Redefining Cancer-Free With ctDNA

Survivorship after the diagnosis and treatment of cancer is a uniquely uncertain state of being, one in which periods of relief are punctuated by uncertainty. Even while all indications suggest the cancer is gone, many patients can’t help but feel a sense of foreboding, as though the cancer had merely gone into hiding.^1,2 Behind this fear is an understanding that what it means to be ”cancer free” is not so much defined by the lack of residual disease but rather our ability to detect it. 

Stated plainly, determining whether a cancer is really gone is challenging. At its core, this challenge is a technical one. Standard of care (SOC) technologies for screening, diagnosing, and monitoring cancer have practical limitations, beyond which residual cancer can persist without notice. Under the best of circumstances, for example, nuclear imaging can only identify tumors once they’ve grown to several millimeters in diameter.^3-5 As such, there is a gap between what our technology detects as cancer-free, and what it means to be biologically cancer-free. 

Fortunately, this gap may be closing with the development of platforms centered on minimal residual disease (MRD) testing. By leveraging next-generation sequencing (NGS) technologies, MRD testing provides an unprecedented view into tumor evolution over time and enables the highly sensitive, albeit indirect, detection of malignant cells. Such sensitivity makes it possible to detect recurrent tumors weeks, months, and sometimes almost a year before they otherwise would have been captured by SOC techniques.^6-9.

In this article, we dive into MRD and how it promises to both address an unmet need in the care of cancer patients and ultimately help patients live longer lives with less uncertainty.

Creating the gap

Tumor recurrence generally refers to the reappearance of a tumor after a prolonged absence. The mechanisms that drive recurrence are complex and not completely understood, but it is clear that recurrent tumors are present long before they’re clinically detectable.^6-9

For most solid tumors, standard practice is to monitor remission or tumor growth using imaging technologies, such as positron emission tomography (PET), X-rays and magnetic resonance imaging (MRI). These approaches have been invaluable in the detection and characterization of various tumor types, but they each have fundamental challenges related to the transmission of information (such as light waves) across many layers of complex biological tissue. 

In the best conditions, most modern imaging modalities can only detect tumors once they’ve grown larger than several millimeters in diameter.^3-5 Additionally, imaging is a hypothesis driven approach, meaning healthcare professionals need to have an idea of where to look for a potential malignant tumor. This can prevent the discovery of an unexpected metastasis. Lastly, imaging can be a laborious and challenging procedure for certain patient types who are elderly and have comorbidities, making it difficult to perform the type of longitudinal testing required to surveil for tumor recurrence. Collectively, these and many other challenges slow down the detection of recurrent tumors. 

Beyond imaging, various blood-based biomarkers are effectively used to detect the recurrence of cancer. However, most tumor types lack a reliable biomarker that is both specific, short-lived (enabling close to real time monitoring), and easily detected in small quantities. However, with the development of MRD testing, this may soon change.

The potential of MRD

MRD testing aims to analyze liquid biopsies (primarily blood samples) for the presence of circulating tumor DNA (ctDNA)—a type of biomarker that can be used for most cancer types and is both non-invasive and informative.^6,9 

Through various mechanisms, fragments of a cell’s genome can leak into its extracellular environment where they’re swept up by the circulatory system. These bits of wayward genetic material can be detected and analyzed with sensitive NGS technologies. And though they’re only about 120 nucleotides long, they can carry telling mutations and epigenetic patterns that point to their malignant origin. 

As ctDNA is leaked from individual cells, it is theoretically possible to detect the presence of a single cancer cell anywhere in the body through a simple blood draw. MRD assays have not yet advanced to this degree of sensitivity, but considerable progress has been made.^6-9 

Research from the TRACERx study, for example, showed that MRD testing was able to identify recurrent tumors in patients with non-small cell lung cancer well before they were detectable using computerized tomography (CT) scanning. In this study, ctDNA identified the presence of malignant cells between 10 and 346 days earlier than CT scanning, including instances of metastatic disease.⁷ 

But while progress has been made, the gap remains. Some of the most sensitive MRD assays to date can only detect ctDNA when it is present in ten parts per million, meaning for every million DNA fragments, ten carry tumor-identifying variants.⁶ This can result in weeks or months of tumor cell regrowth before ctDNA is detectable. To close this gap, data extrapolation suggests that MRD assays will need to be able to detect ctDNA levels as low as one-part-per million (1 ppm) if they’re going to detect residual disease at its lowest levels (e.g., immediately following surgery).

Closing the gap

Blood samples are replete with fragmented DNA, the vast majority of which are derived from non-malignant cells. Following treatment, ctDNA may represent less than 0.1% of the total cell free DNA in a patient sample.⁶ To reliably pick out the tumor-specific DNA, MRD tests must rely on target enrichment panels that are designed to capture pre-defined genetic sequences which are known to be associated with cancer. 

The design of such a panel can have a critical influence over the assay’s sensitivity and specificity. If the panel is designed to be too narrow, such that only a very select few sequences are being captured, it reduces the odds that the panel will encounter a fragment it recognizes (thus decreasing its sensitivity). However, simply increasing the number of targets being captured risks false-positive detection, where fragments of DNA from regular cells are mistaken for ctDNA.⁶ Therefore, the ideal assay would be broad and yet tumor-specific, enabling the capture of a wide number of potential ctDNA sequences while remaining exclusive to tumor-specific targets. 

The most sensitive MRD platform to date is capable of detecting tumor-identifying DNA fragments in quantities as low as 1 ppm. This sensitivity is achieved through superior target enrichment. Panel design is based on results from a biopsy or surgical resection of the patient's tumor prior to treatment. Sequencing of the tumor’s genome enables the design of a patient-specific capture panel that will enrich sequences known to be present in the patient’s tumor. Further, the panel is strengthened by targeting thousands of additional sequences that are known to be associated with cancer. Lastly, the patient’s non-tumor genome is also sequenced to ensure that no sequences on the target capture panel are redundant with germline DNA.

Collectively, this means the platform is able to achieve a highly specific and sensitive enrichment of ctDNA, and in doing so, could make it possible to detect the earliest stages of tumor recurrence. 

Ultimately, continued advances in MRD tests mean that cancer survivors have a better chance of identifying tumor recurrence early and going on to live a truly cancer-free life. 

About the author:

Juan-Sebastian Saldivar is the laboratory medical director at Personalis, where he applies his extensive experience in overseeing clinical laboratory operations, as well as in molecular genetic tests and technologies. Prior to Personalis, he was vice president of Clinical Services and Medical Affairs at Sequenom Laboratories in San Diego, CA, and served as director of Molecular Diagnostics at City of Hope National Medical Center in Duarte, CA.

He is board certified in Clinical Molecular Genetics by the American Board of Medical Genetics and board certified in Molecular Genetic Pathology by the American Board of Pathology. He is also licensed by Laboratory Field Services in the State of California (Clinical Genetic Molecular Biologist) and certified by the NY Department of Health (Certificate of Qualification in Molecular Genetic Testing and Oncology).

Saldivar is also a Fellow of the American College of Medical Genetics, member of the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) and served as an inspector for CAP, as well as contributed as an AMP expert panel member, developing molecular diagnostic guidelines.