Multitemplate Liquid Biopsies

Improvements in liquid biopsy techniques offer a more holistic view of cancer

By Paul W. Dempsey, PhD

In the world of cancer diagnosis and monitoring, liquid biopsy technologies have the potential to be truly revolutionary. It has been known for many years that tumors shed cells, cell fragments, and DNA fragments into the bloodstream. But it is the pairing of these biological mechanisms with modern sample preparation and analysis platforms such as digital polymerase chain reaction (dPCR) or next-generation sequencing that is suddenly making it conceivable to analyze vanishingly rare cancer-derived events in a thicket of normal cells or DNA.

At the simplest level, liquid biopsies are used to hunt for traces of cancer in a blood sample. Such tests have been used to detect evidence of cancer across multiple stages of different types of cancer, including early-stage disease. They may also be used to assess genetic variation in cancer, in order to guide treatment selection and measure changes in the cancer in response to treatment. The idea of being able to monitor even healthy people and catch cancer early in its formation using something as simple as a blood draw has always captured the attention of the oncology community.

Like any new technology, however, liquid biopsy technologies have some hurdles to clear before they can be deployed in mainstream medicine. As scientists amass more and more data from these tests, there is mounting evidence that liquid biopsies represent a very different sampling mechanism from traditional tissue biopsy samples. Liquid biopsies measure signals from cellular and genetic materials released from throughout the tumor. It is now clear that those signals represent a very different sample type from the biopsy that has historically defined the biological target. While more clinical utility trials need to be completed, the information available now strongly suggests that the most reliable, sensitive, and actionable results will be generated by simultaneously sampling multiple biological targets, including circulating tumor cells, cell-free DNA, and tissue biopsy samples.

HETEROGENEITY IN TUMORS AND DATA

One reason for differences between liquid and traditional biopsies is simply that the cancer itself is heterogeneous. It is now widely acknowledged that clonal differences and basic selection—especially in response to treatment—lead to varying cancer profiles within the same tumor. Metastatic sites often show genetic differences from the primary tumor that seeded them. And, of course, new lesions can be formed from cancerous cells ever more removed from the primary tumor.

Experimental data have clearly demonstrated the heterogeneity challenge. Seminal studies in breast, colorectal, kidney, lung, and prostate cancer report the cases of patients on whom multiple biopsies were performed.¹ In one case, all samples harbored unique genetic mutations—even among samples recovered from the same tumor site—making it nearly impossible to fully characterize a patient’s cancer with one simple biological profile.

The challenge of faithfully representing the heterogeneity of a patient’s cancer using liquid biopsies that can produce results not concordant with one another or with other sample types emphasizes the value of the format. For example, several studies of various cancer types have found concordance among the genetic mutations detected with liquid biopsies and tissue biopsies.

For a clinician trying to make a medical decision on the basis of an evidence-based tumor profile, this is par for the course. Tissue biopsy samples frequently produce discordant results, depending on where the sample comes from. For years, the field has been testing biopsy samples repeatedly to detect HER2 amplification, looking for sufficient evidence to support Herceptin treatment. The discordant results often found through such testing are the products of the selection events, both interventional and biological, between primary and metastatic tumors.

With broader studies of emerging liquid biopsy formats, researchers are gradually gaining an improved understanding of what liquid biopsies are measuring. Within the liquid biopsy space, varied biological processes produce measurable tumor targets of distinctly different types. Some liquid biopsies measure circulating tumor cells (CTCs), which are intact cells physically sloughed off a tumor into the bloodstream or selected by the metastatic process to migrate to a new site. Other liquid biopsies analyze circulating cell-free DNA (ccfDNA), the genetic material shed as cancer cells die or are killed by treatment or immune system responses.

Results from CTC-driven and ccfDNA-driven liquid biopsies are complementary, indicating that there is great value for the biomedical community to build evidence-based readouts from as many templates as possible.

INTACT CELLS VERSUS CELL-FREE DNA

First described by Australian physician Thomas Ashworth in 1869, circulating tumor cells can become separated from their source tumor by several different mechanisms. Physical shearing events certainly occur. Additional means of releasing intact tumor cells into circulation include active processes such as mesenchymal transition, which makes cells less sticky, and the programmed release of metastatic stem cells.

Because they can come from either primary or metastatic tumors, CTC samples are biologically selected to represent the range of heterogeneity that is driving a patient’s cancer. CTCs have also been associated with more aggressive and increased numbers of metastases in later stage cancer. And since they are intact cells containing a full complement of proteins, RNA, and DNA, they allow for various types of essential analyses.

Extensive studies have shown that increased numbers of CTCs result in more metastatic events in breast, colon, and prostate cancers, and that these cells predict a worse cancer prognosis. In addition, it has been suggested that the number of CTCs present during the early stages of lung cancer treatment may predict prognosis.

A frustration for the clinical use of CTCs is the typical finding of small numbers of circulating epithelial cells in people with nonmalignant conditions such as chronic inflammation. These events cannot be distinguished using tests that simply count CTCs, and they may therefore lead to a false-positive signal. Since the enumeration technologies for CTCs have been developed in the context of a positive clinical diagnosis, however, the risk of a false-positive test result is irrelevant.

The further elaboration of molecular tools that can classify CTCs as being derived from a tumor now enables researchers to validate cells for the presence of tumor-associated mutations, and to define the relationship of a CTC to a specific population that contains those mutations. With these changes in understanding, we are now able to leverage observations in the substantial and growing body of research demonstrating that populations of tumor cells in blood are responsible for aggressive disease and represent the active portion of cancer.

Liquid biopsies may also target ccfDNA, genetic materials whose existence in blood was discovered almost 70 years ago, but made accessible only recently, as the resolution of DNA analysis technologies has increased. Such ccfDNA fragments are thought to come from all populations of dying or necrotic cells that release their contents into the bloodstream, including cancer cells. Circulating DNA specific to tumor cells can be distinguished by the presence of disease-associated mutations.

The fact that we can detect tumor-derived ccfDNA at all suggests that cancer cells have a higher turnover than other cells in the body. Compared with the amount of normal genetic material in blood, ccfDNA from cancer is incredibly rare, making it difficult to detect in the background of ccfDNA shed by healthy cells.

Multiple studies have shown that ccfDNA can include mutations found in the genetic profiles of tumors. The challenges involved in detecting previously unknown mutations in ccfDNA come in part from the very high sensitivity required to detect a tiny amount of tumor ccfDNA against a background of much greater amounts of normal ccfDNA. Moreover, such tumor-associated mutations found in ccfDNA must also be interpreted in the context of damage to the fragments of DNA that accumulate during the exposed residence of ccfDNA in the bloodstream.

Finding either CTCs or ccfDNA in blood samples can test the detection limits of even advanced technologies, requiring detection levels of 1% or better. Perhaps because templates of these kinds are so hard to detect, multiple studies comparing the sensitivity of technologies for detecting them across many patients have found that CTCs and ccfDNA yield comparable numbers of actionable cancer mutations. This fact, together with the presence of complementary biomarkers available in the two templates, has inevitably driven interest in bringing both biomarkers into the clinic.

COMBINING TARGETS

Releases of CTCs and ccfDNA are caused by different biological processes, and a growing body of data in the liquid biopsy field support the notion that the information they reveal is also different but complementary. Consequently, studying results from CTCs and ccfDNA together is a more powerful approach, providing more medically actionable information, better sensitivity, and more biomarkers than either data set alone.

The first platform to receive FDA clearance for CTC testing is the CellSearch CTC test by Janssen Diagnostics LLC, Raritan, NJ. The test captures and enumerates CTCs of epithelial origin (CD45-; EpCAM+; cytokeratins 8, 18+, 19+), and has been cleared for testing in patients with metastatic breast, colorectal, or prostate cancer. However, the CellSearch test does not support analysis of patients with liver, pancreatic, or soft tissue cancers, which are derived from tissues that do not meet strict epithelial definitions, and therefore have not been successfully evaluated to date.

By way of contrast, a recent study by researchers at Johns Hopkins University analyzed the results of ccfDNA liquid biopsies performed on 640 cancer patients. Within this group, tumor-associated ccfDNA alterations ware found in more than 75% of patients with bladder, breast, ovarian, pancreatic, and other cancers.² However, equivalent ccfDNA markers were found in less than half of patients with brain, kidney, prostate, or thyroid cancers. These findings suggest that the utility of ccfDNA liquid biopsies may be specific to the type of cancer. Furthermore, the ability of the test to detect ccfDNA was also related to the overall tumor burden, with the ccfDNA signal tending to increase with tumor stage.

With molecular tools now available to capture and analyze both CTCs and ccfDNA, it is likely that the defined types of cancer covered by individual tests will gradually broaden. In the future, sequencing of CTCs and ccfDNA will start to address conditions that have previously been underserved.

As an example of this trend, a clinical study published by our group compared the use of CTC and ccfDNA liquid biopsy results to tissue biopsies, which have long been considered the gold standard for evaluating tumors.³ For this analysis, we used the LiquidBiopsy platform by Cynvenio, Westlake Village, Calif, which allows for isolation and mutation analysis of CTCs and ccfDNA from the same patient blood draw.

In this study, next-generation sequencing was performed on samples from 32 patients with metastatic breast cancer, and found significant and complementary overlap among the three data sources.³ For example, the most frequently mutated genes related to breast cancer were TP53 and PIK3CA. Mutations of these genes were defined in 16% and 9% of CTCs, and in 29% and 16% of ccfDNA, respectively. Taken together, the CTC and ccfDNA data showed relevant variant information in 56% of cases—quite comparable to the tissue biopsy rate of 58%, but without the patient risks related to such invasive surgical procedures.

A similar recent study on CTCs and ccfDNA in lung cancer came to the same conclusions, with CTCs, ccfDNA, and tissue biopsies all providing complementary information.

Such findings support the notion that noninvasive liquid biopsies can make it easier for clinicians to perform longitudinal studies of their patients, with findings comparable to tissue biopsies. By developing such monitoring tools, clinicians can begin to treat cancer using practices that resemble current methods of treating infectious disease, which have proven to be a successful model for controlling a wide range of difficult conditions such as HIV infection.

Moving forward, the most comprehensive approach for diagnosing and monitoring cancer will likely turn out to be an initial tissue biopsy—a standard that will continue—paired with ongoing, multitemplate liquid biopsies. Together, these approaches promise to provide the clearest view of cancer biology and progression over time.

NEXT STEPS

In the context of cancer, which is known for its heterogeneous makeup and rapidly shifting genetic profile, liquid biopsies need all the detection power they can get. Early versions of such tests show remarkable promise for early detection of cancer and for routine, noninvasive monitoring of patients throughout the course of their disease. But findings to date also clearly show that each biological target offers only a piece of the diagnostic puzzle.

Liquid biopsies designed to analyze both CTCs and ccfDNA offer a far more holistic view of a patient’s cancer landscape, and can be used in conjunction with traditional tissue biopsies for the most comprehensive analysis of all.

For the fight against cancer, it is possible that we will eventually regard the impact of liquid biopsies as equal to that of the advent of antibiotics for infectious disease. To make the most of this new technology, however, it will be necessary to conduct clinical trials evaluating the utility of these tests in specific cancer situations.

Paul W. Dempsey, PhD, is chief science officer at Cynvenio Biosystems Inc, Westlake Village, Calif. For further information, contact CLP chief editor Steve Halasey via [email protected].

REFERENCES

1. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366(10):883–892; doi: 10.1056/nejmoa1113205. Available at: www.nejm.org/doi/full/10.1056/nejmoa1113205. Accessed June 23, 2016.

2. Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6(224):224ra24; doi: 10.1126/scitranslmed.3007094. Available at: www.ncbi.nlm.nih.gov/pubmed/24553385. Accessed June 23, 2016.

3. Strauss WM, Carter C, Simmons J, et al. Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers. Oncotarget. 2016;7(18):26724–26738; doi: 10.18632/oncotarget.8494. Available at: www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=8494&path%5B%5D=25235. Accessed June 23, 2016.