in breast cancer: open questions for research." Recent Results Cancer Res 195: 3-9. 2. Gendler SJ (July 2001). "MUC1, the renaissance molecule". J. Mammary Gland Biol Neoplasia 6 (3): 339–353. doi:10.1023/A:1011379725811. PMID 11547902 3. Radford KJ, Thorne RF, Hersey P (1996). "CD63 associates with transmembrane 4 superfamily members, CD9 and CD81, and with beta 1 integrins in human melanoma". Biochem. Biophys.

• Res. Commun. 222 (1): 13–18. doi:10.1006/bbrc.1996.0690. PMID 8630057

4. Omary MB, Ku NO, Strnad P, Hanada S (July 2009). "Toward unraveling the complexity of simple epithelial keratins in human disease". J. Clin. Invest. 119 (7): 1794–805. doi:10.1172/JCI37762. PMC 2701867. PMID 19587454 5. Tang DG, Chen YQ, Newman PJ, et al. (1993). "Identification of PECAM-1 in solid tumor cells and its potential involvement in tumor cell adhesion to endothelium.". J. Biol. Chem. 268 (30): 22883–94. PMID 8226797 6. Gentile A, Trusolino L, Comoglio PM (March 2008). "The Met tyrosine kinase receptor in development and cancer". Cancer Metastasis Rev. 27 (1): 85–94. doi:10.1007/s10555-007- 9107-6. PMID 18175071

Thank you for your time and patience!