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Proteomics Notches Significant Successes in 2013, but Clinical Potential Still Largely Unfulfilled

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2013 proved an eventful year for proteomics as the field saw milestones ranging from the release of long-awaited clinical tests to key regulatory clearances to noteworthy new instrument launches.

In particular, the move of mass spec into the clinic continued to gain steam, as both diagnostic firms and tool vendors notched major wins in pushing clinical products to market.

Yet, despite these achievements, a number of established proteomics firms – particularly on the diagnostics side of the business – continued to struggle. Indeed, while clinical proteomic efforts have grown increasingly sophisticated and rigorous, commercial success has largely eluded the field. The developments of the last year hold significant potential, but, for the moment, it remains largely just that.

One notable exception to that judgment is the use of proteomics in clinical microbiology where MALDI mass spectrometry has come to offer dramatic savings in time and money compared to conventional pathogen detection techniques. In 2013, both Bruker's MALDI Biotyper and BioMérieux's Vitek MS systems – the two market leaders – received US Food and Drug Administration 510(k) clearance, making them available for US clinical use.

The platforms identify microbes by matching the protein profiles of sample organisms generated via MALDI mass spec to profiles contained in a proprietary database. Compared to traditional biochemical methods of microbe detection, MALDI-based systems can offer significant improvements in speed, price, and accuracy.

And while the benefits of clinical proteomics are to a large extent still theoretical, in the case of the MALDI microbiology devices, researchers have begun generating real-world data bolstering the case for their utility.

For instance, in a study published in December 2012 in the Archives of Pathology & Laboratory Medicine, clinicians at Houston's Methodist Hospital found that using Bruker's Biotyper as part of that hospital's antibiotic stewardship program reduced average patient stays by 2.6 days and average hospitalization costs from $45,709 to $26,162 per patient.

Even leaving aside such reductions in expenses associated with patient care, MALDI-based devices could save hospital microbiology labs hundreds of thousands of dollars per year in reagent and personnel costs. In an interview last month with ProteoMonitor, Nathan Ledeboer, medical director for the clinical microbiology and molecular diagnostics laboratories at Milwaukee's Froedtert Hospital, estimated that by moving from biochemical-based identification to MALDI, his lab would save an average of $4 to $5 per test. Given Froedtert's test volumes, that amounts to annual savings of around $300,000.

LC-MS milestones

Of course, from a clinical standpoint, detection of protein biomarkers via LC-MS has traditionally been the field's primary focus, and here, instrument vendors notched a variety of regulatory clearances, as well.

In February, AB Sciex released its 3200MD and 3200MD QTRAP instruments as FDA Class I exempt medical devices, making them available for clinical use. In May, these devices received the EU CE-IVD mark, allowing them to be sold in the EU for general in vitro diagnostic use including in hospitals and clinical diagnostic laboratories.

In April, Thermo Fisher Scientific obtained ISO 13485 certification for two of its LC-MS production facilities, setting the stage for the platforms to potentially receive CE-IVD device registration in Europe and Class I medical device listing in the US. In June, Ian Jardine, Thermo Fisher's vice president of global R&D, told ProteoMonitor that the company also planned in 2013 to register its TSQ Endura triple quad as a Class I exempt medical device; however, the company has not yet done so. Jardine added that the company would likely in the future register some of its Orbitrap instruments as Class I medical devices, noting that the Q Exactive and Exactive machines were the likeliest candidates.

The diagnostics side of the proteomics business also made headway in moving LC-MS-based proteomics into the clinic. Notably, Applied Proteomics announced in April that it would be using multiple-reaction mass spec for its clinical proteomics tests, a switch from its original plan to commercialize them on an immunoassay platform. The move was a sign of API's growing confidence in mass spec-based clinical proteomics, a trend that is increasingly visible across the field.

Triple quad-based MRM assays have long been viewed as a likely format for protein biomarker diagnostics due to their high accuracy and multiplexing ability, but implementing them clinically has proven challenging due to issues including precision, throughput, and sensitivity. Recent advances in sample prep automation and reproducibility, target enrichment workflows, chromatography speed, and mass spec performance, however, have pushed the technology closer to clinical feasibility.

In addition to API's shift in direction, Sera Prognostics in August completed enrollment of 5,500 women in its Proteomic Assessment of Preterm Risk clinical study supporting the validation of its PreTRM proteomic test for predicting risk of preterm birth. The company plans to analyze samples from these 5,500 women using MRM mass spec, making it one of the largest protein biomarker clinical validation studies ever undertaken using such a platform.

Most significantly, in October, Integrated Diagnostics launched out of its CLIA lab its Xpresys Lung test, the first multiplexed proteomic test to go to market using MRM-MS on a triple quadrupole instrument. Intended to aid doctors in identifying lung nodules detected via CT scans as likely benign and thereby avoid unnecessary additional screening or procedures, Xpresys uses MRM-MS to quantify the levels of 11 proteins in patient blood samples.

Running on an Agilent 6490 triple quad instrument linked to standard flow HPLC, the test quantifies proteins at concentrations as low as the low nanogram-per-mL range and with coefficients of variation of typically less than 5 percent. Capable of processing around 1,000 samples per month using between four and six mass spec instruments, the assay is still somewhat low-throughput, but nonetheless represents a major step forward for clinical mass spec.

Commercial conundrum

Whether this technical achievement will translate into commercial success, however, remains to be seen. In an interview with ProteoMonitor upon launch of the Xpresys test, Howard West, a thoracic oncologist at Seattle's Swedish Cancer Institute, noted that additional studies would be needed to determine if the test did, in fact, help patients avoid unnecessary procedures following screening for lung nodules.

The test "clearly needs to be validated in a broader setting prospectively to clarify if it leads to less anxiety and fewer unnecessary invasive studies and imaging studies," West said, adding that, currently, "I have zero confidence that that is actually what would happen in practice."

West's comments point toward a major challenge that has faced proteomic diagnostics both in 2013 and years past. While a number of firms have managed to bring products to market, few, if any, have managed to drive enough physician adoption to turn a profit.

The last year saw continued difficulties for companies including Vermillion, BG Medicine, and Healthlinx. The latter, which has attempted unsuccessfully for several years to bring its OvPlex ovarian cancer test to the US market, filed for bankruptcy in May.

BG Medicine, meanwhile, has struggled to maintain its listing on the Nasdaq Global Market. Currently, it is contending with three separate warnings from the exchange due to failure to maintain a minimum $1 closing bid price on its stock; at least $50 million in market value of its listed securities; and the minimum $15 million threshold value of its publicly held shares.

The company did, however, receive a measure of good news last month in that the Centers for Medicare and Medicaid Services set the 2014 Medicare national limitation amount for its galectin-3 test at $30.01, an increase from $17.80 in 2013.

Vermillion last year named a new CEO, Thomas McLain, replacing interim CEO Bruce Huebner, who took over for Gail Page in November 2012. The company also adopted a new sales approach for its OVA1 ovarian cancer diagnostic, seeking to terminate its license agreement with Quest Diagnostics and take more direct control of its commercialization efforts.

Nonetheless, OVA1 sales remained roughly flat with 2012, in the range of 4,000 to 4,300 per quarter.

Discovery highlights

Moving from the clinic to the discovery space, proteomics made steady progress both in terms of adding capabilities and expanding datasets. Among the year's notable achievements was a study by University of Wisconsin-Madison researcher Josh Coon, in which he and his colleagues profiled the entire yeast proteome in one hour.

Identifying around 4,000 proteins, the effort represented a roughly four-fold increase in speed compared to the field's previous best efforts and, Coon noted, offered the prospect of potentially completing comprehensive human proteome analyses in as a few as three to four hours.

The UW-Madison team performed the analysis, which was published in Molecular & Cellular Proteomics, on Thermo Fisher's new Orbitrap Fusion instrument, which the company released last year at the American Society for Mass Spectrometry annual meeting in June. Combining in one device a quadrupole for precursor selection with both an Orbitrap and ion trap mass analyzer, the instrument's unique architecture along with its high resolution and fast scan rates had early users including Coon and Harvard University researcher Steven Gygi predicting that it would enable significant improvements in proteome coverage. The MCP paper offered one of the first published demonstrations of its capabilities.

In addition to new instrumentation, 2013 also saw application of new techniques to shotgun-style mass spec experiments. Perhaps most significantly, the use of sample-specific reference databases grew dramatically, with a number of researchers adopting this approach to improve coverage of variant protein forms and better correlate their proteomic findings with the underlying genomic data.

Typically generated using RNA-seq, sample-specific reference databases can help overcome limitations inherent in the conventional reference databases used for matching experimental mass spectra and peptide sequences to their corresponding proteins. These conventional databases are often incomplete given the vast number of different protein forms in the human proteome and the fact that not all of these forms are necessarily expressed in every cell or tissue type or in every sample, as in the case of altered protein forms arising from genetic mutations. By creating a reference database specific to the sample undergoing analysis, researchers can better capture the various protein forms expressed by that particular material.

The increase in this form of analysis also has potentially interesting implications for one of the biggest life science acquisitions of 2013, Thermo Fisher's purchase of Life Technologies. Because that deal will add Life Tech's sequencing capabilities and expertise to Thermo Fisher's mass spec-based proteomics offerings, it could enable the development of more streamlined workflows for sample-specific shotgun mass spec analysis.

Top-down proteomics also hit new discovery milestones in 2013. In January, a team led by Pacific Northwest National Laboratory researcher Ljiljana Pasa-Tolic presented a study at the American Society for Mass Spectrometry Conference on Top Down Mass Spectrometry in which they identified in Escherichia coli 1,249 gene products and more than 4,000 proteoforms.

That was the largest top-down effort to date until it was surpassed in September by a study led by Northwestern University researcher Neil Kelleher that identified more than 1,220 proteins and more than 5,000 proteoforms in H1299 cells.

Data dump

Several significant new proteomic datasets went public in 2013, as well. These included the first data from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, which in September released proteomic profiles of 95 colorectal cancer samples that were also genomically characterized by The Cancer Genome Atlas effort. The data consist of LC-MS/MS profiles generated at Vanderbilt University's CPTAC Proteome Characterization Center led by researcher Daniel Liebler.

The consortium plans additional releases in the coming months, including a public assay portal for multiple-reaction monitoring mass spec assays and additional proteomic data sets including TCGA breast and ovarian cancer sets.

University of Texas MD Anderson Cancer Center likewise released proteomic analyses of a number of TCGA samples, announcing in April the launch of The Cancer Proteome Atlas, a database containing proteomic profiles of 4,495 tumor samples – most of them from TCGA – and more than 500 cell lines generated by the lab of MD Anderson researcher Gordon Mills using reverse phase protein array technology. In total, the researchers have collected RPPA-based proteomic data on more 70,000 patient samples to date, with primary areas of focus including leukemia as well as lung, head and neck, ovarian, endometrial, and breast cancer.

Previously established proteomics resources also released updates during the year, most notable among them Sweden's Human Proteome Project, which last month issued its twelfth edition. The new release contains antibody-based protein data for more than 80 percent of the human protein-coding genes as well as RNA expression data for more than 90 percent of these genes, including more than 13 million images of protein profiling in 46 different human tissues along with RNA-seq data for 27 of these tissues.

And, looking ahead, the Human Proteome Organization's Chromosome-Centric Human Proteome Project, or C-HPP, announced in October that it expects to achieve its goal of mapping and characterizing the roughly 20,000 proteins in the human proteome by the end of next year. Project researchers said that there currently remained some 3,500 to 4,000 "missing" proteins for them to characterize, down from their estimate of 6,000 such proteins in 2012.

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