1. Introduction
Development of a biomarker for IVD applications typically begins with research showing that testing for a biomarker gives clinically relevant information. Those studies in the vast majority of the cases provide only indication but not evidence of the clinical utility of the biomarker. The process of translation of a biomarker discovered in a research project to routine use in the diagnostic laboratory is relatively complex. It requires a systematic and planned research to generate evidence for the clinical benefits of the biomarker testing procedure as well as verification of safety and performance of that procedure. Researchers in the field of biomarker development should bear in mind the challenges of that process when designing biomarker development studies. This is especially important regarding methylation biomarkers where the translation of the research indications of biomarker clinical utility to diagnostic use is markedly inadequate.
2. Methylation changes as biomarkers and the types of in-vitro diagnostic biomarkers
A biomarker in medicine is a measurable indicator of biological/clinical state. DNA methylation change (hyper- or hypomethylation) is a biomarker of specific phenotype if it leads to a change of gene expression that induces that phenotype. There is ample evidence available that methylation biomarkers can be utilized at all stages of clinical disease management from disease risk assessment of healthy individuals, through diagnosis, treatment management to post-treatment surveillance.
2.1. Risk assessment biomarkers
There is large evidence that exposure to adverse environmental factors induces methylation changes long before disease occurs. For example: we have shown that the exposure to arsenic in drinking water affects methylation patterns of CD4-positive blood cells [Citation1] or the methylation of BRCA1 gene in blood was shown to correlate with breast cancer risk [Citation2]. This type of methylation changes can potentially be utilized to assess the probability of the healthy individual to develop a specific disease.
2.2. Early disease detection biomarkers
This type of biomarkers is especially important in cancer where early detection significantly increases the chance to cure. From the studies of carcinogenesis of various cancer, we know that methylation changes occur early if not initiate neoplastic transformation [Citation3–Citation5]. At the same time, it is well established that DNA from tumor is secreted and can be readily detected in body fluids that come in direct contact with the neoplastic tissue such as e.g. plasma or urine. Those body fluids are referred to as liquid biopsies. Two diagnostic tests, targeting tumor-specific methylation changes in DNA extracted from plasma are already approved to aid the diagnosis of colorectal cancer [Citation6,Citation7]. This type of use of methylation biomarkers is likely to have a significant impact on the management not only cancer but other diseases, especially that liquid biopsy-based diagnostic tests are characterized by minimal invasiveness.
2.3. Clinical disease management biomarkers
The clinical presentation of cancer (and other diseases) depends largely on the specific methylation changes that neoplastic cells acquired during carcinogenesis. Thus, by testing for disease-specific methylation abnormalities we can infer clinical outcomes. Biomarkers used in clinical patients’ management can be either predictive or prognostic depending on the type of information they provide.
The predictive biomarkers estimate the likelihood of the clinical outcome. The association of those biomarkers with clinical outcomes is mainly inferred from observational studies and intrinsic patient characteristics such as the presence of mutations or methylation changes.
The prognostic biomarkers allow for the identification of individuals that are likely to respond to intervention.
To establish that biomarker is predictive only association of the presence of biomarker with clinical outcome needs to be shown. However, to establish a predictive value of a biomarker at least two independent and well-controlled clinical trials are necessary [Citation8]. Those clinical trials in most of the cases need to compare the intervention to control treatment in two groups of patients with and without the biomarker and demonstrate with statistical certainty correlation of the biomarker with the outcomes of intervention. In the literature, hundreds of disease-specific methylation changes or methylation signatures (genome-wide patterns) have been reported to have prognostic significance [Citation9–Citation11]. However, we are still lacking strong methylation-based predictive biomarkers.
2.4. Post-treatment monitoring/surveillance biomarkers
With the increasing number of treatment options and more cancer types becoming a chronic disease, a need for biomarkers enabling identification of relapsing patients also increases. As most of the methylation changes in metastasis reflect primary tumor [Citation12], the application of methylation biomarkers for post-treatment disease monitoring is straightforward. This type of methylation biomarkers-based tests is already in an advanced stage of development in colorectal cancer [Citation13,Citation14].
3. Regulatory path for biomarker use in in-vitro diagnostics
There is no doubt that methylation biomarkers can significantly impact clinical disease management; however, a diagnostic test needs to meet strict regulatory requirements before it can be used in routine clinical practice. After five years transition period which ends on 16th of May 2022, almost all diagnostics tests (referred to as In-Vitro Diagnostic Devices, IVDD), to be lawfully distributed in EU will need to meet the requirements of In Vitro Diagnostic Device Regulation (EU 2017/746). This new legislation defines IVDD as: '….any medical device which is a reagent, reagent product, calibrator, control material, kit, instrument, apparatus, equipment, software or system, whether used alone or in combination, intended by the manufacturer to be used in vitro for the examination of specimens, including blood and tissue donations derived from the human body, solely or principally for providing information on one or more of the following:
concerning a physiological or pathological process or state
concerning congenital physical or mental impairments
concerning the predisposition to a medical condition or a disease
to determine the safety and compatibility with potential recipients
to predict treatment response or reactions
'to define or monitor therapeutic measures.'
Under this regulation, the approval of IVDD for diagnostic use begins with the classification of the device into one of the four classes: A, B, C, or D. The classification is based on the assessment of the risk associated with the use of the device. The devices in category A are of the lowest and in class D the highest risk for the individuals undergoing the testing. Class A in principle will include general laboratory use products. The biomarker testing procedures cannot be classified as A devices and therefore will be classified in class B or higher. The IVDD devices B through D will need to acquire CE (the Conformité Européene) marking, a label indicating that the IVDD is legally distributed in EU. The major part of the process of CE certification is the preparation of the technical documentation which is referred to as ‘a technical file.’ In that document, a producer of the device presents an evidence that device is safe and performs to give the clinically relevant result that it was designed to give. The description of the clinically relevant result that the device provides is a very first part of the technical documentation and is referred to as ‘the intended use.’ The intended use description is followed by data fulfilling all the components of the concept referred in regulation to as the clinical evidence and defined as: ‘clinical data and performance evaluation results, pertaining to a device of sufficient amount and quality to allow a qualified assessment of whether the device achieves the intended clinical benefit and safety, when used as intended by the manufacturer.’ There are three major parts that the technical documentation of IVDD needs to address to fulfill the requirements of the clinical evidence: scientific validity, analytical performance, and clinical performance.
Scientific validity is evidence that the analyte (biomarker/methylation change) which the device is designed to detect is associated with the clinical condition.
Analytical performance documents that IVDD correctly detects and measures the analyte. A significant part of this requirement is an evaluation of the performance of the detection procedure that IVDD utilizes. The studies presented in this part of the technical documentation need to evaluate among other features of IVDD such as: specificity, accuracy, precision, detection limits, quantitation limit, linearity, range, and robustness for each targeted biomarker. Moreover, the studies need to show that the analytical procedure adheres to the general safety and performance requirements .
Clinical performance shows that the detection of the analyte indicating specific condition as described in scientific validity part of the technical documentation indeed yields clinically relevant result in the target population or the interned user of the IVDD.
The accuracy of technical documentation will be controlled by Notified Bodies, an organization designated by legislation in the EU country to assess the conformity of the CE market product with the regulation. The conformity assessment will be performed by Notified Body before product launch but also during product marketing.
4. Summary
This brief text indicates the magnitude of the research and work that needs to be done in planned and controlled fashion to introduce biomarker testing into diagnostic use. The process is complex however, if already biomarker discovery studies are planned with consideration of the above described regulatory framework, the translation of the research evidence to the diagnostic practice will be significantly faster.
Declaration of interest
TKW work is supported by the Polish National Agency for Academic Exchange. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Reviewers Disclosure
Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.
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References
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