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March 15, 2017 (Vol. 37, No. 6)

Microanalyte MDx Goes Mainstream

Nucleic Acid Analytes Are Reliable Sources of Data for Diagnostic Calculations

  • Reference Standards

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    Circulating tumor nucleic acids present opportunities for noninvasive cancer management. Because these nucleic acids may be obtained safely and easily, they facilitate serial monitoring, which may encompass treatment follow-up and detect nascent resistance. [Memorial Sloan Kettering Cancer Center]

    Fundamental shifts in diagnostic technologies have also significantly influenced the development of reference standards that can accurately reflect the true nature of a sample, such as the complexities found in cancer.

    “We’re increasingly looking at many biomarkers in parallel,” says Brian Burke, Ph.D., director of business development, Horizon Discovery, Cambridge, U.K. “This approach helps us understand the heterogeneity of cancer. We’re starting to use multiple analytes—such as solid tumor material, circulating tumor cells, and circulating free DNA—to build a picture of what is going on in the patient.

    “Our reference standards are based on engineering a cancer mutation into a cell line and then having a matched normal cell line without the mutation. Through a combination of different cell lines, we are able recapitulate something that mimics the actual tumor mix with the corresponding ratios of normal genes to nucleotide polymorphisms and other types of genetic variations that drive cancer.”

    The reference standards can be used to validate biomarker assays. Also, these standards contribute to platform development, helping answer questions regarding sensitivity, reproducibility, and specificity.

    “As we move from solid tumor specimens to smaller amounts of cancer in liquid biopsies,” informs Dr. Burke, “we confront the challenge of looking for vanishingly small numbers of molecules in a very complex mixture of blood.”

    The development of highly sensitive and reliable detection methods is facilitated if suitable reference standards are available. “The ideal scenario,” Dr. Burke concludes, “is to develop a universal reference standard that can cover multiple analytes and accurately represent the full complexity of a disease state.”

  • Harmonizing Companion Diagnostics

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    Horizon Discovery provides genetically defined, human genomic reference standards that can help laboratories ensure the sensitivity, specificity, and accuracy of their assays and workflows. The company’s HDx products include cell-free DNA reference standards.

    The idea of a universal standard naturally lends itself to the concept of harmonization between various diagnostic tests. Companion diagnostics has predominately been developed with the view of one diagnostic test supporting one drug to determine therapeutic applicability for one patient.

    “When there was a limited number of drugs directed against the same target, harmonization was a relatively straightforward concept,” recalls Scott D. Patterson, Ph.D., vice president, biomarker sciences, Gilead Sciences. “It was, in any case, necessary to bring the idea of diagnostics supporting therapeutics into play.”

    “However, when multiple drugs are being developed at a similar but asynchronous time frame, each with its own diagnostic, harmonization is a huge challenge,” Dr. Patterson continues. “If all of those drug and diagnostic combinations gain approval, the result is multiple diagnostic tests for different drugs targeting the same biomarker.”

    One good example is PD-L1 expression testing for immuno-oncology applications.

    “There are multiple diagnostics and drugs available, some of which are companion diagnostics, and some of which are complementary diagnostics. So now we have multiple different tests for the same purpose,” Dr. Patterson adds. “You want competition, but you don’t want confusion.”

    One solution is to develop a diagnostic test that encompasses a broad panel of targets and enables simultaneous testing of a patient for different therapies. “In other words, the goal is to develop a testing panel that could support multiple drugs,” explains Dr. Patterson. “That is where the field is trying to go at the moment.”

    “We are always looking at the risk-benefit ratio for any therapeutic decision,” he stresses. “There are some biomarkers that are positive selection biomarkers, and there are others that are negative selection biomarkers. It would be wonderful if there were analytically validated panels that drug developers could use to harmonize different tests and identify patients who could benefit most from specific therapies.”

  • High-Throughput Molecular Clinical Research via LC/MS

    Hemoglobin profiling is an essential technique for a number of clinical research applications, including hemoglobinopathy workflows for the identification of genetic blood disorders such as sickle-cell disease. The genetic defects associated with these diseases result in the formation of abnormal globin chains in the hemoglobin molecule, caused by sequence truncations, omissions, and SNPs during protein translation.

    Blood profiling analysis must be capable of confident characterization and relative quantitation of both normal and abnormal hemoglobin variants.

    With over 1,000 forms of hemoglobin reported to date, traditional targeted research screening approaches are unable to comprehensively analyze all molecules of interest on a convenient timescale, according to Scott Peterman, Ph.D., marketing manager, mass spectrometry, biomarker research, Thermo Fisher Scientific.

    “Furthermore, with so many samples to screen—each one as individual and valuable as the next, it is critical that workflows are reliable, robust, and deliver accurate results each and every time,” adds Dr. Peterman.

    Research Study

    In a recent research study, a high-throughput hemoglobinopathy routine was developed to quantify normal alpha and beta human hemoglobin chains, alongside abnormal variants, in complex mixtures containing human and bovine hemoglobin. The Thermo Scientific Vanquish* Ultra High Performance Liquid Chromatography (UHPLC) system was used to deliver “superior,” in the words of Dr. Peterman, separation power, enabling confident global protein profiling regardless of sample complexity.

    “When used in conjunction with the advanced resolution and dynamic range of the Thermo Scientific Q Exactive Focus* mass spectrometer, the method demonstrated exceptional reproducibility for fast and reliable large-scale analysis,” he continues.

    Automated high-throughput workflows capable of handling large numbers of complex samples is essential. The latest advances in UHPLC and mass spectrometer technology are helping researchers understand how they might analyze complex biological samples more rapidly, robustly, and with greater confidence in their results, says Dr. Peterman.


    * For Research Use Only. Not for use in diagnostic procedures.

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