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MD Anderson Study Pilots Whole-Genome, Exome Sequencing of Exosomal DNA


NEW YORK (GenomeWeb) – In a new study, researchers from MD Anderson have demonstrated the possibility of sequencing whole genomes and exomes from the DNA contained in extracellular vesicles, or exosomes.

The study, which appeared online ahead of print in Annals of Oncology late last week, is a proof of principle for future high-resolution sequencing to identify clinically relevant cancer mutations in blood or other body fluids using these particles, which had previously been investigated mostly for their RNA content, the authors said.

The discovery that exosomes contain high molecular weight, double-stranded DNA is recent. A different research team at MD Anderson, led by Raghu Kalluri, published the first discovery in the Journal of Biological Chemistry in January 2014.  Kalluri has since gone on to start a commercial company called Codiak Biosciences to develop exosome-based products.

Another study, published in Cell Research  by a team at Weill Cornell Medical College in April 2014, showed for the first time that the majority of DNA associated with tumor exosomes is double stranded.

Hector Alvarez, an MD Anderson pathology researcher and the co-senior author of the new Annals of Oncology study, told GenomeWeb that he and his colleagues had independently discovered the presence of high molecular weight DNA in exosomes, about the same time as Kalluri's group.

Based on this first encouraging sign, Alvarez and his colleagues then moved on to try to show that they could conduct high resolution genomic analysis of the DNA and RNA in the exosomal compartment. This was the backbone for their new publication, as well as the basis for a new liquid biopsy program recruiting patients at MD Anderson for exosome-based cancer mutation detection, Alvarez said.

While targeted sequencing is being rapidly advanced as a liquid biopsy approach in the context of cell-free DNA, the degraded nature of DNA particles in the blood makes sequencing at whole-genome resolution difficult.

RNA fares even worse when exposed outside of a cell or other vesicle in circulation, so comparison of matched DNA and RNA sequences in the context of cfDNA is even more problematic, Alvarez said. Exosomes offer a potential solution to these problems because they appear to protect nucleic acids from degradation by DNAases and RNAses in circulation.

As with cfDNA-based liquid biopsy, the presumption is that exosomes may also represent a more complete picture of the cancer genome than tumor tissue biopsy, where heterogeneity makes samples inherently impure.

In their new study, Alvarez and his co-investigators described their results isolating exosomes from three patients with pancreaticobiliary cancers — two pancreatic and one ampullary — and sequencing the DNA and RNA extracted from these vesicles using whole-genome, whole-exome, and transcriptome sequencing protocols on the Illumina HiSeq 2500.

The three patients were part of a larger effort at MD Anderson called the "Moon Shots Program," aimed at rapidly converting scientific discoveries into life-saving advances for patients.

To isolate exosomes from these patients' blood and pleural effusion, the team used an ultracentrifugation approach, rather than a commercial exosome extraction kit. Alvarez explained that in initial unpublished experiments, the team compared a variety of approaches, but that gold standard serial gradient ultracentrifugation gave the cleanest population of extracellular vesicles.

"In our hands, on a face to face comparison with the same input plasma, the quality of information we got from commercial kits was not even close," he said.

Excitingly, Alvarez said, the exosome approach appeared to result in the availability of a very high proportion of tumor to normal DNA. After extracting high molecular weight DNA, the researchers estimated that the tumor fractions ranged from 56 percent to 82 percent.

"The signal is so pure and so enriched … even more than you would expect from regular tissue because when you take a tissue biopsy you always deal with a heterogeneous population of cells since most of the tissue is stroma," Alvarez said. "So we were surprised by the purity of tumor signal even up to 80 percent purity."

When they sequenced the samples, the researchers found that whole exome sequencing resulted in a mean coverage depth of 133x to 490x for about 95 percent to 99 percent of target regions. The team was able to cover up to 91 percent of the human genome with whole-genome sequencing.

The researchers also identified multiple cancer-associated markers, including copy number alterations, point mutations, insertions, deletions, gene fusions, and even some potentially actionable alterations, such as in NOTCH1 and BRCA2.

Moving forward, Alvarez said he and his colleagues have several new goals. First, they've recruited many additional patients at MD Anderson for exosome-based liquid biopsy analysis, as a first step toward collecting enough evidence to reveal whether the approach has clinical utility in monitoring disease, or potentially guiding treatment decisions. The group hopes to publish results in the near future comparing its exosome-based approach with ctDNA-based liquid biopsy using a lower-resolution digital sequencing approach in about 80 patients.

"We want to estimate the amount of molecules that travel in each of these compartments, the cell-free DNA [versus] exosomes, with the hypothesis that the biogenesis of these DNA molecules is different — for example we think that cell-free DNA is probably coming from cancer cells that die, while exosomal DNA may be coming from cells that continually proliferate," Alvarez said. "It is something we are learning as we go, how these two [samples] differentiate and how the different type of information may help manage a patient in different ways."

The team is also collecting high-resolution sequencing data for patients in a serial manner to attempt to show that this type of investigation can reveal important changes in a patient's disease over time, he added.