Complement system analyses and biomarkers.

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1 Complement system analyses and biomarkers

2 MASP1 MASP2 MBL/ficolin Lectin pathway C1r C1 C1s C1q Classical pathway C3 Spontaneous hydrolysis Alternative pathway C3H 2 O FB C4/C2 C3H 2 OBb C4bC2a C3-convertase C3bBb FD Mast cells C3a Monocytes Neutrophils C3aR C5aR Chemotaxis Inflammation C4bC2aC3b C5a C5-convertase Opsonization with ic3b C3bBbC3b Phagocytosis C9 MAC/TCC C8 C7 C5b C6 Cell lysis

3 Complement = inflammation and tissue damage heat redness swelling pain less function

4 Diseases associated with excessive complement activation ischemia/reperfusion (stroke, bypass) transplantation burn injuries asthma glomerulonephritis sepsis age-related macula degeneration Systemic lupus erythematosus (SLE) rheumatoid arthritis hemolytic uremic syndrome hemolytic anemia Alzheimer s multiple sclerosis Guillain-Barre myasthenia gravis

5 MASP1 MASP2 MBL/ficolin Lectin pathway C1r C1 C1s C1q Classical pathway C3 Spontaneous hydrolysis Alternative pathway C3H 2 O FD C4/C2 C3H 2 OBb C4bC2a C3-convertase C3bBb FD Mast cells C3a Monocytes Neutrophils C3aR C5aR Chemotaxis Inflammation C4bC2aC3b C5a C5-convertase Opsonization with ic3b C3bBbC3b Phagocytosis C9 MAC/TCC C8 C7 C5b C6 Cell lysis

6 When is it relevant to test complement function?

7 Warning signs for complement deficiencies meningococcal meningitis at >5 years of age (MAC, properdin) recurrent bacterial infections, esp. with Pneumococcus autoimmune manifestations (C1q/r/s, C4, C2, anti-c1q) angioedema without urticaria (C1-inhibitor) renal inflammatory disorders Hemolytic uremic syndrome (FH, FI, CD46, C3, FB) C3 glomerulopathy (low C3, normal C4, C3NeF)

8 Testing for complement deficiency Hemolytic assays sheep or rabbit erythrocytes incubated with patient sera Quantification of specific components (ELISA, RIA, nephelometry, turbidimetry, TRIFMA) Functional assay for individual components (chromogenic substrate for C1-inh) WIELISA (Eurodiagnostica)

9 To diagnose or follow up complement activation in patients diagnosis of ahus follow up in SLE during treatment with Eculizumab (PNH, ahus, myasthenia gravis)

10 Complement biomarkers Decrease of of C1q, C4, C3, FB levels depend on consumption and synthesis (acute phase proteins) Activation products: C3a, C5a very short half-life (1 min) C3b, C3c, C3d difficult to detect specifically (and not as part of C3) C3bBbP, stcc (soluble MAC) better half-life (1h)

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12 Recommended complement analyses

13 Pre-analytic considerations physiological and pathological complement activation continues ex vivo! samples must be processed fast all samples must be stored in -70 c, even for short-term EDTA at > 10 mm as anticoagulant repeated freezing and thawing not possible

14 The committee for the standardisation and quality assesment of complement measurements References Bergseth G., Ludviksen J.K., Kirschfink M., Giclas P.C., Nilsson B., Mollnes T.E An international serum standard for application in assays to detect human complement activation products. Mol. Immunol. 56, Botto M., Kirschfink M., Macor P., Pickering M.C., Würzner R., Tedesco F Complement in human diseases: Lessons from complement deficiencies. Mol Immunol. 46, Frazer-Abel A, Sepiashvili L, Mbughuni MM, Willrich MA Overview of Laboratory Testing and Clinical Presentations of Complement Deficiencies and Dysregulation.Adv Clin Chem. 2016;77:1-75. Mollnes T.E., Jokiranta T.S., Truedsson L.,Nilsson B, Rodriguez de Cordba S,, Kirschfink M Complement analysis in the 21st century. Mol.Immunol. 44, Nilsson B, Nilsson Ekdahl K Complement diagnostics: concepts, indications, and practical guidelines. Clin. Dev. Immunol. 2012, Prohászka Z, Nilsson B, Frazer-Abel A, Kirschfink M Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control. Immunobiology Nov;221(11):

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16 Assay measuring complement convertases Detects - autoantibodies such as C3Nef - gain of function mutations in C2, C4, C3, FB Blom et al 2014, Clin Exp Immunol, 178,

17 Detection of C3Nef GN32, GN39, GN23, IND4 sera from patients with C3Nef F24 healthy control

18 Detection of mutations #10 p.arg1215gln in FH, benign #11 plys633arg in C3 and anti-fh autoantibodies

19 C4 b-chain g-chain a-chain C4a complement activation C4b What is C4d and why would you like to measure it? Cleavage by factor I C4c C4d antibodies to cleavage neoepitope 45 kda

20 Generation of neoc4d specific Ab

21 Detection with antibodies (abs 490 nm) Specificity of the antibody against C4d Anti C4d Anti C anti -C4d (ours) anti -C4 (Dako) C4 C4b C4b FI C4BP C4b FI mutant C4BP C4 C4b C4b FI C4BP C4b FI mutant C4BP C4 C4b C4d 30 30

22 Our assay Commercial assay Blom et al 2015, Mol Immunol, 66,

23 Increase of C4d after treatment with anti-cd20 (leukemia)

24 Systemic Lupus Erythematosus - SLE Autoimmune 7000 cases in Sweden (450 per year) 90% women Multiple organs affected Flares Autoantibodies against nuclear antigens Complement system involved

25 Enter weight in SLEDAI-2K Score column if descriptor is present at the time of the visit or in the preceding 10 days. 8 Cranial nerve disorder New onset of sensory or motor neuropathy involving cranial nerves. SLE Disease Activity Index (SLEDAI) SLEDAI 2K Descriptor Definition Weight 8 SCORE Lupus headache Severe, persistent headache; may be migrainous, but must be 8 Seizure Recent onset, exclude metabolic, infectious nonresponsive or drug causes. to narcotic analgesia. 8 CVA New onset of cerebrovascular accident(s). Exclude arteriosclerosis. 8 Vasculitis Ulceration, gangrene, tender finger nodules, periungual infarction, splinter hemorrhages, or biopsy or angiogram proof of vasculitis. 4 Arthritis > 2 joints with pain and signs of inflammation (i.e., tenderness, swelling 8 Psychosis Altered ability to function in normal activity due to severe disturbance in the perception of reality. Include hallucinations, incoherence, marked loose associations, impoverished thought content, marked illogical thinking, bizarre, disorganized, or catatonic behavior. Exclude uremia and drug causes 8 Organic brain syndrome Altered mental function with impaired orientation, memory, or other intellectual function, with rapid onset and fluctuating clinical features, inability to sustain attention to environment, plus at least 2 of the following: perceptual disturbance, or incoherent effusion). speech, insomnia or daytime drowsiness, or increased or decreased psychomotor activity. Exclude metabolic, infectious, or drug causes. 8 Visual disturbance Retinal changes of SLE. Include cytoid bodies, retinal hemorrhages, serous exudate or hemorrhages in the choroid, or optic neuritis. Exclude hypertension, infection, or drug causes. 8 Lupus headache Severe, persistent headache; may be migrainous, but must be nonresponsive to narcotic analgesia. 8 CVA New onset of cerebrovascular accident(s). Exclude arteriosclerosis. 8 Vasculitis Ulceration, gangrene, tender finger nodules, cause. periungual infarction, splinter hemorrhages, or biopsy or angiogram proof of vasculitis. 4 Arthritis Proteinuria > 2 joints with pain and signs of >0.5 inflammation gram/24 (i.e., tenderness, hours swelling or effusion). 4 Myositis Proximal muscle aching/weakness, associated with elevated creatine 2 Rash phosphokinase/aldolase or electromyogram changes or a biopsy showing myositis. Inflammatory type rash. 4 Urinary casts Heme-granular or red blood cell casts. 4 Hematuria >5 red blood cells/high power field. Exclude stone, infection or other 2 Mucosal ulcers cause. Oral or nasal ulcerations. 4 Proteinuria >0.5 gram/24 hours 4 Pyuria >5 white blood cells/high power field. Exclude infection. 2 Rash Inflammatory type rash. 2 Alopecia Pericarditis Abnormal, patchy or diffuse loss of hair. 2 Mucosal ulcers Oral or nasal ulcerations. 2 Pleurisy Pleuritic chest pain with pleural rub or effusion, or pleural thickening. 2 Pericarditis Pericardial pain with at least 1 of the following: rub, P effusion, or electrocardiogram or echocardiogram confirmation. 2 Low complement Decrease in CH50, C3, or C4 below the laboratory lower limit of normal for testing laboratory 2 Increased DNA binding Increased DNA binding by Farr assay above normal range for testing laboratory. laboratory. 1 Fever >38 o C. Exclude infectious cause. 1 Thrombocytopenia <100,000 platelets / x10 9 /L, exclude drug causes. 1 Leukopenia < 3,000 white blood cells / x10 9 /L, exclude drug causes. TOTAL SCORE: Low complement is a hallmark of SLE 4 Myositis Proximal muscle aching/weakness, associated with elevated creatine phosphokinase/aldolase or electromyogram changes or a biopsy showing myositis. 8 Cranial nerve disorder New onset of sensory or motor neuropathy involving cranial nerves. 4 Urinary casts Heme-granular or red blood cell casts. 4 Hematuria >5 red blood cells/high power field. Exclude stone, infection or other 4 Pyuria >5 white blood cells/high power field. Exclude infection. 2 Alopecia Abnormal, patchy or diffuse loss of hair. 2 Pleurisy Pleuritic chest pain with pleural rub or effusion, or pleural thickening. 2 Pericardial pain with at least 1 of the following: rub, P effusion, or electrocardiogram or echocardiogram confirmation. 2 Low complement Decrease in CH50, C3, or C4 below the lower limit of normal for testing 2 Increased DNA binding Increased DNA binding by Farr assay above normal range for testing 1 Fever >38 o C. Exclude infectious cause. 1 Thrombocytopenia <100,000 platelets / x10 9 /L, exclude drug causes. 1 Leukopenia < 3,000 white blood cells / x10 9 /L, exclude drug causes.

26 Can we improve assays measuring complement biomarkers in SLE? Currently used: C3 and C4 Problem: concentrations of C3 and C4 depend on both consumption and synthesis Can we measure instead products of complement activation such as C4d?

27 Two SLE sample collections (Lund) Flare & Remission Longitudinal 98 patients sample at highest flare sample in remission 77 healthy controls 69 patients disease duration 7.5 (0 41) years sampling interval 70+/- 30 days follow-up duration 778 ( ) days

28 C4d [mg/l] C4d levels are high in SLE, especially in flare *** Controls SLE Mann-Whitney U p < p < p = p = Martin et al 2017, Art Res Ther, 19, 266

29 Only C4d is a significant marker of disease activity (SLEDAI) ROC-curve analysis C4d AUC = 0.64 ( ) p < Cut-off: C4d high > 0.4 mg/l Clinical reference values C3 low <0.77 g/l and C4 low <0.12 g/l

30 C4d is superior to C4 as marker for flare McNemar s Test C4d high vs C3 low (p = 0.345) C4d high vs C4 low (p < ) n = 37 (C4d high &C3 low ) 17 (C4d high ) n = 22 (C4d high &C4 low ) 32 (C4d high ) 11 (C3 low ) 33 (-) 7 (C4 low ) 37 (-) C4d high C3 low C4 low PPV = Positive Predictive Value NPV = Negative Predictive Value

31 C4d associates with nephritis Nephritis: - most severe complication of SLE - affects 30-50% SLE patients - early detection/prediction of onset = faster adjustment of treatment

32 C4d is superior to C4 as marker for nephritis McNemar s Test C4d high vs C3 low (p = 0.508) C4d high vs C4 low (p = 0.002) n = 21 (C4d high &C3 low ) 6 (C4d high ) n = 12 (C4d high &C4 low ) 15 (C4d high ) 3 (C3 low ) 4 (-) 2 (C4 low ) 5 (-) C4d high C3 low C4 low PPV = Positive Predictive Value NPV = Negative Predictive Value

33 C4d may be used to predict onset of nephritis Is C4d increased in sample preceding nephritis by 2 months? all 69 patients 19 patients which at some point had nephritis (recurring)

34 Benign nodule Lung cancer

35 C4d (AU) Sensitivity C4d-containing fragments ( g/ml) Sensitivity C4d in patients with lung cancer vs matched healthy controls 3 P= Commercial assay AUC=0.69 Control Cancer Specificity P< Our assay AUC=0.82 Control Cancer Specificity Ajona et al 2018, Oncotarget, 9, 6346

36 C4d (AU) Sensitivity C4d-containing fragments ( g/ml) Sensitivity C4d in patients with indeterminate lung nodules P= AUC= Commercial assay Control Cancer Specificity P= AUC= Our assay Control Cancer Specificity

37 C4d (CAU) C3bBbP CAU) TCC (CAU) Complement biomarkers in rheumatic diseases *** *** *** healthy controls rheumatoid arthritis * systemic sclerosis psoriatic arthritis ankylosing spondylitis healthy controls rheumatoid arthritis *** systemic sclerosis psoriatic arthritis ankylosing spondylitis healthy controls rheumatoid arthritis systemic sclerosis psoriatic arthritis ankylosing spondylitis Strong correlation of all biomarkers with scleroderma renal crisis

38 Indications of complement activation in scleroderma renal crisis No C3b deposits C3b in glomerulus C3b in tubuli IgA nephropathy

39 Take home messages Complement diagnostic assays are poorly standardised Pre-analytic issues are of grave importance Complement biomarkers may be used for: - diagnosis of complement deficiency and some disorders such as ahus - selection of patients to be treated with complement inhibitors - follow up of treatment afficacy - identification of novel diseases/patient groups with complement involvement - development of new complement inhibitors antibody against cleavage neo-epitope in C4d has superior specificity C4d may be a good biomarker of nephritis in SLE

40 Thanks to: Myriam Martin Marcin Okroj Birgitta Gullstrand Anders Bengtsson Roger Hesselstrand Tore Saxne Ruben Pio Daniel Ajona Yngve Sommarin

41 C3 Tick-over C3 H2O Bb initial C3 convertase C3a fd fb C3 convertase C2a C4b C1 MBL/ficolin C3b amplification C5 convertase C3b C4b C2a Bacterium MAC C8 C7 C6 C5b C9 C5 C3bC3b Bb C5 convertase C5a properdin C3b Bb C3 convertase fd fb