High serum levels of caspase-cleaved cytokeratin-18 are associated with malignant middle cerebral artery infarction patient mortality

Background There have been found apoptotic changes in brain tissue samples from humans after cerebral ischemia. Caspase-cleaved cytokeratin (CCCK)-18 could appears in blood during apoptosis. High circulating levels of CCCK-18 have been associated with a poor prognosis in patients with cerebral process, such as traumatic brain injury and spontaneous cerebral hemorrhage. However, they have not been explored in patients with ischemic stroke. Thus, the aim of this study was to determine whether there is an association between serum CCCK-18 levels and mortality in patients with severe malignant middle cerebral artery infarction (MMCAI). Methods This was an observational, prospective and multicentre study. We included patients with severe MMCAI. We considered MMCAI as severe when Glasgow Coma Scale (GCS) was lower than 9. We measured serum CCCK-18 levels at the diagnosis moment of the severe MMCAI. Results We found that non-surviving severe MMCAI patients (n = 33) showed lower GCS and platelet count, and higher serum CCCK-18 levels than survivor ones (n = 33). We found an area under the curve (AUC) of serum CCCK-18 levels to predict 30-day mortality of 82% (95% CI = 71%–91%; p < 0.001). In the multiple logistic regression analysis was found that serum CCCK-18 levels were associated with 30-day mortality (OR = 1.023; 95% CI = 1.010–1.037; p = 0.001) after to control for platelet count and GCS. Conclusions To our knowledge, this is the first series reporting data on serum CCCK-18 levels in ischemic stroke patients. The novel findings of our study were that non-surviving severe MMCAI patients had higher serum CCCK-18 levels than surviving patients, and that there is an association between high serum CCCK-18 levels and MMCAI patients mortality.

Cytokeratins (CK) are proteins, until now named as CK-1 to CK-20, existing mainly in the intracytoplasmic cytoskeleton of epithelial tissue. During apoptosis CK-18 is cleaved at various sites by the action of caspases and appears caspase-cleaved cytokeratin (CCCK)-18, which could be released into the blood [14,15].
Previously, there were found higher circulating CCCK-18 levels in patients with sepsis [16][17][18][19][20], liver diseases [21][22][23][24][25], and tumoral diseases [26,27]. In addition, there was found an association between high circulating CCCK-18 levels and a poor prognosis of patients with different cerebral process, such as traumatic brain injury [28] and spontaneous cerebral hemorrhage [29,30]. However, they have not been explored in patients with ischemic stroke. Thus, the aim of this study was to determine whether there is an association between serum CCCK-18 levels and mortality of patients with severe malignant middle cerebral artery infarction (MMCAI).

Design and subjects
This observational prospective multicentre study was carried with the written informed consent from patient legal guardians in 6  We included patients with severe malignant middle cerebral artery infarction (MMCAI). We estimated the severity of MMCAI according to Glasgow Coma Scale (GCS) [31], and we defined a MMCAI as severe when GCS ≤ 8. We excluded patients with age less than 18 years, pregnancy, inflammatory or malignant disease, intracerebral hemorrhage or subarachnoid hemorrhage.
Previously, we determined in some of those patients serum levels of biomarkers related with inflammation, coagulation and oxidation such as substance P [32], soluble CD154 [33] and malondialdehyde [34]. The aim of the current research was to determine serum levels of a biomarker related with apoptosis, such as CCCK-18, in 66 patients with severe MMCAI.

Blood sample collection and serum CCCK-18 analysis
Serum blood samples were collected at the moment of the MMCAI diagnosis to measure serum CCCK-18 levels. All determinations were performed at the Laboratory Department of the Hospital Universitario de Canarias (La Laguna, Tenerife, Spain). We determine serum CCCK-18 levels by enzyme-linked immunosorbent assay (ELISA) using M30 Apoptosense® ELISA kit (PEVIVA AB, Bromma, Sweden). The intra-assay coefficient of variation (CV), inter-assay CV, and detection limit assay were < 10%, < 10% and 25 u/L respectively.

Statistical methods
Continuous and categorical variables were reported as medians (and interquartile ranges) and frequencies (and percentages) respectively. Continuous and categorical variables were compared between groups using Wilcoxon-Mann-Whitney test and chi-square test respectively. We carried out a multiple logistic regression to anlyze the association between serum CCCK-18 levels and mortality at 30 days after to control for platelet count and GCS. We calculated Odds Ratio and its 95% confidence intervals (CI) to measure the clinical impact of predictor variables. We performed receiver operating characteristic (ROC) curve to determine the prediction capacity of serum CCCK-18 levels for mortalty at 30 days. We constructed 30-day mortality Kaplan-Meier curves of patiens with higher and lower serum CCCK-18 levels than 298 u/L. Youden J index was used for the selection of 298 u/L as the optimal prognostic cut-off value of serum CCCK-18 level. All p-values lower than 0.05 were considered statistically significant. We performed statistical analyses using SPSS 17.0 (SPSS Inc., Chicago, IL, USA), LogXact 4.1, (Cytel Co., Cambridge, MA), and NCSS 2000 (Kaysville, Utah).
In the multiple logistic regression was found that serum CCCK-18 levels were associated with mortality at 30 days (OR = 1.023; 95% CI = 1.010-1.037; p = 0.001) after to control for platelet count and GCS (Table 2).

Discussion
To our knowledge, this is the first series reporting data on serum CCCK-18 levels in ischemic stroke patients. The novel findings of our study were that non-surviving severe MMCAI patients had higher serum CCCK-18 levels than surviving patients, and that there is an association between high serum CCCK-18 levels and MMCAI patients mortality.
Previously there has been found apoptotic changes in brain tissue samples from humans after cerebral ischemia [8][9][10][11][12][13]. However, the association between high serum CCCK-18 levels and MMCAI patients mortality found in our study is a novel finding. Those findings are in consonance with those of previous studies, due to that there is be found an association between high serum CCCK-18 levels and poor prognosis of patients with traumatic brain injury [28], acute spontaneous intracerebral haemorrhage [29] and aneurysmal subarachnoid hemorrhage [30].
The interpretation of all those findings is uncertain. Cytokeratin-18 exists mainly in the intracytoplasmic cytoskeleton of epithelial tissue and during apoptosis citokeratin-18 is cleaved by caspases and appears as CCCK-18 into the blood [14,15]. Then the question about the origen of CCCK-18 in patients with traumatic brain injury [28], spontaneous cerebral hemorrhage [29,30], and cerebral infarction (our current study) arise now. There is two posible splanations for that question. First, that there is cytokeratin-18 in brain; and this has been found in a study of patients with pituitary adenomas [36], and in a study of rats with glioma [37]. In the study by Luiciani et al. was found CCCK-18 in cell extracts of patients with pituitary adenomas, and the use of octreotide induced apoptosis in cells of growth hormone-secreting tumors assessed by the increased of CCCK-18 in cell extracts [36]. In the study by Adri et al was found CCCK-18 in cell extracts of glioma from rats, and the use of Parmelia sulcata Taylor (one of the most common lichens that lives mainly in the bark of the trees) induced apoptosis in cell tumors assessed by the increased of CCCK-18 in cell extracts [37]. Second, that MMCAI may cause a systemic inflammatory response syndrome (SIRS), and this could activate sistemic cellular apoptosis. In fact, there are studies reporting SIRS after cerebral infarction [38][39][40], and in SIRS appears different pro-inflammatory cytokines [41] that could activate apoptosis [2][3][4][5][6][7]. The administration of some antiapoptotic agents in ischemic cerebral animal models have reduced brain apoptosis degree and functional deficits [42][43][44].
Some limitations of our study should be recognized. First, data about the evolution of circulating CCCK-18 concentrations during the evolution of non-surviving and surviving patients were not reproted. Second, data about serum CCCK-18 levels in healthy controls were not reported; although, the objective of our study was to determine whether there is an association between serum CCCK-18 levels and mortality in MMCAI patients and was not to determine whether there is an increase of serum CCCK-18 levels in MMCAI patients. Third, we have not explored apoptosis in cerebral samples; although, the objective of our study was to determine whether apoptosis is associated with mortality of MMCAI patients using a technique easily reproducible by other researchers. Fourth, we have not data about how many patients were excluded from the study and the exclusion motivation.

Conclusions
To our knowledge, this is the first series reporting data on serum CCCK-18 levels in ischemic stroke patients. The novel findings of our study were that nonsurviving severe MMCAI patients had higher serum CCCK-18 levels than surviving patients, and that there is an association between high serum CCCK-18 levels and MMCAI patients mortality.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Authors' contributions LL conceived, designed and coordinated the study, participated in acquisition and interpretation of data, and drafted the manuscript. MMM, MA, LR, JSV, JJC, VGM participated in acquisition of data. APC participated in blood determination levels. AJ participated in the interpretation of data. All authors revised the manuscript critically for important intellectual content, made the final approval of the version to be published, and were agree to be accountable for all aspects of the work.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.

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