Best Biomarker Practice Guidelines to (1) select and validate biomarker detection tests and

(2) select and design studies to for clinical biomarker feasibility studies – To increase the number of clinically used biomarkers, CliniMARK will, in collaboration with biomarker initiatives throughout Europe,

improve the quality and reproducibility of clinical biomarker feasibility studies, rather than further increasing the number of biomarker discovery studies.

Implementation of the CliniMARK approach: Demonstrator project in Chronic Obstructive Pulmonary Disease (COPD)

To demonstrate the value of the generated BBP guidelines, COPD will be chosen as a demonstrator project. The CliniMARK network will gather and classify all known COPD biomarkers and select the appropriate research-grade detection tests.

Furthermore, the CliniMARK network will design technical validation studies, and design clinical multi-centre feasibility studies.

Capacity building: New channels for BBP dissemination - CliniMARK aims to develop novel strategies to improve adherence to guidelines. Implementation of BBP guidelines throughout Europe will greatly improve the biomarker research capacity. Therefore CliniMARK will disseminate the results through channels such as publication of guidelines in scientific journals, LinkedIn groups, conferences, flyers, and publication of reports.

However, these channels have proven insufficient to promote strong adherence to guidelines. One new example that will be investigated is an online platform where the expertise of all network members will be compiled and classified. Scientists who aim to study a biomarker can enter this biomarker in the online tool, classify the biomarker according to the BBP guidelines and receive an overview of which studies must be performed for each phase and of experts or existing platforms

(like the EATRIS Biomarker and BBMRI Biobanking platforms) that can provide the necessary expertise, samples and tools for each phase. In addition, the network will seek collaboration with scientific journals on biomarkers to develop a publication policy to stimulate adherence to the BBP. The relationship between the CliniMARK aim, objectives, Action challenge and the multidisciplinary Action network is represented in Figure 2 below.

State-of-the-art technologies to measure protein biomarkers include: Within CliniMARK, the focus will be on protein biomarkers using two key technologies: (1) mass spectrometry based methods like Matrix-Assisted Laser Desorption/Ionization (MALDI), Liquid Chromatography coupled to tandem mass spectrometry (LC-MS/MS) and Selective Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM)/Parallel Reaction Monitoring (PRM), (2) immuno-assay based methods include immunocyto/histochemistry, western blotting, the enzyme-linked immunosorbent assay (ELISA), multiplex assays (e.g. Luminex or affimer/aptamerbased technologies), and plasmon resonance imaging.

Continuous development of these analytical techniques improves their reliability, detection limits, and high-throughput potential. However, since in guidelines for early development no attention is payed to eventual market introduction, different research groups use methods that cannot be used in later phases (e.g. for market introduction). State-of-the-art clinical biomarker feasibility studies. Case-control studies to determine sensitivity, and specificity of biomarkers are mostly performed on human samples from biobanks. The use of biobank materials has significant issues such as low sample amounts, non-standardized sample collection, handling and storage, and lack of uniformity of concomitant clinical data.

Several efforts have been made to provide standardized guidelines on biobanking, and to extend sample cohorts by connecting existing biobanks throughout Europe (e.g. BBMRI). Only limited efforts, however, exist to standardize clinical validation study designs. As a result, the sensitivity and specificity of biomarkers are often reported based on ill-designed studies that lack critical information (e.g. health status, confounding factors like BMI or medication, health care costs, technical details on the used biomarker detection technique). This makes it very difficult to reproduce and compare biomarker validation studies.

This COST Action is the first pan-European initiative to deliver guidelines for ‘Best Biomarker Practice’ that cover the design of biomarker development studies to establish clinical feasibility of the biomarker and connect the different phases in biomarker research. These will include sample handling, detection techniques, laboratory reproducibility assessments, clinical validation designs, and extensive diagnostic assay development of biomarkers. To establish the BBP, the network will focus on one disease: COPD, with the objective to define a general framework that is applicable to more technologies, disease indications, and other biomarker use. CliniMARK will rank known COPD protein biomarkers based on physico-chemical characteristics (e.g. size, charge, concentration), and evaluate all research-grade detection techniques and their shortcomings that are available for each biomarker, related to their clinical use. For each biomarker, CliniMARK will then select the most appropriate detection technique per biomarker with regards to expected analytical sensitivity and specificity, with-in run and between run reproducibility, expected optimization and analytical validation duration, and feasibility to further develop the research-grade test into a clinical-grade test.

Subsequently, CliniMARK will define the requirements for analytical validation for the selected test for each biomarker (e.g. what sample material to use, and limits for variability, and reproducibility). Furthermore, the network will compile and use existing guidelines on biobanking for biobanks in COPD. CliniMARK will classify COPD biomarker classes according to their prospective use (e.g. diagnosis, companion diagnostic, prognosis) and evaluate optimal clinical validation study designs (e.g. sensitivity, specificity, clinical impact, economic impact). For each protein biomarker, a development plan for appropriate feasibility study designs will be developed. In addition, in parallel to the specific development plans for COPD biomarkers, a BBP will be designed, based on the thorough evaluation of available biomarker measurement techniques, clinical feasibility study design and existing guidelines. This process will result in two documents: First, a standardized development plan, (outlining intended use, analytical specificiations, and clinical feasibility study design) for selected COPD biomarkers to efficiently enter into phase 3. And second, during development of the plan for specific biomarkers, all decisions and considerations for these decisions will be documented and integrated to develop a standardized BBP whitepaper on selection and analytical validation of research-grade detection techniques and clinical feasibility studies for biomarkers in general.

Furthermore, the CliniMARK network will identify current technological hurdles of detection techniques (e.g. sample preparation, sensitivity, high-throughput potential, sample preparation methods), and establish development plans to overcome these hurdles. The development plans to pass COPD biomarkers through phase 2 will subsequently be carried out by the CliniMARK network members. For each task in the development, the network member with appropriate expertise will be selected. This will result in the standardized development of 1 selected biomarker from phase 1 into phase 3. In addition, the CliniMARK network will develop a pilot online tool to establish research networks for biomarker feasibility research and development. This tool will be designed according to the BBP and be loaded with information on the CliniMARK network with regards to expertise on biomarker detection, biobanking, medical expertise, data analysis, study design, statistical analysis, and health technology. Members can subsequently enter a biomarker in this tool, provide its class and will subsequently receive a biomarker study plan according to the BBP with suggestions of CliniMARK network members that can provide the necessary expertise and data.

Significant investments have been made in biomarker discovery studies, clinical biomarker feasibility studies, and development of diagnostic tests to increase the amount of validated biomarkers and clinically implemented diagnostic tests.

These investments have resulted in the discovery of thousands of new potential biomarkers, but not in reproducible outcomes of clinical biomarker feasibility studies, nor in a significant increase in clinically implemented diagnostics tests. Therefore, CliniMARK will not focus on discovery or on full diagnostic development but on the intermediate analytical and clinical biomarker development phase (Figure 1 above).

CliniMARK intends to improve the quality and reproducibility of discovered biomarkers through a unique European network that will standardise and connect methods to clinically validate biomarkers.

The main aim of CliniMARK is to develop a European network of analytical, clinical, and industrial biomarker experts that define consensus on selection and validation of biomarker detection techniques and clinical feasibility study designs.

Separated networks at the national level exist but significant efforts by these networks often lead to non-reproducible results. To develop a BBP guideline to increase the number of thoroughly validated, reliable biomarkers,

a pan-European academic-industrial network is not only valuable, but an absolute necessity [15].

The CliniMARK Action is complementary to existing efforts. To achieve its long term goal of increasing the number of reproducibly studied biomarkers, the developed BBP guidelines must be implemented in running projects. To achieve such implementation, CliniMARK follows a 3-phased approach. First, CliniMARK will invite members from current biomarker initiatives throughout Europe to join the CliniMARK Action from the start to help establish BBP.

Identified initiatives include ESFRI landmark infrastructures such as EATRIS, BBMRI, and ELIXIR, the FP7 projects IMPROvED, EPREDICE and DIPROMON, the Horizon 2020 PHC-12 projects, and other projects such as BIOAIR, BIOFINDER. Second, to demonstrate the value of the BBP approach, CliniMARK will implement these guidelines in running projects on COPD as a demonstrator project. COPD has been selected as multiple members of the CliniMARK network are involved in COPD biomarker projects and actual implementation of the BBP guidelines in these projects is feasible.

Third, since members of existing European biomarker initiatives have been invited to CliniMARK network at the start, the network to implement the demonstrated CliniMARK BBP approach can be directly transferred to biomarker validation projects for other medical conditions through the CliniMARK network. In addition, the CliniMARK network will establish close contacts with EMA and FDA initiatives on regulatory guidelines for biomarkers (such as the WRIB meetings and EBF (European Bioanalysis Forum) meetings).