Anyone who has watched family members or friends battle cancer hopes for a technological advance that will help better diagnose and treat this disease. Two things top almost everyone’s wish list: Super sensitive tools for early detection and precise methods to select the best treatment. Circulating tumor DNA (ctDNA) could have a key role in both advances.
“Tumor genomes contain multiple somatic mutations, which when accurately characterized can provide the biomarkers and other information required to understand and potentially treat an individual patient’s cancer,” says Geoff Otto, vice president, clinical product development and strategy at Foundation Medicine. “Analyzing tumor DNA typically requires a tumor biopsy, which is invasive and potentially risky.” In some advanced cancer patients, he notes, a tumor biopsy may not be possible at all.
Nonetheless, tumor cells release small fragments of DNA into the bloodstream, and this is ctDNA. “This means that the genomes of tumors for some cancers can be analyzed using only a blood draw, providing a noninvasive way to identify the clinically relevant somatic mutations and thereby help match a patient to the most appropriate therapy based only upon a liquid biopsy,” Otto explains. “It can also help identify other clinically important genomic variants including resistance mutations to help guide treatment including matching a patient to a relevant clinical trial.”
Beyond the simplicity of a liquid biopsy, it may also provide more qualitative information than other techniques. “Typically, a tissue sample represents only a fraction of a tumor and does not represent the whole tumor, nor does it represent metastatic sites,” says Viresh Patel, marketing director for the digital biology group at Bio-Rad Laboratories. “ctDNA, on the other hand, includes DNA from all tumor cells, including potentially unknown metastases.”
Beyond the simplicity of a liquid biopsy, it may also provide more qualitative information than other techniques.
To achieve the benefits of analyzing ctDNA, scientists need specific tools to analyze it, and some already exist, but they’re not foolproof. “While ctDNA can be detected fairly reliably, a negative result does always mean that the DNA was not there and careful bioinformatics are needed to minimize false positives,” says Hatim Husain, assistant clinical professor at the University of California, San Diego. “Volume of the sample, location of the tumor, and the type of mutation may influence how easy or hard they are to detect.”
Searching the samples
In Husain’s lab, research provides promise. “We have been able to identify cancer-specific DNA in different biospecimens including blood, urine, pleural effusions, and ascites,” he says. In fact, he and his team recently reported that recent improvements in the sensitivity, specificity, and feasibility of ctDNA detection assays allow for the possibility for implementation into clinical practice and appropriate studies are underway.
Next Gen Sequencers
Find, compare and review next gen
sequencers from different suppliers Search
In one study of patients with various types of cancer, Husain and his colleagues applied next-generation sequencing to their plasma, and the researchers found alterations in ctDNA in more than half of the patients. Also, these results correlated with comparative analysis of tissue samples. This suggests that current techniques for liquid biopsies in some cases could be as conclusive as testing the tumor itself.
This research team has even shown that liquid biopsies of ctDNA can reveal specific alterations in genes that indicate that a specific drug might be applicable for some patients. In combination, this research indicates that testing ctDNA might address two of the biggest challenges in fighting cancer: early detection and picking the best treatment.
Technical tools
Scientists at Foundation Medicine developed FoundationACT, which is a liquid biopsy test. “FoundationACT can detect genomic mutations present in ctDNA even at low purities, down to 0.1% mutant allele frequency,” Otto says. “In addition, FoundationACT has demonstrated greater than 99% sensitivity and greater than 99% positive predictive value for base substitutions and insertions/deletions, as well as greater than 99% sensitivity and 98% positive predictive value for rearrangements and fusions.”
Like all analytical tests, FoundationACT depends on sample preparation. Otto points out that enhanced extraction methodology produces a high quantity of high-quality ctDNA. “Proprietary technology then uses synthetic DNA barcodes to accurately identify and isolate unique ctDNA fragments from plasma,” Otto explains. “Hybrid capture-based next-generation sequencing is then combined with proprietary computational algorithms that enable specific and sensitive calling of mutations.”
So far, Otto and his colleagues have validated FoundationACT to study 62 cancer-related genes, and shown that it’s effective for breast, gastrointestinal, lung, and prostate cancer. “Importantly, FoundationACT can provide information to guide therapy and identify resistance mutations,” Otto adds. “In one case, a non-small cell lung cancer patient began responding to a specific targeted therapy after FoundationACT identified a ROS1 fusion, which was previously missed by a different ctDNA assay.”
In another example, FoundationACT revealed a mutation that suggested a targeted treatment. When the patient’s disease, unfortunately, progressed, the assay revealed what Otto describes as “a novel resistance mutation, which helped to explain disease progression.” So, this tool can diagnose cancer, indicate targeted treatment, and help scientists learn more about how cancer mutates in ways that make some drugs fail. The latter will prove crucial in finding ways to keep battling cancer as it adapts to drugs and works around them.
Diagnosing from droplets
Although many scientists explore ctDNA with next-generation sequencing, other tools can also be used. For example, PCR costs less, provides faster results, and requires less overhead and expertise. To provide extremely sensitive results, Droplet Digital PCR (ddPCR) from Bio-Rad Laboratories divides a sample into thousands (or millions) of droplets. As Patel explains, “Next-generation sequencing can be used for primary diagnosis or early-stage detection of biomarkers, and once the biomarker is identified, ddPCR can be used more routinely to monitor the disease.” In addition, this technology can detect just a few target molecules out of the more than 100,000 in a sample.
Ultimately, the technology selected depends on many factors. “Cancer type and stage, urgency for needed information, what is already known and what can be usefully learned about a specific patient’s disease are important factors to consider when selecting either next-generation sequencing or ddPCR,” Patel says.
One thing is certain, the analysis of ctDNA promises better and faster diagnosis of cancer, plus better information about the best treatment for a specific cancer. We keep hearing about personalized medicine, and ctDNA could take us there—providing a particular patient with the precise treatment that is needed. Then, maybe we won’t need to watch our loved ones die from this awful illness.
Image: Shutterstock Images