1145391 research-article2022 ESO0010.1177/23969873221145391European Stroke JournalHonegger et al. Original Research Article European Stroke Journal Serum S-100B adds incremental value for 2023, Vol. 8(1) 309­–319 © European Stroke Organisation 2022 the prediction of symptomatic intracranial Article reuse guidelines: hemorrhage and brain edema after acute sagepub.com/journals-permissions https://doi.org/10.1177/23969873221145391 DOI: 10.1177/23969873221145391 journals.sagepub.com/home/eso ischemic stroke Tim Honegger1 , Juliane Schweizer1, Antonela Bicvic2, Laura P Westphal1, Valerie Schütz1, Corinne Inauen1, Thomas Pokorny1, Katja Bracher1, Marcel Arnold2, Urs Fischer2,3 , Leo H Bonati3 , Gian Marco De Marchis3 , Krassen Nedeltchev4, Timo Kahles4 , Carlo Cereda5, Georg Kägi6, Joan Montaner7,8,9, Alejandro Bustamante10, Elena Palà11, George Ntaios12 , Christian Foerch13, Andreas Luft1, Katharina Spanaus14, Lanja Saleh14, Arnold von Eckardstein14, Markus Arnold1* and Mira Katan1,3* Abstract Background: Early identification of patients developing symptomatic intracranial hemorrhage and symptomatic brain edema after acute ischemic stroke is essential for clinical decision-making. Astroglial protein S-100B is a marker of blood- brain barrier disruption, which plays an important role in the formation of intracranial hemorrhage and brain edema. In this study, we assessed the prognostic value of serum S-100B for the development of these complications. Methods: Serum S-100B levels were measured within 24 h from symptom onset in 1749 consecutive acute ischemic stroke patients from the prospective, observational, multicenter BIOSIGNAL cohort study (mean age 72.0 years, 58.3% male). To determine symptomatic intracranial hemorrhage or symptomatic brain edema, follow-up neuroimaging was performed in all patients receiving reperfusion therapy or experiencing clinical worsening with an NIHSS increase of ⩾4. Results: Forty six patients (2.6%) developed symptomatic intracranial hemorrhage and 90 patients (5.2%) developed symptomatic brain edema. After adjustment for established risk factors, log10S-100B levels remained independently associated with both symptomatic intracranial hemorrhage (OR 3.41, 95% CI 1.7–6.9, p = 0.001) and symptomatic brain edema (OR 4.08, 95% CI 2.3–7.1, p < 0.001) in multivariable logistic regression models. Adding S-100B to the clinical 1  epartment of Neurology, University Hospital Zurich, Zurich, Switzerland D 2 Department of Neurology, Inselspital University of Berne, Switzerland 3 Department of Neurology and Stroke Center, University Hospital Basel, Switzerland 4 Department of Neurology, Kantonsspital Aarau, Switzerland 5 Stroke Center, Neurocenter of Southern Switzerland (NSI), Ospedale Regionale di Lugano, Switzerland 6 Department of Neurology, Kantonsspital St. Gallen, Switzerland 7 Neurovascular Research Laboratory, Vall d’Hebron Institute of Research (VHIR) Barcelona 8 Institute de Biomedicine of Seville, IBiS/Hospital Universitario Virgen del Rocío/CSIC/University of Seville 9 Department of Neurology, Hospital Universitario Virgen Macarena, Seville 10 Stroke Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain 11 Neurovascular Research Laboratory, Vall d’Hebron Institute of Research (VHIR)-Universitat Autònoma de Barcelona, Barcelona, Spain 12 Department of Internal Medicine, University of Thessaly, Larissa, Greece 13 Department of Neurology, Goethe University Frankfurt, Germany 14 Institute of Clinical Chemistry, University Hospital Zurich, Switzerland *These authors contributed equally to this work. Corresponding author: Mira Katan, University Hospital Basel, Neurologie, Petersgraben 4, Basel 4031, Switzerland. Email: mira.katan@usb.ch 310 European Stroke Journal 8(1) prediction model increased the AUC from 0.72 to 0.75 (p = 0.001) for symptomatic intracranial hemorrhage and from 0.78 to 0.81 (p < 0.0001) for symptomatic brain edema. Conclusions: Serum S-100B levels measured within 24 h after symptom onset are independently associated with the development of symptomatic intracranial hemorrhage and symptomatic brain edema in acute ischemic stroke patients. Thus, S-100B may be useful for early risk-stratification regarding stroke complications. Keywords S-100B, stroke complications, intracranial hemorrhage, brain edema, ischemic stroke, serum biomarker Date received: 27 September 2022; accepted: 28 November 2022 Introduction collection blood samples and clinical data for further analysis. In patients with acute ischemic stroke (AIS), secondary neuronal damage can occur in subsequent hours to days, caused by complications such as symptomatic intracranial Study population hemorrhage (sICH) or symptomatic brain edema (sBE). The design of the BIOSIGNAL study has been described in AIS followed by these complications is associated with detail elsewhere.15 In brief, patients older than 18 years who poor functional outcome and higher mortality.1,2 Therefore, were admitted with AIS and gave consent were recruited early risk-stratification is crucial to guide patient manage- within 24 h from symptom onset between October 2014 to ment, such as the decision over intensive care unit place- October 2017. Patients who were diagnosed with primarily ment or intensified monitoring. Moreover, such information hemorrhagic stroke, transient ischemic attack or stroke may facilitate time-sensitive therapy, such as decompres- mimics (e.g. migraine, epilepsy) at hospital discharge, or sive hemicraniectomy. Inversely, identifying low-risk who refused informed consent were excluded from the patients may help to improve health care allocation. study. Recruitment was stopped after 150 recurring cere- Patient history, clinical assessment and neuroimaging on brovascular events occurred, as intended for a different admission provide some prognostic information, but their research question within the BIOSIGNAL study. predictive value for complications is limited.3,4 Serum bio- markers of blood-brain barrier (BBB) disruption could be an important adjunct to predict complications, as BBB dis- Data collection ruption has previously been associated with sBE and sICH.5 Demographic variables, vital signs, vascular risk factors, S-100B, a calcium-binding protein produced predomi- National Institute of Health Stroke Scale (NIHSS) score, nantly by astroglial cells, is an established biomarker of and modified Charlson Comorbidity Index were docu- BBB dysfunction.6,7 Its association with stroke severity, mented on admission. All patients received brain imaging infarction volume and functional outcome in AIS patients is with CT or MRI on admission. Standard etiological work well-researched.8–11 Pilot studies have demonstrated an up included 12-lead ECG, >24 h continuous ECG monitor- association of S-100B with sICH and sBE.12–14 In this study, ing, transthoracic and/or transoesophageal echocardiogra- we aimed to externally validate these associations and eval- phy as well as cerebrovascular ultrasound and/or CT or MR uate the incremental prognostic value of serum S-100B lev- angiography. Per protocol, in all patients who received any els for the development of sICH and sBE during form of reperfusion therapy (i.e. intravenous or intraarterial hospitalization when measured within 24 h after symptom thrombolysis (IVT/IAT) and/or mechanical thrombec- onset in a large multicentric AIS cohort. tomy), control neuroimaging was performed after 24 h. In addition, all patients showing clinical worsening during Methods hospital stay, defined as an increase in NIHSS by ⩾4 points, received control neuroimaging. Patients who Study design and approval deceased, who were treated palliatively or who were trans- The Biomarker Signature of Stroke Etiology (BIOSIGNAL) ferred to a different hospital before control neuroimaging study is a prospective, observational, multicenter, incep- was performed were excluded from the final analysis tion cohort study to evaluate selected blood biomarkers in (n = 10) (Figure 1). patients with confirmed AIS (NCT02274727). The study protocol was approved by each local Ethics Committee Blood sampling and conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was Venous blood was sampled in EDTA-containing plastic obtained from all patients or their welfare guardians for the tubes within 24 h after symptom onset. Samples were Honegger et al. 311 Figure 1. Final study population selection process. immediately centrifuged at 3000g and 4°C for 20 min. To evaluate the association of S-100B levels with pri- Plasma was alliquoted and stored at −80°C until time of mary outcomes, multivariable logistic regression models analysis. S-100B levels (µg/ml) were measured from were fitted to calculate odds ratios (OR) and 95% confi- plasma in a blinded batch analysis with the Roche Elecsys dence interval (95% CI). Models for sICH and sBE were S100 assay running on the e801 unit of the Cobas 8000 adjusted for pre-defined covariates based on previous stud- platform (Roche Diagnostics, Mannheim, Germany) with a ies and pathophysiological considerations. The pre-defined lower limit of detection of 0005 µg/l and a lower limit of model for sICH included age,18,19 sex,19 previous use of quantification of 0.015 µg/l. Inter- and intraassay impreci- antithrombotic drugs (oral anticoagulants and/or antiplate- sion of the assay as expressed by coefficient of variance lets),18 NIHSS on admission,18,19 reperfusion therapy (IVT/ was less than 2.6%. No values below LoQ were observed. IAT and/or mechanical thrombectomy),16 as well as log10S- Analysis can be performed on two different immunoassay 100B levels. The pre-defined model for sBE included age,17 analyzing machines, is completed within 18 min and is sex,20 NIHSS on admission,17,20,21 CRP levels,22,23 reperfu- commercially available. sion therapy,24 affection of middle cerebral artery (MCA),25 and log10S-100B levels. Interaction analyses were per- formed to assess effect modification of log10S-100B by Outcome variables demographic variables and established risk factors. Finally, Primary outcome measures included the occurrence of we assessed the influence of time from symptom onset to sICH and sBE. Based on the ECASS-II study, we defined blood draw on S-100B levels as well as both outcomes with sICH as apparent extravascular blood in the brain or within separate logistic regressions. Patients with wake-up stroke the cranium on imaging in temporal correlation with clini- and unknown symptom onset were excluded from this anal- cal worsening (increase in NIHSS by ⩾4 points) or death ysis, as the time interval could not reliably be determined. due to neurological deterioration.16 sBE was defined as For both outcomes we used additional stepwise model apparent mass effect of the brain on control neuroimaging building strategies for a best out-of-sample predictive as the predominant cause of clinical deterioration (increase model. Variables were selected based on univariate logistic in NIHSS by ⩾4 points) or death as well as by the indica- regression with a cut-off level of p < 0.15 to achieve reten- tion for decompressive hemicraniectomy.17 The neuroradi- tion of possibly meaningful covariates.26 Backward selec- ologist on duty, who was blinded to S-100B levels, tion was performed with a cut-off level of p < 0.05. evaluated the presence of intracranial hemorrhage or brain Receiver Operating Characteristic (ROC) curves were plot- edema at each site separately. ted and Areas under the curve (AUC) were calculated as an overall discriminatory measure. Models with and without S-100B were compared using the likelihood-ratio test. To Statistical analysis assess improvement in classification after adding S-100B Discrete variables are expressed as counts (percentages), to the model, continuous net reclassification improvement continuous variables as medians (interquartile range (NRI) was computed. Subgroup analyses were performed (IQR)). Logarithmic transformation (log10) was used to based on stroke severity as well as treatment with IVT. normalize skewed distributions, including serum levels of Different S-100B cut-off values were evaluated for S-100B. For group comparisons, Fisher’s exact test or inclusion and exclusion of patients at increased risk for Mann-Whitney U test was conducted where appropriate. sICH, respectively sBE. We chose cut-off values with 312 European Stroke Journal 8(1) respective specificity values above 90% and calculated the model increased its discriminatory accuracy from an AUC corresponding sensitivity, negative and positive predictive of 0.72 (95% CI 0.65–0.78) to 0.75 (95% CI 0.68–0.82, values. p = 0.001) (Supplemental Figure 1). Overall continuous In addition, we applied six existing sICH prognostica- NRI was 0.36 (p = 0.02) with event NRI being 0.02 and tion scores for AIS patients after IVT to our cohort: SITS non-event NRI 0.34, meaning that additional 2% of cases score,27 SEDAN score,28 HAT score,29 SPAN-100,30 MSS,31 received higher probabilities to be correctly classified into and GRASPS score.19 Where necessary for calculation of the event group, and additional 34% of cases received the score, analysis was only performed in patients receiving higher probabilities to be correctly classified into the non- CT on admission. Patients without IVT were excluded from event group. this analysis. All statistical analysis was performed using In the best out-of-sample predictive model based on Stata (Version 15.1, StataCorp LLC, College Station, stepwise model building, log10S-100B remained as an inde- Texas). pendent predictor (OR 4.17, 95% CI 2.2–7.8, p < 0.0001 per unit log10S-100B increase; improvement of AUC from 0.65 [95% CI 0.57–0.73] to 0.73 [95% CI 0.64–0.81], Results p < 0.0001). The final model included log10S-100B, reper- Patient baseline characteristics fusion therapy and coronary heart disease. Application of six existing sICH prognostication scores Of 1759 AIS patients enrolled in the study, 10 were excluded after IVT to our cohort revealed AUC values ranging from as pre-defined control neuroimaging could not be per- 0.48 to 0.63 versus 0.68 to 0.77 in the original studies.19,27–31 formed because of a switch to palliative care treatment The addition of log10S-100B to the scores led to an increase (n = 6), transfer to a different hospital (n = 3), or death with- of the AUC, ranging from 0.71 to 0.73. In all scores, this out prior follow up neuroimaging (n = 1). In the remaining increase was significant (p < 0.01). Adjustment for other 1749 patients, blood samples were available in 98.4% with score items revealed that log10S-100B remained indepen- a median S-100B concentration of 0.08 µg/l (IQR 0.05– dently associated with sICH in every score (Table 3). 0.15). The median S-100B concentration in patients who were excluded was 0.09 µg/l (IQR 0.08–0.17), which was not significantly different from that of included cases Predictive value of S-100B for sBE (p = 0.23). Mean age was 72.0 years, 41.7% were female. Serum S-100B levels were significantly higher in patients All patients who suffered apparent clinical worsening and with sBE (0.17 µg/l [IQR 0.08–0.41] vs 0.08 µg/l [0.05– all patients receiving re-canalization therapy received con- 0.14], p < 0.0001). In the predefined multivariable regres- trol neuroimaging. Participating centers were free to extend sion model, only log10S-100B (OR 4.08, 95% CI 2.3–7.1, the indication for follow up neuroimaging, for example fol- p < 0.001 per unit log10S-100B increase), age (OR 0.97, lowing clinical worsening with an increase in NIHSS of 95% CI 0.95–0.98, p < 0.0001) and NIHSS on admission less than four points or as a standard of care. In total, 1584 (OR 1.11, 95% CI 1.1–1.2, p < 0.001) remained indepen- patients (90.6%) received follow up neuroimaging. The dently associated with sBE (Table 4). Adding log10S-100B complication sICH occurred in 46 patients (2.6%), while 90 to the model improved its discriminatory accuracy from an patients (5.2%) developed sBE. Patients undergoing reper- AUC of 0.78 (95% CI 0.73–0.83) to 0.81 (95% CI 0.76– fusion therapy showed higher rates of stroke complications 0.86, p < 0.001) (Supplemental Figure 2). Overall continu- (sICH: 3.9% [n = 32/817] vs 1.5% [n = 14/932], sBE 6.7% ous NRI was 0.44 (p < 0.001) with event NRI being 0.13 [n = 55/817] vs 3.8% [n = 33/932]). Among patients with and non-event NRI 0.31. Interaction analysis revealed no sICH, 32.6% (n = 15) died during hospital stay and among significant interaction between log10S-100B and the inde- patients with sBE, 23.3% (n = 21) received decompressive pendent variables. hemicraniectomy while 46.7% (n = 42) died during the hos- Using stepwise model building, log10S-100B remained as pitalization. Further details on baseline characteristics are an independent predictor in the best out-of-sample model (OR listed separately (Table 1). 4.02, 95% CI 2.4–6.8 p < 0.001 per unit log10S-100B increase; improvement of AUC from 0.77 [95% CI 0.72–0.82] to 0.81 Predictive value of S-100B for sICH [95% CI 0.76–0.86], p < 0.001). The resulting model included log10S-100B, age, NIHSS, and log10creatinine. Serum S-100B levels were significantly higher in patients who developed sICH (0.15 µg/l [IQR 0.08–0.26] vs Subgroup analyses 0.08 µg/l [0.05–0.14], p < 0.0001). In the pre-defined mul- tivariable regression model, only log10S-100B (OR 3.40, For sICH, the interaction analysis showed a significant 95% CI 1.69–6.87 per unit log10S-100B increase, p = 0.001) interaction between log10S-100B and NIHSS on admission and reperfusion therapy (OR 2.18, 95% CI 1.09–4.36, in the multivariable model (p = 0.04). Therefore, we p < 0.05) remained independently associated with sICH performed a subgroup analysis for patients with minor ­ during hospitalization (Table 2). Adding log10S-100B to the stroke (NIHSS ⩽ 5) and non-minor stroke (NIHSS ⩾ 6). Honegger et al. 313 Table 1. Baseline characteristics categorized by sICH and sBE. No. (%) Total sICH No sICH p value sBE No sBE p value 1749 (100) 46 (2.6) 1703 (97.4) 90 (5.15) 1659 (94.85) Demographic data Age, median (IQR) 74.4 (64–82) 76.0 (70–81) 74.3 (64–82) 0.87 70.7 (61–81) 74.5 (64–82) 0.04 Female sex, n (%) 730 (41.7) 20 (43.5) 710 (41.7) 0.88 33 (36.7) 697 (41.7) 0.33 Medical history BMI, kg/m2, median (IQR)* 25.5 (23–28) 24.2 (23–27) 25.6 (23–28) 0.16 25.3 (23–28) 25.6 (23–28) 0.50 Hypertension, n (%) 1272 (72.7) 38 (82.6) 1234 (72.5) 0.18 65 (72.2) 1207 (72.8) 0.90 Dyslipidemia, n (%) 1349 (77.1) 37 (80.4) 1312 (77.0) 0.72 61 (67.8) 1288 (77.6) 0.04 Diabetes mellitus, n (%) 308 (17.6) 12 (26.1) 296 (17.4) 0.17 13 (14.4) 295 (17.8) 0.48 Smoking, n (%) 392 (22.7) 8 (18.2) 384 (22.8) 0.59 20 (23.0) 372 (22.6) 0.90  History of stroke or TIA, 299 (17.1) 11 (23.9) 288 (16.9) 0.23 17 (18.9) 282 (17.0) 0.67 n (%)  Family history of 211 (13.1) 4 (9.5) 207 (13.2) 0.65 9 (12.2) 202 (13.1) 1.00 cardiovascular events, n (%)+ Heart failure, n (%) 111 (6.4) 5 (10.9) 106 (6.2) 0.21 5 (5.6) 106 (6.4) 1.00 Coronary heart disease, n (%) 354 (20.3) 16 (34.8) 338 (19.9) 0.02 16 (17.8) 338 (20.4) 0.69 Atrial fibrillation, n (%) 318 (18.2) 13 (28.3) 305 (17.9) 0.08 25 (27.8) 293 (17.7) 0.02  Peripheral arterial disease, 219 (12.5) 6 (13.0) 213 (12.5) 0.82 12 (13.3) 207 (12.5) 0.75 n (%) Chronic kidney disease, n (%) 512 (29.3) 14 (30.4) 498 (29.3) 0.87 28 (31.1) 484 (29.2) 0.72  Charlson Comorbidity Index, 1 (0–2) 1 (0–3) 0 (0–2) 0.03 0 (0–2) 1 (0–2) 0.48 median (IQR) Stroke severity NIHSS, median (IQR) 5 (2–11) 11 (5–16) 5 (2–11) <0.001 15 (7–17) 5 (2–11) <0.001 Stroke territory MCA affected, n (%) 1371 (78.4) 41 (89.1) 1330 (78.1) 0.10 75 (83.3) 1296 (78.1) 0.29 Left MCA affection 719 (41.1) 26 (56.5) 693 (40.7) 0.03 43 (47.8) 676 (40.7) 0.19 Right MCA affection 605 (34.6) 13 (28.3) 592 (34.8) 0.43 31 (34.4) 574 (34.6) 1.00 Bilateral MCA affection 47 (2.7) 2 (4.3) 45 (2.6) 0.35 1 (1.1) 46 (2.8) 0.51 Acute treatment Reperfusion therapy, n (%) 817 (46.7) 32 (69.6) 785 (46.1) <0.01 55 (61.1) 762 (45.9) <0.01  Intravenous thrombolysis, 705 (40.3) 29 (63.0) 676 (39.7) <0.01 42 (46.7) 663 (40.0) 0.23 n (%)  Intraarterial thrombolysis, 13 (0.7) 1 (2.2) 12 (0.7) 0.29 2 (2.2) 11 (0.7) 0.14 n (%)  Mechanical recanalization, 333 (19.1) 19 (41.3) 314 (18.5) <0.001 39 (43.8) 294 (17.8) <0.001 n (%) Medication before admission Antithrombotic drugs, n (%) 860 (49.2) 29 (63.0) 831 (48.8) 0.07 42 (46.7) 818 (49.3) 0.67 Antihypertensive drugs, n (%) 1042 (59.6) 32 (69.6) 1010 (59.3) 0.17 52 (58.4) 990 (59.7) 0.83 Lipid-lowering drugs, n (%) 552 (31.6) 21 (45.7) 531 (31.2) 0.05 27 (30.3) 525 (31.7) 0.91 Laboratory values, median (IQR) S-100B (µg/l) 0.08 (0.05–0.15) 0.15 (0.08–0.26) 0.08 (0.05–0.14) <0.001 0.17 (0.08–0.41) 0.08 (0.05–0.14) <0.001 Creatinine (µmol/l) 83 (69–99) 86 (74–97) 83 (69–99) 0.55 89 (71–105) 83 (69–99) 0.17 LDL-cholesterol (mmol/l)& 2.7 (2.0–3.4) 2.4 (1.4–3.3) 2.7 (2–3.4) 0.09 2.6 (2–3.3) 2.7 (2–3.4) 0.23 HDL-cholesterol (mmol/l)% 1.3 (1.1–1.6) 1.4 (1.0–1.7) 1.3 (1.1–1.6) 0.86 1.4 (1.0–1.7) 1.3 (1.1–1.6) 0.77 CRP (mg/l) 3.4 (1.3–9.6) 8.4 (1.7–13) 3.3 (1.3–9.3) 0.02 8.4 (1.7–13) 3.3 (1.3–9.3) <0.001 sICH: symptomatic intracranial hemorrhage; sBE: symptomatic brain edema; IQR: interquartile range; BMI: body mass index; NIHSS: National Insti- tute of Health Stroke Scale; MCA: middle cerebral artery; LDL: low density lipoprotein; HDL: high density lipoprotein; CRP: C-reactive protein. Statistics: values are median (IQR) or counts (percentages). Testing performed using the Mann-Whitney U Test for continuous variables and Fisher’s exact test for binary variables. *8.3% missing values. + 7.6% missing values. & 6.6% missing values. % 5.2% missing values. All other variables had less than 2.5% of values missing. Percentages and IQR are based on available data. Bold indicates statistical significance p < 0.05. 314 European Stroke Journal 8(1) Table 2. Multivariable logistic regression and ROC analyses for sICH with and without log10S-100B. Predictors Model with log10S-100B Model without log10S-100B OR 95% CI p Value OR 95% CI p Value Log10S-100B 3.41 1.69–6.87 0.001 - - - Age 0.99 0.97–1.02 0.47 0.10 0.97–1.02 0.68 Female sex 0.88 0.47–1.66 0.70 0.99 0.53–1.85 0.98 NIHSS on admission 1.04 0.99–1.10 0.09 1.08 1.03–1.13 <0.01 Reperfusion therapy 2.18 1.09–4.36 <0.05 1.91 0.97–3.76 0.06 Antithrombotic drugs 1.80 0.93–3.47 0.08 1.81 0.94–3.50 0.08 Predictors AUC 95% CI Likelihood ratio test (p value)1 Log10S-100B 0.67 0.59–0.75 - Model without log10S-100B 0.72 0.65–0.78 - Model with log10S-100B 0.75 0.68–0.82 0.01 sICH: symptomatic intracranial hemorrhage; sBE: symptomatic brain edema; OR: Odds Ratio; 95% CI: 95% Confidence Interval; NIHSS: National Institute of Health Stroke Scale; Reperfusion therapy: intravenous or intraarterial thrombolysis and/or mechanical recanalization; AUC: Area Under the Receiver Operating Characteristic Curve. 1 To compare the AUC of nested (without log S-100B) versus whole model (with log S-100B). Bold indicates statistical significance p < 0.05. Table 3. AUC of prediction scores for sICH after IVT in the original studies, in a comparison of the scores and applied to the BIOSIGNAL study cohort. (a) AUC of sICH prediction scores reported in previous studies. (b) AUC of sICH prediction scores calculated in the BIOSIGNAL study cohort. MSS HAT score SEDAN score GRASPS score SITS score SPAN-100 Original studies MSS31 0.68 - - - - - HAT score29 - 0.74 - - - - SEDAN score27 - - 0.77 - - - GRASPS score19 - - - 0.71 - - SITS score27 - - - - 0.70 - SPAN-10030 - - - - - 0.73 Comparison of scores4 NINDS study 0.62 0.65 0.69 0.67 0.61 0.55 ECASS-II tria 0.63 0.65 0.70 0.67 0.64 0.56 SITS registry 0.66 0.64 0.69 0.67 0.67 0.56 BIOSIGNAL study* Without log10S-100B 0.63 0.52+& 0.63+ 0.61 0.62 0.50 Adding log10S-100B 0.72 0.73+& 0.73+ 0.72 0.72 0.71 p Value <0.01 <0.001 <0.01 <0.01 =0.001 <0.001 AUC: Area under the curve; sICH: symptomatic intracranial hemorrhage; IVT: intravenous thrombolysis. *Including only BIOSIGNAL patients who received intravenous thrombolysis (n = 705). + Including only BIOSIGNAL patients receiving IVT and CT as initial imaging modality (n = 601). & The HAT Score could only partially be reconstructed in BIOSIGNAL patients, as extent of hypodensity on CT was not systematically assessed and documented. Log10S-100B remained as an independent predictor of below 0.04 µg/l showed a specificity of 90.9% for not sICH in patients with minor stroke (OR 18.8 [5.4–65.0], developing sICH (13th percentile of the cohort) (Table 5). p < 0.001), while the association was attenuated in the non- Therefore, it could be used for exclusion of severe clini- minor stroke group (OR 1.9 [0.81–4.32], p = 0.14). In the cal courses after AIS. For inclusion of patients at high bivariate model predicting sICH, both log10S-100B and risk for the development of sICH, a cut off-value of NIHSS on admission remained independently associated S-100B above 0.31 µg/l showed a specificity of 90.2% (p < 0.01 respectively). (89th percentile). Further, we evaluated cut-off values for the inclusion For the outcome variable sBE, log10S-100B remained and exclusion of patients at elevated risk for sICH in significantly associated both in cases with minor and non- patients with minor stroke. A cut off-value of S-100B minor stroke (p < 0.001 respectively). Honegger et al. 315 Table 4. Multivariate logistic regression and ROC analyses for sBE with and without log10S-100B. Model with log S-100B Model without log S-100B Predictors OR 95% CI p Value OR 95% CI p Value Log10S-100B 4.08 2.34–7.14 <0.001 - - - Age 0.97 0.95–0.98 <0.001 0.97 0.96–0.99 <0.001 Female sex 0.62 0.38–1.01 0.06 0.73 0.45–1.17 0.19 NIHSS on admission 1.11 1.07–1.15 <0.001 1.14 1.10–1.18 <0.001 Reperfusion therapy 1.12 0.68–1.85 0.46 1.00 0.61–1.63 1.00 Log CRP 1.19 0.82–1.74 0.91 1.49 1.04–2.15 <0.05 MCA Affected 1.04 0.56–1.95 0.12 0.95 0.51–1.74 0.86 Predictors AUC 95% CI Likelihood ratio test (p value)1 Log10S-100B 0.70 0.64–0.76 - Model without log S-100B 0.78 0.73–0.83 - Model with log10S-100B 0.81 0.76–0.86 <0.001 sBE: symptomatic brain edema; OR: Odds Ratio; 95% CI: 95% Confidence Interval; NIHSS: National Institute of Health Stroke Scale; Reperfusion therapy: intravenous or intraarterial thrombolysis and/or mechanical recanalization; CRP: C-reactive protein; MCA: middle cerebral artery; AUC: Area Under the Receiver Operating Characteristic Curve. 1 To compare the AUC of nested (without log S-100B) versus whole model (with log S-100B). Bold indicates statistical significance p < 0.05 Table 5. Cut-off sensitivities and specificities for sICH in patients with minor stroke (NIHSS ⩽ 5). Inclusion of patients at risk (rule in cut-off) Exclusion of patients at risk (rule out cut-off) S-100B cut-off level (µg/l) >0.7 <0.04 Specificity (%) 98.7 90.9 Sensitivity (%) 36.4 19.8 NPV (%) 99.2 98.9 PPV (%) 26.3 2.8 sICH: symptomatic intracranial hemorrhage; sBE: symptomatic brain edema; NPV: negative predictive value; PPV: positive predictive value. Moreover, subgroup analysis was performed based on large cohort of AIS patients. Adding log10S-100B to the whether patients received IVT. These patients did not show pre-defined prediction models led to an increase in the higher S-100B levels and did not develop sBE more often diagnostic accuracy of both models measured by compari- than patients receiving conservative treatment. However, son of AUC and reclassification indices, showing its incre- patients developed sICH significantly more often after IVT mental value beyond previously established risk factors. than after conservative treatment (4.1% [n = 29] vs 1.6% Of note, interaction analysis revealed only significant [n = 17], p < 0.01). Lastly, we assessed the influence of time effect modification of S-100B levels by clinical stroke to blood draw on S-100B levels. S-100B was not signifi- severity concerning the outcome of sICH. Subgroup analy- cantly lower when blood was drawn before the median time sis showed an attenuated association in patients with of 270 min (IQR 110–900 min) compared to later blood NIHSS ⩾ 6. We hypothesized that the explanatory power of draw (0.07 µg/l [0.05–0.13] vs 0.08 µg/l [0.05–0.14], BBB disruption measured by S-100B is strong especially in p = 0.18). Inclusion of time from symptom onset to blood patients with smaller infarcts at generally lower risk of draw to the models for sBE and sICH did not alter the exist- sICH, whereas the additional explanatory power of S-100B ing association of S-100B with both outcomes (OR 4.66 diminishes in patients with more severe infarcts (according [2.3–9.3] and 4.63 [2.0–10.6] respectively, p < 0.001 for to NIHSS levels) who inherently are at higher risk of sICH. both). Time to blood draw showed no significant associa- This is particularly interesting as the assessment of the risk tion with both outcomes (OR 1.00 [1.0–1.0] for both, for development of sICH is especially challenging in p = 0.47 and 0.15 respectively). patients with mild stroke. Our data suggest that serum S-100B levels over 0.7 µg/l in patients with minor strokes (NIHSS ⩽ 5) are highly specific for sICH and might indi- Discussion cate a need for intensified and prolonged monitoring. This study confirmed the predictive value of serum S-100B Our findings regarding the association of S-100B and levels measured within 24 h after symptom onset for the sICH are in line with those of pilot studies. In a prospective occurrence of sICH and sBE in an unselected, prospective, study of 458 AIS patients without IVT or anticoagulation 316 European Stroke Journal 8(1) therapy, S-100B was independently associated with sICH sICH diagnosis, whereas in our study an increase of NIHSS in multivariable models adjusted for age, NIHSS, platelet of ⩾4 points was required. However, despite various patho- count, hypertension, diabetes, and antiplatelet therapy.12 physiological considerations behind the scores, log10S-100B Another study including 275 AIS patients found S-100B led to significant increase of the AUC in every score (Table levels in the highest quintile compared to the lowest quin- 3). Also, log10S-100B remained independently associated tile to be independently associated with hemorrhagic trans- with sICH after adjustment for the other variables in every formation 24 h after IVT.13 score. This underlines the validity of S-100B as a useful Evidence regarding the association between S-100B lev- prognostic adjunct regarding the development of sICH. els and sBE is scarcer. A pilot study including 51 patients Scores have also been developed for the assessment of with proximal MCA occlusion showed significantly higher malignant MCA infarction. The EDEMA Score was devel- S-100B levels in patients with cerebral herniation or treat- oped based on data from 222 AIS patients with severe ment with decompressive hemicraniectomy. S-100B levels stroke and showed a sensitivity of 0.73 and a specificity of 24 h after symptom onset >1.03 µg/l predicted a malignant 0.70 at a score of 3 for death with midline shift >5 mm or infarct with a specificity of 83%.14 While pre-selection of need for decompressive hemicraniectomy.36 However, as patients with proximal MCA occlusion and different defini- radiological features of brain edema are items of the score, tions limit a direct comparison with this study, our findings it is not predictive in nature. The DASH Score was devel- confirm the previously hypothesized association between oped in a study with 119 AIS patients with ICA- or S-100B and brain edema. M1-occlusion receiving MRI on admission and showed an Aside from S-100B, investigators have strived to predict AUC of 0.88 for the detection of malignant MCA infarction both intracranial hemorrhage12 and brain edema14 using within 48 hours.3 This high diagnostic accuracy likely other blood biomarkers associated with BBB disruption resulted from the pre-selection of stroke patients at risk for including matrix metalloprotease-9,32 glial fibrillary acidic stroke complications. Furthermore, the potentially missing protein,33 neuron-specific enolase,12 and vascular endothe- availability of MRI within the first hours of stroke onset lial growth factor.12 However, external validation in large would probably be a limitation. Alberta Stroke Program studies including unselected AIS patients is lacking, thus it Early CT score (ASPECTS) of 7 was shown to have a sen- remains unclear which biomarkers may be most useful in sitivity of 50% and a specificity of 86% for malignant clinical practice. infarction in 226 patients with MCA infarction.37 The extent of BBB permeability can also be estimated In summary, while several clinical and neuroradiologi- using novel modalities in neuroimaging. In a study includ- cal approaches exist to predict sICH and sBE, none of these ing 106 patients with M1 or ICA occlusion treated with scores included specific serum markers of BBB permeabil- IAT, the contrast volume transfer coefficient (Ktrans) at ideal ity. According to our findings, such markers may be a help- cut-off showed a sensitivity of 55% and a specificity of ful addition in the early assessment of sICH and sBE risk. 68.2% for the prediction of hemorrhagic transformation.34 This study has limitations. We only analyzed single Another study retrospectively assessed an automatically measurements of S-100B, while concentrations are known computed BBB permeability parameter from admission CT to gradually increase 8–10 h after symptom onset and reach- scans and aimed to predict either sBE or sICH in 32 AIS ing maximum plasma concentrations after 2–4 days.10,14,38 patients regardless of therapeutic interventions.35 It showed Biomarker trajectories created from repeated measurements a sensitivity of 100% and a specificity of 79% for the devel- may contain additional information regarding the develop- opment of either outcome. Despite these promising results, ment and extent of BBB dysfunction and therefore the risk an inherent disadvantage of advanced radiological exami- of subsequent sBE or sICH. However, risk-stratification nations is the need for specialized neuroradiologists usually regarding time-sensitive therapy needs to be available in the only available in hospitals involved in tertiary care. Blood- hyperacute phase of stroke, therefore measurements beyond based biomarkers, on the other hand, are easily measurable 24 h are unlikely to be useful for clinical application. and their interpretation does not require special training. Second, control neuroimaging was only performed in in In attempt to predict the development of sICH in the 90.3% of all cases. Per protocol, control imaging was sys- early stages of stroke, numerous scores (HAT Score, SPAN- tematically performed if patients showed clinical deteriora- 100 Score, SEDAN Score, SITS Score, GRASPS Score, tion or if they had received any kind of reperfusion therapy. Multicenter Stroke Survey) were evaluated specifically for This approach was generally agreed to be feasible and sen- the risk of sICH in the subgroup of patients treated with IVT. sible, as ICH or BE in patients who are clinically stable A previous comparison these sICH scores reported AUC would not lead to any intervention. values from 0.55 to 0.70 versus 0.68 to 0.77 in the original Both brain edema and intracranial hemorrhage are studies.4 After application of score items to our cohort, we more likely to occur with increased infarction size39,40 and found AUC values from 0.50 to 0.63, which might be lower higher S-100B levels were shown to correlate with final due to a stricter definition of sICH – four of the scores did infarction volumes.9,10 In clinical practice, infarct size consider any neurological deterioration as sufficient for often cannot be correctly determined in the early phase of Honegger et al. 317 stroke as this would require initial MRI imaging in all Ethical approval patients, which may not routinely be available. Thus, Ethical approval for this study was obtained from the ethics com- lesion size may not be a feasible marker for very early mittees of the Canton of Zurich, Switzerland (BASEC No. risk-stratification in most hospitals. Further, blood draw PB_2016-00672, KEK-ZH-No. 2014-0001), of the Larissa was performed within 24 h of symptom onset – in some University Hospital, Larissa, Greece (No. 46748), of the cases after initial diagnostic and therapeutic measures. University Hospital Vall d’Hebron in Barcelona, Spain (No. Therefore, this study did not evaluate S-100B as a pre- 46228895E) and of the Goethe University Frankfurt am Main, treatment risk assessment tool. Lastly, 97.3% of the Germany (No. 478/14). included patients were Caucasian, limiting the generaliz- ability of our results to other ethnic groups. Guarantor In conclusion, we found that S-100B levels are signifi- MK cantly and independently associated with clinically impor- tant complications after acute ischemic stroke. Our data Contributorship add to the preliminary evidence that S-100B levels may TH: acquisition and analysis of data, drafting a significant portion assist in improving risk prediction and in selecting either of the manuscript or figures, accepts responsibility for conduct of vulnerable individuals for intensified monitoring or stable research, and will give final approval. JS, AB, LW, VS, CI, TP, patients for earlier discharge to a normal ward or rehabili- MA, UF, LB, GM, KN, TK, CC, GK, JM, AB, EP, GN, CF, AL, tation facility. Further studies are required to elaborate KS, LS, AvE: acquisition and analysis of data, contribution of vital effectiveness of measuring S-100B concerning improved reagents/tools/patients, revising the manuscript, accepts responsi- clinical decision-making. bility for conduct of research, and will give final approval. MA: acquisition and analysis of data, drafting a significant portion of the manuscript or figures, analysis or interpretation of data, statisti- Declaration of conflicting interests cal analysis, accepts responsibility for conduct of research, and The author(s) declared the following potential conflicts of interest will give final approval. MK: conception and design of the study, with respect to the research, authorship, and/or publication of this drafting a significant portion of the manuscript or figures and spon- article: TH Nothing to report; JS Nothing to report; AB Nothing soring, revising the manuscript, data acquisition, analysis or inter- to report; LW Nothing to report; VS Nothing to report; CI Nothing pretation of data, statistical analysis, study supervision, contribution to report; TP Nothing to report; MA reports honoraria and board of vital reagents/tools/patients, obtaining funding, accepts respon- involvements outside the submitted work; UF reports grants, con- sibility for conduct of research, and will give final approval. sulting fees, honoraria and board involvements, outside the sub- mitted work; LB Nothing to report; GM Nothing to report; KN ORCID iDs Nothing to report; TK Nothing to report; CC reports fees outside the submitted work; GK reports grants outside the submitted Tim Honegger https://orcid.org/0000-0002-9941-171X work; JM Nothing to report; AB Nothing to report; EP Nothing to Urs Fischer https://orcid.org/0000-0003-0521-4051 report; GN Nothing to report; CF reports a patent Use of GFAP Leo H Bonati https://orcid.org/0000-0003-1163-8133 for identification of intracerebral Hemorrhage US20150247867 Gian Marco De Marchis https://orcid.org/0000-0002-0342- licensed to Banyan Biomarkers; AL reports grants and fees out- 9780 side the submitted work.; KS Nothing to report; LS Nothing to Timo Kahles https://orcid.org/0000-0002-1569-6376 report; AvE reports grants, fees, honoraria and board involve- ments, outside the submitted work; MA Nothing to report; MKv George Ntaios https://orcid.org/0000-0002-0629-9248 reports grants from the Swiss National Science Foundation Markus Arnold https://orcid.org/0000-0002-5524-2301 (142422), the Swiss Heart Foundation, the Göhner Foundation and the Swiss Seaside Foundation as well as non-financial support Supplemental material from Roche diagnostics during the conduct of the study and is Supplemental material for this article is available online. involved in the Advisory Board of AstraZeneca, Bayer, Amgen, BMS/Pfizer and Medtronic outside the submitted work. 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