Paving the Way for MS-based Protein Tests that Target Unmet Clinical Needs Christa Cobbaert (Presenter) Leiden University Medical Center Presenter Bio: Christa Cobbaert is head and professor of the Department of Clinical Chemistry and Laboratory Medicine at the Leiden University Medical Center. Her scientific research focuses on test evaluation with great attention to standardization, harmonization and trueness verification of medical test results, for the sake of better of patient care. Her scientific work is done in close collaboration with the Dutch External Quality Assessment Scheme organizer (SKML) and with international colleagues which collaborate in joint working groups. Her efforts resulted in ca. 150 original publications and several appointments on (inter)national positions because of her expertise on metrological traceability of test results. She is currently vice chair of the International Federation of Clinical Chemistry (IFCC) Scientific Division EC and member of ISO TC 212/ working group 2.

Authors: Christa M. Cobbaert (1), L. Renee Ruhaak (1), Arnoud van der Laarse (1), Wilma E. Mesker (2), Rob A. E. M. Tollenaar (2) Manfred Wuhrer (3), Yuri E.M. van der Burgt (1,3)
Leiden University Medical Center, Leiden, the Netherlands

Proteins in body fluids are routinely tested in clinical laboratories by immunoassays, however diagnostic sensitivity- and specificity may not be sufficient for the intended use. Moreover, the applied immunoassays cannot recognize the different proteoforms, whereas MS-based strategies result in detailed structural characterization of specific protein targets. Furthermore, clinically effective tests that support patient management at an early and curable stage are lacking for a wide variety of diseases. In our institute we define specific unmet clinical needs with the clinicians according to a structured checklist developed by the European Federation of Laboratory Medicine Working Group on Test Evaluation and explore quantitative clinical chemistry proteomics as a strategy to detect and quantitate specific proteins in a multiplexed way.

Introduction

The number of CE- and/or FDA-approved protein tests in medical laboratories has hardly increased over the last two decades and runs out of step with the huge number of “promising biomarkers” and the large unmet clinical needs for patient care. Protein biomarkers that enable amongst others early detection of cancer(s), chronic obstructive pulmonary disease, and kidney damage could help to diminish the burden of disease in a curable stage. MS-based quantitative Clinical Chemistry Proteomics (qCCP) has potential to detect and quantify protein biomarkers of clinical interest in a selective way. In our institute we define specific unmet clinical needs with the clinicians according to a structured checklist developed by the European Federation of Laboratory Medicine (EFLM) Working Group on Test Evaluation that guarantees that only tests that are fit-for-clinical purpose are developed and implemented [1,2]. We then develop specific qCCP tests using a targeted and multiplexed bottom-up proteomics workflow with automated liquid handling and LC-triplequadrupole-MS [3]. Sustainability of the findings is ascertained by introducing the concept of metrological traceability of test results.

Methods

We focus on widely occurring chronic diseases, such as cardiovascular diseases (CVD) and various types of cancer. The assessment of CVD risk is routinely done by measurement of LDL- and HDL-cholesterol. However, the test methods are unreliable in hypertriglyceridemic sera. Moreover, residual risk in coronary artery disease risk is often not explained by serum lipids [4]. In case of breast cancer (BCa) and colorectal cancer (CRC) novel diagnostic and prognostic biomarkers are urgently needed and in case of prostate cancer (PCa) the generally applied measurement of prostate-specific antigen (PSA) requires test refinement. The stage (or extent) of development of appropiate MS-based protein tests varies for our different purposes. Whereas we have multiple years of experience in the quantification of apolipoproteins, the evaluation of BCa and CRC protein candidates is at an early stage. Proteoform analysis of PSA has resulted in promising data that requires further validation in new patient cohorts (see also MSACL-EU abstract Guinevere Kammeijer).

Results

We have applied our multiplex LC-triplequadrupole-MS test for the quantification of six serum / plasma apolipoproteins (A-I, B, C-I, C-II, C-III and E) intermittently during a two-year period. To evaluate performance of the test with regard to precision and bias of test results, two native serum pools were included as internal quality control in 50 batches over this period. The apolipoprotein measurements were compared to assigned values to assess bias and precision by analysis of repeated quality control observations in comparison to predefined intervals [3]. Two lots of trypsin and three HPLC columns were used in the course of this analyses. Similar results were found for both QC samples and could be confirmed for the second peptide of each protein with CVs<10% for all peptides with one exception (peptide ESLSSYWESAK in apoC-II). Assay performance was unaffected by typical laboratory workflow variation and reagent lot changes. Long-term precision traces for high abundant serum apolipoproteins demonstrate robustness of multiplex protein quantification. The clinical relevance in CVD for measuring apoA-I and apoB is clear, whereas the clinical utility of apoCs and apoE needs further exploration. The clinical relevance of the recently described apoCII-deficient patients with normotriglyceridemia and enhanced CVD risk should be further explored (4). In addition, in-depth proteoform knowledge on these latter two apolipoprotein is pursued. Proteoform analysis can be considered as stratification layer additional to quantitative levels of individual proteins or protein panels that serve as potential biomarkers [5].

Conclusions & Discussion

Clinical protein MS is a growing and exciting field, and the number of clinical chemists that acknowledge its potential with regard to being complementary to immunoassays for quantitation of specific proteins is rapidly expanding [6]. Unfortunately the number of new protein markers that made it from MS-based proteomics into the clinic is rather limited [7]. It is stressed that biomarker translation and method transfer go hand-in-hand. Rather than having a technological origin, key-reasons for the translation-lag are the use of invalid samples, lack of thoughtful study designs, silo-thinking of the stakeholders involved and lack of appropriate test evaluation and adequate test standardization. MS-based methods in general and quantitative CCP specifically, will complement or even replace “total” protein read-outs obtained through immunoassays. Furthermore, proteoform analysis provides a stratification layer additional to quantitative levels of individual proteins or protein panels that already serve as biomarkers. An interesting example is the detection of PCa, for which the generally applied measurement of PSA cannot differentiate between benign prostate hypertrophia and maligne cancer. The measurement of specific proteoforms may render MS-based strategies feasible as an add-on test to the corresponding protein quantification test [4].

1. Horvath et al., Test Evaluation Working Group of the European Federation of Clinical Chemistry Laboratory Medicine. From biomarkers to medical tests: the changing landscape of test evaluation.Clin. Chim. Acta, 427: 49-57 (2014)

2. Monaghan et al., Test Evaluation Working Group of the European Federation of Clinical Chemistry and Laboratory Medicine. Biomarker development targeting unmet clinical needs. Clin. Chim. Acta, 460: 211-219 (2016)

3. Ruhaak et al., Robust and Accurate 2-Year Performance of a Quantitative Mass Spectrometry-Based Apolipoprotein Test in a Clinical Chemistry Laboratory. Clin. Chem., 64: 747-749 (2018)

4. Hermans et al., Low levels of apolipoprotein-CII in normotriglyceridemic patients with very premature coronary artery disease: observations from the MISSION! Intervention study. J. Clin. Lipidol., 11:1407–14 (2017).

5. van der Burgt and Cobbaert, Proteoform analysis to fulfill unmet clinical needs and reach global standardization of protein measurands in clinical chemistry proteomics. Clin. Lab. Med., accepted for publication (2018)

6. Annesley et al., Clinical Mass Spectrometry-Achieving Prominence in Laboratory Medicine.Clin. Chem., 62: 1-3 (2016)

7. Anderson, Within sight of a rational pipeline for development of protein diagnostics. Clin. Chem., 58: 28-30 (2012)