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Results from First AML Genome Indicate More Data Needed to Provide Clinical Use

The first fully sequenced tumor genome, published in Nature last week, revealed a small number of mutations in coding regions that likely played a role in causing the cancer, and that could not have been uncovered using other approaches.
But the remainder of the genome could harbor hundreds of other relevant mutations, and researchers and cancer experts believe a lot more tumor genomes must be sequenced in order to understand their effect, and before doctors will be able to use the information to make clinical decisions.
“It’s a remarkable achievement, but clearly, it’s just the tip of the iceberg,” said John McPherson, director of cancer genomics at the Ontario Institute for Cancer Research, who was not involved in the study.
For the project, researchers at Washington University in St. Louis sequenced the genome of leukemia cells and from normal skin cells from an acute myelogenous leukemia patient. Using the Illumina Genome Analyzer, they obtained almost 33-fold coverage for the tumor genome, and 14-fold coverage for the skin genome. The total cost of the project was approximately $700,000 (see In Sequence 10/14/2008). 
After analyzing just the gene-coding portions of the DNA, which makes up just 1 to 2 percent of the total, they found mutations in 10 genes that were specific to the tumor, of which two were already known to contribute to cancer progression and eight had not been linked to AML before. Of these, four were in gene families that are “strongly associated with cancer pathogenesis” and four in genes that have not been associated with cancer before but that function in metabolic pathways that “could act to promote cancer,” according to the authors.
“At first glance, it seems like, ‘Well, that’s a lot of sequencing to find 10 genes,’” said McPherson. But, he stressed, many of these mutations could not have been discovered by resequencing candidate genes that were suspected to play a role in cancer. Also, the number of mutations may actually be higher than 10, because the researchers used very stringent methods in their analysis and might have discarded some actual mutations.
In addition, the number of mutations in the entire tumor genome is likely higher. The scientists estimate that another 500 to 1,000 somatic mutations may be harbored in the remainder of the cancer genome, which they did not analyze. These mutations “will provide a rich source of potentially relevant sequence changes that will be better understood as more cancer genomes are sequenced,” they write.
They also mention that the short unpaired Illumina sequence reads might have missed a number of mutations, such as cryptic translocations. Paired-end reads can be used to survey these and other structural variants in tumor genomes, they note, but paired-end sequencing methods “were not available when we initiated sequencing of this subject’s genome.”

“The most important thing that will happen in the clinical arena in the next few years will be deciding … which of these mutations are relevant to decision-making.”

In any case, sequencing additional AML genomes will be required to interpret the results. “This is a single, isolate genome, and we need to do more” to make sense of the mutations, McPherson said. “It will be interesting to see whether they find the same genes or if they find other genes and identify pathways that are involved,” he said.
The scientists estimate in their paper that “hundreds or perhaps thousands” of additional tumor/normal genome pairs have to be sequenced to put those mutations in a larger context and assess their biological and clinical relevance.
As previously reported, the Wash U researchers have already started sequencing a second AML genome with paired-end reads on the Illumina platform at a predicted total cost of approximately $200,000.
Next year, they plan to sequence five more AML genomes and begin whole-genome sequencing projects for different cancer types, including glioblastoma, lung cancer, and breast cancer.
A cost of $200,000 per genome is still too much for many teams, such as McPherson’s, who would like to sequence 500 pancreatic cancers and their controls. For now, he and his team are sequencing selected parts of the genome and transcriptomes, although he said he is considering entirely sequencing “a couple” of selected samples.
But as the cost of sequencing keeps falling, it may become cheap enough to enable large-scale tumor sequencing projects within a year or two, according to McPherson.
“To extract as much information as [the Wash U team] did, I would want to see it in the $30,000-or-less range before I’d embark on very large scale,” he said. “And that’s not that far off; I really think it’s on the horizon.”
Clinical Applications
Several experts agree that Wash U’s AML genome generated little data that could be applied in the clinic immediately. Rather, the study shows that whole-genome tumor sequencing is now possible in principle.
“I think that’s the main point of the paper,” McPherson said. “It’s less of ‘Here is the underlying cause of leukemia,’ but more of just ‘Here is what you get when you do a whole genome sequence of a tumor.”
Interestingly, when the researchers sequenced the eight newly discovered mutated genes in 187 additional AML tumor samples, they found that none of them harbored any mutations in those genes.
Some researchers find this result disheartening for possible clinical applications. “I think it’s very discouraging for a drug company at the moment,” said Larry Loeb, a professor of biochemistry and pathology at the University of Washington in Seattle and an expert in cancer mutations.
“To tell you what to target, this is not the data you want to end up with,” he said. “What you would have liked is to have these eight [mutated genes] being present in the other people. And without that, you don’t have a common denominator of which way to go.”
Also, because the researchers studied clonal mutations rather than random mutations in single tumor cells, “we won’t know anything about the emergence of drug resistance from their data,” according to Loeb. “For that, you would virtually have to sequence single molecules, and we don’t have that technology yet.”
On the upside, the data could help determine whether a patient’s tumor is already resistant to a drug, he said.
The mutations found in Wash U’s AML patient would likely not have changed her treatment today, according to Ross Levine, a physician in the leukemia service and human oncology and pathogenesis program at Memorial Sloan-Kettering Cancer Center, who was not involved in the study.
But additional tumor-sequencing studies of AML samples could show whether a subset of the mutations may be relevant for clinical care, he said. “The most important thing that will happen in the clinical arena in the next few years will be deciding, using existing treatment and treatment algorithms, which of these mutations are relevant to decision-making. And then, long term, we need to use the insights about the new mutations to design new treatments.”
Even rare mutations could be useful, he said, as long as they fall into clusters of genes with similar biological function. “So even if patients don’t have the same mutations, if they have a certain type of mutation, that predicts something clinically relevant,” Levine said. “And we just can’t, until we see more data, make predictions as to whether that is the way the data falls out.”
Whether or not whole-genome sequencing in the future will become a routine method for characterizing tumors from cancer patients will hinge on both its cost and its usefulness, according to Levine.
If a large study of leukemia discovers, for example, that only a relatively small number of genes are mutated in the disease, a targeted test for mutations in these genes could be designed. However, if it turns out that any of several thousand genes can be affected in AML, “then we might as well do the whole genome,” he added.
As for the cost of whole-genome sequencing, Levine said it must not exceed a few thousand dollars since tumor sequencing will not obviate any of the current diagnostic tests. “It’s going to have to be something that can be integrated into existing clinical care, but not at the expense of not being able to do other things,” he said.

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