Applications for ddPCR in Oncology
New Findings Show Liquid Biopsy Can Guide Treatments
Treatment options for patients with cancer have been increasing in recent years, but not all patients respond to these treatments with the same success. Although precision medicines are now available to more patient populations than ever before, treatment outcomes will improve further when physicians can better predict whether a patient is likely to or is in fact responding favorably to a particular treatment. Liquid biopsies are a non-invasive, rapid method that can aid this effort.
Methods to Monitor Treatment Response
There are several lines of treatment available to a patient with cancer. The first option is typically surgery to remove the tumor if it is not too advanced. After surgery, patients may undergo any of a variety of targeted therapies, immunotherapies, chemotherapies or other adjuvant therapies. However, the effectiveness of these treatments can decline over time due to the emergence of genetic mutations that make the tumor drug resistant . Tumor drug resistance can arise from factors such as tumor genetics and heterogeneity, the state of the tumor microenvironment, as well as the selective pressures exerted by the treatment regimen. These factors make predicting a patient’s treatment response difficult, but through the use of genetic biomarkers underlying tumorigenesis and resistance, it is possible to monitor how the tumor evolves and determine the appropriate targeted treatments.
Traditional diagnosis and monitoring methods include computed tomography (CT) or magnetic resonance imaging (MRI) to determine the size and location of tumors, tissue biopsy to assess the tumor’s cellular, molecular, and genetic profile, and blood tests to identify cancer type by genetic and protein biomarkers. While these methods all provide valuable information, they come with significant limitations. Imaging methods do not always reflect a tumor’s response to certain therapies, for example when beneficial inflammation makes a tumor appear to grow during immunotherapy . Blood tests for protein biomarkers can have limited specificity [3,4], and tissue biopsies are highly invasive and thus not amenable to repeated patient sampling over time.
In searching for a complementary approach that overcomes many of these drawbacks, physicians are increasingly adopting liquid biopsy often powered by droplet-digital PCR (ddPCR) technology to profile circulating tumor DNA (ctDNA) and monitor treatment response and relapse in real time (fig.
1). This highly sensitive method can quantify the presence of genetic or epigenetic alterations associated with specific kinds of cancer. With this data physicians can adjust treatment over time if mutations arise, maximizing their patients’ chances for a positive outcome.
Droplet Digital PCR in Liquid Biopsy
Oncology researchers are using ddPCR-based liquid biopsy to find new ways that ctDNA can be used as a biomarker for different types of cancer to test for residual disease or to monitor for treatment efficacy, likelihood of recurrence or tumor progression. This endeavor is a complex one: every cancer is associated with a unique set of genetic mutations. As this method gains popularity, researchers are testing whether known mutations can be broadly applied to monitor multiple cancers, and they are uncovering new mutations that may become important future biomarkers.
The studies below show how researchers are using ddPCR-powered liquid biopsy to uncover new applications for genetic biomarkers. They show when ctDNA is an effective predictor of treatment response and cancer progression, and when it is not. These studies demonstrate a growing body of evidence that supports the use of ddPCR-powered liquid biopsy during cancer treatment.
Tumor Recurrence in Hepatocellular Carcinoma
It is vital to detect and track new tumors in patients with hepatocellular carcinoma (HCC), because one in three will experience tumor recurrence within one year after surgery. For physicians to administer follow-up therapies in time, they must be able to identify, or even predict, recurrence in a timely manner.
In a recent study , Ya-Chun Wang, PhD, of TCM Biotech International Corporation and colleagues analyzed plasma samples of 50 patients with HCC. Comparing samples before and after surgery, they sought to identify a more sensitive biomarker to monitor tumor recurrence. For this purpose, the researchers studied virus host (vh)-chimera DNA, which arises because hepatitis B (HBV) viral DNA integrates with the chromosome of most patients with HCC.
The researchers used ddPCR to quantify the vh-chimera DNA in the plasma before and after surgery. Of patients that still had chimeric DNA in their plasma two months after surgery, 82% experienced tumor recurrence in the following year. Further, in all but two of these patients, the tumor appeared to have come from the original HCC tumor cells, because the vh-chimera DNA matched that of the original clone.
The authors note that the findings support the use of vh-chimera DNA as a biomarker to complement existing ones in detecting the existence of HBV-HCC and in tracking tumor recurrence based on vh-chimera clonalities.
Importantly, this study highlights a major strength of ddPCR technology – its sensitivity. The researchers noted that ddPCR’s limit of detection as low as two copies per reaction.
Monitoring Melanoma with Mutant BRAF ctDNA
For patients with advanced melanoma, predicting and monitoring treatment efficacy is currently hindered by the lack of any validated blood-based biomarkers to track via liquid biopsy. David Polsky, MD, PhD of NYU Langone Medical Center and his colleagues are one of several teams evaluating how ctDNA can be used as a biomarker to predict and measure treatment efficacy in these patients.
Polsky’s recent study  focused on quantifying ctDNA for a mutated version of the BRAF gene, which is commonly associated with melanoma. He and his team used ddPCR to conduct a quantitative analysis of this mutation in 345 patients undergoing therapy and found that patients who had higher baseline concentrations of BRAF ctDNA were less likely to survive. At the same time, they found patients tended to survive longer if BRAF ctDNA levels dropped to undetectable levels within four weeks of treatment. Thus, this marker may be useful in making and modifying treatment decisions.
Predicting Progression of Melanoma Metastases
Tracking ctDNA can be used to effectively monitor disease progression for many types of cancer, but Jenny Lee, MD, of Macquarie University in Sydney and the Melanoma Institute Australia, and her colleagues discovered the limitations of using ctDNA to analyze the progression of advanced melanoma when the cancer has metastasized specifically to the brain .
Following up on an earlier study , Lee and her colleagues used ddPCR technology to analyze ctDNA levels in 48 patients with advanced melanoma and brain metastases. All the patients were receiving immune checkpoint therapy. Eight had metastases in their brains but nowhere else. The researchers found that an absence of detectable tumor ctDNA at the start or at an early stage of treatment was generally a good sign. Specifically, it was associated with superior progression-free survival (early on in therapy) and overall survival, though this did not apply to responses observed in brain metastases.
As to why that might be the case, Lee noted that the metastases in the brain may have been smaller than those in other parts of the body and that the blood brain barrier could have filtered out ctDNA before it reached the blood.
The team concluded that though ctDNA was useful for monitoring treatment efficacy in metastatic melanoma, it may not be helpful in detecting brain metastasis or monitoring whether the brain tumors respond to immune checkpoint therapy. This study has potentially important implications because up to half of melanoma patients exhibit brain metastases at some point in their treatment.
As evidence demonstrates new applications for ddPCR-based liquid biopsy to monitor, and in some cases predict, how cancer patients will respond to treatments, such testing may become a standard component of diagnostic workflows in the clinic. These noninvasive tests can complement methods used for initial diagnosis by allowing doctors to monitor patients and adjust treatments as needed. Overall, routine monitoring with liquid biopsy powered by ddPCR promises to contribute to improved patient outcomes.
Dr. George Karlin-Neumann
Hercules, CA, USA
1. Masoori B, Mohammadi A, Davudian S, Shirjang S, Baradaran B. The different mechanisms of cancer drug resistance: a brief review. Adv Pharm Bull. 2017;7(3):339–348; doi: 10.15171/apb.2017.041.
2. Thrust SC, van den Bent MJ, Smits M. Pseudoprogression of brain tumors. J Magn Reson Imaging. 2018;48(3):571–589; doi: 10.1002/jmri.26171.
3. Adhyam M, Gupta AK. A review on the clinical utility of PSA in cancer prostate. Indian J Surg Oncol. 2012;3(2):120–129; doi: 10.1007/s1393-012-0142-6.
4. Tsao SCH, Weiss J, Hudson C, et al. Monitoring response to therapy in melanoma by quantifying circulating tumor DNA with droplet digital PCR for BRAF and NRAS mutations. Sci Rep. 2015;5:11198; doi: 10.1038/srep11198.
5. J Clin Oncol 37, 2019 (suppl; abstr 4090)
6. J Clin Oncol 37, 2019 (suppl; abstr 9510)
7. J Clin Oncol 37, 2019 (suppl; abstr 9581)
8. Lee JH, Long GV, et al. Circulating tumour DNA predicts response to anti-PD1 antibodies in metastatic melanoma. Annals of Oncology. 2017 May 1;28(5):1130-1136. doi: 10.1093/annonc/mdx026.