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Cancer Research | Liquid Biopsy

Hunting for traces of
cancer in blood

Cancers shed cells into the blood that can yield clues about how a cancer is evolving. Amin El-Heliebi, Ph.D.,  at the Medical University of Graz, Austria is working towards developing a blood-based test - a liquid biopsy - to help identify prostate cancer patients who stop responding to first-line treatment in real time. The hope is that switching patients to other treatment early on will save more lives.

Tests that detect snippets of cancer in blood - liquid biopsies - carry huge potential to improve the success of cancer treatment. They provide a quick and non-invasive way to sample a tumor using biomarkers circulating in the blood, especially when the location of the tumor isn’t accessible for a biopsy. Many oncologists currently use available liquid biopsy tests as “companion diagnostics” to identify cancer patients who might benefit from targeted cancer therapies. 

But researchers around the globe are hoping that liquid biopsies will soon be able to do more than that. “Several studies have shown that levels of circulating tumor DNA (ctDNA) correlate with how well a cancer patient is responding to treatment,” says Amin El-Heliebi, Ph.D., a cancer researcher in the Division of Cell Biology, Histology and Embryology at the Medical University of Graz, Austria. But every cancer is different. Available tests rely on DNA, for example, but cancers of the kidney, soft tissue and brain shed little to no DNA. Moreover, developing a highly accurate test is challenging. 

In such cases, a multianalyte approach might prove more successful. El-Heliebi has found that analyzing multiple tumor analytes from blood drawn from patients with prostate cancer can give clinicians clues about how well their patients are responding to therapy in real time, and is more informative than DNA alone. The research suggests that analyses of multiple analytes might be a way to improve the accuracy of liquid biopsies for other types of cancers too. 

In patients with advanced cancer, such information can help clinicians decide whether to switch a patient to a different therapy, says El-Heliebi, or confirm whether a treatment is having an effect. El-Heliebi is applying the same concept to other cancers, such as Ewing’s Sarcoma, a cancer of bone and soft tissue, in hopes of improving treatment outcomes. And he is also working to help standardize liquid biopsy workflows to improve reproducibility, and ultimately the accuracy, of results. 

Amin El-Heliebi, Ph.D.
Amin El-Heliebi, Ph.D. is a cancer researcher in the Division of Cell Biology, Histology and Embryology at the Medical University of Graz, Austria. Most cancer deaths are caused by cancer metastasis, which arise from cells that break free of tumors and seed metastasis in other parts of the body. El-Heliebi’s research focuses on studying therapy resistance and circulating tumor cells (CTC) shedding.

Levels of circulating tumor DNA (ctDNA) correlate with how well a cancer patient is responding to treatment.

Amin El-Heliebi, Ph.D.
Research Professor at the Medical University Graz

What liquid biopsy tests can do

The US Food and Drug Administration approved the first liquid biopsy test in 2013 to monitor patients with advanced metastatic breast, colon, and prostate cancers based on the cancers’ level of circulating tumor cells (CTCs). Three years later, the FDA approved the first circulating tumor DNA (ctDNA) blood-based genetic test to detect epidermal growth factor receptor gene mutations in patients with non-small cell lung cancer (NSLC); the test plucks out patients who are likely to respond to a therapy that targets the mutation. In 2021, two other liquid biopsy tests were FDA approved to detect additional mutations in NSLC and specific mutations in prostate, ovarian and breast cancer that might be amenable to targeted therapies.  

Prostate cancer presents a particular challenge. It is the second most common cancer of men worldwide, according to the World Cancer Research Fund. Yet the only non-invasive screening test - blood levels of prostate-specific antigen - has fallen out of favor because it identifies too many men with cancers that grow so slowly, that they would never be life threatening. And although treatment success rates are high - more than 85% of men survive their cancer five years - there is no reliable way to identify cancer patients who might not respond to first-line therapy.

In an effort to understand how to better treat advanced prostate cancer, El-Heliebi and his colleagues used 3 different assays to study mRNA, DNA and CTCs in the blood of 19 patients with prostate cancer. When a single assay was used, researchers were only able to glean information on 44-53% of the patient samples. Combining all three approaches, however, provided tumor-related information on 89% of patients.

“We showed we can get information from pretty much all patients and that this information was relevant.” says El-Heliebi.  “Each assay, mRNA, DNA and CTCs, is cumulative, adding more information than a single assay alone. This was our big find.” The research was published in 2020 in the journal Cancers.

 Liquid biopsy, miRNA, lncRNA

As part of the process of metastasis, many tumors shed cells into the bloodstream. Liquid biopsies try to identify these circulating tumor cells (CTCs). Moreover, all cells, including cancer cells, have a limited lifespan. Liquid biopsies can also identify circulating cell-free tumor DNA (ctDNA) or other biological material released from the breakdown of dead or dying cancer cells.

It can be frustrating when levels of tumor DNA go up…But we can be more vigilant in those cases because a marker in blood is there.

Amin El-Heliebi, Ph.D.
Research Professor at the Medical University Graz

Liquid biopsy for Ewing’s Sarcoma

In a follow-up joint study with the Center for Biomarker Research in Medicine (CBmed) in Graz and in collaboration with QIAGEN and PreAnalytiX, El-Heliebi has built on the research. Although the majority of prostate cancer patients respond well to common anti-androgen treatment, a significant number eventually stop responding. El-Heliebi has found a signature of multiple analytes that correspond to a person’s cancer evolving resistance to therapy. “We can see the tumor changing in real time,” he says. Prostate cancers are driven by androgens so the most common treatments target androgen pathways. But if the cancer is becoming resistant, such therapies won’t work.

The research is still investigational, El-Heliebi says, and needs to be confirmed in a larger patient group. But if the findings hold up, they would open up new ways for clinicians to treat advanced prostate cancer more effectively, by switching to chemotherapy as soon as they see resistance against anti-androgen therapy, for example. 

Additionally, El-Heliebi and his colleagues are also evaluating treatment response in patients with Ewings’ sarcoma - a cancer that forms in bone or soft tissue and that affects young people and children. After sequencing DNA from tumors resected from patients, researchers monitor the blood for the presence of DNA with the same genetic changes as the tumor. They have observed that when DNA markers of the tumor in blood decrease, patient outcomes tend to be better, and vice a versa. 

Such a correlation between blood-based DNA markers and cancer outcomes exists for other cancers too. Several studies have shown that ctDNA in the blood after treatment is a sign that “the disease is still there and that the patient is more likely to relapse,” says El-Heliebi. The effect has been shown in many different tumor types, he adds, and confirmed in large studies. But unfortunately, knowledge that someone is likely to relapse doesn’t always indicate how to better treat the patient, he adds. 

“It can be frustrating when levels of tumor DNA go up, because we can’t always do something about it,” El-Heliebi says. “But we can change the management of patient care.” They can have more extensive testing and imaging, for example. “We can be more vigilant in those cases because a marker in blood is there.” 

In prostate cancer too, monitoring changes in the cancer may in some cases, point to targeted therapies that may better treat the cancer, he adds. 

Female face with blood tubes
Clinicians are using available liquid biopsy tests in three primary ways: to detect relapse in patients already diagnosed with cancer; to find cancers with specific mutations that are likely to respond well to therapies that target those mutations; serially throughout treatment to monitor a tumor and see how well a specific treatment is working.

We showed we can get information from pretty much all patients…We can see the tumor changing in real time.

Amin El-Heliebi, Ph.D.
Research Professor at the Medical University Graz

Too early for intervention

But it’s too early to tell whether interventions based on information from the blood actually improve outcomes, says Don Dizon, Director of Medical Oncology at Rhode Island Hospital in Providence, which is why most clinicians primarily use the tests as companion diagnostics for now, he adds.  “While liquid biopsies enable clinicians to serially check for ctDNA and other markers, it’s still a hypothesis that if you act on that information, you would improve cancer survival. The data isn’t there yet,” he cautions.

And there remain challenges with the technology itself. Blood-based tests still suffer from a lack of standardization, particularly for preanalytic steps such as sample collection, stabilization, transport and extraction methods and analytic criteria, such as determining cut-off values for ctDNA or circulating tumor cell positivity. A 2018 joint review by the American College of Clinical Oncology and the College of American Pathologists on using ctDNA assays for solid tumors concluded that “there is still insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer.”

To help address this issue, El-Heliebi and his colleagues are using the most recent ISO guidelines (ISO 20186-3:2019) which have been determined by large consortia (SPIDA4P and Cancer ID) in collecting their samples. 

“Not everyone knows that you need to invert a sample tube 10 times,” El-Heliebi says. The guidelines serve as a benchmark for quality and have helped researchers to understand the characteristics of samples that are just part of normal variation. For example, about 20% of patient samples show a reddish color of the plasma indicating red blood cell lysis, he says. “But as we follow the ISO guidelines, we can be sure that red blood cell lysis is a common phenomenon in people with advanced prostate cancer rather than artifacts of poor processing.”

Female, Scientist
While researchers are trying to expand the applications of liquid biopsies, plucking out the relatively tiny amounts of tumor material from the much larger amounts of normal biological material in the blood has been challenging. Moreover, not all tumors shed detectable cells or DNA into the bloodstream and researchers are still trying to understand why. Prostate and colon cancers shed high levels of ctDNA in the bloodstream, for example, but renal cancers shed hardly any.

Technologies are advancing rapidly. That’s what drives me and keeps me positive.

Amin El-Heliebi, Ph.D.
Research Professor at the Medical University Graz

Becoming the holy grail of cancer screening

For consistent and reliable results, El-Heliebi depends on QIAGEN workflow solutions. There are many different types of sample collection tubes on the market. All have benefits and drawbacks, he says. “We use PAXgene blood ccfDNA tubes for collection and stabilization of samples while DNA is extracted with a QIAGEN kit and then sequenced with a QIAseq panel.“ 

Regulatory agencies are also increasingly demanding standardized workflows, El-Heliebi adds. “They don’t want different kits and assays mixed together because this is difficult to compare.” 

In the future, he hopes liquid biopsy research will contribute to what would be the “holy grail” of cancer screening—a blood-based assay that could detect many types of cancer early.

Although there are several companies working towards this goal, results have been inconsistent and accuracy isn’t yet high enough to screen for cancer in healthy people. 

But “technologies are advancing rapidly. That’s what drives me and keeps me positive,” El-Heliebi, says.

“We are trying to translate our research into the clinic and we are really proud of that. This is really the most exciting part of the work.”

Liquid Biopsy Research
While much research has been focused on developing a liquid biopsy assay that could be used as a general screening tool to detect cancer early in healthy people, accuracy remains a huge challenge - existing methods either deliver too many false positives, too many false negatives or both. More clinical utility studies are needed to translate liquid biopsies into clinical routine.

July 2023