Circulating tumor cells in oral squamous cell carcinoma-an enigma or reality?

Oral squamous cell carcinoma (OSCC) is ranking 1(st) among males and 4(th) among females in India. In spite of major advances in diagnosis and treatment of OSCC, survival rates, have remained poor. Circulating tumor cells (CTCs) in the blood stream, play an important role in establishing metastases. It is important to identify patients suffering from nonlocalized tumor with "circulating" tumor cells to determine the tailor made, systemic therapy in addition to local resection and irradiation. Thus, detecting metastases at an early stage are needed for better prognosis and survival. CTCs as new prognostic marker to detect the metastatic potential will provide a novel insight into tumor burden and efficacy of therapy. The recent advances and its application in OSCC will be reviewed.

Circulating tumor cells (CTCs) that play a pivotal role in establishing metastases serve as biomarkers for diagnosis, treatment and prognosis of cancer. Death in more than 90% of cancer patients is found to be due to metastasis.[1] Cancer cells are said to be detected in circulation even before the presentation of clinical symptoms.[2] CTCs were reported to be present about 140 years ago.[3]

CellSearch system (Veridex, Raritan, NJ, USA) was introduced in 2004. It is the only device cleared by Food and Drug Administration (FDA) for selecting and enumerating CTCs.[4]

In patients with oral squamous cell carcinoma (OSCC), CTCs are regarded as prognostic markers for the disease – free survival.[5] In the peripheral blood, CTCs are extremely rare events, estimated to be around one cell among a hundred million or billion of circulating blood cells.[6] Thus, an enrichment step is mandatory for efficient detection of these cells.

Development of Metastasis via Circulating Tumour Cells

Epithelial-to-mesenchymal transition occurs when the tumor cells and clusters that are shed from the primary tumor intravasate into circulation. Apoptosis and necrosis kills a majority of CTCs, releasing debris, fragments of cells and intracellular elements (circulating tumor material and ctDNA). Circulating tumor microemboli (CTM) that are clusters of tumor cells, as a result of the collision may release the tumor cells. The CTCs then extravasate, leading to metastasis [Figure 1].[7]

Figure 1

Development of metastases via circulating tumor cells

Circulating Tumor Cells Assays

Involves an initial enrichment step, where tumor cells are isolated following depletion of blood cells

The tumor cells are stained or oncogene probed: The CTCs are labeled by antibodies or aptamers or probed by DNA primers

Tumor cell detection via cytometry, microscopy, conductometry, fiber-optics, reverse transcription- polymerase chain reaction (RT-PCR), fluorescence in situ hybridization and comparative genomic hybridization.

The enrichment methods depend on physical characteristics like size, density or dielectrophoretic mobility that exemplifies CTCs or by the expression of certain surface molecules that are captured by magnetic bead-coated antibodies. Gradient centrifugation is commonly done using ficoll-hypaque that uses density of different cell types. A substantial loss of cell material is however said to occur during enrichment.[8] Monoclonal antibodies are available against different epithelial proteins, namely cytoskeleton keratins, surface adhesion molecules or growth factor receptors.

One of the most advanced methods for detecting and enumerating CTCs from peripheral blood is by the CellSearch system (Veridex, Raritan, NJ, United States) that enriches and immunostains CTCs. It is the first FDA-cleared device for CTC detection in solid tumors and for metastasized prostate, colon, and breast cancers.[910111213] Gröbe et al. detected CTCs in a small number of OSCC patients.[5] The EpCAM positive tumor cells are separated by immunomagnetic bead separation and are immunofluorescent stained with anti-keratin antibodies. The leukocytes are excluded using anti-CD45-antibody. Under fluorescent microscope, nucleated cells with a diameter of 4 μm, keratin positive and CD45 negative are accepted as tumor cells.[14]

Other recently developed EpCAM-based tools include:

Microfluid-based CTC-chips[15]

Surface enhanced Raman spectroscopy with epidermal growth factor receptors as targeting ligand[16]

Detection of CTCs after the leukapheresis.[17]

In Vivo Detection of Circulating Tumour Cells

GILUPI cell collector device consisting of a wire coated with anti-EpCAM antibodies that collects CTCs when inserted into a vein for 30 min. Presence of CTCs and exclusion of leukocytes are confirmed with anti-keratin and ant-CD45 antibodies.[18]

Epithelial Immunospots

Involves detection of viable CTCs after a 48 h culture, where leukocytes are depleted by negative selection using CD45.[19]

Molecular Technologies

They involve PCR amplification based on either DNA or complementary DNA (messenger RNA). RT-PCR approaches use epithelium-specific targets, like keratin 19 encoding targets.[20]

Recent Trends in Circulating Tumour Cells Research

Detecting circulating tumor microemboli

A collective migration of tumor cells leads to the formation of CTM.[21] These collective tumor cells intravasate through leaky blood vessel.[22] CTMs exhibit a highly variable morphology: As clusters, circles or strands.[23]

The likely cause of CTM formation is increased adhesiveness of circulating tumor cells.[24]

Studies indicate CTM presence indicates higher metastatic potential.[25] The suggested explanations are as follows:

CTMs are easily caught in narrow vessels than CTCs[26]

CTMs provide a favorable environment for survival of tumor cells[24]

EGF and VEGF stimulate proliferation and inhibit apoptosis.[27]

Circulating tumor microemboli detection is done using flow cytometry[28] or by cell search.

Circulating tumor cells detection based on telomerase activity

As telomerase activity inactivated in cancer[29] and since tumor cells lose epithelial markers during metastasis, assessing telomerase activity would provide valuable information on CTCs. Since the whole blood sample has to be lysed to measure enzyme activity, CTCs get destroyed, which is a downside for the method.

Circulating tumor cells detection by aptamer technology

Aptamers are single-stranded DNA or RNA molecules.[30] Systemic evaluation of ligands by exponential enrichment automates aptamer production.[31]

Conclusion

With the advent of new technologies to detect CTCs current staging methods of cancer have been refined. Additional treatment options can now be made available for patients suffering from OSCC as collecting samples from peripheral blood is invasive. Tumor cells that undergo an epithelial-to-mesenchymal transition show downregulation of epithelial features. Hence, molecular analysis of CTCs is required. Advances have been made with aptamers and microdevices that minimize technical constraints. CTCs have to be evaluated by proteomics and genomics too.