Liquid gold: blood-based biopsies make headway

Pathologic and, increasingly, molecular analysis of tumor tissue biopsies is the gold standard in initial diagnosis of cancer, but liquid biopsies, which analyze tumor-derived material circulating in the bloodstream are gaining traction. Here, we discuss the current state of development of this complementary and potentially alternative approach to tumor analysis.

Liquid biopsy gaining traction

Biopsies enable oncologists to gather information about a potential or established tumor, including confirmation of the presence of cancerous tissue and determination of its histological characteristics, such as tumor grade and stage, as well as its molecular features, such as the presence of certain gene mutations. Ultimately, this information can be put to use in determining the most appropriate course of treatment.

The current gold standard is a tissue biopsy that typically involves an invasive procedure to permit the collection of a piece of tumor tissue. Yet, tissue biopsies are not always feasible because of the location of the tumor or the poor performance status of many patients with advanced disease. They also provide only a snapshot of the disease at the time at which they were taken and don’t necessarily reflect the genetic heterogeneity or evolution of a tumor over time.

The detection of components that are derived from the tumor circulating in the blood of cancer patients had fueled the idea of blood-based diagnostics in oncology – so-called liquid biopsies. These have rapidly gained traction in the past several decades as a less expensive (the cost of performing genomic analyses on blood samples is at least an order of magnitude less than on tissue samples), less invasive (requiring only a simple blood draw) alternative source of information about tumors.¹

As researchers have refined the ability to exploit liquid biopsies, commercial interest has been piqued. More than 35 companies within the United States alone are developing liquid biopsies, and it’s easy to see why with a market projected to be in the many billions of dollars.²

Seeking out tumor clues in the blood

Liquid biopsies consist of a 10-15 mL blood sample drawn into a tube that contains an anticoagulant and it can contain several different types of tumor-associated material. Thus far, two components – circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) – have formed the cornerstone of liquid biopsies. At present, it is not clear whether these components are released randomly, as a by-product of tumor cell death or if they are released as part of a specific biologic process, such as for the colonization of metastatic sites. It reality, it may be a little of both, and active dissemination may be particularly relevant for CTCs, among which are postulated to be a population of cancer stem cells that can initiate distant metastases.^3,4

The discovery of CTCs dates back to the 1860s, when cells that were morphologically identical to the tumor were identified in the blood of a patient with metastatic cancer. Their potential significance was not fully realized until a few decades ago, when they were found to exist from early on in the course of disease.^3,4

CTCs, which can be either single cells or clusters of cells known as microemboli, have a short half-life in the bloodstream – less than 2 ½ hours – and are also extremely rare (1 mL of blood contains 1-10 CTCs) against a background of many millions of normal cells. Thus the detection and isolation of CTCs presents a significant challenge. More than 40 different platforms are being developed for the isolation and enrichment of CTCs. For the most part, these use a method called positive selection to pick out CTCs.^1,3,4

Positive selection exploits the biological or physical properties that are specific to CTCs and absent in normal cells, for example, the presence of a specific tumor-associated antigen on their surface or differences in size, density or electric charge. The limitations of this method are that, not only do you need to know something about CTCs to begin to understand what makes them truly unique and ensure only isolation of CTCs, but their phenotype is also thought to be continually changing.^1,3,4

In recent years, the focus has shifted toward technologies that use negative depletion, meaning that they target the other types of cells in the blood sample and filter those away until only the CTCs are left behind. The most advanced are devices that use microfluidic technology to sort the cells, such as the CTC-iChip system being developed by researchers at Massachusetts General Hospital in Boston.⁵

ctDNA consists of small fragments of nucleic acids that are not contained within a cell or associated with cell fragments and is thought to be present in 50%-90% of patients, depending on the type of cancer they have. ctDNA has a similarly short half-life in the circulation to CTCs and, like CTCs, ctDNA is present at very low levels in the bloodstream. Although levels of ctDNA have been shown to increase with increasing tumor burden, it is still often obscured by the presence of other cell-free DNA derived from non-tumor cells.

ctDNA can be distinguished from other cell-free DNA by the presence of somatic mutations and a number of highly sensitive methods have been developed to detect them, including the amplification-refractory mutation system (ARMS); digital polymerase chain reaction; and the beads, emulsification, amplification, and magnetics (BEAMing) system. Next-generation sequencing technologies, including tagged-amplicon deep sequencing (TAm-Seq), the Safe-Sequencing System (Safe-SeqS), and cancer personalized profiling by deep sequencing (CAPP-seq), can also be used and the race for ever more sensitive analytical tools is ongoing.^1,3,4,6