November 1, 2022 | Robust levels of large extracellular vesicles (LEVs) were found “hiding in plain sight” in the blood of early-stage breast cancer patients in a recent study led by a team of scientists at the University of Southern California (USC). The LEVs are roughly the size of a cell and found while using a high-definition liquid biopsy platform to stratify normal donors from those with early- or late-stage breast cancer, according to cancer physicist Peter Kuhn, who directs the USC Michelson Convergent Science Institute in Cancer.
The stunning discovery was enabled by a third-generation assay requiring no enrichment that provides a single cell profile of all circulating events, says Kuhn. “I have never before seen such a clean separation between normal and cancer,” says Kuhn, quickly adding that he doesn’t completely understand the underlying biology yet.
A total of five strong signals of early cancer were found by the study, which published in npj Breast Cancer (DOI: 10.1038/s41523-022-00480-4), but the LEVs were the standout indicator. Importantly, the LEVs also had a lower prevalence in late-stage breast cancer and could therefore be a potentially good prognostic indicator.
The “oncosomes” secreted by cancer cells include both large and small extracellular vesicles, but the multianalyte liquid biopsy test used here looks specifically for LEVs, Kuhn points out, at least for now. As the research team has previously shown in a prostate cancer study, LEVs carry the same types of proteins as circulating tumor cells (CTCs), suggesting they come directly from the tumor.
Most liquid biopsy studies focus only on the presence of CTCs using blue-fluorescent DNA stain (DAPI) and algorithms structured to look for the intact nucleus and cytoplasm, says Kuhn. This study, for the first time, looked at both DAPI-positive and DAPI-negative events to detect rare circulating events—including the LEVs found in higher quantities than the CTCs.
In terms of the future for this novel liquid biopsy platform, the “dream scenario” is that blood samples will be collected from women on a routine basis as a complement to and perhaps in lieu of a mammogram that many women find uncomfortable if not painful, to give them confidence in their risk for breast cancer, he says. When breast cancer is first diagnosed, patients and their physicians would also feel more certain about their treatment choices given the ability to derive molecular information from this liquid biopsy.
Breast cancer is the most prevalent form of cancer in the world, affecting one in eight women over their lifetime. Patients today have more treatment options available to them and the drugs are more laser-focused on a particular target as well as more effective, says Kuhn. They are therefore living longer over more lines of therapy and liquid biopsy’s main forte is enabling that choice “because you cannot take tissue biopsies all the time from patients, but you can draw blood fairly frequently.”
To stratify patients as being either normal, early stage, or late stage, Kuhn explains, the research team began by identifying all the rare circulating events in each sample using high-throughput imagery and then categorizing them into eight descriptive color and shape combinations (the biomarkers)—an exercise he likens to sorting the proportion of vehicles on the highway based on their brand or observable descriptions such as pickup, sedan, or SUV.
Next, they determined which best separated the normal blood donors from the ones with cancer, he says. Five of the eight biomarkers, most notably the LEVs, proved to be statistically distinct.
Blood samples were from 130 women (56.9% treatment-naïve, nonmetastatic early-stage, 20% metastatic late-stage, and 23.1% normal donors) who consented to the study over the past 10 years and were processed in a standardized way, says Kuhn. They were stored in a freezer in aliquots and associated with data about the donors over the same period.
The study was conducted in collaboration with Billings Clinic, Duke University, Epic Sciences, and the USC Norris Comprehensive Cancer Center. The team is now automating their rare event detection method for ongoing technical and clinical validation in a second data set, Kuhn says. Early discussions have also begun about a prospective clinical trial that will enroll patients who have not yet been diagnosed with breast cancer, a complicated and expensive undertaking with multiple boundary conditions.
“The critical next step in the evidence build is to ask the question, ‘At the time of a positive mammogram and a simultaneous blood draw, can the combination of the two help with the current diagnostic workup?’ Currently, if a mammogram is sufficiently suspicious it is followed with a biopsy,” which might be termed a “minor surgical intervention” but not a procedure women would likely choose if given a choice between that and a blood draw.
Results of the clinical trial will suggest when the test would be most valuable in guiding clinical decision-making in terms of its utility before, after, or at the same time as the mammogram, says Kuhn.
The drawbacks of mammograms and tissue biopsies are helping to fuel early-detection momentum and late-stage management in the liquid biopsy space. Mammography is not perfect, picking up breast cancer around 87% of the time, according to the Breast Cancer Surveillance Consortium. Women in some resource-poor communities don’t even have access to the imaging test.
A tissue biopsy, in addition to being invasive and painful, is likewise not a fool-proof method, says Kuhn. Since doctors can sample only a small area, it may fail to capture the full extent of a tumor.
The combination of five newly discovered biomarkers of early breast cancer is “absolutely implicated in other cancers,” he adds, although these will be distinct evidence builds because cancers each have different biological starting points and patients also have a unique set of risk factors. USC researchers are already investigating the role of LEVs in multiple disease settings.
Since blood offers a picture of the immune system at work, it should be technically possible to find early signals of every cancer and its interplay with the immune system via liquid biopsies, says Kuhn. But their different origins will likely require separate algorithms to analyze the data that is “finely tuned to specific cancers.”
Working together to improve outcomes and the quality of life for patients and “take the friction out of the system,” which is the goal of the presidential Cancer Moonshot, will make this achievable in our lifetime, he says.