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Serum caspase-cleaved cytokeratin-18 levels and outcomes after aneurysmal subarachnoid hemorrhage
Journal of the Neurological Sciences 359 (2015) 298–304
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Serum caspase-cleaved cytokeratin-18 levels and outcomes after aneurysmal subarachnoid hemorrhage Zi-Gang Yuan, Jian-Li Wang ⁎, Guo-Liang Jin, Xue-Bin Yu, Jin-Quan Li, Tian-Lun Qiu, Rong-Xiao Dai Department of Neurosurgery, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, 568 Zhongxing North Road, Shaoxing 312000, Zhejiang Province, China
a r t i c l e
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Article history: Received 25 September 2015 Received in revised form 3 November 2015 Accepted 11 November 2015 Available online 12 November 2015 Keywords: Caspase-cleaved cytokeratin-18 Subarachnoid hemorrhage Aneurysm Prognosis Biomarker Mortality Functional outcome
a b s t r a c t Objective: Cell apoptosis is involved in acute brain injury after aneurysmal subarachnoid hemorrhage (aSAH). The protein cytokeratin-18 (CK-18) is cleaved by the action of caspases during apoptosis, and the resulting fragments are released into the blood as caspase-cleaved CK (CCCK)-18. Our study examined the relationship between circulating CCCK-18 levels and long-term clinical outcomes among aSAH patients. Methods: We recruited 128 aSAH patients and 128 controls (matched on age and sex). Serum was collected at admission to the emergency department. Unfavorable outcome was defined as the Glasgow Outcome Score scores of 1–3. After a 6-month follow-up period, outcomes were assessed using a logistic regression analyses. The prognostic predictive values were evaluated according to receiver operating curves analysis. Results: aSAH patients had higher plasma CCCK-18 levels compared to controls (235.1 ± 86.8 U/L vs. 25.6 ± 23.4 U/L, P b 0.001). CCCK-18 was independently associated with World Federation of Neurological Surgeons (WFNS) scores (t = 4.460, P b 0.001) and modified Fisher scores (t = 3.781, P b 0.001). Furthermore, CCCK-18 levels were markedly higher among patients with an unfavorable outcome and among non-survivors. CCCK18 was yet identified as an independent prognostic predictor for mortality (odds ratio, 5.769; 95% confidence interval, 1.196–27.832; P = 0.029) and unfavorable outcome (odds ratio, 4.909; 95% confidence interval, 1.521–15.838; P = 0.008), as well as had similar predictive values for them compared with WFNS scores and modified Fisher scores. Conclusions: High circulating CCCK-18 levels were associated with injury severity and a poor clinical outcome after aSAH and CCCK-18 had the potential to be a good prognostic biomarker for aSAH. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Patients with aneurysmal subarachnoid hemorrhage (aSAH) account for a considerable group of patients admitted to an emergency department [1,2]. Because it affects a younger population, the proportion of the potential of years of life lost with aSAH is equivalent to that of the patients with ischemic stroke and intracerebral hemorrhage [3,4]. Brain injury begins early after aSAH [5]. An early activation of endothelial and parenchymal cell apoptosis and neuronal necrosis after SAH has been demonstrated [6]. Cytokeratin-18 (CK-18) is a protein of the intermediate filament group present in most epithelial and parenchymal cells [7]. During apoptosis, CK-18 is cleaved at various sites by the action of caspases, and the resulting fragments, called caspasecleaved CK (CCCK)-18, are released into the blood [8]. Circulating CCCK-18 levels have been studied in patients with apoptosis-related Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; AUC, area under curve; CCCK-18, caspase-cleaved cytokeratin-18; CI, confidence interval; CT, computerized tomography; ELISA, enzyme-linked immunosorbent assay; GCS, Glasgow coma scale; GOS, Glasgow outcome scale; ROC, receiver operating characteristic; WFNS, World Federation of Neurological Surgeons. ⁎ Corresponding author. E-mail address: [email protected] (J.-L. Wang).
http://dx.doi.org/10.1016/j.jns.2015.11.020 0022-510X/© 2015 Elsevier B.V. All rights reserved.
diseases [9–15]. Moreover, circulating CCCK-18 was identified to be a superior marker as compared to creatine kinase and troponin T, for detection of myocardial damage in patients with acute myocardial infarction [16]. Elevated CCCK-18 levels in tumor cytosol can predict the poor survival in patients with breast cancer [17]. Recently, it is confirmed that serum CCCK-18 levels are associated with 30-day mortality after severe traumatic brain injury [18], suggesting that CCCK-18 could be used as a prognostic biomarker in patients with acute brain injury. However, they have not been explored in aSAH patients. Thus, the aim of this study was to determine whether there is an association between serum CCCK-18 levels and long-term clinical outcomes including mortality and functional outcome as well as whether such levels could be used as a biomarker to predict outcomes in aSAH patients. 2. Methods 2.1. Study population In this prospective and observatory study, consecutive aSAH patients were enrolled at the Shaoxing People's Hospital between June 2011 and June 2014. Patients were initially assessed based on the following inclusion criteria: the first-ever non-traumatic SAH, the clinical history of
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SAH within the last 24 h before admission, the single intracranial aneurysms confirmed by computerized tomography (CT) angiography with or without digital subtraction angiography and the treatment through clipping or coiling within the 48 h after admission. Exclusion criteria included rebleeding after admission, suspected pseudoaneurysm, less than 18 years of age, a history of traumatic injuries, recent (within 1 month) infectious diseases, previous neurological diseases like intracerebral hemorrhage and ischemic stroke, previous use of antiplatelet or anticoagulant medication, and other prior systemic diseases such as uremia, liver cirrhosis, malignancy, chronic heart disease, chronic lung disease, diabetes mellitus and hypertension. We also excluded those patients with unavailable biomarker measurements, refusal of participation and loss of follow-up. Healthy controls were recruited from the same hospital as well as were age- and gender- matched to the aSAH patients. This study was approved by the ethic committee of the Shaoxing People's Hospital and the relatives of all patients and the controls signed consent forms. 2.2. Assessment Recorded data included age, gender, aneurysm distribution, mode of aneurysm treatment including clipping or coiling, hydrocephalus, symptomatic cerebral vasospasm, World Federation of Neurological Surgeons (WFNS) grade [19] and modified Fisher grade [20]. Symptomatic cerebral vasospasm was defined as the development of new focal neurological signs, deterioration in level of consciousness, or the appearance of new infarction on CT when the cause was felt to be ischemia attributable to vasospasm after other possible causes of worsening (e.g. hydrocephalus, seizures, metabolic derangement, infection, or oversedation) had been excluded [21,22]. 2.3. Outcome The clinical endpoint was death and unfavorable outcome within 6-months after SAH. The functional outcome was defined by Glasgow outcome scale (GOS) score. GOS was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; and 5 = good recovery. Unfavorable outcome was defined as Glasgow outcome scale score of 1–3 [23]. For follow-up, we used structure telephone interviews performed by 1 doctor, blinded to clinical information. 2.4. Sampling and laboratory analysis Samples were collected from the patients at admission to the emergency department and from the healthy controls at study entry and were stored at −70 °C until measurement. Serum CCCK-18 concentrations were analyzed in duplicate by enzyme-linked immunosorbent assay (ELISA) using M30 Apoptosense ELISA (PEVIVA AB, Bromma, Sweden) in accordance with the manufactures' instructions. Samples were all processed by the same laboratory technician using the same equipment and blinded to all clinical data. The detection limit for the assay was 25 U/L. Thus, all values less than 25 U/L were regarded as zero. 2.5. Statistical analysis SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0 (MedCalc Software, Mariakerke, Belgium) were used to analyze data. Serum CCCK-18 concentrations were analyzed in duplicate in this study and their mean values were accepted for the statistical analysis. The normality of data distribution was assessed by the Kolmogorov–Smirnov test or Shapiro–Wilk test. The categorical variables are presented as frequency and percentage. And the continuous variables are presented as mean ± standard deviation or median (percentile 25–75). For comparison of data between two groups, the significances of inter-group differences were assessed using chi-square test or Fisher exact test for categorical
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data as well as Student t test or Mann–Whitney U test for continuous variables. Bivariate correlations were analyzed by Spearman's correlation coefficient or Pearson's correlation coefficient and then followed by a multivariate linear regression. A logistic regression model was used to identify independent predictors with respect to 6-month mortality and unfavorable outcome. Cutoff values of serum CCCK-18 levels were obtained automatically from receiver operating characteristic (ROC) curves with optimal prognostic predictive sensitivities and specificities. Bonferroni correction was used for the multiple testing. The variables, that univariate analyses revealed to be associated with poor prognosis, were incorporated into multivariate model. All P values lower 0.05 were considered statistically significant. 3. Results 3.1. Patients characteristics During the study period, 174 patients were initially assessed. 46 patients were excluded because of the reasons in Fig. 1. Eventually, 128 aSAH patients were included in this study. The original 174 patients were composed of 99 (56.9%) males and 75 (43.1%) females, and have a mean age of 42.1 ± 10.9 years (range: 22–70 years); the excluded 46 patients, with a mean age of 43.2 ± 12.1 years (range: 22–68 years), included 26 (56.5%) males and 20 (43.5%) females; this group of the eligible 128 aSAH patients consisted of 73 (57.0%) males and 55 (43.0%) females, as well as was aged 41.6 ± 11.6 years (range: 23–70 years). There were no significant differences in gender and age among the three groups. In addition, among this group of the eligible aSAH patients, the admission median WFNS score was 3 (2–3) (range: 1–5) and the admission median Fisher score was 3 (2–3) (range: 2–5). Aneurysmal location is as follows: 35 (27.3%) aneurysms were located at posterior communication artery; 21 (16.4%) aneurysms, internal carotid artery; 31 (24.3%) aneurysms, anterior communication artery; 21 (16.4%) aneurysms, middle cerebral artery; 14 (10.9%) aneurysms, anterior cerebral artery; 5 (3.9%) aneurysms, posterior cerebral artery; 1 (0.8%) aneurysms, vertebral artery. In terms of modes of treatment, 82 (64.1%) patients underwent clipping and 46 (35.9%) patients obtained endovascular coiling. Among these patients, 28 (21.9%) patients had acute hydrocephalus; 17 (13.3%) patients, intraventricular hemorrhage; 37 (28.9%) patients, symptomatic cerebral vasospasm; 31 (24.2%) patients, accepted external ventricular drain. The mean admission time was 10.1 ± 4.1 h (range: 1.0–23.0 h); the mean plasma-sampling time, 12.7 ± 4.7 h (range: 1.5–25.5 h); the mean systolic blood pressure, 146.2 ± 25.9 mmHg (range: 90–200 mmHg); the mean diastolic blood pressure, 89.4 ± 14.2 mmHg (range: 50–120 mmHg); the mean blood glucose level, 12.3 ± 4.6 mmol/L (range: 2.5–23.4 mmol/L); the mean plasma C-reactive protein level, 14.3 ± 3.9 mg/L (range: 4.5–24.4 mg/L). 3.2. The change of serum CCCK-18 levels in aSAH patients The admission CCCK-18 levels were significantly elevated in all patients (235.1 ± 86.8 U/L) compared with healthy controls (25.6 ± 23.4 U/L, P b 0.001). The results of sub-population analysis for CCCK18 concentrations at admission showed that the patients with an unfavorable outcome revealed markedly higher CCCK-18 concentrations (P b 0.001). The values in the patients with an unfavorable outcome were 288.0 ± 75.2 U/L compared to 212.7 ± 81.8 U/L in the patients with a favorable outcome. Similar result was found in non-survivors and survivor of the patients with aSAH (310.1 ± 85.1 U/L vs. 222.8 ± 81.0 U/L, P b 0.001). These comparisons were depicted in Fig. 2. 3.3. Correlation analysis Just shown in Table 1, CCCK-18 levels were highly associated with WFNS scores, Modified Fisher scores, acute hydrocephalus,
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Fig. 1. Flow chart illustrating excluded and included patients with aneurysmal subarachnoid hemorrhage.
intraventricular hemorrhage, symptomatic cerebral vasospasm, external ventricular drain, blood glucose levels and plasma C-reactive protein levels. Using Bonferroni correction, only WFNS scores, Modified Fisher
scores, intraventricular hemorrhage, and external ventricular drain remained to be associated with serum CCCK-18 levels. Finally, a multivariate linear regression demonstrated that CCCK-18 levels were still highly
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Fig. 2. Graph showing the changes of serum caspase-cleaved cytokeratin-18 (CCCK-18) levels after aneurysmal subarachnoid hemorrhage. The data are presented as mean ± standard deviation and intergroup differences were compared using Student t test.
associated with WFNS scores (t = 4.460, P b 0.001) and modified Fisher scores (t = 3.781, P b 0.001). Their associations were shown in Fig. 3. 3.4. Mortality prediction 18 patients (14.1%) died within 6 months after aSAH. According to a ROC curve, serum CCCK-18 levels N 257 U/L predicted 6-month mortality of patients with high area under curve (AUC) in Fig. 4A and Table 2. Based on AUC, its predictive value was similar to WFNS score's [AUC, 0.869; 95% confidence interval (CI), 0.798–0.922; P = 0.148] and modified Fisher score's (AUC, 0.846; 95% CI, 0.772–0.904; P = 0.258). Table 3 showed that the variables associated with death at 6 months included WFNS scores, Modified Fisher scores, acute hydrocephalus, intraventricular hemorrhage, symptomatic cerebral vasospasm, external ventricular drain, blood glucose levels, plasma C-reactive protein levels and serum CCCK-18 levels N 257 U/L. When the above variables were
incorporated into a multivariate model, in addition to the most common determinants for poor outcome (modified Fisher score and WFNS score), the logistic regression analysis identified serum CCCK-18 levels N257 U/L as an independent predictor for mortality. Results of multivariate analysis were displayed in Table 4.
3.5. Poor outcome prediction 38 patients (29.7%) suffered from unfavorable outcome within 6 months after aSAH. According to a ROC curve, serum CCCK-18 levels N246 U/L predicted 6-month unfavorable outcome of patients with high AUC in Fig. 4B and Table 2. Based on AUC, its predictive value was similar to WFNS score's (AUC, 0.847; 95% CI, 0.773–0.905; P = 0.083) and modified Fisher score's (AUC, 0.830; 95% CI, 0.754–0.891; P = 0.168).
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Table 1 The factors correlated with serum caspase-cleaved cytokeratin-18 levels in patients with aneurysmal subarachnoid hemorrhage. Characteristics
r value
P value
Gender (male/female) Age (y) WFNS score on admission Modified Fisher score on admission Aneurysmal location Treatment (clipping/endovascular coiling) Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Symptomatic cerebral vasospasm Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L)
0.092 0.051 0.527 0.477 0.037 0.081 0.232 0.264 0.285 0.200 0.083 0.111 0.049 0.148 0.223 0.233
0.300 0.568 b0.001 b0.001 0.682 0.363 0.008 0.003 0.001 0.024 0.351 0.214 0.579 0.095 0.012 0.008
Bivariate correlations were assessed by Spearman's or Pearson's correlation coefficient. WFNS indicates World Federation of Neurological Surgeons.
Table 5 showed that the variables associated with unfavorable outcome at 6 months included WFNS scores, Modified Fisher scores, acute hydrocephalus, intraventricular hemorrhage, symptomatic cerebral vasospasm, external ventricular drain, blood glucose levels, plasma C-reactive protein levels and serum CCCK-18 levels N246 U/L. When the above variables were incorporated into a multivariate model, in addition to the most common determinants for poor outcome (modified Fisher score and WFNS score), the logistic regression analysis identified serum CCCK-18 levels N246 U/L as an independent predictor for unfavorable outcome. Results of multivariate analysis were displayed in Table 4. Fig. 4. Graph showing receiver operating characteristic curve analysis of serum caspasecleaved cytokeratin-18 (CCCK-18) levels for 6-month mortality (A) and 6-month unfavorable outcome (B) of patients with aneurysmal subarachnoid hemorrhage.
4. Discussion In the current study, we analyzed the medical data of 128 aSAH patients and determined the serum CCCK-18 concentrations on admission. A relationship between this biomarker levels and severity and longterm prognosis of aSAH were yet assessed. The main findings of this study were that serum CCCK-18 levels were indeed elevated compared with healthy controls, and that there was an independent association between this biomarker levels and aSAH severity reflected by WFNS scores and modified Fisher scores, that CCCK-18 could independently predict mortality and functional outcome at 6 months after aSAH. Further, based on a ROC curve, this biomarker had similar predictive values
Table 2 Receiver operating characteristic analysis of clinical outcomes after aneurysmal subarachnoid hemorrhage.
Fig. 3. Graph showing the relationships between serum caspase-cleaved cytokeratin-18 (CCCK-18) levels and modified Fisher scores and World Federation of Neurological Surgeons (WFNS) scores after aneurysmal subarachnoid hemorrhage. Bivariate correlations were analyzed by Spearman's correlation coefficient.
Cutoff values of serum CCCK-18 levels Area under curve (95% CI) Sensitivity (95% CI) Specificity (95% CI l) Positive predicted value (95% CI) Negative predicted value (95% CI) Positive likelihood ratio (95% CI) Negative likelihood ratio (95% CI)
6-month mortality
6-month unfavorable outcome
257 U/L
246 U/L
0.760 (0.677–0.831) 77.8% (52.4% - 93.5%) 64.5% (54.9% - 73.4%) 26.4% (15.3%-40.3%) 94.7% (86.9%-98.5%) 2.19 (1.7–2.9) 0.34 (0.1–0.8)
0.744 (0.660–0.817) 73.7% (56.9% - 86.6%) 70.0% (59.4% - 79.2%) 50.9% (37.1%-64.6%) 86.3% (76.2%-93.2%) 2.46 (1.9–3.1) 0.38 (0.2–0.7)
CCCK-18 indicates caspase-cleaved cytokeratin-18; CI, confidence interval.
Z.-G. Yuan et al. / Journal of the Neurological Sciences 359 (2015) 298–304 Table 3 Comparison of clinical and biochemical characteristics between non-survivors and survivors with aneurysmal subarachnoid hemorrhage.
Female Age (y) WFNS score on admission Modified Fisher score on admission Aneurysmal location Posterior communication artery Internal carotid artery Anterior communication artery Middle cerebral artery Anterior cerebral artery Posterior cerebral artery Vertebral artery Treatment by endovascular coiling Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Symptomatic cerebral vasospasm Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Serum CCCK levels N257 U/L
Non-survivors
Survivors
P value
9 (50.0%) 45.7 ± 13.0 4 (3–4) 4 (3–4)
46 (41.8%) 40.9 ± 11.3 2 (1–3) 2 (2–3)
0.516 0.103 b0.001 b0.001 0.703
6 (33.3%) 3 (16.7%) 2 (11.1%) 4 (22.2%) 3 (16.7%) 0 0 6 (33.3%) 9 (50.0%) 7 (38.9%) 9 (50.0%) 9 (50.0%) 9.9 ± 5.0 11.6 ± 5.1 155.0 ± 23.0 94.0 ± 14.1 14.6 ± 4.8 17.0 ± 4.7 14 (77.8%)
29 (26.4%) 18 (16.4%) 29 (26.4%) 17 (15.4%) 11 (10.0%) 5 (4.5%) 1 (0.9%) 40 (36.4%) 19 (17.3%) 10 (9.1%) 22 (20.0%) 28 (25.5%) 10.2 ± 4.0 12.8 ± 4.7 144.8 ± 26.1 88.7 ± 14.2 11.9 ± 4.5 13.9 ± 3.6 40 (36.4%)
0.804 0.004 0.003 0.014 0.033 0.782 0.318 0.122 0.147 0.019 0.002 0.001
The categorical variables are presented as frequency and percentage, and the continuous variables are presented as mean ± standard deviation or median (percentile 25–75). The significances of inter-group differences were assessed using chi-square test or Fisher exact test for categorical data as well as Student t test or Mann–Whitney U test for continuous variables. WFNS indicates World Federation of Neurological Surgeons; CCCK-18, caspase-cleaved cytokeratin-18.
with WFNS scores and modified Fisher scores. Our findings indicate that CCCK-18 has the potential to assess the severity and prognosis of aSAH. aSAH causes at least 3 pathophysiologically important processes: transient global ischemia due to increased intracranial pressure, acute hypertension and deposition of subarachnoid blood clot. Transient ischemia probably only develops in the subgroup of patients that lose consciousness when the aneurysm ruptures. Acute intracranial and systemic hypertension contributes to blood–brain barrier disruption and cerebral edema. These interact with the subarachnoid clot to produce cerebral vasospasm and possibly various secondary processes [24–26]. To be important, however, these processes must contribute to brain dysfunction. The apoptotic process is one in which cells are actively eliminated via a programmed pathway during morphogenesis, tissue remodeling, and resolution of the immune response [27]. Apoptosis is traditionally known to be present at 24 h after SAH [24]. Recently, it has been demonstrated that an activation of endothelial and parenchymal cell apoptosis and neuronal necrosis is induced at 10 min after SAH [6]. Thus, apoptosis related biomarker may have the potential to be a good prognostic predictor of aSAH. CK-18 is a protein of the intermediate filament family and is present in most epithelial and parenchymal cells [7]. CK-18 is cleaved at various sites by the action of caspases and the resulting fragments are released into the blood [8]. Full-length CK-18 is released into the blood plasma
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Table 5 Comparison of clinical and biochemical characteristics between patients with unfavorable outcome and those with favorable outcome among patients with aneurysmal subarachnoid hemorrhage.
Female Age (y) WFNS score on admission Modified Fisher score on admission Aneurysmal location Posterior communication artery Internal carotid artery Anterior communication artery Middle cerebral artery Anterior cerebral artery Posterior cerebral artery Vertebral artery Treatment by endovascular coiling Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Symptomatic cerebral vasospasm Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Serum CCCK levels N246 U/L
Unfavorable outcome
Favorable outcome
P value
17 (44.7%) 43.3 ± 11.3 3 (3–4) 3 (3–4)
38 (42.2%) 40.9 ± 11.7 2 (1–3) 2 (2–3)
0.793 0.290 b0.001 b0.001 0.471
12 (31.6%) 6 (15.8%) 5 (13.2%) 7 (18.4%) 6 (15.8%) 2 (5.2%) 0 10 (26.3%) 17 (44.7%) 11 (29.0%) 19 (50.0%) 18 (47.4%) 9.3 ± 3.7 12.3 ± 4.5 151.5 ± 24.2 90.0 ± 13.4 14.1 ± 4.7 15.9 ± 4.2 28 (73.7%)
23 (25.6%) 15 (16.7%) 26 (28.8%) 14 (15.6%) 8 (8.9%) 3 (3.3%) 1 (1.1%) 36 (40.0%) 11 (12.2%) 6 (6.7%) 12 (13.3%) 19 (21.1%) 10.4 ± 4.3 12.8 ± 4.8 144.0 ± 26.4 89.2 ± 14.6 11.5 ± 4.4 13.7 ± 3.6 28 (31.1%)
0.140 b0.001 0.001 b0.001 0.003 0.164 0.617 0.132 0.785 0.003 0.003 b0.001
The categorical variables are presented as frequency and percentage, and the continuous variables are presented as mean ± standard deviation or median (percentile 25–75). The significances of inter-group differences were assessed using chi-square test or Fisher exact test for categorical data as well as Student t test or Mann–Whitney U test for continuous variables. WFNS indicates World Federation of Neurological Surgeons; CCCK-18, caspase-cleaved cytokeratin-18.
during necrosis, and CK-18 fragments are released during apoptosis [28,29]. Determination of CK-18 fragments can be carried out by using a monoclonal antibody (M30) that recognizes CCCK-18 fragments, containing the CK-18 Asp 396 neoepitope, without detecting native or intact CK-18 [28,29]. The elevated serum levels of CCCK-18 are found in patients with chronic obstructive pulmonary disease [30]. Circulating levels of CCCK-18 has been studied in patients with sepsis and it is found that serum CCCK-18 levels are associated with mortality in severe septic patients [31]. CCCK-18 is also demonstrated to be a novel sensitive marker for the detection of myocardial damage in acute myocardial infarction [16]. Therefore, apoptosis-specific CCCK-18 may be associated with the prognosis of some critical illness. A recent study has shown that serum CCCK-18 levels are elevated after sever traumatic brain injury. Moreover, it is confirmed that serum CCCK-18 levels are independently associated with short-term prognosis (30-day mortality) [18]. Up to now, no reports have investigated circulating CCCK-18 concentrations in the patients with aSAH. In agreement with the results found in traumatic brain injury, this study showed that elevated CCCK-18 levels in serum emerged in aSAH patients, suggesting that CCCK-18 levels in peripheral blood possibly reflect the severity of acute brain injury. Meanwhile, the current study
Table 4 Multivariate analysis of clinical outcomes after aneurysmal subarachnoid hemorrhage. Variables
WFNS score Modified Fisher score Serum CCCK-18 levels Ncutoff value
6-month mortality
6-month unfavorable outcome
Odds ratio
95% CI
P value
Odds ratio
95% CI
P value
5.267 3.433 5.769
1.963–14.136 1.412–8.350 1.196–27.832
0.001 0.007 0.029
3.804 3.300 4.909
1.883–7.681 1.644–6.626 1.521–15.838
b0.001 0.001 0.008
Logistic-regression models were configured to estimate the independent risk factors associated with prognosis of patients with aneurysmal subarachnoid hemorrhage. WFNS indicates World Federation of Neurological Surgeons; CCCK-18, caspase-cleaved cytokeratin-18; CI, confidence interval. The cutoff value is 257 U/L and 246 U/L in the predictions of 6-month mortality and 6-month unfavorable outcome respectively.
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verified the close association of serum CCCK-18 levels with severity of aSAH indicated by WFNS scores and modified Fisher scores. From this point of view, it is also postulated that serum CCCK-18 levels could be associated with hemorrhagic severity of aSAH. In this study, a multivariate analysis was used to discover the relationship between serum CCCK-18 levels and long-term prognosis of aSAH. The results showed that serum CCCK-18 levels could independently predict 6-month mortality and 6-month unfavorable outcome after aSAH. Interestingly, the predictive value of this biomarker was similar to those of WFNS scores and modified Fisher scores. Thus, serum CCCK-18 concentration has the potential to be a good prognostic predictive biomarker in aSAH. There were two limitations in this study. Firstly, this study was performed in a hospital and the same size was not large enough. Maybe, a multicenter study will be needed to verify the association of serum CCCK-18 levels with clinical outcomes of aSAH. Secondly, we did not perform an analysis of serum CCCK-18 levels at different time points during follow-up. Thus, additional studies are needed to investigate the serial changes of serum CCCK-18 levels after aSAH. In addition, in the current study, cutoff values of serum CCCK-18 levels were obtained automatically from ROC curves with optimal prognostic predictive sensitivities and specificities for 6-month mortality and functional outcome. Although the two cutoff values are very similar, they represent different meanings. 5. Conclusions This study finds a close association between serum CCCK-18 levels and WFNS scores and modified Fisher scores as well as between serum CCCK-18 levels and 6-month mortality and 6-month unfavorable outcome, suggesting that CCCK-18 levels in serum are highly associated with aSAH severity and long-term prognosis. Our findings propose that CCCK-18 has potential to be a good prognostic biomarker of aSAH. Conflict of interest The authors report no conflicts of interest concerning the materials or methods used in this study or the findings specified in this article. Acknowledgments The authors thank all staffs in the Department of Neurosurgery, Shaoxing People's Hospital (Shaoxing, China) for their technical support. References [1] V.L. Feigin, C.M. Lawes, D.A. Bennett, S.L. Barker-Collo, V. Parag, Worldwide stroke incidence and early case fatality reported in 56 population-based studies: a systematic review, Lancet Neurol. 8 (4) (2009) 355–369. [2] N.K. de Rooij, F.H. Linn, J.A. van der Plas, A. Algra, G.J. Rinkel, Incidence of subarachnoid haemorrhage: a systematic review with emphasis on region, age, gender and time trends, J. Neurol. Neurosurg. Psychiatry 78 (12) (2007) 1365–1372. [3] R.V. Krishnamurthi, A.E. Moran, M.H. Forouzanfar, D.A. Bennett, G.A. Mensah, C.M. Lawes, et al., The global burden of hemorrhagic stroke: a summary of findings from the GBD 2010 study, Glob. Heart 9 (1) (2014) 101–106. [4] C.P. Marder, V. Narla, J.R. Fink, K.R. Tozer Fink, Subarachnoid hemorrhage: beyond aneurysms, AJR Am. J. Roentgenol. 202 (1) (2014) 25–37. [5] S. Park, M. Yamaguchi, C. Zhou, J.W. Calvert, J. Tang, J.H. Zhang, Neurovascular protection reduces early brain injury after subarachnoid hemorrhage, Stroke 35 (10) (2004) 2412–2417. [6] V. Friedrich, R. Flores, F.A. Sehba, Cell death starts early after subarachnoid hemorrhage, Neurosci. Lett. 512 (1) (2012) 6–11.
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Serum caspase-cleaved cytokeratin-18 levels and outcomes after aneurysmal subarachnoid hemorrhage Zi-Gang Yuan, Jian-Li Wang ⁎, Guo-Liang Jin, Xue-Bin Yu, Jin-Quan Li, Tian-Lun Qiu, Rong-Xiao Dai Department of Neurosurgery, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, 568 Zhongxing North Road, Shaoxing 312000, Zhejiang Province, China
a r t i c l e
i n f o
Article history: Received 25 September 2015 Received in revised form 3 November 2015 Accepted 11 November 2015 Available online 12 November 2015 Keywords: Caspase-cleaved cytokeratin-18 Subarachnoid hemorrhage Aneurysm Prognosis Biomarker Mortality Functional outcome
a b s t r a c t Objective: Cell apoptosis is involved in acute brain injury after aneurysmal subarachnoid hemorrhage (aSAH). The protein cytokeratin-18 (CK-18) is cleaved by the action of caspases during apoptosis, and the resulting fragments are released into the blood as caspase-cleaved CK (CCCK)-18. Our study examined the relationship between circulating CCCK-18 levels and long-term clinical outcomes among aSAH patients. Methods: We recruited 128 aSAH patients and 128 controls (matched on age and sex). Serum was collected at admission to the emergency department. Unfavorable outcome was defined as the Glasgow Outcome Score scores of 1–3. After a 6-month follow-up period, outcomes were assessed using a logistic regression analyses. The prognostic predictive values were evaluated according to receiver operating curves analysis. Results: aSAH patients had higher plasma CCCK-18 levels compared to controls (235.1 ± 86.8 U/L vs. 25.6 ± 23.4 U/L, P b 0.001). CCCK-18 was independently associated with World Federation of Neurological Surgeons (WFNS) scores (t = 4.460, P b 0.001) and modified Fisher scores (t = 3.781, P b 0.001). Furthermore, CCCK-18 levels were markedly higher among patients with an unfavorable outcome and among non-survivors. CCCK18 was yet identified as an independent prognostic predictor for mortality (odds ratio, 5.769; 95% confidence interval, 1.196–27.832; P = 0.029) and unfavorable outcome (odds ratio, 4.909; 95% confidence interval, 1.521–15.838; P = 0.008), as well as had similar predictive values for them compared with WFNS scores and modified Fisher scores. Conclusions: High circulating CCCK-18 levels were associated with injury severity and a poor clinical outcome after aSAH and CCCK-18 had the potential to be a good prognostic biomarker for aSAH. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Patients with aneurysmal subarachnoid hemorrhage (aSAH) account for a considerable group of patients admitted to an emergency department [1,2]. Because it affects a younger population, the proportion of the potential of years of life lost with aSAH is equivalent to that of the patients with ischemic stroke and intracerebral hemorrhage [3,4]. Brain injury begins early after aSAH [5]. An early activation of endothelial and parenchymal cell apoptosis and neuronal necrosis after SAH has been demonstrated [6]. Cytokeratin-18 (CK-18) is a protein of the intermediate filament group present in most epithelial and parenchymal cells [7]. During apoptosis, CK-18 is cleaved at various sites by the action of caspases, and the resulting fragments, called caspasecleaved CK (CCCK)-18, are released into the blood [8]. Circulating CCCK-18 levels have been studied in patients with apoptosis-related Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; AUC, area under curve; CCCK-18, caspase-cleaved cytokeratin-18; CI, confidence interval; CT, computerized tomography; ELISA, enzyme-linked immunosorbent assay; GCS, Glasgow coma scale; GOS, Glasgow outcome scale; ROC, receiver operating characteristic; WFNS, World Federation of Neurological Surgeons. ⁎ Corresponding author. E-mail address: [email protected] (J.-L. Wang).
http://dx.doi.org/10.1016/j.jns.2015.11.020 0022-510X/© 2015 Elsevier B.V. All rights reserved.
diseases [9–15]. Moreover, circulating CCCK-18 was identified to be a superior marker as compared to creatine kinase and troponin T, for detection of myocardial damage in patients with acute myocardial infarction [16]. Elevated CCCK-18 levels in tumor cytosol can predict the poor survival in patients with breast cancer [17]. Recently, it is confirmed that serum CCCK-18 levels are associated with 30-day mortality after severe traumatic brain injury [18], suggesting that CCCK-18 could be used as a prognostic biomarker in patients with acute brain injury. However, they have not been explored in aSAH patients. Thus, the aim of this study was to determine whether there is an association between serum CCCK-18 levels and long-term clinical outcomes including mortality and functional outcome as well as whether such levels could be used as a biomarker to predict outcomes in aSAH patients. 2. Methods 2.1. Study population In this prospective and observatory study, consecutive aSAH patients were enrolled at the Shaoxing People's Hospital between June 2011 and June 2014. Patients were initially assessed based on the following inclusion criteria: the first-ever non-traumatic SAH, the clinical history of
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SAH within the last 24 h before admission, the single intracranial aneurysms confirmed by computerized tomography (CT) angiography with or without digital subtraction angiography and the treatment through clipping or coiling within the 48 h after admission. Exclusion criteria included rebleeding after admission, suspected pseudoaneurysm, less than 18 years of age, a history of traumatic injuries, recent (within 1 month) infectious diseases, previous neurological diseases like intracerebral hemorrhage and ischemic stroke, previous use of antiplatelet or anticoagulant medication, and other prior systemic diseases such as uremia, liver cirrhosis, malignancy, chronic heart disease, chronic lung disease, diabetes mellitus and hypertension. We also excluded those patients with unavailable biomarker measurements, refusal of participation and loss of follow-up. Healthy controls were recruited from the same hospital as well as were age- and gender- matched to the aSAH patients. This study was approved by the ethic committee of the Shaoxing People's Hospital and the relatives of all patients and the controls signed consent forms. 2.2. Assessment Recorded data included age, gender, aneurysm distribution, mode of aneurysm treatment including clipping or coiling, hydrocephalus, symptomatic cerebral vasospasm, World Federation of Neurological Surgeons (WFNS) grade [19] and modified Fisher grade [20]. Symptomatic cerebral vasospasm was defined as the development of new focal neurological signs, deterioration in level of consciousness, or the appearance of new infarction on CT when the cause was felt to be ischemia attributable to vasospasm after other possible causes of worsening (e.g. hydrocephalus, seizures, metabolic derangement, infection, or oversedation) had been excluded [21,22]. 2.3. Outcome The clinical endpoint was death and unfavorable outcome within 6-months after SAH. The functional outcome was defined by Glasgow outcome scale (GOS) score. GOS was defined as follows: 1 = death; 2 = persistent vegetative state; 3 = severe disability; 4 = moderate disability; and 5 = good recovery. Unfavorable outcome was defined as Glasgow outcome scale score of 1–3 [23]. For follow-up, we used structure telephone interviews performed by 1 doctor, blinded to clinical information. 2.4. Sampling and laboratory analysis Samples were collected from the patients at admission to the emergency department and from the healthy controls at study entry and were stored at −70 °C until measurement. Serum CCCK-18 concentrations were analyzed in duplicate by enzyme-linked immunosorbent assay (ELISA) using M30 Apoptosense ELISA (PEVIVA AB, Bromma, Sweden) in accordance with the manufactures' instructions. Samples were all processed by the same laboratory technician using the same equipment and blinded to all clinical data. The detection limit for the assay was 25 U/L. Thus, all values less than 25 U/L were regarded as zero. 2.5. Statistical analysis SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and MedCalc 9.6.4.0 (MedCalc Software, Mariakerke, Belgium) were used to analyze data. Serum CCCK-18 concentrations were analyzed in duplicate in this study and their mean values were accepted for the statistical analysis. The normality of data distribution was assessed by the Kolmogorov–Smirnov test or Shapiro–Wilk test. The categorical variables are presented as frequency and percentage. And the continuous variables are presented as mean ± standard deviation or median (percentile 25–75). For comparison of data between two groups, the significances of inter-group differences were assessed using chi-square test or Fisher exact test for categorical
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data as well as Student t test or Mann–Whitney U test for continuous variables. Bivariate correlations were analyzed by Spearman's correlation coefficient or Pearson's correlation coefficient and then followed by a multivariate linear regression. A logistic regression model was used to identify independent predictors with respect to 6-month mortality and unfavorable outcome. Cutoff values of serum CCCK-18 levels were obtained automatically from receiver operating characteristic (ROC) curves with optimal prognostic predictive sensitivities and specificities. Bonferroni correction was used for the multiple testing. The variables, that univariate analyses revealed to be associated with poor prognosis, were incorporated into multivariate model. All P values lower 0.05 were considered statistically significant. 3. Results 3.1. Patients characteristics During the study period, 174 patients were initially assessed. 46 patients were excluded because of the reasons in Fig. 1. Eventually, 128 aSAH patients were included in this study. The original 174 patients were composed of 99 (56.9%) males and 75 (43.1%) females, and have a mean age of 42.1 ± 10.9 years (range: 22–70 years); the excluded 46 patients, with a mean age of 43.2 ± 12.1 years (range: 22–68 years), included 26 (56.5%) males and 20 (43.5%) females; this group of the eligible 128 aSAH patients consisted of 73 (57.0%) males and 55 (43.0%) females, as well as was aged 41.6 ± 11.6 years (range: 23–70 years). There were no significant differences in gender and age among the three groups. In addition, among this group of the eligible aSAH patients, the admission median WFNS score was 3 (2–3) (range: 1–5) and the admission median Fisher score was 3 (2–3) (range: 2–5). Aneurysmal location is as follows: 35 (27.3%) aneurysms were located at posterior communication artery; 21 (16.4%) aneurysms, internal carotid artery; 31 (24.3%) aneurysms, anterior communication artery; 21 (16.4%) aneurysms, middle cerebral artery; 14 (10.9%) aneurysms, anterior cerebral artery; 5 (3.9%) aneurysms, posterior cerebral artery; 1 (0.8%) aneurysms, vertebral artery. In terms of modes of treatment, 82 (64.1%) patients underwent clipping and 46 (35.9%) patients obtained endovascular coiling. Among these patients, 28 (21.9%) patients had acute hydrocephalus; 17 (13.3%) patients, intraventricular hemorrhage; 37 (28.9%) patients, symptomatic cerebral vasospasm; 31 (24.2%) patients, accepted external ventricular drain. The mean admission time was 10.1 ± 4.1 h (range: 1.0–23.0 h); the mean plasma-sampling time, 12.7 ± 4.7 h (range: 1.5–25.5 h); the mean systolic blood pressure, 146.2 ± 25.9 mmHg (range: 90–200 mmHg); the mean diastolic blood pressure, 89.4 ± 14.2 mmHg (range: 50–120 mmHg); the mean blood glucose level, 12.3 ± 4.6 mmol/L (range: 2.5–23.4 mmol/L); the mean plasma C-reactive protein level, 14.3 ± 3.9 mg/L (range: 4.5–24.4 mg/L). 3.2. The change of serum CCCK-18 levels in aSAH patients The admission CCCK-18 levels were significantly elevated in all patients (235.1 ± 86.8 U/L) compared with healthy controls (25.6 ± 23.4 U/L, P b 0.001). The results of sub-population analysis for CCCK18 concentrations at admission showed that the patients with an unfavorable outcome revealed markedly higher CCCK-18 concentrations (P b 0.001). The values in the patients with an unfavorable outcome were 288.0 ± 75.2 U/L compared to 212.7 ± 81.8 U/L in the patients with a favorable outcome. Similar result was found in non-survivors and survivor of the patients with aSAH (310.1 ± 85.1 U/L vs. 222.8 ± 81.0 U/L, P b 0.001). These comparisons were depicted in Fig. 2. 3.3. Correlation analysis Just shown in Table 1, CCCK-18 levels were highly associated with WFNS scores, Modified Fisher scores, acute hydrocephalus,
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Fig. 1. Flow chart illustrating excluded and included patients with aneurysmal subarachnoid hemorrhage.
intraventricular hemorrhage, symptomatic cerebral vasospasm, external ventricular drain, blood glucose levels and plasma C-reactive protein levels. Using Bonferroni correction, only WFNS scores, Modified Fisher
scores, intraventricular hemorrhage, and external ventricular drain remained to be associated with serum CCCK-18 levels. Finally, a multivariate linear regression demonstrated that CCCK-18 levels were still highly
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Fig. 2. Graph showing the changes of serum caspase-cleaved cytokeratin-18 (CCCK-18) levels after aneurysmal subarachnoid hemorrhage. The data are presented as mean ± standard deviation and intergroup differences were compared using Student t test.
associated with WFNS scores (t = 4.460, P b 0.001) and modified Fisher scores (t = 3.781, P b 0.001). Their associations were shown in Fig. 3. 3.4. Mortality prediction 18 patients (14.1%) died within 6 months after aSAH. According to a ROC curve, serum CCCK-18 levels N 257 U/L predicted 6-month mortality of patients with high area under curve (AUC) in Fig. 4A and Table 2. Based on AUC, its predictive value was similar to WFNS score's [AUC, 0.869; 95% confidence interval (CI), 0.798–0.922; P = 0.148] and modified Fisher score's (AUC, 0.846; 95% CI, 0.772–0.904; P = 0.258). Table 3 showed that the variables associated with death at 6 months included WFNS scores, Modified Fisher scores, acute hydrocephalus, intraventricular hemorrhage, symptomatic cerebral vasospasm, external ventricular drain, blood glucose levels, plasma C-reactive protein levels and serum CCCK-18 levels N 257 U/L. When the above variables were
incorporated into a multivariate model, in addition to the most common determinants for poor outcome (modified Fisher score and WFNS score), the logistic regression analysis identified serum CCCK-18 levels N257 U/L as an independent predictor for mortality. Results of multivariate analysis were displayed in Table 4.
3.5. Poor outcome prediction 38 patients (29.7%) suffered from unfavorable outcome within 6 months after aSAH. According to a ROC curve, serum CCCK-18 levels N246 U/L predicted 6-month unfavorable outcome of patients with high AUC in Fig. 4B and Table 2. Based on AUC, its predictive value was similar to WFNS score's (AUC, 0.847; 95% CI, 0.773–0.905; P = 0.083) and modified Fisher score's (AUC, 0.830; 95% CI, 0.754–0.891; P = 0.168).
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Table 1 The factors correlated with serum caspase-cleaved cytokeratin-18 levels in patients with aneurysmal subarachnoid hemorrhage. Characteristics
r value
P value
Gender (male/female) Age (y) WFNS score on admission Modified Fisher score on admission Aneurysmal location Treatment (clipping/endovascular coiling) Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Symptomatic cerebral vasospasm Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L)
0.092 0.051 0.527 0.477 0.037 0.081 0.232 0.264 0.285 0.200 0.083 0.111 0.049 0.148 0.223 0.233
0.300 0.568 b0.001 b0.001 0.682 0.363 0.008 0.003 0.001 0.024 0.351 0.214 0.579 0.095 0.012 0.008
Bivariate correlations were assessed by Spearman's or Pearson's correlation coefficient. WFNS indicates World Federation of Neurological Surgeons.
Table 5 showed that the variables associated with unfavorable outcome at 6 months included WFNS scores, Modified Fisher scores, acute hydrocephalus, intraventricular hemorrhage, symptomatic cerebral vasospasm, external ventricular drain, blood glucose levels, plasma C-reactive protein levels and serum CCCK-18 levels N246 U/L. When the above variables were incorporated into a multivariate model, in addition to the most common determinants for poor outcome (modified Fisher score and WFNS score), the logistic regression analysis identified serum CCCK-18 levels N246 U/L as an independent predictor for unfavorable outcome. Results of multivariate analysis were displayed in Table 4. Fig. 4. Graph showing receiver operating characteristic curve analysis of serum caspasecleaved cytokeratin-18 (CCCK-18) levels for 6-month mortality (A) and 6-month unfavorable outcome (B) of patients with aneurysmal subarachnoid hemorrhage.
4. Discussion In the current study, we analyzed the medical data of 128 aSAH patients and determined the serum CCCK-18 concentrations on admission. A relationship between this biomarker levels and severity and longterm prognosis of aSAH were yet assessed. The main findings of this study were that serum CCCK-18 levels were indeed elevated compared with healthy controls, and that there was an independent association between this biomarker levels and aSAH severity reflected by WFNS scores and modified Fisher scores, that CCCK-18 could independently predict mortality and functional outcome at 6 months after aSAH. Further, based on a ROC curve, this biomarker had similar predictive values
Table 2 Receiver operating characteristic analysis of clinical outcomes after aneurysmal subarachnoid hemorrhage.
Fig. 3. Graph showing the relationships between serum caspase-cleaved cytokeratin-18 (CCCK-18) levels and modified Fisher scores and World Federation of Neurological Surgeons (WFNS) scores after aneurysmal subarachnoid hemorrhage. Bivariate correlations were analyzed by Spearman's correlation coefficient.
Cutoff values of serum CCCK-18 levels Area under curve (95% CI) Sensitivity (95% CI) Specificity (95% CI l) Positive predicted value (95% CI) Negative predicted value (95% CI) Positive likelihood ratio (95% CI) Negative likelihood ratio (95% CI)
6-month mortality
6-month unfavorable outcome
257 U/L
246 U/L
0.760 (0.677–0.831) 77.8% (52.4% - 93.5%) 64.5% (54.9% - 73.4%) 26.4% (15.3%-40.3%) 94.7% (86.9%-98.5%) 2.19 (1.7–2.9) 0.34 (0.1–0.8)
0.744 (0.660–0.817) 73.7% (56.9% - 86.6%) 70.0% (59.4% - 79.2%) 50.9% (37.1%-64.6%) 86.3% (76.2%-93.2%) 2.46 (1.9–3.1) 0.38 (0.2–0.7)
CCCK-18 indicates caspase-cleaved cytokeratin-18; CI, confidence interval.
Z.-G. Yuan et al. / Journal of the Neurological Sciences 359 (2015) 298–304 Table 3 Comparison of clinical and biochemical characteristics between non-survivors and survivors with aneurysmal subarachnoid hemorrhage.
Female Age (y) WFNS score on admission Modified Fisher score on admission Aneurysmal location Posterior communication artery Internal carotid artery Anterior communication artery Middle cerebral artery Anterior cerebral artery Posterior cerebral artery Vertebral artery Treatment by endovascular coiling Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Symptomatic cerebral vasospasm Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Serum CCCK levels N257 U/L
Non-survivors
Survivors
P value
9 (50.0%) 45.7 ± 13.0 4 (3–4) 4 (3–4)
46 (41.8%) 40.9 ± 11.3 2 (1–3) 2 (2–3)
0.516 0.103 b0.001 b0.001 0.703
6 (33.3%) 3 (16.7%) 2 (11.1%) 4 (22.2%) 3 (16.7%) 0 0 6 (33.3%) 9 (50.0%) 7 (38.9%) 9 (50.0%) 9 (50.0%) 9.9 ± 5.0 11.6 ± 5.1 155.0 ± 23.0 94.0 ± 14.1 14.6 ± 4.8 17.0 ± 4.7 14 (77.8%)
29 (26.4%) 18 (16.4%) 29 (26.4%) 17 (15.4%) 11 (10.0%) 5 (4.5%) 1 (0.9%) 40 (36.4%) 19 (17.3%) 10 (9.1%) 22 (20.0%) 28 (25.5%) 10.2 ± 4.0 12.8 ± 4.7 144.8 ± 26.1 88.7 ± 14.2 11.9 ± 4.5 13.9 ± 3.6 40 (36.4%)
0.804 0.004 0.003 0.014 0.033 0.782 0.318 0.122 0.147 0.019 0.002 0.001
The categorical variables are presented as frequency and percentage, and the continuous variables are presented as mean ± standard deviation or median (percentile 25–75). The significances of inter-group differences were assessed using chi-square test or Fisher exact test for categorical data as well as Student t test or Mann–Whitney U test for continuous variables. WFNS indicates World Federation of Neurological Surgeons; CCCK-18, caspase-cleaved cytokeratin-18.
with WFNS scores and modified Fisher scores. Our findings indicate that CCCK-18 has the potential to assess the severity and prognosis of aSAH. aSAH causes at least 3 pathophysiologically important processes: transient global ischemia due to increased intracranial pressure, acute hypertension and deposition of subarachnoid blood clot. Transient ischemia probably only develops in the subgroup of patients that lose consciousness when the aneurysm ruptures. Acute intracranial and systemic hypertension contributes to blood–brain barrier disruption and cerebral edema. These interact with the subarachnoid clot to produce cerebral vasospasm and possibly various secondary processes [24–26]. To be important, however, these processes must contribute to brain dysfunction. The apoptotic process is one in which cells are actively eliminated via a programmed pathway during morphogenesis, tissue remodeling, and resolution of the immune response [27]. Apoptosis is traditionally known to be present at 24 h after SAH [24]. Recently, it has been demonstrated that an activation of endothelial and parenchymal cell apoptosis and neuronal necrosis is induced at 10 min after SAH [6]. Thus, apoptosis related biomarker may have the potential to be a good prognostic predictor of aSAH. CK-18 is a protein of the intermediate filament family and is present in most epithelial and parenchymal cells [7]. CK-18 is cleaved at various sites by the action of caspases and the resulting fragments are released into the blood [8]. Full-length CK-18 is released into the blood plasma
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Table 5 Comparison of clinical and biochemical characteristics between patients with unfavorable outcome and those with favorable outcome among patients with aneurysmal subarachnoid hemorrhage.
Female Age (y) WFNS score on admission Modified Fisher score on admission Aneurysmal location Posterior communication artery Internal carotid artery Anterior communication artery Middle cerebral artery Anterior cerebral artery Posterior cerebral artery Vertebral artery Treatment by endovascular coiling Acute hydrocephalus Intraventricular hemorrhage External ventricular drain Symptomatic cerebral vasospasm Admission time (h) Plasma-sampling time (h) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg) Blood glucose level (mmol/L) Plasma C-reactive protein level (mg/L) Serum CCCK levels N246 U/L
Unfavorable outcome
Favorable outcome
P value
17 (44.7%) 43.3 ± 11.3 3 (3–4) 3 (3–4)
38 (42.2%) 40.9 ± 11.7 2 (1–3) 2 (2–3)
0.793 0.290 b0.001 b0.001 0.471
12 (31.6%) 6 (15.8%) 5 (13.2%) 7 (18.4%) 6 (15.8%) 2 (5.2%) 0 10 (26.3%) 17 (44.7%) 11 (29.0%) 19 (50.0%) 18 (47.4%) 9.3 ± 3.7 12.3 ± 4.5 151.5 ± 24.2 90.0 ± 13.4 14.1 ± 4.7 15.9 ± 4.2 28 (73.7%)
23 (25.6%) 15 (16.7%) 26 (28.8%) 14 (15.6%) 8 (8.9%) 3 (3.3%) 1 (1.1%) 36 (40.0%) 11 (12.2%) 6 (6.7%) 12 (13.3%) 19 (21.1%) 10.4 ± 4.3 12.8 ± 4.8 144.0 ± 26.4 89.2 ± 14.6 11.5 ± 4.4 13.7 ± 3.6 28 (31.1%)
0.140 b0.001 0.001 b0.001 0.003 0.164 0.617 0.132 0.785 0.003 0.003 b0.001
The categorical variables are presented as frequency and percentage, and the continuous variables are presented as mean ± standard deviation or median (percentile 25–75). The significances of inter-group differences were assessed using chi-square test or Fisher exact test for categorical data as well as Student t test or Mann–Whitney U test for continuous variables. WFNS indicates World Federation of Neurological Surgeons; CCCK-18, caspase-cleaved cytokeratin-18.
during necrosis, and CK-18 fragments are released during apoptosis [28,29]. Determination of CK-18 fragments can be carried out by using a monoclonal antibody (M30) that recognizes CCCK-18 fragments, containing the CK-18 Asp 396 neoepitope, without detecting native or intact CK-18 [28,29]. The elevated serum levels of CCCK-18 are found in patients with chronic obstructive pulmonary disease [30]. Circulating levels of CCCK-18 has been studied in patients with sepsis and it is found that serum CCCK-18 levels are associated with mortality in severe septic patients [31]. CCCK-18 is also demonstrated to be a novel sensitive marker for the detection of myocardial damage in acute myocardial infarction [16]. Therefore, apoptosis-specific CCCK-18 may be associated with the prognosis of some critical illness. A recent study has shown that serum CCCK-18 levels are elevated after sever traumatic brain injury. Moreover, it is confirmed that serum CCCK-18 levels are independently associated with short-term prognosis (30-day mortality) [18]. Up to now, no reports have investigated circulating CCCK-18 concentrations in the patients with aSAH. In agreement with the results found in traumatic brain injury, this study showed that elevated CCCK-18 levels in serum emerged in aSAH patients, suggesting that CCCK-18 levels in peripheral blood possibly reflect the severity of acute brain injury. Meanwhile, the current study
Table 4 Multivariate analysis of clinical outcomes after aneurysmal subarachnoid hemorrhage. Variables
WFNS score Modified Fisher score Serum CCCK-18 levels Ncutoff value
6-month mortality
6-month unfavorable outcome
Odds ratio
95% CI
P value
Odds ratio
95% CI
P value
5.267 3.433 5.769
1.963–14.136 1.412–8.350 1.196–27.832
0.001 0.007 0.029
3.804 3.300 4.909
1.883–7.681 1.644–6.626 1.521–15.838
b0.001 0.001 0.008
Logistic-regression models were configured to estimate the independent risk factors associated with prognosis of patients with aneurysmal subarachnoid hemorrhage. WFNS indicates World Federation of Neurological Surgeons; CCCK-18, caspase-cleaved cytokeratin-18; CI, confidence interval. The cutoff value is 257 U/L and 246 U/L in the predictions of 6-month mortality and 6-month unfavorable outcome respectively.
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verified the close association of serum CCCK-18 levels with severity of aSAH indicated by WFNS scores and modified Fisher scores. From this point of view, it is also postulated that serum CCCK-18 levels could be associated with hemorrhagic severity of aSAH. In this study, a multivariate analysis was used to discover the relationship between serum CCCK-18 levels and long-term prognosis of aSAH. The results showed that serum CCCK-18 levels could independently predict 6-month mortality and 6-month unfavorable outcome after aSAH. Interestingly, the predictive value of this biomarker was similar to those of WFNS scores and modified Fisher scores. Thus, serum CCCK-18 concentration has the potential to be a good prognostic predictive biomarker in aSAH. There were two limitations in this study. Firstly, this study was performed in a hospital and the same size was not large enough. Maybe, a multicenter study will be needed to verify the association of serum CCCK-18 levels with clinical outcomes of aSAH. Secondly, we did not perform an analysis of serum CCCK-18 levels at different time points during follow-up. Thus, additional studies are needed to investigate the serial changes of serum CCCK-18 levels after aSAH. In addition, in the current study, cutoff values of serum CCCK-18 levels were obtained automatically from ROC curves with optimal prognostic predictive sensitivities and specificities for 6-month mortality and functional outcome. Although the two cutoff values are very similar, they represent different meanings. 5. Conclusions This study finds a close association between serum CCCK-18 levels and WFNS scores and modified Fisher scores as well as between serum CCCK-18 levels and 6-month mortality and 6-month unfavorable outcome, suggesting that CCCK-18 levels in serum are highly associated with aSAH severity and long-term prognosis. Our findings propose that CCCK-18 has potential to be a good prognostic biomarker of aSAH. Conflict of interest The authors report no conflicts of interest concerning the materials or methods used in this study or the findings specified in this article. Acknowledgments The authors thank all staffs in the Department of Neurosurgery, Shaoxing People's Hospital (Shaoxing, China) for their technical support. References [1] V.L. Feigin, C.M. Lawes, D.A. Bennett, S.L. Barker-Collo, V. Parag, Worldwide stroke incidence and early case fatality reported in 56 population-based studies: a systematic review, Lancet Neurol. 8 (4) (2009) 355–369. [2] N.K. de Rooij, F.H. Linn, J.A. van der Plas, A. Algra, G.J. Rinkel, Incidence of subarachnoid haemorrhage: a systematic review with emphasis on region, age, gender and time trends, J. Neurol. Neurosurg. Psychiatry 78 (12) (2007) 1365–1372. [3] R.V. Krishnamurthi, A.E. Moran, M.H. Forouzanfar, D.A. Bennett, G.A. Mensah, C.M. Lawes, et al., The global burden of hemorrhagic stroke: a summary of findings from the GBD 2010 study, Glob. Heart 9 (1) (2014) 101–106. [4] C.P. Marder, V. Narla, J.R. Fink, K.R. Tozer Fink, Subarachnoid hemorrhage: beyond aneurysms, AJR Am. J. Roentgenol. 202 (1) (2014) 25–37. [5] S. Park, M. Yamaguchi, C. Zhou, J.W. Calvert, J. Tang, J.H. Zhang, Neurovascular protection reduces early brain injury after subarachnoid hemorrhage, Stroke 35 (10) (2004) 2412–2417. [6] V. Friedrich, R. Flores, F.A. Sehba, Cell death starts early after subarachnoid hemorrhage, Neurosci. Lett. 512 (1) (2012) 6–11.
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