Vicki Glaser Writer GEN

SISCAPA assays deliver an alternative to conventional immunoassays for protein quantitation.

Clinical proteomics applications rely on the translation of targeted protein quantitation technologies and methods to develop robust assays that can guide diagnostic, prognostic, and therapeutic decision-making. The development of a clinical proteomics-based test begins with the discovery of disease-relevant biomarkers, followed by validation of those biomarkers.

“In common practice, the discovery stage is performed on a MS-based platform for global unbiased sampling of the proteome, while biomarker qualification and clinical implementation generally involve the development of an antibody-based protocol, such as the commonly used enzyme linked ELISA assays,” state López et al. in Proteome Science (2012; 10: 35–45). “Although this process is potentially capable of delivering clinically important biomarkers, it is not the most efficient process as the latter is low-throughput, very costly, and time-consuming.”

“Proteomics is probably 10 years behind genomics in converting these technologies into true clinical tools,” says Paul Beresford, Ph.D., vice president of business development and strategic marketing at Biodesix. “There will be a lot of clinical content in the phenotype, and we’re just starting to tap into that.”

Odilo Mueller, senior director clinical markets at AB Sciex (a business unit of Danaher), observes that in the clinical space, the most common use of LC/MS-based solutions at present is by CLIA-certified labs performing laboratory-developed tests (LDTs).

The API 3200MD™ platform, including the 3200MD QTRAP® LC/MS/MS, is AB Sciex’ first system for use by clinical labs. The company launched three clinical assays this year in Europe—for measuring vitamin D, for monitoring immunosuppressants in the setting of transplantation, and for analyzing panels of amino acids and acylcarnitines for a newborn screening application. It also launched an additional LC/MS in vitro diagnostics (IVD) platform, the IVD MS Analyzer. The company plans to introduce six more tests later in the year.

“The LC-MS platform brings a lot of analytical performance advantages compared to immunoassays, such as better specificity and accuracy, larger detection range, and lower cost per sample,” says Mueller. “But you have to view those in the context of a specific disease or medical question.”

Moving to Mass Spec Platforms

Large hospital labs committed to performing MS-based LDTs typically acquire expertise in complex workflows such as LC-triple quadrupole mass spectrometry, notes Gary Kruppa, vice president of business development at Bruker. MALDI-MS “is a much easier technique” that a bench-level technician can readily learn, says Kruppa. “In the FDA-cleared version of the Biotyper software, you don’t see the mass spectra—there is no need for interpretation [of the data]—you see only the identifications.”

The key advantages of MALDI-MS compared to traditional biochemical tests or the gold-standard next-generation sequencing approaches for diagnosing bacterial infections, for example, are faster results (minutes versus several hours) and lower cost. Bruker developed a benchtop MALDI-TOF platform—the Bruker MALDI Biotyper, which was cleared for IVD use in 2013 by the U.S. FDA and obtained the IVD-CE mark in Europe in 2009, for use by clinical labs to identify infectious microorganisms based on their protein fingerprints.

“Now that we have an FDA-cleared MS device for use in clinical microbiology, we are working with several partners on approaches to develop protein quantitation assays on this platform,” says Kruppa. “We’re optimistic there are things we can do on our MALDI platform with protein quantitation assays applicable to clinical chemistry labs.”

Biodesix developed its clinically validated serum test VeriStrat® to guide treatment decisions in patients with advanced non-small cell lung cancer. The test detects isoforms of acute phase inflammatory proteins that correlate with survival outcomes. It yields a binary test result of VeriStrat Good or VeriStrat Poor that predicts outcome from treatment using the epidermal growth factor receptor inhibitor (EGFRI) erlotinib. Biodesix is currently validating the clinical utility of VeriStrat with additional therapeutics in lung and other solid tumors including colorectal cancer.

The company developed VeriStrat on a MALDI-TOF platform, which allows for a level of detection comparable to immunoassays and for multivariate testing. By measuring multiple analytes and comparing spectra between patients, “we could do true correlational science,” says Dr. Beresford. Biodesix based VeriStrat development on retrospective study samples and, more recently, completed a prospective trial that yielded positive results, demonstrating the test’s predictive value (Lancet 2014; in press).

Protein Biomarkers for Cancer Diagnostics

Al Luderer, CEO of Integrated Diagnostics (Indi®), describes the power of proteomics as the ability to deliver noninvasive, molecular diagnostic tests that can be used early in the process of a patient work-up—in cancer that means before and perhaps instead of a biopsy. According to Luderer, the main limitation in developing proteomic diagnostics based on validated protein biomarkers at present is the availability of well-annotated clinical repositories of plasma samples for the clinical indication being targeted.

“You need the clinical material to support discovery through validation,” notes Luderer. “You need to demonstrate the patient work-up path and to show medical utilization, as that is a key element for reimbursement.”

Indi’s first commercial proteomic test, Xpresys Lung, a blood-based diagnostic test, came on the market in October 2013. Designed to help clinicians assess lung nodules 8–30 mm in size identified on lung CT, the Xpresys Lung test determines the probability that a lung nodule is benign. A test result indicative of a high probability of being benign allows patients to enter a period of watchful waiting without the need for more invasive diagnostic studies or biopsy. Of the approximately 3 million people in the United States who present with a lung nodule on CT each year, about 200,000 will have lung cancer; the rest will have benign lung nodules.

Xpresys Lung is a multiplexed protein assay designed with a high negative predictive value to rule out cancer. To develop the test, Indi measured hundreds of proteins in blood samples from individuals with benign or cancerous lung nodules in prospective studies, looking for significant changes associated with malignant transformation. From these proteins, the company developed an assay consisting of a panel of 11 validated proteins (Science Translational Medicine 2013; 5: 207ra142). Indi developed the test on a multiple reaction monitoring (MRM)-MS platform, which is capable of detection in the sub-nanogram/milliliter range.

In addition to Indi, several companies are applying high-throughput analytical technology to translate protein biomarkers to commercial products. Gus Salem, president and CEO of SISCAPA Assay Technologies (SAT), predicts that “in the next 12 months, we will see another five or six companies have good clinically validated panels on the market in venture-backed CLIA models.”

“There has been no lack of success in proteomic discovery and the identification of protein biomarkers,” continues Salem. “But until now, proteomic discovery wasn’t followed up by the validation needed to translate those biomarkers to the clinic.” That required the analytical technology to do targeted protein quantitation and to compare measurements in large numbers of clinical samples.

Founded in 2011 by proteomics pioneer Leigh Anderson, chairman and CSO of SAT, the company uses Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to develop tools and methods for performing targeted, highly multiplexed protein measurements on a MRM-MS workflow. SAT’s goal is to validate protein biomarkers identified in the research arena and bring them forward into clinical applications.

The company offers an alternative to the traditional workflow of LC-MS, eliminating the need for chromatography through digestion of an entire protein sample up front, followed by the addition of an antibody that can specifically identify and fish out a peptide that serves as a surrogate for the target protein of interest in a disease indication. The result is higher throughput protein detection and quantification that allows for thousands of patient samples to be run in highly multiplexed assays in a day on a single mass spectrometer.

This approach, notes Salem, also overcomes the protein-protein interactions and antibody interference problems that have limited the ability to multiplex on and validate the individual components of traditional immunoassay platforms used to measure protein biomarkers. “In clinical proteomics there will most likely not be a single analyte that will be predictive of disease,” Salem adds. “We believe the high-value clinical answers will come from a panel of proteins.”

The MRM-MS technology and SISCAPA workflow used by the company for protein panel development is already validated and being used in the clinical setting. Earlier this year, Mayo Medical Laboratories began using SISCAPA technology combined with LC and tandem MS to measure serum thyroglobulin for monitoring patients treated for thyroid cancer to detect persistent or recurrent disease. In 20–30% of these patients, the presence of anti-thyroglobulin autoantibodies interferes with the standard immunoassay used in disease monitoring. SISCAPA-MS avoids the autoantibody problem by eliminating the interferences through digestion of the entire sample.

Evolving the Technology

Despite the challenges in converting a research-level technology such as MRM-MS to a commercial application, Indi was “able to track the same technology base from discovery all the way to commercialization,” asserts Luderer.

Waters' customers are primarily using its low-flow or nanoscale ACQUITY UPLC separations systems combined with the Ion Mobility MS platform for biomarker discovery, and the company is focusing on transitioning those technologies into the translational space with its newly introduced ionKey/MS platform, says Jeff Mazzeo, senior director of Waters' health sciences business operations unit.

Jim Langridge, director of health sciences research at Waters, describes several challenges in the ongoing transition from biomarker discovery to translational applications. One is the need for reproducibility of measurements across a large cohort of samples to ensure the clinical relevance of a protein biomarker intended for use in a clinical testing or screening setting. Another is sample prep and the lack of a gold standard for isolating proteins, digesting them into peptides, and doing the necessary quantification. Informatics and interpretation of the data is also an ongoing target of technology and methods development and optimization, and Waters is looking at ways to expand the strength in data visualization it gained with its acquisition of Nonlinear Dynamics into the translational space.

This article was originally published in the June 12 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this new digital publication, go to

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