20.06.13 Circulating tumour cells

Question 1

What are circulating tumour cells (CTCs)

Answer 1

Cells released from primary tumour into blood stream and may result in metastasis into distant organs.

Question 2

What concentration are CTCs present in blood

Answer 2

1-10 cells per ml

Question 3

Enrichment approaches

Answer 3

1. Protein expression-based technologies (positive/ negative enrichment)
2. Physical property based technologies to enrich (filtration, chip, ficoll gradient, electrical field, single spiral microchannel)

Question 4

Detection approaches

Answer 4

1. Immunological technologies (antibodies targeting CTC markers)
2. RNA based technologies (reverse transcription PCR)
3. Functional assays (EPISPOT, Invasion assay, Xeno transplantation)

Question 5

Review of positive enrichment using protein expression based technology

Answer 5

• Requires an assumption about the unknown nature of CTCs in a blood sample (can cause bias)
• Epithelial markers used to distinguish between cancer and normal cells
• e.g. EPCAM (epithelial cell adhesion molecule)
• Also cytoskeletal markers (specific to epithelial cells, e.g. cytokeratins- CK8, CK18) can be used to detect carcinomas. However, some carcinomas undergo epithelial to mesenchymal transition (EMT) so could lead to false negative results.
• Mesenchymal markers= N-Cadherin, vimentin.
• Plastin 3 is a good marker as it is not downregulated by CTCs during EMT and not expressed in blood cells

Question 6

Review of negative enrichment using protein expression based technology

Answer 6

• Blood sample is depleted of leukocytes using antibodies against CD45 (not expressed in carcinomas or solid tumours).
• Benefit as it avoids the loss of CTCs with high phenotypic plasticity (or in various stages of EMT)

Question 7

Review of enrichment technologies using physical properties

Answer 7

• Separating CTCs from normal blood cells via their physical properties.
• E.g. CTCs were initially thought to be larger and less deformable (not true).
• Devices include filtration (size), chip (deformability), ficoll gradient (density), electric field (charge), single spiral microchannel (size).
• Often need further molecular characterisation to define nature of cells.

Question 8

Detection and characterisation of CTCs using functional assays

Answer 8

• Used to identify metastasis initiator cells (MICs)
• Requires CTC isolation prior to testing.
• Therefore, currently limited by the low concentration and yield of CTCs.

Question 9

What functional assays can detect and characterise viable CTCs in patients

Answer 9

• EPISPOT assay. Detects specific proteins secreted during the in vitro culture of CTCs. High sensitivity, but no further analysis possible.
• Invasion assay. Examines the ability of CTCs to digest a fluorescently labelled cell adhesion matrix.
• Xenotransplantation models. Transplantation of patient-derived CTCs into immunodeficient mice can provide in vivo information.

Question 10

What PCR based analysis can be used to study enriched CTCs

Answer 10

• More sensitive than immunocytochemistry
• Specificity is achieved by designing primers to gene of interest.
• RT-PCR used to amplify target mRNA from CTCs.

Question 11

Technical limitations of PCR based analysis of CTCs

Answer 11

• Requires cell lysis, therefore preventing cell counting
• False positives due to illegitimate gene transcription in non-tumour cells
• Amplification of cell free nucleic acid present in blood
• False positive results derived from the use of non-specific markers
• After invasive procedures there may be non-malignant epithelial cells in the blood
• False negative results due to limited expression of tumour marker or presence of PCR inhibitors.
• Limitations can be overcome by using multiplex assays and combining more than one marker.

Question 12

Review of cytometric methods can be used to study enriched CTCs

Answer 12

• Used to isolate and count cancer cells using monoclonal antibodies directed against different antigens (e.g. CKs and EPCAM)
• Lysis not required therefore further downstream analysis is possible
• Limitations: most anti-CK antibodies also bind haematological cells too. EPCAM down regulated in malignant cells during EMT.
• To overcome, existing antibodies combined with anti-CD45 antibody

Question 13

Examples of cytometric methods used to study enriched CTCs

Answer 13

• FAST (Fibre-optic array scanning technology) can analyse large volume of sample without purification (minimises risk of cell loss). Not validated for clinical setting.
• Flow cytometry. High specificity. Can be used to identify CTCs based on size, viability, DNA content, expression of various markers. However, low sensitivity so high amounts of sample need to detect few CTCs.

Question 14

Review of genetic profiling of isolated CTCs

Answer 14

-Genetic analysis may help to understand if CTCs have malignant characteristics or how similar to primary CTC. Important for disease monitoring, prognosis, drug resistance.
-Single CTC analysis overcomes limitations imposed by leukocyte contamination. Enables assessment of heterogeneity, disease evolution (serial CTC analysis).
-Need whole genome amplification as only 6.6pg DNA per cell. Risk of amplification bias or allele drop out.
-Somatic changes will need to be confirmed in multiple independent samples and control tissues to rule out artefacts due to WGA.
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