ddPCR technology drives progress in oncology research

Researchers are using Droplet Digital™ PCR (ddPCR™) to develop liquid biopsy assays for detecting circulating tumor DNA (ctDNA) in a range of human conditions including melanoma and cardiovascular disease.

Dr. Greg Jones, a Professor in the Department of Surgical Sciences at the Dunedin School of Medicine, University of Otago, and Dr. Sandra Fitzgerald, a Research Fellow in the Department of Molecular Medicine and Pathology at the University of Auckland, have significantly contributed to oncology research, particularly through the utilization of ddPCR technology. Their research has not only advanced our understanding of cancer biology but also showcases the exceptional utility of ddPCR for methylation analysis.

We spoke with Dr. Fitzgerald and Dr. Jones about their pioneering work and the essential role of ddPCR in advancing oncological research.

Revolutionizing oncology research with ddPCR

ddPCR technology, notably the QX600™ ddPCR™ system from Bio-Rad, has become a valuable tool in modern oncology research. This advanced system offers six-color multiplexing and absolute quantification of nucleic acids, enabling precise and sensitive detection of genetic variations and mutations. The QX600 ddPCR system’s capabilities have been instrumental in the groundbreaking work conducted by Dr. Jones and Dr. Fitzgerald.

Liquid biopsy and circulating tumor DNA detection

Dr. Fitzgerald’s research focuses on developing liquid biopsy assays to detect ctDNA in melanoma patients undergoing immunotherapy. Utilizing custom next-generation sequencing (NGS) on the Ion Torrent S5 sequencer and ddPCR with the Bio-Rad QX200 system, her team has been able to molecularly profile ctDNA in blood plasma. The combination of NGS and ddPCR has proven invaluable in detecting mutations at the lower limit of detection, revealing a high level of heterogeneity in ctDNA samples. A recent study highlighted the dynamic mutational complexity in ctDNA during immunotherapy, showcasing the clinical relevance of their findings and the essential role of NGS and ddPCR technology in their research¹.

Currently, four ddPCR assays developed by Dr. Fitzgerald’s team are being validated in a diagnostic clinic for advanced lung cancer patients in order to guide treatment decisions in the absence of a tissue biopsy sample for genomic analysis. These assays are expected to play a crucial role in detecting minimal residual disease in patients undergoing surgery for advanced head and neck cancer. The observational study aims to determine if lymphatic fluid, combined with standard pathology markers, could serve as an enriched source of residual disease, thereby informing adjuvant treatment strategies. As Dr. Fitzgerald notes, “This will be using NGS of solid tumor material, and ddPCR will be used to look for residual disease in blood plasma, lymphatic drain fluid and saliva”

Methylation analysis in oncology research

A notable collaboration between Dr. Fitzgerald and Dr. Jones has led to the development of a ddPCR assay to measure the methylation state of the AHRR gene, which is strongly associated with smoking exposure. This assay involves preparing genomic DNA from whole blood, performing a restriction analysis, and conducting ddPCR with probes for AHRR and a housekeeping gene. The results of this approach have shown a strong correlation with patient-reported smoking history and the current gold standard array approach².

"This ddPCR assay is now being used in two further projects," Dr. Fitzgerald states. The first project is an HRC-funded study inviting participants at high risk of developing lung cancer to undergo a low-dose CT scan³. The ddPCR assay is offered as a research test to evaluate its utility. The second project involves using the ddPCR assay alongside ultrasound investigations for potential abdominal aortic aneurysms.

Significant breakthroughs enabled by ddPCR

The rapid and cost-effective assessment of the methylation state of the AHRR gene using ddPCR represents a major breakthrough. This capability has the potential to become a standard test for assessing primary and secondary smoking exposure and environmental toxin exposure for risk modeling. As Dr. Fitzgerald explains, “The speed and low cost at which the methylation state of the AHRR gene can be assessed is the most significant outcome of this work.” Building on this success, Dr. Jones and Dr. Fitzgerald are planning further ddPCR assays to investigate promoter methylation.

The reliability and sensitivity of ddPCR make it ideal for liquid biopsy applications. The ability to detect off-target mutations, such as nucleotide changes just one base away, has been an unexpected yet valuable outcome. The rapid and cost-effective implementation of these assays means that the first clinical tests for liquid biopsy in New Zealand will likely be based on ddPCR technology.

The future of oncology research with ddPCR

The future of oncology research is poised to be significantly shaped by ddPCR technology, particularly with the enhanced multiplexing capabilities of the QX600 system. This technology’s precision and reproducibility make it a powerful tool for both clinical and research settings. The QX600’s ability to quantify up to 12 targets in a single well, combined with its compatibility with existing ddPCR reagents, assays, and consumables, ensures its versatility and broad applicability.

"My biggest advice for lab professionals is that if you can reach out to either a Bio-Rad ddPCR expert or to other researchers doing cutting-edge applications with this technology and ask questions about the technology and how it can be used and manipulated," Dr. Fitzgerald advises. Engaging with experienced scientists can provide valuable insights into the potential applications and optimizations of ddPCR, thereby maximizing its impact on research outcomes.

The contributions of Dr. Jones and Dr. Fitzgerald to the field of oncology, particularly through their innovative use of ddPCR technology, are noteworthy. Their research not only advances our understanding of cancer biology but also demonstrates the exceptional utility of ddPCR for methylation analysis and oncology-related studies. The QX600 ddPCR system from Bio-Rad, with its advanced multiplexing and absolute quantification capabilities, has been instrumental in these advancements. As oncology research continues to evolve, ddPCR technology is set to play a pivotal role in driving further breakthroughs and improving clinical outcomes.

Learn more about Bio-Rad’s QX600™ Droplet Digital™ PCR System which provides ultra-high precision, sensitivity, and absolute quantification of nucleic acid molecules.

References

1. Fitzgerald, S. (2023). Liquid biopsy for ctDNA detection in melanoma. Mol Diagn Ther 27, 537–550, [online] Available at: https://link.springer.com/article/10.1007/s40291-023-00651-4

2. Fitzgerald, S. and Jones, G. (2024). Methylation state analysis of AHRR gene in smoking exposure. Clinical Epigenetics, [online] Available at: https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-024-01659-1

3. Parker, K., Colhoun, S., Bartholomew, K., Sandiford, P., Lewis, C., Milne, D., McKeage, M., McKree Jansen, R., Fong, KM., Marshall, H., Tammemägi, M., Rankin, NM., Hotu, S., Young, R., Hopkins, R., Walker, N., Brown, R., Crengle, S. (2023). Invitation methods for Indigenous New Zealand Māori in lung cancer screening: Protocol for a pragmatic cluster randomized controlled trial. PLoS One,18(8):e0281420. [online] Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10393155/