Improved “Liquid Biopsy” Technique Enhances Tumor DNA Detection

An improved method has been devised to significantly increase the sensitivity of a technique to identify and sequence DNA from cancer cells circulating in a person's blood. The hope is that such "liquid biopsies" of easily obtained blood samples could one day replace the need to surgically obtain tumor tissue for examination.

The new approach works by identifying errors that occur when tumor DNA is captured from the blood and prepared for sequencing. Removing these errors from the sequencing results allows scientists to more accurately identify true cancer-associated mutations from even very small amounts of starting material.

Scientists at Stanford University School of Medicine (CA, USA) collected venous blood samples from cancer patients and healthy volunteers. DNA isolation, shearing of genomic DNA, preparation of precapture sequencing libraries, hybridization-based enrichment, and assessment of library quality and enrichment following hybridization were performed. Sequencing was performed using 2 × 100 or 2 × 150 paired-end reads with an eight-base indexing read on a MiSeq, NextSeq, or HiSeq 2000, 2500, or 4000 (Illumina, San Diego, CA, USA).

The team termed their new, two-pronged approach "integrated digital error suppression," or iDES. It builds upon a method called CAPP-Seq that was previously devised to capture very small 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 scientists were able to detect as few as one tumor DNA molecule in a sea of over 5,000 normal DNA fragments.

The investigators developed a way to tag circulating double-stranded DNA molecules in the blood with "bar codes" that uniquely mark each original molecule. Because the strands of an individual DNA molecule fit together like a zipper, it is possible to predict the sequence of one strand from the sequence of the other. The bar codes therefore allowed the team to match up the two strands and look for discrepancies. Additionally, their approach was designed to minimize the number of molecules that are lost during bar-coding and sample processing, which is particularly important when analyzing the tiny amounts of circulating DNA present in most cancer patients.

Using iDES increased CAPP-Seq's sensitivity for noninvasively identifying a tumor's mutations in the blood by about 15 times. Once telltale tumor-specific mutations have been identified, the augmented technique becomes even more precise, detecting as few as one or two tumor DNA sequences among as many as 400,000 non-tumor DNA fragments. The method enabled biopsy-free profiling of epidermal growth factor receptor (EGFR) kinase domain mutations with 92% sensitivity and greater than 99.99% specificity at the variant level, and with 90% sensitivity and 96% specificity at the patient level. In addition, the approach allowed monitoring of non-small-cell lung carcinoma (NSCLC) circulating tumor DNA (ctDNA) down to four in 105 cell-free DNA (cfDNA) molecules. The study was published on March 28, 2016, in Nature Biotechnology.

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Stanford University School of Medicine
Illumina