Improving the Reliability of Liquid Biopsy in Real-World Settings Through Workflow Standardization: A Discussion with Dr. Matteo Allegretti

Liquid biopsy offers a powerful, noninvasive way to detect and track cancer, but ensuring reliability in real-world settings remains a key hurdle. Variability in workflows and the need for robust verification steps limit widespread adoption.

Dr. Matteo Allegretti, Senior Researcher at the Translational Oncology Research Unit at the IRCCS Regina Elena National Cancer Institute in Rome, is working to address some of these challenges. His team focuses on developing liquid biopsy workflows for translational research applications. He also leads a working group within the INNOVA Consortium, where multidisciplinary teams are advancing liquid biopsy applications across Italy.

Dr. Vishwadeepak Tripathi, Senior Manager in the Global Genetic Sciences Division at Thermo Fisher Scientific, sat down with Dr. Allegretti to discuss how workflows that integrate complementary technologies, such as NGS, digital PCR, and qPCR, are shaping the future of liquid biopsy in translational oncology.

Q: What sparked your interest in translational oncology and led you to pursue what you do today?

A: I became interested in biology and biotechnology during the last years of high school. I initially chose chemistry, but I also had the opportunity to study biotechnology. The turning point came after attending a lecture by Giuseppe Macino, a renowned RNA biologist. His passion had a strong impact on me and ultimately led me to pursue this field.

Today, in my lab, we focus on developing workflows for biomarker identification and verification. This includes noninvasive approaches such as liquid biopsy to monitor cancer over time. We also work on models, such as 3D systems, to study tumor resistance and relapse.

Q: You work with multiple sample types. How do you tailor your workflows for different sample types and application

A: The workflow we choose depends on the biological sample.

For blood samples, we isolate plasma using double-spin centrifugation, store samples in a biobank, and extract nucleic acids using automated systems, such as the KingFisher. We then quantify DNA using instruments such as the Qubit and proceed with downstream analyses. We either apply NGS to profile the full set of genomic mutations in the sample or use a targeted digital PCR approach. For digital PCR, we rely on custom TaqMan assays developed in collaboration with Thermo Fisher to verify specific index mutations identified through NGS.

With urine samples, we focus on generating an epigenetic profile by analyzing changes in DNA methylation during disease evolution. We collect both the cell pellet and the supernatant, and extract DNA from both fractions. After extraction, we assess methylation patterns using targeted digital PCR approaches, focusing on specific CpG islands. Through our collaborators, we also use qPCR platforms such as the QuantStudio 5 to generate methylation scores for translational research applications.

With saliva samples, the focus is on miRNA. We process the sample with a single centrifugation step, stabilize it, and extract RNA using kits specifically designed for saliva. We then perform reverse transcription and use TaqMan Advanced Chemistry to amplify miRNAs in a single step. The cDNA is then analyzed using multiplex digital PCR assays developed in collaboration with Thermo Fisher for targeted miRNA profiling.

Q: It’s encouraging to hear that you are using NGS, digital PCR, and qPCR in a complementary manner. How do you integrate these technologies in practice?

A: In translational research, we mainly face two key challenges: turnaround time and limited sample input. We need to answer specific questions as quickly as possible, often with limited sample material, while still maintaining high sensitivity.

To address this, we combine different technologies on the same sample. We use NGS to obtain a broad genomic profile and identify relevant mutations, and then apply more targeted approaches, such as digital PCR and qPCR, to verify those findings with higher sensitivity.

We are continuously adapting and co-developing our workflows with different vendors. For example, implementing solutions like Genexus in the pathology lab has significantly reduced the delays we previously experienced with manual library preparation and sequencing.

Q. How do you approach workflow design to bring these complementary technologies together?

A. Our focus is on creating a streamlined protocol that allows cross-verification of molecular findings. In many liquid biopsy-based approaches, we use NGS at key time points, such as at the start, during imaging assessments like CT or PET scans, and at relapse. In the meantime, during therapy, we use digital PCR to monitor any fluctuation, even minimal ones that NGS cannot reliably assess. In this way, we can also limit costs and develop a more cost-effective workflow by combining different techniques.

We also use qPCR during assay development. For example, when developing a multiplex assay, we use qPCR to determine the optimal concentrations and proportions of each primer to avoid re-optimization steps.

Q: What do you see as the biggest barriers to translating liquid biopsy methods from research into routine use?

A: Reliability is one of the most important challenges, especially in the liquid biopsy context. Closely related to this is standardization. There are many studies in the literature describing new and potentially more accurate methods, but very few are robust enough to be implemented in routine use.

Only through reliable and reproducible assays can we ensure consistent results across different laboratories. In this sense, reliability serves as an indicator of overall assay quality and is key to accurately assessing specific conditions.

At the same time, combining different technologies within a single workflow is very important. By integrating NGS with qPCR and digital PCR, we can take advantage of the broader genomic information provided by NGS together with the sensitivity and specificity of targeted approaches. These are very precise and accurate solutions, and when combined in a streamlined workflow, they add an additional layer of value to the molecular data.

Q: How easy are Applied Biosystems qPCR and digital PCR systems to use, both in terms of workflow and data interpretation?

A: In our experience, Applied Biosystems solutions are very easy to apply, even when implementing workflows from scratch. We have been working with digital PCR since 2016, and in the early days, using systems like the QuantStudio 3D was quite challenging. The process was manual, involving single chips, and standardization was difficult. The results were also partly influenced by how the chips were prepared.

With newer-generation digital PCR systems, such as the AbsoluteQ, the workflow has become much simpler. It requires only a few pipetting steps with a basic master mix, and the plate can be prepared much faster than older systems. The system also supports a larger number of samples than earlier platforms, and the software has improved significantly, giving more accurate results.

Overall, these advances in digital PCR technology have reduced both workload and hands-on time.

Q: If you had unlimited resources, what would be your vision for advancing your research?

A: I think we need to focus on standardizing workflows much earlier. This is still an open challenge. Many technologies we use, such as whole genome sequencing, generate a large amount of valuable data, but they are not yet ready for routine use.

If I had unlimited resources, I would invest a significant part in assay development and translation research. The goal is to standardize and simplify workflows and adapt them for smaller laboratories. This would help increase accessibility and allow these technologies to be more widely implemented across different settings.

Ultimately, the aim is to close the bench-to-bedside gap. We need to develop new methods and standardize existing ones so they can be effectively applied at the bedside.

Q: What is your message to young scientists entering this field?

A: Research is a very demanding job. It requires highly motivated, fully devoted people. There is a lot of satisfaction in this work, but often it comes unexpectedly. We can spend years trying to develop or identify something, for example, a biomarker, and a lot of effort goes into verification. But when you achieve that goal, all the effort becomes valuable for the community.

Another important point is to be patient and to stay updated. This is the only way to remain competitive and contribute meaningful work, not just to your lab or institute but also to the broader scientific and patient communities.

Watch Dr. Allegretti’s webinar: Advancing cancer research through liquid biopsy and circulating biomarkers

This interview has been edited for brevity and clarity.

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