This article is part of a series of stories related to new technology in cancer research. You can read more articles in the series here.
Whole-Genome Sequencing for Cancer Enters the Clinic
Today, a draft sequence of an entire human genome can be produced in about 10 days, according to Dr. Elaine Mardis, co-director and director of technology development at the Genome Institute.
In less than a decade, researchers have made a "total quantum leap" in technology, Dr. Mardis continued. This leap is bringing the concept of using whole-genome sequencing in everyday clinical decision making for cancer from science fiction-like speculation to reality, with the goals of providing treatment that is better tailored to individual patients and, eventually, improving their outcomes.
A Quantum Leap and Beyond
The Human Genome Project used a process called Sanger sequencing, which is highly labor intensive. In Sanger sequencing, double-stranded DNA is separated into single strands, or templates, and mixed with short, single-stranded pieces of DNA called "primers" that bind to complementary sequences on the templates. An enzyme called DNA polymerase and radioactively labeled versions of the four nucleotides of the genetic code—thymine (T), cytosine (C), guanine (G), or adenine (A)—are then used to extend the length of each bound primer one nucleotide at a time based on the sequence of the template. This process produces numerous labeled extensions of the primers that differ in length by a single nucleotide.
Next, an independent process called gel electrophoresis is used to separate the labeled DNA products on the basis of size. Products ending in T, C, G, or A are loaded into separate lanes on a gel, and shorter molecules travel farther in the gel than longer ones. The final order of the products can be used to reconstruct, or read, the sequence of the DNA template. (See the image to the right.) The use of fluorescent dyes later in the Human Genome Project simplified the gel process slightly but did not substantially shorten the time required to complete the sequencing of an entire genome.
Beginning in 2005, however, technology emerged that eliminated the need for the gel stage of sequencing. In this new technology, as nucleotides are added to the DNA sequence growing from the primer, a chemical reaction takes place that releases a specific frequency of light for each of the four nucleotides of the genetic code. These pulses of light are recorded by a machine, providing an immediate readout of the sequence. This process is sometimes called "next-generation sequencing," but Dr. Mardis prefers the term "massively parallel sequencing," which highlights the capabilities of the new technology.
"In massively parallel sequencing, the sequencing reaction and the detection of the sequencing reaction happen in lock-step," she explained. "This allows you to not only completely eliminate the gel-based separation but examine each of hundreds of thousands or millions of reactions at the same time."
The leap in power provided by massively parallel sequencing can be difficult to grasp. Dr. Cherilynn Shadding of the Genome Institute likes to use a geographical comparison. She tells visitors that it would take 20 Central Parks full of Sanger sequencers to provide the same output as Washington University's two rooms of next-generation sequencing machines.
From Proof-of-Concept to Real Clinical Questions
After the working draft sequence of the human genome was published in 2001, explained Dr. Richard Wilson, director of the Genome Institute, "we figured that we should be able to start applying the reference genome sequence and some of the technology that we had developed to look at regions of the genome that cause disease. We obviously had no shortage of diseases to pick from, but we tried to figure out where we could have the biggest impact, and we decided that was cancer."
In early collaborations with the Washington University School of Medicine and with Memorial Sloan-Kettering Cancer Center, Dr. Wilson and his colleagues launched pilot projects looking for mutations in candidate genes—but not the whole genome—in acute myeloid leukemia (AML) and non-small cell lung cancer. Although the lung cancer work provided some new information (including the fact that certain mutations in the EGFR gene make the cancer sensitive to the drugs erlotinib and gefitinib), "we didn't really discover anything new" when looking at familiar areas of the genome in AML, he recounted.
"In 2005, with some of these next-generation sequencing technologies coming out, we decided that the right way to look at cancer genetics was not just to make a list of our favorite genes and focus on those, but to sequence the whole genome, using both tumor and normal tissue from individual patients, and find all of the mutations," Dr. Wilson continued.
The Washington University researchers published findings from the first cancer genome to be sequenced––that of a patient with AML––in 2008. Follow-up from that study revealed that one of the new mutations discovered, in a gene called DNMT3A, may help identify patients with AML who are at high risk of recurrence. In 2009, the team sequenced its second whole genome from a patient with AML, and, in 2010, the first genome from a woman with inflammatory breast cancer was sequenced.
The cost per whole-genome sequence has dropped rapidly over the past 3 years. The 2008 AML study cost just over $1.5 million. Over half a million of that was devoted just to developing the bioinformatics required to compare the tumor with normal genomes, "because that had never been done before," said Dr. Mardis. Today, she said, the same sequencing costs only $10,000.
This reduction in cost combined with continuing improvements in speed have let researchers move from proof-of-concept studies to larger projects, asking questions about the impact of rare genetic mutations on response to treatment and outcomes. In their Cancer Genome Initiative, the Washington University researchers have sequenced the complete tumor and normal genomes of 150 patients.
The results from 50 of these patients, presented this year at the American Association for Cancer Research annual meeting, compared genetic alterations in women whose tumors responded to treatment with an aromatase inhibitor with those in women whose tumors were resistant to this treatment. The results are being analyzed for genetic clues that might identify resistance before treatment begins.
Another project, in collaboration with St. Jude Children's Research Hospital, is sequencing the genomes of at least 600 children with cancer in hopes of finding new genetic targets for therapy.
The Future Is Now
These studies will provide questions for testing in future clinical trials. An even more futuristic aspect of whole-genome sequencing for cancer is its potential to influence treatment decisions for individual patients—not decades in the future but today.
This May, the Washington University researchers published a case report of the use of whole-genome sequencing to guide the care of a woman with a rare subtype of AML that responds well to a specific targeted therapy, sparing her from more aggressive stem-cell transplantation. The disease subtype could only be identified definitively in her case through whole-genome sequencing.
The analysis, explained Dr. Mardis, proved that whole-genome sequencing can provide important information for clinical decision making at the same cost and in the same time frame as traditional pathologic and cytogenetic techniques that are used to diagnose AML.
"Cytogenetics and limited molecular tests are used now to provide prognostic information for AML patients, but the current tests don't allow us to precisely classify risk for all patients. Further, a complete panel of current tests can cost up to $10,000 per patient, and that cost will go up as new prognostic genes are discovered," said Dr. Timothy Ley, associate director of the Genome Institute.
To improve treatment planning, Dr. Ley and his colleagues are planning to sequence the whole genome of every patient with intermediate-risk AML seen at the Siteman Cancer Center, beginning at the end of this year. These patients are difficult to assign to either chemotherapy or more aggressive stem-cell transplantation based on traditional diagnostic techniques alone.
"More than 200 AML patients have now had their exomes [the portion of the genome that contains protein-coding genes] or whole genomes sequenced, and we will know a lot more about what mutations matter for outcomes by the end of the year," explained Dr. Ley. "All the patients we sequence will be followed to understand whether the decisions we make based on the sequencing data translate into improved survival."
A major issue in looking at the whole genome for cancer is that so many of the mutations discovered so far are not shared among many patients with a single type of cancer or lie in areas of the genome whose function is unknown.
Dr. Wilson thinks the genetic diversity of cancer uncovered so far "actually keeps us going as researchers rather than gets us down. It reminds us that this is not an easy disease that we've decided to work with," he commented. "If it was easy to understand, we wouldn't need whole-genome sequencing."