Setting the Stage for Liquid Biopsy in Breast Cancer Treatment

 

This story is part of our celebration of innovation in 2024.

 

by Dana D’Amico, Connect to Science

Researchers like Dr. Karen Page are setting the stage for the era of the liquid biopsy and tracking tumor evolution through the entire cancer journey.

 

In cancer treatment, understanding the enemy is half the battle – to guide treatment, clinicians need to know what they’re dealing with. The trusted standard for understanding a tumor today is the traditional needle or surgical biopsy, a necessary but invasive process.

Still, it has its limits. Aside from the invasion, cost, and difficulty that might arise when a tumor can’t be accessed safely, the solid tumor biopsy often offers an incomplete picture. The tissue collected represents a moment frozen in space and time, a mere snapshot of an extremely dynamic course of disease. While patients may think of individual tumors as fixed targets for treatment, these accidents of unchecked cellular replication accrue thousands of genetic mutations in a short time. This means the rapid development of ‘subclones,’ or genetically distinct subpopulations within a tumor. Evolving, heterogenous tumors must often be met with evolving, heterogenous treatment.

 

To better understand tumor progression over time, researchers like Dr. Karen Page, the lead for next-generation sequencing (NGS) in Professor Jacqui Shaw’s Lab at the UK’s Leicester Cancer Research Center, have invested decades of work into the potential of a non-invasive ‘liquid biopsy’ for longitudinal and comprehensive study of the disease process. Through a simple blood draw, clinicians and researchers could gain insight into tumors anywhere in the body – even those not yet detectable by imaging.

Now recognized as international experts in liquid biopsy in breast cancer, Page and her colleagues in the Shaw Lab aim to standardize this potentially field-shifting process so that it can make the wider jump from research to clinic and more universal access.

The ab C’s of ctDNA, cfDNA and CTCs 

Liquid biopsy allows the capture of tumor biomarkers through blood plasma. Getting a sample is as simple as a routine blood draw.

These biomarkers can be circulating tumor cells (CTCs) themselves, the seeds of metastasis coursing through the body. But they can also be particles of cell-free DNA (cfDNA), the sea of DNA fragments released into the blood and other bodily fluids from normal cellular processes like apoptosis, necrosis, and secretion. In general, higher levels of cfDNA are associated with cancer as tumor cells multiply out of control and shed a subset of cfDNA called circulating tumor DNA (ctDNA).

This isolated ctDNA holds the key to valuable molecular insights about early, recurring, active, or dormant tumors – sometimes alerting researchers to a tumor’s presence before imaging can. ctDNA analysis can reveal information about how the tumor may shift over the course of disease and treatment: point mutations, copy number alterations, rearrangements, methylation changes, and more.

As Page and others build a more complete library of common metastatic cancer driver mutations and which drugs are most efficacious against them, this genetic knowledge of tumor composition could present a huge advantage for patients and their doctors. The ability to tell if any part of a heterogenous breast cancer tumor is HER2-positive, for example, can help expand known treatment options. Likewise, tracking the levels of ctDNA through treatment can help clinicians understand drug performance and guide any necessary changes.

Researchers’ hope is that liquid biopsy will progress to the point of becoming a universally available, low-cost, non-invasive tool for detecting early cancer or relapse, monitoring treatment response and tumor evolution, identifying treatment resistance, developing novel therapies, and more.

To get there, however, researchers like Page have taken on the challenge of improving and standardizing what can be a highly temperamental process.

Rubbish in, rubbish out

Page’s journey into the world of cfDNA in breast cancer began in more than 20 years ago in 2002.

Though the discovery of cfDNA actually predates that of the double helix, it took until the early 2000s and the advent of high-throughput sequencing and other molecular and genomic tools for applied cfDNA research to really take off. Page got in on the ground floor, and she’s seen the strides liquid biopsy technologies have made over the years.

At that time, the process of analyzing just one locus of DNA took her about two weeks from start to finish.

“It took me that long to isolate the cfDNA, look at the quality, and try to quantitate it accurately. Then, I would need to run a PCR to amplify my region of interest. We used large polyacrimide gels to run those samples and then stain with silver nitrate; even just the gel and staining portion would take around three days,” said Page.

Despite the time sink, Page and colleagues knew that brute force trial and error work was needed to perfect and standardize a protocol over time if it had any chance of helping patients one day. They considered every aspect.

They worked out, for example, that when processing blood samples to plasma, you need to spin and freeze the tubes in just the right way.

“Ideally, you should not have the brake on when centrifuging blood samples, otherwise you could cause cell lysis, interfering with your cfDNA sample. Then, crucially, you need to freeze the DNA before double processing and doing another spin. We did a lot of work with PCR to demonstrate that you can get contaminating genomic DNA if you don’t do that extra spin,” she said.

She tested centrifugation speed and rounds, storage temperature and freeze and thaw cycles, processing time from venipuncture, preservative solutions, collection tube types, and more.

They realized that ctDNA rapidly degrades, so blood samples needed to be stored carefully and processed within hours of collection.

“Research like this is really about getting that first part as optimal as you can,” said Page. “Because if you don’t? It’s rubbish in, rubbish out.”

Back to the future of cancer biomarkers

By 2006, the lab had published a paper detailing the techniques to use in sample processing to improve template DNA and produce better downstream results without genomic DNA contamination.

A few decades later, the core learnings about sample preparation remain but the process looks a lot different.

Today, Page can bring a blood sample through processing, extraction, and sequencing in just four to five days.

“With the custom-designed panels that are available today, we can look at hundreds of hotspot mutations at once. Twenty years ago, you couldn’t even envision what life would be like now with respect to the technology available,” said Page. “Now you can design your own with dual barcodes for sensitivity and look at your variant allele fractions down to such a low level.”

Automated sample preparation tools like the KingFisher™ Flex and MagMAX™ Cell-Free DNA Isolation Kit allow more hands-off isolation of cfDNA from plasma, along with the possibility to capture genomic DNA and CTCs. It’s good news for reproducibility, non-contamination, and the overall standardization that will be necessary to help bring research learnings into the real world of the clinic.

Downstream applications have transformed as well. Page performs NGS and ctDNA analysis using tools like the Ion Torrent™ and Oncomine™ Breast cfDNA Assay, which can detect just one mutant copy of a cancer hotspot gene in a background of 1000 wildtype copies (0.1% level of detection with 20 ng cfDNA input).

She worked on a pilot study confirming that plasma-derived cfDNA collected at genetic medical centers were of sufficient quality and quantity to be used for ctDNA profiling with commercial assays like Oncomine.

“We’ve had a longstanding collaboration with Thermo Fisher Scientific, which has enabled us to test a lot of the NGS and sample prep automation rolling out. That works well for us for cfDNA work,” said Page.

In collaboration with the Thermo Fisher custom R&D team, Page and her colleagues developed a novel custom NGS panel covering 16 known gene mutations and amplifications in metastatic breast cancer cfDNA, including ESR1 mutations that may predict resistance to endocrine therapy. They also discovered, using the Oncomine breast cfDNA assay,  that 34% of patients sampled had at least one identified gene mutation in their cfDNA, with just 3 genes accounting for more than 93% of the mutations in the study.

The more we understand about the genetic drivers of metastatic cancer, the better researchers and clinicians can get ahead of them.

As technology continues to make these assays more accessible and reproducible, researchers like Page continue to advocate for wider patient access from the bench-side by helping to ensure that genetic profiling methods are as reliable, streamlined, and tested as they can be.

Downstream effects

Through all these years that Karen has focused on DNA and breast cancer, not once has her interest in the topic waned. Her career has been driven by questions like, ‘how can we improve on that?’ and ‘what’s next?’

For one, a new generation of scientists is now branching out to consider the synergies of other cancer biomarkers in the liquid biopsy landscape including CTCs, circulating RNAs, and extracellular vesicles. Combining these markers with ctDNA could be the future of early detection.

She’s excited for it all.

“It’s exciting to see how far we’ve come, but really exciting to see how far we can still go in this area to give all patients as much benefit as possible – to move the disease as far back as possible,” she said. “What can we do in the early stage setting of the disease? Can we offer people a lower chance of undergoing surgery or chemotherapy and therefore potentially avoiding its toxic side effects? Can we use this not just for detection, but for monitoring people all the way along their cancer journey?”

“There are so many things that are still to come, and that’s what excites me the most.”

Watch Dr. Karen Page’s presentation about liquid biopsy in breast cancer management  »

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More Reading

• Publication: Shaw et. al. (2021) Circulating Tumor DNA profiling from Breast Cancer Screening Through to Metastatic Disease. JCO Precision Oncology. 5. 1768-1776. 10.1200/PO.20.00522.
• Q+A: On the Leading Edge of cfDNA Discoveries with Dr. Jacqui Shaw
• Tools & Education: Liquid Biopsy Analysis in NGS
• Tools & Education: Cell-Free DNA and FFPE Extraction for Cancer Research
• Cancer Topics: Exploring the Tumor Microenvironment in 3D with Tumoroid Culture

 

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References

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Leicester Cancer Research Centre. University of Leicester. (2020, October 28). https://le.ac.uk/lcrc

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