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Mar 1, 2010 (Vol. 30, No. 5)

Targeted Resequencing Improves Precision

Ability to Focus on Specific Regions of Interest Pushes Technology to the Next Level

  • Healthcare Costs

    Hanlee Ji, M.D., assistant professor of oncology at Stanford University, aims to lower the cost of diagnostics using his group’s resequencing strategy that employs powerful DNA sequencing approaches. The Stanford group is using its skills to design strategies to identify mutations in cancer and, ultimately, put the approach into place for prospective clinical trials.

    Clinical genetics and oncology are the principle areas in which targeted resequencing can lead to improved patient management. Thus it is hardly surprising that Nimblegen, a division of Roche, has focused its efforts on first wave applications of exome resequencing. Using its array-based platforms, the company has used capture oligonucleotide probes to query the entire complement of human exons for the purpose of disease gene discovery. But these array-based technologies are difficult to scale.

    Dr. Ji cites example of mutation testing in colorectal cancer, from the point of view of the gastroenterologist.  “I see a lot of GI cancers including many cases of colon cancer. We have found that K-ras mutation testing is predictive of patient response to specific therapeutics, but to reach the level of validation required for an acceptable diagnostic test one needs a large study, beyond the reach of labor-intensive array screening.”

    In the case of complex disorders with a polygenic determination, one must look at even more individuals in order to build a valid collection of data. Array-based assays are not scalable so the Stanford team is focused on in–solution approaches  employing an aqueous reaction mixture with oligonucleotides in a single tube. Dr. Ji explains that it is practical to manipulate tubes in large-scale studies using 96-well plates, allowing analysis of thousands of patients.

    As an example of the current dynamics of personalized medicine costs, Dr. Ji considers the test for the BRCA1 mutation, which predisposes to a risk of early-onset breast cancer. The cost of the BRCA test is over $2,000 per patient. Dr. Ji believes that his approach could eventually, drastically reduce the costs of this test to around $20.

    “With next-generation sequencing costs plummeting, we can foresee a time when we can screen 100 cancer-related genes for a few hundred dollars. We could analyze a patient’s tumor for less than $100, and in this way, patients and physicians could avoid futile treatments. So we have a win-win situation for everyone, including the insurance providers, since they wouldn’t waste money on treatments that provide no benefit.”

    A number of investigators from large genome centers are attempting to sequence the whole genome of an individual patient or tumor tissue in order to determine genetic profiles. Although genome sequencing costs have dropped precipitously in the last year, they are still too high, at around $10,000, according to Dr. Ji. This is too much for a routine tumor genome screening, so it is essential that the costs be driven down more. He hopes to develop  protocols that could be done in any laboratory in the world.

    Dr. Ji emphasizes that new DNA-based diagnostic tests can be moved rapidly into clinical use. “It’s astounding how fast the field is moving. We are planning for the day when any patient could come into an HMO and provide a family history, and then be tested for susceptibility to specific cancers, such as breast or colon cancer. That would prove to be enormously beneficial, as we identify individuals at high risk of cancer much earlier, which ultimately could be a lifesaver.”

    Jurgen Vanhauwe, Ph.D., senior manager for sequencing market development at Applied Biosystems, part of Life Technologies, will talk about the SOLiD™ platform at the meeting. “This is a highly accurate, massively parallel sequencing platform that delivers a greater than 99.94% base identification accuracy.”

    Accurate base reading is absolutely critical for the success of targeted resequencing strategies. If readings cannot be verified, than errors could be classified as mutations or SNPs, thus rendering the data useless.  The SOLiD platform uses a sequencing methodology based on sequential ligation of dye-labeled oligonucleotide probes whereby each probe assays two base positions at a time (four fluorescent dyes encode 16 possible two-base combinations). This approach, in combination with the nature of the color code, will lead to a sequence of overlapping dimers that allows for error correction.

    The SOLiD platform enables massive sequencing projects, heretofore impossible, Dr. Vanhauwe says. Dr. Peter J. Campbell and his colleagues at the Wellcome Trust and other institutes recently sequenced a small-cell lung cancer cell line, NCI-H209, exploring the mutations within 134 coding exons. They identified a tandem duplication in the gene referred to as CHD7, indicating that this gene may be critical to the transformation process. If it is determined that this alteration is an essential feature of the disease, this would represent an important landmark in our understanding of the mutations brought about by tobacco carcinogens and would point the way to therapeutic agents that could block the effect of these mutations.

    “The exciting feature of this technology is its throughput and accuracy,” says Dr. Vanhauwe. “We could do whole genome sequencing the old way if we had 50 next-generation sequencing machines running in parallel, but with targeting resequencing we can focus the SOLiD platform on the regions of interest. We’re now able to sequence 100 patients in a single run.”

    Instead of a huge, uncharted genetic wilderness, it now possible to focus on critical exons and portions of the genome that determine susceptibility to cardiovascular insult, malignancy, and autoimmune disfunction. But this is only the beginning; within a short time (perhaps months) it should be possible to screen a patient for a large assortment of genetic disorders at a manageable cost.


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