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Oct 15, 2011 (Vol. 31, No. 18)

PCR-Based Diagnostics Put to the Test

Scientists Combine, Enhance, and Tweak the Technique to Fit Desired Dx Applications

  • Melted DNA

    The temperature at which DNA melts can itself be used to determine the methylation patterns.

    Whole-genome methylation microarrays are often used to compare diseased tissue with normal controls, to find areas that are selectively methylated. Once found, the discovery needs be validated “just to confirm array results, because the technology for arrays is too complex to be conclusive,” said Tomasz Wojdacz, Ph.D., a post-doc at the University of Aarhus.

    To do this, Dr. Wojdacz employs a methylation-sensitive high-resolution melting (MS-HRM) protocol in which the melting profiles of bisulfite-treated, PCR-amplified samples are compared to those of both methylated and nonmethylated controls.

    Melting has long been used in methylation studies, he says, but the necessary sensitivity was lacking. Dr. Wojdacz and his colleagues worked for five years to improve this, and were able—in part by using a specific primer design—to bring the sensitivity down to a few cells.

    Their system combines PCR amplification with post-PCR analysis, yielding semiquantitative results that do not require special normalization and are simple to interpret. “It’s just one single PCR followed by melting in the same tube, and we have a precise result on a single gene.”

    In the 30 years or so that methylation biomarkers have been studied, “there’s still only a single test that is sort of clinically applicable at the moment,” Dr. Wojdacz said. He suggested that one issue is having certainty that the proposed markers can indeed distinguish disease from control, and having the technology to do so.

    Assaying thousands of samples and doing statistics on them is one thing, but making a clinical decision based on a single sample is very different. “We think that this technology—MS-HRM—is good enough to be diagnostically applicable.”

  • DNA, Hold the Cells

    Sometimes information from a malignancy is necessary for diagnosis and treatment, but for a host of reasons it’s not convenient or even possible to biopsy the tumor. In such cases researchers can try to identify and assay circulating tumor cells, or perhaps look for biomarkers found in circulating cell-free DNA (CF-DNA).

    Pamela Pinzani, Ph.D., and her colleagues at the University of Florence knew that the BRAFV600E mutation was an early event in most melanomas, and they wanted to find a way to exploit that to create an assay for diagnosis and prediction of response to therapy.

    To find the mutation, they needed to see a difference from wild type of a single base in a minute amount of DNA. Tumor DNA (i.e., BRAF-mutated DNA) typically accounts for around 1–10% total CF-DNA, which, in turn, is typically found in the order of nanograms per mL of plasma.

    A standard real-time PCR assay—the “technique of choice”—turned out not to be specific enough. So they tried to increase the specificity by incorporating locked nucleic acid bases into the mutation-specific probe in place of some normal DNA bases, conferring a higher avidity.

    Yet this, too, was “not sufficient by itself to increase specificity to the level we needed,” Dr. Pinzani recalled. They next turned to a primer that was specific for the mutation, increasing the avidity and efficiency of the amplification reaction of the mutated allele and simultaneously decreasing them for the wild-type sequence. The team was thus able to obtain a standard curve and use this to measure BRAFV600E in plasma samples.

    Most melanoma patients were found to have high levels of BRAFV600E mutation, with a statistical difference between the healthy individuals and melanoma patients. They are now trying to compare it with some other circulating biomarkers.

    “I suppose that more than one circulating biomarker is necessary to have 100% specificity in these patients,” she said. “Because there are some melanomas that do not show this variant, we’ll have to use a multiparameter approach.” Fortunately, the experience with BRAFV600E should provide an excellent place to begin looking for other melanoma-specific markers.

  • Seeking Diagnostics Approval

    Diagnostics are all about getting a reliable, consistent, narrowly focused answer to an equally narrow question, for example, should this individual patient receive therapy X or therapy Y? “From a research perspective you want to generate as much information as possible,” says Stephen Little, vp of personalized healthcare at Qiagen. “But when translating into diagnostics, you want to limit the information to only that which you need to make a treatment decision.”

    A regulatory submission for a diagnostic requires standardization, proof, and rigor. Therefore, when Qiagen sought FDA approval earlier this year for its real-time PCR-based companion diagnostic test to detect mutations in the KRAS gene—indicating whether a colorectal cancer patient was likely to respond to an EGFR inhibitor therapy—it needed to not only prove that its assay could very selectively pick up that mutation against a background of normal cells, but also that the whole system—including the extraction kit and the automated platform that would run the test—was up to the job.

    “The FDA wants to ensure that patients only receive tests that are guaranteed to be accurate and robust with validated manufacturing processes—they set stringent requirements and will only approve or clear diagnostics that can prove this,” Little notes.


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