Liquid Biopsy for Cancer Metastasis Detections
Cancer Metastasis
Cancer metastasis is the phenomenon of cancer cells spreadng from the primary tumor site to form new tumors in distant organs or tissues. It is not random, since they spread to specific organs (breast cancer commonly spreads to bone, brain, lung, liver); this occurrence is called organotropism. Cancer cells after metastasis (metastatic tumors) are stil the same type. For instance, breast cancer cells that spread to the brain are still breast cancer cells, and it does not become brain cancer. Metastasis is responsible for over 90% of deaths. Cancer cells travel along blood and lymph vessels, and while many die, some may travel to different parts of the body and continue to divide, forming metastatic tumors. Steps in metastasis: local invasion (growing in the primary site), intravasation (entering into the blood or lymph vessels), circulation (circulating tumor cells (CTC) face immune attack and shear stress; often travel in clusters through the vessels), extravasation (adhere to endothelial walls and exit vessels), and colonization. Metastasis is usually detected throuh imaging such as a CT scan, MRI, etc. However, this is late detection, as the metastasis has already advanced and had tumors grown to a certain size for the image to detect it.
Circulating Tumor Cells
The existence of CTCs, tumor cells shed into the blodstream, was proposed long ago. In the 1990s-2000s, CTC detection reemerged, especially with the development of enrichment and detection platforms using nanotechnology. In 2004, the CellSearch system (Veridex) became the first and only clinically validated and FDA-cleared test for enumerating CTCs in metastatic breast, prostate, and colorectal cancers.
Inside the Blood
In the blood, red blood cells (RBC) are by far the most numerous (~4.5-6 million per mL). RBCs carry oxygen, have no nuclei, and the size is 6-8 micrometers with thickness of 2 micrometers. White blood cells (WBC) are far fewer (~4,000-11,000 per mL). WBCs are important for immune defense, have nuclei, and are 8 microcmeters larger than the RBC. There are diverse types of WBCs. Platelets are intermediate (~150,000-450,000 per mL). Platelets are important or blood clotting, have no nuclei, and have cell fragments. CTCs occur at <10 cells per mL of blood, making it extremely rare. CTCs have nuclei, cell origins are often epithelial, and the size is larger than RBCs.
CellSearch
CellSearch captures the cancer cells, performing immunomagnetic separation using anti-EpCAM ferrofluids. A cellsafe preservative tube holds a patient’s blood sample, and this sample is put into a microtube and centrifuged to remove plasma. An anti-CAM entibody specifically binds to EpCAM molecules on the CTC surfaces, and the ferrofluids that connect to the EpCAM have a magnetic property that allows the CTCs to be magnetically isolated. Fluorescence staining can be used for identification, usng DAPI to stain the nucleus. The cytokeratin (CK) is typically present in epithelial cells (CTC, CK+), and CD45 is typically expressed in immune cells (WBC) but not in cancer cells (CTC, CD45-).
Kaplan-Meyer Plots
Enumerating the number of CTCs can help to perform statistical methods and make Kaplan-Meyer plots to find survival rates. A Kaplan-Meyer plot is a statistical method to estimate the probability of an event occuring at different points in time. It is frequently usd for clinical trials to observe treatment effectiveness. A non-parametric survival curve is generated by obsering “events” (deaths) at time intervals. To make the plot, it is necessary to follow up with patients and record survival time. From the Kaplan-Meyer plot, it’s seen that the number of CTCs impacts the overall survival; patients with fewer than 5 CTCs have overall survival of 21.9 months, and patients with greater than 5 CTCs have overall survival of 10.9 months.
Here is a Kaplan-Meyer plot of the overall survival of patients with Breast Cancer, using data from The Cancer Genome Atlas (TCGA).
Here is a Kaplan-Meyer plot of the overall survival of patients with Acute Myeloid Leukemia, using data from TCGA.
Liquid Biopsy
A liquid biopsy is a test done on a sample of blood (or sometimes other fluids like urine or cerebrospinal fluid) to detect diseases (for instance, cancer metastasis). The CellSearch system is one kind of liquid biopsy technology, among several technology platforms developed. Metastatic tumors are often hard to reach. Metastatic burden is dynamic (continually changing), and liquid biopsy allows repeated sampling over time. It provides a non-invasive snapshot of the tumor evolution, heterogeneity, and treatment resistance. Liquid Biopsy can detect these biomarker types: CTCs, circulating tumor DNA (ctDNA), cfRNA, exosomes, and tumor educated platelets (TEP).
Other Biotechnology
CellSearch captures CTCs via anti-EpCAM, which is a major limitation, since some cancer types do not have EpCAM expression. Parsortix uses microfluidic and cell deformability. Tumor cells are larger and less deformable than blood cells. A company called Guardant Health is focused on developing detection of ctDNA, fragments of DNA released by tumors into the blood stream.
In the future of liquid biopsy, we look forward to transforming cancer detection and monitoring, earlier detection, real-time monitoring of tumor evolution, non-invasive and broadly accessible biopsies, and assist from BigData AI, where blood could be used to detect cancer before symptoms appear.