Cancer Biomarkers: the Liquid Biopsy Revolution

Biomarkers – measurable biological entities – hold the key to precision medicine. In the cancer arena, people are looking for biomarkers to detect the disease early, monitor therapeutic efficacy, and predict outcomes.

“The earlier we can detect cancer, the better job we can do with treating it – and the sooner you know whether a drug is working the sooner your doctor can modify therapy,” says Marsha A. Moses, PhD, Julia Dyckman Andrus Professor at Harvard Medical School and Director at Boston Children’s Hospital.

Clinical tests based on analyzing a tumor biopsy for genetic or protein-based biomarkers are already playing an increasingly important role in guiding treatment. But researchers are now looking to develop non-invasive tests on body fluids that are easier to collect, such as blood or urine – so-called liquid biopsies.

“Just looking at a few drops of urine can give you lots and lots of information about what’s going on in that patient,” says Moses.

Liquid biopsies would not only be simpler for patients, they would also give doctors the opportunity to get results more rapidly and regularly. And they even offer the potential for self-monitoring.

Protein biomarker discovery

Although researchers use many different platforms for biomarker discovery, mass spectrometry is commonly used for identifying protein-based markers. Due to its sensitivity, it can detect proteins at very low abundance within complex mixtures, enabling researchers to identify extremely tiny differences in their levels in patients versus healthy age-matched controls.

“We’ve looked at a family of enzymes called MMPs  and ADAMs that are implicated in many key steps of tumor development. We’ve found them in both blood and urine – but in urine, they really shouldn’t be there if you’re healthy,” says Moses.

“People argued that the appearance of MMPs in urine must be a mistake as they are high molecular weight proteins and the textbooks said they were above the filtration limit of the kidneys. But it turns out that wasn’t true, it’s just that previous methods weren’t sensitive enough to pick up proteins present in such tiny amounts,” she adds.

Improving quantification

However, although mass spectrometry is a good tool for protein identification, some mass spectrometry-based techniques are not as good at measuring their relative or absolute quantities in a sample – which is critical for clinical applications. But other types, such as isobaric tag for relative and absolute quantitation (iTRAQ) can help improve this capability.

“This technique enables us to analyze disease samples and healthy age-matched controls under the same conditions – so you can you get both identification and quantification at the same time,” explains Moses.

And the development of a powerful new strategy known as sequential window acquisition of all theoretical mass spectra (SWATH-MS) is enabling researchers to obtain consistent and quantitatively accurate proteomic data on a much larger scale.

“This is a game-changing, disruptive technology because it allows you to look at extremely large cohorts with relative quantification of hundreds or thousands of proteins,” enthuses Professor Tony Whetton, Director of the Stoller Biomarker Discovery Centre at the University of Manchester.

However, this creates a new challenge for researchers – generating vast quantities of data that requires high-end bioinformatics for its interpretation.

“It’s a Big Data problem, which is now completely tractable using methodologies that our bioinformaticians and others around the world have now developed,” says Whetton.

Validation is key

Due to the relative ease of discovery studies, there is an ever-increasing pipeline of potential new biomarkers. However, there is a large attrition rate, with many promising candidates not standing up to closer scrutiny in subsequent studies.

“I think this can happen for a variety of reasons, but we all need to do a better job at validating initial findings in large sample cohorts,” says Moses.

With this in mind, researchers developed a key step of guidelines – known by the acronym REMARK – which aim to improve consistency and ensure rigorous standards for reporting tumor biomarker studies.

“Most journals now require that you can document that you’ve followed these recommendations, which I think is a terrific step towards more rigid validation,” says Moses.

Inconsistencies with collecting clinical samples across different sites can also thwart success. As a result, a whole new field of biorepository science has emerged that focuses on developing tools and resources to guide research using biological specimens.

“I think everybody now realizes how critically important sample collection and storage is to successful biomarker discovery and validation,” says Moses.

Bridging the gap

With advances in technology, bioinformatics and improvements in study design and processes, everything is in now place to move more clinical biomarkers through development. And researchers are keen to highlight their success stories.

“By studying blood samples collected from a large prospective study into ovarian cancer, we demonstrated that a signature of proteins measured using mass spectrometry has the ability to predict the risk of disease,” says Whetton.

The team has recently published their encouraging results from the analysis of four proteins, and are now continuing to further refine the predictive nature of their risk algorithm.

“We’ve also undertaken a small discovery study, identifying a panel of proteins that look encouraging for detecting lung cancer earlier,” adds Whetton.

Others are enjoying similar successes with urine samples.

“We looked at a protein called ADAM12 in the urine of women with breast cancer, showing that it is present at the earliest stages of breast cancer establishment and that it increases with disease progression. Since then, we’ve validated this as an important non-invasive biomarker in thousands of urine samples, supporting its potential usefulness in diagnosis and prognosis,” says Moses.

“We went on to look at women with a higher risk of developing breast cancer and found that ADAM12 multiplexed with MMP9 was an excellent  risk predictor,” she adds.     

The power of combinations

There is also much anticipation around the potential of multiplexing proteins with other forms of biomarker – such as circulating tumor cells or tumor DNA in blood samples.

Earlier this year, one such study hit the headlines using a blood test called cancerSEEK that combines a panel of protein and circulating tumor DNA markers to enable the early detection of eight different cancers. And a number of other studies are also using this combination approach.

“I foresee that we will be monitoring people for early stages of cancer with more sophisticated and incisive tools in the future – based on this kind of work,” says Whetton.

Looking to the future

With the stage set to propel more biomarkers from the laboratory to the clinic, researchers are excited about the potential for developing a range of non-invasive tests with a variety of uses for cancer diagnosis and treatment.

“I think the field can achieve impact in monitoring wellness, response to therapy and diagnosis using samples are relatively easy to obtain,” says Whetton.

Due to their relatively low-cost and ease of collection, liquid biopsies also bring new opportunities for widening reach.

“We are also working to develop a smartphone application – which could make a huge difference, particularly in places with a lack of access to state-of-the-art technology,” says Moses.