Abstract
Genomic diagnostics for cancer have reached the clinic, but protein profiling to augment or confirm these analyses remains challenging. Proponents of the proteomics approach say that while it can contribute significantly to diagnostic decision-making, and despite remarkable progress in proteomic methods, these analytical tools have not been established in routine clinical practice.
Limitations preventing integration into the clinic include the high cost of equipment, the need for highly trained personnel and, particularly, the establishment of reliable and accurate protein biomarkers or panels of protein biomarkers for detection of neoplasms.
Investigators continue to cite the urgent need for blood-based molecular tests to assist in the detection and diagnosis of cancers at an early stage, when curative interventions are still possible, and to predict and monitor response to treatment and disease recurrence.
Many scientists believe that targeted proteomics, a sensitive and specific mass spectrometric analysis method to detect and quantitate pre-selected components in a complex sample matrix, such as a proteolytic digest of a plasma or tissue extract, provides a promising approach for exploration of the distinctions among disease states in different types of cancer.
Some, however, have voiced guarded skepticism about the ultimate value of the entire MS-based proteomics approach for biomarker discovery in complicated samples such as blood.
O. John Semmes, Ph.D., of the department of microbiology and cell biology, Eastern Virginia Medica, said in a 2004 article in Cancer Epidemiology, Biomarkers & Prevention (Cancer Epidemiol Biomarkers Prev 2004;13(10):155-57) that despite the tremendous advances made in protein MS following the completion of the genomes of target organisms, “some of the same old obstacles to protein biomarker discovery still exist. The physical hurdles of component complexity, the tremendous range in individual protein concentration, and the dynamic nature of the proteome are still major barriers to overcome.”
Dr. Semmes is among the scientists who agree that that the underlying heterogeneity of cancer necessitates that development of accurate diagnostics will depend on the discovery of a “panel” of proteins that together can discriminate between subtle disease states with population-wide robustness.” This requirement for multiple proteins over a single protein biomarker emphasizes the demand for improved technologies, allowing for measurement of the full complement of the proteome,” he says.
Office of Cancer Clinical Proteomics Research
NIH Program
In 2006, the NIH launched its Clinical Proteomic Technologies for Cancer program to address the pre-analytical and analytical variability issues that form major barriers to the proteomics field. Experimental design; technological and technical aspects of protein identification; variability related to biospecimens collection; data acquisition, analysis, and reporting; and the lack of reproducible proteomic technologies and highly characterized and standardized reagents, the NIH said, had all contributed to the lack of progress.
The initiative was composed of three integrated programs that worked together to overcome these barriers. These included the Clinical Proteomic Technology Assessment for Cancer (CPTAC), which was announced in August 2011. CPTAC comprises a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust, quantitative, proteomic technologies and workflows.
Intended to leverage Proteomic technologies “to bridge the gap between genotype and phenotype,” CPTAC teams include among others the Broad Institute of MIT and Harvard, Fred Hutchinson Cancer Research Center/Massachusetts General Hospital, and a Johns Hopkins University/Memorial-Sloan Kettering team. CPTAC says it combines the requisite in mass spectrometry (MS) and related proteomic technologies, bioinformatics and biostatistics, cancer biology, and clinical/translational cancer research.
A key outcome of the program to date has been improvement of the so-called proteomic biomarker development pipeline. Prior to the initiative, development of biomarkers typically relied on a discovery process followed by a clinical validation (qualification) stage, in which hundreds to thousands of biomarker candidates are detected but only a few are transitioned to full clinical qualification studies due to the prohibitive expense and time of large-scale validation studies for each candidate.
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To address the biomarker issue, consortium researchers designed a two-step workflow for more efficient, timely, and cost-effective development of protein (and peptide) biomarkers prior to clinical qualification studies. These include a “discovery step” in which cancer-specific candidate biomarkers are identified using metrics-driven protein survey technologies that globally interrogate appropriate biospecimens.
A second verification step involves the development of targeted, quantitative assays, commonly multiplexed and suitable for the examination of a larger number of biospecimens to ensure appropriate statistical power.
Other intended primary outputs anticipated from CPTAC were proteome characterization of several tumor types for the understanding of cancer biology and multiplexed quantitative assays for the measurement of candidate protein/peptide biomarkers.
Shotgun Profiling
The CPTAC consortium laboratories perform untargeted (shotgun) profiling of both protein expression and post-translational modifications. Tandem hybrid Orbitrap and time-of-flight mass analyzers enable both deep inventory and quantitative comparisons of complex peptide mixtures from cell, tissue, and biofluid proteomes, according to CPTAC scientists.
Multiple reaction monitoring (MRM) the complementary technology to global profiling enables systematic development of quantitative protein assays through measurements of specific peptides in proteolytic digests. This technology, Matthew J. Ellis, M.D., Ph.D., et al, writing in Cancer Discovery (Cancer Discov. 2013 Oct; 3(10): 1108– 1112.) and speaking for CCPTAC scientists says, overcomes a fundamental limitation of immunochemical methods—the availability of specific antibodies. Moreover, they say, MRM enables selective quantification of variant/mutant or post-translationally modified sequences, both or which are difficult to achieve with antibodies.
MRM assays typically employ triple quadrupole instruments, but similar assays are being deployed on new, hybrid quadrupole-time-of-flight and quadrupole-orbitrap instruments that have higher resolution and mass precision than triple quadrupole instrument.
One aim of the Broad CPTAC team was to establish if MRM-based methods, either alone or coupled with antibody-based approaches, could help overcome the “critical bottleneck” that exists between the initial discovery of candidate biomarkers and their subsequent clinical validation. Because the method provides a unique fragment ion that can be monitored and quantified in the midst of a complicated matrix, it has served as a principal tool for quantification of small molecules in clinical chemistry for number of decades. Broad scientists say that MS-based quantitative assays have the necessary characteristics required for verification studies, namely high specificity, sensitivity, multiplexing capability, and precision.
According to the Broad Institute's Michael A. Gillette, Ph.D., and Steven A. Carr, Ph.D., “To many in the field of proteomics, the pivotal question has been not whether if but when targeted MS will be broadly adopted as a tool for clinical measurement of protein analytes, supplementing not supplanting the current use of immunoassays.” The authors made this comment in an article in January 2013. (Nat Methods. 2013 Jan; 10(1): 28–34).
They add that several studies have now demonstrated the central importance and effective deployment of MRM-MS as a verification tool for candidates in the context of a comprehensive discovery-to-verification biomarker pipeline.
As an example of the tumor-specific characterization referred to in one of the CPTAC goals, Wang et al, working at Johns Hopkins, reported in PNAS (Proc Natl Acad Sci U S A. 2011 Feb 8;108(6):2444-9. doi: 10.1073/pnas.1019203108. Epub 2011 Jan 19) that the altered protein products resulting from somatic mutations can be identified directly and quantified by mass spectrometry. The peptides expressed from normal and mutant alleles were detected by selected reaction monitoring of their product ions using a triple-quadrupole mass spectrometer.
The authors said quantification of the number and fraction of mutant Ras protein present in cancer cells revealed an average of 1.3 million molecules of Ras protein per cell, with the ratio of mutant to normal Ras proteins ranged from 0.49 to 5.6. They further found that mutant Ras proteins could be detected and quantified in clinical specimens such as colorectal and pancreatic tumor tissues as well as in premalignant pancreatic cyst fluids. In addition to answering basic questions about the relative levels of genetically abnormal proteins in tumors, the authors concluded that this approach could prove useful for diagnostic applications.
Consortium Labs Activities
Consortium laboratories continue to report progress in developing and refining MS-based assays for biomarkers. Susan E. Abbatiello, Ph.D., of Harvard's Broad Institute, and colleagues reported results of an interlaboratory Study in Molecular and Cellular Proteomics (Mol Cell Proteomics. 2015 Sep;14(9):2357-74. doi: 10.1074/mcp. M114.047050. Epub 2015 Feb 18) to develop quantitative peptide assays measuring cancer-relevant plasma proteins. These authors had previously shown that that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma. The resulting assays could be transferred across laboratories while maintaining high reproducibility and quantitative precision.
Recently the authors reported that they had extended that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems could develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma.
Writing in Nature Methods, J.J. Kennedy, Ph.D., et al, of the Fred Hutchinson Cancer Research Center, and other CPTAC participant laboratories reported that they had configured and validated across three laboratories 645 novel MRM assays representing 319 proteins expressed in human breast cancer (Nat Methods. 2014 Feb;11(2):149-55. doi: 10.1038/nmeth.2763. Epub 2013 Dec 8). Assays were multiplexed in groups of more than 150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer– related cell lines.
The investigators reported high median assay precision (5.4%), with high interlaboratory correlation (R(2) > 0.96). Peptide measurements in breast cancer cell lines could discriminate among molecular sub-types and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.
To date, the CPTAC initiative illustrates the power of well-funded, focused, multi-laboratory enterprises that incorporate multiple technologies to provide “a fully integrated accounting of DNA, RNA, and protein abnormalities in individual tumors.” Resulting datasests, participating scientists say, will illuminate the complex relationship between genomic abnormalities and cancer phenotypes, thus producing biological insights as well as a wave of novel candidate biomarkers and potential therapeutic targets amenable to verification using targeted mass spectrometry methods.