Colonoscopies are the most effective way to screen for colon cancer. With over 90% accuracy, they enable doctors to find the cancer early, when, most of the time, it can be cured. But colonoscopies are also invasive, can necessitate taking take time off from work and the “prep” required the day before the procedure is famously unpleasant. An alternative, though less accurate, screening method is a stool test, but the awkwardness of these tests presents another deterrent. The result? Many people choose not to be screened at all for an often curable disease.

But now there is a third option. In July 2024, the Food and Drug Administration approved the first blood test that can detect colon cancer. This test, Guardant Health’s Shield, detects telltale DNA fragments that cancerous cells shed into the bloodstream, indicating the presence of cancer. Shield does not match the sensitivity of a colonoscopy — it finds the cancer roughly 10% less of the time — but it’s a viable alternative for people who would not otherwise be screened. All that is required of the patient is having their blood drawn, just as they would at a routine physical examination.

Shield is the latest example of what is known as a liquid biopsy, the use of bodily fluids such as blood, urine and saliva, rather than tissue samples obtained through needles or with a scalpel, to detect disease. From these fluid samples, liquid biopsies identify and isolate molecular evidence — in the form of circulating tumor cells, proteins, cell-free DNA and membrane-bound structures known extracellular vesicles that contain a variety of molecules — that indicate the presence of disease.

“I can’t stress enough the importance that liquid biopsies will have in medicine,” said Steven Soper, Ph.D., Foundation Distinguished Professor of Chemistry and Mechanical Engineering and director of the NIH-funded Center of BioModular Multi-Scale Systems for Precision Medicine at the University of Kansas. This research center, known as CBM2, engineers the plastic microfluidics to create devices that process liquid biopsy samples in an efficient and automated fashion.  “And it is not just for oncology, it’s for a whole gamut of diseases that potentially can be managed.”

Soper said that although the term liquid biopsy is most often associated with cancer, it has begun to be employed more broadly to describe new tools being developed that use these fluids to find other maladies including stroke, infectious diseases and even Alzheimer’s.

These tests also promise to be a key component of precision medicine: The molecular biomarkers found in liquid biopsies not only can help find disease, but also can help physicians determine a patient’s prognosis, decide which treatment is best and, because these tests can easily and cheaply be repeated, monitor how well a patient responds over time to that treatment.

Old idea, novel developments

Soper is looking to the future for sure, but he was also quick to say that liquid biopsies are not a recent invention. Or even anything most of us haven’t already experienced. As he pointed out, if you’ve had a COVID-19 test, which utilizes mucus or saliva, then you’ve had a liquid biopsy. Prostate-specific antigen (PSA) blood-screening tests for prostate cancer date back to the 1990s. And although they are not used as a routine screening tool, blood tests that measure the level of the CA-125 protein in the blood have been in use since the 1980s for women at high risk for ovarian cancer.

“Liquid biopsies, in and of themselves, are not new, but the biomarkers we’re looking at continually change, and the technology that allows us to look deeper and deeper into some of these clinical samples is changing,” said Andrew Godwin, Ph.D., founding director of the Kansas Institute for Precision Medicine and director of the genomics diagnostics division in the Department of Pathology and Laboratory Medicine at KU Medical Center. “We’ve gone from looking for soluble proteins to looking for circulating tumor cells, and now the hot trend is looking at circulating nucleic acids; in other words, DNA and RNA.”

For decades, Godwin has been conducting research related to the biology of liquid biopsies and identifying those biomarkers that can be found in bodily fluids to help diagnose or guide treatment. Recently, he led a study that identified a hormone in the blood that can indicate which patients with HER2-positive breast cancer will benefit most from chemotherapy after they complete endocrine therapy. This information can spare women who would not benefit from chemotherapy from going through it and suffering the side effects.

A big challenge in creating these tests, Godwin said, isn’t just identifying the biomarker, but devising a test that can find it. And that’s where people like Soper and the bioengineering work at CBM2 come into play. Since 2006, Soper has been developing “lab on a chip” technology, a device that isolates specific biomarkers from a blood, urine or saliva sample and conducts the laboratory processing of those cells on a plastic chip the size of a credit card. These chips can detect circulating tumor cells, cell-free DNA, viruses and extracellular vesicles released by biological cells associated with a particular disease.

Soper’s lab collaborates with Children’s Mercy in Kansas City to test the ability of these microfluidic chips to detect circulating leukemia cells in children who have been treated for acute leukemia. The current standard of care is a bone marrow biopsy.

“That’s extremely painful for a little kid,” said Soper. “But with a blood test, we can do much more frequent sampling with much less pain, and with even better sensitivity to look for signs of disease recurrence.”

Bad blood

The sample size of fluid needed in a liquid biopsy varies according to the biomarker of a particular disease. And sometimes, there simply isn’t enough of a particular biological substance in the blood for a test to be able to find.

“In a milliliter of blood, there might be trillions of extracellular vesicles, but not all of them are associated with a specific disease,” said Godwin. “We need to be able to go into that very large haystack and find that very rare needle.”

This brings us to the Theranos debacle. In 2003, Elizabeth Holmes was a young Stanford dropout who founded Theranos, a Silicon Valley start-up that promised to revolutionize diagnostics though a blood-testing machine about the size of a desktop printer. From a pinprick of blood, the “Edison,” as it was called, was supposed to test a person for roughly 100 diseases. Walgreens even launched in-store testing centers equipped with these machines and collected samples. But Theranos never got the Edison to work properly, and employees were secretly processing the blood samples the old-fashioned way — until Holmes was finally convicted of fraud in 2022 and her $9 billion company was liquidated.

Soper said he was always skeptical about the Edison.

“I didn’t even need to know what was under the hood because the whole thing was theoretically impossible,” he said. “A drop of blood is about 100 microliters which is a small amount. And if you have a marker for a disease that shows up in one disease cell per one milliliter of blood, what are the chances that cell is going to show up in a drop of blood? Statistically, it’s just
not feasible.”

Holmes’ deception hurt not just her investors, but also slowed down the field by creating skepticism among today’s potential funders about anything related to blood tests for diagnostics. Godwin said on the upside, it has forced researchers to be extra vigilant about the integrity of their work.

And that work goes on. Liquid biology technology is evolving as new biomarkers are discovered, and that includes at KU Medical Center. In addition to the two precision medicine centers, KU is, along with UCLA and the University of Pennsylvania, one of just three universities in the United States that has a liquid biopsy core, a dedicated laboratory and resources focused on augmenting research and clinical trials. These resources support a number of researchers conducting work related to liquid biopsies across KU Medical Center where, unlike at Theranos, their work is subject to rigorous protocols, thorough peer review and transparent sharing of data.

Early bird

Many liquid biopsy technologies in development are very good at detecting cancers at stage 2 or later, but they are not yet sensitive enough to find cancer in its infancy, when the cancer has the best chance to be cured, noted Stefan Bossmann, Ph.D., University Distinguished Professor and chair of the Department of Cancer Biology at KU Medical Center.

Bossmann is looking to change that. He and his colleague, Obdulia Covarrubias, Ph.D., research assistant professor of cancer biology, have developed a blood test that detects the presence of lung cancer. When caught early, lung cancer often can be treated successfully. But the disease doesn’t cause many symptoms until after it has metastasized to other parts of the body, and by the time it’s diagnosed it’s often too late. CT scans can detect lung cancer sooner, but they are expensive and there are not enough CT scanners in the country to implement population-wide screening.

Bossmann and Covarrubias have developed biosensor technology that detects, in blood serum, protein-digesting enzymes known as proteases. Some proteases can modify lung tissue and make space for a tumor to grow. They then get washed away into the blood. Covarrubias and Bossmann’s technology is a panel of 18 nano-scale sensors made of graphene that releases a fluorescent signal when certain proteases are present in a blood sample, indicating the presence of lung cancer. The test produces results in just one hour and has a 90%-95% probability of detecting lung cancer at stage 1.

Their biosensor technology is much cheaper (for both patients and health systems) than a CT scan, and because the test is cost-effective and could even be administered in a primary care doctor’s office, it has the potential to be widely implemented.

Now the researchers are also applying their technology to other cancers where it makes the most impact to catch it early, including pancreatic, ovarian and breast cancer. In addition to creating the sensors, they do the computer work to identify all the proteases for each cancer type.

“You can think of this as if we are creating a barcode for each cancer,” said Covarrubias.

Preliminary research shows that their nano-sensor technology can identify pancreatic cancer, which has the highest mortality rate of all major cancers, by detecting the activity of nine proteases.

“We can see the transition from pancreatitis to ductal adenocarcinoma,” said Bossmann. “So that’s very, very early. It’s an ideal case when you can see cancer before stage 1 occurs.”

Stroke of genius

Stroke is a leading cause of death and of serious long-term disability in the United States. There are two types: ischemic stroke, which occurs when a blood clot blocks blood vessels to the brain, and hemorrhagic stroke, which happens when an artery in the brain ruptures or leaks blood. Early treatment is critical, but the treatment for each type is different and treating the patient for the wrong type of stroke can kill them. About 87% of strokes are ischemic, according to the Centers for Disease Control and Prevention, and a clot-busting drug must be given within three hours.

The problem is determining the type of stroke quickly. “Time lost is brain lost,”  goes the expression. CT scans and MRIs are the most common way to diagnose stroke, but these take time, require a pathologist to read the results and patients in rural areas may not be able to get to a health care facility with the equipment before it is too late.

Malgorzata Witek, Ph.D., associate research professor of chemistry at KU, researches liquid biopsy biomarkers atCBM2, and the Kansas Institute for Precision Medicine. In collaboration with the SUNY Downstate Medical Center, she and her colleagues are working on a blood biopsy that would isolate certain fragments of nucleic acids from extracellular vesicles that would distinguish the type of stroke. And it would do so within 30 minutes.

“The statistics for stroke are brutal, so this is a very important project,” said Witek.

Untangling Alzheimer’s diagnoses

For decades, physicians have diagnosed Alzheimer’s disease through a combination of cognitive testing, individual and family histories, physical exams and laboratory tests that can rule out other conditions. In the past few years, physicians also have begun to use PET scans and cerebrospinal fluid tests that can detect biomarkers that indicate Alzheimer’s, including amyloid-beta, a protein that builds up in the brain and is a hallmark of the disease.

Early diagnosis is critical for enabling physicians to identify patients that stand to benefit most from the new drugs for Alzheimer’s disease, such as lecanemab (brand name, Leqembi), that remove amyloid plaques in the brain to help slow cognitive decline.

“These new drugs work best for those who are diagnosed in the earliest stages,” said Jeffrey Burns, M.D., co-director of KU Alzheimer’s Disease Research Center. “In fact, there’s a time window; if we don’t diagnose early, they are no longer eligible for the drug.”

Blood tests currently used in research studies have shown promise in diagnosing Alzheimer’s early and accurately, and these tests would be less traumatic for patients than a lumbar puncture to get spinal fluid, and more accessible, as well as less expensive, than a PET scan.

With her colleagues at the KU Alzheimer’s Disease Research Center, Jill Morris, Ph.D., associate professor of neurology, is working on developing such a blood test called ptau217 that detects the phosphorylation of a protein known as tau. This protein is found in the “tau tangles” that accumulate in the brains of people with Alzheimer’s, along with amyloid.

The challenge is creating a test that is as accurate as the spinal fluid tests and PET scans. Morris said the goal in the field for developing blood tests is approximately 90% accuracy for both sensitivity and specificity, meaning not many false positives or false negatives.

For Morris, the accessibility of these tests is also key.

“It’s not necessarily going to be just for people who can access top-tier medical centers,” she said. “It’s going to be a lot easier to put them into the hands of people in primary care.”

Promises and precautions

This kind of progress comes with its own precautions and caveats. For one thing, if a liquid biopsy is able to detect a cancer when it’s very tiny, then there also needs to be imaging capable of finding the exact location of that tumor to remove it. This is one reason that the preclinical imaging core at the KU Cancer Center, which Bossmann directs, will perform research on making imaging that is better, with higher resolution, as well as faster.

“For instance, we will explore new MRI pulse generation and acquisition programs that will save a lot of time during MRI scans,” he said. “This will allow us to image more patients per unit of time with higher precision. We are also developing new attenuators for using micro-CT, which permit the imaging of tumors and metastases without the need for contrast agents. Of course, these methods will have to be translated into the clinic. We are very optimistic that we will be able to do that.”

And there are ethical considerations that will come with these new tests. Just because a test is easy to administer does not necessarily mean it should be.  There is such a thing as overdiagnosis.

“Suppose there’s an asymptomatic patient, and we diagnose her with stage 4 ovarian cancer,” said Godwin. “All we’ve done is put her into the medical system where she is going to have to endure therapies that won’t cure her. That’s one of the reasons we must be very careful about how we’re applying these liquid biopsies and other kinds of tests, because we want to do it so that we can improve the outcomes.”

Improving outcomes is, after all, the endgame.

“The whole area of liquid biopsy is growing tremendously because people are now starting to realize the utility in terms of patient management,” said Soper. “And we are one of the universities on the forefront.”