Liquid Biopsy Sensitivity Dramatically Enhanced

The enormous diagnostic potential to dramatically change clinical medicine that is contained within the technique known as liquid biopsy has ignited a flurry of ideas and designs within the molecular diagnostic field. The ability to take a minimally invasive biological sample from a patient and diagnose or track disease could one day soon replace the need to surgically obtain tissue for study.

One conundrum that has plagued this novel technique during its development is how to minimize inherent DNA damage that occurs when, for instance, tumor DNA is captured from the blood and prepared for sequencing. Removing these errors from the sequencing results would allow researchers to more accurately identify true cancer-associated mutations from even minuscule amounts of starting material.

Now, researchers at the Stanford University School of Medicine believe they have devised a way to significantly increase the sensitivity of a technique to identify and sequence DNA from cancer cells circulating in a person's blood.

“We can detect even more sensitively the presence of specific mutations in the cancer DNA that could help drive treatment choices or detect the presence of residual cancer,” explained co-senior study author Maximilian Diehn, M.D., Ph.D., assistant professor of radiation oncology at Stanford University School of Medicine. “We're getting closer to greatly reducing the need for invasive biopsies to identify tumor mutations or track response to therapies.”

The findings from this study were published today in Nature Biotechnology through an article entitled “Integrated digital error suppression for improved detection of circulating tumor DNA.”

The Stanford researchers used a two-pronged approach that they dubbed integrated digital error suppression (iDES). This method builds upon a previously devised method, from the same researcher group—called CAPP-Seq—to capture tiny amounts of tumor DNA from the blood by looking for a panel of mutations known to be associated with a particular cancer. With CAPP-Seq, the researchers were able to detect as few as one tumor DNA molecule in a sea of over 5,000 standard DNA fragments.

IDES builds upon CAPP-Seq by addressing an inherent technical limitation: the inability to accurately sequence tiny quantities of DNA. Before sequencing can be attempted, PCR amplification is performed for the fragments, which introduces errors in each round of replication.

Moreover, the researchers needed a way to determine whether mutations identified during the sequencing process came from the tumor or were introduced during the sequencing process. They developed a way to tag circulating double-stranded DNA molecules in the blood with bar codes that uniquely mark each original molecule.

“Our technique is a significant advance over prior bar-coding methods because it eliminates more false positives without sacrificing true positives” noted co-senior study author Ash Alizadeh, M.D., Ph.D., assistant professor of oncology at Stanford University School of Medicine. “By tagging DNA molecules at the top of the food chain, so to speak, we can keep track of which molecules have been faithfully reproduced during the sequencing process and which have accumulated errors that were not present in a patient's tumor or bloodstream.”

The researchers combined the bar-coding approach with another approach they termed background polishing. “We discovered that certain sets of sequencing errors are much more likely to occur at specific places in our DNA molecules, even in healthy subjects,” remarked co-lead author Aaron Newman, Ph.D., instructor of medicine and oncology at Stanford University School of Medicine and designer of the computer algorithm to scan the data and flag possible trouble spots for further analysis. Together, the molecular bar-coding and polishing technique allowed them to filter out common sequencing mistakes far more efficiently than either method alone

The authors reported that using iDES increased CAPP-Seq's sensitivity for noninvasively identifying a tumor's mutations in the blood by approximately 15-fold.

“We found that our approach allows highly accurate, noninvasive identification of actionable mutations in lung cancer patients and we are hopeful that the technique will be clinically available soon,” Dr. Diehn stated , who noted that additional clinical studies will be needed to confirm whether iDES-enhanced CAPP-Seq can improve cancer patient outcomes or reduce healthcare costs.

“These same types of tools could be used to detect rare variants in DNA that could signal transplant rejection and antibiotic resistance or aid in prenatal diagnostic tests,” concluded Dr. Alizadeh.