Why assay design is critical for ultra-sensitive ddPCR cancer monitoring

Cancer biology does not change based on where a patient lives, but access to timely molecular diagnostics often does. At a major cancer center, a patient’s tumor may be monitored with tests sensitive enough to detect only a few mutant DNA molecules, while patients elsewhere may wait weeks for even basic testing, if it is available at all.For clinicians and researchers seeking to monitor disease progression, treatment response, and minimal residual disease (MRD), that gap can influence how quickly molecular changes are detected and how broadly precision oncology approaches can be applied.

Diana Vargas-Gold has spent more than 25 years working to close that gap. As Research Scientist at the Rutgers Cancer Institute, Vargas-Gold leads her own assay development research and serves as a co-principal investigator on multidisciplinary translational studies focused on developing and clinically validating ultra-sensitive blood- and cerebrospinal fluid-based liquid biopsy assays for detecting rare cancer mutations. Working with clinical collaborators and industry partners, such as Bio-Rad, her research aims to translate innovative molecular technologies into clinically deployable diagnostics for disease monitoring and minimal residual disease detection.

Speaking with SelectScience^®, Vargas-Gold discusses why assay design, not platform sensitivity alone, determines what a test can detect, how droplet digital PCR (ddPCR) complements next-generation sequencing (NGS) rather than competing with it, and what it will take to bring personalized cancer monitoring to laboratories far beyond major cancer centers.

Same cancer biology, different access to care

Vargas-Gold traces her interest in diagnostic accessibility to her own background. Growing up in Colombia, and later supporting her mother through a cancer diagnosis, shaped how she thinks about precision medicine today. “Cancer doesn't care where someone lives,” she says. “The biology is the same whether a patient is in New Jersey, Bogotá, or a small rural community. What changes is access to timely, high-quality molecular diagnostics.”

When diagnosis or molecular testing is delayed, she notes, the disease has more time to progress, treatment options narrow, and the likelihood of achieving the best possible outcome can decrease. That perspective shapes her research goals. Rather than developing assays that are sensitive alone, she aims to create technologies that are practical, scalable, and feasible to implement across different healthcare environments.

ddPCR plays an important role in that vision. Compared with many sequencing-based approaches, ddPCR offers exceptional analytical sensitivity alongside a relatively straightforward workflow and infrastructure. Detecting a handful of mutant DNA molecules among millions of normal ones is only valuable, Vargas-Gold argues, if the technology can reliably analyze challenging clinical samples and be implemented in more laboratories.

“I want precision medicine to be driven by a patient's biology,” she says. “Not by their ZIP code, country, or the resources available to them.”

How ddPCR extends the value of NGS in cancer monitoring

Vargas-Gold is quick to challenge the common framing of ddPCR and NGS as competing technologies. “It’s a mistake to view ddPCR and NGS as competitors,” she says. “Each has distinct strengths, and together they're much more powerful than either technology alone.”

Sequencing excels at identifying the mutations that define a patient's tumor and revealing biomarkers that may influence treatment decisions. Once those biomarkers have been identified, however, a different clinical question emerges: Are they still present? Are their levels changing over time? Is the treatment working? That's where ddPCR finds its niche.

“NGS discovers the tumor; ddPCR monitors the tumor,” says Vargas-Gold.

Rather than replacing sequencing, ddPCR extends its clinical value by enabling frequent, cost-effective monitoring throughout a patient's cancer journey.” This makes it particularly valuable for longitudinal disease monitoring, MRD assessment, evaluation of treatment response, and detecting signs of recurrence before they become evident through conventional approaches.

Its monitoring capability stems from the technology's underlying design. By partitioning DNA into thousands of individual droplets, ddPCR enables absolute quantification without relying on standard curves, improves precision at very low target concentrations, and increases tolerance to PCR inhibitors that are often present in clinical samples. Those characteristics make the platform well suited to liquid biopsy applications where circulating tumor DNA (ctDNA) may be present at extremely low levels.

Why assay design determines ddPCR sensitivity

While ddPCR platforms are often discussed in terms of sensitivity, Vargas-Gold argues that assay design deserves equal attention. “One of the biggest misconceptions in molecular diagnostics is that analytical sensitivity stems from the platform alone,” she says. “I believe assay design largely determines what the platform can detect.”

Vargas-Gold emphasizes that selecting the appropriate assay design depends on both the biology of the mutation and the clinical question being addressed. For example, in mutational hotspot regions, the optimal approach depends on whether the goal is simply to determine that a hotspot is mutated or to identify the specific sequence variant.

"The technology should be adapted to the biological problem, not the other way around," she says.

For rare mutation detection, specificity is just as important as sensitivity. If an assay cannot reliably distinguish a single mutant molecule from thousands of wild-type molecules, even highly sophisticated instrumentation cannot recover the lost specificity.

Much of Vargas-Gold's research focuses on engineering assays that selectively amplify mutant DNA while suppressing amplification of normal DNA. Her work spans a range of molecular approaches, including SuperSelective Primers, Variant-signaling primers, optimized molecular beacon strategies, and other detection chemistries tailored to specific biological questions.

During the last eight years of her research in Dr. Fred Kramer's laboratory at Rutgers University, Vargas-Gold focused on translating innovative molecular technologies into highly sensitive digital PCR assays. Those experiences helped shape her current philosophy by demonstrating that successful assay development depends not only on innovative primers and probes but also on optimizing the entire analytical system so that assay chemistry and the digital PCR platform work together to achieve the highest possible sensitivity, specificity, and robustness.

SuperSelective Primers exemplify this approach. Designed to preferentially amplify mutant DNA while suppressing wild-type amplification, they separate mutation recognition and target binding into distinct functional domains.

A short “foot” sequence, typically around 10 nucleotides long, sits directly over the mutation of interest. Because the sequence is so short, even a single base mismatch dramatically reduces binding, allowing the primer to discriminate between mutant and normal DNA with high specificity.

A longer “anchor” sequence binds elsewhere within the target gene, positioning the foot correctly and stabilizing primer binding. Connecting the two is a “bridge” sequence designed not to bind the target DNA, creating a physical separation between the two functions.

The result is a primer architecture capable of preferentially amplifying rare mutant molecules while greatly suppressing amplification of abundant wild-type DNA.

“Sometimes, improving performance doesn't require a completely new technology,” Vargas-Gold says. “It requires rethinking how we design the tools we already use.”

How patient-specific ddPCR panels track a tumor’s molecular fingerprint

Because every patient's tumor carries a distinct combination of mutations, Vargas-Gold's laboratory increasingly focuses on multiplexed, patient-specific biomarker panels rather than individual biomarkers. “Every patient's tumor is molecularly unique, and when working with liquid biopsies, every DNA molecule is precious,” she says. This approach becomes particularly valuable when ctDNA levels are very low and sample availability is limited. Rather than monitoring one mutation at a time, multiplex assays allow several patient-specific biomarkers to be tracked simultaneously.

Each additional biomarker creates another independent opportunity to detect tumor-derived DNA, helping reduce the impact of stochastic sampling effects while accounting for tumor heterogeneity and clonal evolution. Together, these factors can increase confidence that molecular signals genuinely reflect residual disease.

“Multiplexing is about more than efficiency,” says Vargas-Gold. “It's about making every molecule and every sample work as hard as possible for the patient.”

In practice, personalized monitoring begins with comprehensive molecular characterization of a patient's tumor. Sequencing identifies tumor-specific variants, which are then prioritized according to their biological relevance, detectability in blood or cerebrospinal fluid, technical feasibility, and the clinical question being addressed. Those variants become personalized molecular biomarkers incorporated into multiplex ddPCR panels that can be repeatedly used throughout treatment.

A key objective of Vargas-Gold's research is developing modular assay designs that allow newly identified biomarkers to be incorporated into standardized workflows without redesigning the entire assay framework.

By maintaining a consistent analytical workflow while allowing biomarker panels to evolve as tumors change or new clinically relevant biomarkers are identified, the approach aims to make personalized molecular monitoring both adaptable and scalable while preserving the consistency required for longitudinal studies and future clinical implementation.

This strategy is particularly valuable in areas such as pediatric oncology and cerebrospinal fluid analysis, where sample volumes can be extremely limited and every microliter matters.

Making personalized cancer monitoring more scalable

For Vargas-Gold, scaling personalized ddPCR workflows is not simply about analytical performance. The greater challenge lies in making personalized molecular testing practical and reproducible across clinical research laboratories. “Standardized workflows, clinical validation, automation, and robust data analysis” are all necessary, she says, if personalized monitoring is to move beyond individual research programs.

A common misconception is that every personalized assay must be developed entirely from scratch. Instead, her group is working to create modular assay frameworks that maintain a consistent workflow while allowing individual biomarkers to change from patient to patient. “Just because the biomarker changes doesn't mean the workflow should,” she says.

Reducing assay costs is another important consideration. One strategy involves universal molecular beacon designs that can be reused across multiple assays, minimizing the need to develop a new patient-specific probe for every biomarker.

She also points to commercially available ddPCR assays, including Bio-Rad assays for clinically relevant mutations and reference gene controls, as resources that can be incorporated into personalized panels instead of requiring every target to be redesigned from the ground up. Ultimately, she believes personalization should occur at the biomarker level rather than the workflow level.

Vargas-Gold notes that developing scalable, personalized molecular diagnostics requires more than innovative assay design. Translating these technologies into clinical practice also depends on a multidisciplinary effort.

At the Rutgers Cancer Institute, she leads translational research in assay development within Dr. Zhiyuan Shen's laboratory while serving as a co-principal investigator on multidisciplinary translational studies. She works closely with an outstanding team of scientists and clinicians, including Dr. Zhiyuan Shen, Dr. Missak Haigentz, Dr. Nehal Parikh, and Dr. Morana Vojnic, whose combined expertise helps guide biomarker selection, assay development, study design, and interpretation of molecular findings.

Together, this collaborative environment helps ensure that assay development is guided by clinically meaningful questions, facilitating the translation of innovative molecular technologies into personalized diagnostics.

Expanding access to precision cancer diagnostics

Looking ahead, Vargas-Gold believes personalized molecular monitoring should eventually become part of routine cancer care rather than remaining confined to specialist laboratories. Achieving that goal will require highly sensitive assays, rigorous clinical validation, standardized implementation strategies, and ongoing collaboration among researchers, clinicians, and industry partners.

The need is substantial. Nearly half of the world's population still lacks access to essential diagnostic services, while many low- and middle-income countries continue to face shortages of pathologists, molecular diagnostic laboratories, and specialized testing capacity.

Meeting that challenge will depend on technologies that can be implemented reliably beyond major academic centers. “High analytical performance should never come at the expense of accessibility,” she says.

For Vargas-Gold, the mission remains personal as well as scientific. “Coming from Colombia and having experienced cancer in my family, this mission is deeply personal,” she says. “To me, high-quality cancer diagnostics should be a human right.”

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