M-protein diagnostics in multiple myeloma patients using ultra-sensitive targeted mass spectrometry and an off-the-shelf calibrator.

OBJECTIVES: Minimal residual disease status in multiple myeloma is an important prognostic biomarker. Recently, personalized blood-based targeted mass spectrometry (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to measure minimal residual disease. However, quantification of MS-MRD requires a unique calibrator for each patient. The use of patient-specific stable isotope labelled (SIL) peptides is relatively costly and time-consuming, thus hindering clinical implementation. Here, we introduce a simplification of MS-MRD by using an off-the-shelf calibrator. METHODS: SILuMAB-based MS-MRD was performed by spiking a monoclonal stable isotope labeled IgG, SILuMAB-K1, in the patient serum. The abundance of both M-protein-specific peptides and SILuMAB-specific peptides were monitored by mass spectrometry. The relative ratio between M-protein peptides and SILuMAB peptides allowed for M-protein quantification. We assessed linearity, sensitivity and reproducibility of SILuMAB-based MS-MRD in longitudinally collected sera from the IFM-2009 clinical trial. RESULTS: A linear dynamic range was achieved of over 5 log scales, allowing for M-protein quantification down to 0.001 g/L. The inter-assay CV of SILuMAB-based MS-MRD was on average 11 %. Excellent concordance between SIL- and SILuMAB-based MS-MRD was shown (R2>0.985). Additionally, signal intensity of spiked SILuMAB can be used for quality control purpose to assess system performance and incomplete SILuMAB digestion can be used as quality control for sample preparation. CONCLUSIONS: Compared to SIL peptides, SILuMAB-based MS-MRD improves the reproducibility, turn-around-times and cost-efficacy of MS-MRD without diminishing its sensitivity and specificity. Furthermore, SILuMAB can be used as a MS-MRD quality control tool to monitor sample preparation efficacy and assay performance.

Semi-Quantitative Multiplex Profiling of the Complement System Identifies Associations of Complement Proteins with Genetic Variants and Metabolites in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. The complement system has been identified as one of the main AMD disease pathways. We performed a comprehensive expression analysis of 32 complement proteins in plasma samples of 255 AMD patients and 221 control individuals using mass spectrometry-based semi-quantitative multiplex profiling. We detected significant associations of complement protein levels with age, sex and body-mass index (BMI), and potential associations of C-reactive protein, factor H related-2 (FHR-2) and collectin-11 with AMD. In addition, we confirmed previously described associations and identified new associations of AMD variants with complement levels. New associations include increased C4 levels for rs181705462 at the C2/CFB locus, decreased vitronectin (VTN) levels for rs11080055 at the TMEM97/VTN locus and decreased factor I levels for rs10033900 at the CFI locus. Finally, we detected significant associations between AMD-associated metabolites and complement proteins in plasma. The most significant complement-metabolite associations included increased high density lipoprotein (HDL) subparticle levels with decreased C3, factor H (FH) and VTN levels. The results of our study indicate that demographic factors, genetic variants and circulating metabolites are associated with complement protein components. We suggest that these factors should be considered to design personalized treatment approaches and to increase the success of clinical trials targeting the complement system.

Quantitative multiplex profiling of the complement system to diagnose complement-mediated diseases.

OBJECTIVES: Complement deficiencies are difficult to diagnose because of the variability of symptoms and the complexity of the diagnostic process. Here, we applied a novel 'complementomics' approach to study the impact of various complement deficiencies on circulating complement levels. METHODS: Using a quantitative multiplex mass spectrometry assay, we analysed 44 peptides to profile 34 complement proteins simultaneously in 40 healthy controls and 83 individuals with a diagnosed deficiency or a potential pathogenic variant in 14 different complement proteins. RESULTS: Apart from confirming near or total absence of the respective protein in plasma of complement-deficient patients, this mass spectrometry-based profiling method led to the identification of additional deficiencies. In many cases, partial depletion of the pathway up- and/or downstream of the absent protein was measured. This was especially found in patients deficient for complement inhibitors, such as angioedema patients with a C1-inhibitor deficiency. The added value of complementomics was shown in three patients with poorly defined complement deficiencies. CONCLUSION: Our study shows the potential clinical utility of profiling circulating complement proteins as a comprehensive read-out of various complement deficiencies. Particularly, our approach provides insight into the intricate interplay between complement proteins due to functional coupling, which contributes to the better understanding of the various disease phenotypes and improvement of care for patients with complement-mediated diseases.

Biosynthetic homeostasis and resilience of the complement system in health and infectious disease.

BACKGROUND: The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection. METHODS: With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens. FINDINGS: In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively. INTERPRETATION: The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. FUND: European Union's Horizon 2020, project PERFORM, grant agreement No. 668303.

Thiopurine methyltransferase in acute lymphoblastic leukaemia: biochemical and molecular biological aspects.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme, catalysing S-methylation of aromatic and heterocyclic sulphhydryl compounds. TPMT activities and genotypes have been determined in patients with acute lymphoblastic leukaemia (ALL) and in control children. Median red blood cell (RBC) TPMT activity in ALL patients at diagnosis was significantly lower than in controls (median 11.5 pmol/10(7) RBC*hr; range 1.7-30.7; n = 191 vs. 14.6 pmol/10(7) RBC*hr; range 1.6-50.7; n = 140). This reduction of TPMT activity in ALL patients was not due to differences in the frequency of mutations in the TPMT gene. In concordance with other authors, we found a higher TPMT activity during maintenance treatment with 6-mercaptopurine (6MP) than at diagnosis and in controls. However, we observed that TPMT activity was already significantly increased after the induction therapy, before the patients received 6MP (median 17.5; range 3.9-40.3 pmol/10(7) RBC*hr; n = 139). In vitro experiments indicate that the early increase of TPMT activity during treatment may be explained by the use of antifolates, e.g., methotrexate and trimethoprim.

Role of 5'-nucleotidase in thiopurine metabolism: enzyme kinetic profile and association with thio-GMP levels in patients with acute lymphoblastic leukemia during 6-mercaptopurine treatment.

Thiopurines are used for treatment of several diseases. Cytotoxicity is caused by the derived compounds 6-thioguanine nucleotides (TGNs) and methyl-6-thioinosine monophosphate (methylthio-IMP). The 6-thiopurine mononucleotides 6-thio-IMP (thio-IMP), 6-thio-GMP (thio-GMP) and methylthio-IMP can be catabolized by purine 5'-nucleotidase. It has been shown that the various 5'-nucleotidases are key enzymes for (6-thio)-purine metabolism. We aimed to investigate whether the overall 5'-nucleotidase (5'NT) activity is correlated with the efficacy and toxicity of 6-thiopurine nucleotides. Substrate affinity of 5'NT for IMP, GMP, AMP, thio-IMP, thio-GMP and methylthio-IMP was studied in human lymphocytes. For each of the substrates, the pH for optimal overall enzyme activity has been determined at a pH range between 6 and 10. At the optimal pH, assays were performed to establish Km and Vmax values. Optimal pH values for the various substrates were between 7 and 8.5. Km values ranged from 33 to 109 microM, Vmax ranged from 3.99 to 19.5 nmol/10(6) peripheral mononuclear cells (pMNC) h, and Vmax/Km ratios ranged from 105 to 250. The results did not show a distinct preference of 5'NT activity for any of the tested thiopurine nucleotides. The enzyme kinetic studies furthermore revealed substrate inhibition by thio-IMP and thio-GMP as a substrate. Inhibition by thio-GMP also seems to occur in patients treated with 6-mercaptopurine (6 MP); subsequently, this may lead to toxicity in these patients.

Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children.

BACKGROUND: Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity. METHODS: We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate. RESULTS: The K(m) values calculated for 6MP were 0.54 (RBC), 0.85 (pMNC) and 0.65 (Molt-F4 cells) mmol/L. The K(m) values for S-adenosylmethionine were 11.9 (RBC), 16.4 (pMNC) and 6.65 (Molt-F4 cells) micro mol/L. The assay variation was 8.2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean +/- standard deviation): female children and young adults, 15.1 +/- 4.8 pmol/10(7) cells per h (n = 44); male children and young adults, 15.8 +/- 6.4 pmol/10(7) cells per h (n = 59). No gender or age differences were found. CONCLUSION: The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.