Molecular Diagnostics

Advances in decoding integrated biological processes; the surge in enabling technologies including the high‐throughput platforms of genomics, proteomics, and metabolomics; and the emergent revolution in targeted drug discovery and development of companion diagnostics place the clinical enterprise on the threshold of individualizing disease management. ¹ , ² , ³ This innovation in health care is predicated on molecular biomarkers enabling disease prediction and prevention, diagnosis, and treatment of individual patients and populations. ⁴ , ⁵ , ⁶ Biomarkers are quantifable disease characteristics that yield information about pathophysiological processes to detect disease progression or predict therapeutic response. ⁷ Traditional biomarkers include surrogate physiological measurements (heart rate, blood pressure, performance status), images (chest X‐ray, mammograms), and individual protein molecules (prostate‐specific antigen [PSA], carcino‐embryonic Antigen [CEA]). A new generation of molecular marker technologies, including single nucleotide polymorphism (SNP) analysis, genomic and proteomic profling, epigenetic profling, and gene expression profiling have the potential to increase disease‐specific sensitivity and specificity to ensure the accuracy necessary for individualized disease management. ² , ³ , ⁵ , ⁶

Although molecular markers represent the envisioned future for individualized medicine, their potential has yet to be realized.

Tis molecular revolution has stimulated a new generation of biotechnology to harness the application of biomarkers to individualized and population medicine. ⁸ However, the potential of biomarker technology has yet to be fully realized, refecting asynchronous development of discovery technologies and paradigms for their validation, early adoption, and broad application. ⁹ , ¹⁰ The paucity in biomarker validation has engendered issues surrounding approval and marketing by regulatory agencies. ⁴ , ⁷ , ¹¹ , ¹² This evolution in regulation, the emergence of requirements for robust analytic validation and clinical qualifcation, and the attendant patient‐ and capital‐intensive resources required have produced substantial barriers to realizing the full potential of biomarkers in clinical practice.

Application in Personalized Patient Management

Individualization of disease management is predicated on biomarkers that subserve specific clinical domains. ³ , ⁴ , ⁵ , ⁷ Preventive biomarkers prospectively identify individuals at increased risk for developing disease. Diagnostic biomarkers identify disease at the earliest stage, before clinical manifestation. Prognostic biomarkers stratify risk of disease progression in patients undergoing definitive therapy. Predictive biomarkers identify patients who are most likely to respond to a specific therapy. Terapeutic biomarkers provide a quantifiable measure of response to therapy in patients undergoing treatment. Finally, biomarkers identify patients at risk for developing adverse reactions to specific therapeutics.

Translation of Technological Advances

The development of analytic technologies for evaluating nucleic acids and proteins, in conjunction with the elaboration of the human genome, has provided the technological impetus to develop molecular biomarkers for disease management. ³ , ⁴ , ⁵ , ⁷ In contrast, conceptual advances in elucidating the molecular mechanisms underlying pathogenesis have yielded a plethora of targets with expanding complexity to satisfy clinical needs for individualization of medical management, providing the associated “pull” for biomarker development. ¹³ Initially, molecular markers evolved in the model of classical protein and genetic markers as single elements related to the presence of disease. ¹⁴ Teir clinical application was potentiated by the development of rapid nucleic acid sequencing technology coupled with mutation‐specific polymerase chain reaction for high‐throughput analyses. These linear initial approaches have dramatically evolved to capture systems‐level alterations underlying pathophysiology. ¹³ , ¹⁴ , ¹⁵ Panels of genetic markers and their disease‐specific mutations are cataloged and their cumulative prognostic or predictive value established. Beyond panels of individual genes, the entire transcriptome can be assessed, distinguishing diseased and normal tissues with different risk profles, to develop patterns of gene expression with prognostic and predictive value. Similar approaches are being examined with patterns of disease‐specific SNPs and epigenetic changes associated with DNA methylation. Most recently, profling the serum proteome using mass spectrometry has been employed to distinguish patients with cancer from those without.

The unmet Promise of Molecular Markers

Although molecular markers represent the envisioned future for individualized medicine, ¹ , ² , ³ , ⁴ , ⁵ , ⁷ their potential has yet to be realized, reflecting issues of technique, study design, and pathophysiology. ⁹ , ¹⁰ , ¹⁶ The technologies producing these markers have been prolific as discovery engines but have not been systematically transitioned to generate robust assay performance consistent with requirements for routine clinical laboratories in the form of analytic validation, and defined disease‐management value in the form of clinical qualification. ⁴ , ⁹ , ¹⁰ , ¹⁶ , ¹⁷ It is not unusual for biomarkers to be assessed with the use of home‐brew assays in individual laboratories that have not undergone rigorous analytic validation to define performance metrics, including reproducibility, sensitivity, and precision. ¹¹ , ¹³ In addition, molecular analytes may be evaluated employing different technical platforms whose performances have not been cross‐validated. ⁴ , ⁷ , ¹⁷ This absence of assay performance standards reflecting rigorous analytic validation and standardization across laboratories and platforms underlies issues of irreproducibility. ⁷ , ¹³ , ¹⁷

Additionally, quantitative and qualitative relationships between analytes and disease management have not undergone rigorous clinical qualification, and the evidence linking a biomarker with biology and clinical endpoints may not be readily available ⁹ , ¹⁰ , ¹⁸ , ¹⁹ These relationships describing the clinical utility of the marker should be assessed in appropriately designed and powered prospective blinded and randomized clinical trials and subsequently validated in follow‐up trials. ⁹ , ¹⁰

The Business of Molecular Markers

Biomarkers can influence the clinical decision making that profoundly affects health care economics. ¹ , ¹⁸ , ¹⁹ Screening for genetic mutations identifies patients at risk for developing breast or colon cancer; those patients then become new customers to the health care system. ²⁰ , ²¹ Prognostic tests to define the risk of recurrence in breast cancer identify patients who may not benefit from expensive chemotherapy. ²² Predictive tests that examine the over‐expression of Her2 receptors in breast tumors identify patients who will respond to expensive monoclonal antibody therapy directed to that target. The impact on clinical outcomes and the associated allocation of limited health care dollars have been used to justify profit margins for molecular diagnostics, comparable with those traditionally reserved for therapeutics. Their emergence as high‐profit products has spurred biotechnology entrepreneurs and venture capitalists to launch new companies focused on developing biomarkers across the disease spectrum. Success depends on whether their products address substantial markets and direct clinical decision making regarding expensive, complex, or dangerous therapeutic interventions. ⁸ At stake is a $5 billion market growing at 25% annually.

Historically, the developmental paradigm for biomarkers was to obtain approval for marketing of test kits by the Food and Drug Administration (FDA) that would then be sold to local clinical laboratories. ⁴ , ¹⁸ , ¹⁹ Now, molecular tests forego FDA approval and implementation in local laboratories and, rather, are run in central laboratories. ⁸ Offering diagnostic tests from a central laboratory, obviating the need for FDA approval, permits shorter and less expensive development timelines. However, these developmental efficiencies are associated with a reciprocal absence of definitive studies analytically validating and clinically qualifying the biomarker, which are mandated by the FDA for marketing approval. ⁷ , ⁹ , ¹⁰ , ¹⁷ It is precisely this failure to provide clinical validation of biomarker value, analogous to safety and efficacy requirements by the FDA for marketing drugs, which contributes to lagging integration of molecular markers into patient management. ¹³ , ¹⁶ , ¹⁷

Regulation of Diagnostic Testing

Although molecular biomarkers have emerged as key indices for disease management, ³ , ⁵ , ⁷ , ¹⁸ oversight and regulation of their safety and validity has not kept pace. ¹¹ , ¹² Today, more than 1000 biomarkers are marketed as diagnostic tests, most offered as home‐brew tests in central laboratories. ¹¹ , ¹² The FDA does not regulate the conduct of diagnostic tests, their analytic validity, or their clinical qualification. Rather, validity, utility, and clinical interpretation of tests are relegated to individual laboratories. In 1988, Congress enacted the Clinical Laboratory Improvement Amendments (CLIA) to certify laboratories testing human specimens and reporting patient results. Under CLIA provisions, laboratories must adhere to requirements for quality control, personnel training, and validation procedures. Moreover, laboratories performing high‐complexity testing must enroll in proficiency testing programs related to the quality of testing services offered. Of significance, there are no specific program or specific quality control, personnel qualification, or proficiency testing requirements for molecular testing. ¹² Indeed, the Centers for Medicare and Medicaid Services (CMS), within the Department of Health and Human Services, is responsible for the quality of CLIA‐approved laboratories. It is significant that one third of CLIA‐certified laboratories performing genetic testing fail to participate in proficiency testing. ¹¹ , ¹² In that context, the inverse relationship between errors in diagnostic analyses and proficiency testing suggest that the current regulatory position may be some cause for concern. ¹¹ , ¹²

The FDA has not had a consistent position regarding jurisdiction over molecular and genetic tests. Indeed, it has authority to regulate them but has exercised enforcement discretion. In 2006, the FDA issued a draft guidance extending regulatory enforcement authority to a subset of home‐brew molecular tests termed in vitro diagnostic multivariate index assays (IVDMIAs). ²³ Multivariate index assays measure multiple analytes and analyze data with algorithms or software programs. The agency targeted IVDMIAs for regulation because the algorithms often are proprietary, making it difficult for physicians to interpret results. Most IVDMIAs will require some level of FDA review, and some will require full regulatory approval before they enter the marketplace. Beyond IVDMIAs, the FDA has not developed an overarching position regarding oversight of home‐brew assays as a class.

Conclusion

Molecular markers offer a clear path from the current empirical, probabilistic model of clinical care to the development and implementation of preemptive, deterministic personalized medicine. ¹ , ³ , ⁵ , ¹⁹ However, their evolution into clinical practice is predicated on the development of strict paradigms focused on analytic validation and clinical qualification. ⁷ , ⁹ , ¹⁰ , ¹⁶ In that regard, biomarker development and clinical application should have an established basis of preclinical and clinical evidence, reflecting clinical trial design, analytical methodologies, and statistical rigor. Moreover, there may be benefits in centralizing federal regulatory oversight of approval, marketing, and quality control in application in the FDA and/or CMS. ⁹ , ¹¹ , ¹² Efforts should focus on collaborations across public and private sectors to facilitate the discovery and application of biomarkers that will support the development of new molecularly‐targeted therapeutics to achieve a truly individualized approach to patient care. ⁴ , ⁷ , ¹⁷ , ¹⁸ , ¹⁹