Digital PCR technology drives progress in early lung cancer diagnosis and personalized therapy

Dr. Gary Pestano, chief development officer at Biodesix, discusses how his team is leveraging Bio-Rad’s advanced Droplet Digital^™ PCR (ddPCR^™) technology to develop novel blood tests to aid the diagnosis and management of lung cancer. Pestano highlights the advantages of ddPCR over orthogonal technologies for circulating tumor DNA (ctDNA) analysis, the exciting potential of ctDNA in early-stage settings, and how Biodesix is collaborating with academia and industry to overcome regulatory obstacles impeding the widespread clinical adoption of ctDNA testing.

The promise of circulating tumor DNA

Over the last decade, the predictive and prognostic utility of blood-based diagnostic testing for patients with solid tumors has been steadily gaining acceptance in clinical practice. One area that has garnered significant interest is the analysis of circulating tumor DNA – fragments of DNA derived from cancerous tumors that are found in a patient’s bloodstream.

ctDNA molecules vary depending on the tumor type, size, location, and stage and have been linked to prognosis, response to therapy, and recurrent disease^1–4. Consequently, ctDNA analysis holds great promise as an approach for cancer diagnosis and monitoring. Moreover, ctDNA measurement offers the advantage of reducing the need for invasive tissue-based tumor biopsies, which can be particularly beneficial in cases where a tumor is difficult to access, such as in lung cancer.

A significant portion of the current research in ctDNA is centered on non-small cell lung cancer (NSCLC), where tissue biopsies are either unavailable or insufficient for as many as one-third of patients. Tumor genotyping through ctDNA analysis is now widely recognized across all phases of clinical assessment for individuals with advanced NSCLC. Furthermore, the administration of therapies such as osimertinib (TAGRISSO^®) based on the detection of targetable mutations by ctDNA analysis has been approved by both the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA).

A focus on early-stage detection

In addition to aiding treatment selection in late-stage lung cancer patients, ctDNA is also emerging as a promising biomarker in other clinical scenarios such as monitoring treatment response, detecting minimal residual disease (MRD), and early cancer screening.

“I think our most challenging application for ctDNA is going to be with early-stage disease,” says Pestano. “As you go into the early stage, there’s much less circulating tumor DNA in the blood, and the technology, timing, and intervention all become a challenge. When to measure, what to measure, how much is present, is it meaningful? These are all the things we're grappling with in the early stage.”

“With ddPCR and our collaboration with Bio-Rad, we think we have one of the more compelling approaches to this question,” he continues. “We’re also working in collaboration with the Memorial Sloan Kettering Cancer Center (MSKCC), so we’re approaching premier cancer institutions, certainly in the US, with this question of early detection with the best available technologies.”

For this work, Biodesix is leveraging Bio-Rad’s QX600^™ ddPCR System, taking advantage of its high sensitivity, specificity, and advanced multiplexing capabilities. “Using the QX600, we're looking at getting after MRD and other questions that need addressing,” says Pestano. “If you gain sensitivity and multiplicity, it's not only applicable to the earliest stages of cancer, but if you can meet the requirements for the earliest stages, then you've got something that really works.”

Advantages of digital droplet PCR for tumor profiling

According to Pestano, there are two main genomic tools used for ctDNA analysis: PCR and next-generation sequencing (NGS). NGS offers an untargeted screening approach with the advantage of being able to simultaneously assess multiple biomarkers and identify novel mutations that could emerge during tumor treatment. However, it relies heavily upon sophisticated bioinformatics platforms, which can be challenging to implement in clinical settings. “With PCR-based testing, we don't have that hurdle,” shares Pestano. “What you get with a ddPCR result is a direct reflection of what you saw – there isn’t a lot of bioinformatics overhead (e.g. thresholding, background subtraction, and data cleaning) – it's a more direct measure.”

“When we started out in 2015, ddPCR was a perfect tool, as there were a limited number of known markers,” he continues. “As time went on, the NGS approaches became more informative, but we also learned that in earlier stage disease, there isn’t a plethora of mutations, there are a few genes and targets which then later drive the cancer. So, ddPCR is useful in the late stage with known targets for monitoring cancer, but it is also extremely valuable in the earlier stage because of its sensitivity and the relative paucity of targets.”

As ctDNA ddPCR tests become more viable for early-stage disease, there is a strong case for their uptake. “NSCLC is curable in its early stages, so the urgency is greater compared to late-stage metastatic cases,” Pestano explains. “The fast turnaround to results, cost-effectiveness, and the now improved sensitivity offered by ddPCR make it the preferred technology to address this challenge. And with the QX600, there's tremendous potential for improving not only sensitivity with ddPCR but also the ability to create bigger panels. It’s not going to be 600 genes in a tube, but with 12 targets per well, and 96 wells, you can get up to quite a number using a QX600, for example.”

Regulatory hurdles

In May 2022, the FDA released draft guidance on the application of ctDNA in early-stage solid tumor clinical trials, including its use in patient selection and patient enrichment, as well as a measure of response and as an early endpoint⁵. While multiple studies have demonstrated the potential of ctDNA analysis for these applications, several regulatory hurdles remain to be overcome before ctDNA can be advanced into routine clinical practice and support drug development.

“One of the ways we’re working to bridge this gap is through our efforts in consortia, including with Friends of Cancer Research,” explains Pestano. “We’re one of many collaborators in a muti-stakeholder project set up by Friends of Cancer Research (FOCR), which is called ctDNA to Monitor Treatment Response (ctMoniTR).” The project, which brings together statisticians, clinicians, and researchers from academia, industry, and government, aims to provide a consensus on how ctDNA can be used to inform treatment decisions, aid drug development, and inform regulatory decision-making. To this end, it is focused on determining whether changes in ctDNA levels are associated with long-term clinical outcomes.

So far, the group has conducted a series of retrospective meta-analyses – the latest of which was presented at this year’s American Society for Clinical Oncology (ASCO) Annual Meeting – that have shown that reductions in ctDNA are strongly associated with better clinical outcomes across multiple measures, including overall survival and progression-free survival. Now in its second stages, the project is expanding its approach to encompass more patients, trials, additional cancer types, and treatments, in addition to exploring the extent to which findings can be generalized across early- and late-stage cancer settings.

As the body evidence demonstrating the analytic validity, clinical validity, and clinical utility of ctDNA continues to accumulate for these applications, blood-based monitoring of solid tumors from early diagnosis through to remission could soon become reality.

Biodesix is a lung cancer diagnostic solutions company addressing the continuum of patient care from early diagnosis of lung nodules through to late-stage cancer. The company develops diagnostic tests addressing important clinical questions by combining simple blood draws and multi-omics with the power of artificial intelligence. Biodesix is the first company to offer three best-in-class tests for patients with lung cancer, and multiple pipeline tests including one with the potential to identify patients who may benefit from immunotherapies. The Biodesix Lung Reflex^® strategy integrates the GeneStrat^® and VeriStrat^® tests to support treatment decisions with results in 72 hours. The Nodify XL2^™ nodule test, evaluates the risk of malignancy, enabling physicians to triage patients to the most appropriate course of action. Biodesix also partners with the world’s leading biotechnology and pharmaceutical companies to solve complex challenges.

References

Tsao, S. C.-H. et al. Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations. Sci. Rep. (2015).

Newman, A. M. et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat. Med. 20, 548–554 (2014).

Bowling, M. et al. Longitudinal monitoring for the emergence of epidermal growth factor C797S resistance mutations in non-small cell lung cancer using blood-based droplet digital PCR. Cancer Drug Resist. (2019).

Mellert H.S., Alexander K.E., Jackson L.P., Pestano G.A. A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction. J Vis Exp. (2018).

https://www.fda.gov/regulatory-information/search-fda-guidance-documents/use-circulating-tumor-deoxyribonucleic-acid-early-stage-solid-tumor-drug-development-draft-guidance