Circulating Tumor Cells (CTCs)

• Circulating biomarkers are assessed in blood of cancer patients.
• Liquid biomarkers can include:
• Circulating tumor cells (CTCs)
• Circulating tumor DNA (ctDNA)
• Cell-free microRNAs (miRNA) and other non-coding RNAs (ncRNAs)
• Extracellular vesicles (exosomes and microvesicles)

CTCs

• CTCs are circulating tumor cells
• CTC clusters are groups of CTCs
• Described by Paget 1889 'seed and soil hypothesis'
• Seed: Cancer cells (tumor cells) act as the "seed."
• Soil: The microenvironment (organ or tissue) where cancer cells spread and grow, acts as the "soil."
• Most CTCs undergo apoptosis or remain dormant
• Less than 0.1% of CTCs can survive to form distant metastases
• CTCs have diameter ranging from 9 to 30μm
• They Very rare events, estimated to be 1 per 10^7 WBC

Methodologies to isolate CTCs

• Antibody based capture can isolate
• EPCAM Uses antibodies that specifically bind to EPCAM (epithelial cell adhesion molecule) or MUC1 (mucin 1) that are commonly expressed on the surface of cancer cells.
• MUC1 Uses antibodies that specifically bind to EPCAM (epithelial cell adhesion molecule) or MUC1 (mucin 1) that are commonly expressed on the surface of cancer cells.
• Physical characteristics such as
• Size filtration, based on their larger size compared to normal blood cells.
• Flow Dynamics, fluid flow to sort or capture CTCs by their ability to interact with the capture surface or pass through filters.
• Membrane capacitance, measures the electrical properties of the cells to distinguish CTCs based on their unique membrane characteristics.
• Other such as CD45 depletion and culture, this removes CD45+ leukocytes (white blood cells), as CD45 is a marker, allowing more isolation of CTCs.

CellSearch

• CellSearch is an EPCAM-based selection
• FDA approved for breast, colon and prostate cancer
• Can detect ~1 cancer cell in 7.5 mls blood
• CellSearch Technology:
• CTCs (Cancer Cells): EpCAM+, CK+, CD45-
• CTCs are isolated from blood samples using magnetic beads coated with EpCAM antibodies.
• Cytokeratins (CKs) are expressed on the surface of epithelial cells, including tumor cells that originate from epithelial tissues.
• CTCs express Cytokeratin (CK) - PE-conjugated CK antibody stains them.
• CD45+ leukocytes are removed to enrich for CTCs, which don't express CD45.
• CTCs are isolated from blood samples using magnetic beads coated with EpCAM antibodies.
• CTCs are rarely detected in healthy individuals
• CTCs predict survival in breast cancer
• CTC count is strong prognostic biomarker for survival in NET -Tumor grade is a key determinant of NET progression and patient outcomes
• Combining CTC count, tumor grade, and CgA levels may improve risk stratification & treatment decisions.
• CTCs may have multiple applications beyond cancer detection.
• Additional applications of CTCs:
• Drug target expression
• Pharmacodynamic assessment in drug development
• Genetic heterogeneity and evolution
• Biology of metastasis
• Strong EPCAM expression in midgut and pancreatic NETs suggests CTC (circulating tumor cell) detection via EPCAM-based methods (e.g., CellSearch) may be effective.
• As a result, they retain EPCAM expression, similar to other epithelial tumors.
• EPCAM promotes proliferation, migration, and invasion, contributing to NET progression.
• Functional studies on identified CTCs from NETs are limited.
• Combining epithelial (CK) and neuroendocrine markers (Synaptophysin, CD56) improves CTC detection sensitivity.
• At diagnosis,
• Low # of CTCs correlated with a better prognosis, however Ki-67 can change over time, affecting prognosis.
• Change in CTC number post treatment predicts survival

Neuroendocrine Tumours (NETs)

• NETs are rare tumours
• NETs: NETs are relatively uncommon types of cancers that arise from neuroendocrine cells, which have characteristics of both nerve and endocrine cells.
• They are made up of 67% Gl tract, 25% Lung, e.g. Alpha cells in pancreas, G cells in stomach
• Many NETs secrete hormonal peptides, which can cause a variety of symptoms depending on the hormone produced which
• May produce hormonal peptides.
  • Gastric ulcers (stimulayes gastric acid)
  • Relaxes smooth muscle, promotes vasodilation
    • Secretion of water & electrolytes in the intestines
• Variable clinical course
• Tumor differentiation impacts survival significantly.
• Better differentiation = Longer survival
• Also well-differentiated neuroendocrine tumors (NETs) have a much better prognosis than poorly differentiated ones.
• Poorly differentiated NETs behave aggressively, requiring more intensive management.
• There are a wide range of Therapeutic Options
• Somatostatin analogues to inhibit the release of hormones from hormones from NETs
• Chemotherapy
• Targeted radiotherapy
• Delivers chemotherapy directly to the tumour via the blood vessels, followed by embolization to block blood flow.
• Clinical Challenges
• Prediction of response
• There are problems with Ki67 such as that they are
• At diagnosis changes overtime therefore affecting prognosis A sampling error
• invasive for monitoring
• and a tumor’s Hetrogeneity may lead to inaccurate grading
• and there is a biopsy and therefore a frequent need to management

ctDNA

• Cell-free fetal DNA (cffDNA) from the placenta enters the maternal bloodstream and is useful in Non-invasive prenatal testing (NIPT) detects fetal genetic abnormalities
• Liquid biopsies provide a minimally invasive method for diagnosing and monitoring diseases.
• Short ~160bp
• Low concentration ~1000-2000 copies of the genome per ml
• You can tell different DNA apart by its sequence (e.g. with a mutation or from a father)
• The fragmentation pattern suggests (foetal) DNA is released by apoptosis
• Short half life (around 1 hour)
• Delayed processing results in white blood cell lysis, releasing genomic DNA, which contaminates ctDNA samples.
• If blood is collected into EDTA tubes it must be centrifuged within a short time frame or white blood cell DNA will be released.
• ctDNA is more reliable in metastatic and late-stage cancers due to increased tumor DNA shedding.
• Detect many different (unknown) mutations with Sequencing ctDNA to detect mutations
• More expensive and less sensitive and specific.
• Because tumour tissue is not always available to determine the sequence of tumour 'exome' from plasma. .
• ctDNA is more reliable in metastatic and late-stage cancers due to increased tumor DNA shedding
• Cytosponge-derived DNA can be sequenced to monitor genetic changes is a less invasive method that Endoscopy

Detection assays

• Detecting and tracking single ctDNA mutations by digital PCR is highly sensitive and specific detection
  • Very sensitive and specific assays can therefore assess mutations

CTC versus ctDNA

• Validated prognostic marker for CTC unlike ctDNA
• Minimally-invasive for both
• Isolation is difficult for CTC and easy for ctDNA
• Sensitivity to early stage detection is Low for CTC and medium for ctDNA
• Suitability of banked material is good for ctDNA but not CTC
• Functional studies can be performed on CTC not ctDNA
• In CTC protein analysis can de performed but not ctDNA
• Overall ctDNA offers more capabilities

This lesson explores circulating tumor cells (CTCs) found in the blood of cancer patients. It covers their role in metastasis, size, frequency, and methods for isolation, including antibody-based capture using EPCAM and MUC1.