Challenges in Delivery of Therapeutic Genomics and Proteomics
A good example for this is the much-maligned PSA test. Identification of the variants at the sequence level is needed to accurately quantify the clinically relevant isoforms and reduce the false positives. Are we ready to move beyond standard immunoassays and if so, why? Henry Rodriguez: While it is safe to say that immunoassays are here to stay, they are specific cases and surely more will come for which their limitations can be complemented by MS-based assays.
The fact of the matter is that immunoassays have been the most commonly used methods in clinical laboratory testing for proteins, and the FDA has extensive experience in the science and regulatory processes for standard-format immunoassays. Furthermore, their economics are well understood cost per assay, start-up costs, and laboratory technician cost to run the assays. Scientifically, there are circumstances where quantification of peptides using MRM will be preferred to traditional immunoassays for mutants, splice variants, and posttranslational modification, as antibodies against these specific changes to the protein primary structures will be difficult to generate.
To circumvent this issue, Hoofnagle and colleagues at ARUP Laboratories and Quest Diagnostics Clinical Chemistry , ;— and ;— developed a peptide immuno-MRM MS assay with acceptable clinical diagnostic performance characteristics, which circumvents the interference of autoantibodies. In terms of economics, MS with its multiplex capability has the potential to drive down cost per assay and with automation platforms coming onboard, can greatly reduce their entry barrier into clinical laboratories. To facilitate the analytical validation of MS platforms, researchers from academia, industry and the National Cancer Institute and FDA published mock k submission documents in Clinical Chemistry in — on a multiplex immunoaffinity MS platform to educate the proteomics community on analytical evaluation requirements for multiplex assays to ensure the safety and effectiveness of these tests for their intended use.
Hoofnagle: Yes, we are ready. Immunoassays as a category are particularly problematic when analyzing human clinical samples. The field of clinical proteomics has spent time identifying reliable methods of calibration, sample handling, and analysis that put clinical chemists in a position to adopt the technology to improve patient care.
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It is not facile and talented laboratory technologists that are needed to ensure success. However, reference laboratories are already harnessing the power of clinical proteomics technologies and I am hopeful that other clinical laboratories will benefit from clinical proteomics technologies very soon.
John Ioannidis: In principle yes, as far as technology is concerned. Newer and more sophisticated is not necessarily better. Mary Lopez: Yes. Standard immunoassays suffer from a variety of pitfalls that typically result in false positives, as mentioned above. This can be due to a lack of specificity of the capture antibody or the heterogeneity of the target molecules or both. It is increasingly clear that most proteins exist in multiple forms due to truncations, posttranslational modifications, or single nucleotide polymorphisms. Many disease-associated proteins can exist in inactive and active forms and antibodies may capture these indiscriminately.
Therefore, specific detection at the sequence level is required to accurately detect and quantify the disease-related isoforms. MS can provide this specificity, even when antibodies are used to enrich low-abundance targets. Are mass spectrometers the next clinical assay platform? Henry Rodriguez: If an assay benefits patient care while being robust, reliable, automated, easy to operate, and cost-effective, it ultimately finds traction in clinical settings.
That said, MS has been playing a pivotal role in a variety of scientific disciplines, and has long been a standard tool at public health laboratories. To me, the question is not whether MS will be the next clinical platform, but when targeted MS will be broadly adopted as a tool for clinical measurement of protein analytes, supplementing current immunoassays. It should be noted that there are no fundamental technical obstacles to its adoption in clinical laboratories albeit not enough biomarkers , so its implementation is largely a matter of engineering.
It will take time for all clinical laboratories to be utilizing MS, but the rapid uptake of targeted MS and the interest by the clinical chemistry profession based upon publications in journals such as Clinical Chemistry , and growth of training webinars and other courses, makes a strong case for MS becoming an integral part of the clinical laboratory.
The best way to view this is that MS assays are not a disruptive technology, but rather a complimentary method to immunoassays that has technological advantages for certain applications. Hoofnagle: They already are. It is now time to put the calibration materials, robust assay reagents, reliable quality assurance programs, sensitive QC procedures, and trained staff in place to drastically change our ability to accurately and precisely characterize human disease one patient at a time. John Ioannidis: This depends on a lot of local parameters that may modulate the extent to which laboratories performing routine tests can use MS reliably, as well as the cost, the training required, and the ease of adoption of the processes in routine practice for flow of information in the hospital and in the clinic.
These are questions that can be addressed with late-stage translational research, translational stage 3 and 4 studies. Until now, this type of research has received hardly any attention in proteomics. Mary Lopez: MS is now, and will certainly continue to be, part of the next revolution in clinical assay platforms.
MS answers a critical need for more specific tests. Not all tests will necessarily be migrated to the MS platforms but where immunoassays fail to deliver, MS will undoubtedly fill the gap. Are you aware of any major successes of clinical proteomics in the clinic? Is diagnosis of microbial infections a good example? Henry Rodriguez: Ask a patient whose diagnosis and treatment from a Staphylococcus aureus microbial infection benefited from clinical proteomics, and the answer is most likely YES. The first clinical MS system cleared by the FDA for rapid identification of disease-causing bacteria and yeast shows the great promise of MS for clinical proteomics.
Such progress in clinical proteomics will pave the way for greater success in the future by accumulating knowledge and experience in the understanding and fulfillment of the validation criteria for multiplex MS-based assays.
And it is in the area of treatment that I think clinical proteomics will also have an impact. Therapeutic compounds are becoming more targeted to defined patient populations, and consequently the expectations for demonstrating benefit in these targeted groups are increasing.
This is especially evident in the field of oncology, where there is a growing need for companion diagnostics CDx , to identify patients with a specific biomarker that is predictive of response. For patients with cancer, for instance, those that are identified as nonresponsive can quickly move on to other, perhaps more effective therapies if they exist. It is obvious that some small proteins are captured in the spectrum of samples from the MALDI target, but it is the pattern of the mass spectrum that matters most. The identification of the proteins and lipids in the spectrum has never been paramount to the success of the platform.
Determining the difference between 2 species of bacteria is often like telling the difference between the 2 sides of the Grand Canyon from a hot air balloon. Further, we must keep in mind that the difference between women with stage 1 ovarian cancer and normal controls is much less, more akin to distinguishing 2 sides of a small creek in the woods from a hot air balloon, and, with MALDI-TOF MS, it is unlikely that we will be able to distinguish the 2. John Ioannidis: At the risk of being called a pessimist, I can't think of something that I would call a major success of clinical proteomics in the clinic to-date.
Genomics and Medicine | NHGRI
For claiming a major success, I would like at a minimum to have some documentation in sufficiently large randomized trials that adoption and implementation of a proteomics technology improved major clinical outcomes for patients. At some point, I would like to see randomized trials performed that would address what we really gain by this and other contester techniques of early diagnosis in clinical terms.
For example, it sounds great that one could identify the microbial species earlier up to 24 h before traditional methods even if this is not accompanied by direct evidence on the antimicrobial susceptibility profile. However, how does this early information translate in terms of intermediate outcomes e. These questions require randomized controlled trials to be answered reliably. Moreover, as several different technologies may eventually compete for earlier diagnosis, one would have to perform head-to-head trials to see which one is the best.
Mary Lopez: One striking example is in the diagnosis of amyloidosis. In , researchers at the Mayo Clinic developed an MS-based test with increased sensitivity and specificity compared to the previously used histopathological tests. Since its development, the assay has been widely adopted because it addresses many problems inherent to histopathology, including high background staining that interferes with subtyping. Are there technological or other advances around the corner that could bring clinical proteomics closer to the clinic?
Henry Rodriguez: Two key areas that come to mind are analytical advances hardware and software and the clinical convergence of genomics with proteomics. Improvements in targeted MS-based proteomic approaches, ranging from automated sample processing to data acquisition, and greater sensitivity with lower load volumes, are resulting in higher throughput and highly multiplexed and sensitive targeted MS analyses, thus providing a viable future for MS-based measurements in clinical laboratories.
Just as our genomics colleagues are employing multiplex DNA in clinical research, targeted multiplex proteomics may soon become a reality. Additional technological advances currently in the research space include assays based on single-cell proteomics e. In terms of the convergence of genomics with proteomics, I see a lot of potential in this area, albeit currently still in the research space.
For example, from a clinical relevance perspective, not every indication will benefit from the increased information by next-generation sequencing, as there are limited therapeutic interventions. Concomitantly, sequencing data using next-generation sequencing does not provide a holistic clinical perspective, and as a result, other types of testing such as proteomics will become ever more relevant.
Hoofnagle: The ability to enrich proteins or tryptic peptides before MS will be essential for the successful translation of clinical proteomics technologies. The development of useful reagents is warranted.
We have had tens of thousands of papers focusing on phase 0 and 1 translational research. What we need is to test the best candidate technologies in real life, with serious clinical and implementation research. Mary Lopez: The increasing sensitivity and resolution of mass spectrometers coupled with new and more efficient sample preparation methods are increasingly facilitating accurate quantification of even low-abundance disease biomarkers. Once these methods are optimized for automation and robust high throughput, they will be increasingly adopted into routine environments because of the added value they deliver with respect to greatly increased specificity.
Can you envision the role of clinical proteomics in clinical medicine 10 years from now? Henry Rodriguez: Of course. In the s, immunoassays were used routinely to measure small molecules. Personally, I'm agnostic to technology. Looking ahead, it is useful to step back and provide realistic timelines in terms of bringing new technologies to market and discovering and developing new protein biomarkers that will drive the use of such technologies in clinical laboratories, such as from new knowledge gained from proteogenomic network and pathway studies.
If one were to use the pharmaceutical industry drug development pipeline as a comparator, some interesting realities come to the forefront. While there are many variables to these numbers, they are stunning. I think the reason there is a heavy focus on timelines in clinical proteomics is because at the beginning there was maybe too much advertising.
But if you look at other fields such as transcriptomics, there currently are not so many tests that are applied in the clinical field.
As a community, we need to set realistic expectations to encourage others basic researchers, clinicians, clinical chemists, and patient advocates in moving the science forward. This type of symbiotic partnership will quickly accelerate the development and deployment of the next-generation clinical proteomics-based assays. It is my hope that new technologies and a better understanding of biology will deliver better care to a patient. Hoofnagle: Yes: automated immunoassays will be replaced with MS assays in certain patients that require a more specific assay to help diagnose, prognose, or manage disease.
John Ioannidis: I don't envision spectacular successes in the next 10 years. However, I can see the possibility of having several applications, where MS or other proteomic technologies may have focused indications, with some incremental benefits in terms of accuracy, rapidity of early diagnosis, or both. I am less optimistic about the prospect of clinically meaningful improvements in predictive ability for common diseases or in outcomes in treated patients. To achieve whatever translational advances can be achieved, we need a shift to support well-designed, collaborative late-translational efforts.
Otherwise, it is possible that 10 years from now and after a few hundred thousands of papers in proteomics, we will still have no well-documented major applications. Mary Lopez: One possible scenario is the existence of clinical analyzers for routine measurement of disease-related protein markers and panels. The automated and routine application of tests for diagnostic and specific biomarker panels will make possible a more personalized approach to medicine.
This technology will provide added benefit to genomic tests that cannot provide information on clinically important protein isoforms and variants. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: a significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; b drafting or revising the article for intellectual content; and c final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: E.